Faculty Bibliography

Introduction: Chlorhexidine gluconate (CHX) is widely used as a preoperative surgical skin-preparation solution and intra-wound irrigation agent, with excellent efficacy against wide variety of bacteria. The cytotoxic effect of CHX on local proliferating cells following orthopaedic procedures is largely undescribed. Our aim was to investigate the in vitro effects of CHX on primary fibroblasts, myoblasts, and osteoblasts. Methods: Cells were exposed to CHX dilutions (0%, 0.002%, 0.02%, 0.2%, and 2%) for either a 1, 2, or 3-minute duration. Cell survival was measured using a cytotoxicity assay (Cell Counting Kit-8). Cell migration was measured using a scratch assay: a "scratch" was made in a cell monolayer following CHX exposure, and time to closure of the scratch was measured. Results: All cells exposed to CHX dilutions of ≥ 0.02% for any exposure duration had cell survival rates of less than 6% relative to untreated controls (p < 0.001). Cells exposed to CHX dilution of 0.002% all had significantly lower survival rates relative to control (p < 0.01) with the exception of 1-minute exposure to fibroblasts, which showed 96.4% cell survival (p = 0.78). Scratch defect closure was seen in < 24 hours in all control conditions. However, cells exposed to CHX dilutions ≥ 0.02% had scratch defects that remained open indefinitely. Conclusions: The clinically used concentration of CHX (2%) permanently halts cell migration and significantly reduces survival of in vitro fibroblasts, myoblasts, and osteoblasts. Further in vivo studies are required to examine and optimize CHX safety and efficacy when applied near open incisions or intra-wound application.

The use of temporomandibular joint (TMJ) implants is considered to be a reliable treatment for some TMJ disorders when TMJ anatomical integrity is compromised. Among all of the designs proposed for these devices, intramedullary approaches are relatively new, and they may offer several advantages compared to those of past models with a lateral approach. In this report, we use finite element analysis (FEA) to calculate stress forces of a TMJ implant featuring a ferrule ring, which is frequently used in engineering as a stress distractor to reduce the splinter effect. Our analysis suggests that the addition of a ferrule ring in the TMJ implant helps to reduce von Mises stresses in the device and displacement forces in the volume and surface of the implant. These results suggest that including a ferrule ring in a TMJ implant may contribute to the stability and outcome of a TMJ implant by reducing component stress and displacement forces.

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.

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

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.

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.

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

OBJECTIVE: Subchondral microdamage may play an important role in post-traumatic osteoarthritis (PTOA) development following ACL rupture. It remains unknown whether this injury mechanism causes subchondral microdamage, or whether its repair occurs by targeted osteoclast-mediated remodeling. If so these events may represent a mechanism by which subchondral bone is involved in PTOA. Our objective was to test the hypothesis that subchondral microdamage occurs, and is co-localized with remodeling, in a novel rat model of ACL rupture. DESIGN: We developed a novel non-invasive rat animal model for ACL rupture and subchondral microdamage generation. By inducing ACL rupture noninvasively rather than surgically, this more closely mimics the clinical injury. MicroCT, MRI and histological methods were used to measure microstructural changes, ligament damage, and cellular/matrix degeneration, respectively. RESULTS: We reproducibly generated ACL rupture without damage to other soft joint tissues. Immediately after injury, increased microdamage was found in the postero-medial aspect of the tibia. Microstructural parameters showed increased resorption at 2 weeks, which returned to baseline. Dynamic histomorphometry showed increased calcein label uptake in the same region at 4 and 8 weeks. Chondrocyte death and protease activity in cartilage was also noted, however whether this was directly linked to subchondral changes is not yet known. Similarly, cartilage scoring showed degradation at 4 and 8 weeks post-injury. CONCLUSIONS: This study shows that our novel model can be used to study subchondral microdamage after ACL-rupture, and its association with localized remodeling. Cartilage degeneration, on a similar time-scale to other models, is also a feature of this system.

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