Search

Objective: To prepare human acellular amniotic membrane (HAAM) and to measure its cytocompatibility and biocompatibility., Methods: HAAM were prepared by chemical detergent-enzymatic extraction. Fresh human amnion was crosslinked with glutaradehyde, shaken in 0.5% SDS for 24 hours, and then treated with 0.25% trypsin for 4 hours. The production were freeze-drying and sterilized using ethylene oxide. Human fibroblasts were isolated from embryo and expanded in vitro. The fibroblasts were seeded in HAAM. HAAM and specimen were stained with HE and Mallory, and observed grossly, under light microscopy and scanning electron microscopy. The HAAM were implanted in the back of SD rats., Results: There were no residues of cells in the HAAM (HE, Mallory staining). One side of HAAM had reticular and porous structure, the other side had compact fibrous structure. Pore size was from 10 to 80 nm. The HAAM could be seeded with expanded fibroblasts in vitro, and fibroblasts had the potential of spread and proliferation. The SD rat in the implant test had no death, convulsions and other abnormal response., Conclusion: The detergent-enzymatic extraction process can remove cells and solvable components effectively and preserve the tissue matrix well and keep the reticular structure. The HAAM can be used as an ideal scaffold of biological membrane for tissue engineering.

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 bio-derived bone in vitro, and the 20 mm tibia defect of goat was made and fixed with plate. Eighteen goats were divided into experimental group, control group and blank group. The defects were not filled with anything in blank group, with tissue engineering bone in experimental group and bio-derived bone in control group. The repair capability was assessed by physical, X-ray and bone mineral density examinations 8, 12, 16, and 24 weeks after operation., Results: In experimental group, the defects were partially repaired 8 weeks, and completely repaired 12 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 24 weeks., 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 bone formation.

Objective: To study the research method of cell survival rate at the procedure of cryopreservation of tissue engineered tendons., Methods: In the 4th generation of human fibroblasts, the dead cells were stained with propidium iodine (PI), while the living cells with Hoechst 33342 (Ho). The living cells and dead cells emitted fluorescence of red and blue respectively after they were stimulated by suitable ultra-violet, then flow cytometry was applied to distinguish them. The seeding cells were collected to make them to be the cell suspension of suitable concentration (6.0 x 10(5) cell/ml) before they were divided into two parts. We cryopreserved and defrosted one part three times to kill the cells and did not cryopreserve the other part, then we made cell suspension at different ratios of cryopreserved cell to non-cryopreserved cells. The fluorescence staining and flow cytometry were used to study the correlation between cell ratios of cryopreserved cell to non-cryopreserved cell and the cell survival rates. We compared the cell survival rates between immediate flow cytometry and that 2 hours after fluorescence staining., Results: The results of flow cytometry showed that correlation between the ratio of cryopreservation and the cell survival rate was significant (r = 0.970, P < 0.05), image analysis study also showed the correlation was significant (r = 0.982, P < 0.05). The cell survival rate decreased by use of flow cytometry two hours after fluorescence staining, but there was no significant difference when compared with that of immediate flow cytometry (P > 0.05). We could also observe the cells on the tissue engineered tendons by fluorescence image directly., Conclusion: Flow cytometry and fluorescence image after PI and Ho staining is a good way in study cell survival rate at the procedure of cryopreservation of tissue engineered tendons.

Objective: To review the research advance of the preparation and characteristics of small intestinal submucosa(SIS)., Methods: Recent original articles related to such aspects of small intestinal submucosa were reviewed extensively., Results: Small intestinal submucosa was an easily obtained biomaterial. SIS was a bio-absorbable and degradable material. SIS had tissue specific regeneration properties., Conclusion: SIS is a suitable bio-derived material for tissue engineering of blood vessel, muscle tendon, urinary bladder and abdomen.

Objective: To construct tissue engineering bone with bio-derived materials and bone marrow stromal cells (MSCs), and to investigate the effect of allogeneic engineering bone implants on healing of segmental bone defects., Methods: MSCs being aspirated aseptically from tibial tuberosities of young rhesus monkeys were induced into osteoblasts in vitro and then were cultured and marked with 5-bromo-2-deoxyuridine (BrdU). Tissue engineering bones were constructed with these labeled osteoblasts being seeded onto bio-derived materials made from fresh human bones which were treated physically and chemically, Then the constructs were implanted in 15 allogeneic monkeys to bridge 2.5 cm segmental bone defects of left radius as experimental groups, bio-derived materials only were implanted to bridge same size defects of right radius as control group. and, 2.5 cm segmental bone defects of both sides of radius were left empty in two rhesus monkeys as blank group. Every 3 monkeys were sacrificed in the 1st, 2nd, 3rd, 6th and 12th weeks postoperatively and both sides of the implants samples were examined macroscopically, histologicaly, and immunohistochemicaly. The two monkeys in blank group were sacrificed in the 12th week postoperatively., Results: Apparent inflammatory reactions were seen around both sides of the implants samples in the 1st, 2nd, 3rd weeks, but it weakened in the 6th week and disappeared at the 12th week. The labeled osteoblasts existed at the 6th week but disappeared at the 12th week. The bone defects in experimental group were repaired and the new bone formed in multipoint way, and osteoid tissue, cartilage, woven bone and lamellar bone occurred earlier when compared with control group in which the bone defects were repaired in 'creep substitution' way. The bone defects in blank group remained same size at the 12th week., Conclusions: Engineering bones constructed with bio-derived materials and MSCs were capable of repairing segmental bone defects in allogeneic monkeys beyond 'creep substitution' way and making it healed earlier. Bio-derived materials being constituted with allogeneic MSCs may be a good option in construction of bone tissue engineering.

Objective: To investigate the feasibility of repairing the whole layer defects of tibial plateau by implanting tissue-engineering cartilage., Methods: The chondrocytes of 2-week-old rabbits were cultured and transferred to the 3rd generation, and mixed with human placenta collagen-sponge. The whole layer defects of tibial plateau in adult rabbits were repaired by the tissue-engineering cartilage in the experimental group; the defects were left un-repaired in control group. The repair results of defects were observed after 4, 12 and 24 weeks., Results: In experimental group, no obvious new cartilage formation was seen 4 weeks after operation; some new cartilage formation was found after 12 weeks. Histological observation showed that chondrocytes had irregular edge, honeycombing structure and that cartilage cavities formed around the chondrocytes. After 24 weeks, obvious new cartilage formation was found with smooth surface, and linked with the tissues around it, but the defect was not repaired completely; histological results showed that cartilage cavities formed and that cartilage matrix was stained positively for toluidine blue. In control group, the defect was not repaired., Conclusion: The tissue-engineering cartilage can repair the defects of the whole layer cartilage of tibial plateau in rabbits, it is feasible to repair the whole layer cartilage defects of tibial plateau by this method.

Objective: To investigate possibility of cartilage cultured in centrifuge tube as graft materials., Methods: Articular chondrocytes isolated from a 3-week-old rabbit formed cartilage after cultivation for 2 weeks. Articular cartilage of humeral head, growth plate of proximal tibia and meniscus were collected from a 6-week-old rabbit. The ultrastructure of chondrocytes and extracellular matrix in the three kinds of cartilages and cultured cartilage were observed by transmission electronic microscopy., Results: Cartilage cultured in centrifuge tube possessed unique ultrastructure and was similar to articular cartilage and growth plate, but it was markedly different from meniscus. The four kinds of cartilages were characteristic of respectively different chondrocytes and extracellular matrix. Cultured cartilage showed typical apoptosis of chondrocytes and "dark chondrocytes" appeared in growth plate. Condrocyte apoptosis was not seen in articular cartilage and meniscus., Conclusion: Cartilage cultured in centrifuge tube has unique ultrastructure and may be used as graft materials for articular cartilage and growth plate.

Objective: To investigate the effects of three-dimentional culture in bioderived material modified by Pluronic F-127 on the growth and function of rabbit periosteal osteoblast in vitro., Methods: Bio-derived materials were from fresh pig ribs and were modified by Pluronic F-127. Then rabbit periosteal osteoblasts were cultured in bio-derived materials(group A), in the modified bio-derived materials(group B) and on the plastic surfaces as a control (group C), respectively. During a 7-day period, the status of growth, cell viability and alkaline phosphatase(ALP) activity were measured., Results: Osteoblasts attached, elongated and grew well on the modified bio-derived materials. There were no significant difference in osteogenesis and ALP activity between group A and group B(P > 0.05). The osteogenesis and ALP activity in groups A, B were less than those in group C (P < 0.01)., Conclusion: Pluronic F-127 can be used for a carrier for bioactive factors to modify bio-derived material.

Objective: To investigate a cryophylactic agent (CPA) to protect tissue engineered tendon (TET) in deep low temperature., Methods: Sixty-four BALB/C inbred nude mice were chosen, which included 4 as blank control group, left sides of 60 as experimental group and their right sides as control group. Transformed human embryonic tendon cells of the 54th passage and artificial materials of carbon fiber (CF) and polyglycolic acid (PGA) were co-cultured in vitro to construct TET. TET was frozen in liquid nitrogen with four kinds of CPA (groups A, B, C, and D) for 2 months. They were thawed quickly and transplanted into hind limbs of nude mice to repair the defects of Achilles tendon, which was 5 mm in length and 65.7% of total Achilles tendon. In control group, no cryopreservation treatment was taken. The morphological, histological, ultrastructure, and immunohistochemistry examinations were made and short tandem repeat loci were detected 2, 4, 6, 8, and 12 weeks later., Results: In the experimental group, the morphological properties of tendon cells resumed gradually and the capability of synthesizing collagen enhanced by degrees. Tendon cells survived and could secret type I collagen and there was less difference between experimental and control groups 12 weeks after transplantation. In group A, vacuole in mitochondrion of tendon cell decreased, tendon cell arranged in order and abundant collagen fibers were found and linked., Conclusion: The cryopreservation agent in group A can protect TET in deep low temperature.

Objective: To determine an optimal co-culture ratio of the rabbit periosteal osteoblasts (RPOB) and rabbit renal vascular endothelial cells(RRVEC) without direct contact for future study of bone tissue engineering., Methods: RPOB and RRVEC in the ratios of 1:0(control group), 2:1(group 1), 1:1(group 2) and 1:2(group 3) were co-cultured by six well plates and cell inserts. Four days later, the proliferation of RPOB and RRVEC were examined through cell count. Differentiated cell function was assessed by alkaline phosphatase (ALP) activity assay and 3H proline incorporation assay., Results: When RPOB and RRVEC were indirectly co-cultured, the proliferation of RPOB and 3H proline incorporation was higher in group 1 than in the other experimental groups and control group (P < 0.05). ALP activity of RPOB was higher in group 1 than in control group and group 3 (P < 0.05), but there was no significant difference between group 1 and group 2 (P > 0.05)., Conclusion: These results suggest that RPOB and RRVEC co-cultured in a ratio of 2:1 is optimal for future study of bone tissue engineering.

Objective: To sum up the clinical results of bio-derived bone transplantation in orthopedics with tissue engineering technique., Methods: From January 2000 to May 2002, 52 cases with various types of bone defect were treated with tissue engineered bone, which was constructed in vitro by allogeneous osteoblasts from periosteum (1 x 10(6)/ml) with bio-derived bone scaffold following 3 to 7 days co-culture. Among them, there were 7 cases of bone cyst, 22 cases of non-union or malunion of old fracture, 15 cases of fresh comminuted fracture of bone defect, 4 cases of spinal fracture and posterior route spinal fusion, 3 cases of bone implant of alveolar bone, 1 case of fusion of tarsotarsal joint. The total weight of tissue engineered bone was 349 g in all the cases, averaged 6.7 g in each case., Results: All the cases were followed up after operation, averaged in 18.5 months. The wound in all the case healed by first intention, but 1 case with second intention. Bone union was completed within 3 to 4.5 months in 50 cases, but 2 cases of delayed union. Six cases were performed analysis of CD3, CD4, CD8, ICAM-1 and VCAM-1 before and after operation, and no obvious abnormities were observed., Conclusion: Bio-derived tissue engineered bone has good osteogenesis. No obvious rejection and other complications are observed in the clinical application.

Objective: To review the recent research progress of bone-marrow stromal stem cells (BMSCs) in the conditions of culture in vitro, chondrogenic differentiation, and the application in cartilage tissue engineering., Methods: Recent original articles related to such aspects of BMSCs were reviewed extensively., Results: BMSCs are easy to be isolated and cultivated. In the process of chondrogenesis of BMSCs, the special factors and interaction between cells are investigated extensively. BMSCs have been identified to form cartilage in vivo. One theory is the committed chondrocyte from BMSCs is only a transient stage., Conclusion: BMSCs are the alternative seeding cells for cartilage tissue engineering. The conditions promoting mature chondrocyte should be further investigated.

Objective: To analysis the biological characteristics of human fibroblasts transfected by human telomerase reverse transcriptase (hTERT) eucaryotic expression plasmid pGRN145., Methods: Fibroblasts from children's foreskin were isolated and cultured in vitro, and the fibroblasts were transfected by pGRN145 with Lipofec-tAMINE PLUS Reagent. After strict screening of hygromycin B, the positive clones were subcultured. The telomerase activity was detected by RT-PCR and TRAP-PCR technique. The cell generation cycle and apoptosis rate were detected by flow cytometry to investigate the proliferative characteristics after transfection, and the chromosome karyotype of transformed cells was analyzed. The collagen secreted by transformed cells was detected by immunohistochemical staining., Results: The morphological properties of fibroblasts did not change obviously after transfection. There were telomerase activity in transfected fibroblasts, while it could not be detected in pre-transfection fibroblasts. The cell generation cycle had no obvious changes between pre-transfection and post-transfection. However, the apoptosis rate of transfected fibroblasts were decreased compared with that of pre-transfection. The fibroblasts transfected by pGRN145 maintained the normal diploid karyotype, as well as the cells could normally secret type I and III collagen., Conclusion: The human fibroblasts transfected by pGRN145 has telomerase activity with prolonged life span of culture, which preliminarily proves the availability of establishing standard seeding cell lines of tissue engineering by hTERT plasmid transfection techniques.

Objective: To present a surgical choice for nonunion and bone defect., Methods: From November 1994 to October 1997, 17 cases of nonunion of fracture and massive bone defect were treated by autogenous iliac bone and fibular bone with vascular anastomosis. Of 17 cases, there were 10 cases of nonunion of bone fracture, 7 cases of bone defect following tumor resection (4 cases of benign and 3 cases of malignant). Autogenous fibular bone grafting with vascular anastomosis, ranging from 12 cm to 29 cm in length, were employed in 12 cases; autogenous iliac bone grafting, ranging from 7 cm x 3 cm to 9 cm x 5 cm in size, were utilized in the other 5 cases. All of 17 cases were followed up for 10 months to 5 years, 3 years and 7 months in average, and were evaluated from clinical manifestation., Results: Bone union was achieved in 10 cases of nonunion of fracture after bone grafting, bony refilling of the bone defect was observed in 4 cases due to benign tumor and 1 case due to malignant tumor. The other 2 cases of malignant tumor died from lung metastasis of the tumor 10 months and 12 months after bone grafting, respectively., Conclusion: Bone grafting is an effective surgical option in treatment of nonunion of fracture and bone defect.

Objective: To study the expression of interleukin-1 receptor in normal mice sciatic nerves and in transected sciatic nerves treated with or without extrinsic interleukin-1 locally at different periods., Methods: Seventy-two KM mice were equally divided into two groups. All the left sciatic nerves were transected. The stumps in experimental group were soaked in liquid with interleukin-1, whereas those in control group without interleukin-1. Then all the stumps were repaired end to end. At the 3rd hour, 1st day, 3rd day, 7th day, 14th day, and 28th day after operation respectively, every proximal stump was dissected and the expression of interleukin-1 receptor was carried out by immunohistochemistry method (LSAB method). The expression level of interleukin-1 of ten normal sciatic nerves of mice was studied, too., Results: Normal nerves showed interleukin-1 receptor expression on the membrane of Schwann cells. After nerve injury, the interleukin-1 receptor expression increased biphasically in both groups, but the intensity of increase was lower in the experimental group than in the control group., Conclusion: Schwann cell is the target cell of interleukin-1.

Objective: To research the protective effect of Schwann cell and extracellular matrix (ECM) gel on neurons in dorsal root ganglion., Methods: 1. Schwann cells were seeded into 30% ECM at 1 x 10(8)/ml and then implanted into PLA hollow fiber conduits to repair 10 mm length defects of rat sciatic nerve, and histological observation was taken at 8 and 12 weeks after operation. 2. To observe the survival of Schwann cells, Schwann cells labeled BrdU were seeded into 30% ECM at 1 x 10(8)/ml and then implanted into PLA hollow fiber conduits to repair 10 mm length defects of rat sciatic nerve. Histological observation and immunohistochemical method stained with BrdU were done at 3 and 6 weeks after operation., Results: 1. When seeded into ECM gel and transplanted into rats, most of the Schwann cells survived to 3 weeks and a part of them survived up to 6 weeks. 2. The survival neuron ratios of Schwann cells with ECM gel group and ECM gel group were 83.5% and 81.3% respectively, and significantly higher than that of saline group (72.9%, P < 0.05)., Conclusion: When seeded into ECM gel and transplanted into rats, most of the Schwann cells survive and protect 83.5% neurons in dorsal root ganglion from retrograde death.

Objective: To investigate the feasibility of freeze-dried demineralized bone matrix (FDBM) as scaffold material in bone tissue engineering., Methods: Osteoblasts which were isolated from cranial periosteum of New Zealand rabbits were cultured as the seeding cells, then the cells were cocultured with heterogenous FDBM in vitro. The cell-material complex was observed under phase microscope, light microscope and electronic scanning microscope in order to evaluate the interaction between cells and FDBM., Results: Eight hours after coculture, the osteoblasts adhered to FDBM scaffolds. Seven days later, the osteoblasts differentiated and proliferated in FDBM network. Extracellular matrix was secreted and calcium nodes were formed among osteoblasts., Conclusion: FDBM is a good scaffold material for the bone tissue engineering.