Treatment of hypertrophic scar in human with autologous transplantation of cultured keratinocytes and fibroblasts along with fibrin glue

Hypertrophic scar involves excessive amounts of collagen in dermal layer and may be painful. Nowadays, we can't be sure about effectiveness of procedure for hypertrophic scar management. The application of stem cells with natural scaffold has been the best option for treatment of burn wounds and skin defect, in recent decades. Fibrin glue [FG] was among the first of the natural biomaterials applied to enhance skin deformity in burn patients. This study aimed to identify an efficient, minimally invasive and economical transplantation procedure using novel FG from human cord blood for treatment of hypertrophic scar and regulation collagen synthesis. In this case series study, eight patients were selected with hypertrophic scar due to full-thickness burns. Human keratinocytes and fibroblasts derived from adult skin donors were isolated and cultured. They were tested for the expression of cytokeratin 14 and vimentin using immunocytochemistry. FG was prepared from pooled cord blood. Hypertrophic scars were extensively excised then grafted by simply placing the sheet of FG containing autologous fibroblast and keratinocytes. Histological analyses were performed using Hematoxylin and eosin [H and E] and Masson's Trichrome [MT] staining of the biopsies after 8 weeks. Cultured keratinocytes showed a high level of cytokeratin 14 expression and also fibroblasts showed a high level of vimentin. Histological analyses of skin biopsies after 8 weeks of transplantation revealed re-epithelialization with reduction of hypertrophic scars in 2 patients. These results suggest may be the use of FG from cord blood, which is not more efficient than previous biological transporters and increasing hypertrophic scar relapse, but could lead to decrease pain rate

Fabrication and characterization of spongy denuded amniotic membrane based scaffold for tissue engineering

As a biological tissue material, amniotic membrane [AM] has low immunogenicity and to date has been widely adopted in clinical practice. However, some features such as low biomechanical consistency and rapid biodegradation is limited the application of AM. Therefore, in this study, we fabricated a novel three-dimensional [3D] spongy scaffold made of the extracellular matrix [ECM] of denuded AM. Due to their unique characteristics which are similar to the skin, these scaffolds can be considered as an alternative option in skin tissue engineering. In this experimental study, cellular components of human amniotic membrane [HAM] were removed with 0.03% [w/v] sodium dodecyl sulphate [SDS]. Quantitative analysis was performed to determine levels of Glycosaminoglycans [GAGs], collagen, and deoxyribonucleic acid [DNA]. To increase the low efficiency and purity of the ECM component, especially collagen and GAG, we applied an acid solubilization procedure hydrochloridric acid [HCl 0.1 M] with pepsin [1 mg/ml]. In the present experiment 1-ethyl-3-[3-dimethyl aminopropyl] carbodiimide hydrochloride [EDC]/N-hydroxysuccinimide [NHS] cross linker agent was used to improve the mechanical properties of 3D lyophilized AM scaffold. The spongy 3D AM scaffolds were specified, by scanning electron microscopy, hematoxylin and eosin [H and E] staining, a swelling test, and mechanical strength and in vitro biodegradation tests. Human fetal fibroblast culture systems were used to establish that the scafolds were cytocompatible. Histological analysis of treated human AM showed impressive removal of cellular components. DNA content was diminished after treatment [39 +/- 4.06 micro g/ml vs. 341 +/- 29.60 micro g/ml]. Differences were observed between cellular and denude AM in matrix collagen [478 +/- 18.06 micro g/mg vs. 361 +/- 27.47 micro g/mg].With the optimum concentration of 1 mM NHS/EDC ratio1:4, chemical cross-linker agent could significantly increase the mechanical property, and resistance to collagenase digestion. The results of 2, 4, 6-Trinitrobenzenesulfonic acid [TNBS] test showed that cross-linking efficiency of AM derived ECM scaffolds was about 65% +/- 10.53. Scaffolds treated with NHS/EDC cross-linker agent by 100 micro g/ml collagenase, lost 75% of their dry weight after 14 days. The average pore size of 3D spongy scaffold was 160 micro m measured from scanning electron microscope [SEM] images that it is suitable for cell penetration, nutrients and gas change. In addition, the NHS/ EDC cross-linked AM scaffolds were able to support human fetal fibroblast cell proliferation in vitro. Extracts and contact prepared from the 3D spongy scaffold of AM showed a significant increase in the attachment and proliferation of the human fetal fibroblasts cells. The extra-cellular matrix of denuded AM-based scaffold displays the main properties required for substitute skin including natural in vitro biodegradation, similar physical and mechanical characterization, nontoxic biomaterial and no toxic effect on cell attachment and cell proliferation

Immunohistochemical and tissue flrray study for comparison of the expression of tumor suppressor genes and intercellular adhesive molecules in colorectal fldenocarcinoma and nontumoral colon

Colorectal Carcinoma is a main health problem in many countries and the third common cancer in Iran. This malignancy at present is the most curable carcinoma of gastrointestinal tract. Variation in the expression of the proteins produced by P53, P2i, Pi6, E-cadherin, and p-catenin genes have been noted in this malignancy and may be important in the prognosis and therapeutic response rate. The aim of this study was to compare the frequency and pattern of expression of these proteins in tumoral and nontumoral colonic mucosa. The correlation with prognostic factors including tumor stage, grade, and vascular and perineural invasion was also determined. The paraffin blocks from tumoral and nontumoral parts of the colon obtained from 58 patients with colorectal adenocarcinoma were studied along with 50 colectomic cases in individuals without malignancy. Cylindrical tissue fragments were obtained from appropriate parts of donor blocks by using a 2.5 mm punch biopsy instrument. Each 30 samples were manually arrayed in one tissue array block. Expression of above genes was investigated after sectioning the blocks and immunohistochemical staining of slides. The expression of P53 in tumor cells was significantly more common than in colonic nontumor cells and colon of individuals without tumor [p<0.001]; expression of this protein in tumoral tissues was directly related to vascular invasion [p=0.017]. The expression frequency of P[2]i and P[16] in tumor cells was less than nontumoral tissues of patients with cancer and patients without cancer [p<0.001]. These two gene products showed no correlation with prognostic factors. The expression frequency of membranous E-cadherin and p-catenin in tumor cells was not different from controls, while the membranous expression of E-cadherin was inversely related to cell differentiation [p=0.023] and vascular invasion [p=0.025]. In addition, the membranous expression of p-catenin was inversely related to vascular invasion [p=0.049]. Cytoplasmic and nuclear expression of p-catenin in tumor cells were significantly higher than their expression in the controls [p<0.001]. Cytoplasmic expression of this marker was inversely related to disease stage [p=0.013], while its nuclear expression was inversely related to cell differentiation [p=0.012]. According to our data, it seems that we are able to predict aggressive capacity of the colorectal tumor by determining the frequency and pattern of expression of P53, E-cadherin and p-catenin proteins. These studies can be done simply on formalin-fixed small biopsy samples before surgery to provide valuable information for surgeons, gastroenterologists, and oncologists to choose the best therapeutic approach and predict the therapeutic response. Manual tissue array method is believed to be an economical technique for similar research projects