Objective To explore the biocompatibility of poly(lacticacid/glycolic acid/asparagic acid-co-polyethylene glycol) biomaterials (PLGA-ASP-PEG) and biological behaviors of cultured marrow stroml stem cells (MSCs) combined with this new type of scaffold in tissue engineering. Methods The PLGA-ASP-PEG tri-block copolymers were obtained through bulk ringopening copolymerization method.MSCs were isolated from the bone marrow of 4 week old New Zealand rabbits. The 3rdgeneration MSCs were cultured combining with PLGA-ASP-PEG in vitro, while cells cultured in PLGA as control group. The cell adhesion rate and the adhesivepower were examined by conventional precipitation method and micropipette aspiration technique respectively. The morphological features were studied by scanning electron microscope. The proliferation behavior of the cells was analyzed by MTT assay. The cell cycle, proliferation index, DNA index and apoptosis of the cells were detected by flow cytometry. The synthesis of protein and collagen were examined by Coomassie Brilliant Blue dyes and 3H-Proline incorporation test. Results The MSCs adhered and grew well on the surface of the biomaterial PLGA-ASP-PEG. The powers of cell adhesion, proliferation and protein and collagen synthesis of the cells were all significantly higher than those of PLGA group (P＜0.05), but the apoptosis rate was significantly lower than that of PLGA group (P＜0.05). The DNA indexes showed the cells of both PLGA-ASP-PEG group and PLGAgroup were normal diploid cells. Conclusion PLGA-ASP-PEG showedgood biocompatibilityand the biological properties improved greatly compared with the PLGA scaffold materials. These results demonstrated that the promise of PLGAASPPEG canbe used as an ideal scaffold material for construction of tissue engineered bone to restore bone defects in bone tissue engineering．
Objective To investigate the ability of plateletrich plasma(PRP) combined with cells and artificial bone in accelerating the repair of bone defect. Methods The marrow stromal stem cells (MSCs) of rabbit were cultured and induced into the osteoblast-like cells in vitro. PRP was produced with low-density twice centrifugations. Forty-eight New Zealand rabbits were made 1.2 cm bilateral radius defect models and divided into 4 groups averagely at random: group A(left:PRP／MSCs／β-tricalcium phosphate(β-TCP), right: MSCs／β-TCP), group B (left:autoradius, right: PRP／MSCs／β-TCP)； group C (left:autoradius,right: MSCs／β-TCP), and group D(left:PRP／β-TCP; right:β-TCP). At 2, 6 and 12 weeks after operation， the repair of bone defect was evaluated by the generalobservation, histology, biomechanics and histomorphology. Results There was a stable platelet concentration in PRP and it was about 5.45±0.23 times of whole blood. In the aspect of bone bridge and conture of the defects, at 2 and 6 weeks, PRP／MSCs／β-TCP and MSCs／β-TCP displayed asimilar outcome and were less than auto in general sample and X-ray;at12 weeks，PRP／MSCs／β-TCP was similar to autoradius and better than MSCs／β-TCP.in the aspect of quantity and quality of bone formation，histology showed that PRP／MSCs／β-TCP and autoradius were better than MSCs／β-TCP（P＜0.05），and there was nosignificantdifference between PRP／MSCs／β-TCP and autoradius（P＞0.05）. At 2 and 6 weeks，there was no significant difference between PRP／β-TCP and β-TCP（P＞0.05）。At 12 weeks，PRP／β-TCP was better than β-TCP（P＜0.05）. In the aspect of intensity f bone formation，at 6 and 12 weeks，PRP／MSCs／β-TCP and autoradiuswere better than MSCs／β-TCP（P＜0.05）. At 6 weeks，autoradius was better than PRP／MSCs／β-TCP（P＜0.05）. At 12 weeks，there was no significant difference between PRP／MSCs／β-TCP and auto（P＞0.05）. PRP／TCP and β-TCP had no significant difference at 12 weeks（P＞0.05）. Conclusion PRP／MSCs／β-TCP demonstrated excellent ability of forming bone in experiment. PRP was most likely to accelerate the repair of bone defect through increasing the activity of proliferation and differentiationof MSCs and osteoblasts.
Objective To construct the recombinant adenovirus bearing human transforming growth factor β1(TGF-β1) and bone morphogenetic protein 7 (BMP-7) genes, and investigate its co-expression in the marrow stromalstemcells (MSCs) and bioactivity effect. Methods Using the replication defective adenovirus AdEasy as a carrier, MSCs were infected by the high-titer-level recombinant adenovirus taking TGF-β1 and BMP-7 genes. Immunocytochemistry, in situ hybridization，reverse transcription-polymerase chain reaction (RT-PCR), and hexuronic acid level test were used to detect the coexpression of the exogenous genes and to analyze their effect transfection on directive differentiation of MSCs. Results The immunocytochemistry staining showed that the brown coarse grains were situated in the cytoplasm of the most MSCs 72 h after infection. Procollagen ⅡmRNA in the cells was detected by the in situ hybridization, and the content of hexuronic acid in the culture mediumwas significantly increased 10 days after infection compared with the level before infecton (Plt;0.01). Conclusion The recombinant adenovirus bearing human TGF-β1 and BMP-7 genes can be constructed, and the exogenous gene can be coexpressed in MSCs, which may offer a novel approach to thelocal combination gene therapy for repairing joint cartilage defects.
Objective To find a new culture system to induce proliferation and osteodifferentiation of marrow stromal cells (MSCs) in vitro for bone tissueeng ineering． Methods There were four groups in this experiment to study effects of Passage 3 osteoblasts derived from the rat cranium and the osteogenic inductor (1 nmol/L dexamethasone，10 mmol/L beta-glycero-phosphate，50 μg/ml retin oic acid) on growth of MSCs isolated from the rat femur and the tibia． MSCs were cultured in the DMEM medium (the c ontrol group) and in the osteoinductive culture medium (the inductor group)；fur thermore, MSCs were co-cultured with the osteoblasts in the DMEM medium (the osteoblast group) and in the osteoinductive culture medium (the combined treatment group)．The cells in the four groups were counted every 2 days for 8 days and alkaline phosphatase (ALP) activity of MSCs at 10 days of cultivation was measured．The MRNA expression of osteocalcin (OC) of MSCs at 2 weeks was assayed with the reverse transcript polymase chain reaction (RT-PCR)． Results There were more cells in the osteoblast group than in the control group(31.73±3.31×104 V S. 24.33±3.04×104, Plt;0.05), but there were fewer cells in the inductor gro up(16.23±2.44×104, Plt;0.05). There was no significant difference in th e cell number between the combined treatment group (21.54±2.29×104) and th e control group（Pgt;0.05）．The ALP activity was higher in the combined trea tment group （2.01±0.56 U）than in the control group (1.27±0.43 U), in the inductor group（1.27±0.43 U）, and in the osteoblast group （0.77±0.19 U）.The osteocalcin mRNA was expressed in the three treat ment groups but was not expressed in the control group. The significantly higher leve l of the osteocalcin mRNA was expressed in the inductor group（0.783±0.094）and in the combined treatment group（0.814±0.071）than in the osteoblast group（0.302±0.026） (Plt;0.05). Conclusion The combined use of t he osteoblast and the inductor can induce marrow stromal cells. Their combined u se does not affect the normal proliferation but can obviously promote the osteodifferentiation of marrow stromal cells. This combined use can become a new culture system of the seed cells for bone tissue engineering．
Objective To investigate the feasibility of repairing goat tibia defect with marrow stromal cells (MSCs).Methods MSCs were cocultured with the bio-derived bone in vitro, and the 20 mm tibia defectswere made and fixed with plate in 35 goats, and they were divided into the experimental group, control group and blank group. The defects on the right side were filled with tissue engineering bone as the experimental group, the defects onthe left side with bio-derived bone as the control group in 33 goats, and the defect on the both sides were not filled with any materials as the blank group in 2 goats. Threpair capability was assessed physically, histopathologically and biomechanically at 2, 4, 6, 8, 12,16 and 24 weeks after operation in 3 groups.Results By physical, histopathological and biomechanical examinations, the bio-derived bone was partially absorbed in the experimental group and was rarely absorbed in the control group in the 4th week; the defects were partially repaired in the experimental group, and in the control group, few new bones were observed in the two ends of the implants, in which there was fibrous tissue. The effects of biomechanics had no statistically significant difference between the experimental group and the control group(P＞0.05) in the 8th week; the defects were perfectly repaired in the experimental group and the effects of biomechanics had statistically significant difference between two groups (P＜0.05) in the 12th weeks. The defects were not repaired in the 24th week in the blank group.Conclusion The tissue engineering bone can efficiently repair bone defect, and itsrepair capability is better than that of bio-derived bone alone both in quantity and in quality of bone formation.
Objective To explore the feasibility of allogeneic marrow stromal stem cells(MSCs) as seed cells to construct tissue engineered bone bydetecting the expressions of interleukin 2(IL-2) and IL-2 receptor in rhesus monkeys after implanting these tissue engineered bones.Methods Engineered bones were constructed with osteoblasts which derived from allogeneic MSCs and bio-derived materials in vitro, and then were implanted to bridge 2.5 cm segmental bone defects of left radius in 15 rhesus monkeys as experimental group, bioderived materials only were implanted to bridge same size defects of right radius as control group. Every 3 monkeys were sacrificed in the 1st, the 2nd, the 3rd, the 6th andthe 12th weeks postoperatively and the expressions of IL-2 and IL-2 receptor in blood and graft samples were detected quantitatively by enzymelinked immuneosorbent assay (ELISA).Results There was no significant difference in the contents of IL-2 and its receptor between 2 groups（P＞0.05）. The contents ofIL-2 and its receptor increased from the 2nd week and maintained high level from the 2nd to the 6th week, but decreased after 6 weeks.ConclusionTissue engineered bones constructed with allogeneic MSCs and bio-derived materials show low immunogenicity. Allogeneic MSCs may be used as seed cells to construct tissue engineered bone.
Objective To investigate the neural markers’ expression in the differentiation of marrow stromal stem cells(MSCs) into neural cells. Methods Rats MSCs were expanded as undifferentiated cells in vitro for 5 to7 generations and cultured in a modified neuronal medium(MNM) after 24 hours of all-trans retinoidacid(ATRA) pretreatment. Immunocytochemistry was used to detect the expression of nestin、neuron-specific nuclear protein(NeuN)、microtubule-associated protein2 (MAP-2) and glial fibrillary acidic protein(GFAP) at different timepoints. Results After ATRA and MNM treatment, MSCs progressively assumed neuronal morphological characteristics. Nestin occurred first after 24 hours of ATRA treatment; then NeuN expressed after 2 hours of MNM treatment; the last one was MAP-2 and it was detected after 9 hours of MNM treatment. Other markers continuously expressed except that the expression of nestin peaked after 18 hours of MNM induction and remarkably decreased after 36 hours. Conclusion ATRA and MNM could promote the differentiation of MSCs into neural cells and the expression of neural-specific markers was consistent with current knowledge regarding the timepoints of markers expression in the neuronal development which provides a good model in vitro for neuronal development research.
Objective To study the feasibility of using mice marrow stromal stem cells(MSCs) as seed cells for tissue engineering cartilage to embed the seed cells in acellular cartilage matrix of human auricle. Methods Acellular cartilage matrix was made from human auricle cartilage. The MSCs were isolated from the nucleated cells fraction of mice marrow by centrifuge.The MSCs were embedded in acellular cartilage matrix. After 10 day’s combined culture, the specimens were observed with optical and electrical microscope.Results The MSCs could well proliferate in the acellular cartilage matrix. The cells were not well-distributed in acellular cartilage matrix. There were more cells in the peripheral part of the matrix than in the central part of the matrix. Most of the cells were in cartilaginous lacunae. There were 1 or 2 cells in every cartilaginous lacunae.Conclusion The MSCs can be used as seed cells of tissue engineering and can well proliferate in the acellular cartilage matrix and become tissue engineering cartilage.
Objective To investigate the ability to repair goat tibia defect with marrow stromal stem cells (MSCs) and bio-derived bone, and the feasibility of the compounds as bone substitute material. Methods MSCs were cultured with the bioderived bone in vitro, and the 20 mm tibia defect of goat was made and fixedwith plate. Eighteen goats were divided into experimental group, control group and blankgroup. The defects were not filled with anything in blank group, with tissue engineering bone in experimental group and bio-derived bone in control group. Therepair capability was assessed by physical, X-ray and bone mineral density examinations8,12,16, and 24 weeks after operation. Results In experimental group, the defects were partially repaired 8 weeks, and completely repaired12 and 16 weeks; there was significant difference in bone density between experimental group and control group (P＜0.05) 8,12 and 16 weeks, but no significant difference 24 weeks. The defects of blank group were not repaired 24weeks. Conclusion The tissue engineering bone can efficiently repair bone defect, and its repair capability is better than that of bio-derived bone alone both in quantity and quality of boneformation.
Objective To construct the recombined DNA pcDNA3.1-hBMP-2 and transfect into human marrow stromal stem cells (MSCs) in vitro, and to explore theeffects of transfection on cellular proliferation and expression of vascular endothelial growth factor (VEGF). Methods The expression of human bone morphogenetic protein 2(hBMP-2) in these cells after transfection was determined by in situ hybridization and immunohistochemical analysis and Western blot analysis. The changes of cell proliferation were observed by flow cytometry. The effects of BMP-2 gene transfection on expression of VEGF in the cells were analyzed by in situ hybridization of VEGF cDNA probe. Results Stable expressionof hBMP-2 in pcDNA3.1-hBMP-2 transfected MSCs was confirmed in the levels of mRNA and protein.Cellular proportion in S period increased, which indicated that the synthesis of cell DNA increased. The expression of VEGF in the cells increased obviously. Conclusion With the help of lipofectamine, the pcDNA3.1-hBMP-2 were transfected into human MSCs successfully. hBMP-2 plays an important role in promoting cellular proliferation and vascular generation during bone repair.