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. Methods. Following Institutional Review Board approval, biomembrane specimens were obtained from 12 patient surgeries for management of segmental bony defects (mean patient age 40.7 years, standard deviation 14.4). Biomembranes from nine tibias and three femurs were processed for morphologic, molecular or stem cell analyses. Gene expression was determined using the Affymetrix GeneChip Operating Software (GCOS). Molecular analyses compared biomembrane gene expression patterns with a mineralising osteoblast culture, and gene expression in specimens with longer spacer duration (> 12 weeks) with specimens with shorter durations. Statistical analyses used the unpaired student t-test (two tailed; p < 0.05 was considered significant). Results. Average PMMA spacer in vivo time was 11.9 weeks (six to 18). Trabecular bone was present in 33.3% of the biomembrane specimens; bone presence did not correlate with spacer duration. Biomembrane morphology showed high vascularity and collagen content and positive staining for the key bone forming regulators, bone morphogenetic protein 2 (BMP2) and runt-related transcription factor 2 (RUNX2). Positive differentiation of cultured biomembrane cells for osteogenesis was found in cells from patients with PMMA present for six to 17 weeks. Stem cell differentiation showed greater variability in pluripotency for osteogenic potential (70.0%) compared with chondrogenic or adipogenic potentials (100% and 90.0%, respectively). Significant upregulation of BMP2 and 6, numerous collagens, and bone gla protein was present in biomembrane compared with the cultured cell line. Biomembranes with longer resident PMMA spacer duration (vs those with shorter residence) showed significant upregulation of bone-related, stem cell, and vascular-related genes. Conclusion. The biomembrane technique is gaining favour in the management of complicated bone defects. Novel data on biological mechanisms provide improved understanding of the biomembrane’s osteogenic potential and molecular properties. Cite this article: Dr H. E. Gruber. Osteogenic, stem cell and molecular characterisation of the human induced membrane from extremity bone defects. Bone Joint Res 2016;5:106–115. DOI: 10.1302/2046-3758.54.2000483

Introduction. Our previous study using microarray analysis showed that Rad (Ras associated with diabetes) was highly expressed in nonunion. The purpose of this study is to investigate the gene expression and immunolocalization of Rad, and other Ras-related G proteins: Rem1 and Rem2 in fracture/nonunion site using rat experimental models. Hypothesis. We hypothesized that Rad had a significant role in nonunion formation. Materials & Methods. For standard healing model, K-wire was inserted into the femur and a closed fracture was created. Nonunion model was produced by periosteal cauterization at the fracture site. At post-fracture days 3, 7, 10, 14, 21, and 28, RNA was extracted from callus or fibrous tissue for real-time PCR. At day 14, specimens were harvested for immunohistochemistry. Results. Significant difference of Rad gene expression was not observed between standard healing fracture and nonunion at the earlier time points. In contrast, significantly higher expression in nonunion was observed at the later time points. There were no significant differences between standard healing fracture and nonunion in gene expression of Rem1 and Rem2. In immunohistochemical analysis, Rad and Rem1 were detected in the fracture site, and Rem2 was not detected. On the other hand, Rad was only detected in fibrous tissue in nonunion. Discussion & Conclusion. Our results suggest a significant role of Rad in fracture healing and nonunion formation. Rad may become a target agent for treatment of nonunion

INTRODUCTION. There is increasing evidence for a multi-stage model of rotator cuff (RC) tendon tears, wherein healing is affected by tear size. The underlying pathophysiology however is not fully understood. Changes in the production and remodeling of the RC extracellular matrix (ECM) are likely to be important determinants of RC tendinopathy as they affect healing and the ability to bear loads. This study aimed to gain greater insight into size related tear pathogenesis by analyzing gene expression profiles from normal, small and massive RC tears. METHODS. The genetic profiles of 28 human RC tendons were analyzed using microarrays representing the entire genome. 11 massive and 5 small torn RC tendon specimens were obtained from tear edges intraoperatively, and compared to 12 age matched normal controls. Semiquantitative real-time polymerase chain reaction (RT-PCR) and immunohistochemistry were performed for validation. RESULTS. Numerous insightful gene changes were detected. Key changes included upregulation of aggrecan in massive tendon tears compared to normal controls, but not in small tears (p < 0.05 and > 2-fold change). Matrix metallopeptidases (MMP)-3,-10,-12,-13,-15,-21,-25 and a disintegrin and metallopeptidase (ADAMs)-12,-15,-22 were significantly upregulated in tears. Aggrecan was upregulated in massive tendon tears but not in small tears. Amyloid was downregulated in the small and massive tear groups when compared to normals. BMP-5 was upregulated in small tears only when compared to normals. As part of the chemotaxis pathway, IL-3,-10,-13,-15,-18 were upregulated in tears, whereas downregulation of IL-1,-8,-11,-27, was seen. RT-PCR and immunohistochemistry confirmed altered gene expression. CONCLUSION. The gene profiles of normal, small and massive RC tear groups suggested they are biologically distinct groups. In addition to confirming altered gene expression in pathways reported in previous studies, this study has identified a number of novel pathways which are affected between the different tendon tear and normal groups. This study identified that RC tear pathogenesis is contributed to by ECM remodeling genes, chemotaxis genes, aggrecan and amyloid. Further investigation is required to determine whether some of these genes may potentially have a role as biomarkers of failure. Modulating these ECM pathways may be a useful treatment strategy for improving clinical outcomes

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

Introduction. The hematoma occurring at a fracture site is known to play an important role in fracture healing. Previously, we demonstrated that fracture hematoma contained multilineage mesenchymal progenitor cells. On the other hand, the process of fracture healing is associated by two different mechanisms, intramembranous and endochondral. However, there are no reports proving the details about cellular analysis in the process of endochondoral ossification. Hypothesis. We hypothesized that one of the cell origins for endochondral ossification after fracture was hematoma. Materials & Methods. Fracture hematoma was obtained during osteosynthesis. Hematoma-derived cells were isolated and cultured for 5-weeks of chondrogenic induction followed by 2-weeks hypertrophic induction using pellet culture system. The pellets were analyzed histologically and immunohistochemically. The gene expression levels of chondrogenic, hypertrophic, osteogenic and angiogenic markers were measured by real-time PCR. Results. The histological and immunohistochemical analysis revealed that the Hematoma-derived cells differentiated into hypertrophic chondrocytes through chondrocytes, and finally differentiate into calcifying chondrocytes. The same trend was seen in the gene expression using real-time PCR analysis. Discussion & Conclusions. Our results suggest that fracture hematoma may be an origin of cells which play key roles in the process of endochondoral ossification during fracture healing

Introduction. It is well known that blood flow is a critical key component of fracture repair. Previously, we demonstrated that transcutaneous application of CO2 increased blood flow in the human body. To date, there has been no report investigating the effect of the carbonated therapy on fracture repair. Hypothesis. We hypothesized that the transcutaneous application of CO2 to fracture site would accelerate fracture repair. Materials & Methods. A closed femoral shaft fracture was produced in rats. Transcutaneous CO2 absorption enhancing hydrogel and CO2 adaptor that sealed the body surface and retained the gas inside were used for CO2 treatment. Rats without CO2 treatment served as control. Radiographic, biomechanical and histological analysis was performed to assess the fracture repair. Gene expression of chondrogenic, hypertrophic, osteogenic and angiogenic markers was measured by real-time PCR at 1, 2, 3, and 4 weeks post-fracture. Results. Union rate, biomechanical properties, and gene expression of chondrogenic, hypertrophic, osteogenic and angiogenic markers was significantly higher in CO2 group compared to control group. Histological evaluation demonstrated that enchondral ossification was promoted in CO2 group. Discussion & Conclusions. Our study indicate that transcutaneous application of CO2 accelerates fracture repair via acceleration of endochondral ossification and vascularization, and may become a novel and useful therapy for promoting fracture repair

Introduction. Recently, some case reports have been published, in which nonunions were successfully healed with parathyroid hormone 1–34 (PTH) administration. Previously, we demonstrated that the intervening tissue at the nonunion site contains multilineage mesenchymal progenitor cells and plays an important role during the healing process of nonunion. We investigated the effect of PTH on osteogenic differentiation of human nonunion tissue-derived cells (NCs) in vitro. Hypothesis. We hypothesized that PTH directly promoted osteogenic differentiation of NCs. Materials & Methods. NCs were isolated from 4 patients, and cultured. The cells were divided into two groups: (1) PTH (−) group: cells cultured in osteogenic medium (OM), (2) PTH (+) group: cells cultured in OM with PTH. Osteogenic differentiation potential was analyzed. Results. Real-time PCR analysis showed that gene expression levels of Runx2, ALP, OC and PTHR1 in PTH (+) group were lower than PTH (−) group at day 14. In both groups, there was no significant difference in ALP activity at days 8 and 14, and in the intensity of Alizarin red S staining at day 20. Discussion. Treatment of PTH did not lead to increase osteogenic differentiation of NCs. Nonunion healing by PTH administration may be caused by other mechanisms such as mobilization and recruitment of osteoprogenitor cells

Introduction. Low-intensity pulsed ultrasound (LIPUS) has been reported to enhance healing of fracture and nonunion. Bone morphogenetic protein-7 (BMP-7) has also been reported to promote bone formation. Recently, we demonstrated progenitor cells with osteogenic/chondrogenic differentiation potential existed in human fracture hematoma and nonunion tissue. Hypothesis. We hypothesised the combined application of LIPUS and BMP-7 would cause major effect on osteogenesis of hematoma-derived cells (HCs) and nonunion tissue-derived cells (NCs). Materials & Methods. HCs and NCs were isolated, and cultured. The cells were divided into two groups: (1) BMP-7 group: cells cultured in osteogenic medium (OM), and (2) BMP-7 + LIPUS group: cells cultured in OM with LIPUS treatment. LIPUS (30 mW/cm2, intensity at 1.5 MHz) was given for 20 minutes daily. Osteogenic differentiation potential and proliferation were analysed. Results. ALP activity, the gene expression of osteogenic genes, and mineralisation of HCs and NCs were shown to be higher in BMP-7 + LIPUS group than in BMP-7 group. There was no significant difference in cell proliferation between the two groups. Discussion. Our findings demonstrated the significant effect of LIPUS on the osteogenic differentiation of HCs and NCs induced by BMP-7. This study may provide significant evidence for the clinical combined application of BMP-7 and LIPUS for the treatment of severe bone fracture and nonunion

Introduction. CXC chemokine receptor 4 (CXCR4) is a specific receptor for stromal-derived-factor 1 (SDF-1). SDF-1/CXCR4 interaction contributes to the regulation of endotherial progenitor cell (EPC) recruitment in ischemic tissues. The purpose of this study is to investigate the mechanistic function of CXCR4 on EPCs for bone fracture healing. Materials and methods. We made CXCR4 gene knockout mice using the Cre/loxP system. A reproducible model of femoral fracture was created in both Tie2-Cre CXCR4 knockout mice (CXCR4KO) and wild type mice (control). To evaluate gain function of the SDF-1/CXCR4 pathway, we set three groups of the SDF-1 intraperitoneally injected group, wild type group, and SDF-1 injected CXCR4 KO group. Results. In morphological examinations, relative callus area at week 2 was significantly greater in control group. Real time RT-PCR analysis showed that the gene expressions of angiogenic and osteogenic markers were higher in wild type group. CXCR4KO group represented a significantly lower perfusion value at fracture site than control group. In gain function study, the fracture in the SDF-1 injected group is significantly faster healed. Conclusion. Our results indicated the significance of SDF-1/CXCR4 signal in EPCs to bone fracture healing. This study also suggested that the promotion of CXCR4/SDF-1 signal on EPCs lead to the acceleration of bone fracture healing for new therapeutic strategies to fracture repair

Introduction. Nonunions pose complications in fracture management that can be treated using electrical stimulation (ES). Bone marrow mesenchymal stem cells (BMMSCs) are essential in fracture healing, although the effects of different clinical ES waveforms available in clinical practice on BMMSCs cellular activities is unknown. Materials and Methods. We compared Direct Current (DC), Capacitive Coupling (CC), Pulsed Electromagnetic wave (PEMF) and Degenerate Wave (DW) by stimulating human-BMMSCs for 5 days for 3 hours a day. Cytotoxicity, cell proliferation, cell-kinetics and cell apoptosis were evaluated after ES. Migration and invasion were assessed using fluorescence microscopy and affected gene and protein expression were quantified. Results. DW had the greatest proliferative and least apoptotic and cytotoxic effects compared to other waveforms and unstimulated cells after 5 days of ES (p < 0.001). DC, DW and CC resulted in significantly more cells in S phase and G2/M phase (p < 0.01) compared to the unstimulated BMMSCs. CC and DW caused more cells to invade collagen and showed increased MMP-2 and MT1-MMP expression (p < 0.001) compared to the other waveforms and unstimulated BMMSCs. DC increased cellular migration in a scratch-wound assay and all ES waveforms increased migration gene expression with DC having the greatest effect (p < 0.01). Conclusion. The ES waveform is vital in influencing BMMSCs cellular activities. Migration and invasion were increased by ES which suggests that the recruitment of BMMSCs to the healing site during a fracture could be increased by ES