Wataru Katagiri

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.

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.

Novel cell-free regeneration of bone using stem cell-derived growth factors.

PURPOSE Tissue engineering and regenerative medicine may now be used for the treatment of maxillofacial defects, but the related procedures have several limitations, including high capital investment, expensive cell culture techniques, and complicated safety and quality management issues. Stem cells secrete many cytokines that can affect cell mobilization and differentiation that accumulate in conditioned media. This study investigated the effects of stem cell-conditioned media from human bone marrow-derived mesenchymal stem cells (MSCs) on bone regeneration and its ability to induce endogeneous stem cell mobilization and bone regeneration. MATERIALS AND METHODS Human MSCs that were 70% to 80% confluent were refed with serum-free Dulbeccos modified Eagle medium, and the cell-cultured conditioned media were collected after 48 hours of incubation. The collected media were defined as cultured conditioned media from MSCs (MSC-CM). Rat bone-marrow-derived stem cells (rMSCs) were cultured with MSC-CM for 48 hours; then, cell mobilization and the expression of osteogenic-related genes were investigated. The presence of cytokines in MSC-CM was determined by enzyme-linked immunosorbent assay. Collagen sponge was then soaked in MSC-CM and grafted into rat calvarial bone defects. At 2 or 4 weeks after implantation of graft materials, implanted sections were evaluated by microcomputed tomography and histologic analysis. RESULTS MSC-CM enhanced the migration, proliferation, and expression of osteogenic marker genes, such as alkaline phosphatase, osteocalcin, and Runx2, of rMSCs in vitro. Cytokines such as insulinlike growth factor-1, vascular endothelial growth factor, transforming growth factor-β1, and hepatocyte growth factor were present in the MSC-CM. MSC-CM significantly increased the migration and expression of osteogenic-related genes of rMSCs. Early bone regeneration in rat calvaria was also observed. CONCLUSION These results suggest that MSC-CM may have potential for novel cell-free regeneration of bone.

First-in-human study and clinical case reports of the alveolar bone regeneration with the secretome from human mesenchymal stem cells

BackgroundSecreted growth factors and cytokines in the conditioned medium from bone marrow-derived mesenchymal stem cells (MSC-CM) have several effects on cell behavior. Our previous studies revealed that MSC-CM enhances bone regeneration by increasing cell mobilization, angiogenesis, and osteogenesis in vitro and in vivo. This clinical study was undertaken to evaluate the safety and use of MSC-CM for alveolar bone regeneration in eight patients who were diagnosed as needing bone augmentation prior to dental implant placement.MethodsThe protocol of this clinical study was approved by the ethics committee of Nagoya University Hospital. MSC-CM was prepared from conditioned medium from commercially available human bone marrow-derived MSCs. Patients were treated with beta-tricalcuim phosphate (β-TCP) or an atelocollagen sponge soaked with MSC-CM. Clinical and radiographic assessments were performed during the follow-up period. Histological assessments were also performed in some cases. Clinical and histological data from patients who underwent the SFE procedure without MSC-CM were also used retrospectively as reference controls.ResultsMSC-CM contained several cytokines such as insulin-like growth factor-1, vascular endothelial growth factor, transforming growth factor-β1, and hepatocyte growth factor in relatively low amounts. No systemic or local complications were reported throughout the study. Radiographic evaluation revealed early bone formation in all cases. Histological evaluation also supported the radiographic findings. Furthermore, infiltration of inflammatory cells was scarce throughout the specimens.ConclusionsMSC-CM was used safely and with less inflammatory signs and appears to have great osteogenic potential for regenerative medicine of bone. This is the first in-human clinical study of alveolar bone regeneration using MSC-CM.

Evaluation of the therapeutic effects of conditioned media from mesenchymal stem cells in a rat bisphosphonate-related osteonecrosis of the jaw-like model

Bisphosphonate-related osteonecrosis of the jaw (BRONJ) is defined as an exposed necrotic bone in the oral cavity that does not heal after appropriate intervention for >8weeks with present or previous bisphosphonate treatment in the absence of radiotherapy. Until now, although several risk factors, including invasive dental procedures, infection, mechanical trauma to the jawbone, and concomitant use of immunosuppressive and chemotherapy drugs have been implicated in the etiology of BRONJ, its underlying mechanisms and treatments remain largely unknown. A study recently showed that intravenous administration of mesenchymal stem cells (MSCs) improved BRONJ, and it was hypothesized that paracrine effects by secretomes from MSCs are the main constituent. Here we used rat BRONJ models to examine the therapeutic effects with serum-free conditioned media from human MSCs (MSC-CM), including various secretomes. We showed that MSC-CM has protected rat MSCs and rat osteoclasts. MSC-CM enhanced the expression of osteogenic-related genes and neovascularization-related genes by real-time reverse-transcriptase polymerase chain reaction analysis in in vitro study. In in vivo study, 5-week-old Wistar/ST male rats received zoledronate (35μg/kg/week) and dexamethasone (1mg/kg/day) subcutaneously for 2weeks. Unilateral maxillary molars were then extracted. Two weeks later, rats were divided into non-treatment, serum-free Dulbeccos modified Eagles medium, and MSC-CM groups. In the MSC-CM group, the open alveolar sockets in 63% of the rats with BRONJ healed with complete soft tissue coverage and socket bones, whereas the exposed necrotic bone with inflamed soft tissue remained in the other groups. Histological analysis showed new bone formation and the appearance of osteoclasts in the MSC-CM group. Osteoclasts were significantly reduced in the non-treatment group. Thus, we concluded that the antiapoptotic and antiinflammatory effects of MSC-CM dramatically regulated the turnover of local bone and indicated therapeutic effects on BRONJ.

Peripheral Nerve Regeneration by Secretomes of Stem Cells from Human Exfoliated Deciduous Teeth

Peripheral nerve regeneration across nerve gaps is often suboptimal, with poor functional recovery. Stem cell transplantation-based regenerative therapy is a promising approach for axon regeneration and functional recovery of peripheral nerve injury; however, the mechanisms remain controversial and unclear. Recent studies suggest that transplanted stem cells promote tissue regeneration through a paracrine mechanism. We investigated the effects of conditioned media derived from stem cells from human exfoliated deciduous teeth (SHED-CM) on peripheral nerve regeneration. In vitro, SHED-CM-treated Schwann cells exhibited significantly increased proliferation, migration, and the expression of neuron-, extracellular matrix (ECM)-, and angiogenesis-related genes. SHED-CM stimulated neuritogenesis of dorsal root ganglia and increased cell viability. Similarly, SHED-CM enhanced tube formation in an angiogenesis assay. In vivo, a 10-mm rat sciatic nerve gap model was bridged by silicon conduits containing SHED-CM or serum-free Dulbeccos modified Eagles medium. Light and electron microscopy confirmed that the number of myelinated axons and axon-to-fiber ratio (G-ratio) were significantly higher in the SHED-CM group at 12 weeks after nerve transection surgery. The sciatic functional index (SFI) and gastrocnemius (target muscle) wet weight ratio demonstrated functional recovery. Increased compound muscle action potentials and increased SFI in the SHED-CM group suggested sciatic nerve reinnervation of the target muscle and improved functional recovery. We also observed reduced muscle atrophy in the SHED-CM group. Thus, SHEDs may secrete various trophic factors that enhance peripheral nerve regeneration through multiple mechanisms. SHED-CM may therefore provide a novel therapy that creates a more desirable extracellular microenvironment for peripheral nerve regeneration.

Conditioned Medium From Mesenchymal Stem Cells Enhances Early Bone Regeneration After Maxillary Sinus Floor Elevation in Rabbits.

Purpose:Conditioned medium from stem cells contains growth factors and has a promising prospect for use in regenerative medicine. In this study, the effects of marrow-derived stem cell–conditioned medium (MSC-CM) on bone regeneration after maxillary sinus floor elevation were examined in rabbits. Materials and Methods:Rabbit bone marrow–derived stem cells (rMSCs) were cultured with MSC-CM for 48 hours, and their mobilization and proliferation were evaluated. Beta–tricalcium phosphate scaffolds were impregnated with MSC-CM and grafted in the rabbit maxillary sinus cavities. At 2, 4, and 8 weeks after transplantation, sections of the newly formed bone were evaluated histologically and by immunohistochemical staining. Results:MSC-CM increased the migration and proliferation of rMSCs and the early bone regeneration in rabbit sinus. Cell proliferation and vascularization were increased at 2 weeks after implantation of grafts impregnated with MSC-CM compared to controls, indicating that MSC-CM is effective at the early phase of bone regeneration. Conclusion:MSC-CM is a promising novel therapeutic agent to promote bone regeneration after maxillary sinus floor elevation.

A defined mix of cytokines mimics conditioned medium from cultures of bone marrow‐derived mesenchymal stem cells and elicits bone regeneration

We previously reported that conditioned medium from cultures of bone marrow‐derived mesenchymal stem cells have strong potential to accelerate bone regeneration. We now examine in vitro and in vivo a defined cytokine cocktail that mimics the effects of conditioned medium on bone regeneration.

Secretomes from mesenchymal stem cells participate in the regulation of osteoclastogenesis in vitro.

ObjectivesThe receptor activator of nuclear factor kappa-B ligand (RANKL) inhibitors are novel clinically effective agents that inhibit osteoclast differentiation, function, and survival by binding to RANKL. Medication-related osteonecrosis of the jaw (MRONJ), caused as a result of treatment using denosumab, is a newly emerging type of bone necrosis, the exact pathogenesis of which is unknown. Several studies recently showed that the intravenous administration of mesenchymal stem cells (MSCs) improved the osteonecrosis of the jaw, and it was hypothesized that paracrine effects by secretomes from MSCs are the main constituent. Our aim was to investigate the effects of serum-free conditioned media from human MSCs (MSC-CM) and RANKL inhibitors on osteoclast differentiation.Materials and methodsCytokines included in MSC-CM were identified using the cytokine array analysis. MSC-CM was added to the culture medium of rat osteoclast precursors containing RANKL inhibitor. Osteoclast differentiation assays, immunohistochemistry, real-time reverse-transcriptase polymerase chain reaction (RT-PCR) analysis, and pit formation assays were performed.ResultsMSC-CM included various cytokines such as the recruitment of cell osteogenesis angiogenesis and cell proliferation. MSC-CM promoted osteoclast differentiation and expression of master regulatory transcriptional factors for osteoclastogenesis. In addition, MSC-CM showed function maintenance in osteoclasts despite the presence of RANKL inhibitors.ConclusionsOur findings suggest that secretomes in MSC-CM were related to the regulation of osteoclast differentiation, which may reduce the effect of RANKL inhibitors.Clinical relevanceNew combinations of drugs using factors included in MSC-CM have effective therapeutic modality for treating patients with MRONJ.

Clinical Study of Bone Regeneration by Conditioned Medium From Mesenchymal Stem Cells After Maxillary Sinus Floor Elevation

Objective: This clinical study was undertaken to evaluate the safety of use of the secretome of bone marrow–derived mesenchymal stem cells (MSC-CM) for maxillary sinus floor elevation (SFE). Materials and Methods: MSC-CM was prepared from conditioned medium from human bone marrow–derived MSCs. Six partially edentulous patients were enrolled in the study. MSC-CM was mixed with porous beta-tricalcium phosphate (&bgr;-TCP) and implanted in 4 patients (experimental group), whereas only &bgr;-TCP was implanted in the other 2 patients (control group). Six months after SFE, bone biopsies and histological assessments were performed. Results: Bone formation was clinically confirmed in all cases. Although Hounsfield units in computed tomography images were not significantly different between the groups, histological analysis revealed a significant difference in newly formed bone area between the groups. In particular, bone volume in the center of the augmented area was significantly greater in the MSC-CM group. Newly formed bone consisted of lamellar bone in the MSC-CM group but woven bone in the &bgr;-TCP group. Conclusion: The secretome of bone marrow–derived mesenchymal stem cells (MSC-CM) was used safely and has great osteogenic potential for regenerative medicine of bone.