Histologic study of artificial skin used in the treatment of full-thickness thermal injury

Integra artificial skin is an effective means of treatment for full-thickness burns. In extensive burn injury the use of such skin substitutes may become the treatment of choice. The artificial skin consists of a dermal substitute of bovine collagen and chondroitin-6-sulfate and an epidermal layer of synthetic polysiloxane polymer (Silastic). Serial biopsy specimens were obtained from 131 patients during a period of 7 days to 2 years after application. In this histologic study, six sequential phases of repair were discerned. In addition, there were occasional unusual histologic features, eosinophilic infiltration, and/or macrophage-derived giant cell formation in the wound area; however, such findings did not clinically correlate with a negative response to Integra artificial skin. Good repair was obtained, with rare exceptions. An intact dermis was achieved as well as definitive closure of a complete epidermal layer with a minimum of scarring.     

Clinical regenerative medicine: the skin

Tissue engineering of skin is classified into acellular artificial skin and cellular artificial skin. Acellular artificial skin or artificial dermis, is composed of an inner collagen sponge and an outer silicone film. When placed on wounds, the collagen sponge is spontaneously converted into a dermis-like connective tissue. Addition of bFGF or cultured fibroblasts accelerates synthesis of the dermis-like tissue. Cultured epidermis often fails to take on a full-thickness skin defect because of lack of dermal component. Cultured skin with both epidermal and dermal components seems to be an ideal skin substitute, but its take rate is still low. Regeneration of complete skin with skin appendages, vascular networks, elastic fibers and so on is desired.     

表皮干细胞联合脱细胞真皮构建人工皮肤促进创面愈合的实验研究

目的探讨表皮干细胞联合脱细胞真皮构建人工皮肤促进创面愈合的可行性。方法（1）用胰蛋白酶和EDTA联合消化法分离表皮,用Ⅳ型胶原快速黏附法分离、纯化人表皮干细胞,以含表皮生长因子、角质细胞无血清培养液等组成人表皮干细胞培养基进行体外培养,测定克隆形成率。（2）将培养人表皮干细胞接种于制备的脱细胞真皮支架中,构建组织工程人工皮肤,移植治疗兔全层皮肤缺损创面,观察创面修复效果。（3）取新西兰白兔常规制作背部全层皮肤缺损创面,随机分为4组：A、B、C组分别用含表皮干细胞的组织工程皮肤、含角质细胞的组织工程皮肤和单纯脱细胞真皮移植于皮肤缺损创面;D组用创面空置为对照。观察创面修复情况、局部炎症反应,并记录创面愈合时间。结果体外培养的人表皮干细胞增殖稳定,克隆形成率明显高于角质细胞对照组（P〈0．05）。移植后A组创面愈合良好,局部炎症反应轻微,无出血、积脓、坏死,创面愈合时间较B、C、D组明显缩短（P〈0．05或P〈0．01）。结论以表皮干细胞作为种子细胞联合脱细胞真皮构建人工皮肤可用于皮肤缺损创面的修复治疗。     

Comparison of a sealed, polymer foam biodegradable temporizing matrix against Integra? dermal regeneration template in a porcine wound model

The aim of this study is to develop and optimize the first stage of a proposed two-stage skin graft replacement strategy. This entails creation of a material that can be applied immediately after burn excision to "temporize" the wound bed, become integrated as a "neodermis," resist contraction and infection, and provide the grounding for the second stage (an autologous, cultured composite skin). Four 8 × 8 cm wounds were generated in six pigs to assess and compare wound contraction using Integra? dermal regeneration template, a biodegradable temporizing polymer matrix (sealed and unsealed), and a secondary intention wound. All dressings were contiguous. Infection resulted in early spontaneous delamination of the Integra? marring the long-term comparison. The wounds treated with the sealed polymer thus contracted significantly less than the wounds treated with Integra? over the 28 days. Histologically, a thick layer of scar developed superficial to the Integra?, unsealed polymer, and in the secondary intention wounds when compared with the sealed polymer, where such a scar layer was characteristically minimal. No clinical signs of infection were observed for any polymer-treated wound. Once the Integra? silicone layer delaminated, wound contraction was aggressive. Optimization of the biodegradable sealing membrane is imminent, and the second stage of composite skin development is under way.     

Construction of skin equivalents for gene therapy of recessive dystrophic epidermolysis bullosa

We have shown that retroviral vectors efficiently transfer the 9-kb collagen type VII cDNA into keratinocytes of dogs with recessive dystrophic epidermolysis bullosa (RDEB) and achieve correction of the RDEB phenotype in vitro. As a next step toward gene therapy applications, we have assessed the suitability of retroviral vectors to transduce human collagen type VII cDNA into primary human RDEB keratinocytes and generate transplantable autologous skin equivalents. The transduced RDEB keratinocytes permanently express high levels of recombinant collagen type VII that assembles into functional homotrimers readily secreted into the extracellular matrix. The recombinant collagen type VII reverts the migration and invasion potential of the transduced RDEB keratinocytes in vitro and is efficiently deposited at the dermal epidermal junction of artificial skin prepared with the reverted cells and artificial dermis made of biomaterial sponges embedded with dermal RDEB fibroblasts. Transplantable fibrin-based skin equivalents made with the transduced RDEB keratinocytes and grafted onto SCID mice either orthotopically or in accordance with the flap method generated cohesive and orderly stratified epithelia with all the characteristics of normal human epidermis, including rapid formation of anchoring fibrils. Because transplantable epithelia are routinely used to cure patients suffering from large skin or mucosal defects, the full phenotypic reversion of primary RDEB epidermal clonogenic cells mediated by recombinant retroviral vectors opens new perspectives in the long-term treatment of genodermatoses.     

Clinical use of injectable bovine collagen: a decade of experience

Bovine collagen has long been recognized as a safe, highly biocompatible material. In 1981 and 1985, two injectable fibrillar suspensions of purified bovine dermal collagen, Zyderm and Zyplast collagen implant, were commercially launched in the USA for use in soft tissue contour irregularities. Since that time more than 750 000 patients have been treated with Zyderm and/or Zyplast collagen implants internationally. Adverse reactions to bovine collagen implants occur in a small percentage of treated patients. The most prevalent adverse reaction is localized hypersensitivity at treatment sites, occurring in 3% of skin tested patients and 1 to 2% of the treated patients. These reactions resolve with time as the implant material is resorbed by the host. Circulating antibodies to bovine collagen can be demonstrated in the sera of a majority of patients (90-100%) with local hypersensitivity. These antibodies are specific for bovine collagen and do not cross react with human type I, II or III collagen. Although the presence of a humoral immune response to bovine collagen can be associated with localized symptoms of hypersensitivity, antibodies to bovine collagen can also be demonstrated in the absence of any clinical manifestation.     

Dermal matrix scaffold engineered with adult mesenchymal stem cells and platelet-rich plasma as a potential tool for tissue repair and regeneration

The purpose of this study was to investigate the efficacy of Integra, an artificial dermal matrix used as a dermal template for skin regeneration, to form a multifunctional scaffold with human bone marrow-derived mesenchymal stem cells (hMSCs) and platelet-rich plasma (PRP) for tissue engineering and regenerative technology. First, we showed that PRP, used as a supplement for growth medium, represented an optimal substitute for animal serum as well as a source of multiple growth factors, was able to satisfactorily support cell viability and cell proliferation and influence stemness gene expression in hMSCs. Moreover, Integra appeared to be a suitable substrate for hMSCs colonization, as judged by two-photon microscopy combined with fluorescence lifetime imaging (FLIM) and confocal analysis. The cells were then seeded on Integra + PRP for 24 and 48 h. Notably, in these conditions, the seeded cells exhibited a greater aptitude to colonize the scaffold, showed improved cell adhesion and spreading as compared with those cultured on Integra alone, and acquired a fibroblast-like phenotype, indicating that the bioengineered scaffold provided an appropriate environment for cellular growth and differentiation. In conclusion, these results, although preliminary, provide clues for the design of new therapeutic strategies for skin regeneration, consisting in the combination of mesenchymal stem cells with engineered biomaterials.     

Multicenter postapproval clinical trial of Integra dermal regeneration template for burn treatment

The safety and effectiveness of Integra Dermal Regeneration Template was evaluated in a postapproval study involving 216 burn injury patients who were treated at 13 burn care facilities in the United States. The mean total body surface area burned was 36.5% (range, 1-95%). Integra was applied to fresh, clean, surgically excised burn wounds. Within 2 to 3 weeks, the dermal layer regenerated, and a thin epidermal autograft was placed. The incidence of invasive infection at Integra-treated sites was 3.1% (95% confidence interval, 2.0-4.5%) and that of superficial infection 13.2% (95% confidence interval, 11.0-15.7%). Mean take rate of Integra was 76.2%; the median take rate was 95%. The mean take rate of epidermal autograft was 87.7%; the median take rate was 98%. This postapproval study further supports the conclusion that Integra is a safe and effective treatment modality in the hands of properly trained clinicians under conditions of routine clinical use at burn centers.     

组织工程皮肤的构建及组织形态学观察

背景：应用于临床的组织工程皮肤具有血管化速度慢、力学强度差以及无法永久性保留等局限,因此,需要对相关技术环节进行改进以制备一种合适的永久性皮肤替代物。目的：建立一种构建有活性的双层组织工程皮肤的方法,并对其组织形态学进行观察。方法：以Ⅳ型胶原和成纤维细胞联合修饰的同种脱细胞真皮基质为支架,与表皮干细胞复合后依次经浸没式培养和气液分离界面培养构建组织工程皮肤,利用光镜和扫描电镜对其组织学特点进行观察和分析。结果与结论：以表皮干细胞和同种脱细胞真皮基质制备的组织工程皮肤具有表、真皮双层结构,其中表皮由多层不同分化程度的表皮细胞组成,真皮为天然三维孔隙结构,胶原纤维完整,且表皮层与真皮层紧密连接,形成整体结构,可满足全层皮肤替代物的基本组织学要求。     

Skin replacement using collagen extracted from bovine hide

This paper reviews the use of biological sponge-shape matrices as dermal replacements in order to orient newly formed wound tissue. Sponge-shape matrices consist of a scaffold made of cross-linked collagen extracted from bovine hide. Other molecules with specific activities on wound tissue ingrowth are bound to collagen. The lamination of sponge with a synthetic material allows this device to be implanted as a temporary skin substitute. For the epidermal cell layer replacement, a biological film-shape matrix can be used in order to cultivate autologous cells during the period that biological sponge-shape matrices are invaded by wound tissue.     

Immunoassay techniques for the detection of circulating antibodies to collagen following the use of collagen medical devices

During the use of collagen medical devices, some adverse clinical reactions occur involving both the cellular and humoral types of the immune response. Thus, the development of immunoassay techniques for measuring the presence and the levels of circulating anticollagen antibodies is required. The authors present their protocol: it is a solid phase radioimmunoassay using collagen coated on polystyrene microplates and labelled protein A as the tracer. An example of the application of the technique is described. Anticollagen antibodies were monitored in 586 patients undergoing bovine collagen implant therapy. In a retrospective study on 166 patients we found a good correlation between the presence of antibodies to collagen and cellular immune reactions such as a positive skin test or adverse clinical reactions after implantation. A prospective study on 420 patients showed that the pretreatment anticollagen serologic test can be useful as an adjunct to skin testing in the conservative management of patients desiring bovine collagen implant therapy. The use of this double test allowed avoidance of any major clinical reaction and reduced minor signs of intolerance.     

Restoring full‐thickness defects with spray skin in conjunction with dermal regenerate template and split‐thickness skin grafting: a pilot study

Complex, full‐thickness soft tissue defects secondary to large burns, trauma and war‐related injuries continue to challenge reconstructive surgeons. To achieve positive surgical outcomes in these patient populations, novel approaches are needed to restore the functional, protective and aesthetic properties of skin. Herein, we provide the first report describing the staged use of a dermal regenerate template (DRT) with a spray‐on epidermal regenerative modality (spray skin) in addition to autologous split‐thickness skin grafting (STSG) in non‐burn trauma and compare these results with those of patients treated with DRT and STSG alone. A pilot study was performed to evaluate whether the use of spray skin technology (ReCell, Avita Medical) as an adjunct to DRT (Integra, Integra Lifesciences) and autologous skin grafting in the treatment of patients with large full‐thickness soft tissue losses impacts donor site burden as well as recipient and donor site re‐epithelialization. In this retrospective study, two patients who were treated with DRT and STSG alone (control group) were compared with two patients who were treated with DRT and spray skin/STSG in combination (experimental group). The mechanisms of injury, total defect and treatment sizes, time to complete re‐epithelialization, lengths of follow‐up, outcomes and complications were reviewed. Our group observed that using a DRT in conjunction with spray skin/STSG can reduce donor site burden and decrease time to complete healing. It can also permit greater or larger meshing ratios, while aiding in improved re‐pigmentation when compared with similar wounds treated with a DRT and autologous skin grafting alone. Copyright © 2017 John Wiley & Sons, Ltd.     

人工真皮修复急性创伤后足背皮肤软组织缺损14例

背景：人工真皮具有硅胶膜和胶原海绵双层结构,是最早开发和应用于临床的组织工程化皮肤替代物,主要应用于皮肤软组织缺损修复。目的：评价人工真皮在足背严重皮肤软组织缺损中的应用。方法：选择北京积水潭医院烧伤科于2009-06/2010-12收治的14例急性外伤后足背严重皮肤软组织缺损患者,均有肌腱或骨外露。所有患者经清创后,以人工真皮移植,待人工真皮成活后以自体断层皮片移植,观察人工真皮和自体皮的成活情况。结果与结论：所有植皮成活,创面愈合良好,供区未见明显瘢痕增生。提示人工真皮联合自体断层皮片移植可以有效修复足背严重皮肤缺损创面,减少供区损伤。     

Skin transplantation.

Transplantation of skin has been recognized as a method to increase the survival of patients who have suffered a massive skin deficit. Until about 30 years ago, a patient who suffered a 60% BSA skin deficit usually died from the effects of overwhelming sepsis. Great strides have since been made in the techniques and technology of skin transplantation. Many different methods are now used to enhance wound closure and to maximize the functioning and cosmesis of the grafted areas. Current methods of permanent wound closure include autografting, Integra with epidermal autografting, cultured epithelial cells, and microskin grafting. Other methods of temporary wound closure include allografts, xenografts, and synthetic materials. These temporary wound coverings act as a "second skin" and keep the wound free from infection while preventing the loss of fluid from the granulation bed. Research continues to strive toward faster wound closure with minimal functional and cosmetic deficits.     

异种去细胞真皮基质在体内转归的初步研究

目的 初步研究异种（猪）去细胞真皮基质在体内的转归。方法 经曲通（Triton）X-100、胰蛋白酶处理得到异种（猪）去细胞真皮基质后，将其包埋于SD大鼠皮下，于术后1、2、4、6、8、12、16、20和30周观察异种（猪）去细胞真皮基质的大体形态及组织学变化。结果 制备的异种（猪）去细胞真皮基质去细胞成分，胶原纤维排列有序，基底膜结构完整。大体观察异种（猪）去细胞真皮基质原始形态随时间延长而逐渐模糊．组织学观察异种（猪）去细胞真皮基质1周以炎性细胞浸润为主，以后成纤维细胞、毛细血管逐渐增多，真皮胶原排列逐渐规则、致密。结论 异种（猪）去细胞真皮基质可在体内作为支架长期存在，并可诱导自身成纤维细胞及毛细血管有序长入。     

组织工程化人工皮肤胶原蛋白和蛋白多糖的代谢

背景:真皮的细胞外基质由胶原蛋白、弹性蛋白和其他基质成份组成。组织工程化人工皮肤作为一种体外皮肤模型应当具有合成、分泌和分解细胞外基质功能。目的:观察组织工程化人工皮肤胶原蛋白和蛋白多糖的代谢。设计:单一样本观察。单位:北京大学深圳医院皮肤科。材料:转铁蛋白;胰蛋白酶;3H-脯氨酸;天狼星红等。方法:实验于2000-06/2004-12在北京大学深圳医院皮肤科和吉林大学再生医学科学研究所完成。①参照文献进行组织工程化人工皮肤的制备。②3H-脯氨酸掺入法测定组织工程化人工皮肤合成胶原蛋白能力,于培养第1,2和3周的人工皮肤培养板孔中,分别加入3H-脯氨酸,培养4h,并与天然皮肤做对照。③苦味酸-天狼星红染色观察组织工程化人工皮肤合成、分泌胶原蛋白的功能;AB-PAS染色测定组织工程化人工皮肤合成、分泌蛋白多糖功能;均于培养第1,2,3,4和6周进行观察。主要观察指标:①组织工程化人工皮肤合成、分泌蛋白多糖测定结果。②组织工程化人工皮肤合成、分泌胶原蛋白的功能。③3H-脯氨酸掺入实验结果。结果:①培养第7,14天的组织工程化人工皮肤3H-脯氨酸掺入值与天然皮肤相近,培养第21天的组织工程化人工皮肤3H-脯氨酸掺入值明显高于天然皮肤。②培养的组织工程化人工皮肤,在偏振光显微镜下可见到有成束的红色双折光物质和细的绿色双折光物质。单纯基质网架只见到红色双折光物质。③培养第1,2周的组织工程化人工皮肤Ab-PAS染色阴性,单纯网架Ab-PAS染色呈阴性,培养第3～6周的组织工程化人工皮肤Ab-PAS染色阳性。结论:构建的组织工程化人工皮肤具有合成、分泌细胞外基质胶原蛋白和蛋白多糖的功能。     

人工真皮修复急性创伤后足背皮肤软组织缺损14例

背景:人工真皮具有硅胶膜和胶原海绵双层结构,是最早开发和应用于临床的组织工程化皮肤替代物,主要应用于皮肤软组织缺损修复.目的:评价人工真皮在足背严重皮肤软组织缺损中的应用.方法:选择北京积水潭医院烧伤科于2009-06/2010-12收治的14例急性外伤后足背严重皮肤软组织缺损患者,均有肌腱或骨外露.所有患者经清创后,以人工真皮移植,待人工真皮成活后以自体断层皮片移植,观察人工真皮和自体皮的成活情况.结果与结论:所有植皮成活,创面愈合良好,供区未见明显瘢痕增生.提示人工真皮联合自体断层皮片移植可以有效修复足背严重皮肤缺损创面,减少供区损伤.     

The utility of collagen-based vehicles in delivery of growth factors for hard and soft tissue wound repair

Bovine demineralized bone powder and reconstituted bovine dermal collagen have been effectively utilized during the past several years to deliver a variety of growth factors in animal models of hard and soft tissue wound repair. Bone morphogenetic proteins have been delivered in a demineralized bone powder matrix to promote ectopic bone formation in the rat subcutaneous model with the objective of studying the process of endochondral bone formation and evaluating the utility of such factors in promoting repair of hard tissue defects. Reconstituted bovine dermal collagen gels and sponges, including composites of collagen and heparin, have been utilized to deliver growth factors such as platelet-derived growth factor (PDGF), transforming growth factor-beta (TGF-beta) and fibroblast growth factor (FGF) to study their effects in subcutaneous and incisional models of dermal wound repair. The results of these experimental animal studies have provided convincing evidence that the rheological properties, biocompatibility and resorbable nature of type I collagen make it an excellent delivery vehicle for evaluation of a variety of growth factors in human clinical studies of hard and soft tissue would repair.