Yutaka Mifune

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.

The effect of platelet-rich plasma on the regenerative therapy of muscle derived stem cells for articular cartilage repair

OBJECTIVE Platelet-rich plasma (PRP) is reported to promote collagen synthesis and cell proliferation as well as enhance cartilage repair. Our previous study revealed that the intracapsular injection of muscle derived stem cells (MDSCs) expressing bone morphogenetic protein 4 (BMP-4) combined with soluble Flt-1 (sFlt1) was effective for repairing articular cartilage (AC) after osteoarthritis (OA) induction. The current study was undertaken to investigate whether PRP could further enhance the therapeutic effect of MDSC therapy for the OA treatment. METHODS MDSCs expressing BMP-4 and sFlt1 were mixed with PRP and injected into the knees of immunodeficient rats with chemically induced OA. Histological assessments were performed 4 and 12 weeks after cell transplantation. Moreover, to elucidate the repair mechanisms, we performed in vitro assays to assess cell proliferation, adhesion, migration and mixed pellet co-culture of MDSCs and OA chondrocytes. RESULTS The addition of PRP to MDSCs expressing BMP-4 and sFlt1 significantly improved AC repair histologically at week 4 compared to MDSCs expressing BMP-4 and sFlt1 alone. Higher numbers of cells producing type II collagen and lower levels of chondrocyte apoptosis were observed by MDSCs expressing BMP-4 and sFlt1 and mixed with PRP. In the in vitro experiments, the addition of PRP promoted proliferation, adhesion and migration of the MDSCs. During chondrogenic pellet culture, PRP tended to increase the number of type II collagen producing cells and in contrast to the in vivo data, it increased cell apoptosis. CONCLUSIONS Our findings indicate that PRP can promote the therapeutic potential of MDSCs expressing BMP-4 and sFlt1 for AC repair (4 weeks post-treatment) by promoting collagen synthesis, suppressing chondrocyte apoptosis and finally by enhancing the integration of the transplanted cells in the repair process.

Enhancement of Tendon-Bone Osteointegration of Anterior Cruciate Ligament Graft Using Granulocyte Colony-Stimulating Factor

Background Whereas anterior cruciate ligament rupture usually requires reconstruction, the attachment between the tendon and the bone is the weakest region in the early posttransplantation period. In this process, the acquisition of appropriate vascularity is a key for early bone-tendon healing. Hypothesis Granulocyte colony-stimulating factor has an effect on the maturation of bone-tendon integration of anterior cruciate ligament reconstruction. Study Design Controlled laboratory study. Methods Twenty-eight healthy adult beagle dogs underwent bilateral anterior cruciate ligament reconstruction using the ipsilateral flexor digitorum superficialis tendon and were divided into 2 groups. A granulocyte colony-stimulating factor-incorporated gelatin surrounded the graft in the granulocyte colony-stimulating factor group, and the same gelatin without granulocyte colony-stimulating factor was used as the control group. Assessment was done at 2 and 4 weeks. Results Histological analysis at week 2 demonstrated that, in addition to more Sharpey fibers, microvessels were significantly enhanced in the granulocyte colony-stimulating factor groups grafts. Computed tomography at week 4 showed a significantly smaller tibial bone tunnel in the granulocyte colony-stimulating factor group. Real-time polymerase chain reaction revealed significantly elevated messenger ribonucleic acid expression levels of vascular endothelial growth factor and osteocalcin in the tibial bone tunnel and graft compared with controls. Furthermore, biomechanical testing of force during loading to ultimate failure at week 4 demonstrated a significant increase in strength in the granulocyte colony-stimulating factor group. Conclusion This study demonstrated that a local application of granulocyte colony-stimulating factor-incorporated gelatin significantly accelerates bone-tendon interface strength via enhanced angiogenesis and osteogenesis. Clinical Relevance Granulocyte colony-stimulating factor has therapeutic potential in promoting an environment conductive to angiogenesis and osteogenesis in bone tunnels.

Local transplantation of human multipotent adipose-derived stem cells accelerates fracture healing via enhanced osteogenesis and angiogenesis.

Adipose tissue is one of the promising sources of multipotent stem cells in human. Human multipotent adipose-derived stem (hMADS) cells have recently been isolated and showed differentiation potential into multiple mesenchymal lineages in vitro and in vivo. On the basis of these evidences, we examined the therapeutic efficacy of hMADS cells for fracture healing in an immunodeficient rat femur non-union fracture model. Local transplantation of hMADS cells radiographically and histologically promoted fracture healing with significant improvement of biomechanical function at the fracture sites compared with local transplantation of human fibroblasts (hFB) or PBS administration. Histological capillary density and physiological blood flow by laser Doppler perfusion imaging were significantly greater in hMADS group than hFB and PBS groups. Expressions of intrinsic (rat) bone morphogenetic protein-2 (BMP-2), vascular endothelial growth factor (VEGF) and angiopoietin-1 in peri-fracture tissue were upregulated in hMADS group than other groups. In addition, presence of BMP-2 or VEGF activated the proliferation and migration of hMADS cells in vitro. These results indicate that hMADS cells stimulate the interaction between the transplanted cells and the resident cells stronger than other cells, and they promote fracture healing more effectively. Furthermore, immunohistochemistry for human-specific antibodies revealed direct differentiation of hMADS cells into osteoblasts or endothelial cells in newly formed callus or vasculature, respectively. RT-PCR for human-specific primers for osteogenic/endothelial markers also disclosed osteogenic and vasculogenic plasticity of the transplanted hMADS cells at the early stage of fracture healing. The present results suggest that transplantation of hMADS cells may become a useful strategy for cell-based bone regeneration in the future clinical setting.

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.

Use of an antifibrotic agent improves the effect of platelet-rich plasma on muscle healing after injury

BACKGROUND Muscle contusions are a common type of muscle injury and are frequently encountered in athletes and military personnel. Although these injuries are capable of healing in most instances, incomplete functional recovery often occurs because of the development of fibrosis in the muscle. We hypothesized that a combination of platelet-rich plasma (PRP) injection and oral administration of losartan (an antifibrotic agent) could enhance muscle healing by stimulating muscle regeneration and angiogenesis and by preventing fibrosis in contusion-injured skeletal muscle. METHODS Contusion injuries were created in the tibialis anterior muscles of mice. Two treatments were tested, alone and in combination: 20 μL of PRP injected into the contusion site one day after injury, and 10 mg/kg/day of losartan administered beginning three days after injury and continuing until the end point of the experiment. Muscle regeneration and fibrosis development were evaluated by histological analysis, and functional recovery was measured by physiological testing. RESULTS Muscle regeneration and muscle function were significantly promoted in the combined PRP + losartan treatment group compared with the other groups. Combined PRP + losartan treatment significantly decreased the expression of phosphorylated Smad2/3 and the development of fibrosis compared with PRP treatment alone, and it increased vascular endothelial growth factor (VEGF) expression and the number of CD31-positive structures compared with losartan treatment alone. Follistatin, a positive regulator of muscle growth, was expressed at a higher level in the PRP + losartan group compared with the other groups. CONCLUSIONS PRP + losartan combinatorial therapy improved overall skeletal muscle healing after muscle contusion injury by enhancing angiogenesis and follistatin expression and by reducing the expression of phosphorylated Smad2/3 and the development of fibrosis. These results suggest that blocking the expression of transforming growth factor (TGF)-β1 with losartan improves the effect of PRP therapy on muscle healing after a contusion injury. CLINICAL RELEVANCE These findings could contribute to the development of biological treatments that aid in the healing of skeletal muscle after injury.

Isolation and characterization of human anterior cruciate ligament-derived vascular stem cells.

The anterior cruciate ligament (ACL) usually fails to heal after rupture mainly due to the inability of the cells within the ACL tissue to establish an adequate healing process, making graft reconstruction surgery a necessity. However, some reports have shown that there is a healing potential of ACL with primary suture repair. Although some reports showed the existence of mesenchymal stem cell-like cells in human ACL tissues, their origin still remains unclear. Recently, blood vessels have been reported to represent a rich supply of stem/progenitor cells with a characteristic expression of CD34 and CD146. In this study, we attempted to validate the hypothesis that CD34- and CD146-expressing vascular cells exist in hACL tissues, have a potential for multi-lineage differentiation, and are recruited to the rupture site to participate in the intrinsic healing of injured ACL. Immunohistochemistry and flow cytometry analysis of hACL tissues demonstrated that it contains significantly more CD34 and CD146-positive cells in the ACL ruptured site compared with the noninjured midsubstance. CD34+CD45- cells isolated from ACL ruptured site showed higher expansionary potentials than CD146+CD45- and CD34-CD146-CD45- cells, and displayed higher differentiation potentials into osteogenic, adipogenic, and angiogenic lineages than the other cell populations. Immunohistochemistry of fetal and adult hACL tissues demonstrated a higher number of CD34 and CD146-positive cells in the ACL septum region compared with the midsubstance. In conclusion, our findings suggest that the ACL septum region contains a population of vascular-derived stem cells that may contribute to ligament regeneration and repair at the site of rupture.

Administrations of Peripheral Blood CD34‐Positive Cells Contribute to Medial Collateral Ligament Healing via Vasculogenesis

Neoangiogenesis is a key process in the initial phase of ligament healing. Adult human circulating CD34+ cells, an endothelial/hematopoietic progenitor‐enriched cell population, have been reported to contribute to neoangiogenesis; however, the therapeutic potential of CD34+ cells for ligament healing is still unclear. Therefore, we performed a series of experiments to test our hypothesis that ligament healing is supported by CD34+ cells via vasculogenesis. Granulocyte colony‐stimulating factor‐mobilized peripheral blood (GM‐PB) CD34+ cells with atelocollagen (CD34+ group), GM‐PB mononuclear cells (MNCs) with atelocollagen (MNC group), or atelocollagen alone (control group) was locally transplanted after the creation of medial collateral ligament injury in immunodeficient rats. Reverse transcriptase‐polymerase chain reaction (RT‐PCR) and immunohistochemical staining at the injury site demonstrated that molecular and histological expression of human‐specific markers for endothelial cells was higher in the CD34+ group compared with the other groups at week 1. Endogenous effect, assessed by capillary density and mRNA expression of vascular endothelial growth factor, was significantly higher in CD34+ cell group than the other groups. In addition to the observation that, as assessed by real‐time RT‐PCR, gene expression of ligament‐specific marker was significantly higher in the CD34+ group than in the other groups, ligament healing assessed by macroscopic, histological, and biomechanical examination was significantly enhanced by CD34+ cell transplantation compared with the other groups. Our data strongly suggest that local transplantation of circulating human CD34+ cells may augment the ligament healing process by promoting a favorable environment through neovascularization.

Therapeutic potential of anterior cruciate ligament-derived stem cells for anterior cruciate ligament reconstruction.

We recently reported that the ruptured regions of the human anterior cruciate ligament (ACL) contained vascular-derived stem cells, which showed the potential for high expansion and multilineage differentiation. In this study, we performed experiments to test the hypothesis that ACL-derived CD34+ cells could contribute to tendon–bone healing. ACL-derived cells were isolated from the rupture site of human ACL by fluorescence-activated cell sorting. Following ACL reconstruction, immunodeficient rats received intracapsular administration of either ACL-derived CD34+ cells, nonsorted (NS) cells, CD34- cells, or phosphate-buffered saline (PBS). We also performed in vitro cell proliferation assays and enzyme-linked immunosorbent assays for vascular endothelial growth factor (VEGF) secretion. We confirmed the recruitment of the transplanted cells into the perigraft site after intracapuslar injection by immunohistochemical staining at week 1. Histological evaluation showed a greater area of collagen fiber formation and more collagen type II expression in the CD34+ group than the other groups at the week 2 time point. Immunostaining with isolectin B4 and rat osteocalcin demonstrated enhanced angiogenesis and osteogenesis in the CD34+ group at week 2. Moreover, double immunohistochemical staining for human-specific endothelial cell (EC) and osteoblast (OB) markers at week 2 demonstrated a greater ability of differentiation into ECs and OBs in the CD34+ group. Microcomputerized tomography showed the greatest healing of perigraft bone at week 4 in the CD34+ cell group, and the failure load of tensile test at week 8 demonstrated the greatest biomechanical strength in the CD34+ group. Furthermore, the in vitro studies indicated that the CD34+ group was superior to the other groups in their cell proliferation and VEGF secretion capacities. We demonstrated that ACL-derived CD34+ cells contributed to the tendon–bone healing after ACL reconstruction via the enhancement of angiogenesis and osteogenesis, which also contributed to an increase in biomechanical strength.