ObjectiveTo review the progress of cell sheet technology (CST) and its application in bone tissue engineering. MethodsThe literature concerning CST and its application was extensively reviewed and analyzed. ResultsCST using temperature-responsive culture dishes is applied to avoid the shortcomings of traditional tissue engineering. All cultured cells are harvested as intact sheets along with their deposited extracellular matrix. Avoiding the use of proteolytic enzymes, cell sheet composed of the cells and extracellular matrix derived from the cells, and remained the relative protein and biological activity factors. Consequently, cell sheet can provide a suitable microenvironment for the bone regeneration in vivo. With CST, cell sheet engineering is allowed for tissue regeneration by the creation of three-dimensional structures via the layering of individual cell sheets, be created by wrapping scaffold with cell sheets, or be created by folding the cell sheets, showing great potential in tissue engineered bone. ConclusionConstructing tissue engineered bone using CST and traditional method of bone tissue engineering will promote the development of the bone tissue engineering.
Objective To summarize the recent progress of construction methods of engineered cell sheet and to forecast the possible prospect. Methods The recent original articles about investigation and appl ication of engineered cell sheet were reviewed. Several common methods were selected and expounded. Results The construction methods of engineered cell sheet mainly include temperature-responsive culture dish, salmon atelocollagen, magnetic force, surface roughness, and polyelectrolytes, which may overcome the l imits of traditional tissue engineering methods. Conclusion The construction methods of engineered cell sheet are feasible and have a bright future in the cl inical appl ication.
ObjectiveTo explore a new method of developing a pre-vascularized cell sheets. MethodsBone marrow mesenchymal stem cells (BMSCs) from 3-week-old Japanese white rabbits were harvested and cultured. Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) were added into the culture medium to differentiate into endothelial like cells (ECs) from BMSCs (experimental group), and non-induced cells served as the control group. The cell morphology was observed; and the von Willebrand factor (vWF) and CD31 immunofluorescent staining was used to identify the induced BMSCs. The 2nd generation BMSCs were seeded on a cell culture dish at a cell density of 9×104cells/cm2 and cultured for 14 days to form a thick cell sheet, and ECs from BMSCs were then seeded on the BMSCs sheet at a cell density of 5×104 cells/cm2 to develop pre-vascularized cell sheets and cultured for 3, 7, and 14 days (group A); non-induced BMSCs sheet and only ECs from BMSCs were used as group B and group C, respectively. The CD31 immunofluorescent staining and histological analysis were performed to evaluate the pre-vascularized cell sheet. ResultsBMSCs changed from long fusiform to cobblestone-like morphology after induced by VEGF and bFGF. The expressions of CD31 and vWF were positive in experimental group, but were negative in control group, which suggested that BMSCs have the ability to differentiate into ECs under this condition. After the ECs were seeded on the BMSCs sheet, the ECs migrated and rearranged; intracellular vacuoles and networks were observed. Furthermore, immunofluorescent staining for CD31 also revealed a developing process of tube formation after the ECs were seeded on the BMSCs sheet. The histological evaluations indicated the microvessel lumen formed. However, no similar change was observed in groups B and C. ConclusionBMSCs have the ability to differentiate into ECs after induced by VEGF and bFGF. ECs from BMSCs can develop into vascular network constructs when seeded on the BMSCs sheet, which provides a new method for engineering pre-vascularized tissue construction.
ObjectiveTo construct a transgenic cell sheet of cartilage-derived morphogenetic protein 1 (CDMP-1) by adenovirus vector in vitro and to identify its biological activity. MethodsThe bone mesenchymal stem cells (BMSCs) were isolated from bone marrow of 1-month-old rabbit, and cultured in vitro. The 3rd-6th generation of BMSCs were used for experiment. The experiment was divided into 3 groups:BMSCs transfected by adenovirus (Ad)-cytomegalovirus (CMV)-human CDMP1 (hCDMP1)-internal ribosome entry site (IRES)-enhanced green fluorescent protein (EGFP) in group A, BMSCs transfected by Ad-CMV-EGFP in group B, and untransfected BMSCs in group C. The expression of green fluorescence was observed in 3 groups under fluorescent inverted microscope. MTT assay was used to detect the proliferation of the cells. The cell sheet was obtained by means of temperature-responsive culture dish for 14 days. The morphological and HE staining observations of the cell sheet were carried out. RT-PCR and Western blot were used to detect the expressions of hCDMP1 and collagen type II at gene and protein levels, while alcian blue staining was used to detect the expression of glycosaminoglycans (GAG). ResultsBright green fluorescence was observed in transfected cells at 72 hours under fluorescent inverted microscope, and the transfection efficiency was up to 90%. MTT assay showed approximate S-shaped growth curves in 3 groups, showing no significant difference in the absorbance (A) value among 3 groups within 9 days (P>0.05). The three-dimensional cell sheets were successfully harvested in vitro. The RT-PCR and Western blot showed that there were positive expressions of hCDMP1 and collagen type II in group A and negative expression in other 2 groups. HE staining and alcian blue staining showed that there were rich fibrous tissues, mass extracellular matrix, and dark blue metachromatic granules in group A, but there was less fibrous tissues and no specific blue metachromatic granules in other 2 groups; and the positive expression area was significantly lower and gray scale of GAG was significantly higher in group A than that in groups B and C (P<0.05). ConclusionA transgenic cell sheet of exogenous recombinant hCDMP1 by adenovirus vector can express collagen type II and GAG, so it has chondrogenic capacity. This technology that overcomes limitations in traditional tissue engineering, such as low cell-attachment efficiency and inflammatory reaction, may be a new tissue engineering approach for hard tissue reconstruction and is hopeful to build a large density of tissue engineered cartilage.
ObjectiveCell sheet technology(CST) demonstrates the innovation and advantage by overcoming some immanent shortcomings of traditional tissue engineering. To review the research progress of CST in oral tissue engineering. MethodsThe related home and abroad literature about CST and its application in stomatology was extensively reviewed and analyzed. ResultsCompared to the traditional tissue engineering technology, CST has the features of high seeding density, abundant matrix, good biological compatibility, and perfect operability, which can improve the survival rate of cell transplantation and promote functional reconstruction. It is reported that CST has been successfully used in the following fields, repair and reconstruction of periodontium, soft tissues of oral mucosa, and bones in maxillofacial region. ConclusionWith the development of CST and combined with the traditional tissue engineering technologies, it will promote the tissue engineering further progress in stomatology.
ObjectiveTo review the development of cell sheet engineering technology in engineering vascularized tissue. MethodsThe literature about cell sheet engineering technology and engineering vascularized tissue was reviewed, analyzed, and summarized. ResultsAlthough there are many methods to engineer vascularized tissue, cell sheet engineering technology provides a promising potential to develop a vascularized tissue. Recently, cell sheet engineering technology has become a hot topic in engineering vascularized tissue. Co-culturing endothelial cells on a cell sheet, endothelial cells are able to form three-dimensional prevascularized networks and microvascular cavities in the cell sheet, which facilitate the formation of functional vascular networks in the transplanted tissue. ConclusionCell sheet engineering technology is a promising strategy to engineer vascularized tissue, which is still being studied to explore more potential.
Objective To review the progress of cell sheet technology and its application in bone and cartilage engineering. Methods The recent literature concerning the cell sheet technology used in treatment of bone and cartilage defects was extensively reviewed and summarized. Results Cell sheet built through many different ways can protect extracellular matrix from proteolytic enzymes. As a three-dimensional structure, cell sheet can repair bone and cartilige defects via folding, wrapping scaffold, or be created by the layering of individual cell sheets. Conclusion The cell sheet technology would have a very broad prospects in bone and cartilage tissue engineering in future.
Primary human hepatocytes (PHH) are the gold standard of in vitro human liver model for drug screening. However, a problem of culturing PHH in vitro is the rapid decline of cytochrome P450 (CYP450) activity, which plays an important role in drug metabolism. In this study, thermo-responsive culture dishes were used to explore the conditions for murine embryonic 3T3-J2 fibroblasts to form cell sheet. Based on the cell sheet engineering technology, a three-dimensional (3D) “sandwich” co-culture system of 3T3-J2 cell sheet/PHH/collagen gel was constructed. The tissue structure and protein expression of the model section were observed by hematoxylin eosin staining and immunofluorescence staining respectively. Phenacetin and bupropion were used as substrates to determine the activity of CYP450. The contents of albumin and urea in the system were determined by enzyme linked immunosorbent assay (ELISA). The results showed that the complete 3T3-J2 cell sheet could be obtained when the cell seeding density was 1.5×106 /dish (35 mm dish) and the incubation time at low temperature was 60 min. Through cell sheet stacking, a 3D in vitro liver model was developed. Compared with the two-dimensional (2D) model, in the 3D model, the cell-cell and cell-matrix connections were tighter, the activities of cytochrome P450 CYP1A2 and cytochrome P450 CYP2B6 were significantly increased, and the secretion levels of albumin and urea were increased. These indexes could be maintained stably for 21 d. Therefore, cell sheet stacking is helpful to improve the level of liver function of 3D liver model. This model is expected to be used to predict the metabolism of low-clearance drugs in preclinical, which is of great significance for drug evaluation and other studies.