ObjectiveTo review the advances in the computational fluid dynamics (CFD) in tissue engineering.MethodsThe latest research of CFD applied to tissue engineering were extensively retrieved and analyzed, the optimization of bioreactor design and the simulation of fluid dynamics and cell growth kinetics during tissue regeneration in vitro were mainly reviewed.ResultsThe simulation and predictive capabilities of CFD can provide important guidance for the optimization of bioreactor design, and the cultivation of engineering tissue. The accuracy of model prediction results can be further improved by combining with experimental research.ConclusionAs a new and effective research tool, CFD has its unique advantages in the application of tissue engineering. However, a more comprehensive and accurate simulation of the whole process of tissue regeneration still needs further studies.
Objective The immunogenicity of tissue engineered skins is still vague, though it has been appl ied cl inically for several years. To observe the evidence of immunologic rejection of tissue engineered skins transplanted to severe combined immunodeficiency (SCID) mice, which are implanted human splenic lymphocytes to construct human immunesystem. Methods Tissue engineered skins and acellular dermic matrix were constructed in vitro. Twenty SCID mice, aging4-6 weeks and weighing 16-17 g, were randomly divided into four groups equally (n=5). The tissue engineered skins, human foreskins from circumcision and acellular dermic matrix were transplanted to groups A, B, and C, respectively; group D was used as a control. After 2 weeks of transplanting, 3 × 107 human splenic lymphocytes were injected into every SCID mouse intraperitoneally. After 4 weeks, the morphology, histology, immunohistochemistry and human IgG immunofluorescence were used to observe immunologic rejection. Results Group A showed that transplanted tissue engineered skins had the bilayer structure of dermis and epidermis, which was similar to the normal human skin structure. Group B showed that the transplanted human foreskins still retained normal structure of human skin. Group C showed that acellular dermic matrix were located in situ and had no sign of degradation. After injecting human splenic lymphocytes into the SCID mice, no inflammatory cells infil itration were observed basically in groups A, C, and D; the inflammatory cells infil itration of group B were significantly higher than that of other 3 groups (P lt; 0.05). The results of anti human keratin 14 monoclonal antibody (mAb) staining and anti human type IV collagen mAb staining were positive in group A; no positive cells for CD3, CD4, and CD8 were observed in groups A, C, and D; and many positive cells for CD3, CD4, and CD8 were observed in group B. The results of IgG immunofluorescence staining was negative in group A, C, and D, and positive in the great vessel wells of group B. Conclusion The immunogenicity of tissue engineered skins is very weak, and tissue engineered skins would not be rejected by host immune system after transplantation.
To investigate the effect of hepatocyte growth factor (HGF) on prol iferation of cultured human eccrine sweat gland epithel ial cells (hESGc) and the involvement of phosphorylation of ERK1/2. Methods hESGc were cultured in keratinocyte serum free medium (KSFM) and the first generation of hESGc was harvested. The expression of C-met was detected by immunocytochemistry. MTT assay was used to detect the effect of HGF on the prol iferation of hESGc. The cells were divided into blank group, control group and experimental group. The culture density was 2 × 103 cells/hole in control group and experimental group. Two hundred μL KSFM with HGF in different levels was added to every hole. hESGcwere cultured in KSFM with HGF at different levels (2, 20, 40 and 80 ng/mL) in experimental group, in KSFM without HGF incontrol group, and in KSFM without HGF and no hESGc in blank group. The cell prol iferation was observed in xperimental group 2 and 4 days later. Western blot was used to detect the expression of phosphorylated ERK1/2 at 40 ng/mL HGF after 0, 5, 30, 90 and 120 minutes. Results The results were positive for anti-C-met staining in the cytoplasm. HGF (40 ng/mL and 80 ng/mL) significantly improved the prol iferation of hESGc (P lt; 0.05). When cultured in the KSFM with 40 ng/mL HGF, the cell prol iferation rate and the absorbance were 74.2%, 0.239 3 ± 0.070 9 at 2 days and 74.8%, 0.287 8 ± 0.074 3 at 4 days; showing significant differences when compared with control group (P lt; 0.05). When cultured in KSFM with 80 ng/mL HGF, the cell prol iferation rate and the absorbance were 54.5%, 0.212 3 ± 0.059 2 at 2 days and 40.3%, 0.231 0 ± 0.056 7 at 4 days; showing significant differences when compared with control group (P lt; 0.05). The expression of p-ERK1/2 reached to the maximum after stimulation of 40 ng/mL HGF for 5 minutes, and relative integral absorbance (RIA) was 0.593 2 ± 0.192 2, increased 8.1 times compared with instant stimulation (P lt; 0.01). Conclusion HGF could induce the prol iferation of hESGc and activate the phosphorylation of ERK1/2 protein.
Objective To investigate the method of constructing a tissue engineered epidermis with human epidermal cells and polycarbonate membrane, and to establ ish a tissue engineered epidermis with barrier function which is intended to be the replacing model in vitro of skin irritation test. Methods The tissue engineered epidermis was constructed by using polycarbonate membrane as scaffold and stratified differentiated epidermis derived from human keratinocytes. The tissue engineered epidermis was cultured on an inert polycarbonate filter at the air-liquid interface. After 13 days of culture, the composition and structure of tissue engineered epidermis were observed by HE staining, immunofluorescence staining of keratin 10 (K10) amp; K13, K14, laminin,involucrin, and filaggrin, and transmission electronic microscope. The half maximal inhibitory concentration of a substance (IC50) of SDS was determined in the penetration test of tissue engineered epidermis cultured in the absence (control group) or the presence (experimental group) of l i pid supplement for 18 hours. Results The constructed epidermis was similar to normalepidermis, which was consisted of a proliferating basal layer, differentiated spinous layer, granular layer, and stratum corneum. The IC50 values of tissue engineered epidermis cultured in the control group and experimental group were 0.072% (2.36 mmol/L) and 0.183% (6.00 mmol/L), respectively. Conclusion The tissue engineered epidermis constructed on polycarbonate membrane has normal composition and structure and barrier function corresponding to the normal epidermis.
Object ive To explore a method to recons t ruct eccr ine sweat gland- l ike s t ructure in vitro. Methods Isolated from the normal axillary full-thickness skin donated by volunteers sweat gland epithel ial cells were cultured in vitro and were observed under inverted phase contrast microscope. These cells at the density of 2 × 105/cm2 were inoculated underneath the Matrigel (group A), on the top of the Matrigel (group B) and in the Matrigel (group C), respectively,for three-dimensional culture. The formation of eccrine sweat gland-l ike structure was observed by confocal laser scanning microscope, HE staining and immunohistochemistry staining. Results Primary epithel ial cells in the secretory portion of sweat gland were attached and spindle-shaped 24 hours after inoculation, and were under polyclonal grain-l ike growth 2-3 days thereafter. Cobblestone-l ike appearances of these cells were evident 14 days after inoculation and the confluent cells were flat and polygonal with relatively big round cell nucleus. Morphologically, subcultured cells at passage 1 were similar to the primary cells; cells at passage 2 were irregular and most of them had long pseudopodium; cells at passage 3 were star-shaped and big and had fusion with adjacent cells. For group A, tubular structure was formed 11 days after three-dimensional culture. For group B, stretched and filamentous-shaped cytoplasm was observed 8 hours after three-dimensional culture, with the formation of lumen or half-lumen structure, but no significant prol iferation was evident. For group C, cell division and prol iferation occurred 2-3 days after three-dimensional culture; the prol iferated cells were closely arranged into tubular structure with obvious lacunae in the middle, which gradually developed into irregular ball-shaped structure with the increase of neonatal cells. The laser scanning confocal microscope observation showed the formation of spherical structure in group C, with tubular structure in the center of cell mass; HE staining testified the spherical structure in group C was tubular structure. The immunohistochemistry staining demonstrated keratin 18 and carcinoembryonic antigen were positively expressed in group C, which was similar to the tubular structure of secretory portion of sweat gland. Conclusion The sweat gland epithel ial cells can be induced to form eccrine sweat gland-l ike structure through three-dimensional culture in Matrigel.