Cell sheet engineering is an important technology to harvest the cultured cells in the form of confluent monolayers using a continuous culture method and a physical approach. Avoiding the use of enzymes, expended cells can be harvested together with endogenous extracellular matrix, cell-matrix contacts, and cell-cell contacts. With high efficiency of cell loading ability and without using exogenous scaffolds, cell sheet engineering has several advantages over traditional tissue engineering methods. In this article, we give an overview on cell sheet technology about its applications in the filed of tissue regeneration, including the construction of soft tissues (corneal, mucous membrane, myocardium, blood vessel, pancreas islet, liver, bladder and skin) and hard tissues (bone, cartilage and tooth root). This techonoly is promising to provide a novel strategy for the development of tissue engineering and regenerative medicine. And further works should be carried out on the operability of this technology and its feasibility to construct thick tissues.
Cells, Cultured ; Extracellular Matrix ; Humans ; Regenerative Medicine ; Tissue Culture Techniques ; Tissue Engineering

The marrow stromal cells of adult New Zealand rabbits cultivated and induced in vitro were used to form MCCAB by mixing,seeding and solidifying methods with the aid of alginate. The MCCABs were auto-transplanted intramuscularly into the rabbits for 4 to 8 weeks. The alginate-cancellous bone matrix composites or the cancellous bone matrix alone were implanted as control. The effectiveness of bone formation was assessed by means of roentgenography and histology.The results showed that the osteogeneses of MCCABs were better than those of the alginate-cancellous bone matrix composites and of the cancellous bone matrix. In the MCCABs, both intramembranous and cartilaginous osteogenesis were seen with the former predominating. In the control, only slight cartilaginous osteogenesis was seen. The results suggested that the osteogenesis of the MCCABs constructed by using tissue engineering method was obvious when transplanted intramuscularly, therefore, this kind of tissue-engineered bone could be an effective way for clinical application.

To investigate the feasibility of using coral and other materials as scaffolds for bone tissue engineering, coral, coral hydroxyapatite(CHA), cancellous bone matrix and other natural biomaterials served as culture scaffolds of osteoblasts were manufactured. The results showed, in addition to PLA, PGA, PLGA and other synthetic polymers, some natural biomaterials are also ideal scaffolds materials for bone tissue engineering.

砄bjective: To study the feasibility of fabrication of trachea cartilage ring by tissue engineering.Methods : PGA non woven mesh was put into the mold of trachea cartilage ring and enforced with polylactic acid. Rabbit chondrocytes were harvested by collagenase type Ⅱdigestion of ear cartilage and seeded into PGA scaffold in the density of 5?10 7/ml.The cell polymer complexes were incubated in vitro for 1 week and then implanted subcutaniously into the back of nude mice. The formation of trachea cartilage ring was observed by gross inspection and histological examination 2 months after implantation. Results: New cartilage tissue in the shape of trachea ring was found 2 months after implantation. The specimens showed the appearance of glisteringly white with good flexibility. Histological examination demonstrated that newly restored tissue was constituted of cartilage. Conclusion: It may be an efficient method to fabricate trachea cartilage ring by seeding chondrocytes in PGA scaffold.

砄bjective: To fabricate tissue engineered bone cartilage composite. Method: Rabbit marrow stem cells (MSCs) were in vitro cultured, expanded and induced to differeciate to osteoblasts. Chondrocytes were obtained by collagenase type Ⅱ digestion of rabbit ear cartilage. Osteoblasts and chondrocytes were co seeded into different part of natural coral scaffold, and then implanted subcutaneously into the back of nude mice. Two months after implantation,the specimens were harvested and bone cartilage composites formation was observed by gross inspection and histologic observation. Results: The newly formed tissue was composed of two parts. One part was glisteringly white and another part was dark red. There was an obvious boundary between the two parts. Microscopic observation revealed successful restoration of bone cartilage composite. Conclusion:Bone cartilage composite can be prepared by co deeding of osteoblasts and chondrocytes into natural coral scaffold.

?Objective: To study the methodology of the culture of Schwann cells derived from degenerated peripheral nerve. Method: Sciatic nerve of adult rats was surgically cut. 14 days after operation, the degenerated nerve tissue was obtained and treated with trypsin and collagenase typeⅡ to prepare single cell suspension,the cells were purified by different speed of attachment and digestion, and incubated on ZQ membrane in the presence of 10 -5 mol/L cytosine arabinoside. The growth of the cells of passage 2 was studied by MTT assay. Schwann cells were identified with anti S100 immumohistochemistry. Results: The cultured cells were spindly in shape and 95% of them were S100 positive. The population doubling time of passage 2 cells was 72 h.The cells attached and stretched on ZQ membrane as well as on the culture vessel surface. Conclusion: Schwann cells can be cultured and purified by predegeneration of the peripheral nerve,different speed of attachment and digestion and the presence of cytosine arabinoside. The cells can grow well on ZQ membrane.

objective: To develop injectable tissue engineered bone through injection of osteoblasts/alginate composite in rabbits. Methods: Bone marrow cells isolated from iliac bone of New Zealand rabbits were cultured and induced to differentiate into osteoblasts.The osteoblasts were mixed with 25 g/L sodium alginate solution to generate osteoblasts/alginate composite with final cellular density of 5?10 6/ml. 0.17 g of sterilized CaSO 4 powder was then added to 2 ml osteoblasts/alginate. The mixture was injected into the dorsal subcutaneous tissue at left side of 6 New Zealand rabbits. The alginate composite without osteoblasts was injected into the right side as the control. 4 and 8 weeks after implantation, the bone formation was evaluated by means of gross, X ray and histological observation. Results: 4 weeks after implantation, cartilage formation was observed and 8 weeks after implantation,new mature bone was observed in the osteoblasts/alginate composites. No new bone formation was observed in all of the control specimens. Conclusion: Calcium alginate can be used as a carrier in injectable bone tissue engineering, and new bone can be created through injection of osteoblasts/alginate composites in immune animals.

砄bjective:To fabricate bone tissue that has similar structural and mechanical characters with normal bone.Methods: Titanium meshes were molded into the shape of column in the length of 12 mm and in the diameter of 8 mm. The column was filled with natural coral granduls.4?10 7 marrow derived osteoblasts in 200 ?l cell culture medium were seeded into each of five scaffolds and incubated in vitro for 2 d to ensure that cells adhere well on the scaffolds. Then the scaffolds were implanted subcutaneously into the back of nude mice. Two months after implantation, the animals were sacrificed and the implanted materials were investigated by gross specimen inspection, X ray examination and histological observation. Results:2 months after in vivo incubation, the newly formed tissue was red and had the gross appearance of bone, and kept the original shape of column. Titanium mesh situated in the surface area. X ray examination showed that large amount of new bone formed in the scaffolds, there was no space between new bone and titanium mesh. Most of coral granduls had been absorbed. Histological observation demonstrated that in the surface area, new bone integrated well with titanium mesh and was enforced by titanium mesh(like cortical bone), and in the middle area large amount of lamellar bone formed.Conclusion: Newly formed bone in this experiment has similar structural with normal cortical bone.

Objective: To investigate the feasibility of chitin as bone substitute material and carrier of rhBMP2.Methods: Porous chitin and chitin/rhBMP2/collagen complex were implanted into calvarial defects in 8 rabbits. Bone repairing ability was assessed by radiographic and histological observation. 2 rabbits without implantation were served as controls. Results: Chitin had certain bone conductive ability. When combined with rhBMP2,a complex possessing both bone conductive activity and bone inductive activity was produced. The complex had greater bone repairing ability than chitin alone. Conclusion: Chitin may be used as a bone substitute material and carrier of BMP. But its mechanical strength and surface activity should be improved.

Objective: To study the biodegradable of coral PLA composite artifical bone combined with bBMP or rhBMP as a new kind of bone substitute material. Methods: The composites were implanted into the muscle pouches of mice after combined with rhBMP-2 or bBMP respectively. Ectopic osteoinductive activity of rhBMP-2 or bBMP was examined and compared by histology and histo-morphometry.Results: rhBMP-2 and bBMP had different osteoinductivety. rhBMP-2 appeared to induce less bone and more angioid tissue and marrow. While bBMP seemed to have opposite effects. Conclusion: bBMP is more osteoinductive than rhBMP-2.