Effects of knockout of the receptor for advanced glycation end-products on bone mineral density and synovitis in mice with intra-articular fractures.

Our group employed the mouse closed intra-articular fracture (IAF) model to test the hypothesis that the innate immune system plays a role in initiating synovitis and post-traumatic osteoarthritis (PTOA) in fractured joints. A transgenic strategy featuring knockout of the receptor for advanced glycation end-products (RAGE -/- ) was pursued. The 42 and 84 mJ impacts used to create fractures were in the range previously reported to cause PTOA at 60 days post-fracture. MicroCT (µCT) was used to assess fracture patterns and epiphyseal and metaphyseal bone loss at 30 and 60 days post-fracture. Cartilage degeneration, synovitis, and matrix metalloproteinase (MMP-3, -13) expression were evaluated by histologic analyses. In wild-type mice, µCT imaging showed that 84 mJ impacts led to significant bone loss at 30 days (p < 0.05), but recovered to normal at 60 days. Bone losses did not occur in RAGE-/- mice. Synovitis was significantly elevated in 84 mJ impact wild-type mice at both endpoints (30 day, p = 0.001; 60 day, p = 0.05), whereas in RAGE-/- mice synovitis was elevated only at 30 days (p = 0.02). Mankin scores were slightly elevated in both mouse strains at 30 days, but not at 60 days. Immunohistochemistry revealed significant fracture-related increases in MMP-3 and -13 expression at 30 days (p < 0.05), with no significant difference between genotypes. These findings indicated that while RAGE -/- accelerated recovery from fracture and diminished synovitis, arthritic changes were temporary and too modest to detect an effect on the pathogenesis of PTOA. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2439-2449, 2018.

The use of autologous adult, allogenic juvenile, and combined juvenile-adult cartilage fragments for the repair of chondral defects.

PURPOSE: The goal of the study was to evaluate the repair of chondral lesions treated with combined autologous adult/allogenic juvenile cartilage fragments, compared with isolated adult and isolated juvenile cartilage fragments. METHODS: Fifty-eight adult (>16 week old) and five juvenile (<6 week old) New Zealand White female rabbits were used. A large osteochondral defect was created in the center of the femoral trochlea of adult rabbits. The rabbits were divided in four groups: Group 1 = untreated defects (controls); Group 2 = adult cartilage fragments; Group 3 = juvenile cartilage fragments; and Group 4 = adult + juvenile cartilage fragments. Killings were performed at 3 and 6 months. The defects were evaluated with ICRS macroscopic score, modified O'Driscoll score, and Collagen type II immunostaining. RESULTS: At 3 months, Group 4 performed better than Group 1, in terms of modified O'Driscoll score (p = 0.001) and Collagen type II immunostaining (p = 0.015). At 6 months, Group 4 showed higher modified O'Driscoll score (p = 0.003) and Collagen type II immunostaining score (p < 0.001) than Group 1. Histologically, also Group 3 performed better than Group 1 (p = 0.03), and Group 4 performed better than Group 2 (p = 0.004). CONCLUSIONS: Mixing adult and juvenile cartilage fragments improved cartilage repair in a rabbit model. In the clinical setting, a new "one-stage" procedure combining the two cartilage sources can be hypothesized, with the advantages of improved chondral repair and large defect coverage, because of the use of an off-the-shelf juvenile allograft. Further studies on larger animals and clinical trials are required to confirm these results.

Biocompatibility and preclinical feasibility tests of a temperature-sensitive hydrogel for the purpose of surgical wound pain control and cartilage repair.

We recently introduced a novel pluronic F127 and hyaluronic acid-based hydrogel (HG) designed to deliver a broad range of therapeutics. The reverse-thermal responsive HG exhibits physical properties that seem to be ideal for the local delivery of drug- and cell-based therapies to specific anatomic sites through percutaneous injection. However, questions related to the HG's safety and efficacy must first be addressed. To address these issues, we performed standard in vitro cytotoxicity and drug release tests and in vivo biocompatibility tests in a rat model. In addition, we determined whether the HG was an effective stem cell carrier in a rat cartilage defect model. We found that the HG showed viability and biocompatibility levels similar to those reported for F127 or hyaluronic acid alone. In vitro drug release studies with bupivacaine, a drug used clinically for local pain relief, revealed that after an initial burst bupivacaine was released continuously for 10 days. Stem cells loaded in the HG were retained in situ and stimulated cartilage regeneration in experimental defects. Taken as a whole, these findings support further efforts to develop the HG as a versatile system for the delivery of a wide range of therapeutic agents in humans. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2013.

Organ culture stability of the intervertebral disc: rat versus rabbit.

There is a need to develop mechanically active culture systems to better understand the role of mechanical stresses in intervertebral disc (IVD) degeneration. Motion segment cultures that preserve the native IVD structure and adjacent vertebral bodies are preferred as model systems, but rapid ex vivo tissue degeneration limits their usefulness. The stability of rat and rabbit IVDs is of particular interest, as their small size makes them otherwise suitable for motion segment culture. The goal of this study was to determine if there are substantial differences in the susceptibility of rat and rabbit IVDs to culture-induced degeneration. Lumbar IVD motion segments were harvested from young adult male Sprague-Dawley rats and New Zealand White rabbits and cultured under standard conditions for 14 days. Biochemical assays and safranin-O histology showed that while glycosaminoglycan (GAG) loss was minimal in rabbit IVDs, it was progressive and severe in rat IVDs. In the rat IVD, GAG loss was concomitant with the loss of notochordal cells and the migration of endplate (EP) cells into the nucleus pulposus (NP). None of these changes were evident in the rabbit IVDs. Compared to rabbit IVDs, rat IVDs also showed increased matrix metalloproteinase-3 (MMP-3) and sharply decreased collagen type I and II collagen expression. Together these data indicated that the rabbit IVD was dramatically more stable than the rat IVD, which showed culture-related degenerative changes. Based on these findings we conclude that the rabbit motion segments are a superior model for mechanobiologic studies.

Comparative digital cartilage histology for human and common osteoarthritis models.

PURPOSE: This study addresses the species-specific and site-specific details of weight-bearing articular cartilage zone depths and chondrocyte distributions among humans and common osteoarthritis (OA) animal models using contemporary digital imaging tools. Histological analysis is the gold-standard research tool for evaluating cartilage health, OA severity, and treatment efficacy. Historically, evaluations were made by expert analysts. However, state-of-the-art tools have been developed that allow for digitization of entire histological sections for computer-aided analysis. Large volumes of common digital cartilage metrics directly complement elucidation of trends in OA inducement and concomitant potential treatments. MATERIALS AND METHODS: Sixteen fresh human knees, 26 adult New Zealand rabbit stifles, and 104 bovine lateral plateaus were measured for four cartilage zones and the cell densities within each zone. Each knee was divided into four weight-bearing sites: the medial and lateral plateaus and femoral condyles. RESULTS: One-way analysis of variance followed by pairwise multiple comparisons (Holm-Sidak method at a significance of 0.05) clearly confirmed the variability between cartilage depths at each site, between sites in the same species, and between weight-bearing articular cartilage definitions in different species. CONCLUSION: The present study clearly demonstrates multisite, multispecies differences in normal weight-bearing articular cartilage, which can be objectively quantified by a common digital histology imaging technique. The clear site-specific differences in normal cartilage must be taken into consideration when characterizing the pathoetiology of OA models. Together, these provide a path to consistently analyze the volume and variety of histologic slides necessarily generated by studies of OA progression and potential treatments in different species.

Biocompatibility and preclinical feasibility tests of a temperature-sensitive hydrogel for the purpose of surgical wound pain control and cartilage repair.

We recently introduced a novel pluronic F127 and hyaluronic acid-based hydrogel (HG) designed to deliver a broad range of therapeutics. The reverse-thermal responsive HG exhibits physical properties that seem to be ideal for the local delivery of drug- and cell-based therapies to specific anatomic sites through percutaneous injection. However, questions related to the HG's safety and efficacy must first be addressed. To address these issues, we performed standard in vitro cytotoxicity and drug release tests and in vivo biocompatibility tests in a rat model. In addition, we determined whether the HG was an effective stem cell carrier in a rat cartilage defect model. We found that the HG showed viability and biocompatibility levels similar to those reported for F127 or hyaluronic acid alone. In vitro drug release studies with bupivacaine, a drug used clinically for local pain relief, revealed that after an initial burst bupivacaine was released continuously for 10 days. Stem cells loaded in the HG were retained in situ and stimulated cartilage regeneration in experimental defects. Taken as a whole, these findings support further efforts to develop the HG as a versatile system for the delivery of a wide range of therapeutic agents in humans.

Rat spinal motion segment in organ culture: a cell viability study.

STUDY DESIGN: This study investigated tissue integrity and viability of cells in an organ culture system of intervertebral disc (IVD) with adjoining vertebral bodies. OBJECTIVE: The goal of this study was to design a methodology to maintain an IVD motion segment in organ culture, thereby preserving viability and tissue architecture. SUMMARY OF BACKGROUND DATA: Study of IVD mechanobiology in vitro necessitates availability of vertebral bodies for controlled application of complex loads. METHODS: IVD motion segments were dissected from rat lumbar segments and maintained in organ culture and cell viability was evaluated histochemically using NitroBlue Tetrazolium. Tissue integrity and morphology were evaluated using conventional histologic techniques. RESULTS: The in vitro organ culture of motion segments maintained the viability and tissue integrity for 14 days. More than 95% viability in all three regions of interest (anulus fibrosus, nucleus pulposus, end plates) was maintained for 14 days in culture. CONCLUSION: Our initial results suggest that long-term motion segment culture is practical, and the inclusion of vertebral bodies will facilitate anchoring during biomechanical stimulation. Thus, we expect the culture system to provide us with an excellent model for studying the pathomechanics of IVD degeneration and the effects of mechanical stimulation on the biology of IVD cells.