Osteoporosis following ovariectomy has been suggested to modulate bone response to polyethylene wear debris. In this work, we evaluate the influence of estrogen deficiency on experimental particle-induced osteolysis. Polyethylene (PE) particles were implanted onto the calvaria of wild-type (WT), sham-ovariectomized (OVX), OVX mice and OVX mice supplemented with estrogen (OVX+E2) (12 mice per group). Sham-implanted mice served as internal controls. After 14 days, seven skulls per group were analyzed with a high-resolution micro-computed tomography (CT) and by histomorphometry, and for tartrate-specific alkaline phosphatase. Five calvariae per group were cultured for the assay of IL-1, IL-6, TNF- and RANKL secretion using quantitative ELISA. The expression of RANKL and OPG mRNA were evaluated using real-time PCR. As assessed by CT and by histomorphometry, PE particles induced an extensive bone resorption and an intense inflammatory reaction in WT, sham-OVX and OVX+E2 mice. In OVX mice group, these features appeared considerably attenuated. In WT, sham-OVX and OVX+E2 mice, PE particles induced an increase in serum IL-6, in TNF-and RANKL local concentrations, and resulted in a two-fold increase in RANKL/OPG mRNA ratio. Conversely, these parameters remained unchanged in OVX mice after PE implantation. The combination of two well-known bone resorptive mechanisms ultimately attenuated osteolytic response, suggesting a protective effect of estrogen deficiency on particle-induced osteolysis. This paradoxical phenomenon was associated with a downregulation of pro-resorptive cytokines. It is hypothesized that excessive inflammatory response was controlled, illustrated by the absence of increase of serum IL-6 in OVX mice after PE implantation

Proliferation of synovial Mesenchymal Stromal/Stem Cells (MSCs) leads to synovial hyperplasia (SH) following Joint Surface Injury (JSI). Uncontrolled Yap activity causes tissue overgrowth due to modulation of MSC proliferation. We hypothesised that YAP plays a role in SH following JSI. A spatiotemporal analysis of Yap expression was performed using the JSI model in C57Bl/6 mice. Synovial samples from patients were similarly analysed. Gdf5-Cre;Yap1fl/fl;Tom mice were created to determine the effect YAP1 knockout in Gdf5 lineage cells on SH after JSI. In patients, Yap expression was upregulated in activated synovium, including a subset of CD55 positive fibroblast-like synoviocytes in the synovial lining (SL). Cells staining positive for the proliferation marker Ki67 expressed active YAP. In mice, Yap was highly expressed in injured knee joint synovium compared to controls. Yap mRNA levels at 2 (p<0.05) and 8 days (p<0.001) after injury were increased. Conditional Yap1 knockout in Gdf5 progeny cells prevented hyperplasia of synovial lining (SL) after JSI. Cellularity was significantly decreased in the SL but not in the sub-lining of injured Yap1 knockout- compared to control mice. The percentage of cells in synovium that were Tom+ increased in response to JSI in control and haplo-insufficient but not in YAP1 knockout mice (p<0.05). Modulation of YAP and proliferation of MSCs in the synovium after JSI provides a system to study the role of SH after trauma in re-establishing joint homeostasis and is a potential novel therapeutic target for the treatment of post traumatic OA

Introduction. The therapeutic potential of hematopoietic stem cells for fracture healing has been demonstrated with mechanistic insight of vasculogenesis and osteogenesis enhancement. Lnk has recently been proved an essential inhibitory signaling molecule in SCF-c-Kit signaling pathway for stem cell self-renewal demonstrating enhanced hematopoietic and osteogenic reconstitution in Lnk-deficient mice. We investigated the hypothesis that down regulation of Lnk enhances regenerative response via vasculogenesis and osteogenesis in fracture healing. Methods. A reproducible model of femoral fracture was created in mice. Immediately after fracture creation, mice received local administration of the following materials with AteloGene, 10μM (1)Lnk siRNA, (2)control siRNA. Results. Lnk group demonstrated more prompt fracture repair than control group. The functional bone healing was also significantly greater in Lnk group. Immunohistochemical staining and the mRNA expressions in fracture sites indicated the superior ability for angiogenesis and osteogenesis in Lnk group. Moreover, Lnk siRNA transfected cells showed high capacity of colony formation in vitro. Conclusion. We clarified that negatively controlled Lnk system contributed to a favorable environment for fracture healing by enhancing vasculogenesis and osteogenesis. These findings suggest that down regulation of Lnk may have a clinical potential for faster fracture healing, which contributes to reduce delayed union or nonunion

Improving periprosthetic bone is essential for implant fixation and reducing peri-implant fracture risk. This studied examined the individual and combined effects of iPTH and mechanical loading at the cellular, molecular, and tissue level for periprosthetic cancellous bone. Adult rabbits had a porous titanium implant inserted bilaterally on the cancellous bone beneath a mechanical loading device on the distal lateral femur. The right femur was loaded daily, the left femur received a sham loading device, and half of the rabbits received daily PTH. Periprosthetic bone was processed up to 28 days for qPCR, histology, and uCT analysis. We observed an increase in cellular and molecular markers of osteoblast activity and decrease in adipocytic markers for both treatments, with small additional effects in the combined group. Loading and iPTH led to a decrease and increase, respectively, in osteoclast number, acting through changes in RANKL/OPG expression. Changes in SOST and beta-catenin mRNA levels suggested an integral role for the Wnt pathway. We observed strong singular effects on BV/TV of both loading (1.53 fold) and iPTH (1.54 fold). Combined treatment showed a small additive effect on bone volume. In conclusion, loading and iPTH act through a pro-osteoblastic/anti-adipocytic response and through control of bone turnover via changes in the RANKL/OPG pathway. These changes led to a small additional, but not synergistic, increase in bone volume with the combined therapy

Introduction. iPSCs represent a promising cell source for bone regeneration. To generate osteoprogenitor cells, most protocols use the generation of embryoid bodies (EBs). However, these protocols give rise to heterogeneous population of different cell lineage. Hypothesis. We hypothesized that a direct plating method without EB formation step could be an efficient protocol for generating a homogeneous population of osteoprogenitor cells from iPSCs. Materials & Methods. Murine iPSC colonies were dissociated with trypsin-EDTA, and obtained single cells were cultured on gelatin-coated plates in MSC medium and FGF-2. Adherent cells obtained by this direct-plating technique were termed as direct-plated cells (DPCs). DPCs were evaluated for cell-surface protein expression using flow cytometry. Expression levels of Oct-3/4 mRNA in iPSCs and DPCs were analyzed by real-time PCR. DPCs were cultured for 14 days in osteogenic medium. Osteogenic differentiation was evaluated by alkaline phosphatase (ALP) activity, real-time PCR, and alizarin red S staining. Results. Expression level of Oct-3/4 in DPCs was robustly down-regulated compared to that in iPSCs. Flow cytometry analysis revealed DPCs had similar characteristics to MSC, suggesting DPCs lost pluripotency. Moreover, the DPCs exhibited high osteogenic potential. Discussion & Conclusion. Our novel direct plating method in the absence of EB formation step could be amenable to large-scale production of osteoprogenitor cells for bone regeneration

Injuries to growth plates may initiate the formation of reversible or irreversible bone-bridges, which may lead to partial or full closure of the growth plate resulting in bone length discrepancy, axis deviation or joint deformity. Blood vessels and vascular invasion are essential for the formation of new bone tissue. The aim of our study was to investigate the spatial and temporal expression VEGF and its receptors R1 and R2 as well as the ingrowth of vessels in the formation of bone bridges in a rat physeal injury model. Quantitative Real Time - Polymerase Chain Reaction (qRT-PCR) was performed for Vascular Endothelial Growth Factor (VEGF) and its R1 and R2 receptors. Samples from the proximal epiphysis, physis and metaphysis of the tibial bone were prepared for immunohistochemical analysis to demonstrate the spatial expression of VEGF and its R1 and R2 receptors as well as laminin. Kinetic expression of VEGF and VEGF-R1 mRNA documented a tendency towards an expression increase on day 7. Histological analysis showed a haematoma containing bone fragments on day 1which was replaced by a bony bridge by day 14. This remodelled and consolidated by day 82. These trabeculae were accompanied by vessel formation. Expression of VEGF was observed on the bone fragments and the haematoma from day 1 through to day 82. Although VEGF-R1 was expressed at all time points the expression of VEGF-R2 was noted until the 14th day. Physeal bone bridge formation is a combination of both enchondral and intramembranous ossification. This is in part triggered by the bony debris observed within the lesion in the first few days. By washing this debris out the likelihood of bone bridge formation may be reduced. We recommend this practice when operating on the physis in order to avoid iatrogenic physeal bar formation

Objectives

The biomembrane (induced membrane) formed around polymethylmethacrylate (PMMA) spacers has value in clinical applications for bone defect reconstruction. Few studies have evaluated its cellular, molecular or stem cell features. Our objective was to characterise induced membrane morphology, molecular features and osteogenic stem cell characteristics.

MicroRNAs (miRNAs ) are small non-coding RNAs that regulate gene expression. We hypothesised that the functions of certain miRNAs and changes to their patterns of expression may be crucial in the pathogenesis of nonunion. Healing fractures and atrophic nonunions produced by periosteal cauterisation were created in the femora of 94 rats, with 1:1 group allocation. At post-fracture days three, seven, ten, 14, 21 and 28, miRNAs were extracted from the newly generated tissue at the fracture site. Microarray and real-time polymerase chain reaction (PCR) analyses of day 14 samples revealed that five miRNAs, miR-31a-3p, miR-31a-5p, miR-146a-5p, miR-146b-5p and miR-223-3p, were highly upregulated in nonunion. Real-time PCR analysis further revealed that, in nonunion, the expression levels of all five of these miRNAs peaked on day 14 and declined thereafter.

Our results suggest that miR-31a-3p, miR-31a-5p, miR-146a-5p, miR-146b-5p and miR-223-3p may play an important role in the development of nonunion. These findings add to the understanding of the molecular mechanism for nonunion formation and may lead to the development of novel therapeutic strategies for its treatment.

Cite this article: Bone Joint J 2015; 97-B:1144–51.