miR-122 and the WNT/ß-catenin pathway inhibit effects of both interleukin-1ß and tumor necrosis factor-α in articular chondrocytes in vitro.

Interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α), and WNT/ß-catenin signaling cause dysregulation of rat primary articular chondrocytes (rArCs), resulting in cartilage extracellular matrix destruction and osteoarthritis (OA) progression. microRNA (miR) miR-122 represses these effects whereas miR-451 exacerbates IL-1ß-stimulated matrix metalloproteinase-13 (MMP-13) and prostaglandin E2 (PGE2) production. The goals of this study were to evaluate crosstalk between these signaling pathways and determine if miR-122 and miR-451 exert their protective/destructive effects through these pathways in an in vitro model of OA. Primary rArCs were treated with IL-1ß or TNF-α for 24 h and total DNA, MMP-13, and PGE2, as well as expression levels of miR-122 and miR-451 were measured. After 24-h transfection with miR-122, miR-451, miR-122-inhibitor, or miR-451-inhibitor, rArCs were treated with or without TNF-α for 24 h; total DNA, MMP-13, and PGE2 were measured. Similarly, cells were treated with WNT-agonist lithium chloride (LiCl), WNT-antagonist XAV-939 (XAV), or PKF-118-310 (PKF) with and without IL-1ß or TNF-α stimulation. Both IL-1ß and TNF-α-stimulation increased MMP-13 and PGE2 production. Transfection with miR-122 prevented TNF-α-stimulated increases in MMP-13 and PGE2 whereas transfection with miR-451 did not change these levels. No differences were found in MMP-13 or PGE2 production with miR-122 or miR-451 inhibitors. LiCl treatment decreased PGE2 production in cultures treated with TNF-α, but not MMP-13. XAV increased TNF-α-stimulated increases in PGE2 but not MMP-13. LiCl reduced IL-1ß-stimulated increases in MMP-13 and PGE2. XAV and PKF increased IL-1ß-stimulated increases in MMP-13 and PGE2. In this in vitro OA model, miR-122 protects against both IL-1ß and TNF-α stimulated increases in MMP-13 and PGE2 production. miR-451 does not act through the TNF-α pathway. The WNT/ß-catenin pathway regulates the effects of IL-1ß and TNF-α stimulation. This study suggests that miR-122 can be used as a treatment or prevention for OA.

Acellular mineralized allogenic block bone graft does not remodel during the 10 weeks following concurrent implant placement in a rabbit femoral model.

OBJECTIVES: Due to bone loss, endosseous implants often require addition of a bone graft to support adequate primary fixation, bone regeneration, and osseointegration. The aim of this study was to compare effectiveness of autogenic and allogenic bone grafts when used during simultaneous insertion of the implant. MATERIALS AND METHODS: 4-mm-diameter rabbit diaphyseal bone autografts or allografts (n = 16/group) with a 3.2-mm pre-drilled hole in the center were placed into a 4 mm defect in the proximal femur of 3.5 kg male New Zealand White rabbits. Machined 3.2 × 10 mm grit-blasted, acid-etched titanium-aluminum-vanadium (Ti6Al4V) implants were placed. Control implants were placed into progressively drilled 3.2-mm holes in the contralateral limbs. Post-insertion day 70, samples were analyzed by micro-CT and calcified histology, or by mechanical torque and push-out testing followed by decalcified histology. RESULTS: Both grafts were integrated with the native bone. Micro-CT showed less bone volume (BV) and bone volume/total volume (BV/TV) in the allograft group, but histology showed no differences in BV or BV/TV between groups. Allograft lacked living cells, whereas autograft was cellularized. No difference was found in maximum removal torque between groups. Compressive loading at the graft-to-bone interface was significantly lower in allograft compared with autograft groups. CONCLUSIONS: There was less bone in contact with the implant and significantly less maximum compressive load in the allograft group compared with autograft. The allograft remained acellular as demonstrated by empty lacunae. Taken together, block allograft implanted simultaneously with an implant produces a poorer quality bone compared with autograft.

Osseointegration and Remodeling of Mineralized Bone Graft Are Negatively Impacted by Prior Treatment with Bisphosphonates.

BACKGROUND: Bisphosphonates limit resorption by inhibiting osteoclast formation and activation. They are removed during preparation of demineralized bone matrix (DBM) particles, but it is not known if osteogenesis and incorporation of mineralized bone allografts from patients treated with oral bisphosphonates are affected in vivo. METHODS: Human block allografts from 3 bisphosphonate-treated donors and 3 age and sex-matched control donors who had not received bisphosphonates were obtained (Musculoskeletal Transplant Foundation); one-half from each donor was demineralized. In the first study, 3 × 2-mm mineralized and demineralized cylindrical grafts were implanted bilaterally in the femoral metaphysis of 56 rats. In the second study, samples from each group were pooled, prepared as particles, and implanted bilaterally in the femoral marrow canal of 24 rats. Osseointegration, defined as native bone in contact with allograft, was assessed at 10 weeks by micro-computed tomography (CT) and histomorphometry. RESULTS: Micro-CT showed greater bone volume in sites treated with demineralized samples compared with the control mineralized and bisphosphonate-exposed mineralized samples. More new bone was generated along the cortical-endosteal interface compared with mineralized samples. Histology showed significantly less new bone in contact with the mineralized bisphosphonate-exposed allograft (10.4%) compared with mineralized samples that did not receive bisphosphonates (22.8%) and demineralized samples (31.7% and 42.8%). A gap was observed between native bone and allograft in the bisphosphonate-exposed mineralized samples (0.50 mm 2 ). The gap area was significantly greater compared with mineralized samples that did not receive bisphosphonates (0.16 mm 2 ) and demineralized samples (0.10 and 0.03 mm 2 ). CONCLUSIONS: Mineralized allografts were osseointegrated, but not remodeled or replaced by living bone, preventing full regeneration of the bone defect. Prior treatment of the donor with bisphosphonates affected osteogenesis, preventing osteointegration and remodeling of the allograft into the regenerating bone. CLINICAL RELEVANCE: Clinical use of mineralized allografts from patients who had received bisphosphonate therapy needs to be evaluated; in this animal model, such grafts were not integrated into the host bone or remodeled, and full regeneration of the bone defects was prevented.

Prophylactic administration of miR-451 inhibitor decreases osteoarthritis severity in rats.

Transfection of chondrocytes with microRNA-451(miR-451), present in growth zone cartilage of the growth plate, upregulates production of enzymes association with extracellular matrix degradation. miR-451 is also present in articular cartilage and exacerbates IL-1ß effects in articular chondrocytes. Moreover, when osteoarthritis (OA) was induced in Sprague Dawley rats via bilateral anterior cruciate ligament transection (ACLT), miR-451 expression was increased in OA cartilage compared to control, suggesting its inhibition might be used to prevent or treat OA. To examine the prophylactic and therapeutic potential of inhibiting miR-451, we evaluated treatment with miR-451 power inhibitor (451-PI) at the onset of joint trauma and treatment after OA had developed. The prophylactic animal cohort received twice-weekly intra-articular injections of either 451-PI or a negative control (NC-PI) beginning on post-surgical day 3. OA was allowed to develop for 24 days in the therapeutic cohort before beginning injections. All rats were killed on day 45. Micro-CT, histomorphometrics, OARSI scoring, and muscle force testing were performed on samples. 451-PI mitigated OA progression compared to NC-PI limbs in the prophylactic cohort based on histomorphometric analysis and OARSI scoring, but no differences were detected by micro-CT. 451-PI treatment beginning 24 days post-surgery was not able to reduce OA severity. Prophylactic administration of 451-PI mitigates OA progression in a post-trauma ACLT rat model supporting its potential to prevent OA development following an ACLT injury clinically.

24R,25-Dihydroxyvitamin D3 Protects against Articular Cartilage Damage following Anterior Cruciate Ligament Transection in Male Rats.

Osteoarthritis (OA) in humans is associated with low circulating 25-hydroxyvitamin D3 [25(OH)D3]. In vitamin D replete rats, radiolabeled 24R,25-dihydroxyvitamin D3 [24R,25(OH)2D3] accumulates in articular cartilage following injection of [3H]-25(OH)D3. Previously, we showed that 24R,25(OH)2D3 blocks chondrocyte apoptosis via phospholipase D and p53, suggesting a role for 24R,25(OH)2D3 in maintaining cartilage health. We examined the ability of 24R,25(OH)2D3 to prevent degenerative changes in articular cartilage in an OA-like environment and the potential mechanisms involved. In vitro, rat articular chondrocytes were treated with IL-1ß with and without 24R,25(OH)2D3 or 1α,25(OH)2D3. 24R,25(OH)2D3 but not 1α,25(OH)2D3 blocked the effects of IL-1ß in a dose-dependent manner, and its effect was partially mediated through the TGF-ß1 signaling pathway. In vivo, unilateral anterior cruciate ligament transections were performed in immunocompetent rats followed by intra-articular injections of 24R,25(OH)2D3 or vehicle (t = 0, 7, 14, 21 days). Tissues were harvested on day 28. Joints treated with vehicle had changes typical of OA whereas joints treated with 24R,25(OH)2D3 had less articular cartilage damage and levels of inflammatory mediators. These results indicate that 24R,25(OH)2D3 protects against OA, and suggest that it may be a therapeutic approach for preventing trauma-induced osteoarthritis.