transforming-growth-factor-beta and Fractures--Bone

Mesenchymal stem cells (MSCs) have been identified in many adult tissues. MSCs can regenerate through cell division or differentiate into adipocytes, osteoblasts and chondrocytes. As a result, MSCs have become an important source of cells in tissue engineering and regenerative medicine for bone tissue and cartilage. Several epigenetic factors are believed to play a role in MSCs differentiation. Among these, microRNA (miRNA) regulation is involved in the fine modulation of gene expression during osteogenic/chondrogenic differentiation. It has been reported that miRNAs are involved in bone homeostasis by modulating osteoblast gene expression. In addition, countless evidence has demonstrated that miRNAs dysregulation is involved in the development of osteoporosis and bone fractures. The deregulation of miRNAs expression has also been associated with several malignancies including bone cancer. In this context, bone-associated circulating miRNAs may be useful biomarkers for determining the predisposition, onset and development of osteoporosis, as well as in clinical applications to improve the diagnosis, follow-up and treatment of cancer and metastases. Overall, this review will provide an overview of how miRNAs activities participate in osteogenic/chondrogenic differentiation, while addressing the role of miRNA regulatory effects on target genes. Finally, the role of miRNAs in pathologies and therapies will be presented.

Topics: Bone Diseases; Bone Morphogenetic Proteins; Chondrogenesis; Core Binding Factor Alpha 1 Subunit; Drug Delivery Systems; Fractures, Bone; Histone Deacetylases; Humans; Matrix Metalloproteinase 13; Mesenchymal Stem Cells; MicroRNAs; Osteogenesis; Repressor Proteins; Signal Transduction; Smad Proteins; Sp7 Transcription Factor; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

A fracture that does not unite in nine months is defined as nonunion. Nonunion is common in fragmented fractures and large bone defects where vascularization is impaired. The distal third of the tibia, the scaphoid bone or the talus fractures are furthermore prone to nonunion. Open fractures and spinal fusion cases also need special monitoring for healing. Bone tissue regeneration can be attained by autografts, allografts, xenografts and synthetic materials, however their limited availability and the increased surgical time as well as the donor site morbidity of autograft use, and lower probability of success, increased costs and disease transmission and immunological reaction probability of allografts oblige us to find better solutions and new grafts to overcome the cons. A proper biomaterial for regeneration should be osteoinductive, osteoconductive, biocompatible and mechanically suitable. Cytokine therapy, where growth factors are introduced either exogenously or triggered endogenously, is one of the commonly used method in bone tissue engineering. Transforming growth factor β (TGFβ) superfamily, which can be divided structurally into two groups as bone morphogenetic proteins (BMPs), growth differentiation factors (GDFs) and TGFβ, activin, Nodal branch, Mullerian hormone, are known to be produced by osteoblasts and other bone cells and present already in bone matrix abundantly, to take roles in bone homeostasis. BMP family, as the biggest subfamily of TGFβ superfamily, is also reported to be the most effective growth factors in bone and development, which makes them one of the most popular cytokines used in bone regeneration. Complications depending on the excess use of growth factors, and pleiotropic functions of BMPs are however the main reasons of why they should be approached with care. In this review, the Smad dependent signaling pathways of TGFβ and BMP families and their relations and the applications in preclinical and clinical studies will be briefly summarized.

Topics: Animals; Bone Morphogenetic Proteins; Bone Regeneration; Fractures, Bone; Humans; Mice; Signal Transduction; Transforming Growth Factor beta

Bone mineral density, a bone matrix parameter frequently used to predict fracture risk, is not the only one to affect bone fragility. Other factors, including the extracellular matrix (ECM) composition and microarchitecture, are of paramount relevance in this process. The bone ECM is a noncellular three-dimensional structure secreted by cells into the extracellular space, which comprises inorganic and organic compounds. The main inorganic components of the ECM are calcium-deficient apatite and trace elements, while the organic ECM consists of collagen type I and noncollagenous proteins. Bone ECM dynamically interacts with osteoblasts and osteoclasts to regulate the formation of new bone during regeneration. Thus, the composition and structure of inorganic and organic bone matrix may directly affect bone quality. Moreover, proteins that compose ECM, beyond their structural role have other crucial biological functions, thanks to their ability to bind multiple interacting partners like other ECM proteins, growth factors, signal receptors and adhesion molecules. Thus, ECM proteins provide a complex network of biochemical and physiological signals. Herein, we summarize different ECM factors that are essential to bone strength besides, discussing how these parameters are altered in pathological conditions related with bone fragility.

Topics: Animals; Bone and Bones; Bone Density; Bone Matrix; Collagen; Extracellular Matrix; Extracellular Matrix Proteins; Fractures, Bone; Homeostasis; Humans; Integrins; Matrix Metalloproteinases; Osteoblasts; Osteoclasts; Osteogenesis Imperfecta; Osteoporosis; Signal Transduction; Transforming Growth Factor beta; Wnt Proteins

Arthrofibrosis is a common complication for patients with bone fracture following external and internal fixation. In this review, we summarize the related factors and significant pathways for joint adhesion following fracture surgery. Moreover, the different types of treatments and related preventive measures are also discussed. Many factors related to the development and treatment of arthrofibrosis are discussed in this review in order to provide possible clues for the prospective targets to develop new medication or treatments for preventing or reducing the joint adhesion following orthopedic surgery.

Topics: Angiogenesis Inhibitors; Bevacizumab; Bone Morphogenetic Protein 2; Debridement; Fibrosis; Fractures, Bone; Humans; Injections, Intra-Articular; Joints; Manipulation, Orthopedic; Mitogen-Activated Protein Kinase 1; Platelet-Derived Growth Factor; Risk Factors; Splints; Substance P; Tissue Adhesions; Transforming Growth Factor beta

The development of therapeutics that target anabolic pathways involved in skeletogenesis is of great importance with regard to disease resulting in bone loss, or in cases of impaired bone repair. This review aims to summarize recent developments in this area.. A greater understanding of how drugs that modulate signaling pathways involved in skeletogenesis exert their efficacy, and the molecular mechanisms resulting in bone formation has led to novel pharmacological bone repair strategies. Furthermore, crosstalk between pathways and molecules has suggested signaling synergies that may be exploited for enhanced tissue formation. The sequential pharmacological stimulation of the molecular cascades resulting in tissue repair is a promising strategy for the treatment of bone fractures. It is proposed that a therapeutic strategy which mimics the natural cascade of events observed during fracture repair may be achieved through temporal targeting of tissue repair pathways.

Topics: Adaptor Proteins, Signal Transducing; Anabolic Agents; Antibodies, Neutralizing; Bone Density Conservation Agents; Bone Morphogenetic Proteins; Bone Remodeling; Bony Callus; Fibroblast Growth Factor 2; Fracture Healing; Fractures, Bone; Fractures, Ununited; Genetic Markers; Humans; Osteogenesis; Platelet-Derived Growth Factor; Signal Transduction; Teriparatide; Transforming Growth Factor beta; Wnt Signaling Pathway

Fracture healing is a complex biological process that requires interaction among a series of different cell types. Maintaining the appropriate temporal progression and spatial pattern is essential to achieve robust healing. We can temporally assess the biological phases via gene expression, protein analysis, histologically, or non-invasively using biomarkers as well as imaging techniques. However, determining what leads to normal versus abnormal healing is more challenging. Since the ultimate outcome of fracture healing is to restore the original functions of bone, assessment of fracture healing should include not only monitoring the restoration of structure and mechanical function, but also an evaluation of the restoration of normal bone biology. Currently few non-invasive measures of biological factors of healing exist; however, recent studies that have correlated non-invasive measures with fracture healing outcome in humans have shown that serum TGFbeta1 levels appear to be an indicator of healing versus non-healing. In the future, developing additional measures to assess biological healing will improve the reliability and permit us to assess stages of fracture healing. Additionally, new functional imaging technologies could prove useful for better understanding both normal fracture healing and predicting dysfunctional healing in human patients.

Topics: Animals; Biomarkers; Extracellular Matrix Proteins; Fracture Healing; Fractures, Bone; Fractures, Ununited; Humans; Mice; Time Factors; Transforming Growth Factor beta

Gene transfer technologies offer the prospect of enhancing bone regeneration by delivering osteogenic gene products locally to osseous defects. In most cases the gene product will be a protein, which will be synthesized endogenously within and around the lesion in a sustained fashion. It will have undergone authentic post-translational processing and lack the alterations that occur when recombinant proteins are synthesized in bioreactors and stored. Several different ex vivo and in vivo gene delivery strategies have been developed for this purpose, using viral and non-viral vectors. Proof of principle has been established in small animal models using a variety of different transgenes, including those encoding morphogens, growth factors, angiogenic factors, and transcription factors. A small number of studies demonstrate efficacy in large animal models. Developing these promising findings into clinical trials will be a long process, constrained by economic, regulatory and practical considerations. Nevertheless, the overall climate for gene therapy is improving, permitting optimism that applications in bone regeneration will eventually become available.

Topics: Bone Morphogenetic Proteins; Bone Regeneration; Fractures, Bone; Gene Transfer Techniques; Genetic Therapy; Genetic Vectors; Humans; Transforming Growth Factor beta; Transgenes; Vascular Endothelial Growth Factor A

Open fractures are plagued by high complication rates, among which infection and nonunion are the most common, leading to higher morbidity and poor patient outcomes. Despite meticulous surgical care and employment of adjunctive therapies, infection rates remain at 20%, due to the limitations of conventional therapies.. Persistent bacteria often survive initial debridement and treatment with antibiotics. Thus, the bone graft subsequently implanted to promote healing can be the nidus for infection. The principles of biofilm theory and the "race to the surface" have been applied to develop dual-purpose bone grafts that are protected by a sustained release of an antibiotic, thereby preventing bacterial colonization. A simultaneous sustained release of a recombinant human growth factor allows the defect to become vascularized and heal.. Current therapies fail to meet the challenges of open fractures. Tissue engineering and drug delivery approaches can address the challenges of healing large bone defects while protecting the implant from infection. When combined as an adjunctive therapy with existing clinical practices for management of open fractures, dual-purpose bone grafts that release both an antibiotic and a growth factor at biologically relevant time scales can potentially reduce infection rates and improve patient outcomes.

Topics: Animals; Anti-Bacterial Agents; Bacterial Infections; Bone Morphogenetic Protein 2; Bone Transplantation; Fractures, Bone; Fractures, Open; Humans; Recombinant Proteins; Transforming Growth Factor beta

Delay in fracture healing is a complex clinical and economic issue for patients and health services.. To assess the incremental effectiveness and costs of bone morphogenetic protein (BMP) on fracture healing in acute fractures and nonunions compared with standards of care.. We searched The Cochrane Library (2008, Issue 4), MEDLINE, and other major health and health economics databases (to October 2008).. Randomised controlled trials (RCTs) and full or partial economic evaluations of BMP for fracture healing in skeletally mature adults.. All clinical and economic data were extracted by one author and checked by another.. Eleven RCTs, all at high risk of bias, and four economic evaluations were included. Apart from one study, the times to fracture healing were comparable between the BMP and control groups. There was some evidence for increased healing rates, without requiring a secondary procedure, of BMP compared with usual care control in acute, mainly open, tibial fractures (risk ratio (RR) 1.19, 95% CI 0.99 to 1.43). The pooled RR for achieving union for nonunited fractures was 1.02 (95% CI 0.90 to 1.15). One study found no difference in union for patients who had corrective osteotomy for radial malunions. Data from three RCTs indicated that fewer secondary procedures were required for acute fracture patients treated with BMP versus controls (RR 0.65, 95% CI 0.50 to 0.83). Adverse events experienced were infection, hardware failure, pain, donor site morbidity, heterotopic bone formation and immunogenic reactions. The evidence on costs for BMP-2 for acute open tibia fractures is from one large RCT. This indicates that the direct medical costs associated with BMP would generally be higher than treatment with standard care, but this cost difference may decrease as fracture severity increases. Limited evidence suggests that the direct medical costs associated with BMP could be offset by faster healing and reduced time off work for patients with the most severe open tibia fractures.. This review highlights a paucity of data on the use of BMP in fracture healing as well as considerable industry involvement in currently available evidence. There is limited evidence to suggest that BMP may be more effective than controls for acute tibial fracture healing, however, the use of BMP for treating nonunion remains unclear. The limited available economic evidence indicates that BMP treatment for acute open tibial fractures may be more favourable economically when used in patients with the most severe fractures.

Topics: Adult; Bone Morphogenetic Protein 2; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Cost-Benefit Analysis; Fracture Healing; Fractures, Bone; Fractures, Malunited; Fractures, Ununited; Health Care Costs; Humans; Radius Fractures; Randomized Controlled Trials as Topic; Recombinant Proteins; Tibial Fractures; Transforming Growth Factor beta

This article is another review of clinical application of the use of bone morphogenetic proteins, specifically rhBMP2 Infuse Bonegraft, in the treatment of both acute and chronic fracture and fusion situations. Overall experience is reported with particular detail to the use of biologics in the treatment of problems involving the tibia, foot, and ankle.

Topics: Adolescent; Adult; Aged; Ankle Injuries; Arthrodesis; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Female; Fibula; Fracture Healing; Fractures, Bone; Fractures, Comminuted; Humans; Male; Middle Aged; Prospective Studies; Recombinant Proteins; Reoperation; Tibial Fractures; Transforming Growth Factor beta; Young Adult

Improvements in resuscitation, dissemination of ATLS protocols, and growth of regional and local trauma centers has increased the survivability after severe traumatic injuries. Furthermore, advances in medical management have increased life expectancy and also patients with orthopaedic injuries. While mechanical stabilization has been a hallmark of orthopaedic fracture care, orthobiologics are playing an increasing role in the management of these patients with complex injuries. Platelet-rich concentrate is an autologous concentration of platelets and growth factors, including transforming growth factor-beta (TGF-beta), vascular endothelial growth factor (VEGF), and platelet-derived growth factor (PDGF). The enhancement of bone and soft tissue healing by the placement of supraphysiologic concentration of autologous platelets at the site of tissue injury or surgery is supported by basic science and clinical studies. Due to the increased concentration and release of these factors, platelet-rich plasma can potentially enhance the recruitment and proliferation of tenocytes, stem cells, and endothelial cells. A better understanding of platelet function and appropriate clinical use is essential in achieving the desired outcomes of platelet-rich concentrate in orthopaedic clinical applications.

Topics: Blood Platelets; Fracture Healing; Fractures, Bone; Humans; Orthopedics; Platelet-Derived Growth Factor; Platelet-Rich Plasma; Soft Tissue Injuries; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

Bone morphogenetic proteins (BMPs) are multi-functional growth factors belonging to the transforming growth factor beta superfamily, especially BMP-2, induce bone formation in vivo, and clinical application in repair of bone fractures and defects is expected. However, appropriate systems to delivery BMPs for practical use need to be developed with the objective to heal cartilage and bone-related diseases in medical, dental and veterinary practice. Thus, the aim of this article was to present an overview of the principals carriers used to delivery BMPs and alternative delivery systems for these proteins.

Topics: Animals; Bone Morphogenetic Proteins; Bone Regeneration; Delayed-Action Preparations; Drug Carriers; Drug Delivery Systems; Fractures, Bone; Transforming Growth Factor beta; Wound Healing

Fracture healing is a complex physiological process involving a coordinated interaction of hematopoietic and immune cells within the bone marrow, in conjunction with vascular and skeletal cell precursors. Multiple factors regulate this cascade of molecular events, which affects different stages in the osteoblast and chondroblast lineage during processes such as migration, proliferation, chemotaxis, differentiation, inhibition, and extracellular protein synthesis. A clear understanding of the cellular and molecular pathways in fracture healing is not only critical for advancing fracture treatment, but it may also enhance further our knowledge of the mechanisms involved within skeletal growth and repair, as well as the mechanisms of aging. An overview of the important molecules involved in fracture healing, including osteogenic autocoids and inhibitory molecules, and their interactions and possible mechanisms of synergy during the healing process is presented in this article.

Topics: Bone Morphogenetic Proteins; Bone Regeneration; Cytokines; Fibroblast Growth Factors; Fracture Healing; Fractures, Bone; Humans; Signal Transduction; Transforming Growth Factor beta

Although fracture healing is a well-optimized biological process that leads to healing, approximately 10-20% of fractures result in impaired or delayed healing and these fractures may benefit from the use of biotechnologies to enhance skeletal repair. Peptide signaling molecules such as the bone morphogenetic proteins have been shown to stimulate the healing of fresh fractures, nonunions, and spinal fusions and side effects from their use appear to be minimal. Other growth factors currently being studied for local application include growth and differentiation factor-5 (GDF-5), vascular endothelial growth factor (VEGF), transforming growth factor beta (TGFbeta), and platelet-derived growth factor (PDGF). Molecules such as prostaglandin E receptor agonists and the thrombin-related peptide, TP508, have shown promise in animal models of fracture repair. Gene therapy using various growth factors or combinations of factors might also aid in fracture repair, particularly as new methods for delivery that do not require viral vectors are developed. Systemic therapy with agents such as parathyroid hormone (PTH), growth hormone (GH), and the HMG-CoA reductase inhibitors are also under investigation. As these and other technologies are shown to be safe and effective, their use will become a part of the standard of care in managing skeletal injuries.

Topics: Dinoprostone; Fracture Healing; Fractures, Bone; Genetic Therapy; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Intercellular Signaling Peptides and Proteins; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

Since the identification of the osteogenic protein-1 (OP-1) gene, also called bone morphogenetic protein-7 (BMP-7), almost 20 years ago, OP-1 has become one of the most characteristic members of the BMP family. The biological activity of recombinant human OP-1 has been defined using a variety of animal models. These studies have demonstrated that local implantation of OP-1 in combination with a collagen matrix results in the repair of critical size defects in long bones and in craniofacial bones and the formation of bony fusion masses in spinal fusions. Clinical trials investigating long bone applications have provided supportive evidence for the use of OP-1 in the treatment of open tibial fractures, distal tibial fractures, tibial nonunions, scaphoid nonunions and atrophic long bone nonunions. Clinical studies investigating spinal fusion applications have provided supportive evidence for the use of OP-1 in posterolateral lumbar models and compromised patients as an adjunct or as a replacement for autograft. Both long bone repair and spinal fusion studies have demonstrated the efficacy and safety of OP-1 by clinical outcomes and radiographic measures. Future clinical investigations will be needed to better define variables, such as dose, scaffold and route of administration. Clearly the use of BMPs in orthopaedics is still in its formative stage, but the data suggest an exciting and promising future for the development of new therapeutic applications.

Topics: Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Bone Transplantation; Fractures, Bone; Fractures, Ununited; Humans; Orthopedic Procedures; Spinal Fusion; Transforming Growth Factor beta

3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) are potent inhibitors of cholesterol biosynthesis. Cholesterol-lowering therapy using statins significantly reduces the risk of coronary heart disease. However, extensive use of statins leads to increases of other undesirable as well as beneficial effects, so-called pleiotropic effects. With respect to these effects, statins augment the expression of bone morphogenetic protein-2, a potent simulator of osteoblast differentiation and its activity, and promote mineralization by cultured osteoblasts, indicating that statins have an anabolic effect on bone. Chronic administration of statins in ovariectomized (OVX) rats modestly increases bone mineral density (BMD) of cancellous bone but not of compact bone. In clinical studies, there are conflicting results regarding the clinical benefits of this therapy for the treatment of osteoporosis. Observational studies suggest an association between statin use and reduction in fracture risk. Clinical trials reported no effect of statin treatment on BMD in hip and spine, and on bone turnover. Statins also may influence oral osseous tissues. Administration of statins in combination with osteoporosis therapy appears to improve alveolar bone architecture in the mandibles of OVX rats with maxillary molar extraction. Statins continue to be considered as potential therapeutic agents for patients with osteoporosis and possibly with periodontal disease. Development of new statins that are more specific and potent for bone metabolism will greatly increase the usefulness of these drugs for the treatment of bone diseases.

Topics: Alveolar Process; Animals; Anticholesteremic Agents; Bone and Bones; Bone Density; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Cell Differentiation; Fractures, Bone; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Osteoblasts; Osteoporosis; Transforming Growth Factor beta

The discovery of bone morphogenetic proteins marks a major step forward in the understanding of bone physiology and in the development of advanced methods in skeletal surgery. The cornerstones for successful growth-factor therapy in skeletal surgery remain biomechanical stability and biological vitality of the bone providing an adequate environment for new bone formation. Knowledge of the biological characteristics, mechanisms of action, and methods of delivery of growth factors will become essential for skeletal surgeons. The current clinical application of bone morphogenetic proteins is safe and efficacious as a result of a well-regulated cascade of events leading to bone formation. Clinical trials have not yet determined whether different clinical indications each require a specific bone-tissue-engineering format or if a single pathway for stimulating bone-healing with growth factors is sufficient.

Topics: Animals; Bone Morphogenetic Proteins; Cell Differentiation; DNA-Binding Proteins; Fracture Healing; Fractures, Bone; Genetic Therapy; Humans; Osteogenesis; Signal Transduction; Smad Proteins; Trans-Activators; Transforming Growth Factor beta

Treatment of fracture non-union is a challenging situation in skeletal surgery. Since the discovery of bone morphogenetic proteins (BMPs) by Urist preclinical research as well as clinical trials has shown the efficacy of these molecules in bone healing enhancement. Recombinant bone morphogenetic protein became available in UK during August 2001. We evaluated the type of indications and the efficacy of BMP-7 in a variety of clinical conditions including persistent fracture non-unions, augmentation of periprosthetic fracture treatment and osteotomies, enhancement of fracture healing following acetalular reconstruction, distraction osteogenesis, free fibular graft and arthrodesis of joints. Out of 653 cases, the overall success rate was 82% (535 cases). No local or systemic adverse effects were encountered. The role of BMP's as a bone stimulating agent is safe, well established and could be considered as a power adjunct in the surgeon's armamentarium for the treatment of these challenging clinical conditions.

Topics: Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Fracture Healing; Fractures, Bone; Humans; Osteogenesis; Transforming Growth Factor beta

Topics: Animals; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Clinical Trials as Topic; Drug Design; Fractures, Bone; Gene Expression Regulation, Developmental; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Intracellular Signaling Peptides and Proteins; Oligopeptides; Osteoblasts; Osteogenesis; Osteoporosis; Proteasome Inhibitors; Protein Serine-Threonine Kinases; rho GTP-Binding Proteins; rho-Associated Kinases; Transforming Growth Factor beta

Recently, several prospective randomized clinical trials have resulted in the publication of critical studies on the efficacy of recombinant human morphogenetic proteins BMP-2 (rhBMP-2) and BMP-7 (OP-1) in spinal fusion and fracture healing. The clinical use of BMPs is now in its infancy and understanding the mechanism and the appropriate application of these proteins is necessary for all practicing orthopedic surgeons. This article will revisit some of the early studies using rhBMPs and review the current literature on their role in fracture healing.

Topics: Animals; Bone Morphogenetic Protein 2; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Fracture Healing; Fractures, Bone; Fractures, Ununited; Humans; Recombinant Proteins; Spinal Fusion; Transforming Growth Factor beta

Topics: Animals; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Bone Resorption; Cell Division; Clinical Trials as Topic; Fractures, Bone; Gene Expression; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Hypolipidemic Agents; Osteoblasts; Osteogenesis; Osteoporosis; Stimulation, Chemical; Transforming Growth Factor beta

Topics: Acyl Coenzyme A; Animals; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Bone Resorption; Cell Differentiation; Diphosphonates; Fractures, Bone; GTP-Binding Proteins; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Mevalonic Acid; Osteoblasts; Osteoclasts; Osteogenesis; Osteoporosis; Polyisoprenyl Phosphates; Protein Prenylation; Sesquiterpenes; Signal Transduction; Transforming Growth Factor beta

Preclinical proof-of-concept, feasibility and efficacy studies in lower animals resulted in the accumulation of data that served as a backbone for clinical trials with the recombinant, osteogenic bone morphogenetic protein-2 (BMP-2). Among the important observations was the dependence of dose and carrier on the outcome for osseous union in relation to the animal model used. Clinical outcome data for spinal applications indicate better overall results compared with traditional controls that utilised autogenous iliac crest bone graft for fusion. Parameters include less blood loss, less operating room time and costs, better fusion outcomes and increased patient satisfaction. At this juncture, the long journey from the identification of BMP-2 in demineralised bone fraction to FDA approval for use in a singular orthopaedic application has been completed. It has been demonstrated to be safe, efficacious and cost-effective, leading to increased patient satisfaction and improved clinical outcome.

Topics: Bone Development; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Clinical Trials as Topic; Fractures, Bone; Humans; Orthopedic Procedures; Recombinant Proteins; Spinal Fusion; Transforming Growth Factor beta

Inefficient healing of bony and cartilaginous defects is a common situation encountered by orthopedic surgeons. Enhancing the regenerative potential of bone and articular cartilage has the potential for profound applications in treatment of nonunions, large segmental bone and cartilage defects, and arthritis. The bone morphogenetic proteins (BMPs) encode a highly conserved class of signaling factors that possess the ability to induce ectopic cartilage and bone formation in vivo. Bone morphogenetic protein family members are expressed during limb development, endochondral ossification, and early fracture and cartilage repair. Loss-of-function and gain-of-function studies have demonstrated the necessity and sufficiency of these genes, respectively, in regulating both cartilage and bone development. Several recent animal studies have demonstrated the potential of BMPs to enhance spinal fusion, repair critical-size defects, accelerate union, and heal articular cartilage lesions. A limited number of clinical trials using BMPs in human beings have been reported, and these agents are currently available for clinical use within and outside the United States. Current challenges to be met are the development of efficient delivery systems to present BMP proteins or genes to target sites and to enhance their duration and function at these locations.

Topics: Animals; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Cartilage, Articular; Cells, Cultured; Drug Delivery Systems; Fractures, Bone; Humans; Osteoarthritis; Spinal Fusion; Transforming Growth Factor beta; Wound Healing

In the US alone, approximately 500,000 patients annually undergo surgical procedures to treat bone fractures, alleviate severe back pain through spinal fusion procedures, or promote healing of non-unions. Many of these procedures involve the use of bone graft substitutes. An alternative to bone grafts are the bone morphogenetic proteins (BMPs), which have been shown to induce bone formation. For optimal effect, BMPs must be combined with an adequate matrix, which serves to prolong the residence time of the protein and, in some instances, as support for the invading osteoprogenitor cells. Several factors involved in the preparation of adequate matrices, specifically collagen sponges, were investigated in order to test the performance in a new role as an implant providing local delivery of an osteoinductive differentiation factor. Another focus of this review is the current system consisting of a combination of recombinant human BMP-2 (rhBMP-2) and an absorbable collagen sponge (ACS). The efficacy and safety of the combination has been clearly proven in both animal and human trials.

Topics: Animals; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Bone Regeneration; Clinical Trials as Topic; Collagen; Drug Carriers; Fractures, Bone; Humans; Oral Surgical Procedures; Recombinant Proteins; Spinal Fusion; Transforming Growth Factor beta

The statins may not only lower cholesterol, they may stimulate bone formation, as suggested by a number of observational studies and animal research. Whether these drugs will be of benefit in treating osteoporosis awaits further clinical trials.

Topics: Animals; Bone Density; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Female; Fractures, Bone; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Mevalonic Acid; Osteogenesis; Osteoporosis; Transforming Growth Factor beta

The use of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) to reduce serum cholesterol is well described. However, the recent finding that statins have direct effects on bone was unexpected. A number of epidemiological studies have recently been published that explore the effects of statins on bone mineral density and risk of fracture in humans. Statins may act by directly stimulating the expression of bone morphogenetic protein-2 and increasing osteoblast differentiation or, like nitrogen-containing bisphosphonates, may have effects on the mevalonate pathway that leads to inhibition of osteoclast activity and osteoblast apoptosis. In addition, the demonstration that statins can inhibit inflammation and encourage angiogenesis offers other possibilities for action.

Topics: Bone and Bones; Bone Density; Bone Development; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Female; Fractures, Bone; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Inflammation; Male; Neovascularization, Pathologic; Osteoblasts; Transforming Growth Factor beta

Topics: Animals; Bone Morphogenetic Proteins; Bone Regeneration; Combined Modality Therapy; Fibroblast Growth Factors; Fracture Fixation; Fracture Healing; Fractures, Bone; Growth Substances; Humans; Platelet-Derived Growth Factor; Sensitivity and Specificity; Somatomedins; Transforming Growth Factor beta

Topics: Animals; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Cartilage, Articular; Clinical Trials as Topic; Disease Models, Animal; Female; Fractures, Bone; Humans; Male; Orthopedics; Osseointegration; Osteogenesis; Prognosis; Rabbits; Sensitivity and Specificity; Transforming Growth Factor beta

Family and twin studies have established a genetic contribution to the etiology of osteoporosis. The genes and allelic variants conferring osteoporotic risk are largely undefined, but the number of candidates has increased steadily in recent years (Table I). Osteoporosis is a complex disease, and allelic variation in many other candidate-genes including those that encode growth factors, cytokines, calciotropic hormones, and bone matrix proteins are likely to also play a role and warrant systematic investigation. Most family and association studies to date have focused on the genetic contributions to bone density, a major determinant of bone strength and fracture risk. Bone density is not the only determinant of skeletal fragility, however, and genetic influences on fracture risk are independent of bone density [Cummings et al., 1995]. The microarchitectural properties and overall size and geometry of bone also influence skeletal strength [Bouxsein et al., 1996], and the genetic influences on these phenotypes should be investigated more rigorously. Even fewer studies have assessed the association between candidate-gene variation and the risk of fracture, the most important clinical outcome of osteoporosis. Large-scale molecular epidemiologic studies will be increasingly necessary in the future to quantify the relative, absolute and attributable risks of fracture associated with specific genetic variants. Osteoporosis is a complex, multifactorial disease, and most candidate-gene association studies have had limited statistical power to assess gene-gene and gene-environment interaction. Although gender plays an important role in the development of osteoporosis, genetic studies have almost exclusively focused on women, and have not tested whether gender modifies the association between genetic variation and osteoporotic risk. Therefore, future genetic studies will need to recruit larger samples of individuals including men. Rapid additional progress in our understanding of the molecular basis of osteoporosis can be expected in the near future as ongoing genome-wide linkage [Spotila et al., 1996] and candidate-gene association analyses are completed. Linkage analyses in families at high-risk for rare metabolic bone diseases should also yield important clues to the pathogenesis of osteoporosis. Recent examples are the mapping of loci for both high [Johnson et al., 1997] and low [Gong et al., 1996] bone mass to chromosome 11q and osteopetrosis to chromosome 1p [

Topics: Animals; Apolipoproteins E; Bone Density; Chromosome Mapping; Collagen; Female; Fractures, Bone; Genetic Predisposition to Disease; Humans; Interleukin-6; Male; Mice; Osteoporosis; Receptors, Calcitriol; Receptors, Estrogen; Risk Factors; Transforming Growth Factor beta

Fracture healing is a specialized form of the reparative process that the musculoskeletal system undergoes to restore skeletal integrity. This biologic process is a consequence of a complex cascade of biologic events that result in the restoration of bone tissue, allowing for the resumption of musculoskeletal function. Several growth-promoting substances have been identified at the site of skeletal injury and appear to play a physiologic role in fracture healing. This article reviews the effects of these osteoinductive growth factors on bone healing as elucidated by both preclinical in vivo fracture and diaphyseal defect healing models.

Topics: Animals; Bone Morphogenetic Proteins; Disease Models, Animal; Fibroblast Growth Factors; Fracture Healing; Fractures, Bone; Human Growth Hormone; Humans; Insulin-Like Growth Factor I; Osteogenesis; Platelet-Derived Growth Factor; Transforming Growth Factor beta

Topics: Animals; Bone Cements; Bone Marrow Transplantation; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Fracture Healing; Fractures, Bone; Genetic Therapy; Humans; Recombinant Proteins; Transforming Growth Factor alpha; Transforming Growth Factor beta

As shown in previous studies, the transforming growth factor beta superfamily of growth factors is involved in many aspects of skeletal development and regulation, including fracture repair and bone regeneration. Several studies have shown transforming growth factor beta messenger ribonucleicacid and protein expression in cells comprising fracture callus. In healing fractures in a chick model, differential isoform expression of the transforming growth factor betas was observed by in situ hybridization, with more prominent expression of the transforming growth factor beta 2 and transforming growth factor beta 3 isoforms. Small amounts of transforming growth factor beta 1 were present in early callus and increased in expression later during chondrogenesis and endochondral ossification. These findings resemble those reported in rat and human fracture callus. Transforming growth factor beta 4 expression was not significant in the chick fracture model. Transforming growth factor beta can function as a morphogen when injected subperiosteally, inducing cartilage and bone formation that morphologically resembles many of the events occurring in fracture callus. Exogenous transforming growth factor beta has been used in several critical size defect models of bone regeneration and fracture healing, with most of the studies showing increased bone or callus formation and increased mechanical stability. Numerous variables, including markedly different dose ranges and differing isoforms, dosing regimens, delivery methods, animal models, and various times and endpoint measures for analysis, make it difficult to comparatively assess the effects of transforming growth factor beta on bone healing. Additional study is necessary to satisfactorily determine the role of transforming growth factor beta in normal fracture healing and its potential for use in augmenting this process.

Topics: Animals; Bone Development; Bone Regeneration; Bony Callus; Cartilage; Chickens; Disease Models, Animal; Fracture Healing; Fractures, Bone; Gene Expression; Humans; Morphogenesis; Osteogenesis; Protein Isoforms; Rats; RNA, Messenger; Transforming Growth Factor beta

Topics: Animals; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Fractures, Bone; Growth Substances; Humans; Insulin-Like Growth Factor I; Platelet-Derived Growth Factor; Transforming Growth Factor beta

Topics: Animals; Cytokines; Fibroblast Growth Factors; Fracture Healing; Fractures, Bone; Humans; Transforming Growth Factor beta

Fractured bones heal by a cascade of cellular events in which mesenchymal cells respond to unknown regulators by proliferating, differentiating, and synthesizing extracellular matrix. Current concepts suggest that growth factors may regulate different steps in this cascade (10). Recent studies suggest regulatory roles for PDGF, aFGF, bFGF, and TGF-beta in the initiation and the development of the fracture callus. Fracture healing begins immediately following injury, when growth factors, including TGF-beta 1 and PDGF, are released into the fracture hematoma by platelets and inflammatory cells. TGF-beta 1 and FGF are synthesized by osteoblasts and chondrocytes throughout the healing process. TGF-beta 1 and PDGF appear to have an influence on the initiation of fracture repair and the formation of cartilage and intramembranous bone in the initiation of callus formation. Acidic FGF is synthesized by chondrocytes, chondrocyte precursors, and macrophages. It appears to stimulate the proliferation of immature chondrocytes or precursors, and indirectly regulates chondrocyte maturation and the expression of the cartilage matrix. Presumably, growth factors in the callus at later times regulate additional steps in repair of the bone after fracture. These studies suggest that growth factors are central regulators of cellular proliferation, differentiation, and extracellular matrix synthesis during fracture repair. Abnormal growth factor expression has been implicated as causing impaired or abnormal healing in other tissues, suggesting that altered growth factor expression also may be responsible for abnormal or delayed fracture repair. As a complete understanding of fracture-healing regulation evolves, we expect new insights into the etiology of abnormal or delayed fracture healing, and possibly new therapies for these difficult clinical problems.

Topics: Animals; Bone Matrix; Calcification, Physiologic; Cartilage; Fibroblast Growth Factor 1; Fibroblast Growth Factor 2; Fractures, Bone; Gene Expression; Growth Substances; Humans; Immunoenzyme Techniques; Osteoblasts; Platelet-Derived Growth Factor; Transforming Growth Factor beta

Natural selection has evolved a process of fracture healing that is characterized by callus formation and is enhanced by physical loading and motion at the fracture site. Internal fixation of fractures alters the biology of fracture healing, especially when rigid fixation using plates is performed. Intramedullary nails that allow some motion and loading usually are associated with callus formation. Plates that practically eliminate interfragmentary motion, however, prevent external periosteal callus formation. Primary osteonal healing can occur with plate fixation, but if interfragmental gaping of greater than 1 mm exists, delayed healing occurs. Closed treatment of fractures occasionally results in nonunion. In addition, physicians' high expectations for skeletal restoration have prompted vigorous research in areas of bone grafting, graft substitutes, and analysis of local and systemic factors that regulate fracture healing. A better understanding of the activity of bone morphogenetic protein and growth factors, such as transforming growth factor beta, will allow manipulation of the biology of healing to enhance the surgical treatment of skeletal trauma.

Topics: Bone Morphogenetic Proteins; Bone Transplantation; Bony Callus; Fracture Fixation, Internal; Fractures, Bone; Growth Substances; Humans; Orthopedic Fixation Devices; Prostaglandins; Proteins; Transforming Growth Factor beta; Weight-Bearing; Wound Healing

 The aim of this study was to assess bone density, bone architecture and clinical function of canine nonunion distal appendicular long bone fractures with a defect treated with fixation, compression-resistant matrix and recombinant human bone morphogenetic protein-2 (rhBMP-2)..  Prospective cohort study with dogs at least 1-year post treatment. Computed tomography was performed and quantitative measurements from previous fracture sites were compared with measurements from contralateral limbs. Subjective evaluation included gait assessment and palpation..  Six patients met the inclusion criteria. The rhBMP-2 treated bone exhibited higher density at the periphery and lower density in the centre, similar to the contralateral limb. All patients were weight bearing on the treated limb and all fractures were healed..  The rhBMP-2-treated bone underwent restoration of normal architecture and density. Acceptable limb function was present in all patients. The results of this study can serve as a basis for long-term response in treating nonunion fractures in veterinary patients.

Topics: Animals; Bone Morphogenetic Protein 2; Bone Regeneration; Dog Diseases; Dogs; Fracture Healing; Fractures, Bone; Fractures, Ununited; Humans; Prospective Studies; Recombinant Proteins; Transforming Growth Factor beta

Topics: Animals; Caveolae; Clathrin; Collagen Type I; Fractures, Bone; Mice; Osteogenesis Imperfecta; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta; X-Ray Microtomography

The management of bone defects and nonunions creates unique clinical challenges. Current treatment alternatives are often insufficient and frequently require multiple surgeries. One promising option is bone morphogenetic protein-2 (BMP-2), which is the most potent inducer of osteogenesis. However, its use is associated with many side effects, related to the delivery and high doses necessary. To address this need, we developed an ex vivo biomimetic hematoma (BH), replicating naturally healing fracture hematoma, using whole blood and the natural coagulants calcium and thrombin. It is an autologous carrier able to deliver reduced doses of rhBMP-2 to enhance bone healing for complex fractures. More than 50 challenging cases involving recalcitrant nonunions and bone defects have already been treated using the BH delivering reduced doses of rhBMP-2, to evaluate both the safety and efficacy. Preliminary data suggest the BH is currently the only clinically used carrier able to effectively deliver reduced doses (∼70% less) of rhBMP-2 with high efficiency, rapidly and robustly initiating the bone repair cascade to successfully reconstruct complex bone injuries without side effects. The presented case provides a clear demonstration of this technology's ability to significantly alter the clinical outcome in extremely challenging scenarios where other treatment options have failed or are considered unsuitable. A favorable safety profile would portend considerable promise for BH as an alternative to bone grafts and substitutes. Although further studies regarding its clinical efficacy are still warranted, this novel approach nevertheless has tremendous potential as a favorable treatment option for bone defects, open fractures, and recalcitrant nonunions.

Topics: Biomimetics; Bone Morphogenetic Protein 2; Fracture Healing; Fractures, Bone; Humans; Recombinant Proteins; Transforming Growth Factor beta; Treatment Outcome

Autologous bone grafts are considered the gold standard grafting material for the treatment of nonunion, but in very large bone defects, traditional autograft alone is insufficient to induce repair. Recombinant human bone morphogenetic protein 2 (rhBMP-2) can stimulate bone regeneration and enhance the healing efficacy of bone grafts. The delivery of rhBMP-2 may even enable engineered synthetic scaffolds to be used in place of autologous bone grafts for the treatment of critical size defects, eliminating risks associated with autologous tissue harvest. We here demonstrate that an osteoinductive scaffold, fabricated by combining a 3D printed rigid polymer/ceramic composite scaffold with an rhBMP-2-eluting collagen sponge can treat extremely large-scale segmental defects in a pilot feasibility study using a new sheep metatarsus fracture model stabilized with an intramedullary nail. Bone regeneration after 24 weeks was evaluated by micro-computed tomography, mechanical testing, and histological characterization. Load-bearing cortical bridging was achieved in all animals, with increased bone volume observed in sheep that received osteoinductive scaffolds compared to sheep that received an rhBMP-2-eluting collagen sponge alone.

Topics: Animals; Biomechanical Phenomena; Bone Morphogenetic Protein 2; Bone Regeneration; Bone Transplantation; Disease Models, Animal; Fracture Healing; Fractures, Bone; Histocytochemistry; Humans; Imaging, Three-Dimensional; Metatarsal Bones; Recombinant Proteins; Sheep; Tissue Scaffolds; Transforming Growth Factor beta; Translational Research, Biomedical; Transplantation, Autologous; X-Ray Microtomography

To study the bone induction and defect repair of true bone ceramics (TBC) combined with rhBMP-2 and Sr.. MC3T3-E1 cells were used to evaluate the bioactivity of the composite. Cell proliferation activity was detected by CCK-8, ALP activity was detected by p-nitrophenyl phosphate (PNPP), and the differences of material surface topography were observed by scanning electron microscopy (SEM). Bone induction was verified by the implantation in nude mice. The rabbit femoral condyle defect model was achieved to verify the bone defect repair ability of the material.. SEM results showed nearly the same surface morphology and cell proliferation quantified by CCK-8 showed that compared with TBC, both TBC&Sr and TBC&BMP-2&Sr had a significant promoting effect (P < 0.05). ALP activity result showed that the ALP activity of TBC&BMP-2&Sr was significantly higher than that of TBC alone (P < 0.05). The bone induction result showed that TBC&Sr had a small amount of new bone formation, and the new bone area was only 2.5 ± 0.11%. The bone induction activity of TBC&BMP-2&Sr was the highest, the new bone area was up to 75.36 ± 4.21%. Histological result of bone defect repair showed that TBC&BMP-2&Sr was also the highest, the new bone area was up to 72.42 ± 3.14%. The repair effect of TBC& BMP-2 was second, and better than that of TBC&Sr.. TBC combined with rhBMP-2 and Sr had the good bioactivity, obvious bone conduction and bone defect repair performance, laying the foundation of clinical application potentially.

Topics: Animals; Bone and Bones; Bone Morphogenetic Protein 2; Bone Regeneration; Bone Substitutes; Cell Differentiation; Cells, Cultured; Ceramics; Coated Materials, Biocompatible; Female; Fractures, Bone; Male; Materials Testing; Mice; Mice, Inbred BALB C; Mice, Nude; Osteogenesis; Rabbits; Recombinant Proteins; Strontium; Tissue Scaffolds; Transforming Growth Factor beta

In experiments on Wistar rats, a simulated defect in the flat bones of the skull was filled with a collagen sponge of animal origin impregnated with BMP-2 or pure sponge; in control rats, the defect was left open. During follow-up, X-ray density of the collagen sponge in the experimental groups differed significantly. The results attest to the absence of spontaneous remodeling of the bone tissue under conditions modeled focal defect. Moreover, stimulation of reparative processes by the collagen matrix did not lead to positive dynamics. Saturation of the collagen sponge with BMP-2 in a concentration of 0.05 mg/ml allowed increasing Xray density of the bone starting from week 4.

Topics: Animals; Biological Dressings; Bone Density; Bone Morphogenetic Protein 2; Bone Regeneration; Collagen; Fractures, Bone; Humans; Male; Osteogenesis; Parietal Bone; Rats; Rats, Wistar; Recombinant Proteins; Transforming Growth Factor beta; X-Ray Microtomography

Multiple myeloma is a plasma cell malignancy that causes debilitating bone disease and fractures, in which TGFβ plays a central role. Current treatments do not repair existing damage and fractures remain a common occurrence. We developed a novel low tumor phase murine model mimicking the plateau phase in patients as we hypothesized this would be an ideal time to treat with a bone anabolic. Using in vivo μCT we show substantial and rapid bone lesion repair (and prevention) driven by SD-208 (TGFβ receptor I kinase inhibitor) and chemotherapy (bortezomib and lenalidomide) in mice with human U266-GFP-luc myeloma. We discovered that lesion repair occurred via an intramembranous fracture repair-like mechanism and that SD-208 enhanced collagen matrix maturation to significantly improve fracture resistance. Lesion healing was associated with VEGFA expression in woven bone, reduced osteocyte-derived PTHrP, increased osteoblasts, decreased osteoclasts, and lower serum tartrate-resistant acid phosphatase 5b (TRACP-5b). SD-208 also completely prevented bone lesion development in mice with aggressive JJN3 tumors, and was more effective than an anti-TGFβ neutralizing antibody (1D11). We also discovered that SD-208 promoted osteoblastic differentiation (and overcame the TGFβ-induced block in osteoblastogenesis) in myeloma patient bone marrow stromal cells in vitro, comparable to normal donors. The improved bone quality and fracture-resistance with SD-208 provides incentive for clinical translation to improve myeloma patient quality of life by reducing fracture risk and fatality. © 2019 American Society for Bone and Mineral Research.

Topics: Alkaline Phosphatase; Animals; Bone Remodeling; Bortezomib; Cancellous Bone; Collagen; Disease Models, Animal; Female; Fractures, Bone; Green Fluorescent Proteins; Humans; Lenalidomide; Mesenchymal Stem Cells; Mice; Mice, SCID; Multiple Myeloma; Organ Size; Osteoblasts; Osteoclasts; Parathyroid Hormone-Related Protein; Pteridines; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A; Wound Healing; X-Ray Microtomography

Acquired heterotopic ossification (HO) is a painful and debilitating disease characterized by extraskeletal bone formation after injury. The exact pathogenesis of HO remains unknown. Here we show that TGF-β initiates and promotes HO in mice. We find that calcified cartilage and newly formed bone resorb osteoclasts after onset of HO, which leads to high levels of active TGF-β that recruit mesenchymal stromal/progenitor cells (MSPCs) in the HO microenvironment. Transgenic expression of active TGF-β in tendon induces spontaneous HO, whereas systemic injection of a TGF-β neutralizing antibody attenuates ectopic bone formation in traumatic and BMP-induced mouse HO models, and in a fibrodysplasia ossificans progressive mouse model. Moreover, inducible knockout of the TGF-β type II receptor in MSPCs inhibits HO progression in HO mouse models. Our study points toward elevated levels of active TGF-β as inducers and promoters of ectopic bone formation, and suggest that TGF-β might be a therapeutic target in HO.

Topics: Achilles Tendon; Adult; Animals; Antibodies, Neutralizing; Becaplermin; Bone Remodeling; Brain Injuries, Traumatic; Cartilage; Case-Control Studies; Disease Models, Animal; Elbow Injuries; Elbow Joint; Female; Fracture Fixation, Internal; Fractures, Bone; Humans; Male; Mesenchymal Stem Cells; Mice; Mice, Knockout; Mice, Transgenic; Middle Aged; Muscle, Skeletal; Myositis Ossificans; Ossification, Heterotopic; Osteoclasts; Osteogenesis; Receptor, Transforming Growth Factor-beta Type II; Spinal Cord Injuries; Tendon Injuries; Tendons; Transforming Growth Factor beta; Transforming Growth Factor beta1; Young Adult

Gerodermia osteodysplastica (GO) is characterized by skin laxity and early-onset osteoporosis. GORAB, the responsible disease gene, encodes a small Golgi protein of poorly characterized function. To circumvent neonatal lethality of the GorabNull full knockout, Gorab was conditionally inactivated in mesenchymal progenitor cells (Prx1-cre), pre-osteoblasts (Runx2-cre), and late osteoblasts/osteocytes (Dmp1-cre), respectively. While in all three lines a reduction in trabecular bone density was evident, only GorabPrx1 and GorabRunx2 mutants showed dramatically thinned, porous cortical bone and spontaneous fractures. Collagen fibrils in the skin of GorabNull mutants and in bone of GorabPrx1 mutants were disorganized, which was also seen in a bone biopsy from a GO patient. Measurement of glycosaminoglycan contents revealed a reduction of dermatan sulfate levels in skin and cartilage from GorabNull mutants. In bone from GorabPrx1 mutants total glycosaminoglycan levels and the relative percentage of dermatan sulfate were both strongly diminished. Accordingly, the proteoglycans biglycan and decorin showed reduced glycanation. Also in cultured GORAB-deficient fibroblasts reduced decorin glycanation was evident. The Golgi compartment of these cells showed an accumulation of decorin, but reduced signals for dermatan sulfate. Moreover, we found elevated activation of TGF-β in GorabPrx1 bone tissue leading to enhanced downstream signalling, which was reproduced in GORAB-deficient fibroblasts. Our data suggest that the loss of Gorab primarily perturbs pre-osteoblasts. GO may be regarded as a congenital disorder of glycosylation affecting proteoglycan synthesis due to delayed transport and impaired posttranslational modification in the Golgi compartment.

Topics: Animals; Bone Diseases; Cell Differentiation; Decorin; Dermatan Sulfate; Disease Models, Animal; Dwarfism; Female; Fractures, Bone; Glycosylation; Golgi Matrix Proteins; Mesenchymal Stem Cells; Mice, Inbred C57BL; Mice, Transgenic; Osteoblasts; Proteoglycans; Signal Transduction; Skin Diseases, Genetic; Transforming Growth Factor beta; Vesicular Transport Proteins

Electromagnetic bone growth stimulators have been found to biologically enhance the bone healing environment, with upregulation of numerous growth factors. The purpose of the study was to quantify the effect, in vivo, of pulsed electromagnetic fields (PEMFs) on growth factor expression and healing time in fifth metatarsal nonunions.. This was a prospective, randomized, double-blind trial of patients, cared for by 2 fellowship-trained orthopedic foot and ankle surgeons. Inclusion criteria consisted of patients between 18 and 75 years old who had been diagnosed with a fifth metatarsal delayed or nonunion, with no progressive signs of healing for a minimum of 3 months. Eight patients met inclusion criteria and were randomized to receive either an active stimulation or placebo PEMF device. Each patient then underwent an open biopsy of the fracture site and was fitted with the appropriate PEMF device. The biopsy was analyzed for messenger-ribonucleic acid (mRNA) levels using quantitative competitive reverse transcription polymerase chain reaction (QT-RT-PCR). Three weeks later, the patient underwent repeat biopsy and open reduction and internal fixation of the nonunion site. The patients were followed at 2- to 4-week intervals with serial radiographs and were graded by the number of cortices of healing.. All fractures healed, with an average time to complete radiographic union of 14.7 weeks and 8.9 weeks for the inactive and active PEMF groups, respectively. A significant increase in placental growth factor (PIGF) level was found after active PEMF treatment (P = .043). Other factors trended higher following active PEMF including brain-derived neurotrophic factor (BDNF), bone morphogenetic protein (BMP) -7, and BMP-5.. The adjunctive use of PEMF for fifth metatarsal fracture nonunions produced a significant increase in local placental growth factor. PEMF also produced trends toward higher levels of multiple other factors and faster average time to radiographic union compared to unstimulated controls.. Level I, prospective randomized trial.

Topics: Bone Morphogenetic Protein 5; Bone Morphogenetic Protein 7; Brain-Derived Neurotrophic Factor; Double-Blind Method; Electromagnetic Fields; Foot Injuries; Fracture Healing; Fractures, Bone; Humans; Metatarsal Bones; Outcome Assessment, Health Care; Prospective Studies; Transforming Growth Factor beta

Traumatic muscle loss (i.e., volumetric muscle loss [VML] injury) impairs adjacent fracture healing but is often left untreated. A promising therapy for this application is a decellularized extracellular matrix (ECM) because of their capacity to regenerate a vascularized tissue bed. This study tested the hypothesis that repair of VML concomitant to fracture with a small intestine submucosa (SIS)-ECM improves musculoskeletal healing.. In male Lewis rats (~375 g), a 3-mm segmental bone defect (SBD) was created in concomitance with a 6-mm, full-thickness VML injury to the adjacent tibialis anterior (TA) muscle. For all rats (n = 10), the SBD was treated with internal plate fixation and delivery of recombinant human bone morphogenetic protein 2 (1 μg) on a collagen sponge. The VML either had no repair or SIS-ECM repair (n = 5/group). Bone regeneration within the SBD (BV/TV [bone volume as a fraction of total volume]) was assessed via in vivo micro-computed tomography at 2, 4, and 6 weeks and histology at 6 weeks after injury. Tibialis anterior muscle in vivo strength and histologic assessments were performed at 6 weeks after injury.. Compared with no repair, SIS-ECM presented -21% (p = 0.09) and -27% (p = 0.004) BV/TV at 4 and 6 weeks after injury, respectively. At 6 weeks, the SBD gap length was shorter for the no repair than that for the SIS-ECM (2.64 ± 0.30 and 3.67 ± 0.41 mm, respectively; p = 0.09), whereas the distances from the end of each cortical segment to the center of the first stabilization screw were longer (1.86 ± 0.25 and 0.85 ± 0.30 mm, respectively; p = 0.035), indicating enhanced resorption in the SIS-ECM group. Both groups presented similar magnitude TA muscle strength deficits compared with their contralateral limbs (10-150 Hz: no repair, -58% to 67%; SIS-ECM, -51% to 74%). The TA muscle of the SIS-ECM group was remarkable for its presentation of fibrosis, edema, and immune cell presence.. Small intestine submucosa-ECM VML repair impaired open fracture healing and failed to improve skeletal muscle strength.

Topics: Animals; Bone Morphogenetic Protein 2; Disease Models, Animal; Extracellular Matrix; Fracture Healing; Fractures, Bone; Intestine, Small; Male; Muscle Strength; Muscle, Skeletal; Rats; Rats, Inbred Lew; Recombinant Proteins; Transforming Growth Factor beta; Treatment Failure

The impaired maturation of bone-forming osteoblasts results in reduced bone formation and subsequent bone weakening, which leads to a number of conditions such as osteogenesis imperfecta (OI). Transplantation of human fetal mesenchymal stem cells has been proposed as skeletal anabolic therapy to enhance bone formation, but the mechanisms underlying the contribution of the donor cells to bone health are poorly understood and require further elucidation. Here, we show that intraperitoneal injection of human amniotic mesenchymal stem cells (AFSCs) into a mouse model of OI (oim mice) reduced fracture susceptibility, increased bone strength, improved bone quality and micro-architecture, normalised bone remodelling and reduced TNFα and TGFβ sigalling. Donor cells engrafted into bones and differentiated into osteoblasts but importantly, also promoted endogenous osteogenesis and the maturation of resident osteoblasts. Together, these findings identify AFSC transplantation as a countermeasure to bone fragility. These data have wider implications for bone health and fracture reduction.

Topics: Amnion; Animals; Bone and Bones; Bone Remodeling; Cell Differentiation; Disease Models, Animal; Female; Flow Cytometry; Fractures, Bone; Gene Expression Profiling; Genetic Markers; Humans; Male; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Mice; Osteoblasts; Osteogenesis; Osteogenesis Imperfecta; Stress, Mechanical; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; X-Ray Microtomography

Much research is now being conducted in order to understand the role of cytokines in the development of the inflammatory response following trauma. The purpose of this study was to evaluate whether serum levels of certain cytokines, measured immediately after initial injury, can be used as potential biomarkers for predicting the development and the degree of severity of the systemic inflammatory response (SIRS) in patients with moderate and severe trauma.. We conducted a prospective study with 71 individuals of whom 13 (18.3 %) were healthy controls and 58 (81.7 %) were traumatized orthopaedic patients who were categorized into two groups: 31 (43.6 %) with moderate injuries and 27 (38.1 %) patients with severe orthopaedic trauma. Thirty cc of heparinized blood were drawn from each individual within a few hours after the injury. Serum levels of pro-inflammatory, regulatory and anti-inflammatory cytokines were measured in each individual participant.. High levels of pro-inflammatory cytokines IL-1β,-6,-8,-12, tumour necrosis factor alpha and interferon gamma were found in all injured patients compared to healthy controls. Only IL-6 and IL-8 were significantly higher in the injured patients. Levels of the regulatory cytokines, transformed growth factor beta (TGF-β) and IL-10 were higher in the injured patients, but significant only for TGF-β. Levels of IL-4 were significantly lower in the injured groups as compared to the controls.. Secretion of large amounts of pro-inflammatory cytokines and decreased level of anti-inflammatory cytokines during the acute phase of trauma may lead to the development of systemic inflammatory response syndrome (SIRS) in unstable polytraumatized patients. SIRS may result in life threatening conditions as acute respiratory distress syndrome (ARDS) and multiple organ failure (MOF). High levels of IL-6, IL-8, TGFβ and low levels of IL-4 were found to be reliable markers for the existence of immune reactivity in trauma patients. More research is needed to study pattern of cytokine levels along the acute period of injury, after surgical interventions and during recovery.

Topics: Adult; Biomarkers; Cytokines; Female; Fractures, Bone; Humans; Interleukin-4; Interleukin-6; Interleukin-8; Male; Middle Aged; Prospective Studies; Systemic Inflammatory Response Syndrome; Transforming Growth Factor beta; Wounds and Injuries; Young Adult

The incidence of osteoporotic fractures increases as our population ages. Until now, the exact biochemical processes that occur during the healing of metaphyseal fractures remain unclear. Diagnostic instruments that allow a dynamic insight into the fracture healing process are as yet unavailable. In the present matched pair analysis, we study the time course of the osteoanabolic markers bone specific alkaline phosphatase (BAP) and transforming growth factor β1 (TGFβ1), as well as the osteocatabolic markers crosslinked C-telopeptide of type-I-collagen (β-CTX) and serum band 5 tartrate-resistant acid phosphatase (TRAP5b), during the healing of fractures that have a low level of bone mineral density (BMD) compared with fractures that have a normal BMD. Between March 2007 and February 2009, 30 patients aged older than 50 years who suffered a metaphyseal fracture were included in our study. BMDs were verified by dual energy Xray absorptiometry (DXEA) scans. The levels of BTMs were examined over an 8-week period. Osteoanabolic BAP levels in those with low levels of BMD were significantly different from the BAP levels in those with normal BMD. BAP levels in the former group increased constantly, whereas the latter group showed an initial strong decrease in BAP followed by slowly rising values. Osteocatabolic β-CTX increased in the bone of the normal BMD group constantly, whereas these levels decreased significantly in the bone of the group with low BMD from the first week. TRAP5b was significantly reduced in the low level BMD group. With this work, we conduct first insights into the molecular biology of the fracture healing process in patients with low levels of BMD that explains the mechanism of its fracture healing. The results may be one reason for the reduced healing qualities in bones with low BMD.

Topics: Aged; Biomarkers; Bone and Bones; Bone Density; Case-Control Studies; Female; Fractures, Bone; Guanine Nucleotide Exchange Factors; Humans; Male; Middle Aged; Time Factors; Transforming Growth Factor beta; Wound Healing

The study was carried out to comparatively analyze metabolic profile and content of growth factors in blood serum of patients with retarded consolidation of fractures of bones of lower extremities. The evaluation was applied to concentration of metabolites, growth factors and enzyme activity of blood serum in 13 patients with retarded consolidation of fractures of thigh and shank bones (main group). The comparative group included 14 patients with solid fractures of thigh and shank bones. The analysis established that as compared to patients with solid fractures of bones, in patients with retarded consolidation of fractures blood serum contained reliably higher concentration of triglycerides, products of glycolysis, epidermal growth factor and transforming growth factors TGF-α and TGF-β2. The content of vitamin E and insullin-like growth factor (IGF-1) was decreased The given markers can be labeled as potential markers of diagnostic and prognosis of development of retarded consolidation of fractures.

Topics: Adult; Biomarkers; Female; Fractures, Bone; Humans; Insulin-Like Growth Factor I; Lower Extremity; Male; Metabolome; Middle Aged; Transforming Growth Factor alpha; Transforming Growth Factor beta; Vitamin E

Nonhealing bone defects are difficult to treat. As the bone morphogenic protein and transforming growth factor beta pathways have been implicated in bone healing, we hypothesized that percutaneous Smad7 silencing would enhance signaling through both pathways and improve bone formation. Critical sized parietal trephine defects were created and animals received percutaneous injection of: agarose alone or agarose containing nonsense or Smad7 small interfering RNA (siRNA). At 12 weeks, SMADs1, 2, 3, 5, 7 and 8 levels were assessed. Smad1/5/8 osteogenic target, Dlx5, and SMAD2/3 angiogenic target, plasminogen activator inhibitor-1 (Pai1), transcription levels were measured. Noncanonical signaling through TGFβ activated kinase-1 (Tak1) and target, runt-related transcription factor 2 (Runx2) and collagen1α1 (Col1α1), transcription were also measured. Micro-computed tomography and Gomori trichome staining were used to assess healing. Percutaneous injection of Smad7 siRNA significantly knocked down Smad7 mRNA (86.3 ± 2.5%) and protein levels (46.3 ± 3.1%). The SMAD7 knockdown resulted in a significant increase in receptor-regulated SMADs (R-SMAD) (Smad 1/5/8 and Smad2/3) nuclear translocation. R-SMAD nuclear translocation increased Dlx5 and Pai1 transcription. Additionally, noncanonical signaling through Tak1 increased Runx2 and Col1α1 target transcription. Compared with animals treated with agarose alone (33.9 ± 2.8% healing) and nonsense siRNA (31.5 ± 11.8% healing), animals treated Smad7 siRNA had significantly great (91.2 ± 3.8%) healing. Percutaneous Smad7 silencing increases signal transduction through canonical and noncanonical pathways resulting in significant bone formation. Minimally invasive gene therapies may prove effective in the treatment of nonhealing bone defects.

Topics: Animals; Bone Morphogenetic Proteins; Cell Nucleus; Disease Models, Animal; DNA-Binding Proteins; Fractures, Bone; Gene Knockdown Techniques; Genetic Therapy; Humans; Male; Mice; Osteogenesis; RNA, Messenger; RNA, Small Interfering; Signal Transduction; Skull; Smad Proteins, Receptor-Regulated; Smad7 Protein; Transforming Growth Factor beta

The reconstruction of segmental bone defects remains an urgent problem in the orthopaedic field, and bone morphogenetic protein-2 (BMP-2) is known for its potent osteoinductive properties in bone regeneration. In this study, chitosan microspheres (CMs) were prepared and combined with absorbable collagen sponge to maintain controlled-release recombinant human bone morphogenetic protein-2 (rhBMP-2). The rhBMP-2-loaded composite scaffolds were implanted into 15 mm radius defects of rabbits and the bone-repair ability was evaluated systematically. CMs were spherical in shape and had a polyporous surface, according to SEM images. The complex scaffold exhibited an ideal releasing profile in vitro. The micro-computed tomographic analysis revealed that the rhBMP-2-loaded composite scaffold not only bridged the defects as early as 4 weeks, but also healed the defects and presented recanalization of the bone-marrow cavity at 12 weeks. These results were confirmed by x-ray. When compared with other control groups, the composite scaffold group remarkably enhanced new bone formation and mechanical properties, as evidenced by bone mineral content evaluation, histological observations and biomechanical testing. Moreover, the biocompatibility and appropriate degradation of the composite scaffold could be obtained. All of these results clearly demonstrated that the composite scaffold is a promising carrier of BMP-2 for the treatment of segmental bone defects.

Topics: Animals; Bone Morphogenetic Protein 2; Bone Regeneration; Capsules; Chitosan; Collagen; Delayed-Action Preparations; Fracture Healing; Fractures, Bone; Male; Rabbits; Recombinant Proteins; Tissue Scaffolds; Transforming Growth Factor beta; Treatment Outcome

Muscle satellite cells make up a stem cell population that is capable of differentiating into myocytes and contributing to muscle regeneration upon injury. In this work we investigate the mechanism by which these muscle progenitor cells adopt an alternative cell fate, the cartilage fate. We show that chick muscle satellite cells that normally would undergo myogenesis can be converted to express cartilage matrix proteins in vitro when cultured in chondrogenic medium containing TGFß3 or BMP2. In the meantime, the myogenic program is repressed, suggesting that muscle satellite cells have undergone chondrogenic differentiation. Furthermore, ectopic expression of the myogenic factor Pax3 prevents chondrogenesis in these cells, while chondrogenic factors Nkx3.2 and Sox9 act downstream of TGFß or BMP2 to promote this cell fate transition. We found that Nkx3.2 and Sox9 repress the activity of the Pax3 promoter and that Nkx3.2 acts as a transcriptional repressor in this process. Importantly, a reverse function mutant of Nkx3.2 blocks the ability of Sox9 to both inhibit myogenesis and induce chondrogenesis, suggesting that Nkx3.2 is required for Sox9 to promote chondrogenic differentiation in satellite cells. Finally, we found that in an in vivo mouse model of fracture healing where muscle progenitor cells were lineage-traced, Nkx3.2 and Sox9 are significantly upregulated while Pax3 is significantly downregulated in the muscle progenitor cells that give rise to chondrocytes during fracture repair. Thus our in vitro and in vivo analyses suggest that the balance of Pax3, Nkx3.2 and Sox9 may act as a molecular switch during the chondrogenic differentiation of muscle progenitor cells, which may be important for fracture healing.

Topics: Animals; Bone Morphogenetic Protein 2; Cell Differentiation; Cells, Cultured; Chick Embryo; Chickens; Chondrogenesis; Fracture Healing; Fractures, Bone; Homeodomain Proteins; Mice; Muscle Development; Muscle Proteins; Paired Box Transcription Factors; PAX3 Transcription Factor; Satellite Cells, Skeletal Muscle; SOX9 Transcription Factor; Transcription Factors; Transforming Growth Factor beta

Traumatic injuries are usually associated with increased secretion of pro-inflammatory cytokines, and are sometimes followed by the development of acute stress symptoms (ASS) and posttraumatic stress symptoms (PTSS).. To measure serum pro- and anti-inflammatory cytokines in accident casualties and to associate it with ASS at hospitalization, and with PTSS 1 month later.. Participants were 48 patients, aged 20-60, hospitalized following various orthopedic injuries including bone fractures, and 13 healthy volunteers matched for gender. At hospitalization (Time 1), 30 ml heparinized venous blood were drawn and cytokines levels in serum were assessed; participants filled out the Acute Stress Disorder Inventory (ASDI), COPE, and injury-related questionnaires. One month later (Time 2), 26 participants filled out the Posttraumatic Disorder Symptom Scale (PDS).. High serum levels of IL-6, IL-8, and TGF-beta and low levels of serum IL-4 and IL-10 were found in injured patients as compared with controls, When controlling for age and severity of injury in the regression analysis, higher levels of IL-6 and IL-8 and lower TGF-beta were predicted by higher ASS and higher use of and emotion-focused coping. Higher PTSS scores at Time 2 were predicted by higher levels of IL-8, lower levels of TGF-beta, and higher ASS measured at Time 1.. High levels of the pro-inflammatory cytokine IL-6 and IL-8 and lower levels of the regulatory cytokine TGF-beta should be further assessed as a possible risk factor or a bio-marker of PTSS in accident casualties.

Topics: Accidents, Traffic; Adaptation, Psychological; Adult; Biomarkers; Cytokines; Emotions; Female; Fractures, Bone; Humans; Interleukins; Male; Middle Aged; Soft Tissue Injuries; Stress Disorders, Post-Traumatic; Stress Disorders, Traumatic, Acute; Transforming Growth Factor beta; Wounds and Injuries; Young Adult

BMP-binding peptide (BBP) enhances the osteogenic activity of recombinant human bone morphogenetic protein-2 (rhBMP-2), but the mechanism underlying the enhancement remains unclear. We aimed to elucidate the potential enhanced efficacy of BBP using critical-sized segmental femoral bone defects in rats. Seventy defects in seven groups of rats were filled with various amounts (0, 2, 5, and 10 microg) of rhBMP-2 with or without 1000 microg BBP. Radiographs were obtained after 4 and 8 weeks. The animals were euthanized at 8 weeks, and femoral specimens were assessed manually, evaluated for bone volume using microcomputed tomography, and subjected to histological or biomechanical analysis. Although 10 microg rhBMP-2 yielded consistent results in terms of bone healing and quality of bone repair across the segmental defect, lower doses of rhBMP-2 failed to induce satisfactory bone healing. However, the combined administration of lower doses of rhBMP-2 and BBP induced the formation of significantly large amounts of bone. Our results suggest that the combined administration of rhBMP-2 and BBP facilitates bone healing and has potential clinical applications.

Topics: Animals; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Bone Regeneration; Dose-Response Relationship, Drug; Drug Therapy, Combination; Femur; Fracture Healing; Fractures, Bone; Male; Models, Animal; Rats; Rats, Inbred Lew; Recombinant Proteins; Transforming Growth Factor beta; Treatment Outcome; X-Ray Microtomography

To report use of recombinant human bone morphogenetic protein-2 (rhBMP-2) as adjunctive therapy for treatment of a comminuted, open, proximal humeral fracture in an avian species.. Clinical report.. A 3.5-month-old male whooping crane (Grus americana).. An open, severely comminuted humeral facture was stabilized with an intramuscular (IM) pin/type IA external skeletal fixator with tie-in configuration. rhBMP-2 was applied in a calcium phosphate matrix (CPM) paste directly to the fracture site as a bone graft substitute. Radiographic evidence of bone healing was monitored for 14 weeks.. Substantial bony callus was evident at 4 weeks and at 8 weeks there was bridging callus with obvious bony remodeling. The fixation was destabilized at 9 weeks by IM pin removal, bone healing progressed and the fixator was removed at 11 weeks. By 14 weeks both cortices had been re-established with continued callus remodeling evident.. rhBMP-2, applied in a CPM paste, was used as a bone graft substitute in the treatment of a comminuted, open humeral fracture in a whooping crane.. Use of rhBMP-2/CPM should be considered in treatment of avian fractures.

Topics: Animals; Animals, Wild; Birds; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Bony Callus; Calcium Phosphates; Fracture Fixation, Internal; Fracture Healing; Fractures, Bone; Fractures, Comminuted; Humeral Fractures; Male; Recombinant Proteins; Transforming Growth Factor beta; Treatment Outcome

Chondrocytes express RANKL, but their role in osteoclastogenesis is not clear. We report that hypertrophic chondrocytes induce osteoclast formation through RANKL production stimulated by BMP2 and Runx2/Smad1 and thus they may regulate resorption of calcified matrix by osteoclasts at growth plates.. Bone morphogenetic protein (BMP) signaling and Runx2 regulate chondrogenesis during bone development and fracture repair and RANKL expression by osteoblast/stromal cells. Chondrocytes express RANKL, and this expression is stimulated by vitamin D3, but it is not known if chondrocytes directly support osteoclast formation or if BMPs or Runx2 is involved in this potential regulation of osteoclastogenesis.. The chondrocyte cell line, ATDC5, primary mouse sternal chondrocytes, and chick sternal chondrocytes were used. Cells were treated with BMP2, and expression of RANKL and chondrocyte marker genes was determined by real-time RT-PCR and Western blot. Chondrocytes and spleen-derived osteoclast precursors +/- BMP2 were co-cultured to examine the effect of chondrocyte-produced RANKL on osteoclast formation. A reporter assay was used to determine whether BMP2-induced RANKL production is through transcriptional regulation of the RANKL promoter and whether it is mediated by Runx2.. BMP2 significantly increased expression of RANKL mRNA and protein in all three types of chondrocytes, particularly by Col X-expressing and upper sternal chondrocytes. Chondrocytes constitutively induced osteoclast formation. This effect was increased significantly by BMP2 and prevented by RANK:Fc. BMP2 significantly increased luciferase activity of the RANKL-luc reporter, and Smad1 increased this effect. Deletion or mutation of Runx2 binding sites within the RANKL promoter or overexpression of a dominant negative Runx2 abolished BMP2- and Smad1-mediated activation of RANKL promoter activity.. Hypertrophic chondrocytes may regulate osteoclastogenesis at growth plates to remove calcified matrix through BMP-induced RANKL expression.

Topics: Animals; Bone Development; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Cell Differentiation; Cell Line, Tumor; Chickens; Cholecalciferol; Chondrocytes; Collagen Type IX; Core Binding Factor Alpha 1 Subunit; Extracellular Matrix; Fractures, Bone; Gene Expression Regulation; Growth Plate; Humans; Mice; Osteoclasts; RANK Ligand; Response Elements; RNA, Messenger; Smad1 Protein; Spleen; Stem Cells; Transforming Growth Factor beta; Vitamins

This study evaluated healing of equine metatarsal osteotomies and ostectomies in response to percutaneous injection of adenoviral (Ad) bone morphogenetic protein (BMP)-2, Ad-BMP-6, or beta-galactosidase protein vector control (Ad-LacZ) administered 14 days after surgery. Radiographic and quantitative computed tomographic assessment of bone formation indicated greater and earlier mineralized callus in both the osteotomies and ostectomies of the metatarsi injected with Ad-BMP-2 or Ad-BMP-6. Peak torque to failure and torsional stiffness were greater in osteotomies treated with Ad-BMP-2 than Ad-BMP-6, and both Ad-BMP-2- and Ad-BMP-6-treated osteotomies were greater than Ad-LacZ or untreated osteotomies. Gene expression of ostectomy mineralized callus 8 weeks after surgery indicated upregulation of genes related to osteogenesis compared to intact metatarsal bone. Expression of transforming growth factor beta-1, cathepsin H, and gelsolin-like capping protein were greater in Ad-BMP-2- and Ad-BMP-6-treated callus compared to Ad-LacZ-treated or untreated callus. Evidence of tissue biodistribution of adenovirus in distant organs was not identified by quantitative PCR, despite increased serum antiadenoviral vector antibody. This study demonstrated a greater relative potency of Ad-BMP-2 over Ad-BMP-6 in accelerating osteotomy healing when administered in this regimen, although both genes were effective at increasing bone at both osteotomy and ostectomy sites.

Topics: Adenoviridae; Animals; Biomechanical Phenomena; Bone Morphogenetic Protein 2; Bone Morphogenetic Protein 6; Bone Morphogenetic Proteins; Bony Callus; Disease Models, Animal; Fracture Healing; Fractures, Bone; Gene Expression Regulation; Gene Transfer Techniques; Genetic Therapy; Horses; Humans; Lac Operon; Metatarsal Bones; Oligonucleotide Array Sequence Analysis; Osteogenesis; Osteotomy; Tomography, X-Ray Computed; Torque; Torsion, Mechanical; Transforming Growth Factor beta

To compare the cost implications of treatment of persistent fracture non-unions before and after application of recombinant human bone morphogenetic protein-7 (BMP-7).. Of 25 fracture non-unions, 9 were treated using BMP-7 alone and 16 using BMP-7 and bone grafting. These patients were prospectively followed up, and the costs incurred were analysed.. The mean number of procedures per fracture performed before application of BMP-7 was 4.16, versus 1.2 thereafter. Mean hospital stay and cost of treatment per fracture before receiving BMP-7 were 26.84 days and pound 13,844.68, versus 7.8 days and pound 7338.4 thereafter. The overall cost of treatment of persistent fracture non-unions with BMP-7 was 47.0% less than that of the numerous previous unsuccessful treatments (p=0.001).. Treating fracture non-unions is costly, but this could be reduced by early BMP-7 administration when a complex or persistent fracture non-union is present or anticipated.

Topics: Adolescent; Adult; Aged; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Bone Regeneration; Bone Transplantation; Costs and Cost Analysis; Drug Costs; Female; Fracture Healing; Fractures, Bone; Fractures, Ununited; Humans; Length of Stay; Male; Middle Aged; Prospective Studies; Recombinant Proteins; Reoperation; Transforming Growth Factor beta; United Kingdom

The rate of bone formation to bridge a fracture gap slows with age. To explore potential pathogenic mechanisms and possible negative-feedback responses by the skeleton to this reduced rate of healing, mRNA transcripts up-regulated more and/or longer were studied in older rats with delayed healing. Female rats at 6 (young), 26 (adult), and 52 (old) weeks of age received unilateral diaphyseal femoral fractures with intramedullary rod stabilization. At 0, 0.4, 1, 2, 4, and 6 weeks after fracture, the fracture site was harvested. Total RNA was extracted, cRNA was prepared, and the cRNA was hybridized to 54 Affymetrix U34A microarrays (three arrays/age/time point). Transcripts for 180 genes were identified as up-regulated more and/or longer in old rats with delayed fracture healing. Of these, 60 were selected for more intense review. Significantly more and/or longer expression was seen in genes related to myofibroblasts, cell proliferation, calcification inhibition, TGF-beta activity, lipid metabolism, cell adhesion, and the cytoskeleton. Further study is needed to determine if these up-regulated transcripts are related to the pathological processes which slow healing or are related to attempts by the fracture tissue to stimulate bone to bridge the fracture gap.

Topics: Actins; Aging; Animals; Apolipoproteins E; Aryl Hydrocarbon Hydroxylases; Bony Callus; Cell Adhesion; Female; Fracture Healing; Fractures, Bone; Lipid Metabolism; Oligonucleotide Array Sequence Analysis; Rats; Rats, Sprague-Dawley; RNA, Messenger; Transforming Growth Factor beta; Up-Regulation

To describe outcome in dogs with insufficient bone healing treated with recombinant human bone morphogenetic protein-2 (rhBMP-2).. Retrospective study.. Four dogs clinically affected with delayed union or nonunion bone healing.. Medical records were reviewed for signalment, clinical problem, treatment, and outcome.. Four dogs that had delayed- or nonunion of bone fracture, osteotomy, or arthrodesis were treated with either minimally invasive, fluoroscopically guided, percutaneous administration or direct surgical application of rhBMP-2. Doses used ranged from 0.2 to 1.6 mg of rhBMP-2. In 3 dogs, a calcium phosphate matrix (CPM) carrier was used whereas in 1 dog commercially prepared rhBMP-2 impregnated in an absorbable collagen sponge (INFUSE Bone Graft) was used. This latter dog had osteomyelitis associated with implant infection before rhBMP-2 administration. Rapid radiographic union was noted in all dogs with excellent long-term outcome. Adverse effects were minimal and included transient worsening of lameness after percutaneous administration of rhBMP-2 in 2 dogs.. rhBMP-2 stimulated rapid bone formation at delayed- or nonunion sites resulting in radiographic bone union with minimal adverse effects and excellent long-term outcome in 4 dogs.. Direct intraoperative administration or fluoroscopically guided, minimally invasive delivery of rhBMP-2 may be an effective treatment modality for bone delayed- or nonunions and could potentially be used to stimulate new bone production in a variety of orthopedic surgical conditions in dogs.

Topics: Animals; Biocompatible Materials; Bone Cements; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Bone Plates; Collagen; Dogs; Female; Femoral Fractures; Fracture Healing; Fractures, Bone; Humans; Humeral Fractures; Male; Osteotomy; Radiography; Recombinant Proteins; Retrospective Studies; Tendon Injuries; Transforming Growth Factor beta; Treatment Outcome

A lyophilization method was developed to locally release adenoviral vectors directly from biomaterials for in situ regenerative gene therapy. Adenovirus expressing a beta-galactosidase reporter gene (AdLacZ) was mixed with different excipient formulations and lyophilized on hydroxyapatite (HA) disks followed by fibroblasts culturing and 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal) staining, suggesting 1 M sucrose in phosphate-buffered saline had best viability. Adenovirus release studies showed that greater than 30% virus remained on the material surface up to 16 h. Lyophilized adenovirus could be precisely localized in defined patterns and the transduction efficiency was also improved. To determine if the lyophilization formulations could preserve viral bioactivity, the lyophilized AdLacZ was tested after being stored at varying temperatures. Bioactivity of adenovirus lyophilized on HA was maintained for greater than 6 months when stored at -80 degrees C. In vivo studies were performed using an adenovirus encoding BMP-2 (AdBMP-2). AdBMP-2 was lyophilized in gelatin sponges and placed into rat critical-size calvarial defects for 5 weeks. Micro-computed tomography (micro-CT) analysis demonstrated that free-form delivery of AdBMP-2 had only modest effects on bone formation. In contrast, AdBMP-2 lyophilized in gelatin sponges led to more than 80% regeneration of critical-size calvarial defects.

Topics: Adenoviridae; Adenoviridae Infections; Animals; beta-Galactosidase; Biocompatible Materials; Bone and Bones; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Bone Regeneration; Durapatite; Fractures, Bone; Freeze Drying; Gelatin Sponge, Absorbable; Gene Expression; Genetic Engineering; Genetic Therapy; Genetic Vectors; Implants, Experimental; Injections; Rats; Rats, Inbred F344; Tomography, X-Ray Computed; Transduction, Genetic; Transforming Growth Factor beta

The direct, local, administration of adenovirus carrying human BMP-2 cDNA (Ad.BMP-2) heals critical-sized femoral bone defects in rabbit and rat models. However, the outcome is suboptimal and the technology needs to provide a more reliable and uniform outcome. To this end, we investigated whether the timing of Ad.BMP-2 administration influenced the formation of mineralized tissue within the defect. Critical-sized defects were created in the femora of 28 Sprague-Dawley rats. Animals were injected intralesionally with a single, percutaneous injection of Ad.BMP-2 (4 x 10(8) plaque-forming units) either intraoperatively (day 0) or 24 h (day 1), 5 days or 10 days after surgery. The femora were evaluated 8 weeks after surgery by X-ray, microcomputed tomography, dual-energy X-ray absorptiometry and biomechanical testing. The incidence of radiological union was markedly increased when administration of Ad.BMP-2 was delayed until days 5 and 10, at which point 86% of the defects healed. These time points also provided greater bone mineral content within the defect site and improved the average mechanical strength of the healed bone. Thus, delaying the injection of Ad.BMP-2 until 5 or 10 days after surgery enables a greater percentage of critical-sized, segmental defects to achieve radiological union, producing a repair tissue with enhanced mineralization and greater mechanical strength.

Topics: Adenoviridae; Animals; Bone and Bones; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Bone Regeneration; Fracture Fixation; Fracture Healing; Fractures, Bone; Genetic Therapy; Genetic Vectors; Male; Models, Animal; Rats; Rats, Sprague-Dawley; Transduction, Genetic; Transforming Growth Factor beta

In articular fractures, both bone and cartilage are injured. We tested whether stem cells transduced with bone morphogenetic protein 2 (BMP2) can promote bone and cartilage repair. Distal femoral articular osteotomies in nude rats were treated with stem cells, either wild-type or transduced with an adenoviral (Ad) BMP2. Cells were delivered in alginate (ALG) carrier or by direct injection in saline solution. Gene expression of these cells at the osteotomy site was confirmed by in vivo imaging. At day 14, only the group that received AdBMP2 stem cells by direct injection showed completely healed osteotomies, while other groups remained unhealed (P < 0.0003). In ALG groups, bone healing was impeded by the development of a chondroid mass, most pronounced in the AdBMP2 ALG group (P < 0.002). We were successful in achieving repair of both bone and cartilage in vivousing direct stem cell injection. Our data suggests that BMP2 augmentation might be critically important in achieving this effect.

Topics: Adenoviridae; Alginates; Animals; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Disease Models, Animal; Female; Fractures, Bone; Gene Transfer Techniques; Genes, Reporter; Genetic Therapy; Glucuronic Acid; Hexuronic Acids; Humans; Male; Mesenchymal Stem Cells; Osteotomy; Rats; Tomography Scanners, X-Ray Computed; Transforming Growth Factor beta

Previous studies have demonstrated the ability of direct adenoviral BMP-2 (Ad.BMP-2) gene delivery to enhance bone repair. Nevertheless, in studies using a rat segmental defect model, it has not proved possible to achieve reliably full osseous union in all animals. To address this issue, we evaluated the effect of vector dose on healing. Critical-size defects were created in the right femora of 27 Sprague-Dawley rats. The defects received a single, intralesional, percutaneous injection of 2.7 x 10(7) (low dose), 2.7 x 10(8) (medium dose), or 2.7 x 10(9) (high dose) plaque-forming units of Ad.BMP-2. After 8 weeks, femora were evaluated by X-ray, dual-energy X-ray absorptiometry, microcomputed tomography (microCT), and histology. The high dose of vector bridged 100%, the medium dose 11%, and the low dose 25% of the defects, as evaluated by X-ray and microCT imaging. Bone mineral content and bone volume of the defects receiving the high dose of vector were significantly higher than those of both groups receiving lower doses. Histologically, defects treated with the high dose were filled by trabecular bone and small amounts of cartilage, whereas large areas of fibrous tissue and cartilage remained in the defects receiving lower doses. However, the newly formed bone lacked the structural organization of native bone, suggesting that further maturation is necessary.

Topics: Animals; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Bone Regeneration; Fracture Healing; Fractures, Bone; Gene Transfer Techniques; Genetic Therapy; Genetic Vectors; Models, Animal; Rats; Rats, Sprague-Dawley; Transforming Growth Factor beta

Collagen/chitosan composite microgranules were fabricated as bone substitutes for the purpose of obtaining high bone-forming efficacy. The microgranules have the flexibility to fill various types of defect sites with closer packing. The interconnected pores formed spaces between the microgranules, which allowed new bone ingrowth and vascularization. In addition, the transforming growth factor-beta 1 (TGF-beta1) was incorporated into the microgranules in order to improve bone-healing efficacy. The collagen/chitosan microgranules were fabricated by dropping a mixed solution into a NaOH/ethanol solution. TGF-beta1 was loaded into the collagen/chitosan microgranules by soaking the microgranules in a TGF-beta1 solution. Scanning electron microscopy (SEM) observations and experiments examining the release of TGF-beta1 from chitosan and the collagen/chitosan microgranules were performed. SEM was used to examine the cell morphologies on the microgranules and cell proliferation was evaluated using a dimethylthiazole tetrazolium bromide assay. The differentiated cell function was assessed by measuring the alkaline phosphatase (ALPase) activity as well as detecting an osteocalcin assay. The in vivo bone-regeneration experiments were performed using a rabbit calvarial defect model. TGF-beta1 was released from the collagen/chitosan microgranules at a therapeutic concentration for 4 weeks. SEM indicated that the seeded osteoblastic cells were firmly attached to the microgranules and proliferated in a multilayer manner. The proliferation of the osteoblasts on the TGF-beta1-loaded microgranules was the highest among the different types of microgranules tested. The ALPase activity and osteocalcin level of all the samples increased during the culture period, and the TGF-beta1-loaded microgranules had a significantly higher ALPase activity and osteocalcin content than the other microgranules. The TGF-beta1-loaded microgranules demonstrated a higher bone-regenerative capacity in the rabbit calvarial defects after 4 weeks than the TGF-beta1-unloaded microgranules.

Topics: Animals; Bone Regeneration; Cell Differentiation; Cell Line; Cell Proliferation; Chitosan; Collagen; Delayed-Action Preparations; Fractures, Bone; Humans; Male; Mice; Microspheres; Osteoblasts; Osteogenesis; Rabbits; Skull; Transforming Growth Factor beta; Transforming Growth Factor beta1

We describe a 13-year-old boy with atrophic tibial pseudarthrosis associated with neurofibromatosis who had undergone nine unsuccessful operations. Eventually, union was obtained by the use of bone morphogenetic protein 7 in conjunction with intramedullary stabilisation and autologous bone graft.

Topics: Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Combined Modality Therapy; Fibula; Follow-Up Studies; Fractures, Bone; Humans; Infant; Male; Neurofibromatoses; Pseudarthrosis; Radiography; Tibia; Tibial Fractures; Transforming Growth Factor beta

The purpose of this study was to evaluate the influence of age on the effectiveness of rhBMP-7 treatment in a fracture with severe periosteal damage that is known to result in non-union formation. Closed stabilized femur fractures were produced in 3-month-old and 18-month-old rats. The fracture site was exposed and 2 mm of the periosteum cauterized circumferentially to impair normal fracture healing. The cauterized fracture site was immediately treated with either 100 microg rhBMP-7 (BMP-7 group), or with 25 microL of vehicle alone (control group). Fracture healing was evaluated with radiographs taken at 3 and 6 weeks. Animals were sacrificed at 3 and 6 weeks and specimens subjected to biomechanical and histological evaluation. In both age groups, none of the control animals healed throughout the 6 weeks experimental duration. All of the rhBMP-7-treated 3-month-old animals were radiographically healed at 3 weeks. In comparison, only 56% (9/16) of the rhBMP-7-treated 18-month-old animals were radiographically healed at 3 weeks. At 6 weeks, however, all of the 18-month-old rhBMP-7-treated animals had healed. Histology revealed slower healing in the 18-month-old animals. Treatment with rhBMP-7 significantly increased all of the biomechanical properties in both age groups. In the 3-month-old animals the mechanical strength approached that of the intact femur at 3 weeks, while in the 18-month-old animals this did not occur until 6 weeks. In conclusion, rhBMP-7 can effectively stimulate fracture repair in both young (3-month-old) and old (18-month-old) rats. However, the effect of rhBMP-7 on the rate of fracture healing is greater in young rats compared to old rats.

Topics: Aging; Animals; Biomechanical Phenomena; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Femoral Fractures; Fracture Healing; Fractures, Bone; Humans; Radiography; Rats; Rats, Inbred F344; Recombinant Proteins; Transforming Growth Factor beta

Critical size defects of bone and delayed fracture healing due to metabolic disorders are still problems in orthopaedic surgery. Adenoviral vectors encoding bone morphogenetic protein-2 (Ad.BMP-2) have been used to stimulate bone formation in small animals. The present study evaluated the use of direct adenoviral gene transfer for inducing bone formation in a large animal. Standardized iliac crest defects were created surgically on both sides of the pelvic bone of white mountain sheep. The efficiency of gene transfer was evaluated using recombinant adenoviruses carrying the cDNA for luciferase. High levels of transgene expression, restricted to the site of injection, were found for the 1st week. Transgene expression then fell considerably, but could still be detected for up to 5 weeks. To investigate the effect on bone healing, Ad.BMP-2 (10(11) particles in 200 mul saline) was unilaterally injected into iliac crest defects and into tibial osteotomies. The contralateral defects remained untreated to evaluate possible systemic effects. The controls were treated with saline solution. Bone formation within the defect, assessed by micro-computed tomography (CT) measurement at 8 weeks, and callus formation after osteotomy were significantly reduced following direct application of Ad.BMP-2. The retardation compared to untreated control animals was additionally found at the contralateral iliac crest indicating a systemic inhibitory effect. Histological analysis confirmed the CT measurement and showed an increased number of inflammatory cells within both defects. Antibodies against the adenovirus and the transgene product were detected in all treated animals. These data show a systemic retardation of bone formation following a single local injection of Ad.BMP-2 in sheep. This finding stands in contrast to the data obtained from small animal models. Further studies are needed to determine the contribution of the immune response to these results, and whether a lower dose of Ad.BMP-2 would be advantageous.

Topics: Adenoviridae; Animals; Antibodies, Viral; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Bony Callus; Female; Fracture Healing; Fractures, Bone; Gene Expression; Genetic Therapy; Genetic Vectors; Hip Fractures; Luciferases; Models, Animal; Osteogenesis; Osteotomy; Sheep; Tibial Fractures; Time Factors; Transduction, Genetic; Transforming Growth Factor beta; Transgenes

Topics: Biomedical Research; Bone Morphogenetic Protein 2; Bone Morphogenetic Protein Receptors; Bone Morphogenetic Proteins; Calcinosis; Fracture Healing; Fractures, Bone; Humans; Orthopedics; Transforming Growth Factor beta; United States

The aim of this study was to examine whether variants in genes for transforming growth factor beta1 (TGF-beta1; Leu10>Pro and Arg25>Pro), plasminogen activator inhibitor 1 (PAI-1; 4G>5G variant) and collagen 1 (COL1A1; Sp1 variant) may be useful in identifying individuals with increased susceptibility to early postmenopausal bone loss within the population of Czech women.. Polymorphisms were genotyped (by PCR and restriction analysis) in 1400 females representatively selected from the Czech population as well as in 218 postmenopausal osteoporotic women 40-70 years of age (mean age 58,7 years) and a 151 postmenopausal females within the same age range (mean age 59,1 years) with normal BMD.. We have not found any statistically significant differences in the frequency of individual genotypes or alleles of analyzed variants between the groups of osteoporotic patients (OP), population group (PG) and control group (CG). The frequency of the individual genotypes in the analyzed groups was as follows 1) TGF-beta1 gene: Leu10Leu10 OP 30.2 %, PG 35.6 %, CG 35.1 %; Leu10Pro10 OP 52.1 %, PG 47.1 %, CG 50.0 %; Pro10Pro10 OP 17.7 %, PG 17.3 %, CG 14.9 %; 2) TGF-beta1 gene Arg25 homozygotes OP 83.8 %, PG 86.1%, CG 89.3 %, Pro25 carriers OP 16.2 %, PG 13.9 %, CG 10.7 %, 3) PAI-1 gene: 4G4G OP 34.9 %, PG 31.8, CG 28.5 %, 5G4G OP 43.6 %, PG 46.7 %, CG 50.3 %, 5G5G OP 21.5 %, PG 21.5%, CG 21.2%, and 4) COL1A-1 ("SS" homozygotes, OP 63.0%, PG 63.7%, CG 64.6 %, "s" carriers OP 37.0 %, PG 36.3 %, CG 35.1 %).. Variants in genes for TGF-beta1 (Leu10>Pro and Arg25>Pro), PAI-1 (4G>5G) and COL1A1 (Sp1 variant) are not associated with low BMD in postmenopausal Czech Caucasian females.

Topics: Adult; Aged; Bone Density; Collagen Type I; Collagen Type I, alpha 1 Chain; Female; Fractures, Bone; Gene Frequency; Genetic Testing; Genotype; Humans; Middle Aged; Osteoporosis; Plasminogen Activator Inhibitor 1; Polymorphism, Genetic; Transforming Growth Factor beta; White People

A hydroxyapatite/type I collagen (HAp/Col) composite, aligning hydroxyapatite nano-crystals along collagen molecules, has been synthesized. The biocompatibility, osteoconductivity and efficacy as an rhBMP-2 carrier of this novel biomaterial implanted in the weight-bearing site have been examined. The HAp/Col implants adsorbing 0 or 400 microg/ml of rhBMP-2 were implanted into bone defects of tibiae in 3 beagle dogs and fixed according to the Ilizarov method. As a control, bone defects of 20 mm remaining in 2 beagle dogs and the dogs were allowed to walk using a Ilizarov external skeletal fixator. The radiological and histological findings suggest that the implants induce bone remodeling units and are a superior carrier of rhBMP-2 due to the stimulation of early callus and new bone formation. As a next step, anterior fusion was carried out on 6 beagle dogs with the implants adsorbing 400 microg/ml of rhBMP-2, and 9 dogs with the implants without rhBMP-2. In 3 dogs of the rhBMP-treated group, as well as 6 dogs of the non-rhBMP-treated group, the implant was fixed with a poly-L-lactide plate. Histological and radiographical analysis suggest that enhancement of callus formation and bone bridging by rhBMP-treatment is effective to prevent collapse of the implant.

Topics: Animals; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Bone Substitutes; Cervical Vertebrae; Collagen; Dogs; Drug Delivery Systems; Durapatite; Fracture Healing; Fractures, Bone; Implants, Experimental; Male; Manufactured Materials; Materials Testing; Osseointegration; Osteogenesis; Radiography; Radius; Spinal Fusion; Transforming Growth Factor beta; Treatment Outcome; Ulna

Severe periosteal and soft tissue disruption at the time of fracture may result in the formation of an atrophic nonunion. We have developed a reproducible atrophic nonunion in an animal model. The purpose of this study was to evaluate whether the immediate application of recombinant human BMP-7 to the fracture site could rescue the healing process in this nonunion model. A total of 56 three month old Fisher 344 rats were utilized. A 1.25 mm diameter K-wire was inserted into the femur in a retrograde fashion, and a mid-diaphyseal closed transverse fracture was created using a standard three point bending device. To create a nonunion, the fracture site was exposed and 2 mm of the periosteum was cauterized on each side of the fracture. The fracture site was immediately treated with either the application of rhBMP-7 50 microg in 25 microl of rat tail tendon collagen buffer (BMP-7 group), or with 25 microl of rat tail tendon collagen buffer only (Control group). Fracture healing was evaluated with serial radiographs every two weeks for an eight weeks period. Specimens at four and eight weeks were subjected to biomechanical and histological evaluation. None of the Control group healed throughout the eight weeks experimental duration. At four weeks 63% of the BMP-7 group had healed, and all had healed by six weeks. This investigation showed pronounced differences between the BMP-7 group and the Control group both histologically and biomechanically. In conclusion, we have demonstrated that the immediate application of BMP-7 may rescue the fracture healing process and prevent the development of nonunion following severe periosteal disruption.

Topics: Animals; Atrophy; Biomechanical Phenomena; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Dose-Response Relationship, Drug; Fracture Healing; Fractures, Bone; Humans; Male; Rats; Rats, Inbred F344; Recombinant Proteins; Transforming Growth Factor beta

One consideration for the practical use and application of osteogenic proteins is an effective method of delivery. This study evaluated a putty-type collagen carrier with recombinant human osteogenic protein-1 (rhOP-1) ability to heal canine critical sized (2.5 cm) ulna segmental defects compared to rhOP-1 with a particulate collagen carrier (OP device). The addition of carboxymethylcellulose (CMC) to the particulate collagen carrier (OPCMC device) to form the putty consistency was evaluated in two doses (3.5 and 1.75 mg rhOP-1/g carrier). The CMC greatly improved the intraoperative handling and site containment of the device. For the one-half dose and full-dose sites there were no statistically significant differences in the radiographic grading of defect healing when treated with the particulate OP-1 device and the device with CMC added. However, there was a dose effect with greater and earlier new bone formation observed with increased rhOP-1. Mechanically, there were no differences between particulate and putty formulations, although again, a significant effect was observed for treatment dose with the full-dose OPCMC device restoring 94% of the strength of the intact ulna compared to only 65% for the identical one-half dose implant. Regardless of rhOP-1 dose, the quality of union grading and total histologic score appearance were improved with the addition of CMC, although differences in histologic scoring were not statistically significant. Overall, the radiographic, mechanical, and histologic bone-healing characteristics with the one-half dose OPCMC device were similar to sites treated with the full-dose OP device. The observed improvement in healing may allow for lesser amounts of the device or a device with less rhOP-1 to be used.

Topics: Animals; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Bone Regeneration; Carboxymethylcellulose Sodium; Dogs; Fracture Healing; Fractures, Bone; Humans; Male; Materials Testing; Radiography; Transforming Growth Factor beta

In this study, hydrogel scaffolds, based on the polymer oligo(poly(ethylene glycol) fumarate) (OPF), were implanted into osteochondral defects in the rabbit model. Scaffolds consisted of two layers-a bottom, bone forming layer and a top, cartilage forming layer. Three scaffold formulations were implanted to assess how material composition and transforming growth factor-beta1 (TGF-beta1) loading affected osteochondral repair. Critical histological evaluation and scoring of the quantity and quality of tissue in the chondral and subchondral regions of defects was performed at 4 and 14 weeks. At both time points, no evidence of prolonged inflammation was observed, and healthy tissue was seen to infiltrate the defect area. The quality of this tissue improved over time with hyaline cartilage filling the chondral region and a mixture of trabecular and compact bone filling the subchondral region at 14 weeks. A promising degree of Safranin O staining and chondrocyte organization was observed in the newly formed surface tissue, while the underlying subchondral bone was completely integrated with the surrounding bone at 14 weeks. Material composition within the bottom, bone-forming layer did not appear to affect the rate of scaffold degradation or tissue filling. However, no bone upgrowth into the chondral region was observed with any scaffold formulation. TGF-beta1 loading in the top layer of scaffolds appeared to exert some therapeutic affect on tissue quality, but further studies are necessary for scaffold optimization. Yet, the excellent tissue filling and integration resulting from osteochondral implantation of these OPF-based scaffolds demonstrates their potential in cartilage repair strategies.

Topics: Absorbable Implants; Animals; Biocompatible Materials; Bone and Bones; Bone Diseases; Bone Substitutes; Cartilage; Cartilage, Articular; Chondrocytes; Coloring Agents; Disease Models, Animal; Fractures, Bone; Gelatin; Hydrogel, Polyethylene Glycol Dimethacrylate; Hydrogels; Inflammation; Lipid Bilayers; Materials Testing; Osteochondritis; Phenazines; Polyesters; Polyethylene Glycols; Rabbits; Regression Analysis; Time Factors; Tissue Engineering; Transforming Growth Factor beta; Transforming Growth Factor beta1; Wound Healing

The characteristic toughness and strength of bone result from the nature of bone matrix, the mineralized extracellular matrix produced by osteoblasts. The mechanical properties and composition of bone matrix, along with bone mass and architecture, are critical determinants of a bone's ability to resist fracture. Several regulators of bone mass and architecture have been identified, but factors that regulate the mechanical properties and composition of bone matrix are largely unknown. We used a combination of high-resolution approaches, including atomic-force microscopy, x-ray tomography, and Raman microspectroscopy, to assess the properties of bone matrix independently of bone mass and architecture. Properties were evaluated in genetically modified mice with differing levels of TGF-beta signaling. Bone matrix properties correlated with the level of TGF-beta signaling. Smad3+/- mice had increased bone mass and matrix properties, suggesting that the osteopenic Smad3-/- phenotype may be, in part, secondary to systemic effects of Smad3 deletion. Thus, a reduction in TGF-beta signaling, through its effector Smad3, enhanced the mechanical properties and mineral concentration of the bone matrix, as well as the bone mass, enabling the bone to better resist fracture. Our results provide evidence that bone matrix properties are controlled by growth factor signaling.

Topics: Animals; Bone and Bones; Bone Density; Bone Matrix; Extracellular Matrix; Fracture Healing; Fractures, Bone; Gene Deletion; Gene Expression Regulation; Kinetics; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microscopy, Atomic Force; Osteoblasts; Phenotype; RNA, Messenger; Signal Transduction; Smad3 Protein; Spectrum Analysis, Raman; Stress, Mechanical; Tomography, X-Ray; Transforming Growth Factor beta

In recent clinical trials demonstrating the efficacy of recombinant human bone morphogenetic protein-2 (rhBMP-2) for the acceleration of bone-healing, investigators used carriers requiring open surgery for administration. In this study, we used a nonhuman primate fibular osteotomy model to evaluate injectable rhBMP-2/carrier formulations that can be administered in closed fractures.. The fibular osteotomy model was first characterized by evaluating surgically harvested fibular segments containing untreated osteotomy sites (controls) from seventy adult male Cynomolgus monkeys at eight weeks (twenty-four monkeys), ten weeks (thirty-four), twelve weeks (six), and fourteen weeks (six). Fibular segments, from twenty-four animals, in which an osteotomy had not been performed served as normal controls (intact). The contralateral limb of twenty-four of the animals was then used to evaluate the effect of rhBMP-2 administered, three hours after the osteotomy, in eight carrier formulations (buffer, calcium phosphate paste, and hyaluronan gel, hyaluronan paste, and gelatin foam formulated with and without tricalcium phosphate granules). Each carrier was used in three monkeys. At ten weeks, the fibulae with the treated osteotomy sites were harvested and were compared with the contralateral, untreated osteotomized fibulae (paired control). The most promising carrier, calcium phosphate paste (alpha bone substitute material, or alpha-BSM), was then evaluated in eleven additional animals. The outcomes included the findings on radiographs made weekly until the time of fibular harvest, the callus area, the biomechanical properties, and the histologic findings.. Radiographic and histologic studies confirmed complete bridging of the control osteotomy sites in most animals by fourteen weeks. The mean torsional stiffness and maximum torque of the control osteotomy sites were 42.7% and 53.7%, 55.2% and 60.4%, 66.7% and 66.4% of the mean torsional stiffness and maximum torque of the intact fibulae at eight, ten, and twelve weeks, respectively, but they were not substantially different from the mean torsional stiffness and maximum torque of the intact fibulae at fourteen weeks (82.3% and 79.8%). In the carrier screening study, outcome measures of healing were more consistently enhanced in the rhBMP-2/alpha-BSM-treated osteotomy sites. In the confirmatory study, the mean callus area, torsional stiffness, and maximum torque were 86%, 72%, and 68% greater in the rhBMP-2/alpha-BSM-treated osteotomy sites than in the paired-control osteotomy sites at ten weeks (p < 0.001). The torsional stiffness and maximum torque in the rhBMP-2/alpha-BSM-treated osteotomy sites were equal to those in the intact fibulae, whereas those parameters in the paired-control osteotomy sites were only 55% and 58%, respectively, of the torsional stiffness and maximum torque of the intact fibulae. Histologic analysis confirmed complete osseous bridging of the rhBMP-2/alpha-BSM-treated osteotomy sites but incomplete bridging of the paired-control osteotomy sites at ten weeks.. A single percutaneous injection of rhBMP-2/alpha-BSM accelerates the healing of fibular osteotomy sites in nonhuman primates by approximately 40% compared with the healing of untreated osteotomy sites.

Topics: Animals; Biomechanical Phenomena; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Calcium Phosphates; Drug Carriers; Fracture Healing; Fractures, Bone; Injections; Macaca fascicularis; Male; Ointments; Osteotomy; Radiography; Time Factors; Transforming Growth Factor beta

Genetic contributions play an important role in determining bone mineral density (BMD) and bone turnover. Transforming growth factor-beta (TGF-beta) is abundant in bone and has been implicated as an important regulator of both bone formation and resorption. Several polymorphisms of the TGF-beta1 gene have recently been suggested to be associated with BMD and susceptibility to osteoporotic spine fractures. To determine the relationship between TGF-beta1 polymorphisms and BMD in southern Chinese women, three SNPs at C(-1348) -T, T29 -C, and T(861-20) -C of TGF-beta1 gene were analyzed in 237 postmenopausal southern Chinese women by RFLP and direct sequencing. BMD at the lumbar spine and hip region, biochemical markers of bone turnover, as well as serum levels of TGF-beta1 were measured. Only the T29 -C polymorphism of TGF-beta1 gene was associated with BMD and fracture risk. The prevalence of fragility fractures was significantly higher in individuals with TC genotype (P < 0.05). Serum alkaline phosphatase and osteocalcin levels as well as urinary N-telopeptide excretion were significantly higher in women with TC than with TT or CC genotypes, and the difference remained significant after adjusting for age and BMI (all P < 0.05). Women with TC genotype had lower BMD at the trochanteric (P < 0.03) and total hip region (P = 0.05). No difference was observed in the serum TGF-beta1 levels among the three genotypes. In conclusion, an association between T29 -C polymorphisms of TGF-beta1 gene and BMD, bone turnover as well as fragility fractures were demonstrated in postmenopausal southern Chinese women.

Topics: Aged; Asian People; Biomarkers; Bone and Bones; Bone Density; Bone Remodeling; Female; Fractures, Bone; Hong Kong; Humans; Osteoporosis, Postmenopausal; Polymerase Chain Reaction; Polymorphism, Genetic; Polymorphism, Restriction Fragment Length; Risk; Transforming Growth Factor beta; Transforming Growth Factor beta1

Topics: Aged; Amino Acid Sequence; Bone Density; Cohort Studies; Female; Fractures, Bone; Genetic Predisposition to Disease; Genotype; Humans; Leucine; Multivariate Analysis; Osteoporosis; Polymorphism, Genetic; Proline; Proportional Hazards Models; Risk Factors; Transforming Growth Factor beta; Transforming Growth Factor beta1; United States; White People

Hydroxyapatite is a synthetic bone graft, which is used for the treatment of bone defects and nonunions. However, it is a rather inert material with no or little intrinsic osteoinductive activity. Recombinant human osteogenic protein-1 (rhOP-1) is a very potent biological agent, that enhances osteogenesis during bone repair. Bone marrow contains mesenchymal stem cells, which are capable of new bone formation. Biosynthetic bone grafts were created by the addition of rhOP-1 or bone marrow to granular porous hydroxyapatite. The performance of these grafts was tested in a sheep model and compared to the results of autograft, which is clinically the standard treatment of bone defects and nonunions. A 3 cm segmental bone defect was made in the tibia and fixed with an interlocking intramedullary nail. There were five treatment groups: no implant (n=6), autograft (n=8), hydroxyapatite alone (n=8), hydroxyapatite loaded with rhOP-1 (n=8), and hydroxyapatite loaded with autologous bone marrow (n=8). At 12 weeks, healing of the defect was evaluated with radiographs, a torsional test to failure, and histological examination of longitudinal sections through the defect. Torsional strength and stiffness of the healing tibiae were about two to three times higher for autograft and hydroxyapatite plus rhOP-1 or bone marrow compared to hydroxyapatite alone and empty defects. The mean values of both combination groups were comparable to those of autograft. There were more unions in defects with hydroxyapatite plus rhOP-1 than in defects with hydroxyapatite alone. Although the differences were not significant, histological examination revealed that there was more often bony bridging of the defect in both combination groups and the autograft group than in the group with hydroxyapatite alone. Healing of bone defects, treated with porous hydroxyapatite, can be enhanced by the addition of rhOP-1 or autologous bone marrow. The results of these composite biosynthetic grafts are equivalent to those of autograft.

Topics: Animals; Biocompatible Materials; Biomechanical Phenomena; Bone Marrow Transplantation; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Bone Transplantation; Combined Modality Therapy; Drug Therapy, Combination; Durapatite; Female; Fracture Healing; Fractures, Bone; Humans; Radiography; Recombinant Proteins; Sheep; Tibia; Torsion Abnormality; Transforming Growth Factor beta; Transplantation, Autologous

Osteoporosis is a disease that is strongly genetically determined and polymorphisms present in a range of candidate genes may be involved. A number of previous studies have shown an association between the T869C functional polymorphism of the gene for transforming growth factor beta (TGF beta) and bone mineral density (BMD) and fracture, but these studies have been limited to relatively small studies of selected subjects. In a population-based study of 1337 white women over age 70 we examined the TGF beta T869 polymorphism in relation to BMD, calcaneal quantitative ultrasound (QUS), and prevalent and incident fracture. The TGF beta C allele was observed in 50% of the subjects and was associated with reduced hip BMD at all sites (2.8% total hip, 2.4% femoral neck, 2.6% intertrochanter, and 3.4% trochanter) compared to the TGF beta TT genotype. The TGF beta C allele was also associated with a reduction in the QUS parameters BUA, SOS, and stiffness of 0.87%, 0.26%, and 2.4%, respectively, compared to the TGF beta TT genotype. After adjustment for body mass index in an analysis of variance model, the effect of the TGF beta C allele remained significant at the total hip, the femoral neck, and the trochanter, and for the QUS SOS and stiffness parameters. The TGF beta C allele was associated with an increase in osteoporosis [T score < or =-2.5 SD; odds ratio (OR) 2.07; 95% confidence interval (CI) 1.19-3.60] and prevalent fracture (1.37; 95% CI 1.06-1.75). After adjustment for BMD and QUS stiffness, the association of the TGF beta C allele with prevalent fracture was still present (OR 1.40; 95% CI 1.04-1.89), suggesting that the effect of the C allele on fracture was independent of a reduction in BMD and QUS stiffness. Subjects with normal BMD and a TGF beta C allele had an increased risk of incident fracture over 3 years compared to subjects with normal BMD and a TGF beta TT genotype (relative risk 3.95; 95% CI 1.52-10.29). This association was not found in osteopenic or in osteoporotic subjects, indicating a BMD-TGF beta C allele interaction in relation to the association of the TGF beta C allele with fracture risk. These findings are of potential clinical usefulness, as the TGF beta T869C genotype could be used, in conjunction with other genetic and clinical information, to determine an individual's risk of osteoporosis.

Topics: Aged; Bone Density; Bone Diseases, Metabolic; Calcaneus; Female; Fractures, Bone; Gene Frequency; Genetic Predisposition to Disease; Humans; Incidence; Osteoporosis; Polymorphism, Genetic; Prevalence; Risk Factors; Transforming Growth Factor beta; Ultrasonography

Of the various growth factors involved in the healing response after a fracture, bone morphogenetic proteins (BMPs) are emerging as key modulators. BMPs exert their effects by binding to a complex of type I and type II receptors leading to the phosphorylation of specific downstream effector proteins called Smads. The current study examined the presence of BMP signaling components in human callus obtained from five nascent malunions undergoing fracture fixation. These callus samples represented various stages of bone healing and a mixture of endochondral and intramembraneous bone healing. We performed immunohistochemistry on the callus, using antibodies for BMP (BMP-2,-3,-4,-7), their receptors (BMPR-IA, -IB, -II), and phosphorylated BMP receptor-regulated Smads (pBMP-R-Smads). Active osteoblasts showed fairly consistent positive staining for all BMPs that were examined, with the immunoreactivity most intense for BMP-7 and BMP-3. Immunostaining for BMPs in osteoblasts appeared to colocalize with the expression of BMPR-IA, -IB, and -II. Positive immunostaining for pBMP-R-Smads suggests that the BMP receptors expressed in these cells are activated. Staining for BMPs in cartilage cells was variable. The immunostaining appeared stronger in more mature cells, whereas staining for BMP receptors in cartilage cells was less ubiquitous. However, the expression of pBMP-R-Smads in cartilage cells suggests active signal transduction. Fibroblast-like cells also had a variable staining pattern. Overall, our findings indicate the presence of BMPs, their various receptors, and activated forms of receptor-regulated Smads in human fracture callus. To the best of our knowledge, this is the first study that documents the expression of these proteins in human fracture tissue. Complete elucidation of the roles of BMP in bone formation will hopefully lead to improved fracture healing care.

Topics: Adolescent; Adult; Bone Morphogenetic Protein 2; Bone Morphogenetic Protein 3; Bone Morphogenetic Protein 4; Bone Morphogenetic Protein 7; Bone Morphogenetic Protein Receptors, Type I; Bone Morphogenetic Protein Receptors, Type II; Bone Morphogenetic Proteins; Bony Callus; DNA-Binding Proteins; Fracture Healing; Fractures, Bone; Humans; Immunohistochemistry; Male; Procollagen; Protein Serine-Threonine Kinases; Receptors, Growth Factor; Smad Proteins; Trans-Activators; Transforming Growth Factor beta

The characterization of protein components produced from bone tissues with fracture healing was investigated. Weanling rats were sacrificed between 1 and 7 days after the femoral fracture. Protein content in the femoral-diaphyseal tissues was markedly elevated by fracture healing. Moreover, when the femoral-diaphyseal tissues with fracture healing were cultured for 24 h in a serum-free medium, many proteins in the bone tissues were released into the medium. Analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that many protein molecules were released from the diaphyseal tissues with fracture healing. Especially, a protein molecule of approximately 66 kDa was markedly increased by fracture healing. This protein molecule was significantly increased, when the diaphyseal tissues with fracture healing were cultured in the presence of zinc acexamate (10(-6)-10(-4) M). Zinc acexamate (10(-4) M)-induced increase in medium 66 kDa protein molecule was significantly inhibited in the presence of actinomycin D (10(-7) M) or cycloheximide (10(-6) M). The zinc effect was completely blocked in the presence of PD98059 (10(-5) M), an inhibitor of MAPK kinase, or staurosporine (10(-6) M), an inhibitor of protein kinase C. The medium 66 kDa protein molecule was significantly elevated in the presence of parathyroid hormone (1-34) (10(-7) M), insulin-like growth factor-I (10(-8) M) or transforming growth factor-beta (10(-11) M), while 17beta-estradiol (10(-9) M) did not have an effect. The effect of these bone-stimulating factors was equal to the zinc effect. Zinc did not significantly enhance the effect of insulin-like growth factor-I in increasing medium 66 kDa protein molecule. The present study demonstrates that fracture healing increases production of the approximately 66 kDa protein molecule which is a major component produced from femoral-diaphyseal tissues of weanling rats, and that this elevation is enhanced by zinc treatment.

Topics: Animals; Bone and Bones; Cycloheximide; Dactinomycin; Electrophoresis, Polyacrylamide Gel; Estradiol; Flavonoids; Fracture Healing; Fractures, Bone; Insulin-Like Growth Factor I; Male; Parathyroid Hormone; Phosphoproteins; Rats; Rats, Wistar; Staurosporine; Time Factors; Transforming Growth Factor beta; Zinc

Distraction osteogenesis is a well-established clinical treatment for limb length discrepancy and skeletal deformities. In our previous studies, we have shown that the tension at the distraction gap correlated with the plasma bone specific alkaline phosphatase activity during distraction. Transforming growth factor-beta1 (TGF-beta1) has been shown to have a regulatory role in alkaline phosphatase activity during fracture healing. This study is to investigate the expression of TGF-beta1 during distraction as a biological response to mechanically stimulated osteoblastic activity by immunohistochemistry. The expression of TGF-beta1 in the distraction callus was compared with that in the fracture callus. During the distraction phase, the osteoblasts and osteocytes expressed a high level of TGF-beta1. Moderate expression of TGF-beta1 was observed in fibroblast-like cells in the fibrous zone of the distraction callus. After the distraction stopped, the expression of TGF-beta1 in different cell types decreased. In fracture healing, the strong expression of TGF-beta1 declined after the first week. Our results showed that the mechanical force induced and sustained TGF-beta1 expression in osteoblasts and fibroblasts-like cells of the distraction callus. Transforming growth factor-beta1 may play a role in transducing mechanical stimulation to biological tissue during in distraction osteogenesis.

Topics: Animals; Bone and Bones; Bone Regeneration; Fibroblasts; Fracture Healing; Fractures, Bone; Goats; Immunohistochemistry; Osteogenesis, Distraction; Osteotomy; Physical Stimulation; Transforming Growth Factor beta; Transforming Growth Factor beta1

Transforming growth factor beta is one of the most abundant growth factors stored in bone. It is known as a potent regulator of osteoblast proliferation and differentiation as well as of production extracellular matrix. We established a highly specific RT-PCR in combination with HPLC for detection and quantification of TGFbeta1 and TGFbeta2 mRNA expression in 89 human bone samples. Levels of TGFbeta1 protein ranged between 27 and 580 ng/g bone (mean 188 +/- 15 ng/g; n=75) and for TGFbeta2 between 7.2 and 35 ng/g bone (mean 14.3 +/- 2.1 ng/g; n=57). TGFbeta1 and TGFbeta2 protein concentrations and TGFbeta isoform mRNA expression in bone were not significantly different between the sexes. TGFbeta isoform mRNA expression as well as protein content in bone declined age dependently. TGFbeta1 and TGFbeta2 protein and mRNA expression were different in bone samples from different sites of the skeleton indicating in part the regulation by mechanical stimuli. In contrast to TGFbeta1, TGFbeta2 mRNA expression was significantly enhanced in osteoarthritic bone compared to unaffected bone. These data are in concordance to previous results concerning the expression of TGFbeta3 in bone. In conclusion, the data suggest distinct patterns' of expression of the TGFbeta isoforms under physiological and pathological conditions in bone.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Aging; Bone and Bones; Chromatography, High Pressure Liquid; Female; Fractures, Bone; Gene Expression; Humans; Male; Middle Aged; Osteoarthritis; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transforming Growth Factor beta; Transforming Growth Factor beta1; Transforming Growth Factor beta2

Topics: Aged; Asian People; Bone Density; Case-Control Studies; Female; Fractures, Bone; Genotype; Humans; Japan; Logistic Models; Middle Aged; Multivariate Analysis; Polymorphism, Genetic; Postmenopause; Prevalence; Spine; Transforming Growth Factor beta

Topics: Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Cholestyramine Resin; Fractures, Bone; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Hypolipidemic Agents; Pravastatin; Risk; Transforming Growth Factor beta

Topics: Animals; Bone Morphogenetic Proteins; Bone Substitutes; Cell Biology; Cell Differentiation; Cell Division; Collagen; Extracellular Matrix Proteins; Fractures, Bone; Fractures, Ununited; Humans; Morphogenesis; Osteogenesis; Protein Binding; Transforming Growth Factor beta

Angiogenesis is essential to both normal and pathological bone physiology. Vascular endothelial growth factor (VEGF) has been implicated in angiogenesis, whereas transforming growth factor-beta1 (TGF-beta1) modulates bone differentiation, matrix formation, and cytokine expression. The purpose of this study was to investigate the relationship between TGF-beta1 and VEGF expression in osteoblasts and osteoblast-like cells. Northern blot analysis revealed an early peak of VEGF mRNA (6-fold at 3 h) in fetal rat calvarial cells and MC3T3-E1 osteoblast-like cells after stimulation with TGF-beta1 (2.5 ng/ml). The stability of VEGF mRNA in MC3T3-E1 cells was not increased after TGF-beta1 treatment. Actinomycin D inhibited the TGF-beta1-induced peak in VEGF mRNA, whereas cycloheximide did not. Blockade of TGF-beta1 signal transduction via a dominant-negative receptor II adenovirus significantly decreased TGF-beta1 induction of VEGF mRNA. Additionally, TGF-beta1 induced a dose-dependent increase in VEGF protein expression by MC3T3-E1 cells (P < 0.01). Dexamethasone similarly inhibited VEGF protein expression. Both TGF-beta1 mRNA and VEGF mRNA were concurrently present in rat membranous bone, and both followed similar patterns of expression during rat mandibular fracture healing (mRNA and protein). In summary, TGF-beta1-induced VEGF expression by osteoblasts and osteoblast-like cells is a dose-dependent event that may be intimately related to bone development and fracture healing.

Topics: Animals; Bony Callus; Cell Line; Culture Media, Conditioned; Dexamethasone; Endothelial Growth Factors; Fetus; Fractures, Bone; Glucocorticoids; Lymphokines; Mice; Osteoblasts; Protein Synthesis Inhibitors; Rats; Rats, Sprague-Dawley; RNA, Messenger; Signal Transduction; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors; Wound Healing

Growth stimulation by growth hormones or by IGF-1 is well known whereas publications about stimulation of fracture healing with these substances are rare. One of the main effects of IGF-1 is angiopoiesis which consequently may be expected to induce and accelerate the soft tissue callus formation. BMP-2 is a stimulator for osteoclasts and osteoblasts and prepares the osseous reparation. RhBMPs, mostly BMP-2, are already used in the reconstruction of the cranial vault. The main disadvantage of these proteins is the quick inactivation after about 20-30 minutes. Local stimulation without systemic effects might be avoided by releasing a low but constant dose of IGF-1 or BMP. We speculate that the combination of a reasonable implant with an integrated drug release system may enlarge the field of application of biodegradable implants in the treatment of fractures. Such an implant might remarkably reduce the time for fracture healing and also lower the costs of postoperative treatment. In this paper we report our animal models in which we have tested constructive principles of biodegradable implants, we present the first clinical experiences with polyactates (PLLA) in the osteosynthesis of children, and we describe our inventions concerning drug releasing implants. 1) PLLA-implants a) The animal models We performed on oblique osteotomy of the right metacarpal bone in 48 juvenile calves and osteoplastic reconstructions of the cranial vault in 28 juvenile Göttinger minipigs. Biodegradable osteosynthesis with PLLA implants led to results as good as with conventional materials in both animal models, so that we proceeded to clinical evaluation in children. b) The clinical application in children 35 children, aged 6 weeks to 17 years, had resorbable osteosynthesis of their cranioplasties for correction of congenital malformations and posttraumatic reconstructions. The follow-up ranged from 5-15 years. 2) Invention of a drug-releasing system IGF-1 or BMP-2 is planned to be incapsulated by evaporation technique into nanospheres of low PDLLA (Poly-DL-Lactite) with a molecular weight of about 20,000 and an expected resorption time of 2-3 weeks (patent pending). 3) Concept of our drug-releasing resorbable implants Plates could be built up in multilayers of PDLLA sheets, separated by a spongiform matrix containing the IGF-1- or BMP-2-loaded nanospheres. The screws are hollow due to an internal driving system. The specially designed thread could also contain nanospheres, whic

Topics: Absorbable Implants; Animals; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Bone Plates; Bone Screws; Cattle; Child; Drug Delivery Systems; Finger Injuries; Fracture Fixation, Internal; Fracture Healing; Fractures, Bone; Humans; Insulin-Like Growth Factor I; Lactic Acid; Metacarpus; Osteotomy; Polyesters; Polymers; Skull Fractures; Swine; Swine, Miniature; Transforming Growth Factor beta

Bone tissue has been shown to contain numerous cell-to-cell signalling peptides called growth factors. These growth factors are thought to have important regulating effects for bone remodeling and bone healing, due to their potent effects on bone cell metabolism. In vivo studies over the last half decade have demonstrated that growth factors candidates for future clinical use in orthopedic surgery. In numerous clinical situations enhanced bone formation and bone healing could lead to improved results of surgical procedures. This thesis describes the most important bone growth factors and their actions in vitro and in vivo. In vitro investigations of growth factor effects on osteoblast chemotaxis and metabolism are described as well as in vivo studies with growth factor stimulation of fracture healing and bone healing to prosthetic-like implants. In vitro results: Several growth factors exhibited chemotactic effects towards human osteoblasts. TGF-beta 1 and PDGF-BB had the strongest chemotactic effects, whereas PDGF-AA, IGF-1, and IGF-2 had less but significant chemotactic effects towards human osteoblasts. TGF-beta 1 exhibited the highest chemotactic potency with maximal activity at 100 pg/mL, whereas the other growth factors had maximal effects at 10-100 ng/mL. BMP-2 was found to have chemotactic effects toward human osteoblasts, human bone marrow osteoprogenitor cells, and U2-OS osteosarcoma cells. BMP-4 and BMP-6 were without any chemotactic effects towards these celltypes. Human bone marrow osteoprogenitor cells were the most responsive celltype to BMP-2 stimulation. Growth factor combinations resulted in synergic stimulative effects on different metabolic functions on human osteoblasts. Combinations with TGF-beta 1 and PDGF-BB strongly stimulated proliferation and chemotaxis. Combinations with TGF-beta 1, PDGF-BB and BMP-2 strongly stimulated an osteoblast differentiation parameter (alkaline phosphatase activity). The different growth factor combinations had no effect on collagen synthesis in human osteoblasts. In vivo results: Continuous application of 1 and 10 micrograms natural TGF-beta to a plated tibial osteotomy in rabbits increased mechanical bending strength and callus formation at 6 weeks observation. Diaphyseal cortical bone remodeling was not affected by the local growth factor application. In a dog model with unloaded implants surrounded by a gap, 0.3 microgram rhTGF-beta 1 adsorbed to gritblasted tricalcium phosphate coated implants, was

Topics: Animals; Bone and Bones; Bone Plates; Cell Culture Techniques; Cell Division; Chemotaxis; Dogs; Fracture Fixation; Fractures, Bone; Growth Substances; Humans; Osteoblasts; Platelet-Derived Growth Factor; Prostheses and Implants; Rabbits; Somatomedins; Transforming Growth Factor beta; Wound Healing

An interdisciplinary study based on animal experiments, cell culture studies, and finite element models is presented. In a sheep model, the influence of the osteotomy gap size and interfragmentary motion on the healing success was investigated. Increasing gap sizes delayed the healing process. Increasing movement stimulated callus formation but not tissue quality. Typical distributions of intramembranous bone, endochondral ossification, and connective tissue in the fracture gap are quantified. The comparison of the mechanical data determined by a finite element model with the histologic images allowed the attribution of certain mechanical conditions to the type of tissue differentiation. Intramembranous bone formation was found for strains smaller than approximately 5% and small hydrostatic pressure (< 0.15 MPa). Strains less than 15% and hydrostatic pressure more than 0.15 MPa stimulated endochondral ossification. Larger strains led to connective tissue. Cell culture studies on the influence of strain on osteoblasts supported these findings. Proliferation and transforming growth factor beta production was increased for strains up to 5% but decreased for larger strains. Osteoblasts under larger strains (> 4%) turned away from the principal strain axis and avoided larger deformations. It is hypothesized that gap size and the amount of strain and hydrostatic pressure along the calcified surface in the fracture gap are the fundamental mechanical factors involved in bone healing.

Topics: Animals; Bony Callus; Cartilage; Cell Division; Cells, Cultured; Connective Tissue; Disease Models, Animal; Finite Element Analysis; Fracture Fixation; Fracture Healing; Fractures, Bone; Humans; Hydrostatic Pressure; Metatarsal Bones; Movement; Osteoblasts; Osteogenesis; Sheep; Stress, Mechanical; Transforming Growth Factor beta

A commonly encountered problem in orthopedics is bone and cartilage tissue injury which heals incompletely or without full structural integrity. This necessitates development of improved methods for treatment of injuries which are not amenable to treatment using current therapies. An already large and growing number of growth factors which play significant roles in bone remodeling and repair have been identified in the past few years. It is well established that bone morphogenic proteins induce the production of new bone and cartilage. An efficient method of delivery of these growth factors by conventional pharmacological means has yet to be elucidated. We wished to evaluate the use of retroviral vector-mediated gene transfer to deliver genes of therapeutic relevance for bone and cartilage repair. To determine the feasibility of using amphotropically packaged retroviral vectors to transduce primary rabbit mesenchymal stem cells of periosteal origin, primary periosteal cells were isolated from New Zealand white rabbits, transduced in vitro with a retroviral vector bearing both the nuclear localized lacZ marker gene and the neo(r) gene, and selected in G418. We used a convenient model for analysis of in vivo stability of these cells which were seeded on to polymer scaffold grafts and implanted into rabbit femoral osteochondral defects. The nuclear localized beta-galactosidase protein was expressed in essentially 100% of selected cells in vitro and was observed in the experimental explants from animals after both 4 and 8 weeks in vivo, while cells transduced with a retroviral vector bearing only the neo(r) gene in negative control explants showed no blue staining. We extended our study by delivering a gene of therapeutic relevance, human bone morphogenic protein 7 (hBMP-7), to primary periosteal cells via retroviral vector. The hBMP-7 gene was cloned from human kidney 293 cell total RNA by RT-PCR into a retroviral vector under control of the CMV enhancer/promoter. Hydroxyapatite secretion, presumably caused by overexpression of hBMP-7, was observed on the surface of the transduced and selected periosteal cells, however, this level of expression was toxic to both PA317 producer and primary periosteal cells. Subsequently, the strong CMV enhancer/promoter driving the hBMP-7 gene was replaced in the retroviral vector by a weaker enhancer/promoter from the rat beta-actin gene. Nontoxic levels of expression of hBMP-7 were confirmed at both the RNA and protein leve

Topics: Animals; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Cartilage; Cells, Cultured; Fractures, Bone; Fractures, Cartilage; Gene Expression; Gene Transfer Techniques; Genetic Therapy; Genetic Vectors; Humans; Male; Periosteum; Rabbits; Retroviridae; RNA; Transforming Growth Factor beta

Tricalcium phosphate (TCP) was combined with amylopectin to form a deliverable carrier paste for recombinant human transforming growth factor beta 1 (rhTGF-beta 1) intended for bone repair applications. Approximately 80% of rhTGF-beta 1 was released from the carrier within 24 h following in vitro incubation in serum. Full biological activity was maintained, suggesting the growth factor was stable in this formulation before and after in vitro release. In vivo efficacy also was assessed, in comparison to a sham control group and a placebo-treated group, using a rabbit unilateral segmental defect model (1 cm). Radiographs of defect sites taken at scheduled intervals and the mechanical testing of treated limbs at 56 days demonstrated a higher incidence of radiographic bone union, in concert with a stronger torque strength, in the rhTGF-beta 1-treated group compared to the placebo group. The short duration of the study and the fact that the model used was not a critical defect may account for the lack of superiority of the rhTGF-beta 1-treated group over the healing of the sham control. The in vivo pharmacokinetics of the growth factor evaluated in the same rabbit model suggested that rhTGF-beta 1 persisted intact at the defect site for more than 21 days. Gamma imaging and radioactivity recovery at defects administered to [131I]- and [125I]-labeled rhTGF-beta 1, respectively, estimated the half-life of rhTGF-beta 1 eliminated from the applied site to be 4-6 days. The present report substantiates the potential of rhTGF-beta 1 and its carrier for treatment of bone defects.

Topics: Amylopectin; Animals; Biocompatible Materials; Bone and Bones; Bone Cements; Calcium Phosphates; Fractures, Bone; Humans; Rabbits; Recombinant Proteins; Transforming Growth Factor beta

Clinical trauma suppresses immunity and experimental wound fluids have been shown to be immunosuppressive. To ascertain whether human wounds contain immunosuppressive cytokines, we assayed serum from fracture/soft-tissue hematomas (FSTH) of 22 patients for interleukin (IL)-10, transforming growth factor (TGF)-beta 1, and IL-4. Results were correlated to concurrent plasma cytokine concentrations in the same patients and in volunteer plasma. IL-10 was present in high concentration (1376 +/- 539 pg/mL) in all (7/7) FSTH < 24 h old. In FSTH > 24 h old, IL-10 was found intermittently and at lower levels (239 +/- 106 pg/mL, p = .011 vs. FSTH < 24 h old). IL-10 was rarely detectable in fracture patient plasma and never detectable (< 20 pg/mL) in normal plasma. No significant variations of IL-4 or total TGF-beta 1 were found in FSTH or plasma. FSTH are significant potential sources of IL-10 activity in trauma patients, which may be overlooked when only plasma is assayed. The potential for a relationship between cytokines found locally at sites of injury and clinical immune modulation in trauma requires further study.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers; Female; Femoral Fractures; Fractures, Bone; Hematoma; Humans; Interleukin-10; Interleukin-4; Internal Fixators; Male; Middle Aged; Pelvic Bones; Reference Values; Retrospective Studies; Tibial Fractures; Transforming Growth Factor beta

Fracture repair offers an opportunity to study the physiology of bone formation at the fracture site. Isolation of growth factors from bone matrix has implicated growth factors as participants in bone physiology. We therefore examined the expression patterns of aFGF, IGF-I, PDGF, and TGF-beta during fracture repair. An animal model has been developed to study repair of tibial fractures. The model provides both reproducible and quantifiable results, allowing the fracture repair process to be divided into four stages (Bourque et al., Lab. Anim. Sci 42: 369-374, 1992). Fractured tibiae were examined immunohistochemically with polyclonal antibodies to four growth factors. PDGF was visualized in macrophages in close proximity to the periosteum during stage 1. aFGF was visualized in cells of the expanded cambial layer and was associated with a rapid increase in the population of fibroblast-like mesenchymal cells during stage 2. IGF-I was visualized in young chondroblasts at the edge of the cartilage mass replacing the fibrous callus during stage 3. TGF-beta was visualized in calcified matrix producing chondrocytes at the edge of ossification fronts penetrating the cartilage callus during stage 4. The immunohistochemical results suggest that these growth factors act as local simulators of the repair process.

Topics: Animals; Cartilage; Fibroblast Growth Factor 1; Fracture Healing; Fractures, Bone; Growth Substances; Immunoenzyme Techniques; Insulin-Like Growth Factor I; Macrophages; Male; Mice; Monocytes; Osteoblasts; Platelet-Derived Growth Factor; Tibia; Transforming Growth Factor beta