Masahito Fujio

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.

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.

Conditioned media from hypoxic-cultured human dental pulp cells promotes bone healing during distraction osteogenesis

Distraction osteogenesis (DO) is a surgical procedure used to correct various skeletal disorders. Improving the technique by reducing the healing time would be of clinical relevance. The aim of this study was to determine the angiogenic and regenerative potential of conditioned media (CMs) collected from human dental pulp cells (hDPCs) grown under different culture conditions. CM collected from cells under hypoxia was used to improve bone healing and the DO procedure in vivo. The angiogenic potentials of CMs collected from hDPCs grown under normoxic (−Nor) and hypoxic (−Hyp) conditions were evaluated by quantitative PCR (VEGF‐A, angiopoietin‐1, angiopoietin‐2, interleukin‐6 (IL‐6) and CXCL12), ELISA assays (VEGF‐A, Ang‐2), tube‐formation and wound‐healing assays, using human umbilical vein endothelial cells. The results demonstrated that hypoxic CM had significantly higher angiogenic potential than normoxic CM. Human fetal osteoblasts (hFOBs) were exposed to CM, followed by alizarin red staining, to assess the osteogenic potential. It was found that CM did not enhance the mineralization capacity of hFOBs. DO was performed in the tibiae of 30 mice, followed by a local injection of 20 µl CM (CM–Nor and CM–Hyp groups) or serum‐free DMEM (control group) into the distraction zone every second day. The mice were sacrificed at days 13 and 27. The CM–Hyp treatment revealed a higher X‐ray density than the control group (p < 0.05). Our study suggests that the angiogenic effect promoted by hypoxic culture conditions is dependent on VEGF‐A and Ang‐2 released from hDPCs. Furthermore, CM–Hyp treatment may thus improve the DO procedure, accelerating bone healing.

Bone Marrow Stromal Cell Paracrine Factors Direct Osteo/Odontogenic Differentiation of Dental Pulp Cells

Growth factors play an important role in osteo/odontogenic differentiation of human dental pulp cells (hDPCs). The aim of this in vitro study was to compare the biological effects of recombinant human growth differentiation factor 5 (rhGDF-5) alone and a cocktail of soluble growth factors (conditioned medium) released from human bone marrow mesenchymal stem cells (hBMMSCs) on the morphology, proliferation and osteo/odontogenic differentiation potential of hDPCs. Passage 4 hDPCs were harvested for culture in four different media: (a) DMEM with 10% FBS, (b) odontogenic induction medium (OM), (c) OM plus 500 ng/mL rhGDF-5, and (d) OM plus conditioned medium (CM). Morphological changes at 48 and 120 h were determined by crystal violet staining. The proliferation rates at 3, 24, 48, and 120 h were assayed by MTT. Using real-time reverse transcription-polymerase chain reaction (RT-PCR), the mRNA levels of dentin sialophosphoprotein (DSPP), dentin matrix protein 1 (DMP1), collagen type I (Col 1), Runt-related transcription factor 2 (Cbfa1/Runx2), alkaline phosphatase (ALP), osteocalcin (OC), β3 tubulin (TUBB3), glial cell-derived neurotrophic factor (GDNF), angiopoietin-1 (Ang1), and vascular endothelial growth factor A (VEGFA), were determined at 2, 5, and 9 days. Protein expression of dental sialoprotein (DSP), DMP1, OC, and TUBB3 was recorded at 5 days, using western blot and immunocytochemistry. The effect of the different culture media on mineralization was determined by ALP staining at day 5 and Alizarin red S staining at days 7 and 14. In response to the different culture media, the shape of the hDPCs varied from spindled to polygonal and cuboidal. CM inhibited the cellular proliferation rate, while rhGDF-5 had no effect at early time points, but promoted cellular proliferation at 120 h of culture. In the CM group, the mRNA levels of Cbfa1/Runx2, Col 1, ALP, VEGFA, Ang1, and TUBB3 decreased and the levels of GDNF and OC increased. The mRNA levels of DSPP and DMP1 were inconsistent at the time points evaluated. The staining assays also demonstrated that compared with the other groups, the CM group exhibited lower expression of ALP and higher mineralization levels. Protein expression of DSP, DMP1, OC, and TUBB3 was pronounced by the CM-treated cells. It is concluded that under these in vitro conditions, CM released from hBMMSCs have a greater osteo/odontogenic inductive effect on hDPCs than rhGDF-5.

Proteomic analysis of bone proteins adsorbed onto the surface of titanium dioxide

Osseointegration is the structural and functional connection between bone tissues and implants such as titanium dioxide (TiO2). The bone-TiO2 interface is thought to contain proteoglycans. However, exhaustive analysis of the proteins in this layer has not been performed. In this study, we evaluated the bone protein adhered on the surface of TiO2 comprehensively. Pig bone protein was extracted by sequential elutions with guanidine, 0.1 M EDTA, and again with guanidine. The proteins obtained from these extractions were allowed to adhere to an HPLC column packed with TiO2 and were eluted with 0.2 M NaOH. The eluted proteins were identified by LC/MS/MS and included not only proteoglycans but also other proteins such as extracellular matrix proteins, enzymes, and growth factors. Calcium depositions were observed on TiO2 particles incubated with bone proteins, guanidine-extracted proteins adhered to TiO2 displayed significantly high amounts of calcium depositions.

An Experimental Study on Guided Bone Regeneration Using a Polylactide-co-glycolide Membrane-Immobilized Conditioned Medium.

PURPOSE To investigate whether bone regeneration can be accelerated by using a conditioned medium (CM) and guided bone regeneration (GBR) technique. MATERIALS AND METHODS CM was harvested from rat bone marrow stromal cells (BMSCs). The components of CM were immobilized using a polylactide-co-glycolide (PLGA) membrane treated with and without 0.5 mol/L sodium hydroxide (NaOH) to elevate the hydrophilicity. Four experimental groups were prepared: PLGA membrane treated with (1) phosphate-buffered saline (PBS; PBS-M), (2) PBS and 0.5 mol/L NaOH (hydrophilic treatment; PBS-HM), (3) CM (CM-M), and (4) CM and 0.5 mol/L NaOH (CM-HM). These experimental membranes were observed using scanning electron microscopy. Proteins derived from BMSCs immobilized on the PLGA membrane were detected with liquid chromatography-tandem mass spectrometry (LC/MS/MS). Cell proliferation and alkaline phosphatase (ALP) activity were measured to analyze the effect of CM on the BMSCs. Experimental membranes were transplanted into a rat calvarial bone defect model. Microcomputed tomography and histologic analyses were performed 4 and 8 weeks after transplantation. RESULTS The CM derived from BMSCs can be immobilized on the PLGA membrane. Hydrophilic treatment of the PLGA membrane enhanced the amount of CM immobilization. LC/MS/MS analysis showed that the immobilized proteins on the surface of PLGA membrane were extracellular matrix, such as collagen, decorin, and fibronectin. The proteins in the CM, which were released from the PLGA membrane, enhanced cell proliferation and ALP activity in rat BMSCs. Newly formed bone area at the bone defects that had been treated with CM-HM was significantly high compared with those at bone defects treated with the other membranes. CONCLUSION The PLGA membrane treated with 0.5 mol/L NaOH and CM promoted bone regeneration in this rat calvarial defect model.

Activated FGFR3 promotes bone formation via accelerating endochondral ossification in mouse model of distraction osteogenesis

Achondroplasia (ACH) is one of the most common short-limbed skeletal dysplasias caused by gain-of-function mutations in the fibroblast growth factor receptors 3 (FGFR3) gene. Distraction osteogenesis (DO) is a treatment option for short stature in ACH in some countries. Although the patients with ACH usually show faster healing in DO, details of the newly formed bone have not been examined. We have developed a mouse model of DO and analyzed new bone regenerates of the transgenic mice with ACH (Fgfr3ach mice) histologically and morphologically. We established two kinds of DO protocols, the short-DO consisted of 5days of latency period followed by 5days of distraction with a rate of 0.4mm per 24h, and the long-DO consisted of the same latency period followed by 7days of distraction with a rate of 0.3mm per 12h. The callus formation was evaluated radiologically by bone fill score and quantified by micro-CT scan in both protocols. The histomorphometric analysis was performed in the short-DO protocol by various stainings, including Villanueva Goldner, Safranin-O/Fast green, tartrate-resistant acid phosphatase, and type X collagen. Bone fill scores were significantly higher in Fgfr3ach mice than in wild-type mice in both protocols. The individual bone parameters, including bone volume and bone volume/tissue volume, were also significantly higher in Fgfr3ach mice than in wild-type mice in both protocols. The numbers of osteoblasts, as well as osteoclasts, around the trabecular bone were increased in Fgfr3ach mice. Cartilaginous tissues of the distraction region rapidly disappeared in Fgfr3ach mice compared to wild-type mice during the consolidation phase. Similarly, type X collagen-positive cells were markedly decreased in Fgfr3ach mice during the same period. Fgfr3ach mice exhibited accelerated bone regeneration after DO. Accelerated endochondral ossification could contribute to faster healing in Fgfr3ach mice.