iridoids and Intervertebral-Disc-Degeneration

Biomaterial-based therapy that can restore annulus fibrosus (AF) function in early stage and promote endogenous repair of AF tissues is a promising approach for AF tissue repair. In this study, we established a genipin-crosslinked decellularized AF hydrogels (g-DAF-G) that are injectable and could manifest better in situ formability than noncrosslinked decellularized AF hydrogel, while preserving the capacity of directing differentiation of human bone mesenchymal stem cells (hBMSCs) towards AF cells. Hematoxylin and eosin staining, 4',6-diamidino-2-phenylindole staining, and so forth showed that the majority of cellular components were removed, whereas extracellular matrix and microstructure were largely preserved. The storage modulus increased from 465.5 ± 9.4 Pa to 3.29 ± 0.24 MPa after 0.02% genipin crosslinking of decellularized AF hydrogels (DAF-G) to form g-DAF-G. AF-specific genes (COL1A1, COL5A1, TNMD, IBSP, FBLN1) were significantly higher in DAF-G and g-DAF-G groups than that in control group after 21 days of culturing. g-DAF-G significantly restored nucleus pulposus water content and preserved intervertebral structure in vivo. Summarily, we produced a novel AF regeneration biomaterial, g-DAF-G, which exhibited well biocompatibility, great bioactivity, and much higher mechanical strength than DAF-G. This study will provide an easy and fast therapeutic alternative to repair AF injuries or tears.

Topics: Animals; Annulus Fibrosus; Bone Marrow Cells; Cattle; Cell Differentiation; Cross-Linking Reagents; Humans; Hydrogels; Intervertebral Disc Degeneration; Iridoids; Mesenchymal Stem Cells

Microdiscectomy is the current standard surgical treatment for intervertebral disc (IVD) herniation, however annulus fibrosus (AF) defects remain unrepaired which can alter IVD biomechanical properties and lead to reherniation, IVD degeneration and recurrent back pain. Genipin-crosslinked fibrin (FibGen) hydrogel is an injectable AF sealant previously shown to partially restore IVD motion segment biomechanical properties. A small animal model of herniation and repair is needed to evaluate repair potential for early-stage screening of IVD repair strategies prior to more costly large animal and eventual human studies. This study developed an ex-vivo rat caudal IVD herniation model and characterized torsional, axial tension-compression and stress relaxation biomechanical properties before and after herniation injury with or without repair using FibGen. Injury group involved an annular defect followed by removal of nucleus pulposus tissue to simulate a severe herniation while Repaired group involved FibGen injection. Injury significantly altered axial range of motion, neutral zone, torsional stiffness, torque range and stress-relaxation biomechanical parameters compared to Intact. FibGen repair restored the stress-relaxation parameters including effective hydraulic permeability indicating it effectively sealed the IVD defect, and there was a trend for improved tensile stiffness and axial neutral zone length. This study demonstrated a model for studying IVD herniation injury and repair strategies using rat caudal IVDs ex-vivo and demonstrated FibGen sealed IVDs to restore water retention and IVD pressurization. This ex-vivo small animal model may be modified for future in-vivo studies to screen IVD repair strategies using FibGen and other IVD repair biomaterials as an augment to additional large animal and human IVD testing.

Topics: Animals; Biomechanical Phenomena; Fibrin Tissue Adhesive; Hydrogels; Intervertebral Disc; Intervertebral Disc Degeneration; Iridoids; Rats

Understanding the effect of impact loading on the mechanical response of the intervertebral disc (IVD) is valuable for investigating injury mechanisms and devising effective therapeutic modalities. This study used 24 porcine thoracic motion segments to characterize the mechanical response of intact (N = 8), degenerated (Trypsin-denatured, N = 8), and repaired (Genepin-treated, N = 8) IVDs subject to impact loading. A meta-model analysis of poroelastic finite element simulations was used in combination with ex-vivo creep and impact tests to extract the material properties. Forward analyses using updated specimen-specific FE models were performed to evaluate the effect of impact duration. The maximum axial stress in the IVDs, Von-Mises stress in the endplates, and intradiscal pore pressure (IDP) were calculated, under a 400 N preload, subject to a sequence of impact loads for 10 impact durations (10-100 ms). The results were in good agreement with both creep and impact experiments (error < 10%). A significant difference was found in the maximum axial stress between the intact and degenerated disc groups. The IDP was also significantly lower in the degenerated disc group. The Von Mises stress in the adjacent endplates significantly increased with degeneration. It is concluded that the disc time-dependent response significantly changes with disc degeneration. Cross-linker Genipin has the potential to recover the hydraulic permeability and can potentially change the time dependent response, particularly in the IDP. This is the first study, to our best knowledge, which explores the effect of impact loading on the healthy, degenerated and repaired IVD using both creep and impact validation tests.

Topics: Animals; Biomechanical Phenomena; Computer Simulation; Finite Element Analysis; Intervertebral Disc; Intervertebral Disc Degeneration; Iridoids; Models, Biological; Pressure; Stress, Mechanical; Swine; Weight-Bearing

Degenerative disc disease, associated with discrete structural changes in the peripheral annulus and vertebral endplate, is one of the most common pathological triggers of acute and chronic low back pain, significantly depreciating an individual's quality of life and instigating huge socioeconomic costs. Novel emerging therapeutic techniques are hence of great interest to both research and clinical communities alike. Exogenous crosslinking, such as Genipin, and platelet-rich plasma therapies have been recently demonstrated encouraging results for the repair and regeneration of degenerated discs, but there remains a knowledge gap regarding the quantitative degree of effectiveness and particular influence on the mechanical properties of the disc. This study aimed to investigate and quantify the material properties of intact (N = 8), trypsin-denatured (N = 8), Genipin-treated (N = 8), and platelet-rich plasma-treated (N = 8) discs in 32 porcine thoracic motion segments. A poroelastic finite element model was used to describe the mechanical properties during different treatments, while a meta-model analytical approach was used in combination with ex vivo experiments to extract the poroelastic material properties. The results revealed that both Genipin and platelet-rich plasma are able to recover the mechanical properties of denatured discs, thereby affording promising therapeutic modalities. However, platelet-rich plasma-treated discs fared slightly, but not significantly, better than Genipin in terms of recovering the glycosaminoglycans content, an essential building block for healthy discs. In addition to investigating these particular degenerative disc disease therapies, this study provides a systematic methodology for quantifying the detailed poroelastic mechanical properties of intervertebral disc.

Topics: Animals; Biomechanical Phenomena; Computer Simulation; Cross-Linking Reagents; Disease Models, Animal; Elasticity; Finite Element Analysis; Humans; In Vitro Techniques; Intervertebral Disc Degeneration; Iridoids; Models, Biological; Platelet-Rich Plasma; Regenerative Medicine; Sus scrofa

Annulus fibrosus (AF) defects from annular tears, herniation, and discectomy procedures are associated with painful conditions and accelerated intervertebral disc (IVD) degeneration. Currently, no effective treatments exist to repair AF damage, restore IVD biomechanics and promote tissue regeneration. An injectable fibrin-genipin adhesive hydrogel (Fib-Gen) was evaluated for its performance repairing large AF defects in a bovine caudal IVD model using ex vivo organ culture and biomechanical testing of motion segments, and for its in vivo longevity and biocompatibility in a rat model by subcutaneous implantation. Fib-Gen sealed AF defects, prevented IVD height loss, and remained well-integrated with native AF tissue following approximately 14,000 cycles of compression in 6-day organ culture experiments. Fib-Gen repair also retained high viability of native AF cells near the repair site, reduced nitric oxide released to the media, and showed evidence of AF cell migration into the gel. Biomechanically, Fib-Gen fully restored compressive stiffness to intact levels validating organ culture findings. However, only partial restoration of tensile and torsional stiffness was obtained, suggesting opportunities to enhance this formulation. Subcutaneous implantation results, when compared with the literature, suggested Fib-Gen exhibited similar biocompatibility behaviour to fibrin alone but degraded much more slowly. We conclude that injectable Fib-Gen successfully sealed large AF defects, promoted functional restoration with improved motion segment biomechanics, and served as a biocompatible adhesive biomaterial that had greatly enhanced in vivo longevity compared to fibrin. Fib-Gen offers promise for AF repairs that may prevent painful conditions and accelerated degeneration of the IVD, and warrants further material development and evaluation.

Topics: Animals; Bioreactors; Cattle; Chondrogenesis; Compressive Strength; Fibrin Tissue Adhesive; Hydrogels; Intervertebral Disc; Intervertebral Disc Degeneration; Iridoids; Nitric Oxide; Organ Culture Techniques; Rats; Rats, Sprague-Dawley; Regeneration; Stress, Mechanical; Tensile Strength; Torque

An in situ study using whole-organ culture system.. To study the effect of disc degeneration at different stages on its rheological and dynamic properties and to investigate the efficacy of exogenous cross-linking therapy.. Disc degeneration can involve protein denaturation or microdefects to the disc's collagen fiber network. A disc degeneration model using whole-organ culture technique can be effectively used for the screening of treatments of degenerated discs. Exogenous cross-linking therapy has been shown to enhance the mechanical properties of the disc by cross-linking collagen. However, the efficacy of this therapy on the degenerated disc is unclear.. A total of 40 porcine thoracic discs were assigned to 5 groups: intact discs, moderately degenerated discs, moderately degenerated discs with cross-linker augmentation, severely degenerated discs, and severely degenerated discs with cross-linker augmentation. The disc degeneration was simulated by trypsin digestion and mechanical fatigue loading. Rheological properties, dynamic properties, water content, and histological analysis were conducted after a 7-day incubation.. The mechanical properties of moderate degenerated discs significantly decrease both in rheological and dynamic properties, and laminate structure disorganization was observed. Mechanical defects of severely degenerated discs resulted in disc height loss, an increase in the aggregate modulus and stiffness modulus, and a decrease in the damping coefficient, hydraulic permeability, and water content. Cross-linker augmentation significantly recovered mechanical properties of moderately degenerated discs and restored the water content compared with the intact disc. However, the augmentation did not fully repair the severely degenerated discs.. Trypsin-induced extracellular matrix damage resulted in a change of the disc's biomechanics. Cross-linker augmentation recovers the rheological and dynamic properties of moderately degenerated discs but not of the severely degenerated discs. The genipin cross-linker may be able to improve the proteoglycan depletion effect in the nucleus pulposus but may not be effective to restore the structural damage in the collagen molecule of the anulus fibrosus.

Topics: Animals; Biomechanical Phenomena; Collagen; Computer Simulation; Cross-Linking Reagents; Intervertebral Disc; Intervertebral Disc Degeneration; Iridoids; Models, Biological; Organ Culture Techniques; Orthopedic Procedures; Proteoglycans; Rheology; Severity of Illness Index; Stress, Mechanical; Swine; Thoracic Vertebrae; Treatment Outcome; Trypsin

Biochemical studies aimed at optimization of protein crosslinking formulations for the treatment of degenerative disc disease and subsequent biomechanical testing of tissues treated with these formulations.. To optimize protein crosslinking formulations for treatment of degenerating spinal discs.. Nonsurgical exogenous crosslinking therapy is a potential new, noninvasive technology for the treatment of degenerative disc disease. The technology is based on the injection of protein crosslinking reagents into the pathologic disc to restore its mechanical properties and also to potentially increase the permeability of the tissue and so facilitate the exchange of waste products and nutrients.. Diffusion of genipin (GP) was monitored following injection into spinal discs and the effects of surfactants on diffusion studied. Formulations for GP and methylglyoxal (MG) were biochemically optimized and used to treat bovine spinal discs. Their effects on bovine anulus tissue were evaluated using a circumferential tensile test, while the GP formulation was also tested with respect to its ability to reduce disc bulge under load.. GP exhibited a distinct time-dependent diffusion and sodium-dodecyl-sulfate, but not Tween-20, enhanced diffusion by 30%. Two crosslinkers, GP and MG, were inhibited by amines but enhanced by phosphate ions. Both formulations could enhance a number of physical parameters of bovine anulus tissue, while the GP formulation could reduce disc bulge following injections into spinal discs.. Formulations lacking amines and containing phosphate ions appear to be promising candidates for clinical use of the crosslinkers GP and MG.

Topics: Animals; Biomechanical Phenomena; Cattle; Cross-Linking Reagents; Diffusion; In Vitro Techniques; Injections, Spinal; Intervertebral Disc; Intervertebral Disc Degeneration; Iridoid Glycosides; Iridoids; Lumbar Vertebrae; Polysorbates; Pyruvaldehyde; Sodium Dodecyl Sulfate; Surface-Active Agents; Tensile Strength; Time Factors