Etsuko Okumachi

Profiling microRNA expression during fracture healing

BackgroundThe discovery of microRNA (miRNA) has revealed a novel type of regulatory control for gene expression. Increasing evidence suggests that miRNA regulates chondrocyte, osteoblast, and osteoclast differentiation and function, indicating miRNA as key regulators of bone formation, resorption, remodeling, and repair. We hypothesized that the functions of certain miRNAs and changes to their expression pattern may play crucial roles during the process of fracture healing.MethodsStandard healing fractures and unhealing fractures produced by periosteal cauterization at the fracture site were created in femurs of seventy rats, with half assigned to the standard healing fracture group and half assigned to the nonunion group. At post-fracture days 3, 7, 10, 14, 21, and 28, total RNA including miRNA was extracted from the newly generated tissue at the fracture site. Microarray analysis was performed with miRNA samples from each group on post-fracture day 14. For further analysis, we selected highly up-regulated five miRNAs in the standard healing fracture group from the microarray data. Real-time PCR was performed with miRNA samples at each time point above mentioned to compare the expression levels of the selected miRNAs between standard healing fractures and unhealing fractures and investigate their time-course changes.ResultsMicroarray and real-time polymerase chain reaction (PCR) analyses on day 14 revealed that five miRNAs, miR-140-3p, miR-140-5p, miR-181a-5p, miR-181d-5p, and miR-451a, were significantly highly expressed in standard healing fractures compared with unhealing fractures. Real-time PCR analysis further revealed that in standard healing fractures, the expression of all five of these miRNAs peaked on day 14 and declined thereafter.ConclusionOur results suggest that the five miRNAs identified using microarray and real-time PCR analyses may play important roles during fracture healing. These findings provide valuable information to further understand the molecular mechanism of fracture healing and may lead to the development of miRNA-based tissue engineering strategies to promote fracture healing.

Profiling microRNA expression in fracture nonunions: Potential role of microRNAs in nonunion formation studied in a rat model

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.

EFFICIENT DERIVATION OF OSTEOPROGENITOR CELLS FROM INDUCED PLURIPOTENT STEM CELLS FOR BONE REGENERATION

PurposeThere has been great interest in the use of induced pluripotent stem cells (iPSCs) in bone regenerative strategies. To generate osteoprogenitor cells from iPSCs, the most widely used protocol relies on an intermediate using embryoid body (EB) formation. We hypothesized that an osteoprogenitor cell population could be efficiently generated from iPSCs by employing a “direct-plating method” without the EB formation step.MethodsMurine iPSC colonies were dissociated with trypsin-EDTA, and obtained single cells were cultured on gelatin-coated plates in MSC medium and FGF-2. Adherent homogeneous fibroblast-like cells obtained by this direct-plating technique were termed as direct-plated cells (DPCs). Expression levels of Oct-3/4 mRNA were analysed by real-time PCR. DPCs were evaluated for cell-surface protein expression using flow cytometry. After osteogenic induction, osteogenic differentiation ability of DPCs was evaluated.ResultsThe expression level of Oct-3/4 in DPCs was significantly down-regulated compared to that observed in iPSCs, suggesting that the cells lost pluripotency. Flow cytometry analysis revealed that DPCs exhibited cell-surface antigens similar to those of bone marrow stromal cells. Furthermore, the cells proved to have a high osteogenic differentiation capacity, which was confirmed by the significant increase in alkaline phosphatase activity, the expression levels of osteogenic genes, and calcium mineralization after 14-day osteogenic induction.ConclusionsThese findings indicate that our novel direct-plating method provides a clinically applicable, simple, and labour-efficient system for generating large numbers of homogeneous iPSC-derived osteoprogenitor cells for bone regeneration.

Topical Cutaneous CO2 Application by Means of a Novel Hydrogel Accelerates Fracture Repair in Rats

BACKGROUND We previously demonstrated that topical cutaneous application of CO2, by means of a hydrogel in which the CO2 readily dissolves, increases blood flow and oxygen dissociation from hemoglobin in the soft tissues surrounding bone. In the present study, we utilized a rat fracture model to test the hypothesis that application of this treatment to fractured limbs would accelerate fracture repair. METHODS A closed femoral shaft fracture was created in each rat. Topical cutaneous application of CO2 by means of a hydrogel was performed five times a week for up to four weeks in the CO2/hydrogel group (n = 60). Sham treatments were performed in the control group (n = 60). Radiographic, histological, immunohistochemical, laser Doppler perfusion imaging, real-time polymerase chain reaction, and biomechanical assessments were performed. RESULTS Radiographic fracture union was evident at week 3 in twelve (86%) of fourteen animals in the CO2/hydrogel group compared with five (36%) of fourteen in the control group (p < 0.05; 95% CI [confidence interval] for the difference in union rate, 2.26% to 99.64%). Histological assessment revealed promotion of endochondral ossification in the CO2/hydrogel group. Immunohistochemical assessment at week 2 showed significantly greater capillary density in the CO2/hydrogel group (p < 0.05; 95% CI for the difference, 161 to 258 per mm(2)). Laser Doppler perfusion imaging demonstrated that the blood flow in the fractured limb was significantly greater at weeks 2 and 3 in the CO2/hydrogel group (p < 0.05; 95% CI for the difference, 8.4% to 22.4% and 6.7% to 19.0%, respectively). Gene expression of chondrogenic, osteogenic, and angiogenic markers was significantly greater in the CO2/hydrogel group at several time points. Ultimate stress, extrinsic stiffness, and failure energy (relative to the contralateral limb) were significantly greater in the CO2/hydrogel group at week 3 (p < 0.05; 95% CI for the difference, 24.8% to 67.5%, 4.0 % to 22.7%, and 9.6% to 58.8%, respectively). There were no significant differences between the groups with respect to any outcome measure at week 4. CONCLUSIONS Topical cutaneous application of CO2 by means of a hydrogel accelerated fracture repair in association with the promotion of angiogenesis, blood flow, and endochondral ossification. .

Comparative analysis of rat mesenchymal stem cells derived from slow and fast skeletal muscle in vitro

PurposeSkeletal muscle comprises different kinds of muscle fibres that can be classified as slow and fast fibres. The purpose of this study was to compare the yield, proliferation, and multi-potentiality of rat mesenchymal stem cells (MSCs) from the tibialis anterior (TA; fast muscle) and soleus (SO; slow muscle) in vitro.MethodsThe TA and SO muscles were harvested, and isolated cells were plated. After two hours, the cells were washed extensively to remove any cell that did not adhere to the cell culture plate. The adherent cells, namely MSCs, were then cultured. Both types of MSCs were differentiated toward the osteogenic, chondrogenic and adipogenic lineages using lineage specific induction factors.ResultsThe colony-forming unit fibroblast (CFU-F) assay revealed that the SO contained significantly higher quantities of MSCs than the TA. The self-renewal capacity of MSCs derived from the TA was significantly higher at later passages (passage 9–11). Both types of MSCs exhibited similar cell surface antigens to bone marrow (BM)-derived MSCs and were positive for CD29, CD44, and CD90 and negative for CD11b, CD34, and CD45. TA-derived MSCs were superior in terms of osteogenic differentiation capacity, but there was no significant difference in chondrogenic and adipogenic differentiation capacity.ConclusionOur results demonstrated significant differences in the properties of muscle-derived MSCs from different muscle types (i.e. fast or slow muscles). The greater expandability and osteogenic differentiation ability of TA-derived MSCs suggests that fast muscle may be a better source for generating large numbers of MSCs for bone regeneration.

Venous thromboembolism in Japanese patients with fractures of the pelvis and/or lower extremities using physical prophylaxis alone

Purpose. To investigate the rate of venous thromboembolism (VTE) in Japanese patients with fractures of the pelvis and/or lower extremities using physical prophylaxis alone. Methods. Records of 66 men and 60 women aged 15 to 95 (mean, 57) years with fractures of the pelvis and/or lower extremities were retrospectively reviewed. They were screened for VTE based on D-dimer values. Contrast-enhanced computed tomography and/or ultrasonography were performed when the D-dimer value did not decline predictably or exceeded 20 μg/ml even 5 days after injury or surgery. Physical prophylaxis for VTE in terms of graduated compression stockings and intermittent pneumatic compression were applied for all patients. Results. Of the 126 patients, 24 were detected to have VTE (10 of 29 with multiple fractures and 14 of 97 with single fractures). Six patients were detected to have asymptomatic pulmonary thromboembolism (PTE), whereas 20 patients were detected to have deep vein thrombosis (bilaterally in 7). The rates of VTE were high in patients with multiple fractures (35%), pelvic fractures (18%), and femoral shaft fractures (50%). The rate of PTE was high in patients with pelvic fractures (12%). Conclusion. The rate of VTE in the Japanese patients was similar to that in western populations. Our screening method was useful for preventing fatal PTEs. Surgeons should be vigilant for VTE during the first 2 weeks after injury, especially in patients with multiple and pelvic fractures.

Effects of parathyroid hormone 1‐34 on osteogenic and chondrogenic differentiation of human fracture haematoma‐derived cells in vitro

Parathyroid hormone (PTH) 1‐34 has been shown to accelerate fracture healing. Previously, we reported that progenitor cells with osteogenic and chondrogenic potential exist in human fracture haematoma, suggesting that the fracture haematoma‐derived progenitor cells (HCs) contribute to fracture healing. However, there has been no study investigating the effect of PTH on HCs. We investigated the effect of pulsatile and continuous PTH treatment on human fracture HCs in vitro. HCs were isolated from seven patients. The HCs were divided into four groups: growth medium; control [osteogenic medium (OM) without PTH]; PTH‐C (OM with continuous PTH); and PTH‐P (OM with pulsatile PTH) groups. Osteogenic differentiation potential and proliferation of HCs were compared among the four groups. For chondrogenesis, the HCs were divided into two groups: control [chondrogenic medium (CM) without PTH]; and PTH‐C (CM with continuous PTH) groups, and chondrogenic differentiation potential was analysed. PTH treatment did not affect cell proliferation, regardless of the mode of administration. Osteogenic activity was also not significantly affected by continuous PTH treatment but significantly inhibited by pulsatile PTH treatment. Conversely, chondrogenic differentiation was significantly inhibited by continuous PTH treatment. Our results revealed that PTH treatment on HCs, either continuous or pulsatile, does not exhibit any positive effect, and indicates that exogenous PTH administration after fracture has no effect on HCs. PTH may not have a positive effect at the fracture site during the early stage of fracture healing in which haematoma formation occurs. Copyright

Altered expression of microRNA during fracture healing in diabetic rats

Objectives Diabetes mellitus (DM) is known to impair fracture healing. Increasing evidence suggests that some microRNA (miRNA) is involved in the pathophysiology of diabetes and its complications. We hypothesized that the functions of miRNA and changes to their patterns of expression may be implicated in the pathogenesis of impaired fracture healing in DM. Methods Closed transverse fractures were created in the femurs of 116 rats, with half assigned to the DM group and half assigned to the control group. Rats with DM were induced by a single intraperitoneal injection of streptozotocin. At post-fracture days five, seven, 11, 14, 21, and 28, miRNA was extracted from the newly generated tissue at the fracture site. Microarray analysis was performed with miRNA samples from each group on post-fracture days five and 11. For further analysis, real-time polymerase chain reaction (PCR) analysis was performed at each timepoint. Results Microarray analysis showed that there were 14 miRNAs at day five and 17 miRNAs at day 11, with a greater than twofold change in the DM group compared with the control group. Among these types of miRNA, five were selected based on a comparative and extended literature review. Real-time PCR analysis revealed that five types of miRNA (miR-140-3p, miR-140-5p, miR-181a-1-3p, miR-210-3p, and miR-222-3p) were differentially expressed with changing patterns of expression during fracture healing in diabetic rats compared with controls. Conclusions Our findings provide information to further understand the pathology of impaired fracture healing in a diabetic rat model. These results may allow the potential development of molecular therapy using miRNA for the treatment of impaired fracture healing in patients with DM. Cite this article: S. Takahara, S. Y. Lee, T. Iwakura, K. Oe, T. Fukui, E. Okumachi, T. Waki, M. Arakura, Y. Sakai, K. Nishida, R. Kuroda, T. Niikura. Altered expression of microRNA during fracture healing in diabetic rats. Bone Joint Res 2018;7:139–147. DOI: 10.1302/2046-3758.72.BJR-2017-0082.R1.

Altered expression of SDF-1 and CXCR4 during fracture healing in diabetes mellitus

PurposeDiabetes mellitus (DM) is known to impair fracture healing. The purpose of this study was to elucidate and compare the gene expression patterns and localization of stromal cell-derived factor 1 (SDF-1) and CXC chemokine receptor 4 (CXCR4) during fracture healing of the femur in rats with and without DM.MethodsClosed transverse fractures were created in the femurs of rats equally divided into a DM group and control group; DM was induced by streptozotocin. At post-fracture days five, seven, 11, 14, 21 and 28, total RNA was extracted from the fracture callus and mRNA expression levels of SDF-1 and CXCR4 were measured by real-time polymerase chain reaction. Localization of SDF-1 and CXCR4 proteins at the fracture site was determined by immunohistochemistry at days 21 and 28.ResultsSDF-1 expression was significantly lower in the DM group than in the healthy group on days 21 and 28, and showed a significant difference between days 14 and 21 in the healthy group. There was no significant difference in CXCR4 expression levels between the healthy and DM groups at any time point. On day 21 immunoreactivity of SDF-1 and CXCR4 was detected at the fracture site of the healthy group but no immunoreactivity was observed in the DM group. On day 28, immunoreactivity of SDF-1 and CXCR4 was detected at the fracture site in both groups.ConclusionGene expression and localization of SDF-1 and CXCR4 was altered during fracture healing, which may contribute to the impaired fracture healing in DM.

Human pseudoarthrosis tissue contains cells with osteogenic potential

INTRODUCTION Nonunion is a challenging problem that may occur after certain bone fractures. The treatment of nonunion is closely related to its type. To develop an effective treatment strategy for each type of nonunion, biological analysis of nonunion tissue is essential. Pseudoarthrosis is a distinct pathologic entity of nonunion. To understand the pathology of pseudoarthrosis, we investigated the cellular properties of pseudoarthrosis tissue-derived cells (PCs) in vitro. PATIENTS AND METHODS PCs were isolated from four patients with pseudoarthrosis and cultured. Cells were evaluated for cell-surface protein expression by using flow cytometry. Osteogenic differentiation capacity was assessed by using Alizarin Red S staining, alkaline phosphatase (ALP) activity assay, and reverse transcription polymerase chain reaction (RT-PCR) after osteogenic induction. Chondrogenic differentiation capacity was assessed via Safranin O staining and RT-PCR after chondrogenic induction. RESULTS PCs were consistently positive for the mesenchymal stem cell-related markers CD29, CD44, CD105, and CD166, but were negative for the haematopoietic-lineage markers CD31, CD34, CD45, and CD133. Alizarin Red S staining revealed that PCs formed a mineralised matrix that was rich in calcium deposits after osteogenic induction. ALP activity under osteogenic conditions was significantly higher than that under control conditions. Gene expression of ALP, Runx2, osterix, osteocalcin, and bone sialoprotein was observed in PCs cultured under osteogenic conditions. Induced pellets were negatively stained by Safranin O staining. Gene expression of aggrecan, collagen II, collagen X, SOX5, and SOX9 was not observed. CONCLUSION We have shown for the first time the properties of cells in patients with pseudoarthrosis. Our results indicated that osteogenic cells existed in the pseudoarthrosis tissue. This study might provide insights into understanding the pathology of pseudoarthrosis and improving the treatment for pseudoarthrosis.