OBJECTIVE: To explore a new method of preparing the composite of DL-polylactic acid (PDLLA), hydroxyapatite(HA), decalcium bone matrix (DBM), and to observe the degradation characteristics of PDLLA/HA/DBM in vitro. METHODS: An emulsion blend method was developed to prepare the composite of PDLLA/HA/DBM based on the weight rate of PDLLA:HA:DBM = 1.5-2:1-1.5:1. The characteristics of the particles was observed by scanning electron microscope. In vitro, PDLLA/HA/DBM and PDLLA were put into PBS(pH7.4) respectively; the pH value, weight and biomechanics of them were determined during the degradation. RESULTS: Without heating, the emulsion blend method could be developed to prepare PDLLA/HA/DBM. Scanning electron microscope showed that the gap diameter in the compound material was 100 to 400 microns, and the porosity was 71.3%; During degradation, the pH value of PDLLA decreased little within 2 weeks, then decreased obviously and decreased to 4.0 at the end of the 4th week; while the pH value of PDLLA/HA/DBM kept quite steady and was 6.4 at the end of the 12th week. The weight of PDLLA decreased little within 4 weeks, then decreased obviously and remained 50% of its prime weight at the end of the 12th week; while the weight of PDLLA/HA/DBM decreased little within 5 weeks, then decreased obviously and remained 60% of the prime at the end of the 12th week. The prime biomechanical strength was 1.33 MPa in PDLLA and 1.71 MPa in PDLLA/HA/DBM. There was significant difference between them (P lt; 0.05). The strength of PDLLA decreased little within 3 weeks, then decrease obviously and was 0.11 MPa at the end of the 12th week; the strength of PDLLA/HA/DBM decreased little within 4 weeks, then decrease obviously and was 0.21 MPa at the end of the 12th week. CONCLUSION: The emulsion blend method is a new method to prepare bone repair materials. As a new bone repair material, PDLLA/HA/DBM is suitable for bone tissue engineering for its good characteristics of porosity and degeneration.
OBJECTIVE: To compare the clinical results of repairing bone defect of limbs with tissue engineering technique and with autogeneic iliac bone graft. METHODS: From July 1999 to September 2001, 52 cases of bone fracture were randomly divided into two groups (group A and B). Open reduction and internal fixation were performed in all cases as routine operation technique. Autogeneic iliac bone was implanted in group A, while tissue engineered bone was implanted in group B. Routine postoperative treatment in orthopedic surgery was taken. The operation time, bleeding volume, wound healing and drainage volume were compared. The bone union was observed by the X-ray 1, 2, 3, and 5 months after operation. RESULTS: The sex, age and disease type had no obvious difference between groups A and B. all the wounds healed with first intention. The swelling degree of wound and drainage volume had no obvious difference. The operation time in group A was longer than that in group B (25 minutes on average) and bleeding volume in group A was larger than that in group B (150 ml on average). Bone union completed within 3 to 7 months in both groups. But there were 2 cases of delayed union in group A and 1 case in group B. CONCLUSION: Repair of bone defect with tissue engineered bone has as good clinical results as that with autogeneic iliac bone graft. In aspect of operation time and bleeding volume, tissue engineered bone graft is superior to autogeneic iliac bone.
Objective To observe effects of the direct impaction onthe cell survival and the bone formation of the tissue engineered bone modified by the adenovirus mediated human bone morphogenetic protein 2 (Adv-hBMP2) gene and to verify the feasibility of the impacted grafting with it. Methods The marrow stromal cells (MSCs) were separated from the canine bone marrow and were cultured. MSCs were transfected with the Adv-hBMP2 gene and combined with the freeze-dried cancellous bone (FDB) to form the tissue engineered bone. Four days after the combination, the tissue engineered bone was impacted in a simulated impactor in vitro and implanted in the mouse. The cell survivals were evaluated with SEM 1 and 4 days after the combination, immediately after the impaction, and 1 and 4 days after the impaction, respectively. The bone formation and the allograft absorption were histologically evaluated respectively. Results There were multiple layers of the cells and much collagen on FDB before the impaction. Immediately after the impaction, most of the cells on the direct contact area disappearedand there was much debris on the section. Some of the cells died and separatedfrom the surface of FDB at 1 day, the number of the cells decreased but the collagen increased on the surface at 4 days. Histologically, only the fibrous tissue was found in FDB without the cells, the bone formation on FDB was even in distribution and mass in appearance before the impaction, but declined and was mainly on the periphery after the impaction in the AdvhBMP2 modified tissue-engineered bone. Conclusion The simulated impaction can decrease the cells survival and the bone formation of the AdvhBMP-2 modified tissue-engineered bone. The survival cells still function well.It is feasible to use the tissue engineered bone in the impaction graft.
Objective To observe the changes in the peripheral blood T lymphocyte subsets and the histomorphology of the transplanted tissues in the rabbits in the early stage after transplantation of the tissue engineered boneconstituted by the biologically-derived scaffold and to confirm the feasibility of the biologicallyderived materials as a scaffold in the bone tissue engineering. Methods Forty-eight healthy New Zealand rabbits (weight, 2.0-2.5 kg) with a 1-cm defect were equally and randomly divided into 4 groups: Groups A-D. The partial demineralized freeze-dried bone (PDFDB), the tissue engineered bone constructed by the osteoblasts derived from the lactant rabbit periosteum as a seeding cell, the xenogeneic cancellous bone undergoing the antigen self-digestion, partial demineralization and freeze-driedprocess as a scaffold, and the fresh xenogeneic allografting bone were respectively transplanted into the segmental defects of the rabbit radii in Groups A-D.To examine the effects of the 4 different materials, the flow cytometry was used to observe the changes in the T lymphocyte subsets in the rabbit peripheral blood at 1, 2, and 4 weeks after the operations and to examine the osteogenesis achieved by the 4 materials, the histological observations were also performed at 2, 4, 8, and 12 weeks after the operations. Results Two weeks after the tissue engineered bone transplantation in Group B, the osteoblasts and chondroblasts were found in the apertures of the scaffold, the new bone formation could be observed, the osteoclasts could be seen in the peripheral zone, and some of the netlike frameworks were destroyed and absorbed. Four weeks after the operation, the histological observation revealed that the osteocartilagionous callus turned into a woven bone. The peripheral blood T lymphocyte subsets of CD4+ and CD8+ were significantly greater in number 1-2 weeks after the operations and in Groups A and B than before the operations and in the other groups (.Plt;0.05)；4 weeks after the operations the T lymphocyte subset of CD4+ was only slightly greater in number than before the operations, but with no statistically significant difference (Pgt;0.05). In Group C, the increase of the T lymphocyte subsets of CD4+ and CD8+ was not significant after the operation (Pgt;0.05). The T lymphocyte subsets of CD4+ and CD8+ were significantly greater in number 1, 2 and 4 weeks after the operations and in Group D than before the operation and in the other groups (Plt;0.05). Conclusion The tissue engineered bone constructed by the partial demineralized freezedried bone as a scaffold does not cause a serious immunologic rejection in the early stage after the transplantation and does not affect its good ability to repair the bone defect. The biologicallyderived bone canbe used as a scaffold in the bone tissue engineering.
Objective To investigate the effect of dexamethasone, recombinant human fibroblast growth factor (rhFGF) and recombinant human bone morphogenetic protein 2 (rhBMP-2) on the proliferation and differentiation of marrow stromal stem cells (MSCs) for their further application in tissue engineering. Methods MSCs were isolated and cultured in vitro, and then exposed to different dose of dexamethasone (10-8 mol/L,10-7 mol/L,10 -6 mol/L), rhFGF (50 ng/ml,200 ng/ml,500 ng/ml) and rhBMP-2 (50 ng/ml,500 ng/ml,1 000 ng/ml) respectively. The total protein and alkaline phosphatase (ALP) activity of each group was measured on 4th and 7th day. Results Exposure of MSCs with 10-6mol/L dexamethasone inhibited protein synthesis without obvious effects on ALP expression. The application of rhFGF significantly promoted cell proliferation but inhibited ALP activity. In comparison, ALP expression was significantly enhanced by treatment of rhBMP-2 at concentration of 500 ng/ml，1 000 ng/ml. Conclusion The exposure of dexamethasone as well as rhBMP-2 to MSCs with an appropriate concentration promotes osteogenic expression without reverse effects on cell proliferation, which indicates the great potential value in cell-based strategy of bone tissue engineering.
Objective To study the adhesion characteristic in vitrobetween porous biphasic calcium phosphate(BCP) nanocomposite and bone marrow mesenchymal stem cells (MSCs) that have been induced and proliferated. Methods MSCs obtained from SD ratbone marrow were in vitro induced and proliferated. After their osteoblastic phenotype were demonstrated, MSCs were seeded onto prepared porous BCP nanocomposite(experiment group)and common porous hydroxyapatite (control group). Their adhesion situation was analyzed by scanning electron microscope. The initial optimal cell seeding density was investigated between new pattern porous BCP nanocomposite and MSCs by MTT automated colormetric microassay method. Results The differentiation of MSCs to osteoblastic phenotype were demonstrated by the positive staining of mineralized node, alkaline phosphatase (ALP) and collagen typeⅠ, the most appropriate seeding density between them was 2×106／ml. The maximal number which MSCs could adhere to porous BCP nanocomposite was 1.28×107/cm3. Conclusion MSCs can differentiate to osteoblastic phenotype.The MSCs were well adhered to porous BCP nanocomposite.
【Abstract】 Objective To produce a new bone tissue engineered carrier through combination of xenograft bone (X)and sodium alginate (A) and to investigate the biological character of the cells in the carrier and the abil ity of bone-forming in vivo, so as to provide experimental evidence for a more effective carrier. Methods BMSCs were extracted from 2-week-old New Zealand rabbits and the BMSCs were induced by rhBMP-2 (1 × 10-8mol/L). The second generation of the induced BMSCs was combined with 1% (V/W) A by final concentration of 1 × 105/mL. After 4-day culture, cells in gel were investigated by HE staining. The second generation of the induced BMSCs was divided into the DMEM gel group and the DMEM containing 1% A group. They were seeded into 48 well-cultivated cell clusters by final concentration of 1 × 105/mL. Seven days later, the BMP-2 expressions of BMSCs in A and in commonly-cultivated cells were compared. The second generation of the induced BMSCs was mixed with 2% A DMEM at a final concentration of 1 × 1010/mL. Then it was compounded with the no antigen X under negativepressure. After 4 days, cells growth was observed under SEM. Twenty-four nude mice were randomly divided into 2 group s (n=12).The compound of BMSCs-A-X (experimental group) and BMSCs-X (control group) with BMSCs whose final concentrat ion was 1 × 1010/mL was implanted in muscles of nude mice. Bone formation of the compound was histologically evaluated by Image Analysis System 2 and 4 weeks after the operation, respectively. Results Cells suspended in A and grew plump. Cell division and nuclear fission were found. Under the microscope, normal prol iferation, many forming processes, larger nucleus, clear nucleolus and more nuclear fission could be seen. BMP-2 expression in the DMEM gel group was 44.10% ± 3.02% and in the DMEM containing 1% A group was 42.40% ± 4.83%. There was no statistically significant difference between the two groups (P gt; 0.05). A was compounded evenly in the micropore of X and cells suspended in A 3-dimensionally with matrix secretion. At 2 weeks after the implantation, according to Image Analysis System, the compound of BMSCs-A-X was 5.26% ± 0.24% of the totalarea and the cartilage-l ike tissue was 7.31% ± 0.32% in the experimental group; the compound of BMSCs-X was 2.16% ± 0.22% of the total area and the cartilage-l ike tissue was 2.31% ± 0.21% in the control group. There was statistically significant difference between the two groups (P lt; 0.05). At 4 weeks after the operation, the compound of BMSCs-A-X was 7.26% ± 0.26% of the total area and the cartilage-l ike tissue was 9.31% ± 0.31% in the experimental group; the compound of BMSCs-X was 2.26% ± 0.28% of the total area and the cartilage-l ike tissue was 3.31% ± 0.26% in the control group. There was statistically significant difference between the two groups (P lt; 0.05). Conclusion The new carrier compounding A and no antigen X conforms to the superstructural principle of tissue engineering, with maximum cells load. BMSCs behave well in the compound carrier with efficient bone formation in vivo.
OBJECTIVE: To investigate the effect of compound pattern of ceramic bovine bone (CBB) and hydrogel(HG) on attachment, proliferation and differentiation of bone marrow stromal cell (MSC), and to find out the best way of constructing tissue engineered bone. METHODS: CBB, HG and MSC was compounded in different patterns and sequences to form CBB/HG/MSC (group A), HG/MSC/CBB (group B), CBB/MSC/HA (group C) and CBB/MSC (control group). Attachment and morphology of MSC were observed by scanning electronic microscope; the proliferation of MSC was evaluated by cell count; alkaline phosphatase(ALP) activity was examined by histochemistry and type I collagen synthesis was examined by immunohistochemistry staining 5 and 10 days later. RESULTS: In group A, MSC spread better, and ALP activity of group A was significantly higher than that of group B and control group(P lt; 0.01); but there was no significant difference between group A and group C(P gt; 0.05). There was no significant difference in type I collagen synthesis between four groups on the 5th day; but mean gray scale of type I collagen in group B was significantly higher than that in the other groups on the 10th day(P lt; 0.01). CONCLUSION: Different compound patterns of CBB, HG and MSC affect attachment, proliferation, differentiation of MSC. The compound pattern of CBB/HG/MSC is better than the others.
Objective The combined appl ication of green fluorescent protein (GFP) and confocal laser scanning microscope three-dimensional reconstruction (CLSM-3DR) were used to monitor the construction and in vivo transplantation of tissue engineered bone (TEB), to provide for technology in selection of scaffolds and three-dimensional constructional methods. Methods After bone marrow mesenchymal stem cells (BMSCs) were isolated from a 2-year-old green goat by a combination method of density gradient centrifugation and adherent culture, and the expressions of CD29, CD60L, CD45, and CD44 in BMSCs were detected by flow cytometry. Plasmid of pLEGFP-N1 was ampl ified, digested by enzymes (Hind III, BamH I, Sal I, and Bgl II), and identified. Transfection of pLEGFP-N1 into PT67 cells was performed under the help of l iposome. Positive PT67 cells were picked out with G418, and prol iferated for harvesting virus. Based on the titre of virus, after BMSCs were infected by virus containing pLEGFP-N1, GFP positive BMSCs were collected and prol iferated for seeding cells. TEB was fabricated by GFP positive BMSCs and decalcified bone matrix (DBM) and observed by CLSM-3DR for the evaluation of the distribution and prol iferation of seeding cells. After TEB was transplanted in the defect of goat femur, CLSM was used for observing the survival and distribution of GFP positive cells in the grafts. Results The isolated cells were fibroblast-l ike morphous, with the positive expression of CD29 and CD44, and negative expression of CD60L and CD45. The digested production of pLEGFP-N1 was collected for ionophoresis, whose results showed the correct fragment length (6 900 bp). The virus of pLEGFP-N1 was harvested by transfection of pLEGFP-N1 into PT67 cells and used for further infection to obtain GFP positive BMSCs. The prol iferated GFP positive BMSCs and DBM were used for fabrication of TEB. The distribution, prol iferation, and migration of BMSCs in TEB were observed by CLSM-3DR. GFP positive cells also were observed in images of TEB graft in goat femur 28 days after transplantation. Conclusion The BMSCs labeled by GFP in three-dimensional scaffold in vivo were monitored well by CLSM-3DR. It suggests a wide use potency in monitoring of three-dimensional cultured TEB.
Objective To investigate the morphology and biomechanics of in vivo osteogensis after repairing rabbit skull defects with plastic engineered bone which was prefabricated with alginate gel, osteoblasts and bone granules. Methods Twenty-eight rabbits were divided into group A (n=16), group B(n=8) and group C(n=4).The bilateral skull defects of 1 cm in diameter were made. Left skull defects filled with alginate gel-osteoblasts-bone granules(group A1) and right skull defects filled withalginate gel-bone granules(group A2).The defects of group B was left, as blank control and group C had no defect as normal control. The morphological change and bone formation were observed by methods of gross, histology and biomechanics. Results In group A1, the skull defects were almost entirely repaired by hard tissue 12 weeks after operation. The alginate gel-osteoblasts-bone granule material had changed into bone tissue with fewbone granules and some residuary alginate gel. The percentage of bone formation area was 40.92%±19.36%. The maximum compression loading on repairing tissue ofdefects was 37.33±2.95 N/mm; the maximum strain was 1.05±0.20 mm; andloading/strain ratio was 35.82±6.48 N/mm. In group A2, the alginate and bone granules material partially changed into bone tissue 12 weeks after operation. The percentage of bone formation area was 18.51%±6.01%. The maximum compression loading was 30.59±4.65 N; the maximum strain was 1.35±0.44 mm; and the loading/strainratio was 24.95±12.40 N/mm. In group B, the skull defects were mainly repaired bymembrane-like soft tissue with only few bone in marginal area；the percentage of bone formation area was 12.72%±9.46%. The maximum compression loading was 29.5±2.05 N; the maximum strain was 1.57±0.31mm；and the loading/strainratio was 19.90±5.47 N/mm.In group C, the maximum compression
loading was 41.55±2.52 N; the maximum strain was 095±017 mm; and the l
oading/strain ratio was 47.57±11.22 N/mm. 〖
WTHZ〗Conclusion〓〖WTBZ〗The plastic engineered bone prefabricated with algina
te gelosteoblastsbone granule may shape according to the bone defects and ha
s
good ability to form bone tissue, whose maximum compression loading can reach 89
%
of normal skull and the hardness at 12 weeks after operation is similar to that
of normal skull.
