Takahiro Niikura

Local transplantation of G-CSF-mobilized CD34(+) cells in a patient with tibial nonunion: a case report.

Although implantation of crude bone marrow cells has been applied in a small number of patients for fracture healing, transplantation of peripheral blood CD34+ cells, the hematopoietic/endothelial progenitor cell-enriched population, in patients with fracture has never been reported. Here, we report the first case of tibial nonunion receiving autologous, granulocyte colony stimulating factor mobilized CD34+ cells accompanied with autologous bone grafting. No serious adverse event occurred, and the novel therapy performed 9 months after the primary operation resulted in bone union 3 months later without any symptoms including pain and gait disturbance.

Local Transplantation of Granulocyte Colony Stimulating Factor-Mobilized CD34+ Cells for Patients With Femoral and Tibial Nonunion: Pilot Clinical Trial

Most bone fractures typically heal, although a significant proportion (5%–10%) of fractures fail to heal, resulting in delayed union or persistent nonunion. Some preclinical evidence shows the therapeutic potential of peripheral blood CD34+ cells, a hematopoietic/endothelial progenitor cell‐enriched population, for bone fracture healing; however, clinical outcome following transplantation of CD34+ cells in patients with fracture has never been reported. We report a phase I/IIa clinical trial regarding transplantation of autologous, granulocyte colony stimulating factor‐mobilized CD34+ cells with atelocollagen scaffold for patients with femoral or tibial fracture nonunion (n = 7). The primary endpoint of this study is radiological fracture healing (union) by evaluating anteroposterior and lateral views at week 12 following cell therapy. For the safety evaluation, incidence, severity, and outcome of all adverse events were recorded. Radiological fracture healing at week 12 was achieved in five of seven cases (71.4%), which was greater than the threshold (18.1%) predefined by the historical outcome of the standard of care. The interval between cell transplantation and union, the secondary endpoint, was 12.6 ± 5.4 weeks (range, 8–24 weeks) for clinical healing and 16.1 ± 10.2 weeks (range, 8–36 weeks) for radiological healing. Neither deaths nor life‐threatening adverse events were observed during the 1‐year follow‐up after the cell therapy. These results suggest feasibility, safety, and potential effectiveness of CD34+ cell therapy in patients with nonunion.

The effect of transcutaneous application of carbon dioxide (CO2) on skeletal muscle

In Europe, carbon dioxide therapy has been used for cardiac disease and skin problems for a long time. However there have been few reports investigating the effects of carbon dioxide therapy on skeletal muscle. Peroxisome proliferators-activated receptor (PPAR)-gamma coactivator-1 (PGC-1α) is up-regulated as a result of exercise and mediates known responses to exercise, such as mitochondrial biogenesis and muscle fiber-type switching, and neovascularization via up-regulation of vascular endothelial growth factor (VEGF). It is also known that silent mating type information regulation 2 homologs 1 (SIRT1) enhances PGC-1α-mediated muscle fiber-type switching. Previously, we demonstrated transcutaneous application of CO(2) increased blood flow and a partial increase of O(2) pressure in the local tissue known as the Bohr effect. In this study, we transcutaneously applied CO(2) to the lower limbs of rats, and investigated the effect on the fast muscle, tibialis anterior (TA) muscle. The transcutaneous CO(2) application caused: (1) the gene expression of PGC-1α, silent mating type information regulation 2 homologs 1 (SIRT1) and VEGF, and increased the number of mitochondria, as proven by real-time PCR and immunohistochemistry, (2) muscle fiber switching in the TA muscle, as proven by isolation of myosin heavy chain and ATPase staining. Our results suggest the transcutaneous application of CO(2) may have therapeutic potential for muscular strength recovery resulting from disuse atrophy in post-operative patients and the elderly population.

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.

Tactile Surgical Navigation System for Complex Acetabular Fracture Surgery

The authors describe a tactile surgical navigation system using custom 3-dimensional (3D) models of the bony pelvis for complex acetabular fracture surgery. The bone area of interest was extracted from the Digital Imaging and Communications in Medicine (DICOM) data of computed tomography scans. A standard triangulated language file was used to create 3D models of the bony pelvis by layered manufacturing using a 3D printer and non-cytotoxic, sterilizable, acrylic-based photopolymers. No infections and no toxic or other adverse events were observed. The models were useful for preoperative assessment, planning, and simulation; intraoperative assessment; obtaining informed consent; and education.

Causative factors of fracture nonunion: the proportions of mechanical, biological, patient-dependent, and patient-independent factors

BackgroundUnderstanding the causative factors of fracture nonunion leads to both prevention and improvements in treatment. The purpose of this study was to understand the clinical characteristics and causative factors of nonunion in a case series.MethodsOne hundred two consecutive patients with fracture nonunions of the extremities who were surgically treated in our hospital over the last decade were analyzed. Data were collected by reviewing medical charts. Radiographs were reviewed to classify the nonunion by radiographic appearance. Causative factors of nonunions were identified for each patient. Factors relating to inadequate mechanical stability or reduction and those relating to a decline in biological activity were investigated. Mechanical factors included inappropriate dynamization, inappropriate reduction, inappropriate surgical management, insufficient fixation, and conservative treatment. Surgical technical errors were identified through careful review by three experienced trauma surgeons. Biological activity factors included comminution and bone loss, open fracture, excessive surgical exposure, infection, previous radiation therapy, alcohol abuse, diabetes mellitus, smoking, genetic disorders, and metabolic disease or endocrine pathology. We also classified the causative factors as patient-dependent or patient-independent factors.ResultsOf the 102 nonunions, 47 were oligotrophic, 22 were hypertrophic, 17 were atrophic, 12 were defect types, and 4 were comminuted. Twenty-four cases had factors of inadequate mechanical stability or reduction, 23 cases had biological factors, and 55 cases had both types of factors. Four cases had patient-dependent factors, 40 cases had patient-independent factors, and 58 cases had both types of factors.ConclusionsOur results demonstrated that there were a considerable number of nonunions with causative factors which can be improved, such as inadequate fracture management.

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.

Osteogenic activity of human fracture haematoma-derived progenitor cells is stimulated by low-intensity pulsed ultrasound in vitro

The haematoma occurring at the site of a fracture is known to play an important role in bone healing. We have recently shown the presence of progenitor cells in human fracture haematoma and demonstrated that they have the capacity for multilineage mesenchymal differentiation. There have been many studies which have shown that low-intensity pulsed ultrasound (LIPUS) stimulates the differentiation of a variety of cells, but none has investigated the effects of LIPUS on cells derived from human fracture tissue including human fracture haematoma-derived progenitor cells (HCs). In this in vitro study, we investigated the effects of LIPUS on the osteogenic activity of HCs. Alkaline phosphatase activity, osteocalcin secretion, the expression of osteoblast-related genes and the mineralisation of HCs were shown to be significantly higher when LIPUS had been applied but without a change in the proliferation of the HCs. These findings provide evidence in favour of the use of LIPUS in the treatment of fractures.

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.