Faculty Bibliography

The purpose of this study was to examine the influence of topical vancomycin on cell migration and survival of tissue healing cells. Human osteoblasts, myoblasts and fibroblasts were exposed to vancomycin at concentrations of 1, 3, 6, or 12 mg/cm² for either a 1-h or 48-h (continuous) duration. Continuous exposure to all vancomycin concentrations significantly reduced cell survival (<22% cells survived) and migration in osteoblasts and myoblasts (P < 0.001). 1-h vancomycin exposure reduced osteoblast and myoblast survival and migration only at 12 mg/cm² (P < 0.001). Further in vivo studies are warranted to optimize the dosage of intrawound vancomycin.

Objective Focal chondral defects alter joint mechanics and cause pain and debilitation. Microfracture is a surgical technique used to treat such defects. This technique involves penetration of subchondral bone to release progenitor cells and growth factors from the marrow to promote cartilage regeneration. Often this results in fibrocartilage formation rather than structured hyaline cartilage. Some reports have suggested use of growth hormone (GH) with microfracture to augment cartilage regeneration. Our objective was to test whether intra-articular (IA) GH in conjunction with microfracture, improves cartilage repair in a rabbit chondral defect model. We hypothesized that GH would exhibit a dose-dependent improvement in regeneration. Design Sixteen New Zealand white rabbits received bilateral femoral chondral defects and standardized microfracture repair. One group of animals ( n = 8) received low-dose GH by IA injection in the left knee, and the other group ( n = 8) received high-dose GH in the same manner. All animals received IA injection of saline in the contralateral knee as control. Serum assays, macroscopic grading, and histological analyses were used to assess any improvements in cartilage repair. Results Peripheral serum GH was not elevated postoperatively ( P = 0.21). There was no improvement in macroscopic grading scores among either of the GH dosages ( P = 0.83). Scoring of safranin-O-stained sections showed no improvement in cartilage regeneration and some evidence of increased bone formation in the GH-treated knees. Conclusions Treatment with either low- or high-dose IA GH does not appear to enhance short-term repair in a rabbit chondral defect model.

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.

Annexin A6 (AnxA6) is expressed in articular chondrocytes at levels higher than in other mesenchymal cell types. However, the role of AnxA6 in articular chondrocytes is not known. Here we show that complete lack of AnxA6 functions resulted in increased ß-catenin activation in Wnt3a-treated murine articular chondrocytes, whereas AnxA6 expressing articular chondrocytes showed decreased ß-catenin activation. High expression of AnxA6 in human articular chondrocytes showed the highest inhibition of Wnt/ß-catenin signaling. Inhibition of Wnt/ß-catenin signaling activity by AnxA6 together with cytosolic Ca2+ was achieved by interfering with the plasma membrane association of the Wnt signaling complex. AnxA6 also affected the cross-talk between Wnt/ß-catenin signaling and NF-κB signaling by decreasing ß-catenin activity and increasing NF-κB activity in Wnt3a-, interleukin-1beta (IL-1ß)-, and combined Wnt3a/IL-1ß-treated cells. Wnt3a treatment increased the mRNA levels of catabolic markers (cyclooxygenase-2, interleukin-6, inducible nitric oxide synthase) to a much lesser degree than IL-1ß treatment in human articular chondrocytes, and decreased the mRNA levels of matrix metalloproteinase-13 (MMP-13) and articular cartilage markers (aggrecan, type II collagen). Furthermore, Wnt3a decreased the mRNA levels of catabolic markers and MMP-13 in IL-1ß-treated human articular chondrocytes. High expression of AnxA6 resulted in decreased mRNA levels of catabolic markers, and increased MMP-13 and articular cartilage marker mRNA levels in Wnt3a-treated human articular chondrocytes, whereas leading to increased mRNA levels of catabolic markers and MMP-13 in human articular chondrocytes treated with IL-1ß, or combined Wnt3a and IL-1ß. Our findings define a novel role for AnxA6 in articular chondrocytes via its modulation of Wnt/ß-catenin and NF-κB signaling activities and the cross-talk between these two signaling pathways.

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.