Periodontal tissue regeneration using the cytokine cocktail mimicking secretomes in the conditioned media from human mesenchymal stem cells.

Secretomes in the conditioned media from human mesenchymal stem cells (MSC-CM) were previously demonstrated to promote periodontal tissue regeneration. By mixing insulin-like growth factor-1, vascular endothelial growth factor-A, and transforming growth factor-ß1 which were included in MSC-CM, we made the cytokine cocktail (CC) mimicking MSC-CM, and then evaluated its efficacy on periodontal tissue regeneration. In vitro, CC promoted the migration of dog bone marrow-derived stem cells and periodontal ligament cells, and the tube formation of human umbilical vein endothelial cells. In vivo, class II furcation defects were surgically created at premolars in dogs. After 4 weeks of vinylpolysiloxane-induced inflammation, defects were filled with or without CC mixed in hydroxypropyl cellulose, or enamel matrix derivative (EMD). After 8 weeks, periodontal tissues were evaluated histologically and immunohistochemically. CC showed promotional effects on angiogenesis and formation of new bone and cementum. Osteogenesis by CC was greater than that by EMD and cementogenesis by CC was as well as that by EMD. CC may be promising for periodontal tissue regeneration.

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-ß1 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.

Secreted Frizzled-Related Protein Promotes Bone Regeneration by Human Bone Marrow-Derived Mesenchymal Stem Cells.

Secreted frizzled-related protein (sFRP)-3 is a negative regulator of Wnt signaling in human mesenchymal stem cells (hMSCs). The present study investigated the effects sFRP-3 on osteogenic differentiation by assessing osteogenic gene expression in hMSCs in vitro and by examining bone regeneration in a rat bone defect model. sFRP-3 treatment induced osteogenic differentiation in hMSCs as determined by alkaline phosphatase, collagen type I, osteocalcin, and Runt-related transcription factor 2 gene expression. hMSCs with or without sFRP-3 were implanted into a rat calvarial bone defect; a radiographic analysis by micro-computed tomography and histological analysis 4 and 8 weeks after implantation showed greater bone regeneration in the sFRP(+) than in the sFRP(-) group. These results suggest that modulation of Wnt signaling contributes to osteogenic differentiation in hMSCs. Specifically, sFRP-3 induces osteoblastic differentiation of cultured MSCs and bone regeneration in a calvarial bone defect, suggesting that it can be a useful agent for the treatment of bone defects.

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-ß1, 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.

Efficacy of semi-solidification of enteral nutrients for postoperative nutritional management with a nasogastric tube.

Postoperative nutritional management with a nasogastric tube is often used to prevent malnutrition after oral and maxillofacial surgery. However, enteral nutrients (EN) may cause various complications due to their liquid formulation. In this study, we retrospectively evaluated the efficacy of semi-solid EN with a xanthan gum thickener through a nasogastric tube and examined patients' complications, nutritional status, and quality of life. We established two groups: an L group (n=20) to which we administered liquid EN, and an SS group (n=20) to which we administered semi-solidified EN. The primary outcome was the occurrence of gastrointestinal complications. The secondary outcome was a change in nutritional status based on body weight and controlling nutritional status. The other outcome was the improvement in the patients' quality of life, assessed by the administration time. During nutritional management with a nasogastric tube, the median daily administration time in the L group was 9.0 hours, and 9 patients experienced diarrhea. In the SS group, the median daily feeding time was 2.3 hours, and only 2 patients experienced diarrhea. Both groups exhibited a decrease in body weight while controlling nutritional status scores were maintained. Semi-solidification of EN may be useful for postoperative nutritional management after oral and maxillofacial surgery by reducing complications, maintaining nutritional status, and shortening administration time.

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

OBJECTIVES: 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. MATERIALS AND METHODS: A cocktail of recombinant human insulin-like growth factor-1, vascular endothelial growth factor-A and transforming growth factor-ß1 was prepared at concentrations similar to those in conditioned medium. Conversely, these cytokines were depleted from conditioned medium, and the effects of the cocktail, the conditioned medium and the cytokine-depleted conditioned medium on bone regeneration were evaluated in vitro and in vivo. RESULTS: The cytokine cocktail and conditioned medium enhanced cell migration, tube formation, and expression of osteogenic and angiogenic genes. Depletion of cytokines significantly decreased the effects of conditioned medium in vitro. Similarly, the cytokine cocktail and conditioned medium, but not cytokine-depleted medium, increased bone regeneration in damaged rat calvarial bone. Immunohistochemistry indicated that the cytokine cocktail and conditioned medium strongly enhanced recruitment of endogenous stem cells and endothelial cells. CONCLUSIONS: The data indicate that the cytokine cocktail and conditioned medium enhance the migration of stem cells and endothelial cells to damaged bone, and elicit osteogenesis and angiogenesis.

Angiogenesis in newly regenerated bone by secretomes of human mesenchymal stem cells.

BACKGROUND: For an effective bone graft for reconstruction of the maxillofacial region, an adequate vascular network will be required to supply blood, osteoprogenitor cells, and growth factors. We previously reported that the secretomes of bone marrow-derived mesenchymal stem cells (MSC-CM) contain numerous growth factors such as insulin-like growth factor (IGF)-1, transforming growth factor (TGF)-ß1, and vascular endothelial growth factor (VEGF), which can affect the cellular characteristics and behavior of regenerating bone cells. We hypothesized that angiogenesis is an important step for bone regeneration, and VEGF is one of the crucial factors in MSC-CM that would enhance its osteogenic potential. In the present study, we focused on VEGF in MSC-CM and evaluated the angiogenic and osteogenic potentials of MSC-CM for bone regeneration. METHODS: Cytokines in MSC-CM were measured by enzyme-linked immunosorbent assay (ELISA). Human umbilical vein endothelial cells (HUVECs) were cultured with MSC-CM or MSC-CM with anti-VEGF antibody (MSC-CM + anti-VEGF) for neutralization, and tube formation was evaluated. For the evaluation of bone and blood vessel formation with micro-computed tomography (micro-CT) and for the histological and immunohistochemical analyses, a rat calvarial bone defect model was used. RESULTS: The concentrations of IGF-1, VEGF, and TGF-ß1 in MSC-CM were 1515.6 ± 211.8 pg/mL, 465.8 ± 108.8 pg/mL, and 339.8 ± 14.4 pg/mL, respectively. Tube formation of HUVECs, bone formation, and blood vessel formation were increased in the MSC-CM group but decreased in the MSC-CM + anti-VEGF group. Histological findings suggested that new bone formation in the entire defect was observed in the MSC-CM group although it was decreased in the MSC-CM + anti-VEGF group. Immunohistochemistry indicated that angiogenesis and migration of endogenous stem cells were much more abundant in the MSC-CM group than in the MSC-CM + anti-VEGF group. CONCLUSIONS: VEGF is considered a crucial factor in MSC-CM, and MSC-CM is proposed to be an adequate therapeutic agent for bone regeneration with angiogenesis.

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

BACKGROUND: Secreted 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. METHODS: The 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-tricalcium phosphate (ß-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. RESULTS: MSC-CM contained several cytokines such as insulin-like growth factor-1, vascular endothelial growth factor, transforming growth factor-ß1, 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. CONCLUSIONS: MSC-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 Dulbecco's modified Eagle's 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 Dulbecco's modified Eagle's 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.

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 Dulbecco's 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-ß1, 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.