Hideharu Hibi

Human dental pulp-derived stem cells promote locomotor recovery after complete transection of the rat spinal cord by multiple neuro-regenerative mechanisms

Spinal cord injury (SCI) often leads to persistent functional deficits due to loss of neurons and glia and to limited axonal regeneration after injury. Here we report that transplantation of human dental pulp stem cells into the completely transected adult rat spinal cord resulted in marked recovery of hind limb locomotor functions. Transplantation of human bone marrow stromal cells or skin-derived fibroblasts led to substantially less recovery of locomotor function. The human dental pulp stem cells exhibited three major neuroregenerative activities. First, they inhibited the SCI-induced apoptosis of neurons, astrocytes, and oligodendrocytes, which improved the preservation of neuronal filaments and myelin sheaths. Second, they promoted the regeneration of transected axons by directly inhibiting multiple axon growth inhibitors, including chondroitin sulfate proteoglycan and myelin-associated glycoprotein, via paracrine mechanisms. Last, they replaced lost cells by differentiating into mature oligodendrocytes under the extreme conditions of SCI. Our data demonstrate that tooth-derived stem cells may provide therapeutic benefits for treating SCI through both cell-autonomous and paracrine neuroregenerative activities.

Translational research for injectable tissue-engineered bone regeneration using mesenchymal stem cells and platelet-rich plasma: from basic research to clinical case study

Translational research involves application of basic scientific discoveries into clinically germane findings and, simultaneously, the generation of scientific questions based on clinical observations. At first, as basic research we investigated tissue-engineered bone regeneration using mesenchymal stem cells (MSCs) and platelet-rich plasma (PRP) in a dog mandible model. We also confirmed the correlation between osseointegration in dental implants and the injectable bone. Bone defects made with a trephine bar were implanted with graft materials as follows: PRP, dog MSCs (dMSCs) and PRP, autogenous particulate cancellous bone and marrow (PCBM), and control (defect only). Two months later, dental implants were installed. According to the histological and histomorphometric observations at 2 months after implants, the amount of bone–implant contact at the bone–implant interface was significantly different between the PRP, PCBM, dMSCs/PRP, native bone, and control groups. Significant differences were also found between the dMSCs/PRP, native bone, and control groups in bone density. These findings indicate that the use of a mixture of dMSCs/PRP will provide good results in implant treatment compared with that achieved by autogenous PCBM. We then applied this injectable tissue-engineered bone to onlay plasty in the posterior maxilla or mandible in three human patients. Injectable tissue-engineered bone was grafted and, simultaneously, 2–3 threaded titanium implants were inserted into the defect area. The results of this investigation indicated that injectable tissue-engineered bone used for the plasty area with simultaneous implant placement provided stable and predictable results in terms of implant success. We regenerated bone with minimal invasiveness and good plasticity, which could provide a clinical alternative to autogenous bone grafts. This might be a good case of translational research from basic research to clinical application.

Injectable Tissue-Engineered Bone Using Autogenous Bone Marrow–Derived Stromal Cells for Maxillary Sinus Augmentation: Clinical Application Report from a 2–6-Year Follow-Up

This clinical study used injectable tissue-engineered bone, along with bone marrow-derived stromal cells (BMDSCs) and platelet-rich plasma (PRP), to conduct maxillary sinus floor augmentation by the simultaneous placement of bone graft and dental implants and to examine the state of regenerated bone after functional loading in 16 sinus augmentations in 12 patients whose alveolar crestal bone height was 2-10 mm. We used PRP as an autologous scaffold-which provides signal molecules-with in vitro expanded BMDSCs to enhance osteogenesis. All 41 dental implants prepared with the materials were clinically stable after second-stage surgery. The height of mineralized tissue at 2 years showed the mean increases of 8.8 +/- 1.6 mm compared to preoperative values, and no adverse effects and remarkable bone absorption were seen in the 2-6-year follow-up time. Although these results are preliminary, injectable tissue-engineered bone would stably predict the success of bone formation and dental implants, reduce patient burden, and provide minimally invasive cell therapy for patients.

Injectable bone applied for ridge augmentation and dental implant placement: human progress study.

Purpose:The aim of this study was to clinically evaluate the success of implants placed in conjunction with a new material, tissue-engineered bone, and the stability of the regenerated bone after functional loading on a long-term basis. Methods:The tissue-engineered bone was applied to 14 cases, in which 6 patients were with partially or totally edentulous arches scheduled for sinus floor grafting and 8 patients underwent concurrent onlay plasty. Results:This study showed that the injectable bone formation induced bone in this anatomical site in 100% of the patients. The results also indicate that it might be possible to achieve the osseointegration of simultaneous implant placements with the grafts. Conclusions:It may be possible that injectable bone can shorten the period of implant treatment and reduce the patient’s burden and expect good long-term prognosis.

Mesenchymal stromal cells of human umbilical cord Wharton's jelly accelerate wound healing by paracrine mechanisms

BACKGROUND AIMS Mesenchymal stromal cells (MSC) can be isolated from the perivascular connective tissue of umbilical cords, called Whartons jelly. These human umbilical cord perivascular cells (HUCPVC) might provide therapeutic benefits when treating skeletal or cutaneous malformations in neonatal patients. METHODS HUCPVC were isolated, and their proliferation rate, marker expression and multilineage differentiation potential determined. HUCPVC or their conditioned medium (HUCPVC-CM) was injected into the excisional wound of a mouse splinted-wound model. The effects of the treatment on wound closure were examined by morphohistochemical and gene expression analyses. RESULTS HUCPVC expressed typical MSC markers and could differentiate into osteoblastic and adipogenic lineages. HUCPVC transplanted into the mouse wound accelerated wound closure. Immunohistologic analysis showed that the HUCPVC accelerated wound healing by enhancing collagen deposition and angiogenesis via paracrine mechanisms. Furthermore, treatment with HUCPVC-CM alone significantly enhanced wound closure. HUCPVC-CM increased the number of anti-inflammatory M2 macrophages expressing resistin-like molecule (RELM)-α/CD11b and promoted neovessel maturation. Quantitative polymerase chain reaction (PCR) analysis showed that HUCPVC-CM increased the expression of tissue-repairing cytokines interleukin (IL)-10, transforming growth factor (TGF)-β1, vascular endothelial growth factor (VEGF)-1 and angiopoietin-1 at the healing wound. CONCLUSIONS Our results show that HUCPVC promotes wound healing via multifaceted paracrine mechanisms. Together with their ability to differentiate into the osteogenic linage, HUCPVC may provide significant therapeutic benefits for treating wounds in neonatal patients.

Stem cell-conditioned medium accelerates distraction osteogenesis through multiple regenerative mechanisms

Distraction osteogenesis (DO) successfully induces large-scale skeletal tissue regeneration, but it involves an undesirably long treatment period. A high-speed DO mouse model (H-DO) with a distraction speed twice that of a control DO model failed to generate new bone callus in the distraction gap. Here we demonstrate that the local administration of serum-free conditioned medium from human mesenchymal stem cells (MSC-CM) accelerated callus formation in the mouse H-DO model. Secretomic analysis identified factors contained in MSC-CM that recruit murine bone marrow stromal cells (mBMSCs) and endothelial cells/endothelial progenitor cells (EC/EPCs), inhibit inflammation and apoptosis, and promote osteoblast differentiation, angiogenesis, and cell proliferation. Functional assays identified MCP-1/-3 and IL-3/-6 as essential factors in recruiting mBMSCs and EC/EPCs. IL-3/-6 also enhanced the osteogenic differentiation of mBMSCs. MSC-CM that had been depleted of MCP-1/-3 failed to recruit mBMSCs, and consequently failed to promote callus formation. Taken together, our data suggest that MSCs produce a broad repertoire of trophic factors with tissue-regenerative activities that accelerate healing in the DO process.

Mandibular lengthening by distraction osteogenesis using osseointegrated implants and an intraoral device: A preliminary report

PURPOSE This study investigated an approach to distraction osteogenesis of the mandible using osseointegrated implants and an intraoral device. MATERIALS AND METHODS Five adult dogs were used for this experiment. After the extraction of the left mandibular premolar and molar teeth, two osseointegrated implants were placed. Abutment connection, attachment of the intraoral distraction device, and an osteotomy in the region between the implants were performed 3 months after implantation. The distraction was done at rate of 1 mm/day for 10 consecutive days to elongate the mandible 10 mm. The animals were killed 2, 3, and 4 weeks after the distraction was completed, and radiographic and histologic examinations were done. RESULTS The longer the time after completion of distraction, the more uniform the new bone that was observed radiographically and histologically in the gap created by the distraction. The titanium implants remained stable during the course of mandibular lengthening. CONCLUSION An intraoral device using osseointegrated dental implants can serve as a mechanism for distraction osteogenesis in the maxillofacial skeleton.

Novel application of stem cell-derived factors for periodontal regeneration.

The effect of conditioned medium from cultured mesenchymal stem cells (MSC-CM) on periodontal regeneration was evaluated. In vitro, MSC-CM stimulated migration and proliferation of dog MSCs (dMSCs) and dog periodontal ligament cells (dPDLCs). Cytokines such as insulin-like growth factor, vascular endothelial growth factor, transforming growth factor-β1, and hepatocyte growth factor were detected in MSC-CM. In vivo, one-wall critical-size, intrabony periodontal defects were surgically created in the mandible of dogs. Dogs with these defects were divided into three groups that received MSC-CM, PBS, or no implants. Absorbable atelo-collagen sponges (TERUPLUG®) were used as a scaffold material. Based on radiographic and histological observation 4 weeks after transplantation, the defect sites in the MSC-CM group displayed significantly greater alveolar bone and cementum regeneration than the other groups. These findings suggest that MSC-CM enhanced periodontal regeneration due to multiple cytokines contained in MSC-CM.

Stromal cell-derived factor-1 enhances distraction osteogenesis-mediated skeletal tissue regeneration through the recruitment of endothelial precursors.

Distraction osteogenesis (DO) is a unique therapy that induces skeletal tissue regeneration without stem/progenitor cell transplantation. Although the self-regeneration property of DO provides many clinical benefits, the long treatment period required is a major drawback. A high-speed DO mouse model (H-DO), in which the distraction was done two times faster than in control DO (C-DO) mice, failed to generate new bone callus in the DO gap. We found that this was caused by the unsuccessful recruitment of bone marrow endothelial cells (BM-ECs)/endothelial progenitor cells (EPCs) into the gap. We then tested the ability of a local application of stromal cell-derived factor-1 (SDF-1), a major chemo-attractant for BM-ECs/EPCs, to accelerate the bone regeneration in H-DO. Our data showed that, in H-DO, SDF-1 induced callus formation in the gap through the recruitment of BM-ECs/EPCs, the maturation of neo-blood vessels, and increased blood flow. These results indicate that the active recruitment of endogenous BM-ECs/EPCs may provide a substantial clinical benefit for shortening the treatment period of DO.

Secretomes from bone marrow–derived mesenchymal stromal cells enhance periodontal tissue regeneration

BACKGROUND AIMS Periodontal tissue regeneration with the use of mesenchymal stromal cells (MSCs) has been regarded as a future cell-based therapy. However, low survival rates and the potential tumorigenicity of implanted MSCs could undermine the efficacy of cell-based therapy. The use of conditioned media from MSCs (MSC-CM) may be a feasible approach to overcome these limitations. The aim of this study was to confirm the effect of MSC-CM on periodontal regeneration. METHODS MSC-CM were collected during their cultivation. The concentrations of the growth factors in MSC-CM were measured with the use of enzyme-linked immunoassay. Rat MSCs (rMSCs) and human umbilical vein endothelial cells cultured in MSC-CM were assessed on wound-healing and angiogenesis. The expressions of osteogenetic- and angiogenic-related genes of rMSCs cultured in MSC-CM were quantified by means of real-time reverse transcriptase-polymerase chain reaction analysis. In vivo, periodontal defects were prepared in the rat models and the collagen sponges with MSC-CM were implanted. RESULTS MSC-CM includes insulin-like growth factor-1, vascular endothelial growth factor, transforming growth factor-β1 and hepatocyte growth factor. In vitro, wound-healing and angiogenesis increased significantly in MSC-CM. The levels of expression of osteogenetic- and angiogenic-related genes were significantly upregulated in rMSCs cultured with MSC-CM. In vivo, in the MSC-CM group, 2 weeks after implantation, immunohistochemical analysis showed several CD31-, CD105-or FLK-1-positive cells occurring frequently. At 4 weeks after implantation, regenerated periodontal tissue was observed in MSC-CM groups. CONCLUSIONS The use of MSC-CM may be an alternative therapy for periodontal tissue regeneration because several cytokines included in MSC-CM will contribute to many processes of complicated periodontal tissue regeneration.