Diane R. Wagner

In vivo rabbit models that deliver mechanical impacts to knee cartilage are important for studying injury-induced osteoarthritis, but variability in impact characteristics limit the repeatability of the models. In this study, the mechanical impact load delivered to a cadaveric rabbit posterior medial condyle was characterized, with particular emphasis on the influence of placement of the k-wire that stabilized the joint during impact. K-wire position was not prescribed and varied across specimens (7.85 ± 2.28 mm anterior and 2.61 ± 3.47 mm distal to the impact site) and was treated as a source of variability. Impact parameters and subchondral bone failure metrics were quantified, and their relationship with k-wire placement were evaluated using multiple linear regression. Peak stress, loading rate, impact duration and work were significantly associated with k-wire location in the sagittal plane, with these associations primarily driven by the anterior-posterior position of the k-wire. In contrast, the proximal-distal position was not a significant predictor for individual impact parameters. Consistency of the k-wire placement and resulting impact parameters were also assessed. With effort to reproduce a fixed placement, the coefficients of variation of the impact parameters were less than ~15%, indicating relatively consistent impacts. Additionally, regression analysis indicated that a substantial portion of fracture variability was accounted for by k-wire placement, suggesting that caution must be exercised in positioning the k-wire to prevent fractures. Collectively, these findings indicate that the stiff k-wire alters the compliance of the impacted tissue and influences the characteristics of the mechanical impact load and the susceptibility to subchondral bone fracture. These results highlight the importance of precise joint stabilization in impact-based osteoarthritis models and provide a foundation for future studies on cartilage injury, post-traumatic osteoarthritis disease progression, and novel therapeutic interventions.

Post-Traumatic Osteoarthritis (PTOA) is a prevalent, degenerative, and painful progressive joint disease that typically arises after significant joint trauma, affecting over 6 million Americans. Impact-induced cartilage trauma results in a chondrocyte injury response that culminates in PTOA. Understanding the molecular events associated with chondrocyte injury is necessary to develop therapies to prevent PTOA or slow its progression. To facilitate this, an ex vivo cartilage impact model that was sublethal for 24 h was developed. Osteochondral explants were harvested from bovine metacarpophalangeal joints, and a drop tower was used to deliver impact loads to the explants. To ensure that the stress and impact were applied relatively uniformly to the articular cartilage, explants were positioned with the bone surface facing upward, and the bone was impacted from above. Different loads were applied to the explants from carriage heights of 4.0 cm, 4.5 cm, and 5.0 cm, resulting in average peak stresses of 10.87 ± 1.84 MPa, 11.69 ± 0.67 MPa, and 12.98 ± 0.75 MPa, respectively. The study analyzed the role of subchondral bone presence in cartilage during post-impact culture and the effect of using either a fitted or loose holder during impact, assessing cell viability and apoptosis. Articular cartilage impacted from 4.5 cm, combined with a fitted holder and immediate bone removal, provided the optimal model to induce cartilage injury with minimal cell death at 24 h post-impact.

Post-Traumatic Osteoarthritis (PTOA) is a prevalent, degenerative, and painful progressive joint disease that typically arises after significant joint trauma, affecting over 6 million Americans. Impact-induced cartilage trauma results in a chondrocyte injury response that culminates in PTOA. Understanding the molecular events associated with chondrocyte injury is necessary to develop therapies to prevent PTOA or slow its progression. To facilitate this, an ex vivo cartilage impact model that was sublethal for 24 h was developed. Osteochondral explants were harvested from bovine metacarpophalangeal joints, and a drop tower was used to deliver impact loads to the explants. To ensure that the stress and impact were applied relatively uniformly to the articular cartilage, explants were positioned with the bone surface facing upward, and the bone was impacted from above. Different loads were applied to the explants from carriage heights of 4.0 cm, 4.5 cm, and 5.0 cm, resulting in average peak stresses of 10.87 ± 1.84 MPa, 11.69 ± 0.67 MPa, and 12.98 ± 0.75 MPa, respectively. The study analyzed the role of subchondral bone presence in cartilage during post-impact culture and the effect of using either a fitted or loose holder during impact, assessing cell viability and apoptosis. Articular cartilage impacted from 4.5 cm, combined with a fitted holder and immediate bone removal, provided the optimal model to induce cartilage injury with minimal cell death at 24 h post-impact.

Purpose To evaluate the mechanical wear of cartilage with different types of degradation. Methods Bovine osteochondral explants were treated with interleukin-1β (IL-1β) to mimic inflammatory conditions, with chondroitinase ABC (ChABC) to specifically remove glycosaminoglycans (GAGs), or with collagenase to degrade the collagen network during 5 days of culture. Viscoelastic properties of cartilage were characterized via indentation. Biochemical assays were performed to quantify the cartilage matrix loss to the media during culture and from an accelerated, ex vivo wear test. The coefficient of friction during the wear test was measured. Distribution of GAGs in the tissue was assessed histologically. Results All three degradative treatments decreased the cartilage modulus values and depleted GAGs in histological sections. However, wear was not uniform among the different treatments. Collagen loss from the tissue due to mechanical wear was only higher with IL-1β and collagenase treatment, while collagen loss due to wear with ChABC treatment was similar to untreated controls. In addition, less GAG was released due to mechanical wear in all degraded groups than the controls, likely because GAGs had already been depleted from these tissues during culture. As no significant differences in the coefficient of friction were observed between groups, changes in wear were attributed to altered tissue composition and structure rather than to changes in frictional forces. Conclusions Results suggest that cartilage with a degraded collagen network is more susceptible to mechanical wear, but that cartilage wear may be relatively unaffected by the loss of GAGs. Furthermore, exacerbated mechanical wear could be an additional mechanism by which inflammatory cytokines induce cartilage breakdown.

Post-traumatic osteoarthritis (PTOA) is responsible for 12% of all osteoarthritis cases in the United States. PTOA can be initiated by a single traumatic event, such as a high-impact load acting on articular cartilage, or by joint instability, as occurs with anterior cruciate ligament rupture. There are no effective therapeutics to prevent PTOA currently. Developing a reliable animal model of PTOA is necessary to better understand the mechanisms by which cartilage damage proceeds and to investigate novel treatment strategies to alleviate or prevent the progression of PTOA. This protocol describes an open, drop tower-based rabbit femoral condyle impact model to induce cartilage damage. This model delivered peak loads of 579.1 ± 71.1 N, and peak stresses of 81.9 ± 10.1 MPa with a time-to-peak load of 2.4 ± 0.5 ms. Articular cartilage from impacted medial femoral condyles (MFCs) had higher rates of apoptotic cells (p = 0.0058) and possessed higher Osteoarthritis Research Society International (OARSI) scores of 3.38 ± 1.43 compared to the non-impacted contralateral MFCs (0.56 ± 0.42), and other cartilage surfaces of the impacted knee (p < 0.0001). No differences in OARSI scores were detected among the non-impacted articular surfaces (p > 0.05).

A better understanding of molecular events following cartilage injury is required to develop treatments that prevent or delay the onset of trauma-induced osteoarthritis. In this study, alterations to SIRT1 activity in bovine articular cartilage explants were evaluated in the 24 h following a mechanical overload, and the effect of pharmacological SIRT1 activator SRT1720 on acute chondrocyte injury was assessed. SIRT1 enzymatic activity decreased as early as 5 min following the mechanical overload, and remained suppressed for at least 24 h. The chondrocyte injury response, including apoptosis, oxidative stress, secretion of inflammatory mediators, and alterations in cartilage matrix expression, was prevented with pharmacological activation of SIRT1 in a dose-dependent manner. Overall, the results implicate SIRT1 deactivation as a key molecular event in chondrocyte injury following a mechanical impact overload. As decreased SIRT1 signaling is associated with advanced age, these findings suggest that downregulated SIRT1 activity may be common to both age-related and injury-induced osteoarthritis.

Objective: The objective of this study was to evaluate photochemical crosslinking using Al(III) phthalocyanine chloride tetrasulfonic acid (CASPc) and light with a wavelength of 670 nm as a potential therapy to strengthen articular cartilage and prevent tissue degradation. Design: Changes in viscoelastic properties with indentation were used to identify 2 crosslinking protocols for further testing. Crosslinked cartilage was subjected to an in vitro, accelerated wear test. The ability of the crosslinked tissue to resist biochemical degradation via collagenase was also measured. To better understand how photochemical crosslinking with CASPc varies through the depth of the tissue, the distribution of photo-initiator and penetration of light through the tissue depth was characterized. Finally, the effect of CASPc on chondrocyte viability and of co-treatment with an antioxidant was evaluated. Results: The equilibrium modulus was the most sensitive viscoelastic measure of crosslinking. Crosslinking decreased both mechanical wear and collagenase digestion compared with control cartilage. These beneficial effects were realized despite the fact that crosslinking appeared to be localized to a region near the articular surface. In addition, chondrocyte viability was maintained in crosslinked tissue treated with antioxidants. Conclusion: These results suggest that photochemical crosslinking with CASPc and 670 nm light holds promise as a potential therapy to prevent cartilage degeneration by protecting cartilage from mechanical wear and biochemical degradation. Limitations were also evident, however, as an antioxidant treatment was necessary to maintain chondrocyte viability in crosslinked tissue.