[In vitro biocompatibility of novel absorbable hydroxyapatite and AO artificial bone beta-tricalcium phosphate with rhesus bone marrow stromal cells].

OBJECTIVE: To study the in vitro biocompatibility of novel hydroxyapatite (HA) and AO artificial bone beta-tricalcium phosphate (beta-TCP) with rhesus bone marrow stromal cells (rBMSCs) . METHODS: The third passage of rBMSCs were cultured with HA and beta-TCP respectively, with the cells cultured without the materials as the control. The morphology and proliferation of cells were observed by inverted phase-contrast microscope and scanning electron microscope (SEM). MTT assay was used to semiquantitatively evaluate the cell proliferation. RESULTS: The rBMSC cocultured with HA exhibited good growth as observed under inverted phase-contrast microscope, without significant difference from the cells in the control group. Some small particles were seen pealing off from beta-TCP, and some of the cells died. Under SEM, rBMSCs showed good adhesion to HA with obvious proliferation, but the ratio of adhesive cells was not as high as that in beta-TCP group. MTT assay showed no significant difference in the cell number between HA and the control groups, but the cell number in beta-TCP group was notably less than that of control group. CONCLUSION: Novel HA has good biocompatibility with rBMSCs for bone tissue engineering, and AO artificial bone still needs improvement to serve as scaffold material for BMSCs.

[Bone defect repair with a new tissue-engineered bone carrying bone morphogenetic protein in rabbits].

OBJECTIVE: To construct a new tissue-engineered bone with poly (D, L-lactide-co-glycolide) (PLGA), bone morphogenetic protein (BMP) and bone marrow-derived stem cells (BMSCs) and observe its effect in repairing segmental bone defects. METHODS: A 15-mm bone defect in the right radius was induced in New Zealand white rabbits, and the models were randomized into three groups to receive implantation of the tissue-engineered bone grafts constructed with PLGA carrying 5 mg BMP and about 1 x 10(6) BMSCs (experimental group), grafts of PLGA with about 1 x 10(6) BMSCs (control group), or grafts of exclusive PLGA (blank control group), respectively. The osteogenesis in the bone defect after the implantation on was evaluated X-ray films, and the histological changes of the tissues sampled from the bone defect 4, 8, and 12 weeks after operation were observed and new bone formation was measured by image analysis. RESULTS: The bone defect was completely repaired in the experimental group 12 weeks after the implantation, showing the best results among the 3 groups. The bone defects in the blank control group was filled with only fibrous and connective tissues at 12 weeks. CONCLUSION: This tissue-engineered bone constructed with PLGA, BMP and BMSCs possesses good ability in repairing segmental bone defect.

[The method of accelerating osteanagenesis and revascularization of tissue engineered bone in big animal in vivo].

OBJECTIVE: To study whether tissue engineered bone can repair the large segment bone defect of large animal or not. To observe what character the fascia flap played during the osteanagenesis and revascularization process of tissue engineered bone. METHODS: 9 Chinese goats were made 2 cm left tibia diaphyseal defect. The repairing effect of the defects was evaluated by ECT, X-ray and histology. 27 goats were divided into three groups: group of CHAP, the defect was filled with coral hydroxyapatite (CHAP); group of tissue engineered bone, the defect was filled with CHAP + bone marrow stroma cells (BMSc); group of fascia flap, the defect was filled with CHAP + BMSc + fascia flap. After finished culturing and inducing the BMSc, CHAP of group of tissue engineered bone and of fascia flap was combined with it. Making fascia flap, different materials as described above were then implanted separately into the defects. Radionuclide bone imaging was used to monitor the revascularization of the implants at 2, 4, 8 weeks after operation. X-ray examination, optical density index of X-ray film, V-G staining of tissue slice of the implants were used at 4, 8, 12 weeks after operation, and the biomechanical character of the specimens were tested at 12 weeks post operation. RESULTS: In the first study, the defect showed no bone regeneration phenomenon. 2 cm tibia defect was an ideal animal model. In the second study, group of CHAP manifested a little trace of bone regeneration, as to group of tissue engineered bone, the defect was almost repaired totally. In group of fascia flap, with the assistance of fascia flap which gave more chance to making implants to get more nutrient, the repair was quite complete. CONCLUSIONS: The model of 2 cm caprine tibia diaphyseal defect cannot be repaired by goat itself and can satisfy the tissue engineering's demands. Tissue engineered bone had good ability to repair large segment tibia defect of goat. Fascia flap can accelerate the revascularization process of tissue engineered bone. And by this way, it augment the ability of tissue engineered bone to repair the large bone defect of goat.

[Biocompatibility of adult human osteoblasts with coral-derived hydroxyapatite in vitro].

OBJECTIVE: To study the biocompatibility of the osteoblasts from adult human bone marrow with coral-derived hydroxyapatite (CHA) in in vitro culture. METHODS: Bone marrow was obtained from healthy adult subjects and cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10 % fetal bovine serum. The subsequent cell passaging was conducted in conditioned medium containing dexamethasone, beta-sodium glycerophosphate and ascorbic acid, with the osteoblasts in culture then divided into CHA group (in which the cells were cultured with CHA) and osteoblasts group (without CHA). The proliferation and differentiation of all the cultured cells were observed at different time points under inverted phase contrast microscope, optical microscope with HE staining and scanning electron microscope respectively. Proliferation of the cultured cells were evaluated by MTT assay, and the activity of alkaline phosphatase and total micro-protein contents in these cultured osteoblasts were quantitatively detected. RESULTS: The osteoblasts from adult human bone marrow grow well in vitro, regardless of the presence of CHA, with biological and morphological characteristics similar to those of normal osteoblasts. CHA improved the adhesion, growth and proliferation of the cultured cells, showing no adverse effects on the cell functions. CONCLUSION: CHA is an optimal scaffold material for bone tissue engineering, which may potentially find clinical application for bone defect repair.

[Green fluorescent protein as a tracer of bone marrow stromal cells in bone tissue engineering in rhesus].

OBJECTIVE: To observe the role of green fluorescent protein (GFP) in tracing rhesus bone marrow stromal cells (rBMSCs) during tissue-engineered bone formation in vivo. METHODS: Ad5.CMV-GFP was amplified by infecting QBI-293A cells, and the bone marrow was harvested from the ilium of adult male rhesus to obtain rBMSCs, which were cultured and passaged in vitro. GFP was transfected into the third-passage rBMSCs via adenovirus vector and the labeled cells were inoculated into absorbable HA scaffold and cultured for 3 days, with untransfected rBMSCs as control, before the cell-matrix compounds were implanted into the latissimus dorsi muscles of rhesus. Samples were harvested at 6 week and embedded in paraform, and ground sections of the bone tissue were prepared to observe green fluorescence under laser scanning confocal microscope. Propidium iodide staining of the sections was also performed for observation. RESULTS: The rBMSCs grew well after GFP transfection, and green fluorescence could be seen 24 h after the transfection and became stronger till 48 h, with a positive transfection rate beyond 80%. Six weeks after cell implantation, the rBMSCs labeled by GFP-emitted green fluorescence were detected in the bone tissue under laser scanning confocal microscope. CONCLUSION: GFP can effectively trace BMSCs during bone tissue engineering, and the transplanted BMSCs constitute the main source of bone-forming cells in bone tissue engineering.

[Perfusion-weighted magnetic resonance imaging for monitoring vascularization in tissue-engineered bone in rhesuses].

OBJECTIVE: To assess the value of perfusion-weighted magnetic resonance (MR) imaging (PWMRI) in monitoring vascularization in tissue-engineered bone graft. METHODS: Tibial diaphyseal defect of 20 mm was induced in 25 lower limbs of 13 rhesuses and fixed with an AO reconstruction plate with 7 holes. The monkeys were randomized into 5 groups according to the materials used for defect filling: group A, with beta-tricalcium phosphate (beta-TCP), bone marrow stromal cells (BMSCs) and blood vessel bundles; group B, with beta-TCP and blood vessel bundles; group C, with beta-TCP and BMSCs; group D, with beta-TCP, and group E without filling. PWMRI, X-ray, and radionuclide imaging were carried out at weeks 4, 8, 12 postoperatively. The maximum slope rates of the single intensity-time curve (SS(max)) and the baseline values (SI(baseline)) on the same time points were calculated. Transmittances on the X-ray films and isotope counts in the region of interest (ROI) were assessed and calculated. RESULTS: Compared with other groups, group A showed the highest SS(max) at weeks 4, 8, and 12 postoperatively, and its SS(max) at week 8 was significantly higher than that at week 4 (P=0.003). The SS(max) was positively related to isotope counts in ROI at week 8 after operation (r(s)=0.899, P=0.038), and inversely related to transmittance on X-ray films at week 12 (r(s)=-0.892, P=0.042). CONCLUSION: The SS(max) of the single intensity-time curve can accurately reflect the vascularization of the tissue-engineered bone graft, and PWMRI allows sensitive, quantitative, noninvasive and radiation-free vascularization monitoring.