Development of primary osteoarthritis during aging in genetically diverse UM-HET3 mice.

BACKGROUND: Primary osteoarthritis (OA) occurs without identifiable underlying causes such as previous injuries or specific medical conditions. Age is a major contributing factor to OA, and as one ages, various joint tissues undergo gradual change, including degeneration of the articular cartilage, alterations in subchondral bone (SCB) morphology, and inflammation of the synovium. METHODS: We investigated the prevalence of primary OA in aged, genetically diverse UM-HET3 mice. Articular cartilage (AC) integrity and SCB morphology were assessed in 182 knee joints of 22-25 months old mice using the Osteoarthritis Research Society International (OARSI) scoring system and micro-CT, respectively. Additionally, we explored the effects of methylene blue (MB) and mitoquinone (MitoQ), two agents that affect mitochondrial function, on the prevalence and progression of OA during aging. RESULTS: Aged UM-HET3 mice showed a high prevalence of primary OA in both sexes. Significant positive correlations were found between cumulative AC (cAC) scores and synovitis in both sexes, and osteophyte formation in female mice. Ectopic chondrogenesis did not show significant correlations with cAC scores. Significant direct correlations were found between AC scores and inflammatory markers in chondrocytes, including matrix metalloproteinase-13, inducible nitric oxide synthase, and the NLR family pyrin domain containing-3 inflammasome in both sexes, indicating a link between OA severity and inflammation. Additionally, markers of cell cycle arrest, such as p16 and ß-galactosidase, also correlated with AC scores. In male mice, no significant correlations were found between SCB morphology traits and cAC scores, while in female mice, significant correlations were found between cAC scores and tibial SCB plate bone mineral density. Notably, MB and MitoQ treatments influenced the disease's progression in a sex-specific manner. MB treatment significantly reduced cAC scores at the medial knee joint, while MitoQ treatment reduced cAC scores, but these did not reach significance. CONCLUSIONS: Our study provides comprehensive insights into the prevalence and progression of primary OA in aged UM-HET3 mice, highlighting the sex-specific effects of MB and MitoQ treatments. The correlations between AC scores and various pathological factors underscore the multifaceted nature of OA and its association with inflammation and subchondral bone changes.

Cytokine Receptor-like Factor 1 (CRLF1) and Its Role in Osteochondral Repair.

BACKGROUND: Since cytokine receptor-like factor 1 (CRLF1) has been implicated in tissue regeneration, we hypothesized that CRLF1 released by mesenchymal stem cells can promote the repair of osteochondral defects. METHODS: The degree of a femoral osteochondral defect repair in rabbits after intra-articular injections of bone marrow-derived mesenchymal stem cells (BMSCs) that were transduced with empty adeno-associated virus (AAV) or AAV containing CRLF1 was determined by morphological, histological, and micro computer tomography (CT) analyses. The effects of CRLF1 on chondrogenic differentiation of BMSCs or catabolic events of interleukin-1beta-treated chondrocyte cell line TC28a2 were determined by alcian blue staining, gene expression levels of cartilage and catabolic marker genes using real-time PCR analysis, and immunoblot analysis of Smad2/3 and STAT3 signaling. RESULTS: Intra-articular injections of BMSCs overexpressing CRLF1 markedly improved repair of a rabbit femoral osteochondral defect. Overexpression of CRLF1 in BMSCs resulted in the release of a homodimeric CRLF1 complex that stimulated chondrogenic differentiation of BMSCs via enhancing Smad2/3 signaling, whereas the suppression of CRLF1 expression inhibited chondrogenic differentiation. In addition, CRLF1 inhibited catabolic events in TC28a2 cells cultured in an inflammatory environment, while a heterodimeric complex of CRLF1 and cardiotrophin-like Cytokine (CLC) stimulated catabolic events via STAT3 activation. CONCLUSION: A homodimeric CRLF1 complex released by BMSCs enhanced the repair of osteochondral defects via the inhibition of catabolic events in chondrocytes and the stimulation of chondrogenic differentiation of precursor cells.

Development of primary osteoarthritis during aging in genetically diverse UM-HET3 mice.

Background: Primary osteoarthritis (OA) occurs without identifiable underlying causes such as previous injuries or specific medical conditions. Age is a major contributing factor to OA, and as one ages, various joint tissues undergo gradual change, including degeneration of the articular cartilage, alterations in subchondral bone (SCB) morphology, and inflammation of the synovium. Methods: We investigated the prevalence of primary OA in aged, genetically diverse UM-HET3 mice. Articular cartilage (AC) integrity and SCB morphology were assessed in 182 knee joints of 22-25 months old mice using the Osteoarthritis Research Society International (OARSI) scoring system and micro-CT, respectively. Additionally, we explored the effects of methylene blue (MB) and mitoquinone (MitoQ), two agents that affect mitochondrial function, on the prevalence and progression of OA during aging. Results: Aged UM-HET3 mice showed a high prevalence of primary OA in both sexes. Significant positive correlations were found between cumulative AC (cAC) scores and synovitis in both sexes, and osteophyte formation in female mice. Ectopic chondrogenesis did not show significant correlations with cAC scores. Significant direct correlations were found between AC scores and inflammatory markers in chondrocytes, including matrix metalloproteinase-13, inducible nitric oxide synthase, and the NLR family pyrin domain containing-3 inflammasome in both sexes, indicating a link between OA severity and inflammation. Additionally, markers of cell cycle arrest, such as p16 and ß-galactosidase, also correlated with AC scores. In male mice, no significant correlations were found between SCB morphology traits and cAC scores, while in female mice, significant correlations were found between cAC scores and tibial SCB plate bone mineral density. Notably, MB and MitoQ treatments influenced the disease's progression in a sex-specific manner. MB treatment significantly reduced cAC scores at the medial knee joint, while MitoQ treatment reduced cAC scores, but these did not reach significance. Conclusions: Our study provides comprehensive insights into the prevalence and progression of primary OA in aged UM-HET3 mice, highlighting the sex-specific effects of MB and MitoQ treatments. The correlations between AC scores and various pathological factors underscore the multifaceted nature of OA and its association with inflammation and subchondral bone changes.

The Role of Hyaluronan/Receptor for Hyaluronan-Mediated Motility Interactions in the Modulation of Macrophage Polarization and Cartilage Repair.

Hyaluronan (HA), a negatively charged linear glycosaminoglycan, is a key macromolecular component of the articular cartilage extracellular matrix. The differential effects of HA are determined by a spatially/temporally regulated display of HA receptors, such as CD44 and receptor for hyaluronan-mediated motility (RHAMM). HA signaling through CD44 with RHAMM has been shown to stimulate inflammation and fibrotic processes. This study shows an increased expression of RHAMM in proinflammatory macrophages. Interfering with HA/RHAMM interactions using a 15-mer RHAMM-mimetic, HA-binding peptide, together with high-molecular-weight (HMW) HA reduced the expression and release of inflammatory markers and increased the expression of anti-inflammatory markers in proinflammatory macrophages. HA/RHAMM interactions were interfered in vivo during the regeneration of a full-thickness cartilage defect after microfracture surgery in rabbits using three intra-articular injections of 15-mer RHAMM-mimetic. HA-binding peptide together with HMWHA reduced the number of proinflammatory macrophages and increased the number of anti-inflammatory macrophages in the injured knee joint and greatly improved the repair of the cartilage defect compared with intra-articular injections of HMWHA alone. These findings suggest that HA/RHAMM interactions play a key role in cartilage repair/regeneration via stimulating inflammatory and fibrotic events, including increasing the ratio of proinflammatory/anti-inflammatory macrophages. Interfering with these interactions reduced inflammation and greatly improved cartilage repair.

Development of primary osteoarthritis during aging in genetically diverse UM-HET3 mice.

This study investigated the prevalence and progression of primary osteoarthritis (OA) in aged UM-HET3 mice. Using the Osteoarthritis Research Society International (OARSI) scoring system, we assessed articular cartilage (AC) integrity in 182 knee joints of 22-25 months old mice. Aged UM-HET3 mice showed a high prevalence of primary OA in both sexes. Significant positive correlations were found between cumulative AC (cAC) scores and synovitis in both sexes, and osteophyte formation in female mice. Ectopic chondrogenesis did not show significant correlations with cAC scores. Significant direct correlations were found between AC scores and inflammatory markers in chondrocytes, including matrix metalloproteinase-13 (MMP-13), inducible nitric oxide synthase (iNOS), and the NLR family pyrin domain containing-3 (NLRP3) inflammasome in both sexes, indicating a link between OA severity and inflammation. Additionally, markers of cell cycle arrest, such as p16 and ß-galactosidase, also correlated with AC scores. Using micro-CT, we examined the correlations between subchondral bone (SCB) morphology traits and AC scores. In male mice, no significant correlations were found between SCB morphology traits and cAC scores, while in female mice, significant correlations were found between cAC scores and tibial SCB plate bone mineral density. Finally, we explored the effects of methylene blue (MB) and mitoquinone (MitoQ), two agents that affect mitochondrial function, on the prevalence and progression of OA during aging. Notably, MB and MitoQ treatments influenced the disease's progression in a sex-specific manner. MB treatment significantly reduced cAC scores at the medial knee joint, while MitoQ treatment reduced cAC scores, but these did not reach significance. In conclusion, our study provides comprehensive insights into the prevalence and progression of primary OA in aged UM-HET3 mice, highlighting the sex-specific effects of MB and MitoQ treatments. The correlations between AC scores and various pathological factors underscore the multifaceted nature of OA and its association with inflammation and subchondral bone changes.

Excess Growth Hormone Triggers Inflammation-Associated Arthropathy, Subchondral Bone Loss, and Arthralgia.

Growth hormone (GH) is a key mediator of skeletal growth. In humans, excess GH secretion due to pituitary adenoma, seen in patients with acromegaly, results in severe arthropathies. This study investigated the effects of long-term excess GH on the knee joint tissues. One year-old wild-type (WT) and bovine GH (bGH) transgenic mice were used as a model for excess GH. bGH mice showed increased sensitivity to mechanical and thermal stimuli, compared with WT mice. Micro-computed tomography analyses of the distal femur subchondral bone revealed significant reductions in trabecular thickness and significantly reduced bone mineral density of the tibial subchondral bone-plate associated with increased osteoclast activity in both male and female bGH compared with WT mice. bGH mice showed severe loss of matrix from the articular cartilage, osteophytosis, synovitis, and ectopic chondrogenesis. Articular cartilage loss in the bGH mice was associated with elevated markers of inflammation and chondrocyte hypertrophy. Finally, hyperplasia of synovial cells was associated with increased expression of Ki-67 and diminished p53 levels in the synovium of bGH mice. Unlike the low-grade inflammation seen in primary osteoarthritis, arthropathy caused by excess GH affects all joint tissues and triggers severe inflammatory response. Data from this study suggest that treatment of acromegalic arthropathy should involve inhibition of ectopic chondrogenesis and chondrocyte hypertrophy.

Sexual dimorphic impact of adult-onset somatopause on life span and age-induced osteoarthritis.

Osteoarthritis (OA), the most prevalent joint disease, is a major cause of disability worldwide. Growth hormone (GH) has been suggested to play significant roles in maintaining articular chondrocyte function and ultimately articular cartilage (AC) homeostasis. In humans, the age-associated decline in GH levels was hypothesized to play a role in the etiology of OA. We studied the impact of adult-onset isolated GH deficiency (AOiGHD) on the life span and skeletal integrity including the AC, in 23- to 30-month-old male and female mice on C57/BL6 genetic background. Reductions in GH during adulthood were associated with extended life span and reductions in body temperature in female mice only. However, end-of-life pathology revealed high levels of lymphomas in both sexes, independent of GH status. Skeletal characterization revealed increases in OA severity in AOiGHD mice, evidenced by AC degradation in both femur and tibia, and significantly increased osteophyte formation in AOiGHD females. AOiGHD males showed significant increases in the thickness of the synovial lining cell layer that was associated with increased markers of inflammation (IL-6, iNOS). Furthermore, male AOiGHD showed significant increases in matrix metalloproteinase-13 (MMP-13), p16, and ß-galactosidase immunoreactivity in the AC as compared to controls, indicating increased cell senescence. In conclusion, while the life span of AOiGHD females increased, their health span was compromised by high-grade lymphomas and the development of severe OA. In contrast, AOiGHD males, which did not show extended life span, showed an overall low grade of lymphomas but exhibited significantly decreased health span, evidenced by increased OA severity.

Protective Effects of a Hyaluronan-Binding Peptide (P15-1) on Mesenchymal Stem Cells in an Inflammatory Environment.

Mesenchymal stem cells (MSCs) obtained from various sources, including bone marrow, have been proposed as a therapeutic strategy for the improvement of tissue repair/regeneration, including the repair of cartilage defects or lesions. Often the highly inflammatory environment after injury or during diseases, however, greatly diminishes the therapeutic and reparative effectiveness of MSCs. Therefore, the identification of novel factors that can protect MSCs against an inflammatory environment may enhance the effectiveness of these cells in repairing tissues, such as articular cartilage. In this study, we investigated whether a peptide (P15-1) that binds to hyaluronan (HA), a major component of the extracellular matrix of cartilage, protects bone-marrow-derived MSCs (BMSCs) in an inflammatory environment. The results showed that P15-1 reduced the mRNA levels of catabolic and inflammatory markers in interleukin-1beta (IL-1ß)-treated human BMSCs. In addition, P15-1 enhanced the attachment of BMSCs to HA-coated tissue culture dishes and stimulated the chondrogenic differentiation of the multipotential murine C3H/10T1/2 MSC line in a micromass culture. In conclusion, our findings suggest that P15-1 may increase the capacity of BMSCs to repair cartilage via the protection of these cells in an inflammatory environment and the stimulation of their attachment to an HA-containing matrix and chondrogenic differentiation.

A Hyaluronan-binding Peptide (P15-1) Reduces Inflammatory and Catabolic Events in IL-1ß-treated Human Articular Chondrocytes.

Inflammation plays a critical role in osteoarthritis (OA). It stimulates catabolic events in articular chondrocytes and prevents chondrogenic precursor cells from repairing cartilage lesions, leading to accelerated cartilage degradation. Therefore, the identification of novel factors that reduce catabolic events in chondrocytes and enhances chondrogenic differentiation of precursor cells in an inflammatory environment may provide novel therapeutic strategies for the treatment of OA. The goal of this study was to determine whether a hyaluronan (HA)-binding peptide (P15-1), via interacting with high molecular weight (HMW)HA can enhance the anti-inflammatory properties of HMWHA and decrease catabolic events in interleukin-1beta (IL-1ß)-treated human articular chondrocytes. Treatment with P15-1 decreased catabolic events and stimulated anabolic events in articular chondrocytes cultured in an inflammatory environment. P15-1 pre-mixed with HMWHA was more effective in inhibiting catabolic events and stimulating anabolic events than P15-1 or HMWHA alone. Our findings suggest that P15-1 together with HMWHA inhibits catabolic events in articular chondrocytes via the inhibition of p38 mitogen-activated protein kinases (MAPK) and increasing the thickness of the pericellular matrix (PCM) around chondrocytes thereby decreasing catabolic signaling. Finally, conditioned medium from IL-1ß and P15-1-treated human articular chondrocytes was less inhibitory for chondrogenic differentiation of precursor cells than conditioned medium from chondrocytes treated with IL-1ß alone. In conclusion, P15-1 is proposed to function synergistically with HMWHA to enhance the protective microenvironment for chondrocytes and mesenchymal stem cells during inflammation and regeneration.

Extracellular Vesicles Released From Articular Chondrocytes Play a Major Role in Cell-Cell Communication.

The purpose of this investigation was to determine the role of extracellular vesicles (EVs), released from articular chondrocytes in a physiological or pathological state, in cell-cell communication with other articular chondrocytes or chondrocyte precursor cells. The conditioned medium from interleukin-1ß (IL-1ß)-treated human articular chondrocytes stimulated catabolic events and inhibited type II collagen expression in articular chondrocytes to a much greater degree than medium from IL-1ß-treated chondrocytes after complete removal of EVs. The vehicle-treated and IL-1ß-treated human articular chondrocytes released EVs of similar size; however, the number of EVs released by IL-1ß-treated chondrocytes was markedly higher than the number of EVs released from the vehicle-treated cells. Furthermore, our findings demonstrate that similar to medium from IL-1ß-treated chondrocytes containing EVs, EVs isolated from medium of IL-1ß-treated chondrocytes stimulated catabolic events in articular chondrocytes, whereas EVs isolated from the medium of vehicle-treated chondrocytes inhibited catabolic events and increased messenger RNA levels of aggrecan and type II collagen in IL-1ß-treated chondrocytes. Furthermore, the medium containing EVs from vehicle-treated articular chondrocytes or EVs isolated from this medium stimulated chondrogenesis of C3H10T1/2 cells, whereas medium containing EVs from IL-1ß-treated chondrocytes or EVs isolated from this medium inhibited chondrogenesis. Our findings suggest that EVs released by articular chondrocytes play a key role in the communication between joint cells and ultimately in joint homeostasis, maintenance, pathology, and repair. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:731-739, 2020.

Role of Hyaluronan in Inflammatory Effects on Human Articular Chondrocytes.

Hyaluronan (HA) fragments have been proposed to elicit defensive or pro-inflammatory responses in many cell types. For articular chondrocytes in an inflammatory environment, studies have failed to reach consensus on the endogenous production or effects of added HA fragments. The present study was undertaken to resolve this discrepancy. Cultured primary human articular chondrocytes were exposed to the inflammatory cytokine IL-1ß, and then tested for changes in HA content/size in conditioned medium, and for the expression of genes important in HA binding/signaling or metabolism, and in other catabolic/anabolic responses. Changes in gene expression caused by enzymatic degradation of endogenous HA, or addition of exogenous HA fragments, were examined. IL-1ß increased the mRNA levels for HA synthases HAS2/HAS3 and for the HA-binding proteins CD44 and TSG-6. mRNA levels for TLR4 and RHAMM were very low and were little affected by IL-1ß. mRNA levels for catabolic markers were increased, while type II collagen (α1(II)) and aggrecan were decreased. HA concentration in the conditioned medium was increased, but the HA was not degraded. Treatment with recombinant hyaluronidase or addition of low endotoxin HA fragments did not elicit pro-inflammatory responses. Our findings showed that HA fragments were not produced by IL-1ß-stimulated human articular chondrocytes in the absence of other sources of reactive oxygen or nitrogen species, and that exogenous HA fragments from oligosaccharides up to about 40 kDa in molecular mass were not pro-inflammatory agents for human articular chondrocytes, probably due to low expression of TLR4 and RHAMM in these cells.

Alterations in Synovial Fluid Biomarker Levels in Knees With Meniscal Injury as Compared With Asymptomatic Contralateral Knees.

BACKGROUND: Changes in the joint microenvironment after an injury to the articular surface of the knee have been implicated in the pathogenesis of osteoarthritis. While prior studies focused on changes in this microenvironment after anterior cruciate ligament ruptures, few have explored the biomarker changes that occur in the setting of meniscal injuries. PURPOSE: To determine whether meniscal injury results in significant alterations to synovial fluid biomarker concentrations as compared with noninjured contralateral knees. Additionally, to explore the relationship between synovial fluid biomarkers and the degree of cartilage injury seen in these patients. STUDY DESIGN: Cross-sectional study; Level of evidence, 3. METHODS: Patients undergoing surgery for unilateral meniscal injury were prospectively enrolled from October 2011 to December 2016, forming a cohort that had synovial fluid samples collected from both the injured knee and the contralateral uninjured knee at the time of meniscal surgery. Synovial fluid samples were collected just before incision, and the concentrations of 10 biomarkers of interest were determined with a multiplex magnetic bead immunoassay. The concentrations of synovial fluid biomarkers from the operative and contralateral knees were compared. Additionally, the synovial fluid biomarker concentrations of operative knees from patients with associated high-grade cartilage lesions were compared with those with low-grade lesions. RESULTS: The current analysis included synovial fluid samples from 82 knees (41 operative and 41 contralateral) from 41 patients undergoing arthroscopic surgery to treat a symptomatic meniscal injury. The mean ± SD age of patients was 49.86 ± 11.75 years. There were significantly greater concentrations of 4 of the 5 proinflammatory biomarkers (IL-6, MCP-1, MIP-1ß, and MMP-3) in symptomatic knees as compared with asymptomatic knees when controlling for the duration of symptoms, body mass index, age, and the random effects of by-patient variability. In the injured knees, associated high-grade cartilage lesions were predictive of elevated MCP-1, MIP-1ß, and VEGF levels. Low synovial fluid concentration of TIMP-1 or a greater ratio of MMP-3 to TIMP-1 was associated with the presence of synovitis. Increasing age was found to be an independent predictor of increased IL-6, MCP-1, and VEGF concentrations in the setting of symptomatic meniscal injury. CONCLUSION: The authors identified 4 proinflammatory synovial fluid biomarkers whose concentrations were significantly different after meniscal injury as compared with uninjured contralateral knees. Furthermore, they describe the effects of associated cartilage damage, synovitis, and patient age on biomarker concentrations.

Cytotoxicity evaluation of chlorhexidine gluconate on human fibroblasts, myoblasts, and osteoblasts.

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.

Annexin A6 regulates catabolic events in articular chondrocytes via the modulation of NF-κB and Wnt/ß-catenin signaling.

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.

Topical vancomycin and its effect on survival and migration of osteoblasts, fibroblasts, and myoblasts: An in vitro study.

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.

Engineered Coiled-Coil Protein for Delivery of Inverse Agonist for Osteoarthritis.

Osteoarthritis (OA) results from degenerative and abnormal function of joints, with localized biochemistry playing a critical role in its onset and progression. As high levels of all- trans retinoic acid (ATRA) in synovial fluid have been identified as a contributive factor to OA, the synthesis of de novo antagonists for retinoic acid receptors (RARs) has been exploited to interrupt the mechanism of ATRA action. BMS493, a pan-RAR inverse agonist, has been reported as an effective inhibitor of ATRA signaling pathway; however, it is unstable and rapidly degrades under physiological conditions. We employed an engineered cartilage oligomeric matrix protein coiled-coil (CccS) protein for the encapsulation, protection, and delivery of BMS493. In this study, we determine the binding affinity of CccS to BMS493 and the stimulator, ATRA, via competitive binding assay, in which ATRA exhibits approximately 5-fold superior association with CccS than BMS493. Interrogation of the structure of CccS indicates that ATRA causes about 10% loss in helicity, while BMS493 did not impact the structure. Furthermore, CccS self-assembles into nanofibers when bound to BMS493 or ATRA as expected, displaying 11-15 nm in diameter. Treatment of human articular chondrocytes in vitro reveals that CccS·BMS493 demonstrates a marked improvement in efficacy in reducing the mRNA levels of matrix metalloproteinase-13 (MMP-13), one of the main proteases responsible for the degradation of the extracellular cartilage matrix compared to BMS493 alone in the presence of ATRA, interleukin-1 beta (IL-1ß), or IL-1 ß together with ATRA. These results support the feasibility of utilizing coiled-coil proteins as drug delivery vehicles for compounds of relatively limited bioavailability for the potential treatment of OA.

A competitive alphascreen assay for detection of hyaluronan.

A method for specific quantification of hyaluronan (HA) concentration using AlphaScreen® (Amplified Luminescent Proximity Homogeneous Assay) technology is described. Two types of hydrogel-coated and chromophore-loaded latex nanobeads are employed. The proximity of the beads in solution is detected by excitation of the donor bead leading to the production of singlet oxygen, and chemiluminescence from the acceptor bead upon exposure to singlet oxygen. In the HA assay, the donor bead is modified with streptavidin, and binds biotin-labeled HA. The acceptor bead is modified with Ni(II), and is used to bind a specific recombinant HA-binding protein (such as HABP; aggrecan G1-IGD-G2) with a His-tag. Competitive inhibition of the HA-HABP interaction by free unlabeled HA in solution is used for quantification. The assay is specific for HA, and not dependent on HA molecular mass above the decasaccharide. HA can be quantified over a concentration range of approximately 30-1600 ng/mL using 2.5 µL of sample, for a detectable mass range of approximately 0.08-4 ng HA. This sensitivity of the AlphaScreen assay is greater than existing ELISA-like methods, due to the small volume requirements. HA can be detected in biological fluids using the AlphaScreen assay, after removal of bound proteins from HA and dilution or removal of other interfering proteins and lipids.

The Effect of Growth Hormone on Chondral Defect Repair.

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.

Use of Ferrule Rings as Stress Dissipators in Temporomandibular Joint Intramedullary Implants: A Finite Element Analysis Study.

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.

Povidone-iodine Solutions Inhibit Cell Migration and Survival of Osteoblasts, Fibroblasts, and Myoblasts.

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 postoperative 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.