Aaron M. Stoker

A Review of Translational Animal Models for Knee Osteoarthritis

Knee osteoarthritis remains a tremendous public health concern, both in terms of health-related quality of life and financial burden of disease. Translational research is a critical step towards understanding and mitigating the long-term effects of this disease process. Animal models provide practical and clinically relevant ways to study both the natural history and response to treatment of knee osteoarthritis. Many factors including size, cost, and method of inducing osteoarthritis are important considerations for choosing an appropriate animal model. Smaller animals are useful because of their ease of use and cost, while larger animals are advantageous because of their anatomical similarity to humans. This evidence-based review will compare and contrast several different animal models for knee osteoarthritis. Our goal is to inform the clinician about current research models, in order to facilitate the transfer of knowledge from the “bench” to the “bedside.”

Differences in interleukin-1 response between engineered and native cartilage.

Unlike native cartilage explants that are used in autologous tissue transfer procedures, engineered cartilage constructs are typically highly fragile when first formed and must rely on cellular activity to develop over time. However, inflammatory cytokines such as interleukin-1alpha (IL-1alpha) are often present in target joints and may interfere with this development process. Herein we examine to what extent nascent engineered tissue is susceptible to chemical perturbations by IL-1alpha (10 ng/mL), especially when compared to native explants, and whether in vitro preconditioning may promote sufficient integrity to lessen this impact. The studies were carried out using a chemically defined medium supplemented with or without the antiinflammatory steroid dexamethasone. We find that engineered tissue (bovine chondrocytes in agarose hydrogel) at early time points (days 0 and 14) does not grow when exposed to the cytokine even temporarily, but both bovine explants and more developed engineered tissue (day 28) are able to withstand the same exposure without degradation of properties. We argue therefore that some in vitro preconditioning may be necessary to promote both sufficient mechanical integrity and the chemical fortitude without which insufficiently developed engineered constructs will not survive the harsh mechanochemical environment within the joint.

The Effect of Bupivacaine and Morphine in a Coculture Model of Diarthrodial Joints

PURPOSE To perform a controlled laboratory study to evaluate the effect of bupivacaine and morphine on chondrocytes and synovium in a coculture model of diarthrodial joints. METHODS A proven coculture model that allows for the assessment of cartilage and synovium exists. The model allows for simulation of the diarthrodial joint in both health and disease by using culture media with or without the addition of interleukin-1. Effects of the presence of bupivacaine and morphine were evaluated by measuring media concentration of glycosamino glycans (GAG), nitric oxide (NO), and prostaglandin E(2) (PGE(2)), and tissue concentration of GAG, water, and collagen. Cell viability was determined through the use of confocal microscopy on days 1 and 2. RESULTS Cell viability 2 days after exposure to 0.5% bupivacaine was significantly less in the presence of bupivacaine than in the other groups, nearing a 100% decrease in viability. There was little effect of bupivacaine on cartilage water content or the tissue concentration of GAG and collagen. Morphine and bupivacaine both inhibited the expected rise in NO and PGE(2) when interleukin-1 was added to the media. CONCLUSIONS Continuous 0.5% bupivacaine exposure has a clear detrimental effect on chondrocytes in this in vitro study. Both bupivacaine and morphine appear to have anti-inflammatory effects. Continuous morphine exposure does not cause gross chondrotoxicity in vitro and presents itself as a potential alternative intra-articular analgesic. CLINICAL RELEVANCE Intra-articular bupivacaine infusion is an effective analgesic strategy and is frequently used in both office and outpatient surgical settings. This study provides evidence that the continued usage of postoperative bupivacaine continuous infusion pumps may have a detrimental effect on chondrocytes. Morphine has been shown to be an effective intra-articular analgesic, and its anti-inflammatory role seen in this study makes it a potential alternative to bupivacaine.

Animal models of cartilage repair

Cartilage repair in terms of replacement, or regeneration of damaged or diseased articular cartilage with functional tissue, is the ‘holy grail’ of joint surgery. A wide spectrum of strategies for cartilage repair currently exists and several of these techniques have been reported to be associated with successful clinical outcomes for appropriately selected indications. However, based on respective advantages, disadvantages, and limitations, no single strategy, or even combination of strategies, provides surgeons with viable options for attaining successful long-term outcomes in the majority of patients. As such, development of novel techniques and optimisation of current techniques need to be, and are, the focus of a great deal of research from the basic science level to clinical trials. Translational research that bridges scientific discoveries to clinical application involves the use of animal models in order to assess safety and efficacy for regulatory approval for human use. This review article provides an overview of animal models for cartilage repair. Cite this article: Bone Joint Res 2014;4:89–94.

Mechanical and biochemical characterization of cartilage explants in serum-free culture.

Allografts of articular cartilage are both used clinically for tissue-transplantation procedures and experimentally as model systems to study the physiological behavior of chondrocytes in their native extracellular matrix. Long-term maintenance of allograft tissue is challenging. Chemical mediators in poorly defined culture media can stimulate cells to quickly degrade their surrounding extracellular matrix. This is particularly true of juvenile cartilage which is generally more responsive to chemical stimuli than mature tissue. By carefully modulating the culture media, however, it may be possible to preserve allograft tissue over the long-term while maintaining its original mechanical and biochemical properties. In this study juvenile bovine cartilage explants (both chondral and osteochondral) were cultured in both chemically defined medium and serum-supplemented medium for up to 6 weeks. The mechanical properties and biochemical content of explants cultured in chemically defined medium were enhanced after 2 weeks in culture and thereafter remained stable with no loss of cell viability. In contrast, the mechanical properties of explants in serum-supplemented medium were degraded by ( approximately 70%) along with a concurrent loss of biochemical content (30-40% GAG). These results suggest that long-term maintenance of allografts can be extended significantly by the use of a chemically defined medium.

Effects of growth factors on equine synovial fibroblasts seeded on synthetic scaffolds for avascular meniscal tissue engineering.

Across species, the avascular portion of the knee meniscus cannot heal spontaneously if severely injured. The most common treatment is meniscectomy which results in osteoarthritis. The objective of this study was to assess the fibrochondrogenic potential of equine fibroblast-like synoviocytes (FLS) seeded on scaffolds under the influence of growth factors in vitro to determine the potential of developing a novel cell-based repair strategy. Cultured FLS were seeded onto synthetic scaffolds in a rotating bioreactor under the influence of three growth factor regimens: none, basic fibroblast growth factor (bFGF) alone, and bFGF plus transforming growth factor (TGF-beta(1)) and insulin-like growth factor (IGF-1). Constructs were analyzed for mRNA expression and production of fibrochondroid extracellular matrix constituents. Type II collagen and aggrecan mRNA were significantly higher in growth factor-treated groups (p<0.05). Despite sub-optimal extracellular matrix production, FLS can exhibit fibrochondral characteristics and may have potential for cell-based tissue engineering for avascular meniscal regeneration.

Improved Osteochondral Allograft Preservation Using Serum-Free Media at Body Temperature

Background: Osteochondral allografts (OCAs) are currently preserved at 4°C and used within 28 days of donor harvest. The window of opportunity for implantation is limited to 14 days due to a 2-week disease testing protocol. Hypothesis: Osteochondral allograft tissues stored at 37°C will have significantly higher chondrocyte viability, as well as superior biochemical and biomechanical properties, than those stored at 4°C. Study Design: Controlled laboratory study. Methods: Osteochondral allografts from 15 adult canine cadavers were aseptically harvested within 4 hours of death. Medial and lateral femoral condyles were stored in Media 1, similar to the current standard, or Media 2, an anti-inflammatory and chondrogenic media containing dexamethasone and transforming growth factor−β3, at 4°C or 37°C for up to 56 days. Chondrocyte viability, glycosaminoglycan (GAG) and collagen (hydroxyproline [HP]) content, biomechanical properties, and collagen II and aggrecan content were assessed at days 28 and 56. Five femoral condyles were stored overnight and assessed the next day to serve as controls. Results: Storage in Media 1 at 37°C maintained chondrocyte viability at significantly higher levels than in any other media-temperature combination and at levels not significantly different from controls. Osteochondral allografts stored in either media at 4°C showed a significant decrease in chondrocyte viability throughout storage. Glycosaminoglycan and HP content were maintained through 56 days of storage in OCAs in Media 1 at 37°C. There were no significant differences in elastic or dynamic moduli among groups at day 56. Qualitative immunohistochemistry demonstrated the presence of collagen II and aggrecan throughout all layers of cartilage. Conclusion: Osteochondral allograft viability, matrix content and composition, and biomechanical properties were maintained at “fresh” levels through 56 days of storage in Media 1 at 37°C. Osteochondral allografts stored at 4°C were unable to maintain viability or matrix integrity through 28 days of storage. These findings suggest that storage of OCAs in a defined media at 37°C is superior to current protocols (4°C) for tissue preservation prior to transplantation. Clinical Relevance: Storage of OCAs in serum-free chemically defined media at 37°C can increase the “window of opportunity” for implantation of optimal tissue from 14 days to 42 days after disease testing clearance.

Effects of Dexamethasone on the Functional Properties of Cartilage Explants During Long-Term Culture

Background Intact articular cartilage tissue is used clinically in the form of osteochondral allografts and experimentally as explants in modeling the physiologic behavior of chondrocytes in their native extracellular matrix. Long-term maintenance of allograft tissue is challenging. Hypothesis By carefully modulating the preservation environment, it may be possible to preserve osteochondral allograft tissue over the long term while maintaining its original mechanical and biochemical properties. Study Design Controlled laboratory study. Methods In this study, juvenile bovine, mature bovine, and canine cartilage explants were cultured in chemically defined media with or without supplementation of dexamethasone for up to 4 weeks. Results The mechanical properties and biochemical content of juvenile bovine explants cultured in the presence of dexamethasone were significantly enhanced after 2 weeks in culture and remained stable with sustained cell viability thereafter. In contrast, the mechanical properties and biochemical content of juvenile bovine explants cultured in the absence of the dexamethasone significantly decreased after 2 weeks of culture. The mechanical and biochemical content of mature bovine and canine explants were not significantly affected by the presence of dexamethasone and maintained initial (day 0) mechanical and biochemical properties throughout the entire culture period with or without supplementation of dexamethasone. Conclusion These results suggest that juvenile and mature cartilage explants respond differently to dexamethasone. The functional properties of juvenile cartilage explants can be maintained in vitro through the addition of dexamethasone to culture media. Functional properties of mature cartilage can be preserved for at least 4 weeks in culture regardless of the presence of dexamethasone. Clinical Relevance Biochemical and biomechanical properties of osteochondral allograft tissue may be enhanced by the addition of dexamethasone to culture media. These findings may translate to longer shelf life of preserved osteochondral allograft transplantation tissue and increased clinical availability of grafts.

Tissue-engineered articular cartilage exhibits tension–compression nonlinearity reminiscent of the native cartilage

The tensile modulus of articular cartilage is much larger than its compressive modulus. This tension-compression nonlinearity enhances interstitial fluid pressurization and decreases the frictional coefficient. The current set of studies examines the tensile and compressive properties of cylindrical chondrocyte-seeded agarose constructs over different developmental stages through a novel method that combines osmotic loading, video microscopy, and uniaxial unconfined compression testing. This method was previously used to examine tension-compression nonlinearity in native cartilage. Engineered cartilage, cultured under free-swelling (FS) or dynamically loaded (DL) conditions, was tested in unconfined compression in hypertonic and hypotonic salt solutions. The apparent equilibrium modulus decreased with increasing salt concentration, indicating that increasing the bath solution osmolarity shielded the fixed charges within the tissue, shifting the measured moduli along the tension-compression curve and revealing the intrinsic properties of the tissue. With this method, we were able to measure the tensile (401±83kPa for FS and 678±473kPa for DL) and compressive (161±33kPa for FS and 348±203kPa for DL) moduli of the same engineered cartilage specimens. These moduli are comparable to values obtained from traditional methods, validating this technique for measuring the tensile and compressive properties of hydrogel-based constructs. This study shows that engineered cartilage exhibits tension-compression nonlinearity reminiscent of the native tissue, and that dynamic deformational loading can yield significantly higher tensile properties.

Enhanced Fracture and Soft-Tissue Healing by Means of Anabolic Dietary Supplementation

BACKGROUND Malnutrition is common in hospitalized injured patients. It contributes to delayed fracture-healing and increased morbidity. However, relatively little attention has been directed toward nutritional strategies for augmenting musculoskeletal recovery after a fracture. This animal study was designed to examine the effects of dietary protein intake and the role of conditionally essential amino acids in muscle and bone-healing after a fracture. METHODS One hundred adult male rats were used. Ten rats served as controls and received a 15% protein diet throughout the study. The remaining ninety rats received a 6% protein diet for five weeks to induce protein malnutrition. The rats underwent intramedullary nailing and closed midshaft fracture of one femur. After the fracture, they were separated into three isocaloric dietary groups. Group P6 received a diet with 6% protein; Group P15, a diet with 15% protein; and group P30, a diet with 30% protein with conditionally essential amino acids. At two, four, and six weeks after surgery, ten animals from each group were killed and the femora were evaluated with dual x-ray absorptiometry, histomorphometric assessment of callus, and torsional testing. The quadriceps muscles were analyzed for total mass, total protein content, and for mRNA expression of insulin-like growth factor-1 (IGF-1), IGF-2, IGF receptors, actin, myosin, and vascular endothelial growth factor (VEGF). RESULTS The P30 group demonstrated elevations in albumin, body mass, muscle mass, total protein content of muscle, and bone mineral density in the fracture callus compared with the P6 diet group at six weeks (p < 0.05). Molecular analysis of muscle revealed that IGF-1, IGF-2, IGF receptors, myosin, actin, and VEGF gene expression were significantly (p < 0.001) higher in the P6 group compared with the P30 group. Biomechanical testing of the femora, however, showed no significant differences. CONCLUSIONS Dietary supplementation with conditionally essential amino acids in malnourished animals had anabolic effects on bone mineralization, body mass, and muscle mass.