transforming-growth-factor-beta and Femoral-Fractures

Gene therapy represents a promising approach for delivering regenerative molecules to specific tissues including bone. Several laboratories have shown that virus-based BMP expression vectors can stimulate osteoblast differentiation and bone formation in vivo. Both in vivo and ex vivo transduction of cells can induce bone formation at ectopic and orthotopic sites. Adenovirus and direct DNA delivery of genes encoding regenerative molecules can heal critical-sized defects of cranial and long bones. Although osteogenic activity can be demonstrated for individual BMP vectors, substantial synergies may be achieved using combinatorial gene therapy to express complimentary osteogenic signals including specific combinations of BMPs or BMPs and transcription factors. Further control of the bone regeneration process may also be achieved through the use of inducible promoters that can be used to control the timing and magnitude of expression for a particular gene. Using these types of approaches, it should be possible to mimic natural processes of bone development and fracture repair and, in so doing, be able to precisely control both the amount and type of bone regenerated.

Topics: Adenoviridae; Animals; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Bone Regeneration; Cell Differentiation; Femoral Fractures; Fibroblasts; Gene Transfer Techniques; Genetic Therapy; Genetic Vectors; Humans; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Mice; Neoplasm Proteins; Osteoblasts; Osteogenesis; Rats; Skull Fractures; Transcription Factors; Transforming Growth Factor beta

Surgical revision concepts for the treatment of aseptic humeral, femoral, and tibial diaphyseal nonunion were evaluated. It was analyzed if the range of time to bone healing was shorter, and if clinical and radiological long-term outcome was better following application of additional recombinant human Bone Morphogenetic Protein-7 (rhBMP-7) compared to no additional rhBMP-7 use.. In a retrospective comparative study between 06/2006 and 05/2013, 112 patients diagnosed with aseptic diaphyseal humerus (22 patients), femur (41 patients), and tibia (49 patients) nonunion were treated using internal fixation and bone graft augmentation. For additional stimulation of bone healing, growth factor rhBMP-7 was locally administered in 62 out of 112 patients. Follow-up studies including clinical and radiological assessment were performed at regular intervals as well as after at least one year following nonunion surgery.. One hundred and two out of 112 (humerus: 19, femur: 37, tibia: 47) nonunion healed within 12 months after revision surgery without any significant differences between the cohort groups. According to the DASH outcome measure for the humerus (p = 0.679), LEFS for the femur (p = 0.251) and the tibia (p = 0.946) as well as to the SF-12 for all entities, no significant differences between the treatment groups were found.. Aseptic diaphyseal nonunion in humerus, femur, and tibia healed irrespectively of additional rhBMP-7 application. Moreover, the results of this study suggest that successful nonunion healing can be linked to precise surgical concepts using radical removal of nonunion tissue, stable fixation and restoration of axis, length and torsion, rather than to the additional use of signaling proteins.. This clinical trial was conducted according to ICMJE guidelines as well as to the approval of the National Medical Board (Ethics Committee of the Bavarian State Chamber of Physicians; TRN: 2016-104) and has been retrospectively registered with the German Clinical Trails Register (TRN: DRKS00012652 ).

Topics: Adult; Aged; Bone Morphogenetic Protein 7; Bone Screws; Bone Transplantation; Diaphyses; Female; Femoral Fractures; Follow-Up Studies; Fracture Fixation, Internal; Fracture Healing; Fractures, Ununited; Humans; Humeral Fractures; Male; Middle Aged; Radiography; Recombinant Proteins; Reoperation; Retrospective Studies; Tibial Fractures; Time Factors; Transforming Growth Factor beta; Young Adult

In the clinical treatment of fractures, rhBMP-2 administration is associated with a well-established profile of side-effects, including osteolysis and ectopic bone formation, which are driven by pro-inflammatory processes triggered by the use of high doses. Immunomodulatory strategies could minimize the incidence of side-effects by enabling the use of lower, and safer, rhBMP-2 doses. This study investigated whether interleukin-1 receptor antagonist (IL-1Ra) can enhance the therapeutic efficacy of a low dose of rhBMP-2 in a weight-bearing femoral fracture healing model. Exogenous IL-1Ra, in combination with rhBMP-2, was delivered using a collagen-hydroxyapatite scaffold (CHA) to attenuate IL-1β produced in response to fracture. Femoral defects were treated with CHA scaffolds alone, or loaded with IL-1Ra (2.5 µg), rhBMP-2 (1 µg), IL-1Ra (2.5 µg) in combination with rhBMP-2 (1 µg). Bone healing was assessed over 14 weeks in comparison to control groups, empty defect, and a higher dose of rhBMP-2 (5 µg), which were recently demonstrated to lead to non-union, and successful bridging of the defect, respectively. The combination of IL-1Ra and rhBMP-2 led to significantly faster early bone formation, at both week 4 and 6, compared to a low dose of rhBMP-2 alone. By 14 weeks, the combination of IL-1Ra and a rhBMP-2 promoted full bridging of femurs, which were 3-fold more mechanically reliable compared to the femurs treated with a low dose of rhBMP-2 alone. Taken together, this study demonstrates that IL-1Ra can significantly enhance femoral bone healing when used in combination with a low dose of rhBMP-2. STATEMENT OF SIGNIFICANCE: Enabling the use of lower and safer doses of rhBMP-2, a potent inducer of bone formation, is of clinical relevance in orthopaedic medicine. In this study, the immunomodulatory interleukin-1 receptor antagonist (IL-1Ra) was investigated for its capacity to enhance the therapeutic efficacy of rhBMP-2 when used at lower doses in a weight-bearing femoral fracture healing model. The combination of IL-1Ra and rhBMP-2 led to significantly faster early bone formation, and resulted in more mechanically reliable healed femurs, compared to a low dose of rhBMP-2 alone. This demonstrates for the first time in a rat long bone healing model that IL-1Ra can significantly enhance bone healing when used in combination with a low dose of rhBMP-2.

Topics: Animals; Bone Morphogenetic Protein 2; Femoral Fractures; Fracture Healing; Interleukin 1 Receptor Antagonist Protein; Rats; Receptors, Interleukin-1; Recombinant Proteins; Transforming Growth Factor beta; Weight-Bearing

Fibronectin (FN) functions as a potent stimulator of osteogenic differentiation, and bone fracture healing. In FN family, FN1 acts as an interactive protein gene product to mediate chondrocyte adhesion. However, its effect on fracture healing remains elusive. Therefore, we aimed to investigate the involvement of FN1 in fracture healing. Hard callus formations were found at fracture site with thicker periosteum in lateral cortical bone area outside the fracture site in model mice. The decreased number of osteogenic cells in the middle of the callus region and increased extracellular matrix were suggestive of successful induction. Immunoblotting and RT-qPCR revealed that expression of FN1 was increased in tissues of fracture mice. As displayed by Safranin-fast green staining hematoxylin-eosin staining, the overexpression of FN1 at fracture site promoted osteoid formation and chondrocyte differentiation. The stimulating role of FN1 in collagen production was evidenced by increased levels of Col2, Col1, ColX, Osteonectin, and Osteocalcin and enhanced BMD, BV, BV/TV and Tb.Th values verified by immunoblotting and immunohistochemical staining. Additionally, the upregulation of FN1 contributed to promoted TGF-β, c-Caspase-9/t-Caspase-9 ratio and NF-κB p65 protein expression as well as lowered p-PI3K/PI3K and p-AKT/AKT ratios, implying the positive correlation between FN1 and the TGF-β/PI3K/Akt signaling pathway. The key findings of the present study provided evidence indicating that overexpression of FN1 contributes to fracture healing by activation of the TGF-β/PI3K/Akt signaling pathway.

Topics: Animals; Cell Differentiation; Chondrocytes; Collagen; Femoral Fractures; Fibronectins; Mice; Mice, Inbred C57BL; Mice, Nude; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Transforming Growth Factor beta

Biodegradable implant coatings promote proliferation and expression of BMP-2, VEGF, and TGF-β2 genes and enhance BMP-2, VEGF, and TGF-β2 regulatory effects at different stages of reparative osteogenesis.. To study the topography and ratio of PCNA-, VEGF-, BMP-2-, and TGF-β2-immunoreactive cells in rat femoral bone after closed fracture and implantation of titanium implants with biodegradable calcium phosphate and hydroxyapatite coatings.. Standard titanium implant screws and similar implants with bioactive coatings were used. A total of 18 rats were randomly divided into three groups, two experimental and a control one. The rats in the first experimental group were implanted with implants without specific coating, while those in the second group, with implants with specific coatings. The control rats were subjected to the same fracture as the experimental ones without subsequent implantation. On days 7, 14, and 30 of experiment, the rats were sampled for histological examination. Histological sections were prepared and processed for PCNA, BMP-2, VEGF, and TGF-β2 immunoreactivity.. In the regeneration zone, PCNA-immunoreactive cells substantially outnumbered other immunoreactive cell types. During the first two weeks after fracture, in the immediate vicinity of implant surface, the rate of VEGF production increased in osteoblast subpopulations and level of TGF-32 immunoreactivity decreased in chondroblasts. The level of TGF-32 was maximum on day 30 of experiment. BMP-2-immunoreactive osteocytes were found in the zone of external general plates. They accumulated at implants with calcium phosphate coating. Their number gradually increased by day 30 of experiment.. The present data suggest that biodegradable implant coatings promote proliferation and expression of BMP-2, VEGF, and TGF-β2 genes and enhance BMP-2, VEGF, and TGF-β2 regulatory effects at different stages of reparative osteogenesis.

Topics: Animals; Bone Morphogenetic Protein 2; Calcium Phosphates; Cell Proliferation; Coated Materials, Biocompatible; Durapatite; Femoral Fractures; Femur; Male; Osteogenesis; Prostheses and Implants; Rats; Surface Properties; Titanium; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

Substantial evidence exists demonstrating the individual effectiveness of both rhBMP-2 and -7 in the treatment of nonunions, data comparing the clinical effectiveness of adjunct rhBMP-2 and -7 remains scarce. Therefore, we examined our large single-center case series to compare the clinical effectiveness of both rhBMP-2 and -7 in non-union therapy aiming to answer: - Does a certain type of BMP have an advantageous effect on radiological outcome of applied lower limb non-union therapy? - Does application of a certain type of BMP have an advantageous effect on radiological outcome of infected lower limb nonunions? - Are there any additional risk factors associated with inferior outcome in context with an adjunct BMP treatment?. Both BMPs have the same effect on the radiological outcome of surgically treated lower limb nonunions.. Single-center retrospective database analysis of a case series of patients with lower limb long bone nonunions receiving either a one- or two-stage (Masquelet-) procedure based on the "diamond concept" with application of rhBMP-2 or -7. The "diamond concept" summarizes core factors that need to be present to achieve bone healing. In particular, these factors relate to the optimization of the mechanical (stability) and biological environment (sufficient osteogenic and angiogenic cells, osteoconductive scaffolds and growth factors). All medical data from patients that received surgical treatment between 01/01/2010 and 31/12/2016 were assessed. In total, 356 patients were treated with BMPs and 156 patients 18 years or older with non-union of their tibia or femur having a follow-up of at least 1 year were included. Consolidation in context with type of rhBMP was compared and the influence of relevant risk factors assessed.. Consolidation rate was significantly higher in patients treated with rhBMP-2 (rhBMP-2: 42/46 (91%) vs. rhBMP-7: 64/110 (58%); p<0.001). In particular, application of rhBMP-2 increased the likelihood of consolidation for tibial nonunions (OR 32.744; 95%CI: 2.909-368.544; p=0.005) and when used in two-stage therapy (OR 12.095; 95% CI: 2.744-53.314; p=0.001). Furthermore, regression modeling revealed a higher correlation between application of rhBMP-2 and osseous consolidation in infected nonunions (OR 61.062; 95% CI: 2.208-1688.475; p=0.015) than in aseptic nonunions (OR 4.787; 95% CI: 1.321-17.351; p=0.017). Risk factors negatively influencing the outcome of non-union treatment in context with rhBMPs were identified as active smoking (OR 0.357; 95% CI: 0.138-0.927; p=0.024), atrophic nonunion (OR 0.23; 95% CI: 0.061-0.869; p=0.030), higher BMI (OR 0.919; 95% CI: 0.846-0.998; p=0.046) and a larger defect size (OR 0.877; 95% CI: 0.784-0.98; p=0.021).. Patients who received rhBMP-2 for the treatment of tibial nonunions and as part of the two-stage treatment had a significantly higher rate of healing compared to patients treated with rhBMP-7 regardless of infection.. III, retrospective case-control study.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Atrophy; Body Mass Index; Bone Morphogenetic Protein 2; Bone Morphogenetic Protein 7; Bone Transplantation; Chemotherapy, Adjuvant; Female; Femoral Fractures; Femur; Fracture Fixation, Internal; Fracture Healing; Fractures, Ununited; Humans; Male; Middle Aged; Radiography; Recombinant Proteins; Retrospective Studies; Risk Factors; Smoking; Tibia; Tibial Fractures; Transforming Growth Factor beta; Treatment Failure; Young Adult

Reconstruction of a segmental fracture with massive bone loss is still a challenge for orthopaedic surgeons. The aim of our study was to develop a suitable biodegradable thermosensitive hydrogel system as a carrier for bone morphogenetic protein (BMP)-2 delivery in the treatment of critical-sized femoral defects.. A block copolymer composed of monomethoxypoly(ethylene glycol) (mPEG), poly(lactic-co-glycolic acid) (PLGA) and 2, 2'-Bis (2-oxazolin) (Box) was synthesized by ring opening polymerization. The synthesized block copolymer was characterized by (1)H-NMR spectroscopy and gel permeation chromatography (GPC). Different biophysical and biochemical properties of the synthesized copolymer, including temperature-induced structure changes, degradation rate, pH changes during hydrolytic degradation, cell toxicity, and the release profile of BMP-2, were also evaluated and/or were compared with those of a well-characterized mPEG-PLGA copolymer. In animal testing, rabbits (n = 36) that received critically sized (10 mm) femoral defects were divided into 6 groups. These experimental groups included an untreated group, autograft, and groups treated with the synthesized copolymer carrying different concentrations of BMP-2 (0, 5, 10, and 20 μg/ml). Bone repair was evaluated using X-ray radiography, histological staining, micro-computed tomography (μCT), biomarker examination and biomechanical testing in a 12-week treatment period.. A new thermosensitive mPEG-PLGA/Box/mPEG-PLGA block copolymer, or named as BOX copolymer, was successfully prepared. Compared to the reported mPEG-PLGA in vitro, the prepared BOX copolymer at the same weight percent concentrations exhibited wider temperature ranges of gelation, slower degradation rates, higher the pH values, as well as less cytotoxicity. Furthermore, the BMP-2 release from BOX hydrogel exhibited a near-linear release profile in vitro. In animal experiments, treatment of critical-sized bony defects with 25 wt% BOX hydrogel carrying BMP-2 effectively promoted fracture healing during the 12-week trial period and higher concentrations of BMP-2 treatment correlated with better bone quality. Most importantly, clinical outcome and bone healing in the BOX-hydrogel group with 20 μg/ml BMP-2 were nearly equivalent to those in the autograft group in a 12-week treatment course.. These data support that the use of BOX hydrogel (25 wt%) as a drug delivery system is a promising method in the treatment of large bone defects.

Topics: Animals; Autografts; Biodegradable Plastics; Biomarkers; Biomechanical Phenomena; Bone Morphogenetic Protein 2; Bone Transplantation; Cell Line; Disease Models, Animal; Drug Carriers; Femoral Fractures; Femur; Fracture Healing; Fractures, Ununited; Humans; Hydrogels; Mice; Polyesters; Polyethylene Glycols; Polyglactin 910; Rabbits; Radiography; Recombinant Proteins; Temperature; Transforming Growth Factor beta; X-Ray Microtomography

Many patients of all ages are admitted to hospital due to bone fractures. The etiology of fracture has a very wide spectrum, ranging from motor accidents to pathological conditions such as tumors, osteoporosis, and others. Bone fracture healing is a well-programmed and well-organized process, but is also long and intractable. The outcome of this process is therefore affected by many factors, such as the patient's age, ethnicity, nutritional status, and extent of the fracture. At present, regional analgesic techniques are frequently applied in order to avoid the complications of systemic opioid administration, central block applications. Femoral block is one of the regional analgesic techniques frequently applied by anesthesiologists when the lower extremities are involved. In this study, we evaluated the effect of femoral nerve block on the healing of an experimental non-stabilized femur fracture via expression of TGF-β, VEGF, and β-catenin and bone histomorphometry in rats.. In the control group, only the femoral fracture was performed and the bone was not fixated, similarly as in other groups. In the One-Day Block group, a one-time femoral nerve block was applied after the femoral fracture. In the Three-Day Block group, a daily femoral nerve block was performed for three days after the femoral fracture. On Days 4, 7, and 13, femurs were excised. The bone sections were stained with hematoxylin-eosin to evaluate bone tissue and Safranin O to assess callus tissue, cartilaginous tissue, and new bone areas. TGF-β, VEGF, and β-catenin were assessed by immunohistochemistry.. Histomorphometric analysis revealed that femoral block application had a positive impact on bone healing. TGF-β expression in the One-Day and Three-Day Block Groups was significantly higher than in the control group at all times, as was also the case with VEGF expression. On day 13, β-catenin expression was significantly higher in the Three-Day Block group than the others.. The results of the study suggests that the applications of a femoral nerve block for perioperative analgesia, for either one day or three days, resulted in better and more rapid bone healing.

Topics: Animals; beta Catenin; Bony Callus; Cartilage; Disease Models, Animal; Femoral Fractures; Femoral Nerve; Fracture Healing; Immunohistochemistry; Intercellular Signaling Peptides and Proteins; Male; Nerve Block; Rats; Transforming Growth Factor beta

Effective repair of critical-size long bone defects presents a significant clinical challenge. Electrospun scaffolds can be exploited to deliver protein therapeutics and progenitor cells, but their standalone application for long bone repair has not been explored. We have previously shown that electrospun composites of amphiphilic poly(d,l-lactic acid)-co-poly(ethylene glycol)-co-poly(d,l-lactic acid) (PELA) and hydroxyapatite (HA) guide the osteogenic differentiation of bone marrow stromal cells (MSCs), making these scaffolds uniquely suited for evaluating cell-based bone regeneration approaches. Here we examine whether the in vitro bioactivity of these electrospun scaffolds can be exploited for long bone defect repair, either through the participation of exogenous MSCs or through the activation of endogenous cells by a low dose of recombinant human bone morphogenetic protein-2 (rhBMP-2). In critical-size rat femoral segmental defects, spiral-wrapped electrospun HA-PELA with preseeded MSCs resulted in laminated endochondral ossification templated by the scaffold across the longitudinal span of the defect. Using GFP labeling, we confirmed that the exogenous MSCs adhered to HA-PELA survived at least 7 days postimplantation, suggesting direct participation of these exogenous cells in templated bone formation. When loaded with 500 ng of rhBMP-2, HA-PELA spirals led to more robust but less clearly templated bone formation than MSC-bearing scaffolds. Both treatment groups resulted in new bone bridging over the majority of the defect by 12 weeks. This study is the first demonstration of a standalone bioactive electrospun scaffold for templated bone formation in critical-size long bone defects.

Topics: Animals; Bone and Bones; Bone Density; Bone Marrow Cells; Bone Morphogenetic Protein 2; Bone Regeneration; Cell Differentiation; Cell Survival; Durapatite; Femoral Fractures; Lactates; Male; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Osteogenesis; Polyethylene Glycols; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Tissue Engineering; Tissue Scaffolds; Tomography, X-Ray Computed; Transforming Growth Factor beta

Electron beam melting (EBM)-fabricated porous titanium implants possessing low elastic moduli and tailored structures are promising biomaterials for orthopedic applications. However, the bio-inert nature of porous titanium makes reinforcement with growth factors (GFs) a promising method to enhance implant in vivo performance. Bone-morphogenic protein-2 (BMP-2) and vascular endothelial growth factor (VEGF) are key factors of angiogenesis and osteogenesis. Therefore, the present study is aimed at evaluating EBM-fabricated porous titanium implants incorporating GF-doped fibrin glue (FG) as composite scaffolds providing GFs for improvement of angiogenesis and osteogenesis in rabbit femoral condyle defects. BMP-2 and VEGF were added into the constituent compounds of FG, and then this GF-doped FG was subsequently injected into the porous scaffolds. In five groups of implants, angiogenesis and osteogenesis were evaluated at 4 weeks post-implantation using Microfil perfusion and histological analysis: eTi (empty scaffolds), cTi (containing undoped FG), BMP/cTi (containing 50 μg rhBMP-2), VEGF/cTi (containing 0.5 μg VEGF) and Dual/cTi (containing 50 μg rhBMP-2 and 0.5 μg VEGF). The results demonstrate that these composite implants are biocompatible and provide the desired gradual release of the bioactive growth factors. Incorporation of GF delivery, whether a single factor or dual factors, significantly enhanced both angiogenesis and osteogenesis inside the porous scaffolds. However, the synergistic effect of the dual factors combination was observable on angiogenesis but absent on osteogenesis. In conclusion, fibrin glue is a biocompatible material that could be employed as a delivery vehicle in EBM-fabricated porous titanium for controlled release of BMP-2 and VEGF. Application of this method for loading a porous titanium scaffold to incorporate growth factors is a convenient and promising strategy for improving osteogenesis of critical-sized bone defects.

Topics: Alloys; Animals; Biocompatible Materials; Bone Morphogenetic Protein 2; Bone Regeneration; Cell Survival; Coculture Techniques; Delayed-Action Preparations; Drug Delivery Systems; Elastic Modulus; Electrons; Femoral Fractures; Fibrin Tissue Adhesive; Human Umbilical Vein Endothelial Cells; Humans; Materials Testing; Mesenchymal Stem Cells; Neovascularization, Physiologic; Osteogenesis; Porosity; Prostheses and Implants; Rabbits; Rats; Recombinant Proteins; Tissue Scaffolds; Titanium; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

Recombinant human bone morphogenetic protein (rhBMP)-2 is a potent osteoinductive agent; however, its clinical use has been reduced because of safety and efficacy concerns. In preclinical studies involving a critical-sized defect in a rat model, sclerostin antibody (Scl-Ab) treatment increased bone formation within the defect but did not result in reliable healing. The purpose of the current study was to evaluate bone repair of a critical-sized femoral defect in a rat model with use of local implantation of rhBMP-2 combined with systemic administration of Scl-Ab.. A critical-sized femoral defect was created in rats randomized into three treatment groups: local rhBMP-2 and systemic Scl-Ab (Scl + BMP), local rhBMP-2 alone, and collagen sponge alone (operative control). The Scl + BMP group received local rhBMP-2 (10 μg) on a collagen sponge placed within the defect intraoperatively and then twice weekly injections of Scl-Ab (25 mg/kg) administered postoperatively. The femora were evaluated at twelve weeks with use of radiography, microcomputed tomography (microCT), histomorphometric analysis, and biomechanical testing.. At twelve weeks, all Scl + BMP and rhBMP-2 only samples were healed. No femora healed in the operative control group. Histomorphometric analysis demonstrated more bone in the Scl + BMP samples than in the samples treated with rhBMP-2 alone (p = 0.029) and the control samples (p = 0.003). MicroCT revealed that the Scl + BMP group had a 90% greater bone volume within the defect region compared with the rhBMP-2 group and a 350% greater bone volume compared with the operative control group (p < 0.001). Biomechanical testing showed that the group treated with Scl + BMP had greater torsional strength and rigidity compared with the rhBMP-2 group (p < 0.001 and p = 0.047) and the intact femoral control group (p < 0.001). Torque to failure was lower in the rhBMP-2 group compared with the intact femoral control group (p < 0.002). Mean energy to failure was higher in the Scl + BMP samples compared with the rhBMP-2 only samples (p = 0.001).. In a critical-sized femoral defect in a rat model, local rhBMP-2 combined with systemic administration of Scl-Ab resulted in more robust healing that was stronger and more rigid than results for rhBMP-2 alone and intact nonoperative femora.. Our study demonstrated that combining an osteoinductive agent with a systemically administered antibody that promotes bone formation can enhance bone repair and has potential as a therapeutic regimen in humans.

Topics: Adaptor Proteins, Signal Transducing; Animals; Bone Density Conservation Agents; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Chemotherapy, Adjuvant; Drug Administration Schedule; Drug Therapy, Combination; Femoral Fractures; Fracture Fixation, Internal; Fracture Healing; Genetic Markers; Humans; Injections, Subcutaneous; Male; Radiography; Random Allocation; Rats; Recombinant Proteins; Transforming Growth Factor beta

Topics: Animals; Bone Density Conservation Agents; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Femoral Fractures; Fracture Fixation, Internal; Humans; Male; Transforming Growth Factor beta

This study investigated the fracture-healing effects of α-lipoic acid (α-LA), which was applied orally once daily in preventive treatment mode during 1 month after fracture induction. Rats were randomly divided into sham-operated group (group 1), femoral fracture control (group 2), femoral fracture + 25 mg/kg α-LA (group 3), and femoral fracture + 50 mg/kg α-LA (group 4). Rats in the experimental groups were orally administered 25 or 50 mg/kg α-LA once daily for 30 days starting from postoperative day 1. Thirty days postoperatively, the rats underwent X-ray imaging and were then euthanized for blood and tissue collection. Histopathological, biochemical, molecular, computed tomography (CT), and mechanical strength tests were performed on samples. The serum levels of osteocalcin (OC), osteopontin (OP), tumor necrosis factor alpha (TNF-α), and interleukin-6 (IL-6) did not differ significantly between groups 2 and 3. Serum OC, OP, TNF-α, and IL-6 levels in group 4 were significantly lower than those in group 3. From X-ray images, staging for fracture healing was scored as <2 in group 2, >2 in group 3, and >3 in group 4. In group 2, the average score of less than 2 suggests insufficient fracture healing; those of both the α-LA groups were >2, indicating progression of healing. Transforming growth factor beta (TGF-β) messenger RNA (mRNA) levels were significantly higher in the sham group than in the femoral fracture control. Both doses of α-LA increased TGF-β mRNA expression compared to the fracture group. CT results and biomechanical testing at 4 week after fracture demonstrated that α-LA has fastened bone healing, which was confirmed by stereological analyses in which 50 mg/kg α-LA increased the number of osteoclasts. Our findings indicate that α-LA supplementation promotes healing of femoral fractures in rats.

Topics: Administration, Oral; Animals; Disease Models, Animal; Dose-Response Relationship, Drug; Female; Femoral Fractures; Fracture Healing; Gene Expression Regulation; Rats; Rats, Wistar; RNA, Messenger; Thioctic Acid; Tomography, X-Ray Computed; Transforming Growth Factor beta; Treatment Outcome

The further development in research of bone regeneration is necessary to meet the clinical demand for bone reconstruction. Plasminogen is a critical factor of the tissue fibrinolytic system, which mediates tissue repair in the skin and liver. However, the role of the fibrinolytic system in bone regeneration remains unknown. Herein, we investigated bone repair and ectopic bone formation using plasminogen-deficient (Plg⁻/⁻) mice. Bone repair of the femur is delayed in Plg⁻/⁻ mice, unlike that in the wild-type (Plg⁺/⁺) mice. The deposition of cartilage matrix and osteoblast formation were both decreased in Plg⁻/⁻ mice. Vessel formation, macrophage accumulation, and the levels of vascular endothelial growth factor (VEGF) and transforming growth factor-β (TGF-β) were decreased at the site of bone damage in Plg⁻/⁻ mice. Conversely, heterotopic ossification was not significantly different between Plg⁺/⁺ and Plg⁻/⁻ mice. Moreover, angiogenesis, macrophage accumulation, and the levels of VEGF and TGF-β were comparable between Plg⁺/⁺ and Plg⁻/⁻ mice in heterotopic ossification. Our data provide novel evidence that plasminogen is essential for bone repair. The present study indicates that plasminogen contributes to angiogenesis related to macrophage accumulation, TGF-β, and VEGF, thereby leading to the enhancement of bone repair.

Topics: Animals; Bone Regeneration; Female; Femoral Fractures; Fracture Healing; Macrophages; Male; Mice; Mice, Knockout; Neovascularization, Physiologic; Plasminogen; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

Use of recombinant human bone morphogenetic protein-2 (rhBMP-2) is expensive and may cause local side effects. A small synthetic molecule, SVAK-12, has recently been shown in vitro to potentiate rhBMP-2-induced transdifferentiation of myoblasts into the osteoblastic phenotype. The aims of this study were to test the ability of SVAK-12 to enhance bone formation in a rodent ectopic model and to test whether a single percutaneous injection of SVAK-12 can accelerate callus formation in a rodent femoral fracture model.. Collagen disks with rhBMP-2 alone or with rhBMP-2 and SVAK-12 were implanted in a standard athymic rat chest ectopic model, and radiographic analysis was performed at four weeks. In a second set of rats (Sprague-Dawley), SVAK-12 was percutaneously injected into the site of a closed femoral fracture. The fractures were analyzed radiographically and biomechanically (with torsional testing) five weeks after surgery.. In the ectopic model, there was dose-dependent enhancement of rhBMP-2 activity with use of SVAK-12 at doses of 100 to 500 μg. In the fracture model, the SVAK-12-treated group had significantly higher radiographic healing scores than the untreated group (p = 0.028). Biomechanical testing revealed that the fractured femora in the 200 to 250-μg SVAK-12 group were 43% stronger (p = 0.008) and 93% stiffer (p = 0.014) than those in the control group. In summary, at five weeks the femoral fracture group injected with SVAK-12 showed significantly improved radiographic and biomechanical evidence of healing compared with the controls.. A single local dose of a low-molecular-weight compound, SVAK-12, enhanced bone-healing in the presence of low-dose exogenous rhBMP-2 (in the ectopic model) and endogenous rhBMPs (in the femoral fracture model).. This study demonstrates that rhBMP-2 responsiveness can be enhanced by a novel small molecule, SVAK-12. Local application of anabolic small molecules has the potential for potentiating and accelerating fracture-healing. Use of this small molecule to lower required doses of rhBMPs might both decrease their cost and improve their safety profile.

Topics: Animals; Biomechanical Phenomena; Bone Morphogenetic Protein 2; Bony Callus; Dose-Response Relationship, Drug; Drug Therapy, Combination; Enzyme Inhibitors; Femoral Fractures; Fracture Healing; Fractures, Closed; Injections, Intralesional; Male; Models, Animal; Radiography; Random Allocation; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Transforming Growth Factor beta; Treatment Outcome; Triazines; Ubiquitin-Protein Ligases; Vinyl Compounds

Large segmental defects in bone do not heal well and present clinical challenges. This study investigated modulation of the mechanical environment as a means of improving bone healing in the presence of bone morphogenetic protein (BMP)-2. Although the influence of mechanical forces on the healing of fractures is well established, no previous studies, to our knowledge, have described their influence on the healing of large segmental defects. We hypothesized that bone-healing would be improved by initial, low-stiffness fixation of the defect, followed by high-stiffness fixation during the healing process. We call this reverse dynamization.. A rat model of a critical-sized femoral defect was used. External fixators were constructed to provide different degrees of stiffness and, importantly, the ability to change stiffness during the healing process in vivo. Healing of the critical-sized defects was initiated by the implantation of 11 μg of recombinant human BMP (rhBMP)-2 on a collagen sponge. Groups of rats receiving BMP-2 were allowed to heal with low, medium, and high-stiffness fixators, as well as under conditions of reverse dynamization, in which the stiffness was changed from low to high at two weeks. Healing was assessed at eight weeks with use of radiographs, histological analysis, microcomputed tomography, dual x-ray absorptiometry, and mechanical testing.. Under constant stiffness, the low-stiffness fixator produced the best healing after eight weeks. However, reverse dynamization provided considerable improvement, resulting in a marked acceleration of the healing process by all of the criteria of this study. The histological data suggest that this was the result of intramembranous, rather than endochondral, ossification.. Reverse dynamization accelerated healing in the presence of BMP-2 in the rat femur and is worthy of further investigation as a means of improving the healing of large segmental bone defects.. These data provide the basis of a novel, simple, and inexpensive way to improve the healing of critical-sized defects in long bones. Reverse dynamization may also be applicable to other circumstances in which bone-healing is problematic.

Topics: Absorptiometry, Photon; Analysis of Variance; Animals; Biopsy, Needle; Bone Morphogenetic Protein 2; Bone Regeneration; Disease Models, Animal; External Fixators; Femoral Fractures; Femur; Fracture Fixation; Fracture Healing; Humans; Immunohistochemistry; Male; Pilot Projects; Random Allocation; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Tensile Strength; Transforming Growth Factor beta; X-Ray Microtomography

Segmental bone loss remains a challenging clinical problem. A frequent mitigating factor is inadequate blood supply. Small molecules that activate the hypoxia-inducible factor pathway can be used to stimulate angiogenesis. We investigated an approach to promote healing using angiogenic and osteogenic compounds in combination with a biodegradable, weightbearing scaffold.. Adult rats underwent removal of a 5-mm segment of femur stabilized by a cylindrical biodegradable implant and intramedullary fixation. Treatment groups included 1) saline (negative control); 2) desferrioxamine (DFO, a hypoxia-inducible factor activator; 3) low-dose recombinant human bone morphogenetic protein-2 (rhBMP-2) (5 μg); 4) DFO and low-dose rhBMP-2 (5 μg); or 5) rh-BMP-2 (10 μg). Angiography was used to evaluate vascularity. Bone healing was assessed by radiographs, microcomputed tomography, histology, and biomechanical testing.. Increased vascularity was seen at 6 weeks in the DFO treatment group. There appeared to be increased bone bridging as assessed by radiographic scores and microcomputed tomography in the BMP groups, although the quantification of bone volume did not show statistically significant differences. Biomechanical testing revealed improved stiffness in the treatment groups.. DFO improved angiogenesis and stiffness of bone healing in segmental defects. BMP improved radiographic scores and stiffness. Use of angiogenic compounds in segmental bone loss is promising.. Activation of the hypoxia-inducible factor pathway may prove useful for bone defects, particularly where impaired blood supply exists.The low-cost approach could be useful in segmental bone defects clinically.

Topics: Angiogenesis Inducing Agents; Animals; Bone Morphogenetic Protein 2; Deferoxamine; Dose-Response Relationship, Drug; Femoral Fractures; Fracture Healing; Neovascularization, Physiologic; Polypharmacy; Radiography; Rats; Recombinant Proteins; Transforming Growth Factor beta; Treatment Outcome

Bone repair involves a complex set of regulated signaling pathways that control the formation of new bone matrix and the resorption of damaged bone matrix at the fracture site. It has been reported that the optimal time point for single-dose zoledronic acid (ZA) administration systemically increased the strength of bone morphogenetic protein (BMP)-7-mediated callus. However, its repair mechanism during bone fracture healing remains unknown. We aimed to investigate the synergic effect of recombinant human (rh) BMP-2 and ZA in a rat femoral fracture model. Fifty-eight rats were divided into 4 groups. Group I (n=14) animals were implanted with a carrier alone. Group II (n=15) animals were implanted with a carrier containing 1-μg rhBMP-2. Group III (n=14) animals were implanted with a carrier and a subcutaneous systemic ZA injection 2 weeks after surgery. Group IV (n=15) animals were implanted with a carrier containing 1-μg rhBMP-2 and ZA subcutaneous injection 2 weeks after surgery. The rats were euthanized after 6 weeks and their fractured femurs were explanted and assessed by manual palpation, radiographs, and high-resolution micro-computerized tomography (micro-CT) and were subjected to biomechanical and histological analysis. The fusion rates in Group IV (93.3%) were considerably higher than those in Groups I (28.6%), II (53.3%), and III (57.1%). Additionally, the radiographic scores of Group IV were higher than those in Groups I, II, and III. In micro-CT analysis, the tissue volume (TV) of the callus was higher in Group IV than in Groups I and II (p<0.05). New bone volume (BV) and trabecular spacing (Tb.Sp) also showed essentially the same trend as that of TV. The ratio of BV to TV (BV/TV), the trabecular number (Tb.N), and the trabecular thickness (Tb.Th) was higher in Groups III and IV than in Groups I and II (p<0.05). In biomechanical analysis, the ultimate loads at failure and stiffness in Groups III and IV were on average higher than those in Groups I and II (p<0.05), while the energy absorption of Group IV was higher than those of Groups I and II (p<0.05). The synergic effect of rhBMP-2 and ZA given systemically as a single dose at the optimal time was efficacious for fracture repair and significantly enhanced bone fusion. Our results suggest that this combination facilitates bone healing and has potential clinical application.

Topics: Anabolic Agents; Animals; Biomechanical Phenomena; Bone Morphogenetic Protein 2; Diphosphonates; Disease Models, Animal; Drug Therapy, Combination; Femoral Fractures; Humans; Imidazoles; Male; Palpation; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Transforming Growth Factor beta; X-Ray Microtomography; Zoledronic Acid

Recombinant human bone morphogenetic protein-2 (rhBMP-2) has been shown to enhance new bone formation in fracture and bone defect models in both normal and diabetic rats. Effects of rhBMP-2 in a segmental femoral defect model in diabetes mellitus (DM) BB Wistar rats have not been reported.. Collagen sponge soaked with either buffer or rhBMP-2 was inserted in a mid-diaphyseal 3.0-mm defect fixed with polyimide plate and stainless steel screws, in 62 DM BB Wistar rats. Progress of new bone formation in the defect was monitored with serial radiographs every 2 weeks. Histomorphometric analysis of the new bone formation was done on undecalcified sections of the extracted femurs at 3 and 6 weeks post surgery. Further analysis of the new bone was done by assessment of neoangiogenesis using immunohistochemical staining for Platelet endothelial cell adhesion molecule-1. Mechanical testing was performed at 9 weeks to assess the new bone with respect to 4 different parameters of mechanical and structural properties of bone.. Radiographs assessed over a 6-point grading system showed statistically significant improvement in scores in rhBMP-2-treated rats at 6 weeks (P < 0.001). Histomorphometric analysis showed statistically significant increase in area of new bone formation between rats treated with rhBMP-2 compared with buffer at both 3 and 6 weeks (P < 0.001). On Platelet endothelial cell adhesion molecule-1 staining at 3 weeks, the mean number of vessels in rhBMP-2-treated DM rats was 12.76 +/- 5.43/mm(2) compared with 4.49 +/- 1.89/mm(2) in buffer treated DM rats (P = 0.034). On mechanical testing, all 4 DM/buffer rats had nonunion. In DM/rhBMP-2 rats, the torque to failure and torsional rigidity values were 393.57 +/- 233.3 (P < 0.03) and 29,711 +/- 6224 (P < 0.002), respectively.. Clearly, although DM has a known impact on osseous healing, its negative effects are ameliorated with the application of the rhBMP-2-collagen carrier and demonstrates the potential clinical role of this adjunct in the clinical arena.

Topics: Animals; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Diabetes Complications; Disease Models, Animal; Drug Implants; Femoral Fractures; Fracture Healing; Humans; Male; Rats; Rats, Wistar; Recombinant Proteins; Transforming Growth Factor beta; Treatment Outcome

Platelet-rich plasma (PRP) is a common therapy for acceleration of maxillofacial and spinal fusion bone-graft healing. This study analyzes the therapeutic role of PRP during long-bone fracture healing evaluated Lewis rats. Following creation of unilateral open femur fractures, either 500 microL thrombin-activated PRP (PRP treated group) or 500 microL saline (control group) were applied once to the fracture site. Fracture healing was analyzed after 1 and 4 weeks. Following 4 weeks of fracture healing, radiographic analysis demonstrated higher callus to cortex width ratio (P < 0.05) in the PRP group (PRP: 1.65 +/- 0.06; control: 1.48 +/- 0.05). Three-point load bearing showed increased bone strength following PRP treatment (PRP: 60.85 +/- 6.06 Newton, control: 47.66 +/- 5.49 Newton). Fracture histology showed enhanced bone formation in the PRP group. Immunohistochemistry and Western-blotting demonstrated healing-associated changes in transforming growth factor (TGF)-beta1 and bone morphogenetic protein (BMP)-2. Our results suggest that PRP accelerates bone fracture healing of rat femurs via modulation of TGF-beta1 and BMP-2 growth factor expression.

Topics: Analysis of Variance; Animals; Blood Transfusion; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Bony Callus; Femoral Fractures; Fracture Healing; Male; Platelet-Rich Plasma; Radiography; Rats; Rats, Inbred Lew; Transforming Growth Factor beta; Transforming Growth Factor beta1; Weight-Bearing

The objective of this study was to compare the efficacy of adenoviral and lentiviral regional gene therapy in a rat critical sized femoral defect model. The healing rates and quality of bone repair of femoral defects treated with syngeneic rat bone marrow cells (RBMCs) transduced with either lentiviral vector (Group I) or adenoviral vector (Group II) expressing bone morphogenetic protein-2 (BMP-2) gene were assessed. RBMCs transduced with the adenoviral vectors produced more than 3 times greater (p<0.001) BMP-2 when compared to RBMCs transduced with lentiviral vectors in an in vitro evaluation. Serial bioluminescent imaging demonstrated short duration luciferase expression (less than 3 weeks) in defects treated with RBMCs co-transduced with two adenoviral vectors (Group IV; adenovirus expressing BMP-2 and luciferase [Ad-BMP-2+Ad-Luc]). In contrast, the luciferase signal was present for 8 weeks in defects treated with RBMCs co-transduced with two lentiviral vectors (Group III; lentivirus expressing BMP-2 and luciferase gene [LV-BMP-2+LV-Luc]). There were no significant differences with respect to the radiological healing rates (p=0.12) in defects treated with lentiviral versus adenoviral mediated BMP-2 gene transfer. Biomechanical testing of healed Group I femoral specimens demonstrated significantly higher energy to failure (p<0.05) when compared to Group II defects. Micro CT analysis revealed higher bone volume/tissue volume fraction (p=0.04) in Group I defects when compared to Group II defects. In conclusion, prolonged BMP-2 expression associated with lentiviral mediated gene transfer demonstrated a trend towards superior quality of bone repair when compared to adenoviral mediated transfer of BMP-2. These results suggest that the bone repair associated with regional gene therapy is influenced not just by the amount of protein expression but also by duration of protein production. This observation needs validation in a more biologically challenging environment where differences in healing rates and quality of bone repair are more likely to be significantly different.

Topics: Adenoviridae; Animals; Biomechanical Phenomena; Bone Marrow Cells; Bone Marrow Transplantation; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Elasticity; Femoral Fractures; Fracture Healing; Genetic Therapy; Genetic Vectors; Intercellular Signaling Peptides and Proteins; Lentivirus; Luciferases; Luminescent Measurements; Male; Rats; Rats, Inbred Lew; Recombinant Proteins; Transfection; Transforming Growth Factor beta

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

Demineralized bone matrix (DBM) has been shown to possess osteoinductive capability and one of the specific bone morphogenetic proteins (BMPs) found within DBM that has been attributed with this osteoinductive ability is BMP-7, also known as osteogenic protein-1 (OP-1). The specific aims of this study were (1) to compare the treatment of segmental bone defects with OP-l and DBM in a rat femur model and (2) to determine the effects of the two treatments given at high and low doses via sustained release drug delivery. Animals in Group 1 acted as the control and Group 2 had a created segmental defect with plating and placement of a calcined tricalcium phosphate lysine (TCPL) capsule containing antibiotic (sham). Group 3 and 4 animals had a created segmental defect and received a TCPL carrier containing antibiotic along with DBM or OP-1, respectively. After 4 weeks post-implantation, animals were sacrificed before the retrieval of the bone. The femora were analyzed radiographically and histologically for bone growth. Analysis of the gross specimens showed considerable bone regeneration at low and high doses for both DBM and OP-1 when compared to the shams. At low levels bone regeneration between DBM and OP-1 was very similar. However, at high doses, OP-1 was shown to cause bone overgrowth with a greater curvature and an increased thickness of the distal and proximal ends of the femur. The stained slides showed the defects treated with DBM and OP-1 to be bridged with lamellar and woven bone that was continuous with the original bone. Histologically, the experimental femora demonstrated natural remodeling processes with new osteons and angiogenesis.

Topics: Animals; Anti-Bacterial Agents; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Bone Substitutes; Delayed-Action Preparations; Disease Models, Animal; Drug Combinations; Femoral Fractures; Femur; Fracture Healing; Radiography; Rats; Rats, Sprague-Dawley; Tobramycin; Transforming Growth Factor beta; Transplantation, Homologous; Treatment Outcome

Osteogenic Protein-1 (OP-1, BMP-7) acts locally on connective tissue progenitors (CTPs) to induce bone formation. The response to OP-1 and similar agents is potentially limited by the number of local CTPs. This study tested the hypothesis that supplementing local CTPs using autogenous bone marrow will enhance bone formation at an OP-1 implant. Four 1.0-cm diameter unicortical cylindrical defects in the left proximal femur were grafted in each of seven dogs. Radial ingrowth of new bone formation was assessed at 4 weeks using micro CT. The OP-1 (3.5 mg rhOP-1 in 1 g bovine collagen I matrix) was implanted in each site combined with either clotted blood or aspirated bone marrow (BM). Bone formation was increased in the group augmented with transplanted marrow. These data suggest that increasing the local population of cells and CTPs using aspirated bone marrow can enhance the performance of OP-1, but may not eliminate the effects of site variation on the response to OP-1 and similar agents. The canine multiple femoral defect model defined in this study is well suited to quantitatively evaluate strategies for augmenting bone repair using local cell targeting and cell transplantation strategies.

Topics: Animals; Bone Marrow Transplantation; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Disease Models, Animal; Dogs; Femoral Fractures; Femur; Fracture Healing; Implants, Experimental; Male; Osteogenesis; Osteotomy; Tomography, X-Ray Computed; Transforming Growth Factor beta

Reconstruction of a major bone loss remains a challenge for the orthopaedic surgeon. Most of the bone defects result from a bone tumour resection whereas a post-traumatic bone loss is more rare due to the numerous options available for bone fixation. However in high-energy trauma, the injury to bone may be so extensive as to justify removal of fragmented bone. A 57-year-old man presented with a severe injury at the thigh after a hunting accident, including a comminuted fracture of the femoral shaft. After thorough debridement, he was left with a large diaphyseal bone defect which was subsequently treated with a structural bone allograft, autogenous graft and rhBMP-7. Bone healing was achieved after several months.

Topics: Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Bone Transplantation; Debridement; Femoral Fractures; Fractures, Comminuted; Humans; Male; Middle Aged; Transforming Growth Factor beta; Transplantation, Autologous; Transplantation, Homologous; Wounds, Gunshot

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

Large segmental defects in bones can result from tumor removal, massive trauma, congenital malformation, or non-union fractures. Such defects often are difficult to manage and require multiple-phase surgery to achieve adequate union and function. In this study, we propose a novel design of bone morphogenetic protein 2 (BMP-2) carrier for tissue engineering of segmental defect regeneration. The tube-shaped BMP-2 carrier was fabrication from a poly(propylene fumarate)/tricalcium phosphate (PPF/TCP) composite via casting technique developed in our laboratory. An in vitro evaluation showed that the compressive strength of the carrier decreased about 48% in 12 weeks while maintained a pH in the 6.8-7.4 range. In vivo study was conducted by implanting carriers loaded with 10 microg of BMP-2 in 5 mm rat femur gap model for 15 weeks. X-ray evidence of bridging was first found in the BMP group at 3 weeks. Bridging in all animals (N = 4) in the BMP group was found at 9 weeks. No x-ray evidence of bridging was found in the No BMP group (N = 3). pQCT analysis indicated that the bone mineral density of the callus in the BMP group has reached the level of native femur at 15 weeks after implantation, while the callus in the No BMP group has a bone mineral density at a lower level of 84% to the native femur. Histology analysis shows that a normal fatty bone marrow was restored and mineralized callus formed and bridged the segmental defect.

Topics: Animals; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Bone Regeneration; Calcification, Physiologic; Drug Carriers; Drug Evaluation, Preclinical; Drug Implants; Femoral Fractures; Fracture Healing; Pilot Projects; Rats; Rats, Long-Evans; Transforming Growth Factor beta; Treatment Outcome; Weight-Bearing

Bone morphogenetic proteins (BMPs) are osteoinductive proteins used intensively in clinical investigations involving various bone-related treatments. Owing to their high potential in new bone formation they require local application at the treatment site. For this purpose various controlled delivery systems with BMPs as the excipients have been prepared in recent years. Focusing on this clinical need a disc-shaped BMP carrier was designed as a local delivery system using soluble collagen and chondroitin sulfate. In situ release studies carried out with a model protein (FITC-labeled Protein A) presented a very high rate of release; with most of the protein content being released within 24 h. This rate could be decreased by providing a poly(L-lactide) (PLLA) and sucrose acetate isobutyrate-based (SAIB-based) coat around the release system, applied after BMP loading. In this way, it was possible to extend the release period from 24 h to about 12 days. In situ release of BMP from the same carriers, as quantitated using an ELISA kit, was even slower, with 50% of the protein being released in 15 days. In order to be able to secure the BMP delivery system at the bone defect site and to provide support a mesh knitted using Vicryl sutures and bonded with poly(L-lactide-co-glycolide) (PLGA) was tested in in vivo. Two time periods, 1 and 3 weeks, were used to evaluate the healing process. Osteoinduction by the BMP carrier system was assessed by histology-based bone scoring and X-ray examinations. PLLA-SAIB-coated collagen discs containing BMP presented good biocompatibility and optimum osteogenic stimulation. Structural changes in histological micrographs at week 1 indicated dose-dependent periosteal ossification. At the end of week 3 histological findings with both BMP (1 and 2 microg) doses were almost the same.

Topics: Animals; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Bone Regeneration; Chondroitin Sulfates; Coated Materials, Biocompatible; Collagen Type I; Delayed-Action Preparations; Diffusion; Elasticity; Femoral Fractures; Fracture Healing; Hardness; Lactic Acid; Male; Materials Testing; Polyesters; Polymers; Rats; Rats, Sprague-Dawley; Sucrose; Tensile Strength; Transforming Growth Factor beta; Treatment Outcome

Recombinant human bone morphogenetic protein (rhBMP)-2 in a block copolymer composed of poly-D,L-lactic acid with randomly inserted p-dioxanone and polyethylene glycol (PLA-DX-PEG) as a carrier and porous beta-tricalcium phosphate (beta-TCP) blocks were used to generate a new fully absorbable osteogenic biomaterial. The bone regenerability of the rhBMP-2/PLA-DX-PEG/beta-TCP composite was studied in a critical-sized rabbit bone defect model. In an initial study, a composite of PLA-DX-PEG (250 mg) and beta-TCP (300 mg) loaded with or without rhBMP2 (50 microg) was implanted into a 1.5 cm intercalated bone defect created in a rabbit femur. Defects were assessed by biweekly radiography until 8 weeks postoperatively. The bony union of the defect was recognized only in the BMP-loaded group. To obtain further data on biomechanical and remodeling properties, another BMP-loaded composites group was made and observed up to 24 weeks. All defects were completely repaired without residual traces of implants. Anatomical and mechanical properties of the repaired bone examined by histology, 3-dimensional CT (3D-CT) and mechanical testing were essentially equivalent to the nonoperated-on femur at 24 weeks. These experimental results indicate that fully absorbable rhBMP-2/PLA-DX-PEG/beta-TCP is a promising composite having osteogenicity efficient enough for repairing large bone defects.

Topics: Absorbable Implants; Absorptiometry, Photon; Animals; Biocompatible Materials; Biomechanical Phenomena; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Bony Callus; Calcium Phosphates; Femoral Fractures; Femur; Fracture Healing; Guided Tissue Regeneration; Humans; Lactates; Osteogenesis; Polydioxanone; Polyethylene Glycols; Rabbits; Recombinant Proteins; Tomography, Spiral Computed; Transforming Growth Factor beta

Osteogenic proteins (OP-1) promote osteoinduction. Formation of new bone growth in patients receiving OP-1 is not consistent, and is possibly due to the short half-life of the drug. In order to test the capacity of OP-1 to consistently produce bone in a fracture model, a drug delivery system was developed to prolong the action of OP-1. Fifteen Sprague Dawley male rats were randomly divided into three equal groups; Animals in group 1 served as control. Animals in groups 2 and 3 had a 5mm defect created in the left femur using a number six dental burr and a drug delivery capsule (TCPL) containing either antibiotic alone (sham) or antibiotic +OP-1. Body weights, blood, and X-rays were taken weekly. Femurs and organs were harvested 30 days post-op, and processed for histomorphometry. Data was analyzed using ANOVA and significant difference between the groups was determined using Student Newman Kuels (p < 0.05). The results showed complete bone healing in the OP-1 group with an evident callus formation. The osteoid tissue exhibited a proliferation of osteoblasts, which differentiated from the vascularized mesenchymal tissue. The complete bone healing using OP-1 sharply contrasted sham treatment, where an obvious injury was still seen at 30 days. Histologically sham animals exhibited the early stage of repair with evidence of blood clotting and mesenchyme with early formation of osteoblasts. Overall, OP-1 delivered in a sustained manner for 30 days caused increased bone formation in a defect model.

Topics: Animals; Anti-Bacterial Agents; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Drug Combinations; Drug Implants; Femoral Fractures; Fracture Healing; Male; Osteitis; Osteogenesis; Rats; Rats, Sprague-Dawley; Transforming Growth Factor beta; Treatment Outcome; Vancomycin

Although the mechanisms of osteoinduction by bone morphogenic proteins (BMPs) are increasingly understood, the most appropriate time to administer BMPs exogenously is yet to be clarified. The purpose of this study was to investigate when BMP may be administered to a fracture arena to maximise the enhancement of healing. Forty mice with externally fixed left femoral fractures were randomised into four groups: Group I, the control group was given a placebo of 30 microl saline at day 0; Groups II, III and IV were given 30 microl saline plus 2.5 microg rhBMP-2, at post-operative days 0, 4 or 8, respectively. Sequential radiographs were taken at days 0, 8, 16. On day 22 the mice were sacrificed and both femora were harvested for biomechanical assessment in 3-point bending and histological evaluation. Radiographic analysis indicated that healing of fractures in Groups II and III was significantly greater (p < 0.05) than those in Groups I and IV, at both 16 and 22 days post-fracture. The highest median bone mineral content at the fracture site was evidenced in Group III and II. Furthermore, Group III also had the highest relative ultimate load values, followed by Groups II, IV and I. Greater percentage peak loads were observed between Group I and both Groups II and III (p < 0.05). Histological examination confirmed that at 22 days post-fracture, only fractures in Groups II and III had united with woven bone, and Groups I and IV still had considerable amounts of fibrous tissue and cartilage at the fracture gap. Data presented herein indicates that there is a time after fracture when rhBMP administration is most effective, and this may be at the time of surgery as well as in the early fracture healing phases.

Topics: Animals; Biomechanical Phenomena; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Femoral Fractures; Humans; Male; Mice; Osteogenesis; Recombinant Proteins; Time Factors; Transforming Growth Factor beta

Bone repair involves both anabolic and catabolic responses. We hypothesized that anabolic treatment with OP-1 (BMP-7) and anti-catabolic treatment with zoledronic acid could be synergistic. In a rat critical defect, this combination therapy produced significant increases in new bone volume and strength.. When used to augment bone healing, osteogenic protein 1 (OP-1/BMP-7) and other BMPs stimulate the anabolic response, inducing osteoblast recruitment, differentiation, and bone production. However, BMPs can also upregulate catabolism by direct stimulation of osteoclasts and indirectly by osteoblasts through RANKL/RANK. We hypothesized that if such osteoclastic upregulation were modulated by zoledronic acid (ZA), the combination of OP-1 and ZA should produce increased new bone over OP-1 alone.. Rats with a surgically induced 6-mm femoral critical size defect were separated into five dosing groups: Carrier, Carrier + ZA, OP-1, OP-1 + ZA, and OP-1 + ZA administered 2 weeks after surgery (2W). Carrier +/- 50 microg OP-1 was placed in the defect, and 0.1 mg/kg ZA or saline was administered subcutaneously. Bone repair was analyzed by radiographs, QCT, mechanical testing, histology, and histomorphometry.. Carrier alone and Carrier ZA groups did not unite by 8 weeks. Radiological union occurred in all OP-1 groups but was tenuous in some animals treated with OP-1 alone. BMC was increased by 45% in the OP-1 ZA group and 96% in the OP-1 ZA 2W group over OP-1 alone (p < 0.01). Callus volume increased over OP-1 alone by 45% and 86% in the OP-1 ZA and OP-1 ZA 2W groups, respectively (p < 0.01). The increased callus volume in the OP-1 ZA 2W group translated to increases in strength of 107% and stiffness of 148% (p < 0.05). BFR was not significantly different between OP-1 groups regardless of ZA treatment.. ZA treatment significantly increased the BMC, volume, and strength of OP-1-mediated callus in a critical size defect in rats at 8 weeks. Thus, modulation of both anabolic and catabolic responses may optimize the amount and mineral content of callus produced, which could be of clinical benefit in obtaining bone union.

Topics: Animals; Biomechanical Phenomena; Bone Density; Bone Density Conservation Agents; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Bone Regeneration; Bony Callus; Cell Count; Diphosphonates; Elasticity; Femoral Fractures; Femur; Fracture Healing; Histocytochemistry; Imidazoles; Male; Osteoclasts; Rats; Rats, Wistar; Stress, Mechanical; Transforming Growth Factor beta; Zoledronic Acid

The purpose of this study was to evaluate the efficacy and safety of recombinant bone morphogenetic protein 7 (rhBMP-7 or OP-1) as a bone-stimulating agent in the treatment of persistent fracture non-unions. Twenty-five consecutive patients [19 males, mean age 39.4 years (range: 18-79)] with 26 fracture non-unions were treated with rhBMP-7. There were 10 tibial non-unions, eight femoral, three humeral, three ulnar, one patellar, and one clavicular non-union. The mean follow-up was 15.3 months. The mean number of operations performed prior to rhBMP-7 application was 3.2, with autologous bone graft and bone marrow injection being used in 10 cases (38.5%). Both clinical and radiological union occurred in 24 (92.3%) cases, within a mean time of 4.2 months and 5.6 months, respectively. Of the remaining two cases, one patient ultimately underwent a below knee amputation, secondary to recurrence of deep sepsis. The other patient with recalcitrant ulnar non-union although the radiological union was incomplete, declined further intervention, as he was asymptomatic. No complications or adverse effects from the use of rhBMP-7 were encountered. This study supports the view that the application of rhBMP-7 as a bone-stimulating agent is safe and a power adjunct to be considered in the surgeon's armamentarium for the treatment of these challenging clinical conditions.

Topics: Adolescent; Adult; Aged; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Bone Transplantation; Female; Femoral Fractures; Femur; Follow-Up Studies; Fracture Fixation, Internal; Fracture Healing; Fractures, Ununited; Humans; Humeral Fractures; Humerus; Knee Injuries; Male; Middle Aged; Patella; Radiography; Recombinant Proteins; Shoulder Fractures; Tibial Fractures; Transforming Growth Factor beta; Ulna Fractures

Regenerated cellulose sponges were coated biomimetically with hydroxyapatite to increase their osteogenic properties. Induction of apatite precipitation was carried out with bioactive glass in simulated body fluid (SBF) for 24 h and the final coating was carried out in 1.5 x concentrated SBF for 14 days. Biomimetically mineralized and non-mineralized sponges were then implanted into standard size femoral cortical defects of rats, and the invasion of bone into the implant was followed up to one year. The apatite coating did not improve the osteoconductive property of cellulose in this rat cortical defect model. In fact, it generated a strong and highly cellular inflammatory reaction and less osteoid tissue. The biomimetic implants contained more immunodetectable TGFbeta1 (a strong stimulator of fibroblast activity) than untreated implants, and also bound more TGFbeta1 in vitro, which could, at least in part, explain the fibrotic invasion of biomimetically mineralized sponges.

Topics: Animals; Bone Substitutes; Cellulose; Coated Materials, Biocompatible; Durapatite; Femoral Fractures; Male; Materials Testing; Osteogenesis; Rats; Rats, Sprague-Dawley; Transforming Growth Factor beta; Transforming Growth Factor beta1; Treatment Outcome

The influence of mechanical tissue strain caused by flexible fracture fixation on the systemic occurrence of systemic mitogens during callus healing was investigated. For this purpose the mitogenic capacity and growth factor concentration of sera from patients undergoing fracture treatment were determined. Sera from 9 patients whose fractures had been stabilized by external fixation were collected before and during fracture treatment. The sera were added to cell culture media of the osteoblastic cell line SaOS-2. After 5-6 days cell proliferation was measured. Transforming growth factor-beta1 (TGF-beta1) and insulin-like growth factor-I (IGF-I) concentrations were analyzed in serum samples from different healing stages.. paired Wilcoxon-test. Sera from fracture patients decreased SaOS-2 proliferation in the first week after surgery (p<0.05) compared to sera obtained prior to surgery. In the fourth or fifth week proliferation increased significantly (p<0.03). The increased proliferation of the SaOS-2 cells was associated with elevated levels of TGF-beta and IGF-I (p<0.05). The higher mitogenic activity of sera suggests an increased level of circulating mitogens. In a previous study this increase had also been observed in patients during distraction osteogenesis treatment but not in patients with primary bone healing by a stable fixated plate. It is therefore assumed that their release from the fracture site is a consequence of mechanical stimulation by interfragmentary movement of fracture ends.

Topics: Adult; Bony Callus; Cell Division; Cell Line; Female; Femoral Fractures; Femur; Fracture Fixation; Fracture Healing; Humans; Insulin-Like Growth Factor I; Male; Middle Aged; Mitogens; Osteoblasts; Osteogenesis, Distraction; Tibia; Tibial Fractures; Transforming Growth Factor beta; Transforming Growth Factor beta1

Total hip arthroplasty (THA) has become an almost standard procedure for the treatment of various hip lesions. However, one of the limitations has been the mechanical loosening of the prosthesis, a condition termed peri-prosthetic osteolysis. Consequently, at revision surgery, various grades of bone defect are often noted. Alternative approaches aimed at overcoming this problem have included a special design of the revision prosthesis and allo- or autogeneic bone grafting in combination with or without biomaterials. In a further attempt to address the loosening of the prosthesis, we have combined human bone morphogenetic protein-2, produced by DNA recombination (rhBMP-2) with a new synthetic biodegradable polymer (poly-D,L-lactic-acid-para-dioxanone-polyethyleneglycol block co-polymer; PLA-DX-PEG). We present data on the efficacy of the rhBMP-2 laden prosthesis to reconstruct a bone defect in a canine model. In this model, medial half of the proximal femur was surgically resected to create a bone defect that was repaired with the rhBMP-2/PLA-DX-PEG composite. Twelve weeks after implantation, the original bone defects in the rhBMP-2 treatment groups had been repaired. Thus, this type of 'hybrid' prosthesis may provide a new modality to repair bone defects or restore lost bone mass encountered in revision arthroplasty.

Topics: Absorbable Implants; Animals; Arthroplasty, Replacement, Hip; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Coated Materials, Biocompatible; Combined Modality Therapy; Dogs; Equipment Failure Analysis; Femoral Fractures; Hip Prosthesis; Joint Instability; Lactates; Male; Polyethylene Glycols; Porosity; Prosthesis Design; Radiography; Reoperation; Surface Properties; Transforming Growth Factor beta

Fracture-healing slows with age. While six-week-old rats regain normal bone biomechanics at four weeks after a fracture, one-year-old rats require more than twenty-six weeks. The objective of this study was to examine the possible role of altered mRNA gene expression in this delayed union.. Closed midshaft femoral fractures were created in six-week-old and one-year-old Sprague-Dawley female rats. The animals were killed at zero-time (unfractured) or at 0.4, one, two, three, four, or six weeks after the fracture. mRNA levels were measured by reverse transcription-polymerase chain reaction in the fracture callus for twenty-seven matrix, cytokine, and cytokine-receptor genes for the seven animals per time-point per age-group.. The younger rats healed radiographically by four weeks after the fracture, whereas none of the older rats had healed by the sixth week. Despite the difference in healing rates, the levels of mRNA gene expression, in general, followed the same pattern in both age-groups. The mRNA expression levels increased to a peak at one to two weeks after the fracture and then decreased to very low or undetectable levels at four and six weeks after the fracture for both age-groups. Significantly lower levels of mRNA for Indian hedgehog (Ihh) and bone morphogenetic protein-2 (BMP-2) were detected in the fracture calluses of the older rats (p < 0.01 and p < 0.05, respectively).. All genes studied were up-regulated by the fracture in both age-groups. Thus, the failure of the older rats to heal promptly was not due to the lack of expression of any of the studied genes. The increase in mRNA for Ihh and BMP-2 in the older rats was smaller than that in the younger rats, which may contribute to slower fracture repair. The return of mRNA gene expression to baseline in the older rats prior to healing may contribute to the delayed union. The slower healing response of the older rats did not stimulate a negative-feedback increase in the mRNA expression of stimulatory cytokines.

Topics: Age Factors; Aggrecans; Aging; Animals; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Bony Callus; Collagen Type II; Cytokines; Disease Models, Animal; Extracellular Matrix Proteins; Female; Femoral Fractures; Fracture Healing; Fractures, Ununited; Gene Expression; Hedgehog Proteins; Lectins, C-Type; Osteocalcin; Proteoglycans; Radiography; Rats; Rats, Sprague-Dawley; Receptors, Cytokine; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sequence Analysis, RNA; Trans-Activators; Transforming Growth Factor beta; Up-Regulation

Recombinant human bone morphogenetic protein-2 (rhBMP-2), surgically implanted with a matrix material, has been shown to induce bone formation and enhance fracture repair. The purpose of this investigation was to test the hypothesis that a single, local, percutaneous injection of rhBMP-2 would accelerate fracture-healing in a standard rat femoral fracture model.. Fractures were created, following intramedullary pinning, in the femora of 144 male Sprague-Dawley rats. The animals were divided into three groups of forty-eight each. Six hours after the fracture, one group received an injection of 80 micro g of rhBMP-2 in 25 micro L of buffer vehicle, one received an injection of 25 micro L of buffer vehicle alone, and one did not receive an injection. Twelve animals from each of these three groups were killed at one, two, three, and four weeks after treatment, and the femora were harvested for torsional biomechanical testing. An additional cohort of seventy-two animals, in which a fracture was also created, was divided into the same three treatment groups; six animals from each of these groups was killed at one, two, three, and four weeks; and the femora were processed for qualitative histological analysis.. Torsional biomechanical testing indicated that the stiffness of the rhBMP-2-treated fractures was twice that of both control groups at the two, three, and four-week time-points. The strength of the rhBMP-2-treated fractures was 34% greater than that of the buffer-treated controls (p = 0.03) at three weeks and, at four weeks, was 60% and 77% greater than that of the buffer-treated controls and that of the untreated controls, respectively (p < 0.005). At four weeks, the stiffness and strength of the rhBMP-2-treated fractures were equal to those of the intact contralateral femora, whereas the buffer-treated and untreated fractures were significantly weaker than the intact femora. At two and three weeks, large areas of bone formation, typically spanning the fracture, were observed histologically in the rhBMP-2-treated sites. In contrast, the control fractures exhibited primarily soft cartilaginous callus at these time-points. By four weeks, remodeling of the hard callus and recorticalization were observed in the rhBMP-2-treated fracture sites, whereas cartilage and/or soft tissue was still present in the control fracture sites.. These data demonstrate that a single, local, percutaneous injection of rhBMP-2 accelerates fracture repair in this rat femoral fracture model. This effect appears to result from a combination of the induction of bone formation at the fracture site and acceleration of the rate at which the fracture callus matures.

Topics: Animals; Biomechanical Phenomena; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Bone Nails; Dose-Response Relationship, Drug; Femoral Fractures; Femur; Fracture Healing; Injections; Male; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Transforming Growth Factor beta

Transforming growth factor-beta 1 (TGF-beta1)is a polypeptide growth factor which has been shown to increase bone formation in experimental studies. In this study it was combined to a bioabsorbable self-reinforced poly-LD-lactic acid fracture fixation pin. To assess the effect of TGF-beta1 on the healing of a bone defect, the pins were implanted in the rat distal femur next to a bone defect filled with a viscose cellulose sponge. The pins used in the study group (13 rats) contained 50 microg of TGF-beta1, whereas in the control group of nine rats an identical pin without the growth factor was used. In the histologic examination at 1, 3 and 6 weeks no difference was detected in the amount of bone inside the viscose cellulose sponge between the rats treated with TGF-beta1 and those with no added growth factor. At 3 weeks there was more fibroblast-rich mesenchymal tissue inside the viscose cellulose sponge in the rats treated with TGF-beta1. In the radiographic examination at 3 weeks there was an increase in the amount of new periosteal bone on the bone defect in the TGF-beta1-treated rats.

Topics: Absorbable Implants; Animals; Biocompatible Materials; Bone and Bones; Bone Nails; Cellulose; Coated Materials, Biocompatible; Femoral Fractures; Fracture Fixation, Internal; Fracture Healing; Lactic Acid; Male; Polyesters; Polymers; Radiography; Rats; Rats, Wistar; Time Factors; Transforming Growth Factor beta; Transforming Growth Factor beta1

The utility of cortical allografts in repairing large bone defects is limited by their slow and incomplete incorporation into host bone. In order to determine the effects of recombinant human osteogenic protein-1 (rhOP-1) impregnation on allograft incorporation, we used a canine intercalary bone defect model. Bilateral resection of a 4 cm segment of the femoral diaphysis and reconstruction with structural bone allografts were performed. In one limb, the allograft was soaked in solution with rhOP-1 for 1 h before implantation. In the other limb, the allograft was soaked in the same solution without rhOP-1. Dynamic load-bearing, radiographic analysis, biomechanical testing, and histomorphometric analysis were conducted. Radiographic analysis showed significantly larger periosteal callus area in the rhOP-1 treated group at week 2. The rhOP-1 significantly increased allograft bone porosity and significantly increased the number of active osteons in the allografts. There were no significant differences between the rhOP-1 treated and non-treated allografts in load bearing and biomechanical analyses. These findings indicate that rhOP- I increases intercalary allograft remodeling without deleterious effects in mechanical and functional strength.

Topics: Animals; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Bone Transplantation; Disease Models, Animal; Dogs; Femoral Fractures; Internal Fixators; Radiography; Recombinant Proteins; Torsion Abnormality; Transforming Growth Factor beta; Transplantation, Homologous; Weight-Bearing

The goal of this study was to use a segmental defect model in the rat femur to determine if osteogenic protein-1 (OP-1) is capable of inducing bone formation in the presence of bacterial contamination. A 6 mm segmental defect was surgically created and stabilized with a polyacetyl plate and Kirschner wires in one femur in each of 126 Sprague-Dawley rats. The animals were divided into eight groups in which the defect was either left untreated, or subjected to various combinations of OP-1 (11 or 50 microg), lyophilized bovine type I collagen (carrier for the OP-1), and 10(5) colony-forming units of Staphylococcus aureus. The animals were euthanized at either 2, 4, or 9 weeks. Quantitative radiographic and histologic analyses were performed on the harvested tissue. The initial contamination progressed to infection in all animals receiving bacteria, as determined by qualitative bacteriology. There was very little, if any, bone formation in the untreated defects, and in the contaminated defects with or without collagen carrier. Bone formation was significantly greater in contaminated defects with either dose of OP-1, compared with contaminated defects without OP-1. The 50 microg dose of OP-1 induced significantly more bone formation than the 11 microg dose, both with and without bacteria. This investigation has demonstrated that OP-1 maintains its osteoinductive capability in a contaminated segmental defect. OP-1 may potentially be used in the clinical management of contaminated fractures.

Topics: Animals; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Femoral Fractures; Femur; Male; Osteitis; Osteogenesis; Rats; Rats, Sprague-Dawley; Staphylococcal Infections; Transforming Growth Factor beta

Long-term clinical data have shown that reconstruction using bone allografts provide adequate function after extensive tumor surgery. Complications such as nonunion of allograft-host interface, infection, and allograft fracture often require major revision surgeries. Allograft fractures usually do not induce the same repair process that is seen in normal fracture healing. The authors did an experimental study to test whether bone morphogenetic protein-2 can induce and achieve osseous repair in an allograft osteotomy model. Recombinant human bone morphogenetic protein-2 was applied at femoral intercalary allograft osteotomy sites in 20 rats. Forty additional rats served as controls (carrier alone and sham). Specimens in all groups were examined histologically and radiographically at 4 and 8 weeks. Specimens in the control groups showed only fibrosis by 8 weeks. In contrast, none of 10 specimens in the experimental group showed radiographic union at 8 weeks. New bone formation and integration with underlying allografts were seen in the experimental group as early as 4 weeks. These data suggest that fracture repair in the allograft bone can be triggered by a biologic regulator that is expressed during normal fracture healing.

Topics: Animals; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Bony Callus; Femoral Fractures; Fracture Healing; Models, Animal; Osteotomy; Rats; Rats, Inbred Lew; Recombinant Proteins; Transforming Growth Factor beta

The effect of zinc, a stimulator of bone formation, on bone protein components in the femoral-diaphyseal tissues with fracture healing was investigated. Rats were sacrificed between 1 and 7 days after the femoral fracture, and the diaphyseal tissues were cultured in a serum-free Dulbecco's modified Eagle's medium for 24 h. Protein content in the femoral-diaphyseal tissues was markedly elevated by fracture healing. The amount of protein in the medium cultured with the diaphyseal tissues obtained from fracture healing rats was markedly elevated as compared with that of normal rats, indicating that bone protein components were secreted into culture medium. Analysis with sodium dodecyl sulfate-polyacrylamide gel elecrophoresis (SDS-PAGE) showed that many protein molecules were secreted from the diaphyseal tissues with fracture healing. Especially, protein molecule of about 66 kDa was markedly secreted by fracture healing. The presence of zinc acexamate (10(-5) and 10(-4) M) in culture medium induced a significant elevation of medium protein content; the zinc effect was enhanced by culture with the diaphyseal tissues of fracture healing rats. Also, the culture of diaphyseal tissues with fracture healing caused a significant increase in insulin-like growth factor-I (IGF-I) and transforming growth factor-beta1 (TGF-beta1) in culture medium. The production of IGF-I and TGF-beta1 from bone tissues with fracture healing was significantly enhanced in the presence of zinc acexamate (10(-6)-10(-4) M). Moreover, the addition of IGF-I (10(-8) M) or TGF-beta1 (10(-10) M) in a culture medium caused a significant elevation of protein content in the medium cultured with the femoral-diaphyseal tissues from normal and fracture healing rats. The effect of IGF-I or TGF-beta1 was significantly enhanced in the presence of zinc acexamate (10(-4) M). Also, deoxyribonucleic acid (DNA) content in the diaphyseal tissues from normal and fracture healing rats was significantly raised by the culture with IGF-I or TGF-beta1. The effect of IGF-I was significantly enhanced by zinc. The present study demonstrates that fracture healing increases production of bone IGF-I and TGF-beta1, and that this elevation is enhanced by zinc treatment.

Topics: Aminocaproates; Aminocaproic Acid; Animals; Bone and Bones; Cells, Cultured; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Femoral Fractures; Fracture Healing; Growth Substances; Insulin-Like Growth Factor I; Male; Proteins; Rats; Rats, Wistar; Transforming Growth Factor beta; Transforming Growth Factor beta1; Zinc; Zinc Compounds

Expression of the genes encoding bone morphogenetic proteins (BMPs), BMP type IA receptor (BMPR-1A), and rat distal-less homolog (rDlx) was studied in bone, callus, and the surrounding soft tissue following rat femoral closed fracture, using RT-PCR-based techniques. Before fracture, the genes encoding BMP-5, BMP-6, and BMPR-1A were found to be expressed in both bone and the surrounding soft tissue, whereas the BMP-2 gene was expressed only in bone and BMP-7 was not expressed in either tissue. Expression of these genes was unaffected by fracture. The gene encoding BMP-4 was also expressed in both bone and the surrounding soft tissue before fracture. Moreover, although unchanged in bone, 6 h after fracture BMP-4 expression was increased tenfold in the surrounding soft tissue. The increased BMP-4 expression was transient and returned to prefracture levels within 72 h. Expression of rDlx was also increased in bone after fracture, but at later times than were observed with BMP-4: elevated rDlx expression was detected after 48 h and persisted for 30 days or more. No expression of rDlx was observed in the surrounding soft tissue before or after fracture. These findings indicate that BMP-4 and rDlx are selectively expressed following femoral fracture in the rat, and also suggest that they are involved in the formation of the callus at an early point during the postfracture healing of bone.

Topics: Alkaline Phosphatase; Animals; Blotting, Southern; Bone Morphogenetic Protein 2; Bone Morphogenetic Protein 4; Bone Morphogenetic Protein 5; Bone Morphogenetic Protein Receptors, Type I; Bone Morphogenetic Proteins; Femoral Fractures; Fracture Healing; Gene Expression Regulation; Homeodomain Proteins; Isoenzymes; Male; Osteocalcin; Protein Serine-Threonine Kinases; Rats; Rats, Sprague-Dawley; Receptors, Growth Factor; Reverse Transcriptase Polymerase Chain Reaction; Transcription Factors; Transforming Growth Factor beta

This study evaluated the ability of gene transfer to enhance bone healing. Segmental defects were created surgically in the femora of New Zealand white rabbits. First generation adenoviruses were used as vectors to introduce into the defects genes encoding either human bone morphogenetic protein-2 (BMP-2) or, as a negative control, firefly luciferase. Representative specimens were evaluated histologically after 8 weeks. Healing of the defects was monitored radiographically for 12 weeks, after which time the repair tissue was evaluated biomechanically. By radiological criteria, animals receiving the BMP-2 gene had healed their osseous lesions after 7 weeks, whereas those receiving the luciferase gene had not. Histologic examination of representative rabbits at 8 weeks confirmed ossification across the entire defect in response to the BMP-2 gene, whereas the control defect was predominantly fibrotic and sparsely ossified. At the end of the 12-week experiment, the control femora still showed no radiological signs of stable healing. The difference in radiologically defined healing between the experimental and control groups was statistically significant (P < 0. 002). Biomechanical testing of the femora at 12 weeks demonstrated statistically significant increases in the mean bending strength (P < 0.005) and bending stiffness (P < 0.05) of the animals treated with the BMP-2 gene. Direct, local adenoviral delivery of an osteogenic gene thus led to the healing of an osseous lesion that otherwise would not do so. These promising data encourage the further development of genetic approaches to enhancing bone healing. Gene Therapy (2000) 7, 734-739.

Topics: Adenoviridae; Animals; Biomechanical Phenomena; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Femoral Fractures; Femur; Genetic Therapy; Rabbits; Radiography; Transfection; Transforming Growth Factor beta; Wound Healing

A beta-tricalcium phosphate-monocalcium phosphate monohydrate (beta-TCP-MCPM) cement was evaluated as an effective carrier of recombinant human bone morphogenetic protein-2 (rhBMP-2) in rat femoral critical-size defects. Hard cement cylinders (4 x 5 mm) impregnated with two different doses of rhBMP-2 (1.26 or 6.28 microg) were implanted into each defect, and the results were compared with those in rats that had implantations of cylinders only. Implantation of the 6.28 microg dose of rhBMP-2 caused a large bone shell to form around the defect, resulting in osseous union in all cases within 3 weeks. Except for beta-TCP granules, the cement was resorbed and replaced by bone tissue at 6 weeks. A torsion test at 9 weeks showed that the failure torque and bone stiffness had recovered 99% and 141%, respectively, compared with the intact contralateral femur. The defects that received 1.26 microg of rhBMP-2 resulted in 40% union and 41% of the failure torque at 9 weeks. However, no instances of union were observed in the defects implanted with cylinders only. In conclusion, the beta-TCP-MCPM cement was shown to be effective as a rhBMP-2 carrier. Combined with rhBMP-2, this cement was rapidly resorbed and completely healed the defects.

Topics: Animals; Biomechanical Phenomena; Bone Cements; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Bone Nails; Bone Plates; Calcium Phosphates; Drug Carriers; Femoral Fractures; Fracture Healing; Humans; Male; Materials Testing; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Transforming Growth Factor beta

Although growth factors have been demonstrated during bone healing, their presence has not yet been confirmed in callus distraction. Therefore, in 3 patients we searched for cytokines during callus distraction. Bone biopsies were immuno-histochemically stained for TGF-beta1, IGF-I, TGF-beta type II receptor, IGF receptor, and proliferating cell nuclear antigen (PCNA). Histologically we found immature woven bone in the middle of the callus zone and increasing calcification and lamellar bone in the re-modelling zone. Osteoblasts and fibroblast-like cells in the middle zone, and osteoblasts in all zones stained for TGF-beta and its receptor. The number of positive staining cells related to proliferous activity as assessed both by PCNA, and by bone density in radiographs. IGF-I could be detected everywhere. In conclusion, growth factors are present in bone formation and in areas of re-modelling during callotasis. Their relation to proliferous activity and radiographic density supports their involvement in osteogenesis.

Topics: Adult; Bony Callus; Female; Femoral Fractures; Femur; Fracture Healing; Humans; Immunohistochemistry; Insulin-Like Growth Factor I; Male; Osteitis; Osteogenesis, Distraction; Reoperation; Tibia; Transforming Growth Factor beta

The effect of a bioabsorbable pin containing transforming growth factor-beta(1) on fracture healing was studied in a rat model. The growth factor was mixed into a bioabsorbable polymer paste (a blend of an l-lactic acid oligomer and a copolymer of epsilon-caprolactone and dl-lactide) that was used to fill the grooves of a self-reinforced fracture fixation pin made of a poly-ld-lactic acid copolymer. In an in vitro assay, sustained release of the growth factor from the pins over a 7-day period was demonstrated. A distal femoral osteotomy was made in 60 rats and stabilized with the fracture fixation pin in 48 of them; In the remaining 12 rats, no fixation was performed. The pin used in the study group contained either 5 microg (15 rats) or 50 microg (15 rats) of the growth factor, while in a control group of 18 rats an identical pin without the growth factor was used. After a follow-up of 1, 3, or 6 weeks, the femurs were examined radiographically, histologically, histomorphometrically, and microradiographically, and also used in tetracycline labeling studies. Faster callus formation was evident in the growth factor-treated rats but no acceleration in the healing of the osteotomy was detected.

Topics: Absorbable Implants; Animals; Bone Nails; Caproates; Chi-Square Distribution; Coated Materials, Biocompatible; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Femoral Fractures; Fracture Fixation, Internal; Fracture Healing; Lactic Acid; Lactones; Male; Microradiography; Osteotomy; Polyesters; Polymers; Rats; Rats, Wistar; Statistics, Nonparametric; Transforming Growth Factor beta

Middiaphyseal 2.5-cm segmental defects in the right femurs of 12 sheep were stabilized with stainless steel plates and implanted with (1) 2 mg recombinant human bone morphogenetic protein 2 and poly[D,L-(lactide-co-glycolide)] bioerodible polymer with autologous blood (n = 7), (2) 4 mg recombinant human bone morphogenetic protein 2 and poly[D,L-(lactide-co-glycolide)] and blood (n = 3), or (3) poly[D,L-(lactide-co-glycolide)] and blood only (n = 2). Bone healing was evaluated for 1 year using clinical, radiographic, gross pathologic, and histologic techniques. Union occurred in three sheep in Group 1, two in Group 2, and none in Group 3. In the animals that healed, new bone first was visible radiographically between Weeks 2 and 6 after implantation; new bone mineral content equaled that of the intact femur not surgically treated by Week 16; recanalization of the medullary cavity approached completion at Week 52; and at necropsy the surgical treated femurs were rigidly healed, the poly[D,L-(lactide-co-glycolide)] was resorbed completely, and woven and lamellar bone bridged the defect site. In two Group 1 sheep euthanized at Weeks 2 and 6, polymer particles were permeated by occasional multinucleated giant cells. Some plasma cells, lymphocytes, and neutrophils were present locally. The poly[D,L-(lactide-co-glycolide)] tended to fragment during surgical implantation. Despite these observations, the recombinant human bone morphogenetic protein 2/poly[D,L-(lactide-co-glycolide)] implant was able to heal large segmental bone defects in this demanding model.

Topics: Animals; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Bony Callus; Disease Models, Animal; Femoral Fractures; Prostheses and Implants; Recombinant Proteins; Sheep; Transforming Growth Factor beta; Wound Healing

This is the first report on successful treatment of spontaneous nonunion fracture of a dog by use of recombinant human bone morphogenetic protein-2 (rhBMP-2). A 4-year-old Pomeranian dog with a 2-year history of femoral nonunion fracture was treated with implantation of rhBMP-2. The dog had received surgical correction twice prior to the admission but radiography of the affected limb revealed a typical figure of nonunion fracture. Glossly, the fractured ends were sclerotic and the area between the ends was filled with fibrous tissue. After debridement the femoral shaft was fixed by an 8-hole plate. rhBMP-2 at a total dose of 256 micrograms was implanted with a synthetic carrier into the 8-mm defect formed by the debridement. Callus formation responding to rhBMP-2 was radiographically observed at 2 weeks after implantation and the defect reached radiographic union by 8 weeks after implantation. The plate was removed at 9 months after implantation. Any complications were not observed for 5 months after removal of the plate.

Topics: Animals; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Bone Plates; Dog Diseases; Dogs; Femoral Fractures; Humans; Male; Radiography; Recombinant Proteins; Transforming Growth Factor beta

The effect of an absorbable pin containing transforming growth factor beta 1 on fracture healing was studied in a rat model of delayed osteotomy fixation. Transforming growth factor beta 1 was mixed into a blend of L-lactide oligomer and a copolymer of epsilon-caprolactone and DL-lactide that was placed in the grooves of a self reinforced fracture fixation pin made of poly-LD-lactic acid copolymer. A distal femoral osteotomy was made in 54 rats and left untreated. A week later surgery was performed to fix the osteotomy with a fracture fixation pin in 48 rats. In the remaining six rats no fixation was performed. The pin that was used in the study group contained either 5 micrograms (15 rats) or 50 micrograms (15 rats) of the growth factor, while in the control group of 18 rats, an identical pin without growth factor was used. The femurs were examined radiographically, histologically, histomorphometrically, and microradiographically. Tetracycline labeling studies were used after a followup of 1, 3, and 6 weeks. Faster callus formation in the transforming growth factor beta 1 treated animals but no acceleration in the healing of the osteotomy is reported. The addition of bone growth factors to bioabsorbable fracture fixation materials may enhance bone healing.

Topics: Animals; Biocompatible Materials; Bone Nails; Combined Modality Therapy; Disease Models, Animal; Drug Evaluation, Preclinical; Femoral Fractures; Fracture Fixation, Internal; Fracture Healing; Male; Materials Testing; Osteotomy; Polyesters; Radiography; Rats; Rats, Wistar; Transforming Growth Factor beta

We studied the pharmacological effects of dalteparin (low-molecular-weight heparin) and heparin on bone metabolism in rats. After their 28 days of consecutive intravenous injections, significant loss of bone weight and mineral contents was observed in the heparin-treated rats, whereas dalteparin slightly reduced bone mass. By the end of the experiment, the femora of 7 out of 8 rats fractured in the heparin (10,000 U/kg/day)-treated group, but none had broken in the control and dalteparin-treated groups. Serum osteocalcin levels were significantly decreased in the former group. The growth plate width of the tibia was increased in a dose-dependent manner, especially in the heparin-treated group. Histomorphometric assessment of tibia showed that the osteoid surface and mineral apposition rates were significantly reduced in the heparin-treated group, whereas the eroded surface was significantly increased in the heparin-treated group. The above results suggest that heparin not only augments bone resorption but also suppressed bone formation and that dalteparin has a weaker suppressive effect on bone formation compared with heparin.

Topics: Animals; Anticoagulants; Bone Density; Bone Marrow; Densitometry; Femoral Fractures; Heparin; Heparin, Low-Molecular-Weight; Male; Minerals; Organ Size; Osteocalcin; Radiography; Rats; Rats, Sprague-Dawley; 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

The effects of ultrasound stimulation on various parameters of bone repair after diaphyseal injury were assessed in a standard rat femur fracture model. Bilateral closed femoral fractures were made in 79 skeletally mature male Long-Evans rats. An ultrasound signal consisting of a 200 microsecond burst sine wave of 0.5 MHz repeating at 1 kHz, with an intensity of 50 or 100 mW/cm2 spatial and temporal average, was applied to one fracture in each animal. The contralateral fracture was not exposed to ultrasound and served as a control. Mechanical testing of the healing fracture was performed 3 weeks after injury. In fractures treated with a 50 mW/cm2 ultrasound signal, the average maximum torque (223.5 +/- 50.5 Nmm compared with 172.6 +/- 54.9 Nmm, p = 0.022, paired t test) and average torsional stiffness (13.0 +/- 3.4 Nmm/degree compared with 9.5 +/- 2.9 Nmm/degree, p = 0.017) were significantly greater in treated than in control fractures. In animals treated with a 100 mW/cm2 ultrasound signal, the average maximum torque and torsional stiffness were greater in treated than in control fractures, but this trend did not reach statistical significance. Biochemical analysis of callus in ultrasound-treated and control fractures failed to demonstrate significant differences in cell number, collagen content, or calcium content. Evaluation of gene expression in fractures treated with 50 mW/cm2 ultrasound demonstrated a shift in the expression of genes associated with cartilage formation; aggrecan gene expression was significantly higher on day 7 after fracture and significantly lower on day 21 (p = 0.033 and 0.035, respectively). alpha 1(II) procollagen gene expression was similarly modified, but this trend did not reach statistical significance. Expression of genes coding for bone-related proteins, including alpha 1(I) procollagen, bone gamma-carboxyglutamic acid protein, alkaline phosphatase, and transforming growth factor-beta 1, did not differ between ultrasound-treated and control fractures. These data suggest that ultrasound stimulation increased the mechanical properties of the healing fracture callus by stimulating earlier synthesis of extracellular matrix proteins in cartilage, possibly altering chondrocyte maturation and endochondral bone formation.

Topics: Aggrecans; Alkaline Phosphatase; Animals; Calcium; Chondroitin Sulfate Proteoglycans; Collagen; Disease Models, Animal; Evaluation Studies as Topic; Extracellular Matrix Proteins; Femoral Fractures; Femur; Gene Expression; Lectins, C-Type; Male; Osteocalcin; Osteogenesis; Procollagen; Proteoglycans; Rats; Rats, Inbred Strains; RNA, Messenger; Stress, Mechanical; Transforming Growth Factor beta; Ultrasonic Therapy

Topics: Animals; Bony Callus; Cell Division; Extracellular Matrix Proteins; Femoral Fractures; Fibroblast Growth Factors; Gene Expression; Growth Substances; Male; Organ Culture Techniques; Platelet-Derived Growth Factor; Rats; Rats, Inbred Strains; Suramin; Transforming Growth Factor beta; Wound Healing