Faculty Bibliography

Purpose: The intrawound application of lyophilized vancomycin has been reported to significantly decrease the rates of perioperative infection in arthroplasty and spine procedures. The local effect of clinically used supra-therapeutic concentration of intra wound vancomycin on surrounding healing tissue has been a topic of continued investigation. The purpose of this study was to examine the in vitro cytotoxicity profile of vancomycin hydrochloride on osteoblasts, fibroblast, and myoblasts. Methods: Human primary osteoblasts (Lonza), fibroblasts (Lonza), and myoblasts (DV Biologics) were expanded and passaged in sterile polystyrene tissue culture flasks and plated at a density of 10,000 cells/cm2. Cells were exposed to vancomycin hydrochloride (Sigma-Aldrich) at concentrations of 1, 3, 6, or 12 mg/cm2. To assess the effect of vancomycin on cell migration, a scratch assay was performed, in which a "scratch" was made in a cell monolayer following vancomycin exposure, and images were subsequently captured at regular intervals until cellular closure of the scratch. Cell survival was measured 48 hours post-vancomycin exposure using a cell cytotoxicity assay (Cell Counting Kit-8, Dojindo). Results: Vancomycin concentrations greater than or equal to 1 mg/cm2 decreased survival of myoblasts and osteoblasts to less than 11% relative to control. Vancomycin greater than or equal to 3 mg/ cm2 decreased fibroblast survival to less than 8% relative to control (Fig. 1). Vancomycin concentrations of 1 mg/cm2 did not significantly affect the survival of fibroblasts. Closure of the scratch defect was observed in less than 24 hours for all control conditions. In myoblasts and osteoblasts, the scratch defect remained open indefinitely following exposure to vancomycin concentrations greater than or equal to 1 mg/cm2. Closure of the scratch defect in fibroblasts was observed in less than 36 hours following exposure to vancomycin of 1 mg/cm2, and remained opened indefinitely following exposure to vancomycin greater than or equal to 3 mg/cm2. Conclusions: Vancomycin has a significant cytotoxic effect on proliferating osteoblasts and myoblasts at concentrations greater than (Figure Presented) or equal to 1 mg/cm2.Vancomycin has a pronounced cytotoxic effect on fibroblasts at concentrations greater than or equal to 3 mg/cm2. Further in vivo studies are warranted to investigate the effect of high local concentrations of vancomycin on infection, bony fusion, and wound healing

Introduction: Damage to the articular cartilage is common, especially through a trauma or injury to the knee joint. Because of the lack of intrinsic capacity to heal, chondral defects remain a major challenge to repair. Current methods used for cartilage regeneration generally result in poorly repaired defects leading to early onset of posttraumatic osteoarthritis (PTOA) and subsequently requiring joint replacement (1). The use of mesenchymal stem cells (MSCs) derived from ones own bone marrow or adipose tissues has been suggested to be used for cartilage repair (2). The transplantation of stem cells for tissue repair requires cell settlement, proliferation and differentiation. The local tissue microenvironment or stem cell niche plays a key role for the successful transplantation of stem cells for tissue repair (3). Very little, however, is known about the stem cell niche required for the successful transplantation of stem cells for cartilage repair. In addition, stem cell settlement and chondrogenesis in cartilage repair has to occur in an unfriendly inflammatory environment in response to injury. In this study, we determined how a novel peptide (NP-0100) that binds to hyaluronan (HA) affects MSC attachment, proliferation and chondrogenic differentiation under normal and inflammatory conditions. Previously we have shown that NP-0100 inhibited catabolic events and stimulated the expression of articular cartilage markers in human articular chondrocytes cultured in an inflammatory environment (4). Therefore, we hypothesized that NP-0100 together with high molecular HA (HMWHA) will enhance cartilage repair by optimizing the stem cell niche for precursor cells to repair cartilage and reduce inflammation. Methods: Chondrogenesis of the multipotential murine C3H/10T1/2 cell line was induced in micromass cultures in the presence of BMP-2 (100ng/ml). In addition, the micromass cultures were treated with NP-0100 or cultured in conditioned media from untreated and IL-1beta-treated articular chondrocytes in the absence or presence of NP-0100. Chondrogenesis was determined by alcian blue staining and real time PCR analysis of chondrocyte marker genes. Cell attachment and proliferation was assessed on tissue culture plates or tissue culture plates with immobilized high molecular HA (HMWHA) or HMWHA together with NP- 0100. Cell attachment was determined by DAPI staining. Cell proliferation was determined using the CCK-8 kit. Results: C3H10T1/2 cells better attached to HMWHA-coated tissue culture plates than to uncoated tissue culture plates. The largest number of cells, however, attached to tissue culture plates that were coated with both HMWHA and NP-0100. In addition, cells showed the highest proliferation rate on HMWHA/NP- 0100-coated plates followed by HMWHA-coated plates. The lowest proliferation rate was detected on uncoated tissue culture plates. Furthermore, NP-0100 stimulated the expression of articular cartilage markers (aggrecan and type II collagen) and Sox-9, a master transcription factor that regulates chondrogenesis. NP-0100-treated C3H/10T1/2 micromass cultures also stained more intensely with Alcian blue, which is indicative of increased levels of sulfated proteoglycans than micromass cultures not treated with NP-0100. Chondrogenesis was markedly inhibited when the micromass cultures were cultured in the presence of conditioned media from IL-1beta-treated human articular chondrocytes compared to conditioned media from untreated human articular chondrocytes. However, conditioned media from human articular chondrocytes treated with IL-1beta in combination with NP-0100 showed a reduced inhibition of chondrogenesis compared to conditioned media from IL-1beta-treated human articular chondrocytes. Discussion: Our findings show that a novel peptide NP-0100 in the presence of HMWHA stimulated attachment, proliferation and chondrogenic differentiation of precursor cells, and this suggests a potential therapeutic role for NP-0100 in promoting cartilage repair. The improved attachment and proliferation of precursor cells on tissue culture plates that were coated with HMWHA and NP-0100 rather than HMWHA alone suggests that NP-0100 stabilizes or crosslinks HMWHA to create a more favorable microenvironment (stem cell niche) for the precursor cells to adhere and proliferate. This notion is supported by a previous study showing that a cross-linked HMWHA is required for the formation of the stem cell niche for precursor cells to repair muscle after injury (5). NP-0100 not only supported the formation of the stem cell niche but also stimulated chondrogenesis of C3H10T1/2 cells in high-density micromass culture. Furthermore, the peptide was able to protect the micromass cultures from an inflammatory environment that otherwise inhibited chondrogenesis. Future studies have to determine the mechanisms by which NP-0100 together with HMWHA stimulates chondrogenesis even in an inflammatory environment

STUDY DESIGN: In vitro laboratory study. OBJECTIVE: The purpose of this study was to identify the effect of dilute povidone-iodine (PVI) solutions on human osteoblast, fibroblast and myoblast cells in vitro. SUMMARY OF BACKGROUND DATA: Dilute PVI wound lavage has been used successfully in spine and joint arthroplasty procedures to prevent post-operative surgical site infection, but their biologic effect on host cells is largely unknown. METHODS: Human primary osteoblasts, fibroblasts, and myoblasts were expanded in cell culture and subjected to various concentrations of PVI (0%, 0.001%, 0.01%, 0.1%, 0.35%, 1%) for 3 minutes. To assess the effect of PVI on cell migration, a scratch assay was performed, in which a "scratch" was made by a standard pipette tip in a cell monolayer following PVI exposure, and time to closure of the scratch was evaluated. Cell survival and proliferation was measured 48 hours post-PVI exposure using a cell viability and cytotoxicity assay. RESULTS: Closure of the scratch defect in all cell monolayers was achieved in < 24 hours in untreated controls and following exposure to PVI concentrations < 0.1%. The scratch defect remained open indefinitely following exposure to PVI concentrations of >/= 0.1%. PVI concentrations < 0.1% did not have significant effect on survival rates compared with control for all cell types. Cells exposed to PVI >/= 0.1% had cell survival rates of less than 6% (p < 0.05). CONCLUSIONS: Clinically used concentration of PVI (0.35%) exerts a pronounced cytotoxic effect on osteoblasts, fibroblast, and myoblasts in vitro. Further investigation is required to systematically study the effect of PVI on tissue healing in vivo and also determine a safe and clinically potent concentration for PVI lavage. LEVEL OF EVIDENCE: N/A.

The progressive ankylosis protein (ANK) is a transmembrane protein that transports intracellular pyrophosphate (PPi) to the extracellular milieu. In this study we show increased fatty degeneration of the bone marrow of adult ank/ank mice, which lack a functional ANK protein. In addition, isolated bone marrow stromal cells (BMSCs) isolated from ank/ank mice showed a decreased proliferation rate and osteogenic differentiation potential, and an increased adipogenic differentiation potential compared to BMSCs isolated from wild type (WT) littermates. Wnt signaling pathway PCR array analysis revealed that Wnt ligands, Wnt receptors and Wnt signaling proteins that stimulate osteoblast differentiation were expressed at markedly lower levels in ank/ank BMSCs than in WT BMSCs. Lack of ANK function also resulted in impaired bone fracture healing, as indicated by a smaller callus formed and delayed bone formation in the callus site. Whereas 5weeks after fracture, the fractured bone in WT mice was further remodeled and restored to original shape, the fractured bone in ank/ank mice was not fully restored and remodeled to original shape. In conclusion, our study provides evidence that ANK plays a critical role in the adipogenic/osteogenic fate decision of adult mesenchymal precursor cells. ANK functions in precursor cells are required for osteogenic differentiation of these cells during adult bone homeostasis and repair, whereas lack of ANK functions favors adipogenic differentiation.

PURPOSE: We establish a mechanical injury model for articular cartilage to assess the sensitivity of diffusion tensor imaging (DTI) in detecting cartilage damage early in time. Mechanical injury provides a more realistic model of cartilage degradation compared with commonly used enzymatic degradation. METHODS: Nine cartilage-on-bone samples were obtained from patients undergoing knee replacement. The 3 Tesla DTI (0.18 x 0.18 x 1 mm3 ) was performed before, 1 week, and 2 weeks after (control zero, mild, and severe) injury, with a clinical radial spin-echo DTI (RAISED) sequence used in our hospital. We performed stress-relaxation tests and used a quasilinear-viscoelastic (QLV) model to characterize cartilage mechanical properties. Serial histology sections were dyed with Safranin-O and given an OARSI grade. We then correlated the changes in DTI parameters with the changes in QLV-parameters and OARSI grades. RESULTS: After severe injury the mean diffusivity increased after 1 and 2 weeks, whereas the fractional anisotropy decreased after 2 weeks (P < 0.05). The QLV-parameters and OARSI grades of the severe injury group differed from the baseline with statistical significance. The changes in mean diffusivity across all the samples correlated with the changes in the OARSI grade (r = 0.72) and QLV-parameters (r = -0.75). CONCLUSION: DTI is sensitive in tracking early changes after mechanical injury, and its changes correlate with changes in biomechanics and histology. Magn Reson Med, 2016. (c) 2016 Wiley Periodicals, Inc.

INTRODUCTION: Osteoarthritis (OA) is a degenerative disease of the entire joint.¹ In healthy joints the articulating surfaces are encased in a smooth layer of hyaline cartilage and are further protected by the surrounding synovial fluid. High molecular weight hyaluronan (HMWHA) is present at relatively high levels in the synovial fluid and cartilage matrix.'²' HMWHA provides viscoelastic protection and lubrication of the cartilage surfaces, and has also been shown to have important anti-inflammatory properties.'³-⁵' During inflammation hyaluronan (HA) can be degraded to lower molecular weight HA (LMWHA) by the increased levels of free radicals and endogenous hyaluronidases present in the inflamed joint.'⁶' Interestingly, LMWHA, which can signal through a number of different receptors (TLR-2 and 4, CD44 and RHAMM), was suggested to act as potent inflammatory mediator in the joint.'⁷' Our collaborators identified a 15-mer peptide that binds to LMWHA and reduced inflammation and fibrogenesis in excisional skin wounds.^<8' In this study we hypothesized that LMWHA generated in the OA joint is a key mediator for stimulating catabolic and inflammatory events in joint cells and that interfering with LMWHA signaling using the novel peptide inhibitor will attenuate joint inflammation. Methods: Human articular chondrocytes were isolated from articular cartilage samples obtained from patients (donor age range 48 - 67) undergoing total knee replacement surgery at NYU Hospital for Joint Diseases. Knee cartilage was harvested from regions with no macroscopically evident degeneration. The collection of tissue from patients undergoing knee replacement surgery was approved by the Institutional Regulatory Board at NYU School of Medicine. Human chondrocytes were isolated from these cartilage samples and cultured as described previously.'⁹' Cultured chondrocyte were switched to serum-free medium for 24 h and then treated with inflammatory stimuli (interleukin-lbeta (IL-lp), LMWHA; average molecular weight of lOkDa). In addition, serum-starved cells were treated with the 15-mer peptide at various concentrations. A synovial fibroblast cell line (SW982) was also used and was cultured as described previously.'¹⁰' The analysis of HA concentrations in the culture medium was done as described by us previously.'¹¹' Western blotting and real time PCR analysis was performed as described by us.^<12) Results: In the present study we used human articular chondrocytes and a synovial fibroblast cell line (SW982) to determine the involvement of LMWHA promoting inflammation in the joint cavity. We show that LMWHA stimulated catabolic markers (Cox-2, IL-6, iNOS, MMP-13) and inhibited the expression of articular cartilage markers (aggrecan, type II collagen) in human articular chondrocytes and SW982 synovial cells. LMWHA treatment stimulated the NF-KB and the MAP kinase (ERK, JNK, p38) signaling pathways in human chondrocytes similar to IL-lp treatment. Whereas the stimulation of these signaling pathways occurred within the first hour of treatment with IL-lp, the stimulation of these pathways by LMWHA occurred at later time points (6h and 24h). The novel peptide inhibitor markedly reduced the activation of these signaling pathways in LMWHA-treated chondrocytes. In addition, it markedly decreased the expression of catabolic markers and increased the expression of articular cartilage markers in LMWHA-treated chondrocytes. The peptide also inhibited the expression of catabolic markers in IL-1 p-treated human articular chondrocytes and SW982 cells and increased articular cartilage marker expression in IL-1 p-treaded chondrocytes. Treatment of human articular chondrocytes with IL-lp resulted in a marked increase of HA released into the culture medium over time. This increase in HA release correlated with an increase in catabolic markers and a reduction in anabolic markers over time, similar to the effects of the LMWHA treatment. Discussion: Our findings demonstrate that LMWHA stimulates inflammatory and catabolic events in joint cells via activation of NF-Kb and ERK, JNK and p38 signaling pathways. In addition, we demonstrate that a novel peptide that binds to LMWHA and interferes with LMWHA signaling attenuates inflammatory and catabolic events in joint cells mediated by LMWHA and IL-lp. More specifically, we have shown that the novel peptide inhibitor dramatically reduced IL-6 and Cox-2 levels in articular chondrocytes and synovial fibroblasts. Both of these catabolic markers have been previously shown to be associated with increased pain levels in patients with OA.'¹³' These findings suggest that LMWHA plays a key role in mediating inflammation in joint cells during OA pathology, and that a novel peptide inhibitor of LMWHA signaling may act as a novel compound to specifically reduce inflammation and pain in the joint and ultimately slow down cartilage degradation during OA. SIGNIFICANCE: This study identified LMWHA as a key mediator of inflammatory and catabolic events in joint cells. In addition, we determined that interfering with LMWHA signaling using a novel peptide that binds LMWHA may provide a novel therapeutic strategy to reduce inflammation in the OA joint and ultimately slow down cartilage degradation during OA

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: 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

Purpose: Previous studies have shown the potential of diffusion tensor imaging (DTI) in capturing damage of the cartilage. DTI indices, such as mean diffusivity (MD) and fractional anisotropy (FA) are potential biomarkers for cartilage composition and structure. Our hypothesis is that the DTI biomarkers can detect early changes in articular cartilage after mechanical injury. We use a model to replicate the mechanism of injury in the cartilage during ACL rupture, and compare the DTI biomarkers against histology and biomechanics. The study is designed to make the results translational to clinical practice, as we use a clinical scanner, employ the same imaging sequence, and use a protocol which can be optimized for clinical studies. Materials and Methods: Nine cylindrical articular cartilage-on-bone plugs from a knee replacement surgery were incubated at 37C with culture medium. Over a 3-week long protocol, cartilage MRI scanning is combined with biomechanical and histological tests. We first perform a baseline MRI and stress-strain test on each sample. MRI was performed at 3 Twith an in-house butterfly coil specifically designed for the extracted ex-vivo samples. The MRI protocol included a radial spin echo DTI (RAISED) sequence that we normally use for human in-vivo applications. We acquired two b = 0 and 12-direction b = 300 s/mm2 images at a resolution of 0.18 x 0.18 x 1.2 mm3 and derived FA and MD parameters. After MRI and the biomechanical tests, we imparted a zero (n=3), intermediate (n = 3), or high (n = 3) overload to simulate the traumatic injury of cartilage. Two weeks after, the samples underwent a third MRI and biomechanical testing. Cartilage samples were then processed for histology with safranin-O to show the PG density and distribution. Safranin-O slides were graded via OARSI scoring, varying from 0 (heathy) to 6 (bone remodelling). Results: The DTI biomarkers are sensitive to the changes on articular cartilage after injury. In severely injured samples, the MD increased by 15 % in the 2 weeks, from an average of 1.36 mum2/ms at the baseline, whereas the FAdecreased by 43%froma baseline of 0.25.Mildly injured samples showed a MD trend towards higher values, increasing by 9 % frombaseline 1.28 mum2/ms, whereas the FA did not change. As expected, the biomechanics tests reflect a trend of decreased stiffness (Young's modulus) with increasing injury. The change in MD is correlated with the changes in the biomechanics with rho = 0.75 (p = 0.04), and correlated with the OARSI (rho = 0.65, p = 0.07). The OARSI score of the baseline samples was 0.83 +/- 0.44. Severe injury samples increased their OARSI score by 2.53 +/- 1.30, while the mild injury had only a moderate increase in OARSI (0.62 +/- 1.30). Zero injury samples neither changed their score, nor their biomechanical parameters. Conclusion: DTI biomarkers do capture the early signs of damage in articular cartilage. The use of clinically feasible MRI protocols provide new biomarkers for the early diagnosis and monitoring of early stages of post-traumatic OA