OBJECTIVE: To fabricate artificial human skin with the tissue engineering methods. METHODS: The artificial epidermis and dermis were fabricated based on the successful achievements of culturing human keratinocytes(Kc) and fibroblasts (Fb) as well as fabrication of collagen lattice. It included: 1. Culture of epidermal keratinocytes and dermal fibroblasts: Kc isolated from adult foreskin by digestion of trypsin-dispase. Followed by comparison from aspects of proliferation, differentiation of the Kc, overgrowth of Fb and cost-benefits. 2. Fabrication of extracellular matrix sponge: collagen was extracted from skin by limited pepsin digestion, purified with primary and step salt fraction, and identified by SDS-PAGE. The matrix lattice was fabricated by freeze-dryer and cross-linked with glutaraldehyde, in which the collagen appeared white, fibrous, connected and formed pores with average dimension of 180 to 260 microns. 3. Fabrication artificial human skin: The artificial skin was fabricated by plating subcultured Kc and Fb separately into the lattice with certain cell density, cultured for one week or so under culture medium, then changed to air-liquid interface, and cultured for intervals. RESULTS: The artificial skin was composed of dermis and epidermis under light microscope. Epidermis of the skin consisted of Kc at various proliferation and differentiation stages, which proliferated and differentiated into basal cell layer, prickle cell layer, granular layer, and cornified layer. Conifilament not only increased in number, but also gathered into bundles. Keratohyalin granules at different development stages increased and became typical. The kinetic process of biochemistry of the skin was coincide with the changes on morphology. CONCLUSION: Tissue engineered skin equivalent has potential prospects in application of repairing skin defect with advantages of safe, effective and practical alternatives.
Objective To investigate the possible mechanism of the fibroblasts inducing the vascularization of dermal substitute. Methods Fibroblasts were seeded on the surface of acellular dermal matrix and cultivated in vitro to construct the living dermal substitute. The release of interleukin 8 (IL 8) and transfonming growth factor β 1(TGF β 1) in culture supernatants were assayed by enzyme linked immunosorbent assay, the mRNA expression of acid fibroblast growth factor (aFGF) and basic fibroblast growth factor (bFGF) were detected by RT-PCR. Then, the living substtute was sutured to fullth ickness excised wound on BALBouml;C m ice, and the fate of fibroblast w as observed by using in situ hybridizat ion. Results Fibroblasts cultured on acellular dermalmat rix p ro liferated and reached a single2layer confluence. Fibroblasts could secret IL 28 (192. 3±15. 9) pgouml;m l and TGF-B1 (1. 105±0. 051) pgouml;m l. There w as the mRNA exparession of aFGF and bFGF. Fibroblasts still survived and proliferated 3 weeks after graft ing. Conclusion Pept ides secreted by fibroblasts and its survival after graft ing may be relat ive to the vascularizat ion of the dermal subst itute.
Objective To comment on the recent advances of production and application of the bio-derived scaffold in the tissue engineered peripheral nerve. Methods The recent articles were systematically analyzed, and then the production methods of the bio-derived scaffold and its application to the tissue engineered peripheral nerve were evaluated and prospected. Results B iological tissues were processed by some methods to produce the bio-derived materials. These mat erials could maintain the structure and components of the tissues. Moreover, the immunogenicity of these materials was reduced. Conclusion Application of the bio-derived materials is a trend in the fabricating scaffold of the tissue en gineered peripheral nerve.
OBJECTIVE: From the point of view of material science, the methods of tissue repair and defect reconstruct were discussed, including mesenchymal stem cells (MSCs), growth factors, gene therapy and tissue engineered tissue. METHODS: The advances in tissue engineering technologies were introduced based on the recent literature. RESULTS: Tissue engineering should solve the design and preparation of molecular scaffold, tissue vascularization and dynamic culture of cell on the scaffolds in vitro. CONCLUSION: Biomaterials play an important role in the tissue engineering. They can be used as the matrices of MSCs, the delivery carrier of growth factor, the culture scaffold of cell in bioreactors and delivery carrier of gene encoding growth factors.
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 lt; 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 study the expression of type I collagen and its receptor system-integrin alpha 2 beta 1 in different passages of osteoblasts. METHODS: The expression of type I collagen and integrin alpha 2 beta 1 in the primary, sixth and fifteenth passage of osteoblasts were detected by S-P immunohistological staining technique, and their mRNA expression by quantity RT-PCR technique. RESULTS: Type I collagen and integrin alpha 2 beta 1 were expressed in different passages of osteoblasts and there was no significant difference among three passages by immunohistological technique. Their mRNA expression was gradually decreased with subculture. CONCLUSION: Type I collagen promotes the adhesion and phenotype expression of osteoblasts through its receptor-integrin alpha 2 beta 1. The reductive expression of type I collagen-receptor system will decline the phenotype of osteoblasts.
OBJECTIVE: To evaluate the cellular compatibility of three natural xenogeneic bone derived biomaterials. METHODS: Three types of natural xenogeneic bone derived biomaterials were made with physical and chemical treatment, composite fully deproteinized bone(CFDB), partially deproteinized bone(PDPB) and partially decalcified bone(PDCB). Three types biomaterials were cocultured with human embryonic periosteal osteoblasts. The cell growth, attachment, cell cycle, alkaline phosphatase activity were detected to evaluate the cellular compatibility to biomaterials. RESULTS: Osteoblasts attached on all three biomaterials and grew well, the effect of three biomaterials on cell proliferation was PDCB gt; PDPB gt; CFDB. The cell cycle was not obviously affected by three biomaterials. The effect of three biomaterials on alkaline phosphatase activity of osteoblasts was PDCB gt; PDPB gt; CFDB. CONCLUSION: CFDB,PDPB,PDCB have good cellular compatibility without cytotoxic and tumorigenicity, CFDB is the best. The three biomaterials can be used as scaffold materials of bone tissue engineering.
Objective To construct a tissue engineering skin containing capillary-like network by employing tissue engineering method.Methods The numan umbilical vein endothelial cells(HUVECs) were isolated from a new-born umbilical cord. The keratinocytes and dermal fibroblasts were isolated from a new-born foreskin biopsy. After thecollagen gelwas prepared, the fibroblasts and the vascular endothelial cells were added in a ratio of 1 to 1 to construct a skin substitute containing capillary-like network. The skin substitute was observed by HE staining and immuno histochemical staining (Ⅷ factor). The reconstructed skin containing capillary-like network was used to repair the nude mice skin defects in the experimental group. The tissue engineering skin containing no vascular endothelial cells was used in control group.Results Capillary-like network could be observed inthe dermal layer of the tissue engineering skin, and the nude mice skin defectswere repaired by the skin substitutes in the experimental group. In control group, no capillary-like network was found.Conclusion The tissue engineering skin containing capillary-like network is successfully constructed in vitro and can be used to repair the full-thickness skin defects.
Objective To study the biological behavior of osteoblast and vascular endothelial cell culture. Methods The osteoblasts and vascular endothelial cells were obtained from calvarial bone and renal cortox of 2-week rabbits respectively. The experiment were divided into group A (osteoblasts), group B (vascular endothelial cells) and group C(co-cultured osteoblasts and vascular endothelial cells). The cells were identified with cytoimmunochemical staining. The cellular biological behavior and compatibilitywere observed under inverted phase contrast microscope and with histological staining. The cells viability and alkaline phosphatase(ALP) activity were measured. Results The cytoimmunochemical staining showed that the cultured cells were osteoblasts and vascular endothelial cells .The cellular compatibility of osteoblasts and vascular endothelial cells was good. The ALP activity was higher in group C than in group A and group B(P＜0.01), and it was higher in group A than in group B(P＜0.05). In group C, the cellproliferation were increased slowly early, but fast later. Conclusion Thecellular compatibility of osteoblasts and vascular endothelial cells were good. The vascular endothelial cells can significantly increased the osteoblast viability and ALP activity,and the combined cultured cells have greater proliferation ability.
Objective To explore the possibilityof constructing tissue engineering muscles by combining allogeneic myoblasts with small instestinal submucosa(SIS) in rabbits.Methods A large number of purified myoblasts were obtained with multiprocedure digestion and repeated attachment method from skeletal muscles taken from extremities of immature rabbits which were born 7 days ago. The myoblasts were labeled with BrdU, and then combined with SIS to construct tissue engineering muscles. This kind of tissue engineering muscles were grafted into the gastrocnemius muscle defect (1.5 cm in length, 1.0 cmin width) of fifteen rabbits as the experimental group. The SIS was grafted into the same position in the control group. The rabbits were sacrificed 4, 6, 8 weeks after operation. The tissue engineering muscles were evaluated by macroscopic、histological and immunohistochemical observations, and by quantitative analysis of local immunocyte in the grafting site. Results Allogeneic myoblasts with SIS were combined perfectly in vitro. The SIS was connected tightly to surrounding skeletal muscles and inflammation response was obvious 4 weeks after grafting.The SIS began to break down and inflammation response became slight 6 and 8 weeks after operation. Compared with that of 8th week, the quantitative analysis oflocal immunocyte in 4th and 6th week in both experimental and control group hassignificance(Plt;0.05). Newly formed muscle tissues were found around SIS in the experimental group in 4th, 6th, and 8th week. Expression of BrdU and myosin immunohistochemical staining were positive in the experimental group and negative inthe control group.Conclusion Tissue engineering muscles of rabbits which are constructed by combining allogeneic myoblasts with SIS can survive and proliferate.