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

Objective: To study the role of activating transcription factor 2 (ATF-2) in the growth of mandibular condyle cartilage. Methods: Primary chondrocytes of condyle were cultured. Expression plasmid of ATF-2 and plasmid bcl-2 promoter were transfected into chondrocytes. Luciferase assay and Western blot were used. Results: The absence of ATF-2 in mandibular condyle chondrocytes resulted in a decline in bcl-2 promoter activity, reduction in bcl-2 protein level. Conclusion: The results strongly imply that ATF-2 is required for adequate bcl-2 expression, and play a significant role in controlling growth plate chondrocyte progression.

The cuttlebones, harvested from cuttles, undergo the chemical reaction in high temperature and high pressure for a certain time. The products are qualitatively analysed, and spacial structure observation and cytocompatibility are tested. The results show that the chemical component of the cuttlebone is CaCO3 and the crystal type is aragonite. Cuttlebones undergo a hydro-thermal reaction, and thus transform into hydroxyapatite-that is, the cuttlebone-transformed hydroxyapatite(CBHA). The CBHA materials have the interconnected microporous network structures. Under the high magnification, CBHAs appear to have many micro-spheres, thus construct a new self-assembled nano-material system. The marrow stromal osteoblasts can adhere to and proliferate well on the surface of the CBHAs. These results show that CBHAs have good biocompatibility. Therefore, it can be a potential candidate scaffold for bone tissue engineering.
Animals ; Bone Substitutes ; chemical synthesis ; chemistry ; toxicity ; Cells, Cultured ; Durapatite ; chemical synthesis ; chemistry ; toxicity ; Materials Testing ; Osteoblasts ; cytology ; drug effects ; Rabbits ; Sepia ; anatomy & histology ; Spine ; anatomy & histology ; chemistry ; Tissue Engineering

OBJECTIVETo study the effects of artificial bone composite of bicoral, rhBMP-2 and PLA in repairing calvarial critical-size defects.

METHODSCalvarial defects in 24 rabbits were surgically made and then half of the defects were repaired with the artificial composite bone. Another half of them were repaired with bicoral/PLA composite and served as controls. Four rabbits in each group were sacrificed at 4, 8, 12 weeks after operation, respectively. The treatment effects were evaluated with scanning electron microscopy and mechanical strength testing.

RESULTSNew bone was observed not only in the periphery, but also inside the artificial bone in both groups, but earlier and more new bone formation was observed in treatment group compared with control group. The mechanical strength test showed that the artificial bone in two groups, which had same mechanical strength before implantation, had significant different mechanical strength after operation. The strength of the artificial composite bone was higher than that of controls and was same with normal rabbit calvarial bone.

CONCLUSIONThe artificial composite bone possess a highly repairing ability, and the healing in bone defects may be accomplished by both osteoinductive and osteoconductive mechanism. The material may be used as a good substitute for bone grafting.

Animals ; Anthozoa ; Biocompatible Materials ; Bone Morphogenetic Protein 2 ; Bone Morphogenetic Proteins ; pharmacology ; therapeutic use ; Bone Regeneration ; drug effects ; Bone Substitutes ; therapeutic use ; Implants, Experimental ; Lactic Acid ; therapeutic use ; Polyesters ; Polymers ; therapeutic use ; Rabbits ; Recombinant Proteins ; pharmacology ; therapeutic use ; Skull ; injuries ; surgery ; Transforming Growth Factor beta

OBJECTIVESeed cell study is an essential area in the research of tissue engineering. To evaluate the potentiality of marrow stromal cell(MSCs) as seed cell in the regeneration of tissue engineered cartilage, formation of cartilage nodules by culture expanded MSCs pellets under the induction of TGF-beta was investigated.

METHODSMSCs were cultured and expanded in vitro. Cell pellets containing 1 x 10(6) MSCs were obtained by centrifuging MSCs solution at 1,000 r/min in 5 ml centrifugation tube. Pellets were exposed to cell culture media containing 20 ng/ml TGF-beta for 7 days and then cultured for another 7 and 21 days. The nodules were moved out of the tube and cartilage formation was observed by stereomicroscope, light microscope and electronic microscope.

RESULTS10 days after exposure to TGF-beta, pellets contracted and formed small and round nodules on the bottom of the tubes. The nodules grew bigger slowly and reached maximal diameter of 1.8 mm in 28 days. The surface of the nodules was smooth and bright white. Histological examination showed that extra cellular matrix formed in 14 days and in some areas cells situated in lacuna. In 28 days' specimens, a lot of cells situated in lacuna could be observed and the histological appearance looked much similar to cartilage. Electronic microscope observation demonstrated that in 28 days' specimens a large amount of collagen fiber existed.

CONCLUSIONUnder the induction of TGF-beta, MSCs could differentiate into chondrogenesis cell and form cartilaginous nodules in vitro. This indicated that MSCs could be the potential seed cells in the regeneration of cartilage employing method of tissue engineering.

Animals ; Bone Marrow Cells ; cytology ; metabolism ; Cartilage ; cytology ; Cell Differentiation ; Cell Separation ; Cells, Cultured ; Chondrogenesis ; drug effects ; Rabbits ; Stromal Cells ; cytology ; metabolism ; Tissue Engineering ; Transforming Growth Factor beta ; pharmacology

OBJECTIVETo investigate the feasibility of using marrow stromal osteoblast-cancellous bone matrix compound artificial bone (MCCAB) as tissue-engineered bone, the osteogenesis of MCCAB in the cranial defect was observed in the experiment.

METHODSThe in vitro cultivated and induced marrow stromal cells of adult New Zealand rabbits were seeded into the alginate-cancellous bone matrix to form MCCAB. The MCCAB was then implanted into the cranial defect for 4 to 8 weeks. The cancellous bone matrix (CBM) alone or the marrow stromal osteoblasts (MSOs) alone was implanted as the control. The effectiveness of bone formation was assessed by histological and roentgenographic analysis.

RESULTSThe osteogenesis of MCCAB was better than CBM or MSOs and superior to the blank group.

CONCLUSIONMCCAB can effectively repair cranial defect. It could be used clinically to restore large bone defects.

Animals ; Bone Marrow Cells ; cytology ; physiology ; Bone Matrix ; cytology ; Cells, Cultured ; Feasibility Studies ; Male ; Osteoblasts ; cytology ; physiology ; Rabbits ; Skull ; abnormalities ; Stromal Cells ; cytology ; physiology

OBJECTIVETo investigate the feasibility of using marrow stromal osteoblast (MSO) as bone derived cell and using cancellous bone matrix (CBM) as scaffold for bone tissue engineering, the subcutaneous osteogenesis of MSO-CBM compound artificial bone (MCCAB) was observed in the experiment.

METHODSThe marrow stromal cells of adult New Zealand rabbits cultivated and induced in vitro were used to form MCCAB by mixing, seeding and solidifying methods assisted by alginate. The MCCABs were auto-transplanted subcutaneously 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, histology and computerized histomorphometry.

RESULTSThe osteogenesis of MCCABs was better than that of the alginate-cancellous bone matrix composites and of the cancellous bone matrixes. In the MCCABs, both intramembranous and cartilaginous osteogeneses were seen but the former was obvious. In the control, only slight cartilaginous osteogeneses were seen.

CONCLUSIONSThe osteogeneses of the MCCABs constructed by using tissue engineering method were obvious when transplanted subcutaneously. The MSO and CBM can be used as good bone-derived cell and scaffold material respectively for tissue-engineered bone construction.

Animals ; Bone Marrow Transplantation ; Bone Matrix ; transplantation ; Bone Transplantation ; methods ; Male ; Osteoblasts ; transplantation ; Osteogenesis ; Rabbits ; Tissue Engineering

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.

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.