Effect of particulate allogeneic acellular dermal matrix (PADM) combined with autologous split-thickness skin (STS) on wound healing in rats / 中华烧伤杂志

<p><b>OBJECTIVE</b>To evaluate the effects of mixed grafting of allogeneic PADM and autologous STS on wound healing of full-thickness defect in rats.</p><p><b>METHODS</b>Full-thickness defects with size of 6 cm x 4 cm were produced on the back of 12 SD rats, and they were divided into E group (n = 6) and C group (n = 6) according to the random number table. The wounds in E group were grafted with a mix of allogeneic PADM (expansion rate 10: 5) and autologous STS with thickness of 0.2 mm, while those in C group were grafted with autologous STS in the same thickness. The wound healing rate, survival rate, contraction rate, and expansion rate of transplanted skin were observed at post operation week (POW) 2, 3, 4, 6, 8, 12, 20. Tissue samples form wounds and surrounding normal skin were harvested at POW 20 for histopathological observation as follows. The structure of collagen fiber bundle was observed by HE staining, the diameter and gap rate of collagen fiber bundle were also measured. The distribution of type I and III collagen was observed by sircus red staining, and the contents of type I, III collagen and their ratio were also examined. Data were processed with independent samples t test, Levene test, and t' test.</p><p><b>RESULTS</b>Survival rate of transplanted skin in E group at POW 2 [(76.1 +/- 13.1)%] was obviously lower than that in C group [(94.5 +/- 1.3)%, t' = 3.440, P = 0.018]. Contraction rate of transplanted skin in E, C groups at POW 3 showed significant difference [(34 +/- 8)% vs. (16 +/- 12)%, t = -3.211, P = 0.009]. Compared with those in peri-wound normal skin, collagen fiber bundles in C group showed signs of homogenization, and collagen fibers were thin with irregular arrangement. Collagen fiber structure and arrangement of composite skin in E group were similar to those surrounding normal skin with incomplete degradation of PADM. Diameter of collagen fiber bundle [(9.6 +/- 0.8) microm], gap rate between collagen bundle [(24 +/- 5)%], content of type I collagen [(80.2 +/- 5.4)%] and the ratio of type I to type III collagen (4.3 +/- 1.2) in E group were all increased as compared with those in C group [(7.3 +/- 1.4) microm (t = -3.562, P = 0.005), (17 +/- 4)% (t = -2.760, P = 0.020), (68.1 +/- 8.4)% (t = -2.981, P = 0.014), 2.3 +/- 1.0 (t = -3.204, P = 0.009)], while content of type III collagen [(19.8 +/- 5.4)%] in E group was lower than that in C group [(32.0 +/- 8.4)%, t = 2. 981, P = 0.014]. Above-mentioned indexes of collagen in wound of E group were similar to those of normal skin surrounding the wound.</p><p><b>CONCLUSIONS</b>Allogeneic PADM used as dermal regeneration template is beneficial in improving collagen fiber bundle structure in dermis layer of rats with full-thickness skin wounds when repaired with autologous STS, and it accelerates maturation of regenerative dermal tissue.</p>

Clone screening and interference efficiency of seed cells with CCL20 gene knockdown for tissue-engineered skin / 中华外科杂志

<p><b>OBJECTIVE</b>To screen stable cell clones of CCL20 gene knockdown and assess their interference effects, recombinant lentivirus vectors with CCL20 gene specific shRNA were applied to infect human immortal keratinocyte line (HaCaT).</p><p><b>METHODS</b>The three pHSER-CCL20-shRNA-GFP vectors (pHCG-1 and pHCG-2 were CCL20 gene specific, and pHCG-3 was used as mismatch control) have been previously constructed. The virus packaging cell line 293FT was transfected with these vectors by using CaCl2 methods to produce lentiviral particles. After the viral titers of these three harvested cell supernatants were determined by flow cytometry, HaCaT cells were transfected by these viruses and screened under the pressure of G418. The CCL20 mRNA from HaCaT cell clones and the CCL20 protein levels in the supernatants of HaCaT cell clones were detected by Real-time RT-PCR and ELISA, respectively.</p><p><b>RESULTS</b>The titers of three lentiviruses were 7.08 x 10(5) transduced units (TU)/ml, 1.88 x 10(5) TU /ml and 2.08 x 10(5) TU/ml, respectively. Two HaCaT cell clones from each lentiviral vectors were obtained after G418 screening for 5 - 8 weeks. Four CCL20 gene specific clones showed stable interference effect in both Real-time RT-PCR and ELISA. The mRNA expression and protein level of CCL20 gene specific clones were down regulated significantly.</p><p><b>CONCLUSIONS</b>The four human immortal keratinocyte clones with long term CCL20 gene knockdown have been screened by recombinant lentivirus vectors with CCL20 gene specific shRNA. These clones might be served as seed cells for novel tissue-engineered skin with lower rejection.</p>

Study on influencing factors in efficiency of introducing gene into human keratinocyte (KC) / 中华烧伤杂志

<p><b>OBJECTIVE</b>To observe the effect of plasmids in different size and gene transfection protocol on efficiency of introducing gene into human KC.</p><p><b>METHODS</b>Four plasmids in different size, inclu-ding pSUPER-enhanced green fluorescent protein (EGFP), pEGFP-N2, pHSER-green fluorescent protein (GFP) and ploxP-EGFP, were transfected into immortal human KC line (HaCaT) and human embryo kid-ney cell line (293FT) separately following transfection protocols of liposome (LTP), cation polymerizer (CPTP), electroporation combined with nucleus transfection agent (ETP) and lentivirus. 293FT was used as control. GFP expression was observed under inverted fluorescence microscope. The transfection efficiency (TE) was calculated.</p><p><b>RESULTS</b>(1) The four plasmids could be introduced into HaCaT (TE, 1.0%-3.3%) and 293FT (TE, 80.0%-84.7% ) following LTP. (2) The four plasmids could also be introduced into HaCaT (TE, 1.0%-3.7% ) and 293FT (TE, 81.3%-86.7% ) following CPTP. (3) Two shorter plasmids (pSUPER-EGFP and pEGFP-N2) could be introduced into HaCaT by ETP with higher TE than the othr two longer plasmids (pHSER-GFP and ploxP-EGFP), which were 22.3% and 19.0% vs. 4.0% and 3.3%, respectively. (4) pHSER-GFP packaged by lentivirus could be introduced into HaCaT with the TE reaching 97.0%, which surpassed the above three protocols.</p><p><b>CONCLUSIONS</b>It is difficult to introduce exogenous gene into human KC by LTP or CPTP; TE of lentivirus transfection protocol apparently surpasses</p>

Morphological observation of human keratinocytes tridimensionally cocultured with xenogenic acellular dermal matrix / 中华烧伤杂志

<p><b>OBJECTIVE</b>To establish the tridimensional culture method for tissue-engineered skin to observe the histomorphological change in human immortal KC strain (HacaT)cocultured with xenogenic acellular dermal matrix (ADM).</p><p><b>METHODS</b>The ADM was prepared from SD rats by a modified method. HaCaTs were cultured in defined KC-serum free medium. HaCaTs in log growth phase were inoculated on ADM at the cell density of 2 x 10(5)/cm(2). They were submergedly cultured for 5 days and then changed to air-liquid phase culture for another 5 days. ADM and growth of HaCaTs on day 1 and 5 after cocultured with ADM were observed with scanning electron microscope. The histological change in ADM and HaCaTs on day 1, 5, and 10 after cocultured with ADM were examined by HE staining.</p><p><b>RESULTS</b>The gross appearance of ADM was white with smooth and soft texture, and intact collagen bundles without cellular residue. HaCaTs adhered and stretched out pseudopodia on the surface of the ADM on day 1 after combined culture, and a monolayer of cells was formed on day 5, growing into 3-6 layers of cells on day 10 with a tendency to grow into ADM.</p><p><b>CONCLUSIONS</b>SD rats ADM is benefit for the adhesion of HaCaTs and the permeation of nutrient solution, from which an engineered multiple-layered human skin can be obtained within 10 days.</p>