KERATINOCYTE-DRIVEN CONTRACTION OF RECONSTRUCTED HUMAN SKIN

We have previously reported that reconstructed human skin, using deepidermized acellular sterilized dermis and allogeneic keratinocytes and fibroblasts, significantly contracts in vitro. Contracture of split skin grafts in burns injuries remains a serious problem and this in vitro model provides an opportunity to study keratinocyte/mesenchymal cell interactions and cell interactions with extracted normal human dermis. The aim of this study was to investigate the nature of this in vitro contraction and explore several approaches to prevent or reduce contraction. Three different methodologies for sterilization of the dermal matrix were examined: glycerol, ethylene oxide and a combination of glycerol and ethylene oxide. While the nature of the sterilization technique influenced the extent of contraction and thinner dermal matrices contracted proportionately more than thicker matrices, in all cases contraction was driven by the keratinocytes with relatively little influence from the fibroblasts. The contraction of the underlying dermis did not represent any change in tissue mass but rather a reorganization of the dermis which was rapidly reversed (within minutes) when the epidermal layer was removed. Pharmacological approaches to block contraction showed forskolin and mannose-6-phosphate to be ineffective and ascorbic acid-2-phosphate to exacerbate contraction. However, Galardin, a matrix metalloproteinase inhibitor and keratinocyte conditioned media, both inhibited contraction.     

Assessment of the biological properties of human split skin allografts disinfected with peracetic acid and preserved in glycerol

Skin allografts derived from cadaveric human donors are widely used in the treatment of serious burn injuries and other conditions, such as ulcers. In order to render these allografts safe for clinical use, and to enable them to be preserved and banked for long periods, effective methods of decontamination and preservation are required. These methods must not adversely affect graft properties essential for clinical performance. We have investigated the application of a peracetic acid (PAA) disinfection protocol, coupled with preservation in either glycerol or propylene glycol to achieve these goals. An effective decontamination procedure, comprising of a 3h exposure to 0.1% (v/v) PAA in phosphate buffered saline (PBS) at pH 7.0, was developed and had no significant detrimental effects on the structure of skin. Cadaveric skin allografts were then treated with this disinfection protocol and subsequently preserved in either 85% (v/v) glycerol or propylene glycol in PBS, and the biological properties of the allografts thought to be essential to successful clinical performance were assessed. The cytotoxicity of the grafts was assessed using both extract and contact assays; damage to the skin collagen was assessed using a collagenase susceptibility assay and the capacity of the grafts to elicit an inflammatory response in vitro was assessed by quantifying the production of the pro-inflammatory cytokine TNF-alpha by human peripheral blood mononuclear phagocytes. Neither the disinfection protocol nor either of the preservation techniques rendered the grafts cytotoxic or pro-inflammatory. The PAA disinfection and glycerol preservation protocol had no effects on collagenase susceptibility, whereas the disinfection protocol in combination with propylene glycol rendered some of the test samples significantly more susceptible to collagenase digestion. Therefore, this study has demonstrated that PAA disinfection combined with glycerol preservation is suitable for skin allografts. The use of propylene glycol as a preservation agent for skin requires further development.     

不同消毒方法对去细胞跟腱-骨结构的生物力学影响

[目的]研究60^Co和环氧乙烷消毒对化学去细胞跟腱-骨结构生物力学的影响。[方法]切取新西兰大白兔双侧跟腱-骨共12条，1％磷酸三丁酯浸泡48h，去离子水和酒精洗涤，冷冻干燥后，分别采用。Co和环氧乙烷消毒，然后进行力学测试，新鲜兔跟腱-骨为阳性对照。[结果]环氧乙烷消毒跟腱-骨生物力学性能与新鲜兔跟腱一骨相比无显著性差异（P〈0．05），60^Co消毒后兔跟腱-骨力学性能明显减弱。[结论]环氧乙烷消毒法在维持兔去细胞跟腱-骨的力学性能方面优于60^Co消毒法。     

人工真皮替代物的构建及其生物相容性评价

目的　构建人工真皮替代物并评价其生物相容性。方法　取自健康小白猪的异种皮肤先用 0 .5 %安多福浸泡消毒后 ,在无菌操作下 ,取厚度为 0 .3～ 0 .4mm ,经胰蛋白酶消化、冷冻干燥及戊二醛等处理后检测其理化性能和生物学相容性。结果　去细胞真皮底物具有良好的弹性、强度和组织学结构 ;人包皮成纤维细胞均能在 5～ 10min内很好地粘附 ,且生长良好 ,说明人工真皮对细胞生长无毒性 ;人工真皮在大鼠体内的免疫排斥反应轻微 ,组织相容性好。结论　本实验所构建的人工真皮具有良好的理化性能 ,生物相容性好 ,免疫排斥反应小 ,且成本低、来源广 ,是构建组织工程皮肤理想的真皮材料。     

Gamma irradiation and ethylene oxide in the sterilization of native reindeer bone morphogenetic protein extract.

BACKGROUND AND AIMS: For human use, it is necessary to sterilize bone morphogenetic proteins (BMPs), in order to reduce the risk of infections and associated complications. We compared the effects of ethylene oxide and gamma irradiation in the sterilization of native reindeer BMP extract with regard to bone induction in the Balb/C mouse thigh muscle pouch model. MATERIALS AND METHODS: BMP extract, sterilized with ethylene oxide gas (Steri-Vac 4XL, temperature 29 degrees C, exposure time 4 h, ethylene oxide concentration 860 mg/l), or gamma irradiation at doses of 3.15 MRad was administered in implants containing 5 or 10 mg of BMP extract with collagen carrier. Non-sterilized collagen implants served as controls. New bone formation was evaluated based on the incorporation of Ca45 and radiographically three weeks after implantation. RESULTS: The collagen was not able to induce new bone visible in radiographs. The mean Ca45 incorporation in the gamma sterilized group containing 5 mg of BMP extract was 30% (p = 0.04) and that containing 10 mg of BMP extract was 60% (p = 0.02) higher than seen in the corresponding ethylene oxide sterilized groups. The mean new bone areas were 45% higher in the gamma sterilized groups than in the corresponding ethylene oxide sterilized groups, but the differences were not significant. The mean optical density of new bone in the gamma sterilized group containing 5 mg of BMP extract was 75% (p = 0.00) and in that containing 10 mg of BMP extract was 70% (p = 0.00) higher than seen in the corresponding ethylene oxide sterilized groups. CONCLUSION: Native reindeer BMP extract is more sensitive to the effects of ethylene oxide gas sterilization than gamma irradiation. These results suggest that gamma irradiation is recommendable for the sterilization of BMP extracts.     

低浓度胰蛋白酶消化加反复冻融法制备猪脱细胞真皮基质

目的建立一种新的制作脱细胞猪真皮基质的方法。方法采用0．5g／L胰蛋白酶消化断层猪皮，去除表皮和真皮中部分细胞成分，然后反复冻融进一步去除真皮内残留的细胞成分。分别行大体观察、组织学观察及免疫组织化学等检测。结果制备的猪脱细胞真皮基质内细胞成分可被完全去除，表面保留完整的基底膜，胶原结构排列紧密。结论低浓度胰蛋白酶消化加反复冻融，是制备异种脱细胞真皮基质较为简便、有效的方法。     

聚羟基丁酸酯可吸收缝线灭菌方法的实验研究

目的：研究不同的灭菌方法对聚羟基丁酸酯（PHB）可吸收缝线的降解性和力学特性的影响。方法：将聚羟基丁酸酯（PHB）可吸收缝线进行酒精浸泡、环氧乙烷、紫外线照射三种方法进行灭菌处理后,用细菌培养检测灭菌效果,用粘度法测定聚合物分子量从而观察缝线的降解性,用拉伸试验测定缝线的力学特性。结果：①酒精浸泡灭菌组有10%检出细菌,环氧乙烷及紫外线灭菌组未检出细菌;②紫外线照射灭菌和酒精浸泡灭菌组可使缝线黏度下降并具有显著性差异,环氧乙烷对缝线黏度下降的影响没有显著性差异;③经酒精和紫外线灭菌后缝线的断裂强度降低明显,差异具有显著性,经环氧乙烷灭菌后缝线的断裂强度降低无显著性差异。经紫外线灭菌后缝线的断裂伸长率明显降低,差异具有显著性,经酒精和环氧乙烷灭菌后缝线的断裂伸长率无明显降低。结论：环氧乙烷灭菌对PHB可吸收缝线的降解性和力学特性影响较小,是PHB可吸收缝线较理想的灭菌方法。     

选择性脱细胞猪皮研制及早期临床应用

目的介绍选择性脱细胞猪皮覆盖大面积烧伤早期创面的研制方法及临床应用效果. 方法 2001年1月～2002年5月,对1例深Ⅱ度15%、Ⅲ度25%烧伤患者的右前臂和右小腿,行选择性脱细胞中厚猪皮早期覆盖.戊二醛交联后表皮面黏贴于容器,边缘包埋.在含0.25%胰酶的PBS液中37℃消化2小时,去污剂处理24小时后,漂洗备用.将处理后猪皮应用于1例切削痂自体微粒植皮创面覆盖约2%,大体和光镜观察其功能和外观恢复情况. 结果组织学观察见表皮层基本完整,真皮内无细胞.初步临床观察显示,选择性脱细胞猪皮的真皮可在创基成活,失活表皮可被宿主自体表皮替代. 结论选择性脱细胞猪皮有望替代现有材料用于大面积烧伤切削痂创面的早期覆盖.     

Care and sterilization of endourologic instruments

All endourologic instruments must be handled properly if they are to continue to function properly. Care must be taken in washing and sterilizing of this equipment, as not all endoscopic equipment can endure all methods, and people who are working with these instruments must be taught the proper care and sterilization methods of each. For example, fiberoptic telescopes and light cables must never be autoclaved; ethylene oxide is the method of choice. Disinfectant is an alternative. Loops, sheaths, high-frequency cables, resectoscopes, and working elements should be sterilized by ethylene oxide. Loops and high-frequency cables should not be soaked in a disinfectant, but other instruments may be soaked. All instruments should be dried before sterilization. Too much emphasis cannot be placed on the importance of proper storage of these very fragile fiberoptic instruments. With proper care and sterilization, these instruments will need fewer repairs and function properly for a longer time.     

不同方法消毒口腔高速手机效果的比较研究

目的探讨不同方法消毒口腔高速涡轮手机的效果.方法将90支使用后的口腔高速涡轮手机随机分为1、2、3三个消毒组,分别采用2%戊二醛溶液擦拭浸泡30 min、环氧乙烷气体消毒60 min及预真空高压蒸汽消毒10 min的方法消毒;消毒前、后对手机表面及其内部水、气管腔采样进行细菌学检测.结果三组消毒后手机表面灭菌率分别为99.99%、100.00%和100.00%.手机内部水、气管腔冲洗液细菌检测2%戊二醛溶液消毒灭菌率为31.96%,环氧乙烷为100.00%,预真空高压蒸汽灭菌法为100.00%.结论高压蒸汽灭菌法消毒手机耗时短、效果好,可作为口腔高速手机消毒的首选方法.     

Enhancement of keratinocyte performance in the production of tissue-engineered skin using a low-calcium medium

The success of laboratory-expanded autologous keratinocytes for the treatment of severe burn injuries is often compromised by their lack of dermal remnants and failure to establish a secure dermo-epidermal junction on the wound bed. We have developed a tissue-engineered skin substitute for in vivo use, based on a sterilized donor human dermis seeded with autologous keratinocytes and fibroblasts. However, culture rates are currently too slow for clinical use in acute burns. Our aim in this study was to increase the rate of production of tissue-engineered skin. Two approaches were explored: one using a commercial low-calcium media and the other supplementing well-established media for keratinocyte culture with the calcium-chelating agent ethylene glutamine tetra-acetic acid (EGTA). Using commercial low-calcium media for both the initial cell culture and subsequent culture of tissue-engineered skin did not produce tissue suitable for clinical use. However, it was possible to enhance the initial proliferation of keratinocytes and to increase their horizontal migration in tissue-engineered skin by supplementing established culture medium with 0.04 mM EGTA without sacrificing epidermal attachment and differentiation. Enhancement of keratinocyte migration with EGTA was also maximal in the absence of fibroblasts or basement membrane.     

Peracetic Acid and Its Application to Medical Instrument Sterilization

Abstract: Peracetic acid is recognized as a powerful germicidal agent. However, heretofore its corrosive nature has prevented its direct use for medical instrument sterilization. A specially formulated buffered peracetic acid sterilant with anticorrosives has been developed for use in the STERIS PROCESS^TM. The STERIS SYSTEM 1^TM Processor and the STERIS 20^TM Sterilant Concentrate have met or exceed the rigorous requirements of the Environmental Protection Agency and Food and Drug Administration for making claims related to sterilization. With this system, medical devices including endoscopes are sterilized and ready for use in less than 30 min.     

Promising therapy for congenital giant pigmented nevi using acellular autograft nevi-dermal matrix.

As promising new therapy for congenital giant pigmented nevi, the authors investigated the potential use of an acellular autograft nevi-dermal matrix in combination with a split-thickness skin graft. To address whether the processed acellular nevi-dermal matrix from frozen skin could be reconstituted as a viable dermal base, the authors grafted it onto full-thickness skin defects in nude rats. Fibroblast infiltration and neovascularization into the acellular nevi-dermal matrix were observed. However, because the disappearance of the residual melanotic granules of the grafted dermis took 16 weeks, the authors excised with scissors the superficial layer of the acellular nevi-dermal matrix containing a large quantity of melanin. The appearance after using this method was relatively superior even compared with the full-thickness skin graft. The success of their experimental animal model using the acellular nevi-dermal matrix covered with split-thickness skin grafts confirms the potential value for the clinical application of this treatment for congenital giant nevi.     

Ethylene oxide sterilization of bone grafts: Residual gas concentration and fibroblast toxicity

We examined the concentration of ethylene oxide in bone allografts after gas sterilization. Chips of the human femoral head were investigated. Residual gas concentration was determined by gas chromatography after the bone chips had been subjected to defatting and freeze-drying, followed by ethylene oxide gas sterilization. Bones were prepared in various ways in an attempt to reduce the concentration of residual ethylene oxide. The concentration was higher when gas sterilization was performed before freeze-drying than when it was done afterwards. An experiment performed with fibroblasts showed the high toxicity of residual ethylene oxide in bone chips, even when the concentration was very low. The growth of fibroblast was reduced more in medium which had been shaken with bones sterilized with ethylene oxide before freeze-drying than in medium which had been shaken with bones sterilized after freeze-drying. The higher residual ethylene oxide concentrations resulted in a decrease in fibroblastic culture activity. Our experiment showed the importance of reducing the residual ethylene oxide gas concentration. Defatting and freeze-drying result in lower residual ethylene oxide concentrations.     

Transplantation of acellular dermis and keratinocytes cultured on porous biodegradable microcarriers into full-thickness skin injuries on athymic rats

In search of an optimal transplantation regime for sufficient dermal and epidermal regeneration after a full-thickness skin injury, wounds on athymic rats were grafted with split-thickness skin grafts or acellular human dermis followed by transplantation with human keratinocytes either in single-cell suspension or cultured on porous biodegradable microcarriers. After 2 weeks, all wounds grafted with acellular human dermis showed a well organised and vascularised dermal component and reepithelialisation on the grafted dermal matrix was complete 21 days after transplantation with human keratinocytes. Wounds grafted with human keratinocytes seeded on biodegradable microcarriers or split-thickness skin grafts displayed over time (i.e. 16-21 days post-transplantation) a significantly thicker epithelial cell layer in comparison to wounds grafted with keratinocytes in single-cell suspensions or microcarriers not seeded with cells. Furthermore, measurements of dermal thickness in the closed wounds 21 days after grafting showed a significantly thicker and well organised neodermal component in wounds transplanted with keratinocytes seeded on microcarriers or split-thickness skin grafts compared to all other wounds. Positive immunostaining towards von Willebrand factor revealed the plausible proangiogenic effects of transplantation with keratinocytes seeded on microcarriers. Analysis of representative tissue sections after fluorescence in situ hybridisation visualised that grafted human keratinocytes were present in the epidermal layers covering the wounds 16 and 21 days after transplantation, strongly indicating preservation of cell viability. These results shows that the use of biodegradable microcarriers in the culture of autologous keratinocytes for treatment of full-thickness wounds not only facilitate the cultivation, transportation and transplantation processes but also enhances the dermal regeneration induced by a dermal scaffold which results in a clinical result that is significantly superior to the one obtained when keratinocytes are transplanted in a single-cell suspension.     

PDGF-B基因表达在组织工程化皮肤移植后血管重建中的作用

目的构建含有血小板衍化生长因子B(PDGFB)基因的组织工程化皮肤,进行动物移植实验,研究PDGFB基因表达在真皮血管重建中的作用。方法构建PDGFB真核表达质粒,用脂质体LipofectAMINE介导转染成纤维细胞。分别构建3种不同类型的组织工程化皮肤角质形成细胞 脱细胞猪真皮基质(A组),角质形成细胞 脱细胞猪真皮基质 成纤维细胞(B组),角质形成细胞 脱细胞猪真皮基质 PDGFB基因转染的成纤维细胞(C组),分别移植于大鼠背部创面,观察术后2、4、6周真皮血管重建情况。结果术后2周C组真皮浅层内可见较多新生毛细血管长入,B组次之,A组毛细血管长入较少(P<005);术后4周各组真皮浅层内毛细血管数逐渐增加,但C组毛细血管数仍明显高于B、A两组(P<005);术后6周各组织工程化皮肤内毛细血管数差异无显著性意义。结论PDGFB基因在组织工程化皮肤移植后早期真皮血管重建中发挥了重要的作用,为移植后皮片成活提供了保障。     

Development of a reconstructed human skin model for angiogenesis

We have previously shown that reconstructed human skin engineered from autologous keratinocytes, fibroblasts, and sterilized donor allodermis stimulates angiogenesis within 5-7 days when placed on well-vascularized wound beds in nude mice. When this reconstructed skin was used clinically in more demanding wound beds, some grafts were lost, possibly due to delayed vascularization. As this reconstructed skin lacks any endothelial cells, our aim in this study was to develop an angiogenic reconstructed skin model in which to explore strategies to improve angiogenesis both in vitro and in vivo. We report that culture of small-vessel human dermal microvascular endothelial cells (HuDMECs) was achieved using magnetic beads coated with an antibody to platelet cell adhesion molecule as a means of purifying the culture. Keratinocytes, fibroblasts, and HuDMECs could be cultured from the same skin biopsy. Initial studies culturing HuDMECs and other sources of endothelial cells with the tissue-engineered skin showed that these cells were capable of slowly entering the dermis under standard culture conditions in vitro. In conclusion, this provides us with a model in which to explore strategies for improving angiogenesis in vitro and also establishes the culture methodologies for the production of reconstructed skin containing autologous keratinocytes, fibroblasts, and endothelial cells.     

Promotion of acellular dermal matrix resolution in vitro by matrix metalloproteinase-2

OBJECTIVE: To determine whether acellular human dermis is degraded by matrix metalloproteinases (MMPs), a large class of matrix-degrading enzymes. METHODS: The degradation of acellular human dermis specimens was evaluated in vitro. Wild-type murine fibroblasts with a broad-spectrum MMP inhibitor, GM6001, and MMP-2-deficient fibroblasts were placed on the basement membrane and dermal surfaces of acellular human dermis. Matrix degradation and fibroblast infiltration into the matrix were assessed after a 20-day incubation period. RESULTS: The basement membrane thickness of the specimens cultured with wild-type fibroblasts was significantly less than that of specimens cultured with GM6001 (P<.001), and the infiltration of fibroblasts into the dermal surface was limited by the addition of GM6001 (P=.002). To determine whether MMP-2 was involved in this in vitro phenotype, MMP-2-deficient fibroblasts were assessed in comparison with wild-type fibroblasts. Wild-type fibroblasts degraded the basement membrane surface (P<.001) and infiltrated the dermal surface (P = .003) more efficiently than did MMP-2-deficient fibroblasts. CONCLUSIONS: The results from our in vitro experiments suggest that MMPs and specifically MMP-2 may play an important role in the resorption of acellular human dermis. Addition of MMP inhibitors to implanted dermal matrices may slow fibroblast infiltration and improve their longevity in vivo.     

Creation of an acellular dermal matrix from frozen skin

At present, one of the treatments of choice for closure of full-thickness skin loss is to use a cultured epidermal autograft when skin loss is extensive. In this study, we investigated a simple method of processing frozen surplus skin to produce an acellular, structurally intact, dermal matrix. First, the acellular dermal matrix prepared from normal human skin (ADM) we processed was observed using a transmission electron microscope and a scanning electron microscope. The matrix maintained the basement membrane complex and the extracellular matrix structure of the dermis despite frozen skin being used. Next, using an animal model, we transplanted the ADM and Pelnac, which is used as a contrast in full-thickness wounds onto nude rats. The dermal matrix supported fibroblast infiltration and neovascularization. These results suggest that skin processed by our simple method has the potential to be used as a dermal template together with the cultured epidermis in the closure of full-thickness wounds.     

Infectious diseases and the anaesthetist

The methods of dealing with various items of anaesthetic equipment in order to assure a fresh supply for each patient have been discussed. These consist of using disposable items, steam sterilization, disinfection by both chemical methods and pasteurization and the use of ethylene oxide sterilization. The use of disposable bacterial and viral filtres to protect ventilators and soda lime cannisters is discussed. These can then be sterilized by ethylene oxide at less frequent intervals, i.e., weekly. Protection of the anaesthetists' skin from contact with body fluids by the use of barrier methods are stressed. Methods to avoid penetration of the skin by needlestick and sharp objects are discussed. The increasing number of persons being treated for opportunistic infections makes it likely that anaesthetists will encounter increasing numbers of patients infected with HIV. The more common infections encountered in the operating room in North America have been included, with methods of avoiding possible infection from them. Constant vigilance and the use of universal precautions when caring for all patients is therefore required by the anaesthetist in the operating room in order to avoid contacting infection from patients.