Key Engineering Materials Vols. 330-332

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.

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: Since 2001, we have started tissue engineered approach for hard tissue repair using mesenchymal stromal cells (MSCs) derived from patient’s bone marrow. MSCs were culture expanded on culture dish, then applied on various ceramics including hydroxyapatite (HA) ceramics. The MSCs on the ceramics were further cultured in osteogenic media to induce osteognenic differentiation. The differentiation resulted in appearance of bone forming osteoblasts as well as bone matrix on the ceramics, thus we could fabricate the tissue engineered bone. We have reported that the tissue engineered bone is effective for treatment of large bone defect, which is difficult to repair only with artificial materials such as HA ceramics. The present study focused on osteogenic capability of cryopreserved human MSCs derived from patients who already were treated by the tissue engineered bone. The MSCs showed high alkaline phosphatase activity together with abundant bone matrix formation when cultured in osteogenic media. The MSCs also showed in vivo new bone formation when implanted at subcutaneous sites of athymic nude rats. Based on the results, we concluded that the tissue engineering approach is a reliable method to be used in hard tissue regeneration.

Abstract: Calcium phosphate (CaP) hybridized to a whole tendon graft delayed cell repopulation in anterior cruciate ligament (ACL) reconstruction in rabbits. However, a tendon graft masked with an adhesive tape at the intra-articular (IA) portion to prevent CaP hybridization did not delay cell repopulation. Synovial tissues can adhere to the tendon graft and can invade the tendon graft masked at the IA portion. The masking induced an effect similar to that of the unhybridized tendon graft. The CaP hybridized tendon grafts masked at the IA portion showed cell repopulation 2 weeks earlier than the unmasked CaP hybridized tendon grafts.

Abstract: The purpose of this study was to explore the feasibility of repairing massive bone defect with in vivo tissue engineering(TE) bone, and to provide experimental evidence for the application of in vivo TE bone into clinic in the future. Six calcium phosphate ceramics (Ca-P ceramics) columns were prepared, and then immersed in dynamic revised simulated body fluid (RSBF). 72 hours later, the bone-like apatite was formed on the surface and pore walls of ceramics. Three dogs were used in this study. Two ceramic columns were implanted bilaterally in the femoral muscles of each dog to construct living bone graft of in vivo TE bone. 6 weeks after implantation, they were transplanted to the box-like bone defects sites created in bilateral mandible of the same animals. The dogs were sacrificed at 8, 12 week after operation respectively. Samples were harvested for gross observation, X-ray examination, tetracycline fluorescence labeling, SPECT and histological observation. These results demonstrated that as a living bone graft, in vivo TE bone participated in the bone metabolism of host, and integrated with the host bone. It is feasible to reconstruct box-like bone defect of mandible with the in vivo TE bone.

Abstract: To investigate the interaction of the adherent cell and shear flow, a compound drop model was developed to simulate a living adherent cell adhered to a smooth substrate, and a two dimensional computational fluid dynamics (CFD) was conducted to solve the model equations. The results showed that the deformability of the cell increases with Reynolds number and initial contact angle. The nucleus deforms with the cell, and the deformation index of the cell is greater than that of the nucleus. The cell is more deformable while the nucleus is more capable of resisting external shear flow. The cell and the nucleus are not able to deform infinitely with the increase of Reynolds number and the deformation index reaches a maximum. We conclude that the nucleus plays a particular role in the mechanical properties of the adherent cell.

Abstract: Poly(lactide-co-glycolide) (PLGA) and alginate(AG) are the most promising scaffolds in the bone tissue engineering for their stable mechanical characters and three-dimensional porous structure. This study aimed to assay the in vivo osteogenesis potentials by loading the autogenous bone marrow stromal cells (BMSCs) on PLGA or AG. The results suggested that PLGA and AG are both ideal bone tissue engineering scaffold. BMSCs/AG has stronger osteogenesis potentials in vivo than BMSCs/PLGA.

Abstract: Porous & rubbery organic-inorganic hybrids were synthesized from tetraethoxysilane (TEOS) and polydimethylsiloxane (PDMS) through a sol-gel route using sieved sucrose granules as a porogen. The porous hybrids with a high content of PDMS behaved like polymer sponge. The porosity was over 90% irrespective of the hybrid composition and the pore diameter ranged from 100 to 500 μm. In the three-dimensional cell culture, mammalian cells were well cultured in the porous hybrids. The present results indicate that the hybrids may be a promising scaffold for developing such functional culture methods.

Abstract: It was well recognized that mechanical strain plays a crucial role in periodontal tissues remodeling. The aim of this study was to investigate the effect of mechanical strain on osteoblastic precursor cells in a collagen type I gel scaffold. Rat MSCs were isolated and cultured according to the established method. Cells were induced with osteogenic medium, then seeded in a collagen type I gel and mechanically stretched by application of cyclic biaxial strain 24h later. Strain cycle was set to 1 cycle/min (0.017Hz), and strain magnitude was set to 2%, 5%, 7% elongation. Cells were collected in 0h, 3h, 6h, 9h, 12h, 24h and 48h respectively. ODF and ICAM-1 mRNA were analyzed by RT-PCR assay. The results shown that 2-7% elongation strain, either dynamic or static, inhibited ICAM-1and ODF expression of osteoblastic precursors, and the effects were relative tightly to strain magnitude. The inhibition effects of dynamic strain loading group exceeded the corresponding static strain. This work suggested that appropriate mechanical strech may suppress differentiation of osteoclasts through inhibiting expression of ICAM-1 and ODF. Application of mechanical stress might have a beneficial effect on quantity of generated bone tissue and might be a important factor in tissue engineering of periodontal tissues.

Abstract: A novel porous composite scaffold of nano-HA/poly (lactic-co-glycolic) (PLGA) was fabricated by solvent casting/particulate leaching method. Chondrocytes were isolated from the knee articular joints of a rabbit, and then seeded in the scaffolds. The cell-loaded scaffolds were cultured in vitro for 5 days before implantation. Full-thickness articular cartilage defects were created in rabbits, and filled with and without the cell-loaded nano-HA/PLGA scaffolds. The implants were harvested after in vivo incubation of 2 and 5 weeks. Cartilaginous tissues were observed at defects repaired with the cell-loaded scaffolds, while only fibrous tissues were found for the control groups. The repaired tissues were evaluated histologically by hematoxylin and eosin staining. Results revealed that nano-HA/PLGA composite scaffolds facilitated adheration of cells in vitro, and the nano-HA particles could prevented the scaffolds from collapsing and promoted the formation of cartilaginous tissue in vivo.