Takashi Iwakura

In vitro multipotentiality and characterization of human unfractured traumatic hemarthrosis-derived progenitor cells: A potential cell source for tissue repair.

Mesenchymal progenitor cells (MPCs) are a very attractive tool in the context of repair and regeneration of musculoskeletal tissue damaged by trauma. The most common source of MPCs to date has been the bone marrow, but aspirating bone marrow from the patient is an invasive procedure. In an attempt to search for alternative sources of MPCs that could be obtained with minimal invasion, we looked into traumatic hemarthrosis of the knee. In this study, we determined whether a population of multipotent MPCs could be isolated from acute traumatic knee hemarthrosis in the absence of intra‐articular fractures. Mononuclear cells were isolated from the aspirated hemarthrosis by density gradient separation, and cultured. We were able to obtain plastic adherent fibroblast‐like cells from the mononuclear cell fractions. Flow cytometry analysis revealed that the adherent fibroblast‐like cells were consistently positive for CD29, CD44, CD105, and CD166, and were negative for CD14, CD34, and CD45. These were similar to control bone marrow stromal cells. These cells could differentiate in vitro into osteogenic, adipogenic, and chondrogenic cells in the presence of lineage‐specific induction factors. In conclusion, acute unfractured traumatic hemarthrosis of the knee contains MPCs with multipotentiality. Because knee hemarthrosis is easy to harvest with minimal pain and without unnecessary invasion, we regard hemarthrosis‐derived cells as an additional progenitor cell source for future tissue engineering and cell‐based therapy in knee injuries. J. Cell. Physiol. 210: 561–566, 2007.

Efficient Cell-seeding into Scaffolds Improves Bone Formation

Bone marrow stromal cells (BMSCs)/β-tricalcium phosphate (β-TCP) composites have attracted a great deal of attention in bone tissue engineering. If more effective bone regeneration is to be achieved, efficient cell-seeding systems need to be clarified. In this study, we investigated the number of cells contained in composites, and the in vitro/vivo osteogenic differentiation capacity of composites using 4 conventional systems of seeding rat BMSCs into β-TCP: soak, low-pressure, pipette, and syringe systems. The highest number of cells was contained in the composites from the syringe group. Moreover, after two-week osteogenic induction in vitro, the composites in the syringe group exhibited the highest osteogenic potential, which continued at 8 weeks after subcutaneous implantation in vivo. Our results indicated that efficient and appropriate cell-seeding could improve in vitro/vivo bone formation in composites and thus make a potential clinical contribution to successful bone tissue engineering. Abbreviations: BMSCs, bone marrow stromal cells; β-TCP, β-tricalcium phosphate; S-D, Sprague-Dawley; kPa, kilopascal; ALP, alkaline phosphatase; N, Newton; DNA, deoxyribonucleic acid; OCN, osteocalcin; ANOVA, analysis of variance; PLSD, protected least-significant difference; and HE, hematoxylin and eosin.

Human hypertrophic nonunion tissue contains mesenchymal progenitor cells with multilineage capacity in vitro.

Hypertrophic nonunion usually results from insufficient fracture stabilization. Therefore, most hypertrophic nonunions simply require the stabilization of the nonunion site. However, the reasons why union occurs without treating the nonunion site directly is not well understood biologically. In this study, we hypothesized that the intervening tissue at the hypertrophic nonunion site (nonunion tissue) could serve as a reservoir of mesenchymal progenitor cells and investigated whether the cells derived from nonunion tissue had the capacity for multilineage mesenchymal differentiation. After nonunion tissue was obtained, it was cut into strips and cultured. Homogenous fibroblastic adherent cells were obtained. Flow cytometry revealed that the adherent cells were consistently positive for mesenchymal stem cell related markers CD13, CD29, CD44, CD90, CD105, CD166, and negative for the hematopoietic markers CD14, CD34, CD45, and CD133, similar to control bone marrow stromal cells. In the presence of lineage‐specific induction factors, the adherent cells differentiated in vitro into osteogenic, chondrogenic, and adipogenic cells. These results demonstrated for the first time that hypertrophic nonunion tissue contains multilineage mesenchymal progenitor cells. This suggests that hypertrophic nonunion tissue plays an important role during the healing process of hypertrophic nonunion by serving as a reservoir of mesenchymal cells that are capable of transforming into cartilage and bone forming cells.

Ankle Arthrodesis Using Antegrade Intramedullary Nail for Salvage of Nonreconstructable Tibial Pilon Fractures

The treatment of nonreconstructable tibial pilon fractures for which the optimal timing for reduction and fixation has been missed is challenging. Ankle arthrodesis may be a treatment option in such cases. We treated 2 patients with nonreconstructable tibial pilon fractures using ankle arthrodesis with an antegrade intramedullary nail. Our method included exposing of the ankle joint through a lateral approach; excising the distal fibula, comminuted fragments, and remaining articular cartilage; inserting an intramedullary nail in an antegrade fashion from the proximal tibia into the talus; insertion of 2 interlocking screws in the talus and the proximal tibia; and autologous bone grafting using the excised distal fibula. At latest follow-up at 2 and 1 year respectively, fusion was complete, and both patients were pain free and could walk without support. When ankle arthrodesis is performed for a tibial pilon fracture, an intramedullary nail is thought to be superior to a plate, which is bulky and may impede soft tissue healing. Moreover, insertion of an intramedullary nail in an antegrade fashion can preserve the subtalar joint, and is therefore preferred over placement in a retrograde fashion. Ankle arthrodesis using this technique can be a useful salvage procedure for a nonreconstructable tibial pilon fracture.

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.

A Novel System for Transcutaneous Application of Carbon Dioxide Causing an “Artificial Bohr Effect” in the Human Body

Background Carbon dioxide (CO2) therapy refers to the transcutaneous administration of CO2 for therapeutic purposes. This effect has been explained by an increase in the pressure of O2 in tissues known as the Bohr effect. However, there have been no reports investigating the oxygen dissociation of haemoglobin (Hb) during transcutaneous application of CO2 in vivo. In this study, we investigate whether the Bohr effect is caused by transcutaneous application of CO2 in human living body. Methods We used a novel system for transcutaneous application of CO2 using pure CO2 gas, hydrogel, and a plastic adaptor. The validity of the CO2 hydrogel was confirmed in vitro using a measuring device for transcutaneous CO2 absorption using rat skin. Next, we measured the pH change in the human triceps surae muscle during transcutaneous application of CO2 using phosphorus-31 magnetic resonance spectroscopy (31P-MRS) in vivo. In addition, oxy- and deoxy-Hb concentrations were measured with near-infrared spectroscopy in the human arm with occulted blood flow to investigate O2 dissociation from Hb caused by transcutaneous application of CO2. Results The rat skin experiment showed that CO2 hydrogel enhanced CO2 gas permeation through the rat skin. The intracellular pH of the triceps surae muscle decreased significantly 10 min. after transcutaneous application of CO2. The NIRS data show the oxy-Hb concentration decreased significantly 4 min. after CO2 application, and deoxy-Hb concentration increased significantly 2 min. after CO2 application in the CO2-applied group compared to the control group. Oxy-Hb concentration significantly decreased while deoxy-Hb concentration significantly increased after transcutaneous CO2 application. Conclusions Our novel transcutaneous CO2 application facilitated an O2 dissociation from Hb in the human body, thus providing evidence of the Bohr effect in vivo.

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.

Breakage of a Third Generation Gamma Nail: A Case Report and Review of the Literature

The use of intramedullary nails to treat trochanteric fractures of the femur has increased with the increasing size of the elderly population. The third generation Gamma nail is currently one of the most popular devices for the treatment of trochanteric fractures. Nail breakage is a rare complication, possibly resulting from fatigue fracture of the implant. We present the first reported case of breakage of a third generation Gamma nail that was not used to treat a pathological fracture. An 83-year-old woman with an unstable trochanteric fracture of the femur was treated using a third generation Gamma nail. She was referred to our hospital 14 months postoperatively with nail breakage at the opening for the lag screw. The breakage was secondary to nonunion, which was thought to be mainly due to insufficient reduction of the fracture. The broken nail was removed, and the patient underwent cemented bipolar hemiarthroplasty. At followup 18 months later, she was mobile with a walker and asymptomatic with no complications. This case shows that inadequate operation such as insufficient reduction of the trochanteric fracture may result in nonunion and implant breakage, even when using a high-strength, well-designed implant.

Mandibular Hematoma Cells as a Potential Reservoir for Osteoprogenitor Cells in Fractures

PURPOSE We hypothesized that cells within the mandibular fracture hematoma played an important role in mandibular fracture healing. The objective of this study was to analyze cells in human mandibular fracture hematoma. PATIENTS AND METHODS We isolated and analyzed human mandibular fracture hematoma cells (MHCs) and investigated whether MHCs had multilineage mesenchymal differentiation capacity in vitro, similar to bone marrow stromal cells (BMSCs). RESULTS Cell-surface markers showed that the adherent MHCs expressed mesenchymal stem cell-related markers, namely CD29, CD44, CD105, and CD166, while lacking hematopoietic markers CD14, CD34, CD45, and CD133. The proliferative potential, osteogenic potential, and adipogenic potential of MHCs were comparable to those of BMSCs. In contrast, the chondrogenic potential of MHCs was inferior to that of BMSCs. CONCLUSIONS The role of the mandibular fracture hematoma could be as a presumptive local reservoir for osteogenic progenitors and thus contribute to intramembranous bone healing. Our findings may provide new insights into the mechanism of intramembranous bone healing in membranous bone fractures.

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.