Faculty Bibliography

Tissue-engineering techniques have been successful in developing cartilage-like tissues in vitro using cells from animal sources. The successful translation of these strategies to the clinic will likely require cell expansion to achieve sufficient cell numbers. Using a two-dimensional (2D) cell migration assay to first identify the passage at which chondrocytes exhibited their greatest chondrogenic potential, the objective of this study was to determine a more optimal culture medium for developing three-dimensional (3D) cartilage-like tissues using human cells. We evaluated combinations of commonly used growth factors that have been shown to promote chondrogenic growth and development. Human articular chondrocytes (AC) from osteoarthritic (OA) joints were cultured in 3D environments, either in pellets or encapsulated in agarose. The effect of growth factor supplementation was dependent on the environment, such that matrix deposition differed between the two culture systems. ACs in pellet culture were more responsive to bone morphogenetic protein (BMP2) alone or combinations containing BMP2 (i.e. BMP2 with PDGF or FGF). However, engineered cartilage development within agarose was better for constructs cultured with TGFbeta3. These results with agarose and pellet culture studies set the stage for the development of conditions appropriate for culturing 3D functional engineered cartilage for eventual use in human therapies

Objectives Degeneration of articular cartilage is central to OA pathology; however, the molecular mechanisms leading to these irreversible changes are still poorly understood. Here, we investigated how changes in the chondrocyte translational apparatus may contribute to the pathology of OA. Methods Normal and OA human knee cartilage was used to analyze the activity of different components of the translational machinery. Chondrocytes isolated from lesional and non-lesional areas of OA cartilage were used to estimate relative rate of protein synthesis by metabolic labeling. Experimental OA was induced by transection of the anterior cruciate ligament in rats to investigate changes in the translational apparatus associated with OA. The role of IL-1beta signaling was assessed in vitro using rat articular chondrocytes. Expression of mRNAs was analyzed by qPCR and protein levels by immunohistochemistry and Western blotting. Results We identified several novel traits of OA chondrocytes, including upregulation of the Serine/Threonine kinases AKT2 and AKT3 at the post-transcriptional level and increased rate of total protein synthesis, likely due to inactivation of 4E-BP1, a known repressor of cap-dependent translation. We found that 4E-BP1 inactivation is mTOR-dependent and crucial for upregulation of protein synthesis in general and in particular for MMP13 and ADAMTS5 expression. In addition, IL-1beta treatment led to 4E-BP1 inactivation and upregulation of protein synthesis in articular chondrocytes. Conclusions Precise control of protein synthesis is vital for cartilage homeostasis and its dysregulation contributes to the molecular pathology of OA. Our study therefore identifies a novel set of potential therapeutic targets

INTRODUCTION: An increase in the concentration of loose cartilage debris in the joint space has been associated with aging, overuse and obesity resulting in osteoarthritis (OA) [1]. Cartilage and bone particulates have been observed bound to the surface or embedded within the synovium of OA patients who also present with synovial hyperplasia and metaplasia [2]. While the involvement of cartilage debris in synovial inflammation has been long suspected in the clinic and demonstrated in animal models [3], the mechanism of action behind the interaction remains to be elucidated. In the current study, we hypothesize that synovial fibroblasts (SF) have an inflammatory response to sub-lOum cartilage debris that can be further modulated by the presence of pro-inflammatory cytokines and is also mediated by phagocytosis. Using similar sized latex beads as a non-biologic control, we also sought to examine and characterize differences in biosynthetic activity of SF to co-culture with biologic and non-biologic wear particulates in the presence or absence of cytokines. METHODS: Tissue Harvest: Synovial tissue and articular cartilage explants were harvested from the knee joints of bovine calf knees (n=3). Synovial tissue was digested with collagenase IV, and the isolated SF were plated and expanded for two passages with 5 ng/mL bFGF-2. Cartilage explants were maintained in serum free media [4]. Particulate Generation: Cartilage particles were sterilely generated from the superficial and middle zones by manually abrading explants (n=2) with 120 grit sandpaper. The particle solution was filtered with lOum nylon mesh filter. Latex and cartilage wear particles were counted and sized as in [5] (Figure 1 A,B). Imaging: SF were cultured with FITC labeled latex particles or cartilage particles labeled with dichlorotriazinyl aminofluorescein and counterstained with Calcein Red-Orange AM after 48 hours. Dosage Optimization: SF were plated overnight prior to treatment. The optimal particle dosage was defined as the lowest dosage of particles that yielded a significant increase in MTT (metabolism) absorbance normalized to the 0 particle dosage group after 5 days of culture (Figure 1C). Dense Monolayer Co-Culture: SF were plated at a high density overnight and co-cultured with +/- 10 ng/mL IL-la or TNF-a and +/- latex or cartilage particles for 5 days (n=6-8). Particles alone were cultured +/-10 ng/mL IL-la (not shown) or TNF-a. Biochemistry: DNA glycosaminoglycan (GAG), collagen (COL) and nitric oxide (NO) were determined using the Picogreen dsDNA, DMMB dye binding, OHP and Griess assays, respectively. Biochemical content of wear particles alone was subtracted from the measured values of the resulting SF sheet with particles to determine the contribution of SF alone. Statistics: Comparisons were analyzed using ANOVA with Fisher's LSD post-hoc test (p<0.05). RESULTS: Latex and cartilage wear particles had average diameters of 1.05 +/- 0.28 um and 0.85 +/- 0.45 um, respectively. For both latex and cartilage wear particles the lowest concentration that increased cellular metabolism was 250 particles per cell or <0.002%v/v for both particle types. Latex particles were readily phagocytosed by the majority of SF, with 5-20 particles internalized per cell (Figure 2A). Meanwhile, cartilage wear particles were selectively phagocytosed by SF, with positive cells only internalizing 1-5 particles (Figure 2A). Cartilage wear particles were also observed on the surface of the SF (not shown). With cartilage particle co-culture, cellular proliferation was significantly increased compared to non-treated controls or latex treated cells regardless of cytokine treatment (Figure 3A). The DNA content of the cartilage wear particles themselves constituted <1% that of the DNA of the final cell monolayer (not shown). Cell monolayer GAG and GAG synthesis decreased with cytokine treatment and was increased after cartilage wear particle treatment without cytokines only (Figure 3B). Cellular COL was significantly elevated in cartilage particle treated groups. However, COL synthesis was decreased compared to the control for latex and cartilage particle treated groups. Cell monolayer COL and COL synthesis per cell decreased with cytokine treatment across all groups. However, with TNF treatment, only cartilage particle synthesis was increased compared to the control (Figure 3C). Media GAG increased with cartilage treatment and cytokine treatment. However, in TNF treated groups, media GAG per cell was decreased in latex and cartilage treated groups (Figure 3D). NO in the media increased with cytokine and cartilage particle treated groups (Figure 3E). Across all cell and media biochemical measurements, cartilage particle treated groups had a muted response to cytokine treatment, compared to control and latex treated groups. DISCUSSION: Based on the results, we reject our hypothesis that the inflammatory response caused by cartilage wear particles is synergistic with cytokine insult, implicating perhaps a shift in the anabolic-catabolic balance of treated SF. Notably, the proliferative response of SF to cartilage wear particles resulted in an overall increase in extracellular matrix content, which may be suggestive of synovial hyperplasia often reported in OA. Treatment of SF with cartilage wear particles had an inflammatory effect, resulting in decreased anabolic synthesis (less overall matrix normalized by DNA) and increased catabolic activity (increased media GAG/DNA and NO/DNA). This observation is consistent with previous work that reported co-culture with cartilage particles results in increased TNF-a gene expression [1] and the release of proteinases [6]. This effect was not observed following phagocytosis of latex particles, suggesting that the inflammatory response of SF to cartilage wear particles is mediated by a mechanism beyond non-specific phagocytosis. Further characterization of the inflammatory response will investigate other factors upregulated in OA including prostaglandin E2, IL-6, and matrix metalloproteinases. Efforts to better understand the cartilage particle-induced proliferative response is ongoing, including study of the cadherin-11, which regulates synovial architecture [8]. SIGNIFICANCE: Cartilage wear debris play a role in the onset of synovium-mediated inflammation in OA. An in vitro model that can explore the interaction between cartilage particles and the synovium will foster the development of effective strategies to mitigate their negative interactions

Background/Purpose: We aimed to understand the mechanism by which membrane-type 1 matrix metalloproteinase (MT1-MMP, MMP-14) controls bone and cartilage homeostasis. MT1-MMP, a cell-membrane-bound proteinase with an extracellular catalytic site and a 20-amino acid cytoplasmic tail, plays a key role in postnatal bone formation. The genetic deficiency of MT1-MMP in the mouse causes dwarfism, osteopenia and severe arthritis. Deletion of MT1-MMP in bone marrow-derived mesenchymal progenitor cells (BM-MSC) recapitulates this phenotype, showing that MT1-MMP controls osteogenic differentiation in MSC. The phenotype of MT1-MMP-/- mice has been proposed to result from lack of MT1-MMP proteolytic activity. However, mounting evidence shows a variety of proteolysis-independent signaling functions of MT1-MMP. The unique tyrosine (Y573) in the MT1-MMP cytoplasmic tail is fundamental for the control of intracellular signaling. Methods: We generated a mouse with the Y573D mutation in MT1-MMP (MT1-MMP Y573D) and characterized its skeletal phenotype by histological and microCT analyses. Isolated BM-MSC were induced to differentiate into osteoblasts, chondrocytes and adipocytes, using qRT-PCR to analyze gene expression. Mouse C3H10T1/2 MSC were transfected with MT1-MMP cDNA and analyzed for Wnt signaling by luciferase reporter assays. Results: MT1-MMP Y573D mice had increased trabecular bone relative to wt littermates, marked thinning of articular cartilage with disorganized tissue architecture, clustering and cloning of chondrocytes, and pronounced decrease in bone marrow-associated and total body fat. We induced BM-MSC from wt and MT1-MMP Y573D littermates to differentiate into osteoblast and chondrocytes, and myeloid precursors into osteoclasts. The Y573D mutation dramatically increased MSC expression of osteoblast markers and strongly downregulated chondrocyte and osteoclast markers. These findings indicated that Wnt signaling is upregulated in MT1-MMP Y573D-expressing MSC. Therefore, we analyzed Wnt signaling. We transiently transfected C3H10T1/2 MSC cells in osteoblast medium with the cDNAs for wt MT1-MMP and MT1-MMP Y573D. As controls the cells were transfected with the empty vector (pcDNA) or with MT1-MMP E240A, a mutant devoid of proteolytic activity. MT1-MMP Y573D dramatically upregulated Wnt signaling relative to wt MT1-MMP and MT1-MMP E240A. Conclusion: MT1-MMP controls Wnt signaling by a mechanism independent of extracellular proteolysis and mediated by its cytoplasmic tail. MT1-MMP is a bifunctional protein, with an extracellular proteolytic activity that promotes bone formation through ECM remodeling and a cytoplasmic tail that controls osteogenesis by interacting with a key pro-osteogenic signaling pathway

Background/Purpose: We and others have previously shown that periostin (Postn) expression is dramatically elevated in cartilage and sub-chondral bone in OA patients and surgical models of OA (medial meniscectomy and anterior crucial ligament resection or PMX, partial meniscectomy) in rodents. In vitro Postn promotes collagen and proteoglycan degradation in human chondrocytes by upregulating MMP-13 and ADAMTS4 expression. Postn controls gene expression in bone cells by interacting with avb3 integrin. However, the nature of periostin receptor(s) in chondrocytes is unknown. DDR1, a collagen-binding receptor tyrosine kinase highly expressed in chondrocytes, controls MMP-13 expression during chondrogenesis. Therefore, we hypothesized that the effect of Postn on chondrocytes is mediated by DDR1 and Postn-deficient mice (Postn^-/-) are protected from surgically-induced post-traumatic OA. Methods: (Postn^-/-) mice were purchased from Jackson Laboratory (B6;129-Postntm1Jmol/J Stock No: 009067). We subjected 3- months old littermates (Postn+/+, Postn+/- and Postn^-/-) to partial medial meniscectomy (PMX) or sham surgery, and harvested the knee joints 8 week post-surgery for histological assessment of OA progression. Human OA chondrocytes cultures were incubated in the presence or absence of the DDR1 inhibitor DDR1-IN-1 dihydrochloridein (100-500 nM) for 2 h before addition of Postn (1 mug/ml) or control vehicle to the culture medium. MMP-13 levels were determined by ELISA 24 h post stimulation. Results: We observed abundant expression of DDR1 mRNA in human chondrocytes and we found comparable levels of DDR1 in OA and normal cartilage. However, Postn expression was 3-4 times as high in OA than in normal cartilage. Pre-incubation of human cartilage explants or cultured chondrocytes with DDR1-IN-1 dihydrochloridein inhibited both constitutive and Postn-induced MMP-13 expression in a dose-dependent manner. In contrast, neutralizing antibody to alphavbeta3 integrin had no effect on Postn induction of MMP-13 expression. Co-immunoprecipitation experiments showed that Postn physically interacts with DDR1 in human chondrocytes. Furthermore, Postn^-/- mice showed reduced PMX-induced cartilage degeneration and osteophyte formation, and both Postn+/- and Postn^-/- mice had reduced subchondral bone thickening, relative to Postn+/+ mice. Conclusion: Postn^-/- mice are protected from surgically-induced post-traumatic OA, showing that Postn promotes cartilage degeneration. DDR1 mediates the stimulatory effect of Postn on MMP-13 expression. Further studies are in progress to investigate the potential of periostin as a druggable target for the treatment of OA

Background/Purpose: Growing numbers of studies show increased expression in Osteoarthritis (OA) of inflammatory cytokines, such as IL-1beta and TNFalpha, in joint tissues and peripheral blood mononuclear (PBM) cells. The IL1 receptor antagonist (IL1RN) gene cluster region has been associated with susceptibility to knee OA, thereby further implicating inflammation in OA pathogenesis. In these studies, we examined the association of IL-1RN haplotype with the radiographic severity of symptomatic knee OA (SKOA). Methods: Genomic DNA from SKOA patients from three cohorts (NYU I, NYU-II and O

Background/Purpose: Obesity is a common risk factor for knee osteoarthritis (KOA). While it is intuitive that bariatric weight loss improves knee pain, it is not clear how much is due to decreased mechanical load vs metabolic changes. Methods: Patients were screened for knee pain prior to sleeve gastrectomy, gastric bypass, or laparoscopic gastric banding. We required pain for >15 days/month and VAS pain > 30, excluding lupus, inflammatory arthritis, crystal disease, psoriasis, and bilateral knee replacement. Enrolled patients took standing knee xrays for Kellgren-Lawrence (KL) grading. We measured BMI and used the Knee Injury and Osteoarthritis Outcome Score (KOOS) questionnaire at baseline and 1, 3, 6 and 12 months, calculating % excess weight loss (%EWL) and deltaKOOS. We collected blood at baseline and followup to study biomarkers for predicting KOOS scores. Results: Of 536 patients considering bariatric surgery, we found 308 with knee pain and enrolled 176 (91.5% female; BMI 43.6 kg/m2+/-7, 32-61; age 42 +/-11, 18-73) well distributed in xray severity (KL0-4). For the 150 patients who had surgery, knee improvement paralleled weight loss at the followups. At 1 year, %EWL correlated well with deltaKOOS pain (R = .262, n = 114, p = 0.005), similar to other intervals and to other KOOS measures. The sleeve and bypass (n=72 and 27) vs banding (n=15) resulted in higher deltaKOOS pain at 1 year: 32.9 +/-21.3 and 30.7 +/-22.6 vs 10.2 +/-21.4, p=0.001. Sleeve and bypass patients also achieved a higher % of their potential deltaKOOS pain improvement than did banding (65.2% and 60.1% vs 16.8% of remaining KOOS points to 100), and a higher % of patients improved to any degree (93.1% and 88.9% vs 66.7%). Radiographic severity did not predict deltaKOOS at 1 year, nor did the presence of key comorbidities. Patients lost weight in a near-linear fashion through 1 year (Fig. 1), but their KOOS improvements plateaued at 1 month. This held true in sleeve and bypass subgroups (with altered anatomy), while banding showed less consistent deltaKOOS despite a similar trend in %EWL. Baseline leptin levels in obese KOA were higher than non-obese KOA and non-obese/non-OA controls from other cohorts (100.2 +/-61.9 vs 26.2 +/-16.7 and 15.4+/-13.8, p<0.001). Similarly, IL-1Ra, a potential marker of OA progression, was much higher than non-obese KOA or controls (1123+/-940 vs 324.0+/-145.6 and 272+/-130.0, p<0.001). Within obese KOA, higher leptin levels predicted worse xrays (KL0/1 vs KL2/3/4, p = 0.037). After 1 year, mean leptin and IL1-Ra from obese KOA patients had decreased (p<0.001). Conclusion: Bariatric surgery improves knee OA symptoms proportionally to %EWL. Most relief occurs during the 1st month before much weight loss, suggesting a metabolic impact beyond mechanical load reduction on joints - at least with the sleeve and bypass that alter digestive anatomy. Leptin and IL-1Ra serum levels are elevated in obese KOA vs non-obese KOA and controls - and fall after bariatric surgery which could contribute to knee pain relief