Masahiko Miwa

Circulating endothelial/skeletal progenitor cells for bone regeneration and healing

An emerging strategy in the regeneration and repair of bone is to use stem cells, including bone marrow mesenchymal stem cells, which are the most investigated and reliable source for tissue engineering, as well as circulating skeletal stem/progenitor cells, which are receiving abundant attention in regenerative medicine due to their ease of isolation and high osteogenic potential. Because failures in fracture healing are largely due to poor vascularization among many environmental factors, we highlight the first proof-of-principle experiments that elucidated the collaborative multi-lineage differentiation of circulating CD34 positive cells - a cell-enriched population of endothelial/hematopoietic progenitor cells - into not only endothelial cells but also osteoblasts. These cells develop a favorable environment for fracture healing via vasculogenesis/angiogenesis and osteogenesis, ultimately leading to functional recovery from fracture. This review will also highlight current concepts of circulating stem/progenitor cell-based therapy and their potential application for bone repair.

Fracture induced mobilization and incorporation of bone marrow-derived endothelial progenitor cells for bone healing†

We recently reported that systemic administration of peripheral blood (PB) CD34+ cells, an endothelial progenitor cell (EPC)‐enriched population, contributed to fracture healing via vasculogenesis/angiogenesis. However, pathophysiological role of EPCs in fracture healing process has not been fully clarified. Therefore, we investigated the hypothesis whether mobilization and incorporation of bone marrow (BM)‐derived EPCs may play a pivotal role in appropriate fracture healing. Serial examinations of Laser doppler perfusion imaging and histological capillary density revealed that neovascularization activity at the fracture site peaked at day 7 post‐fracture, the early phase of endochondral ossifification. Fluorescence‐activated cell sorting (FACS) analysis demonstrated that the frequency of BM cKit+Sca1+Lineage− (Lin−) cells and PB Sca1+Lin− cells, which are EPC‐enriched fractions, significantly increased post‐fracture. The Sca1+ EPC‐derived vasuculogenesis at the fracture site was confirmed by double immunohistochemistry for CD31 and Sca1. BM transplantation from transgenic donors expressing LacZ transcriptionally regulated by endothelial cell‐specific Tie‐2 promoter into wild type also provided direct evidence that EPCs contributing to enhanced neovascularization at the fracture site were specifically derived from BM. Animal model of systemic administration of PB Sca1+Lin− Green Fluorescent Protein (GFP)+ cells further confirmed incorporation of the mobilized EPCs into the fracture site for fracture healing. These findings indicate that fracture may induce mobilization of EPCs from BM to PB and recruitment of the mobilized EPCs into fracture sites, thereby augment neovascularization during the process of bone healing. EPCs may play an essential role in fracture healing by promoting a favorable environment through neovascularization in damaged skeletal tissue. J. Cell. Physiol. 215: 234–242, 2008.

Local Delivery of Granulocyte Colony Stimulating Factor-Mobilized CD34-Positive Progenitor Cells Using Bioscaffold for Modality of Unhealing Bone Fracture

We recently reported that i.v. transplantation of adult human circulating CD34+ cells, an endothelial/hematopoietic progenitor‐enriched cell population, contributes to fracture healing through the enhancement of vasculogenesis and osteogenesis. However, the scarcity of CD34+ cells in the adult human is a critical issue for the future clinical application of this method. To overcome this issue, we assessed in vitro and in vivo capacity of granulocyte colony‐stimulating factor‐mobilized peripheral blood (GM‐PB) human CD34+ cells for vasculogenesis and osteogenesis. First, we confirmed the differentiation capability of GM‐PB CD34+ cells into osteoblasts in vitro. Second, local transplantation of GM‐PB CD34+ cells on atelocollagen scaffold was performed in nude rats in a model of unhealing fractures. Immunostaining for human leukocyte antigen‐ABC of tissue samples 1 week after fracture and cell therapy showed the superior incorporation after local transplantation compared with systemic infusion. Third, the effects of local transplantation of 105 (Hi), 104 (Mid), or 103 (Lo) doses of GM‐PB CD34+ cells or phosphate‐buffered saline (PBS) on fracture healing were compared. Extrinsic vasculogenic and osteogenic differentiation of GM‐PB CD34+ cells, enhancement of the intrinsic angio‐osteogenesis by recipient cells, augmentation of blood flow recovery at the fracture sites, and radiological and histological confirmation of fracture healing were observed only in the Hi and Mid groups but not in the Lo and PBS groups. These results strongly suggest that local transplantation of GM‐PB CD34+ cells with atelocollagen scaffold is a feasible strategy for therapeutic vasculogenesis and osteogenesis needed for fracture healing.

An in vitro study demonstrating that haematomas found at the site of human fractures contain progenitor cells with multilineage capacity

We isolated multilineage mesenchymal progenitor cells from haematomas collected from fracture sites. After the haematoma was manually removed from the fracture site 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 CD29, CD44, CD105 and CD166, and were negative for the haemopoietic markers CD14, CD34, CD45 and CD133 similar to bone-marrow-derived mesenchymal stem cells. In the presence of lineage-specific induction factors the adherent cells could differentiate in vitro into osteogenic, chondrogenic and adipogenic cells. Our results indicate that haematomas found at a fracture site contain multilineage mesenchymal progenitor cells and play an important role in bone healing. Our findings imply that to enhance healing the haematoma should not be removed from the fracture site during osteosynthesis.

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.

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.

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.

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.