Papers by Keyword: Mesenchymal Stem Cell (MSC)

Abstract: Since 2001, we have clinically utilized human bone marrow-derived mesenchymal stem cells (MSCs) for bone regeneration. The osteogenic ability of MSCs has been assessed by measurement of alkaline phosphatase activity and calcium deposition. As for the detection of in vitro calcium deposition, we have established the method using calcein, which is a calcium-binding fluorescence material. Using this fluorescence material, we could observe the calcium deposition and then estimate the value of calcium deposition. In this report, we cultured rat MSCs on culture plate as well as transparent β-TCP, and calcium deposition was visualized and quantitated using an image analyzer. After 2 weeks differentiation of rat MSCs to osteoblasts, calcium deposition on β-TCP was observed as a signal of calcium-binding fluorescence. This fluorescence signal was also quantitated with an image analyzer.

Abstract: Cellular activities of human mesenchymal stem cells (MSCs) and osteoblasts (HOBs) on a silicon-releasable scaffold, which is siloxane-doped poly(lactic acid) / vaterite composite coated with hydroxycarbonate apatite (SPV-H), were estimated using two types of media, with or without organic factors, dexamethasone (Dex) and β-glycerophosphate (β-GP). The culture tests using MSCs shows that the level of alkaline phosphatase (ALP) activity in the cells cultured on SPV-H increased for 21-day culturing in medium without Dex and β-GP. The proliferation of MSCs on SPV-H was significantly higher than that on a poly(lactic acid) / vaterite composite coated with hydroxyapatite (PV-H) at all time points. In the case of supplementing Dex and β-GP to the medium, the level of ALP activity in MSCs cultured on SPV-H was higher in comparison with that on PV-H at all time points. Scanning electron microscopy showed that there were some agglomerates in HOBs cultured on the SPV-H surface after 21-day culturing in the medium without the factors, while there are no agglomerates on PV-H. The agglomerates were regarded from laser Raman spectroscopy as bone nodules. This result implies stimulation to HOBs by silicon species in SPV-H. SPV-H is expected to be useful as the scaffold for bone tissue engineering.

Abstract: There is a clinical and socio-economic need to produce synthetic alternatives to autologous or allogenic bone grafts. Bioactive glasses and glass-ceramics offer great potential in this area. The aims of this study were to optimise production of apatite-wollastonite (A-W) glassceramic scaffolds produced by selective laser sintering, in terms of their physical and biological properties and to look at how human Mesenchymal Stem Cells (MSCs) responded to these 3-D scaffolds in vitro. An indirect selective laser sintering process successfully produced strong, porous scaffolds. Depending upon particle size(s) and infiltration of the porous structure, flexural strengths between 35 MPa and 100 MPa were obtained. Following static seeding of A-W scaffolds with MSCs, fluoresecent actin and nuclei staining, as observed by confocal microscopy, showed that these scaffolds supported the adherence of human MSC’s at time periods of up to 21 days. As such these seeded scaffolds show great potential for use in bone regenerative medicine.

Abstract: Recently, nanomaterials have received considerable attention because of their potential applications in the biomedical field. In the present study, we investigated the effects of nano-sized calcium metaphosphate (CMP) particles (50 nm) compared with micro-sized CMP particles (200-500 nm and 10 μm) on the proliferation and osteoblastic differentiation of human bone marrow stem cells (BMSCs). BMSCs were challenged with CMP particles with different sizes for 3, 5, and 7 days. An analysis of the proliferation revealed that the nano-sized CMP particles (50 nm) stimulated the proliferation of BMSCs up to 27.79% compared to the untreated control. This stimulatory effect of the nano-sized CMP particle was dose-dependent. CMP particles appeared to adhere on the surface of BMSCs but this did not cause distinguishable morphological changes. Moreover, all CMP particles (50 nm to 10 μm) were capable of stimulating an osteoblastic differentiation of BMSCs as accessed by alkaline phosphatase (ALP) and von Kossa stainings. Further molecular analysis revealed that all the CMP particles induced an expression of osteoblast-related genes such as osteocalcin (OC) and collagen I (Col I). Taken together, our data demonstrate that nano-sized CMP particles have the potential to stimulate the proliferation and osteoblastic differentiation of BMSCs.

Abstract: In the field of tissue engineering for bone regeneration, there have been many studies that examined in the bone forming ability of the porous biomaterials with mesenchymal stem cells (MSCs). To promote the tissue engineering approach in clinical situation, there is a need for the establishment and standardization of evaluation methods for measuring the in vivo bone forming ability. In this study, we examined the seeding process using rat MSCs to ascertain whether it is a valid protocol for various materials. Our results showed that the cell seeding process for the fabrication method of MSCs/materials composite influenced the number and distribution of the MSCs in the materials, therefore the process is a key to show new bone formation which derived from the seeded MSCs. Here, we describe the detailed process which can show consistent new bone formation in pores of the materials.

Abstract: Mesenchymal stem cells (MSCs) from bone marrow seem to be the one of best candidates to regenerate injured tissue. However, recent advances in application of MSCs toward large tissue regeneration are faced with lack of vascularity. In this study, endothelial cells differentiated from MSCs were applied for constructing tissue-engineered bone and cartilage. It was found that endothelial cells from MSCs play an important role of providing vasculature.

Abstract: Poly-L-lactic acid (PLLA) is a desirable and very attractive polymer for fabricating porous scaffolds. As of now, a solvent casting method with organic solvents has been used in scaffold fabrication process. However, residual organic solvents in the scaffolds have the problems of decreasing the effect of osteogenic induction due to the hindrance of bioceramic by polymer solution and it’s harmfulness in vivo. To avoid these disadvantages of scaffolds by organic solvent casting method, we developed a new method fabricating polymer (PLLA)/ceramic (β -TCP) composite scaffolds by baking method without using solvent, and then we tested properties of scaffolds on animals. As the result, non-toxicity has been proved through animal experiment and newly fabricated polymer/ceramic composites by a novel sintering method were induced rapid bone regeneration through enhancing the interaction of cells and a bone induction factor without any host immune response.

Abstract: Grafting of autologous iliac crest and decortication approach in posterior spinal fusion surgery has been the “gold standard”. However, the limited source of autograft has prompted extensive research into bone substitute and biological enhancement of the fusion mass. In this study, the application of stem cell therapy by tissue engineering method was investigated to enhance posterior spinal fusion with
-tricalcium phosphate ceramics in rabbit model. Rabbit bone marrow derived mesenchymal stem cells were aspirated from trochanter region of proximal femur. The mesenchymal stem cells were grown and directed to differentiate into osteogenic cells by osteogenic supplement (ascorbic acid,
-glycerophosphate and dexamethasone) in basal medium (10% FBS in DMEM). The osteogenic cells were seeded on tricalcium phosphate ceramics for one day (MSC group, n=6). The cell-ceramics composite was implanted onto autologous L5 and L6 transverse processes with decortication approach in posterior spinal fusion. The cell free ceramics acts as control (Control group, n=6) and iliac crest autograft as positive control (Autograft group). The spinal segments were harvested at week 7 post-operation. Manual palpation was performed with spinal segments to assess any movement of L5-L6 vertebral joint. The stiffness of the joint was considered as solid fusion. The specimens then were fixed by formalin and transferred to 70% ethanol. The BMC and volume of fusion transverse processes of L5 and L6 was measured by peripheral quantitative computed tomography. In manual palpation, 50% solid fusion was found in MSC group, 60% in autograft group but none in control group. Moreover, the BMC of L5 and L6 transverse processes in MSC group was greater than autograft and control group (45%, 40% respectively, p<0.01). The volume of transverse processes in MSC group was greater than autograft by 45% (p<0.01) and control group by 26% (p<0.05). In conclusion, the mesenchymal stem cells derived osteogenic cells augmented spinal fusion and bone mineralization.

Abstract: Bone grafts have been used to fill bone defects caused by disease or trauma. The amount of autografts is limited and allogenic bone grafts may transmit diseases and cause immune responses. Numerous materials have been proposed and used as scaffolds for bone tissue reconstruction. In this study, we tested nanophase PLGA/HA composite with mesenchymal stem cells in vitro to examine its biological response and cellular activity. The nanophase composite was compared to conventional polystyrene on cytocompatibility by cell attachment, proliferation, alkaline phosphotase activity test and scanning electron microscopy (SEM) analysis. The results demonstrated that human mesenchymal cells showed more cell attachment and higher cell proliferation rate when growing on nanophase PLGA/HA composite than those growing on polystyrene alone. And the composite also promoted MSC cells differentiate to osteoblast cells as compared with control. It was suggested that the combination of bone marrow mesenchymal cells with artificial materials or differentiation factors may enhance bone formation and regeneration, nanophase PLGA/HA composite might therefore be a promising scaffold material for bone tissue substitute in clinical application.

Abstract: The limited source of autograft has prompted extensive research on bone substitute and biological enhancement of the fusion mass in spinal fusion. Biomaterials impregnated with bone marrow aspirate has been applied to spinal fusion surgery. In this study, the effect of stem cell therapy in enhancing posterior spinal fusion was compared with the bone marrow aspirate method in a standard rabbit model. Bone marrow was aspirated from rabbit proximal femur (BMA group, n=6) and loaded on β-tricalcium phosphate ceramics (β-TCP) in excess. The composite was then implanted onto L5 and L6 transverse processes of the same animal in posterior spinal fusion operation with decortication on the same day. For stem cell therapy group, mesenchymal stem cells (MSCs) were isolated from bone marrow aspirate by adherence on plastic culture-ware. The MSCs were treated with osteogenic supplements (OS) during ex vivo cell expansion (MSC group, n=6). The osteogenic cells were seeded on β-TCP ceramics and cultured for one day. The cell-ceramics composite was implanted into the same rabbit as BMA group. The ceramics acted as control (n=6). Three fluorochromes, tetracycline, xyelonol orange and caclein were injected at week 2, 4 and 6 sequentially. The spinal segments were harvested at week 7 post-operation. The manual palpation of vertebral joint was assesses for solid fusion. The gap distance of inter-transverse process was measured by microCT and the bone mineral content (BMC) and volume of transverse processes by peripheral quantitative computed tomography. The specimens were undergone undecalcified histological analysis. The mineralization process was examined by fluorescent microscopy. By manual palpation, 50% of MSC group samples were found to have solid fusion in comparison with the incomplete fusion observed in the BMA and control group. The gap distance of inter-transverse processes in MSC group was the shortest. The volume of the transverse processes in MSC group was significantly greater than BMA and control group by 16% and 26% respectively. The BMC of transverse processes in MSC group was 40% greater than control (p<0.05) and 8% greater than BMA group. In fluorescent microscopy, both green fluorescent signal (labeled at week 6) and orange fluorescent signal (labeled at week 4) were observed in MSC group compare with the predominantly green fluorescent signal in the BMA group. In conclusion, the augmentation of MSC derived osteogenic cells is superior to bone marrow aspirate in rabbit posterior spinal fusion.