Objective To investigate the effect of homograft of marrow mesenchymal stem cells (MSCs) seeded onto poly-L-lactic acid (PLLA)/gelatin on repair of articular cartilage defects. Methods The MSCs derived from36 Qingzilan rabbits, aging 4 to 6 months and weighed 2.5-3.5 kg were cultured in vitroand seeded onto PLLA/gelatin. The MSCs/ PLLA/gelatin composite was cultured and transplanted into full thickness defects on intercondylar fossa. Thirty-six healthy Qingzilan rabbits were made models of cartilage defects in the intercondylar fossa. These rabbits were divided into 3 groups according to the repair materials with 12 in each group: group A, MSCs and PLLA/gelatin complex(MSCs/ PLLA/gelatin); group B, only PLLA/gelatin; and group C, nothing. At 4,8 and 12 weeks after operation, the gross, histological and immunohistochemical observations were made, and grading scales were evaluated. Results At 12 weeks after transplantation, defect was repaired and the structures of the cartilage surface and normal cartilage was in integrity. The defects in group A were repaired by the hylinelike tissue and defects in groups B and C were repaired by the fibrous tissues. Immunohistochemical staining showed that cells in the zones of repaired tissues were larger in size, arranged columnedly, riched in collagen Ⅱ matrix and integrated satisfactorily with native adjacent cartilages and subchondral bones in group A at 12 weeks postoperatively. In gross score, group A(2.75±0.89) was significantly better than group B (4.88±1.25) and group C (7.38±1.18) 12 weeks afteroperation, showing significant differences (P＜0.05); in histological score, group A (3.88±1.36) was better than group B (8.38±1.06) and group C (13.13±1.96), and group B was better than group C, showing significant differences (P＜0.05). Conclusion Transplantation of mesenchymal stem cells seeded onto PLLA/gelatin is a promising way for the treatment of cartilage defects.
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.
ObjectiveTo investigate the ability of autologous peripheral blood endothelial progenitor cells (EPCs) in promoting neovascularization of tissue engineered bone and osteogenesis of bone marrow mesenchymal stem cells (BMSCs). MethodThe peripheral blood EPCs and BMSCs from No. 1-9 New Zealand rabbits were isolated, cultured, and identified. According to the cell types, the third generation of cells were divided into 3 groups:EPCs (group A), BMSCs (group B), and co-cultured cells of EPCs and BMSCs (group C, EPCs:BMSCs=1:2) . Then cells were seeded on the partially deproteinised bone (PDPB) packaged with fibronectin to construct tissue engineered bone. After 4 days, autologous heterotopic transplantation of tissue engineered bone was performed in the rabbit's muscles bag of groups A, B, and C (the right arm, left arm, right lower limb respectively, 2 pieces each part). At 2, 4, and 8 weeks after transplantation, the growth of tissue engineered bone was observed, and the rate of bone ingrowth was calculated by HE staining; the expressions of CD34, CD105, and zonula occludens protein 1(ZO-1) were compared by immunohistochemical staining at each time point in tissue engineered bone among 3 groups. ResultsThe EPCs and BMSCs were isolated and identified successfully; immunofluorescent staining showed that EPCs were positive for CD34, CD133, and von Willebrand factor (vWF), and BMSCs were positive for CD29 and CD90 and were negative for CD34. The tissue engineered bone constructed in 3 groups was transplanted successfully. At 2, 4, and 8 weeks after autologous heterotopic transplantation, the general observations showed that the soft tissue around the tissue engineered bone increased and thickened gradually in each group with time passing; the boundary between bone and soft tissue was not clear; the pore space of tissue engineered bone gradually was filled, especially in group C, the circuitous vascular network could be seen in the tissue engineered bone. HE staining showed capillaries and collagen fibers increased gradually, tissue engineered bone ingrowth rate was significantly higher in group C than groups A and B at 4 and 8 weeks (P<0.05) , and group B was significantly higher than group A (P<0.05) . Immunohistochemical staining showed that the expressions of CD34, CD105, and ZO-1 in tissue engineered bone of 3 groups all increased with the extension of time, showing significant differences between groups at each time point (P<0.05) . At 2 weeks after transplantation, the expression of CD105 in group C was significantly higher than that in groups A and B (P<0.05) ; at 4 and 8 weeks, CD34, CD105, and ZO-1 expressions showed significant differences between 2 groups (P<0.05) ; the expression was the highest in group C, and was the lowest in group B. ConclusionsAutologous peripheral blood EPCs and BMSCs have synergistic effect, and can promote neovascularization and osteogenesis of tissue engineered bone in vivo.
【Abstract】 Objective To search for a feasibil ity of repairing full-thickness cutaneous deficiency with tissueengineered skin substitute composited by human epidermal stem cells and fibroblasts in fibrin frame. Methods Epidermal stem cells and fibroblasts were harvested from human epidermis and dermis by trypsin digestion. Cells were cultured and subcultured in non-serum medium. Epidermal stem cells (5×104/mL) and dermal fibroblasts (1×104/mL) in 0.5 mL medium were coagulated in 0.5 mL fibrin frame to construct tissue engineered skin substitute. The tissue engineered skin substitute was grafted onto full-thickness cutaneous deficiency of nude mice. Forty-five male mice, 4-5 week old, weighted 20 g on average, were randomly divided into 5 groups. Oil yarn (group C), fibrin frame membrane without cell inoculation (group F), composite skin substitute with epidermal stem cells (group S) and composite skin substitute with fibroblasts (group Fb) were used as controls, while tissue engineered skin substitute (group T) was experimental group. The wounds were observed 1, 3, 6, 8 weeks after surgery. Samples were harvested 3, 6, 8 weeks after surgery, and were examined by means of histology、immunohistochemistryand scanning electron microscopy (SEM). Results Four weeks after cell culture, there were some round cells in the culture capsule of epidemic cells, and some fusiform cells in the culture capsule of fibroblast. Six days after cells were cultured in the BrdU culture medium, there were some BrdU positive cells appeared. There were some CK19 positive cells and Nestin positive cells appeared in the chaff of group T before transplanting. The new formed skin of group T grew faster and had less scar than other groups. Six weeks after surgery, the average thickness of new formed skin was (0.460 ± 0.049) mm in group C, (0.480 ± 0.055) mm in group F, (0.540 ± 0.043) mm in group S, (0.510 ± 0.032) mm in group Fb, (0.660 ± 0.047) mm in group T. The thickness of new formed skin in group T was thicker than other groups (P lt; 0.05). By histology and SEM observation, 3, 6, 8weeks after surgery, the new formed cuticular layer, fibroblast and blood vessels in the group T were more than those in theother groups. The al ignment of blood vessels and collagen fibers in group T were much regular than those in the other groups. Three weeks after surgery, the new formed skin of group T had a continuous color zone of positive collagen Ⅳ staining, while no continuous color zone was found in the other groups. Six weeks after surgery, CK14 positive cells appeared in the new formed skin of group T, while no positive cell was found in the other groups. Conclusion Tissue engineered skin substitute which is composited with epidermal stem cells and fibroblasts in fibrin frame has potential prospects in appl ication of repairing fullthickness cutaneous deficiency with advantage of faster wound heal ing.
Objective To evaluate the feasibility and the value of the layered cylindric collagenhydroxyapatite composite as a scaffold for the cartilage tissue engineering after an observation of how it absorbs the chondrocytes and affe cts the cell behaviors. Methods The chondrocytes were isolated and multiplied in vitro, and then the chondrocytes were seeded onto the porous collagen／h ydro xyapatite composite scaffold and were cultured in a three-dimensional environme n t for 3 weeks. The effects of the composite scaffold on the cell adhesivity, proliferation, morphological changes, and synthesis of the extracellular matrix were observed by the phase-contrast microscopy, histology, scanning electron micros copy, and immunohistochemistry. Results The pore diameter of the upper layer of the collagen-hydroxyapatite composite scaffold was about 147 μm. and the porosity was 89%; the pore diameter of the bottom layer was about 85 μm and the porosity was 85%. The layered cylindric collagenhydroxyapatite composite scaffold had good hydrophilia. The chondrocytes that adhered to the surface of the scaffold, proliferated and migrated into the scaffold after 24 hours. The chondrocytesattached to the wall of the microholes of the scaffold maintained a rounded morphology and could secrete the extracellular matrix on the porous scaffold. Conclusion The layered cylindric collagenhydroxyapatite composite scaffold has a good cellular compatibility, and it is ber in the mechanical property than the pure collagen. It will be an ideal scaffold for the cartilage tissue enginee ring.
Objective To explore the histological changes of bio-derived bone prepared by different methods after implantation, and to provide the scaffold material from xenogeneic animal for tissue engineering. Methods Theextremities of porcine femur were cut into 0.5 cm×0.5 cm×0.5 cm. Then they were divided into 5 groups according to different preparation methods: group A was fresh bone just repeatedly rinsed by saline; group B was degreased; group C was degreased and decalcificated; group D was degreased, acellular and decalcificated; group E wasdegreased and acellular. All the materials were implantated into femoral muscle pouch of rabbit after 25 kGy irradiation sterilization. The cell counting ofinflammatory cells and osteoclasts, HE and Masson staining, material degradation, collagen and new bone formation were observed at 2, 6, and 12 weeks postoperatively. Results The residue level of trace element in biomaterials prepared by different methods is in line with the standards. All the animals survived well. There were no tissue necrosis, fluid accumulation or inflammation at all implantation sites at each time point. The inflammatory cells counting was most in group A, and there was significant difference compared with other groups(P＜0.05). There was no significant difference in osteoclasts counting among all groups. For the index of HE and Masson staining, collagen and new bone formation, groups C and D were best, group E was better, and groups A and B were worse. Conclusion The degreased, acellular and decalcificated porcine bone is better in degradation,bone formation, and lower inflammatory reaction, it can be used better scaffold material for tissue engineered bone.
ObjectiveTo investigate the mechanism of muscle-derived cells (MDCs) in repairing sciatic nerve defects in mice by observing the early growth of damaged peripheral nerves.MethodsThe hind limb skeletal muscles of mice carrying enhanced green fluorescent protein (EGFP) was collected to extract and culture EGFP-MDCs to P1 generation for later experiments. Five-mm-long nerve defects were created in the right sciatic nerves of C57BL/6 mice to establish a peripheral nerve defect model. The two stumps of sciatic nerve were bridged with 7-mm-long polyurethane (PUR) conduit. For the MDC group, EGFP-MDCs were injected into the PUR conduit. The PUR group without EGFP-MDCs was used as the negative control group. At 1 and 2 weeks after operation, the proximal and distal nerve stumps of the surgical side were collected to generally observe the early growth of nerve. Immunofluorescence staining of S100β, the marker of Schwann cells, was performed on longitudinal frozen sections of nerve tissues to calculate the maximum migration distance of Schwann cells, and observe the source of the Schwann cells expressing S100β. Immunofluorescence staining of phosphorylated erb-b2 receptor tyrosine kinase 2 (p-ErbB2) and phosphorylated focal adhesion kinase (p-FAK) in transverse frozen sections of nerve tissue was performed to calculate the positive rates of both proteins.ResultsThe general observation showed that the proximal and distal stumps of the surgical side in PUR group were not connected at 1 and 2 weeks after operation, while the bilateral nerve stumps in the MDC group were connected at 2 weeks after operation. Immunofluorescence staining showed that the Schwann cells expressing S100β in proximal and distal nerve stumps of PUR group and MDC group was not connected at 1 week after operation. At 2 weeks after operation, the Schwann cells expressing S100β in the two nerve stumps of the MDC group were connected, but not in the PUR group. At 2 weeks after operation, the sum of the maximum migration distance of Schwann cells in the regenerated nerve in both two groups was significantly increased when compared with that in each group at 1 week after operation, and that of MDC group was significantly higher than that in the PUR group at both 1 and 2 weeks after operation, the differences were all significant (P<0.05). At 1 week after operation, the positive rates of p-ErbB2 and p-FAK in the proximal nerve stump of MDC group were significantly higher than those in PUR group (P<0.05). There was no significant difference in the positive rate of p-ErbB2 of proximal stump between the two groups at 2 weeks after operation (t=0.327, P=0.747), while the positive rate of p-FAK of MDC group was significantly higher than that of PUR group (t=4.470, P=0.000). At 1 and 2 weeks after operation, the positive rates of p-ErbB2 and p-FAK in the distal stump of MDC group were significantly higher than those in PUR group (P<0.05). At 1 and 2 weeks after operation, part of Schwann cells expressing S100β, which were derived from EGFP-MDCs, could be observed in the regenerated nerves of MDC group.ConclusionMDCs can promote the phosphorylation of ErbB2 and FAK in the nerve stumps of mice, and promote the migration of Schwann cells. MDCs can be differentiated into cells expressing the Schwann cell marker S100β, or as other cellular components, to involve in the early repair of peripheral nerves.
Objective Mechanical stimulation and inductive factors are both crucial aspects in tissue engineered cartilage. To evaluate the effects of mechanical stimulation combined with inductive factors on the differentiation of tissue engineered cartilage. Methods Bone marrow mesenchymal stem cells (BMSCs) were isolated from newborn porcine (aged7 days and weighing 3-6 kg) and expanded in vitro. The BMSCs at passage 2 were seeded onto a scaffold of poly (lactic-coglycol ic acid) (PLGA) in the concentration of 5 × 107/mL to prepare cell-scaffold composite. Cell-scaffold composites were cultivated in a medium with chondrocyte-inducted factors (group A), in a vessel with mechanic stimulating only (group B), or mechanic stimulating combined with chondrocyte-inducted factors (group C) (parameters of mechanics: 1 Hz, 0.5 MPa, and 4 hours/day). Cell-scaffold composite and auto-cartilage served as positive control (group D) and negative control (group E), respectively. After 4 weeks of cultivation, the thickness, elastic modulus, and glycosaminoglycan (GAG) content of composites were measured. Additionally, BMSCs chondrogenic differentiation was assessed via real-time fluorescent quantitative PCR, immunohistochemistry, and histological staining. Results The thickness, elastic modulus, and maximum load in group C were significantly higher than those in groups A and B (P lt; 0.05). In groups A, B, and C, cartilage lacuna formation, GAG expression, and positive results for collagen type II were obsersed through HE staining, Safranin-O staining, and immunohistochemistry staining. The dyeing depth was deeper in group A than in group B, and in group C than in groups A and B; group C was close to group E. The GAG content in group C was significantly higher than that in groups A and B (P lt; 0.05). Real-time fluorescent quantitative PCR revealed that mRNA expressions of collagen type I, collagen type II, and GAG in group C were significantly higher than those in groups A and B (P lt; 0.05), and in group A than in group B (P lt; 0.05). Conclusion Mechanical stimulation combined with chondrocyte inductive factors can enhance the mechanical properties of the composite and induce higher expression of collagen and GAG of BMSCs.
Objective Tissue engineering advance in supplying the reparative and reconstructive medicine with promising tissue engineered medical products(TEMPs) and the new therapy alternative. The related supervision and administration of TEMPs is being developed and the standard research of TEMPs is also in progress. The Food and Drug Administration(FDA) of the United States has treated TEMPs as combined products and supervised them according to the level of risk to patients. Lately, FDA has determined that the Center for Devices and Radiological Health (CDRH) should take charge of examination and approval of TEMPs, with the cooperation of the Center for Biological Evaluations and Research(CBER). The regulatory controls have been established respectively in European Union and Japan. In China, TEMPs are identified as medical devices combined with cells. The Department of Medical Device of the State Food and Drug Administration (SFDA) is responsible for the examination and approval of TEMPs, and National Institute for the Control of Pharmaceutical amp; Biological Products(NICPBP) is responsible for evaluation tests. The standards of TEMPs are formulated mainly by the American Society of Testing Materials(ASTM) and International Standardization Organization(ISO).
OBJECTIVE: To evaluate the result of clinical application in the repair of coracoclavicular ligament injury by tissue engineered tendon using the technique of short tandem repeat loci examination. METHODS: In september 1999, human embryonic tendon cells and artificial materials were co-cultured in vitro to construct tissue engineered tendon, which repaired coracoclavicular ligament injury. After 6 months of operation, micro-tissue were sampled during the operation of removal of internal fixation, and morphological characteristics were examined by HE staining, DNA of tissues were extracted to examine D3S1754 and Cyar04 gene loci. RESULTS: The shoulder function of the patient was recovered well after operation, and no local or systemic immunological rejection were occurred. The electrophoresis typing showed 13/14 at D3S1754 and 8/9 at Cyar04 in the tissue of tissue engineered tendon, while the autogenous ligament were 13/13 and 8/8 at D3S1754 and Cyar04 loci respectively, which suggested that the tissue engineered tendon was survived in vivo. CONCLUSION: The examination of short tandem repeat loci is a better index to evaluate the survival situation of tissue engineered tissue after transplantation in clinical application.