背景:作者在对人发角蛋白的机制研究和临床应用中提出了一个伞新的组织工程学概念--在体,原位组织工程.在该理论的指导下,拟以人发角蚩白-胶原海绵为支架,复合一种可作为药物缓释载体的聚甲基丙烯酸羟乙酯,探讨其在体内原位诱导周围组织细胞构建真皮的可行性.目的:以人发角蛋白胶原海绵为敷料内层,复合包裹药物虎杖的聚甲基丙烯酸羟乙酯载体,制各一种双层复合生物材料,观察其对烧伤创面的促愈合作用.设计、时间及地点:随机对照动物实验,于2005-03/12在南方医科大学组胚教研室完成.材料:选用雄性SD大鼠15只制备烧伤动物模型,将模型鼠完全随机分为3组:实验组,阳性对照组及阴性对照组.方法:①将在体内具有慢、中、快3种吸收速度的人发角蛋白组分材料编织成网孔为1 mm×1 mm的网格,与从牛跟腱中抽提Ⅰ型胶原溶液混合后放入模具,经真空冷冻干燥后制成海绵状膜(作复合内层敷料).②用聚合法制备聚甲基丙烯酸羟乙酯,再与药物虎杖一同成膜,制备药物缓释载体膜(作复合外层敷料).③用SD大鼠制备深Ⅱ度烧伤动物模型,烧伤后3 d做清创处理,清创后实验组用与创面相同大小的人发角蛋白胶原海绵-聚甲基丙烯酸羟乙酯复合敷料覆盖创面,阳性对照组用戊二醛猪皮覆盖创面.阴性对照组为单纯无菌纱布覆盖.主要观察指标:①术后以创面完全上皮化为标准,记录创面愈合时间并分别计算7,14,21 d愈合率.②于覆敷料后1,2,4,6,8周切取整个刨面及其周围组织,光学显微镜观察肉牙组织生长情况和免疫组织化学染色观察新生组织中的胶原纤维和弹性纤维形态.结果:①实验组创面覆盖后渗出明显减少且保持一定湿度,而阳性对照组创面较干燥:实验组及阳性对照组的创面愈合时间较阴性对照组提前(P=0.000);创面在7,14,21 d的愈合率均较阴性对照组高(P=0.000),且实验组在14 d的愈合率较阳性对照组高(P＜0.05).②覆敷料后2周,光镜下观察可见创面肉芽组织中有较细小的胶原纤维填充创面.实验组较其他2组明显.胶原蛋白及弹性蛋白的免疫组织化学染色显示:第2周,实验组创面真皮基质中,Ⅰ型胶原呈棕黄色细密条带状;且有少量破染成棕黄色细丝状的弹性纤维,阳性对照组不明显,而阴性对照组中弹性蛋白无阳情表达.第8周,3组创面均愈合良好.实验组及阳性对照组胶原纤维束改造塑形,无瘢痕形成趋势.阴性对照组真皮层组织排列紊乱.结论:人发角蛋白-胶原海绵聚甲基内烯酸羟乙酯/虎杖复合生物敷料可促进深Ⅱ度烧伤实验大鼠创面的愈合.
揹景:作者在對人髮角蛋白的機製研究和臨床應用中提齣瞭一箇傘新的組織工程學概唸--在體,原位組織工程.在該理論的指導下,擬以人髮角蚩白-膠原海綿為支架,複閤一種可作為藥物緩釋載體的聚甲基丙烯痠羥乙酯,探討其在體內原位誘導週圍組織細胞構建真皮的可行性.目的:以人髮角蛋白膠原海綿為敷料內層,複閤包裹藥物虎杖的聚甲基丙烯痠羥乙酯載體,製各一種雙層複閤生物材料,觀察其對燒傷創麵的促愈閤作用.設計、時間及地點:隨機對照動物實驗,于2005-03/12在南方醫科大學組胚教研室完成.材料:選用雄性SD大鼠15隻製備燒傷動物模型,將模型鼠完全隨機分為3組:實驗組,暘性對照組及陰性對照組.方法:①將在體內具有慢、中、快3種吸收速度的人髮角蛋白組分材料編織成網孔為1 mm×1 mm的網格,與從牛跟腱中抽提Ⅰ型膠原溶液混閤後放入模具,經真空冷凍榦燥後製成海綿狀膜(作複閤內層敷料).②用聚閤法製備聚甲基丙烯痠羥乙酯,再與藥物虎杖一同成膜,製備藥物緩釋載體膜(作複閤外層敷料).③用SD大鼠製備深Ⅱ度燒傷動物模型,燒傷後3 d做清創處理,清創後實驗組用與創麵相同大小的人髮角蛋白膠原海綿-聚甲基丙烯痠羥乙酯複閤敷料覆蓋創麵,暘性對照組用戊二醛豬皮覆蓋創麵.陰性對照組為單純無菌紗佈覆蓋.主要觀察指標:①術後以創麵完全上皮化為標準,記錄創麵愈閤時間併分彆計算7,14,21 d愈閤率.②于覆敷料後1,2,4,6,8週切取整箇鑤麵及其週圍組織,光學顯微鏡觀察肉牙組織生長情況和免疫組織化學染色觀察新生組織中的膠原纖維和彈性纖維形態.結果:①實驗組創麵覆蓋後滲齣明顯減少且保持一定濕度,而暘性對照組創麵較榦燥:實驗組及暘性對照組的創麵愈閤時間較陰性對照組提前(P=0.000);創麵在7,14,21 d的愈閤率均較陰性對照組高(P=0.000),且實驗組在14 d的愈閤率較暘性對照組高(P＜0.05).②覆敷料後2週,光鏡下觀察可見創麵肉芽組織中有較細小的膠原纖維填充創麵.實驗組較其他2組明顯.膠原蛋白及彈性蛋白的免疫組織化學染色顯示:第2週,實驗組創麵真皮基質中,Ⅰ型膠原呈棕黃色細密條帶狀;且有少量破染成棕黃色細絲狀的彈性纖維,暘性對照組不明顯,而陰性對照組中彈性蛋白無暘情錶達.第8週,3組創麵均愈閤良好.實驗組及暘性對照組膠原纖維束改造塑形,無瘢痕形成趨勢.陰性對照組真皮層組織排列紊亂.結論:人髮角蛋白-膠原海綿聚甲基內烯痠羥乙酯/虎杖複閤生物敷料可促進深Ⅱ度燒傷實驗大鼠創麵的愈閤.
배경:작자재대인발각단백적궤제연구화림상응용중제출료일개산신적조직공정학개념--재체,원위조직공정.재해이론적지도하,의이인발각치백-효원해면위지가,복합일충가작위약물완석재체적취갑기병희산간을지,탐토기재체내원위유도주위조직세포구건진피적가행성.목적:이인발각단백효원해면위부료내층,복합포과약물호장적취갑기병희산간을지재체,제각일충쌍층복합생물재료,관찰기대소상창면적촉유합작용.설계、시간급지점:수궤대조동물실험,우2005-03/12재남방의과대학조배교연실완성.재료:선용웅성SD대서15지제비소상동물모형,장모형서완전수궤분위3조:실험조,양성대조조급음성대조조.방법:①장재체내구유만、중、쾌3충흡수속도적인발각단백조분재료편직성망공위1 mm×1 mm적망격,여종우근건중추제Ⅰ형효원용액혼합후방입모구,경진공냉동간조후제성해면상막(작복합내층부료).②용취합법제비취갑기병희산간을지,재여약물호장일동성막,제비약물완석재체막(작복합외층부료).③용SD대서제비심Ⅱ도소상동물모형,소상후3 d주청창처리,청창후실험조용여창면상동대소적인발각단백효원해면-취갑기병희산간을지복합부료복개창면,양성대조조용무이철저피복개창면.음성대조조위단순무균사포복개.주요관찰지표:①술후이창면완전상피화위표준,기록창면유합시간병분별계산7,14,21 d유합솔.②우복부료후1,2,4,6,8주절취정개포면급기주위조직,광학현미경관찰육아조직생장정황화면역조직화학염색관찰신생조직중적효원섬유화탄성섬유형태.결과:①실험조창면복개후삼출명현감소차보지일정습도,이양성대조조창면교간조:실험조급양성대조조적창면유합시간교음성대조조제전(P=0.000);창면재7,14,21 d적유합솔균교음성대조조고(P=0.000),차실험조재14 d적유합솔교양성대조조고(P＜0.05).②복부료후2주,광경하관찰가견창면육아조직중유교세소적효원섬유전충창면.실험조교기타2조명현.효원단백급탄성단백적면역조직화학염색현시:제2주,실험조창면진피기질중,Ⅰ형효원정종황색세밀조대상;차유소량파염성종황색세사상적탄성섬유,양성대조조불명현,이음성대조조중탄성단백무양정표체.제8주,3조창면균유합량호.실험조급양성대조조효원섬유속개조소형,무반흔형성추세.음성대조조진피층조직배렬문란.결론:인발각단백-효원해면취갑기내희산간을지/호장복합생물부료가촉진심Ⅱ도소상실험대서창면적유합.
BACKGROUND: Based on our previous researches in mechanism studies and clinical applications of human hair keratin (HHK), a new concept "in vivol in situ tissue engineering" has been proposed. Under the guidance of this theory, a scaffold of HHK-collagan sponge (inner layer) combined with poly (2-hydroxyethyl methacrylate) (PHEMA) (outer layer as a drug delivery carrier) would be developed to investigate its feasibility to be as a dermal dressing. OBJECTIVE: To develop a scaffold composed of HHK-collagan sponge (inner layer) combined with PHEMA film containing polydatin(PD)(outer layer as a drug delivery carrier) and to evaluate the therapeutic efficacy of the HHK-collagen sponge-PHEMA/PD complex on burn wound healing. DESIGN, TIME AND SETTING: A randomized controlled animal experiment was performed at the Department of Histology and Embryology, Southern Medical University between March and December 2005. MATERIALS: Burn was induced in 15 male Sprague-Dawiey (SD) rats, Rat models of burn were evenly randomized to 3 groups: experimental, positive control, and negative control. METHODS: ①HHK-collagen sponge was prepared through combination of a HHK meshwork (1mm × 1 mm in size for each grid) made up of three components (determined according to biochemical procedures of various degrees, i.e., light, medial, and severe) at a ratio of 4:3:3 with primary collagen sponge extracted from bovine tendons in a mould. Sponge film (used as inner layer dressing) was made by vacuum freeze-drying. ② PHEMA was prepared by polymerization. Than PD was added to prepare PHEMNPD film (used as outer layer dressing).③ Degree Ⅱ burn wound models were established in SD rats by scalding, Superficial necrotic tissue was removed from burn wounds at postnatal 3 days and leave the denatured dermis remained. The wounds were either covered with human HHK-collagen- PHEMNPD complex in the experimental group, or with glutaraldehyde-treated porcine skin in the positive control group, and sterile absorbent gauze was used in the negative control group. MAIN OUTCOME MEASURES: ① Complete epithelization was taken as the standards, and at postoperative 7, 14, and 21 days, wound healing was respectively calculated. ② At postoperative 1, 2, 4, 6, and 8 weeks, the whole wound surface and its peripheral tissue were dissected for observing granulation tissue growing under an optical microscope and detecting the collagen fiber and elastic fiber in the newly formed tissue by immunohistochemical staining. RESULTS: ① Gross observation results revealed that in the experimental group, the volume of the diffusate under the ideal moisture was less compared with the positive control group; the healing time was slightly shorter in both the experimental group and the positive control group than in the negative control group (P= 0.000); At postoperative 7, 14, and 21 days, the healing rate was higher in the experimental and positive control groups than in the negative control group (P=0.000), in addition, the experimental group exhibited higher healing rate than the positive control group at postoperative 14 days ( P ＜ 0.05). ②Optical microscope results showed that at postoperative 2 weeks, a small quantity of collagen fibers were found in the wound granulation tissue in all 3 groups, in particular in the experimental group. Immunohistochemical staining results regarding collagen protein and elastin revealed that at postoperative 2 weeks, both the fine strip-like type Ⅰ collagen fibers and a few silk-like elastic fibers were stained yellowish-brown in the dermal matrix in the experimental group, which were weakly positive in the positive control group, while there was no elastin detectable in the negative control group; at postoperative 8 weeks, burn wounds in all the 3 groups were fully recovered. Remodeling of collagen fibers was more obvious in the experimental and positive control groups than in the negative control group, while the tendency to scar formation with derangement of epithelial cells and collagen fibers in dermis was more prominent in the negative control group than in the remaining two groups. CONCLUSION: HHK-collagen sponge-PHEMA/PD complex may be a new burn dressing via in vivo construction of tissue engineered epidermis, in which PHEMA may be a feasible drug-delivery carrier.
