Experimental study of repairing fat defect with human hair keratin material / 中华整形外科杂志

<p><b>OBJECTIVE</b>To investigate the feasibility of adipogenesis from human hair keratin (HHK) material, so as to provide a new method for fat defect and depression deformity.</p><p><b>METHODS</b>3 Tibet mini-pigs were used. 8 fat defects (1.5 cm in diameter) were made bilaterally on the back. The ball-shaped HHK material was implanted to repair the defects at one side. The defects at contralateral side were as controls. The absorption of the HHK material and adipogenesis were studied histologically.</p><p><b>RESULTS</b>2 weeks after implantation, connective tissue and capillary grew into the porous HHK material. 4 weeks after implantation, HHK material was almost totally absorbed, leaving some material debris and foreign body granuloma. Around them, there were clusters of adipocyte. 6 weeks after implantation, the HHK material was totally degraded and the granuloma was disappeared, and then de novo adipose tissue was observed. Its volume was close to the volume of peripheral HHK material that was planted originally. 10 weeks later, the new-formed fat tissue had less fibres and was very similar to the normal fat.</p><p><b>CONCLUSIONS</b>New adipose tissue can be formed after HKK material implantation. It can also be remodeled to be similar to normal fat.</p>

Induced differentiation of rat adipose-derived stem cells into Schwann-like cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To assess the differentiation potential of rat adipose-derived stem cells (ADSCs) into Schwann-like cells in vitro.</p><p><b>METHODS</b>ADSCs isolated from adult SD rats were cultured in vitro and identified with the cell surface antigens CD44, CD49d and CD106 by immunocytochemistry. The ADSCs of the sixth to eighth passages were inoculated in polylysine-coated culture plate and cultured for 12 days in DMEM/F12 culture medium containing 10% fetal bovine serum, 5 ng/ml platelet-derived growth factor, 10 ng/ml basic fibroblast growth factor, 14 micromol/L Forskolin and 200 ng/ml Heregulin to induce their differentiation in vitro. Immunocytochemistry was performed to identify the expression of the cell surface markers nestin, glial fibrillary acidic protein (GFAP), S-100, and P75.</p><p><b>RESULTS</b>The isolated and purified ADSCs were positive for CD44 and CD49d expressions but negative for CD106. After 12 days of culture in the conditional culture medium, most of the cells showed positive expressions of GFAP, S-100, and P75, the specific protein markers of Schwann cells.</p><p><b>CONCLUSION</b>Adult rat ADSCs are confirmed to have potentials of neuroglial differentiation and capable of differentiating into Schwann-like cells in vitro.</p>

Biological dressing with human hair keratin-collagen sponge-poly 2-hydroxyethyl methacrylate composite promotes burn wound healing in SD rats / 南方医科大学学报

<p><b>OBJECTIVE</b>To develop a composite material containing human hair keratin (HHK), collagen sponge (inner layer) and poly 2-hydroxyethyl methacrylate (PHEMA) film that allows sustained release of polydatin and test its effect as a biological dressing in promoting burn wound healing in SD rats.</p><p><b>METHODS</b>Three HHK materials with fast, moderate, and low degradation rates were mixed at the ratio of 4:3:3 to prepare a reticular structure, which was processed into a composite material with bovine tendon-derived collagen sponge, and further complexed with HEMA film containing PD prepared by polymerization. Degree II burn wound was induced in SD rats by scalding and within postburn day 2-5, the wounds were cleansed and covered with the composite material or with glutaraldehyde-treated porcine skin (positive control). At week 1, 2, 4, 6 and 8 following wound dressing, 6 full-thickness skin samples were harvested from the wounds for histological observation and immunohistochemical detection of collagen and elastic fibers, and the wound healing time and healing rate were recorded.</p><p><b>RESULTS</b>The prepared collagen sponge film was transparent and porous (50-300 microm in diameter) and allowed sustained PD release into normal saline within 48 h. Compared with the porcine skin, the composite material reduced exudation and maintained ideal moisture of the wound, and significantly shortened the wound healing time (P=0.000). On day 7, 14, and 21 following dressing, the composite material and porcine skin significantly increased the wound healing rate as compared with the negative control group (P=0.000), and on day 14, the composite achieved significantly greater healing rate than the porcine skin (P<0.05).</p><p><b>CONCLUSION</b>HHK-collagen sponge-PHEMA/PD composite as a dressing material promotes burn wound healing in rats by allowing in vivo construction of tissue engineered epidermis. PHEMA is feasible for sustained drug delivery in this composite.</p>

Toxicity evaluation of chicken calamus keratin conduit as a tissue-engineering scaffold biomaterial / 南方医科大学学报

<p><b>OBJECTIVE</b>To evaluate the toxicity of chicken calamus keratin (CCK) conduit as a tissue-engineered scaffold material.</p><p><b>METHODS</b>The chemical composition of the leaching solution of CCK was determined by means of ultraviolet spectrometry, and the toxic effects of the solution was evaluated by skin sensitization test in rats, intracutaneous stimulation test in rabbits, acute systemic toxicity test in mice, and cytotoxicity test in L929 cells.</p><p><b>RESULTS</b>The leaching solution of CCK consisted mainly of middle-molecular-weight peptides with a small quantity of macromolecular proteins. Skin sensitization test in rats showed that application of the CCK leaching solution caused no obvious skin reddening, regional edema, or skin necrosis. Intracutaneous injection of the leaching solution in rabbits did not induce obvious skin stimulation manifested by intradermal erythema or edema. In acute systemic toxic test, administration of the leaching solution in mice caused no death, organ dysfunction, cyanosis, tremor, severe peritoneal irritation, ptosis, or dyspnoea. In vitro cytotoxicity test indicated that the cell toxicity of the CCK leaching solution was approximately at 0 level.</p><p><b>CONCLUSION</b>CCK contained in the treated chicken calamus easily undergoes hydrolysis to release mainly some peptides which do not induce obvious toxic effects, suggesting the safe potential applications of CCK conduit as a tissue-engineering biomaterial.</p>

Preliminary study of chicken calamus conduit as a scaffold material for tissue engineering / 南方医科大学学报

<p><b>OBJECTIVE</b>To observe the unique structural features of chicken calamus keratin (CCK) conduit as a candidate scaffold material for tissue engineering and its in vivo degradation and histocompatibility after its implantation into living tissues.</p><p><b>METHODS</b>Chicken calami were taken from healthy chickens and treated through sequential, controllable physical and biochemical procedures for preparation of three types of CCK conduits, namely CCK-I (mildly treated), CCK-II (moderately treated) and CCK-III (intensely treated). Light microscopy (LM) and scanning electron microscopy (SEM) were performed for morphological observation. Each of these three types of CCK pieces (experimental group) and the untreated ones (control group) was implanted into the dorsal muscular tissue on both sides of SD rats, respectively. Routine tissue sectioning and HE stain were performed to identify the morphological changes under light microscope. Each of the CCK threads (experimental group) and the untreated chicken calamus threads (control group) was also grafted within the sciatic nerve bundles of SD rats, respectively.</p><p><b>RESULTS</b>The wall of the chicken calamus was composed of 4 compact parts from inside to outside on cross sections, namely the innermost basophilic homogenous coarse line, 3-5 layers of acidophilic corneum, 60-100 layers of circular keratin tracts containing massive pigment granules, and 10-20 outmost layers of keratin tracts with only a few pigment granules. The three-dimensional surface features of chicken calamus identified by SEM, as compared with untreated chicken calamus, was characterized by loose arrangement containing horizontal and vertical keratins with obvious pores of different sizes and depths on its surface. At 8 weeks after implantation into the muscular tissue in experimental groups, the CCK grafts were degraded into thin filaments or/and dispersed pieces and fine granules with the appearance of blood vessels, which facilitated the absorption of the degradation products; at 12 weeks, the grafts were markedly degraded into tiny fragments. In the control group, in contrast, the grafts remains intact throughout the experiment. After implantation of the material into the nerve bundles, similar cell infiltration and tissue responses to the grafts were observed as compared to those occur in intramuscular grafting. The degradation products did not seem to cause nerve tissue degeneration or necrosis.</p><p><b>CONCLUSIONS</b>Fresh chicken calamus is a natural tube composed of multi-layered compact keratin tracts with pigment granules and small amount of matrix, and is non-absorbable in vivo, and therefore does not favor the purpose for use directly as a candidate biological scaffold. After proper treatment, the chicken calamus becomes loosely arranged porous material, and can be degraded and absorbed in vivo without resulting in tissue degradation or necrosis, suggesting its potential for applications in tissue engineering.</p>

Collagen gel coating or cyclosporine A for improving histocompatibility of chicken calamus keratin / 南方医科大学学报

<p><b>OBJECTIVE</b>To improve the histocompatibility of chicken calamus keratin (CCK) graft by collagen-gel coating or using of cyclosporine A (CsA).</p><p><b>METHODS</b>Thirty SD rats were equally randomized into 5 groups, and in 4 of them, CCK implantation into the bilateral erector spinae was performed on different treatment protocols. In group A, the rats received daily intraperitoneal injection of CsA (5 mg/kg) for two consecutive weeks after CCK implantation; in group B, CCK was soaked in CsA (2.5 mg/ml) solution at 4 degrees Celsius; for 48 h before grafting; in group C, CCK coated with collagen gel was grafted; and in group D, only CCK was implanted. Rats in the fifth group received only cutaneous incision as well as muscular dissection to serve as the blank control. CCK degradation and its effect on the surrounding tissues were observed at 2, 4 and 8 weeks after grafting. Immunohistochemistry was performed to identify T lymphocyte infiltration in the host tissues.</p><p><b>RESULTS</b>All the rats survived the operation. Numerous macrophages, especially multinucleated giant cells occurred on the peripheral of the CCK grafts, and small degraded CCK pieces were observed in their cytoplasm. Only a few inflammatory cells were seen in the host tissues. At 2, 4 and 8 weeks after CCK implantation, only a few CD3-positive cells were found in all the groups, and in group A and B, the density of T lymphocytes was significantly lower than that in group D, and there was no significant difference between group A and the blank control group.</p><p><b>CONCLUSIONS</b>CsA significantly improves the histocompatibility of CCK material, and short-term systemic CsA administration achieves the best results. Macrophages, especially multinucleated giant cells participate in CCK degradation in vivo.</p>

Preparation and bioactivity of human hair keratin-collagen sponge, a new type of dermal analogue / 南方医科大学学报

<p><b>OBJECTIVE</b>To develop a three-dimensional porous film of human hair keratin (HHK)-collagen sponge complex for use as a dermal substitute.</p><p><b>METHODS</b>The three components F, B, and Z derived from healthy human hair were weaved into a meshwork and integrated with purified soluble type I collagen extracted from bovine tendons to prepare a highly porous film with vacuum freeze-drying followed by secondary cross-linking with glutaraldehyde. The film was grafted beneath the dorsal skin in 21 SD rats (experimental group), with simple collagen sponge serving as the negative control. The rats receiving surgical operation but without graft served as the blank control. The graft and its surrounding tissue were harvested on days 3, 7 and at weeks 2, 4, 6, 8, 12 after implantation for evaluation of tissue compatibility, vascularization and degradation.</p><p><b>RESULTS</b>The prepared collagen sponge film was semitransparent and porous. Three to 7 days after grafting, inflammatory reaction was relieved gradually, and several fibroblasts and blood vessels were found adherent to the grafts in the experimental groups. At week 4, the wounds healed in the experimental groups, and the fibroblasts were actively secreting collagen and the film degraded obviously with the appearance of elastic fibers. At weeks 6 and 8, new collagen fibers thickened and assumed regular arrangement, and the collagen sponge films disappeared completely. In the control groups, the changes were less obvious and total HHK degradation occurred till week 12.</p><p><b>CONCLUSION</b>The degradable and absorbable HHK-collagen sponge film has relatively satisfactory tissue compatibility and can accelerate wound healing by stimulating cell proliferation and vascularization, showing the potential as an optimal dermal substitute.</p>