Altered microRNA profile during fracture healing in rats with diabetes.

BACKGROUND: MicroRNAs (miRNAs) are a class of small non-coding RNA molecules that regulate gene expression. There is increasing evidence that some miRNAs are involved in the pathology of diabetes mellitus (DM) and its complications. We hypothesized that the functions of certain miRNAs and the changes in their patterns of expression may contribute to the pathogenesis of impaired fractures due to DM. METHODS: In this study, 108 male Sprague-Dawley rats were divided into DM and control groups. DM rats were created by a single intravenous injection of streptozotocin. Closed transverse femoral shaft fractures were created in both groups. On post-fracture days 5, 7, 11, 14, 21, and 28, miRNA was extracted from the newly generated tissue at the fracture site. Microarray analysis was conducted with miRNA samples from each group on post-fracture days 5 and 11. The microarray findings were validated by real-time polymerase chain reaction (PCR) analysis at each time point. RESULTS: Microarray analysis revealed that, on days 5 and 11, 368 and 207 miRNAs, respectively, were upregulated in the DM group, compared with the control group. The top four miRNAs on day 5 were miR-339-3p, miR451-5p, miR-532-5p, and miR-551b-3p. The top four miRNAs on day 11 were miR-221-3p, miR376a-3p, miR-379-3p, and miR-379-5p. Among these miRNAs, miR-221-3p, miR-339-3p, miR-376a-3p, miR-379-5p, and miR-451-5p were validated by real-time PCR analysis. Furthermore, PCR analysis revealed that these five miRNAs were differentially expressed with dynamic expression patterns during fracture healing in the DM group, compared with the control group. CONCLUSIONS: Our findings will aid in understanding the pathology of impaired fracture healing in DM and may support the development of molecular therapies using miRNAs for the treatment of impaired fracture healing in patients with DM.

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

PURPOSE: Diabetes 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. METHODS: Closed 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. RESULTS: SDF-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. CONCLUSION: Gene 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.

Profiling microRNA expression during fracture healing.

BACKGROUND: The 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. METHODS: Standard 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. RESULTS: Microarray 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. CONCLUSION: Our 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.

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 © 2013 John Wiley & Sons, Ltd.

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

PURPOSE: Skeletal 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. METHODS: The 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. RESULTS: The 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. CONCLUSION: Our 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.

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. .

Efficient derivation of osteoprogenitor cells from induced pluripotent stem cells for bone regeneration.

PURPOSE: There 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. 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 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. RESULTS: The 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. CONCLUSIONS: These 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.

Effect of low-intensity pulsed ultrasound on bone morphogenetic protein 7-induced osteogenic differentiation of human nonunion tissue-derived cells in vitro.

OBJECTIVES: Low-intensity pulsed ultrasound (US) has been shown to have positive effects on the healing of nonunions, and bone morphogenetic protein 7 (BMP-7) is known to be a strong stimulator of osteogenic differentiation. Recently, we showed that nonunion tissue contains multilineage mesenchymal progenitor cells, suggesting that nonunion tissue-derived cells may play an important role during the healing process of nonunions. In this study, we investigated whether low-intensity pulsed US promoted BMP-7-induced osteogenic differentiation of nonunion tissue-derived cells in vitro. METHODS: Nonunion tissue-derived cells were isolated from 7 patients. The cells were divided into two groups: (1) BMP-7 alone, consisting of nonunion tissue-derived cells cultured in osteogenic medium containing BMP-7 without low-intensity pulsed US treatment; and (2) BMP-7 + low-intensity pulsed US, consisting of nonunion tissue-derived cells cultured in osteogenic medium containing BMP-7 with low-intensity pulsed US treatment. The osteogenic differentiation potential and proliferation of nonunion tissue-derived cells were compared between the two groups. RESULTS: The alkaline phosphatase activity, gene expression levels of alkaline phosphatase and runt-related transcription factor 2, and mineralization were higher in the BMP-7 + low-intensity pulsed US group than in the BMP-7-alone group. There was no significant difference in cell proliferation between the two groups. CONCLUSIONS: These findings show a significant effect of low-intensity pulsed US on the osteogenic differentiation of nonunion tissue-derived cells induced by BMP-7. This study may provide substantial evidence for the clinical combined application of BMP-7 and low-intensity pulsed US for nonunion treatment.

In vitro hypertrophy and calcification of human fracture haematoma-derived cells in chondrogenic differentiation.

PURPOSE: The haematoma at a fracture site plays an important role in fracture healing. Previously, we demonstrated that a fracture haematoma contains multilineage mesenchymal progenitor cells. We postulated that the haematoma provided a source of chondrogenic cells for endochondral ossification during fracture healing and preservation of the cells contributed to biological fracture healing. In this study, we investigated whether haematoma-derived cells (HCs) could differentiate into hypertrophic chondrocytes and finally induce calcification of the extracellular matrix in vitro. METHODS: Fracture haematomas were obtained from four patients. HCs were cultured for five weeks under conditions that induce chondrogenic differentiation, followed by two weeks of hypertrophic induction using a pellet culture system. The pellets were analysed 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 analyses revealed that HCs differentiated into chondrocytes and hypertrophic chondrocytes, followed by calcification of the extracellular matrix. This sequential differentiation was also reflected in the gene expression profiles. After chondrogenic induction, expression of osteogenic and angiogenic markers was not significantly upregulated. However, the expression of these markers was significantly upregulated following hypertrophic induction. These in vitro observations mimicked the process of endochondral ossification during fracture healing. CONCLUSIONS: Our results suggest that the fracture haematoma may offer a source of cells with chondrogenic potential that play key roles in endochondral ossification during fracture healing. These findings support the opinion that the haematoma should be preserved for biological fracture healing.

Incidence of venous thromboembolism in fractures around and below the knee with physical prophylaxis.

Fractures occurring at the distal part of the lower extremities are recognized to have a relatively lower risk of venous thromboembolism (VTE); however, few detailed reports exist on the subject. The purpose of this study was to investigate the incidence of VTE in fractures around and below the knee. Overall, 109 consecutive patients with fractures around and below the knee who were surgically treated at the authors' hospital were analyzed retrospectively. Physical prophylaxis was performed in all patients. Until April 2009, VTE screening was performed by contrast-enhanced computed tomography or ultrasonography when the D-dimer value did not decline predictably, exceeded 20 µg/mL 5 days after trauma and surgery, or increased above 20 µg/mL after a period of decline. After April 2009, ultrasonography was routinely performed pre- and postoperatively irrespective of the D-dimer value. The patients were divided into 2 groups based on the absence or presence of accompanying injuries, including head, chest, abdominal, or spinal injury and other fractures of the pelvis and lower extremities. Overall, VTE and pulmonary thromboembolism were detected in 28 (25.7%) patients and 5 (4.6%) patients, respectively. All cases were asymptomatic. The VTE incidence rates were 8.6% (former screening) and 40% (newer screening) in patients with isolated fractures and 25% (former screening) and 41.7% (newer screening) in patients with accompanying injuries. The pulmonary thromboembolism incidence rates were 2.9% (former screening) and 0% (newer screening) in patients with isolated fractures and 3.2% (former screening) and 25.0% (newer screening) in patients with accompanying injuries. Surgeons should be vigilant for symptoms of VTE in patients with fractures occurring at the distal part of the lower extremities.

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.

Technique to prepare the bed for autologous bone grafting in nonunion surgery.

This article describes a technique for preparing the bed for autologous bone grafting in nonunion surgery. The procedure is divided into 2 steps. First, both ends of the fracture fragments are chipped into small pieces using an osteotome and hammer without peeling off the periosteum, creating pathways into the bone marrow. Second, cancellous bone harvested from the iliac crest is grafted into the aperture created by the previous bone chipping treatment. The technique is easy to perform and is a promising approach for enhancing bone healing in nonunion and delayed union.

Incidence of venous thromboembolism in pelvic and acetabular fractures in the Japanese population.

BACKGROUND: There are no detailed reports of the incidence of venous thromboembolism (VTE) in pelvic and acetabular fractures in the Asian population. The purpose of this study was to investigate the incidence of VTE in pelvic and acetabular fractures in the Japanese population. METHODS: Forty-six Japanese patients with pelvic and acetabular fractures treated at our hospital from February 2004 to April 2011 were analyzed retrospectively. Until April 2009, VTE screening was performed by contrast-enhanced computed tomography (CT) or ultrasonography (US) when the D-dimer value did not decline predictably, still exceeded 20 µg/ml at 5 days after trauma and surgery, or increased >20 µg/ml after a period of decline. After April 2009, contrast-enhanced CT and US were performed routinely irrespective of the D-dimer value. Physical prophylaxis was performed in all patients. The effects of the presence of pelvic and acetabular fractures, fracture types, accompanying injuries, and screening strategies on the incidences of VTE and pulmonary thromboembolism (PTE) were investigated. RESULTS: Overall, 19 patients (41.3%) were diagnosed with VTE and PTE in ten (21.7%). All were asymptomatic. Compared with trauma patients without pelvic and acetabular fractures treated during the same period, significantly higher incidences of VTE and PTE were observed in patients with pelvic and acetabular fractures. No significant differences were observed in the incidences of VTE and PTE between pelvic and acetabular fractures or between patients with and without accompanying injuries. Compared with the previous screening strategy, the detection rates of VTE and PTE were higher for the newer screening strategy; however, the difference did not reach statistical significance. CONCLUSIONS: We should be vigilant for the high incidence of VTE, especially PTE, in patients with pelvic and acetabular fractures in the Japanese population.