T. Waki

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.

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.

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.

Minimally Invasive Treatment for Tibial Malrotation after Locked Intramedullary Nailing

Rotational malreduction is a potential complication of intramedullary nailing for tibial shaft fractures. We experienced a symptomatic case of a 24° externally rotated malunion that we treated with minimally invasive corrective osteotomy. A 49-year-old man sustained a tibial shaft spiral fracture with a fibula fracture. He had been initially treated elsewhere with a reamed statically locked intramedullary nail. Bone union had been obtained, but he complained of asymmetry of his legs, difficulty walking and running, and the inability to ride a bicycle. We decided to perform corrective osteotomy in a minimally invasive fashion. After a 1 cm incision was made at the original fracture site, osteotomy for the affected tibia was performed with an osteotome after multiple efforts at drilling around the nail with the aim of retaining it. Fibula osteotomy was also performed at the same level. Two Kirschner wires that created an affected rotational angle between the fragments were inserted as a guide for correction. The distal locking screws were removed. Correct rotation was regained by matching the two wires in a straight line. Finally, the distal locking screws were inserted into new holes. The patient obtained bony union and has returned to his preinjury activities with no symptoms.