A model for testing the effects of injury and repair using engineered cartilage tissue analogs

s / Osteoarthritis and Cartilage 20 (2012) S54–S296 S130 Conclusions: In this work, we showed for the first time that multiscale large field imaging (Macroscopy) combined to multimodality approaches (SHG-TCSPC) could be an innovative and non invasive technique to monitor the state of network collagen in biomedical studies such as cartilage tissue engineering. 247 A MODEL FOR TESTING THE EFFECTS OF INJURY AND REPAIR USING ENGINEERED CARTILAGE TISSUE ANALOGS B. Mohanraj, W.C. Strutz, R.L. Mauck, G.R. Dodge. Univ. of Pennsylvania, Philadelphia, PA, USA Purpose: Articular cartilage consists of unique extracellular matrix (ECM) which function to distribute loads in synovial joints. When injured, it cannot repair effectively and is increasingly thought that injury or trauma, even the smallest may initiate degenerative changes (i.e. OA). The goal was to devise a platform to test chondrocyte's response to injury using a cartilage tissue analogue (CTA) after a rapid blunt force trauma and/or laceration. The aimwas tomeasure biosynthetic and catabolic responses in chondrocytes, in a reproducible in vitro model, following injury. The long term goal was to provide a platform to study chondrocyte function and to test therapeutics. Methods: CTA PreparationChondrocytes were isolated from juvenile bovine (1-3 months old) knees and CTA generated. Briefly, cartilage was dissected and digested overnight with collagenase. The CTAmodel is based on using plates coated with hydrogel, poly (2-hydroxyethyl methacrylate), which prevents cell adherence. The cells coalesce and form a single mass which grows quickly and maintains a cartilage phenotype for long periods in culture. In these experiments chondrocytes were seeded at 1-2x106 cells/ well in 96 well plate and cultured in DMEM with 10% FBS for at least 6 -10 weeks to allow for substantial ECM production. Sample CTA Groups(1) Uninjured control, (2) Uninjured treated with 10 ng/mL IL-1a (positive control), (3) Cut CTAs, and (4) Impacted CTAs, were harvested 5 hours and 5 days post-injury. Injuryan Instron 5848 was used to determine the CTA height while resting on a flat surface. The impact injury regimen was 75% strain over 1 sec., held for 3 sec., then released. 3 full thickness lacerations were made through the CTA (‘Cut’). After impact or laceration, CTAs were transferred to medium for additional culture periods or immediately flash frozen for gene expression analysis. At corresponding harvest times CTA were rushed into a powder, and RNA was purified followed by qPCR using the Bio-Rad CFX384 and iQ Sybr GreenSupermix. Data was analyzed using 2T method to obtain relative expressionwith data presented as percent of untreated or un-injured controls. Results: The effects of impact injury on ECM gene expression over an extended period of three time points 0, 24, and 120 h was evaluated.Collagen and aggrecan were down-regulated at 24 h following impact and IL-1 treatment. However, at 120 h, collagen was markedly upregulated after injury, as was aggrecan but to a lesser extent. IL-1a treatment continued to down-regulate aggrecan at 120h, while collagen showed a slight increase (24%). Perlecan was up-regulated in response to injury at 120h (w30%) and to IL-1a at both time points. Comparisons between cut and impacted CTAs at 5h showed an upregulation of collagen and aggrecan expression w3-fold and w2.5-fold, respectively. (Fig 1). Ă However, this upregulation was diminished at 120h. In contrast, in cut CTAs, aggrecan and collagen II was only slightly up-regulated and remained at constant expression levels at 5 and 120h. The stress related gene iNOS was markedly up-regulated at 5h in impacted CTAs (w 4.5X), and this increasewas reduced nearly to uninjured at 120 h. In contrast, cut CTAs had low level expression of iNOS at 5h, but was up-regulated 2.5-fold at 120h. ADAM-TS4 expression was similarly unaffected at 5 h for both cut and impacted CTAs butwas up-regulated in impacted samples (w2.6X) at 120h. Conclusion: A single, rapid, uniaxial compressive load on an engineered cartilage construct was used to generate a model of injury and demonstrate the changes on chondrocyte gene expression. While proteases tested and stress indicators are involved in the cellular injury response after both injury types, chondrocytes appear to respond to compressive injury by increasing the biosynthesis of certain ECM genes at longer time points tested. Extending the recovery periods will reveal to what degree these mechanically-induced injuries heal or become degenerative. This model establishes a platform to test engineered cartilage surrogate's response to injury and ultimately identify pathways involved in repair and test therapeutics. 248 CARTILAGE REPAIR FOR STEROID-INDUCED OSTEONECROSIS OF THE KNEE JOINT H. Tsukiyama, M. Kobayasi, R. Arai, H. Harada, Y. Nakagawa. Kyoto Univ.