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 find new ways for wound healing and tissue expansion by reviewing of progress in recent years in functional molecules which are used for signaling channels of mechanical stress perception and mechanotransduction of keratinocyte. Methods The domestic and international articles were reviewed to summarize the functional molecules and signaling channels of mechanical stress perception and mechanotransduction of keratinocytes. Results The mechanism of mechanical stress perception includes mechano-sensitive channels, growth factor receptor-mediated mechanical stress perception, and mechanical stress perception by protein deformation. The mechanism of mechanotransduction includes cell adhesion-mediated signaling, mitogen-activated protein kinase signaling, the cytoskeleton and extracellular matrix, and so on. Conclusion Keratinocytes can response to the mechanical stress and transfer the effective information to undergo shaping, migration, proliferation, differentiation, and other biological behavior in order to adjust itself to adapt to the new environment.
Objective To review the latest research progress on keratinocyte growth factor (KGF), to thoroughlyunderstand its basic characteristics and appl ication methods and to lay a sol id foundation for the research and development of new KGF medicines and improving the qual ity of skin substitutes. Methods Domestical and international l iteratures on KGFin recent years were extensively reviewed and analyzed. Results KGF was secreted by mesenchymal cells and its receptors were distributed in epithel ium to promote the prol iferation, migration and differentiation of epithel ial cell specifically, which closely related to the organ development, wound heal ing, tumorigenesis and immune reconstruction. Conclusion KGF can be used to improve wound heal ing and the performance of skin substitutes. However, the structure of KGF needs to be changed to el iminate its side effects and purify its promoting effect on epithel ial cell growth.
OBJECTIVE To search an ideal carrier of transferred keratinocytes for transplantation. METHODS The transferred keratinocytes were seeded on the surfaces of the artificial dermis and the silicone membrane and cultured in vitro for 2 weeks. The growth of the keratinocytes was observed by microscope and scanning electron microscope. RESULTS The keratinocytes implanted on the artificial dermis began to rupture and died after 2 to 3 days. While the keratinocytes adhered well on the surface of silicone membrane with pseudopodia formation after 1 week under scanning electron microscope, and the cells kept normal morphological and proliferative properties 2 weeks later. CONCLUSION The silicone membrane can be applied as an useful carrier for the keratinocytes transplantation.
ObjectiveTo investigate the expression of keratinocyte growth factor (KGF) and cyclooxygen-ase-2 (COX-2) protein and microvessel density (MVD), and to explore their function and mechanism in the multistep process of gastric cancer. MethodsThe expressions of KGF and COX-2 protein in 64 samples of gastric cancer and 30 cases of normal gastric mucosa tissues were detected by immunohistochemistry. The MVD was detected by staining the endothelial cells in microvessles using anti-CD34 antibody. ResultsThe positive rate of KGF and COX-2 protein expression in gastric cancer were 65.6% (42/64) and 79.7% (51/64), respectively, which was significantly higher than that in normal gastric mucosa tissues 〔(23.3%, 7/30), P=0.046; (13.3%, 4/30), P=0.008〕. The MVD of gastric cancer was 31.8±8.0, which was significantly higher than that of normal gastric mucosa tissues (14.3±6.1), P=0.000. The MVD in gastric cancer with coexpressive KGF and COX-2 protein was 35.9±5.7, which was significant higher than that with non-coexpressive KGF and COX-2 protein (25.7±7.0), P=0.000. Both the expression of KGF and COX-2 protein were related to the invasion of serosa, lymph node metastasis and TNM staging (Plt;0.05, Plt;0.01). The MVD of gastric cancer tissues was related to lymph node metastasis and TNM staging (Plt;0.05), but unrelated to patient’s age, gender, and differentiation of tumor (Pgt;0.05). The co-expression of KGF and COX-2 protein was frequently found in patients with deeper invasion of serosa, lymph node metastasis, and higher TNM staging (Plt;0.05), but which was not associated withpatient’sage, gender, and differentiation of tumor (Pgt;0.05). The expression of KGF protein was positively correlated to the expression of COX-2 protein (r=0.610, P=0.000). There was positive correlation between MVD and the expression of KGF (r=0.675, P=0.000) and COX-2 protein (r=0.657, P=0.000) in gastric cancer, respectively. ConclusionKGF and COX-2 highly expressed by gastric cancer, which may be involved in the invasion and metastasis of gastric cancer by synergisticly promoting the angiogenesis.
Objective To find a feasible method that can fast isolate seed cells, keratinocyte stem cell and fibroblasts, for composite tissue engineered skin. Methods The foreskin could be attained from posthectomy, the subcutaneous tissue was removed completely, and the full-thick skin was cut into pieces, 2 mm×2 mm in size, then the pieces were submerged into a centrifuge tube containing collagenase Ⅰ in a oscillator. After 3-hour digestion at 37℃, the dermis was dissolved completely with all the fibroblasts in the digestion solution and the epidermis could be separated easily.With more than 10minute digestion in trypsin at 37℃, the epidermal cells could be harvested. Then flowcytometry and FITCimmunofluorescence for cytokeratin 19 of epidermal cells and FITC-immunofluorescence for vimentin of fibroblast were conducted to identify keratinocyte stem cells in the epidermal cells and fibroblasts in the digestion solution. Moreover, epidermal cells and fibroblasts were cultured in vitro for 7 days to investigate their biological behavior. Results Using collagenase Ⅰ combined with trypsin, epidermal cells andfibroblasts could be isolated at one time within 3 hours. Up to 17% cells demonstrated cytokeratin 19 positive in the epidermal cells, with fibroblast vimentin positive. The amount of fibroblast could be enlarged to more than 100 times within 6 days, but the putative keratinocyte stem cells were difficult for subculture. Conclusion Seed cells for composite tissue engineered skin could be harvested fast at one time, that made it possible to reconstruct composite tissue-engineered skin in vitro.
OBJECTIVE: To investigate the skin regeneration using cultured human keratinocytes with collagen sponge transplanted into thickness wound of nude mice. METHODS: Human foreskin from foreskin ectomy procedures was detached with 0.5% Dispase II. Epidermis sheets were separated from dermis and digested with 0.05% Trypsin into single cell suspension. Keratinocytes were cultured and seeded into collagen sponge during logarithmic growth phase. After 3 days, the keratinocytes-collagen sponge were grafted on full thickness wound of nude mice, compared with simple collagen sponge without keratinocytes. The histological, immunohistochemical examination and electron microscopy were detected. RESULTS: After the epidermal substitute was grafted onto wound, the human keratinocytes were able to further proliferate and differentiate and develop into new epithelia. Compared with the control group, the wound healed earlier and contracted less, epithelia matured earlier, and the collagen fiber was less beneath epithelia. CONCLUSION: Keratinocytes can grow on collagen sponge and migrate onto wound to develop into stratified epithelia and inhibit wound contract. The keratinocyte graft can be used to repair skin defect.
To understand the reason of hyperpigmentation following full-thickness skin autograft, 54 guinea pigs were selected as skin autograft models. The changes of density of epidermal keratinocyte (KC) and the amount and distribution of melanin in the epidermal KC were observed by histological, histochemical and autoradiographic techniques. The results showed: (1) The histological changes of KC were in fact a wound-repair process; (2) The increased melanin in KC coordinated with "multi-overlapping screen" effect, a result of multiple layers of "supranuclear cap", was the direct cause of hyperpigmentation of the graft and (3) The hydrolytic disturbance of melanin in KC and the longer life span of the KC were probably the important reasons for the increase of melanin in the epidermis.
【Abstract】 Objective To observe the effects of Angelica dahurica extracts on the biological characteristics of human keratinocytes (KC) in vitro and to explore the possible mechanism in promoting wound healing. Methods HaCaT cells of passage 5 from KC were used during the experiment. Different concentrations (5 × 10-2, 5 × 10-3, 5 × 10-4, and 5 × 10-5 g/L) of Angelica dahurica extracts, which was obtained by 95% ethanol from Angelica dahurica raw material, were prepared by DMEM containing 0.25% fetal bovine serum (FBS). After the extracts at different concentrations were respectively used for KC culture for 5 days, the cell proliferation activities were detected by MTT, and DMEM containing 0.25% FBS served as the negative control. According to the cell proliferation activity, the optimal concentration was determined. KC was further treated with Angelica dahurica extracts of the optimal concentration (experimental group) or with DMEM containing 0.25% FBS (control group) for 48 hours. The cell cycle was tested by flow cytometry. Cyclin D1 and Caspase-3 mRNA levels were also detected by real-time fluorescent quantitative PCR technique. Results Angelica dahurica extracts at concentrations of 5 × 10-4, 5 × 10-3,and 5 × 10-2 g/L could significantly enhance KC proliferation, showing significant differences in absorbance (A) values compared with that of control group (P lt; 0.05) with an optimal concentration of 5 × 10-3 g/L. At this concentration, an increased percentage of S and G2/M phase cells and a decreased percentage of G0/G1 phase cells were detected, showing significant differences when compared with control group (P lt; 0.05). Real-time fluorescent quantitative PCR revealed that the cyclin D1 and Caspase-3 mRNA levels of experimental group was significantly down-regulated, showing significant differences when compared with control group (P lt; 0.05). Conclusion Angelica dahurica extracts can promote the proliferation of KC, accelerate the cell cycle of KC by down-regulating mRNA expressions of cyclin D1, and inhibit apoptosis by down-regulating mRNA expressions of Caspase-3. These effects might enhance the process of wound healing by expediting the process of epithelization.
OBJECTIVE: To investigate the selection and identification of human keratinocyte stem cells(KSC) in vitro. METHODS: According to the characteristics of KSC which can adhere to extracellular matrix very fast, we selected 3 groups of different time(5 minutes, 20 minutes and 60 minutes) and unselected as control group. And the cells were identified by monoclone antibody of beta 1-integrin and cytokeratin 19 (Ck19), then the image analysis was done. Furthermore we analyzed the cultured cells with flow cytometer(FCM) and observed the ultrastructure of the cell by transmission electron microscope(TEM). RESULTS: The cell clones formed in all groups after 10 to 14 days, while the cells of 5 minute group grew more slowly than those of the other groups, however, the clones of this group were bigger. The expression of beta 1-integrin and Ck19 were found in all groups. The positive rate of beta 1-integrin was significant difference between 5 minute group and the other groups (P lt; 0.05). And the expression of Ck19 was no significant difference between 5 minute group and 20 minute group(P gt; 0.05), and between 60 minute group and control group. But significant difference was observed between the former and the later groups(P lt; 0.05). The result of FCM showed that most cells of the 5 minute group lied in G1 period of cell cycle, which was different from those of the other groups. At the same time, the cells of 5 minute group were smaller and contained fewer organelles than those of the other groups. CONCLUSION: The above results demonstrate that the cells of 5 minute group have a slow cell cycle, characteristics of immaturity, and behaving like clonogenic cells in vitro. The cells have the general anticipated properties for KSC. So the KSC can be selected by rapid attachment to extracellular matrix and identified by monoclone antibody of beta 1-integrin and Ck19.