Novel application method of artificial dermis: one-step grafting procedure of artificial dermis and skin, rat experimental study

BACKGROUND: Currently, to treat skin defects with artificial dermis (AD), two surgical procedures where the artificial dermis grafting and another secondary skin grafting are required. The purpose of this study was to achieve simultaneous grafting of the artificial dermis and the split-skin. To enhance the wound angiogenesis, cultured endothelial cells, fibroblasts and PDWHF (platelet derived wound healing factor) were employed. METHODS: The experiment consists of following two parts: (1) Investigation to obtain faster angiogenesis into the bilayer artificial dermis: full-thickness wounds created on the back of the rats were treated with the artificial dermis (Terudermis, with silicone sheet, TERUMO Co., Japan). Prior to the artificial dermis grafting, following four groups were established; control group (AD alone, n=6), PDWHF group (AD treated with PDWHF, n=6), cultured cells group (AD treated with cultured endothelial cells and fibroblasts, n=6), combination group (AD treated with PDWHF and cultured cells, n=6). (2) Trial of one-stage grafting of the AD and the skin: simultaneous grafting of the artificial dermis and skin was performed using the same rat model. Before making skin defects, split thickness skin were harvested. Then the skin grafting was carried out immediately after the AD grafting. To allow grafting of the skin onto the artificial dermis, the AD without silicone sheet (Terudermis without silicone sheet, TERUMO Co., Japan) were used. Two groups, control group (AD alone, n=3) and treatment group (AD with PDWF and cultures, n=3) were established. RESULTS: (1) When the artificial dermis were treated with PDWHF, cultured endothelial cells and fibroblasts, vascular invasion into the artificial dermis was observed 5 days after the surgery. (2) In the treatment group, the skin grafted immediately after the artificial dermis grafting was completely taken. CONCLUSIONS: The present study revealed that treatment with PDWHF, combined with cultured endothelial cells and fibroblasts, accelerated wound angiogenesis. By this method, one-step grafting procedure of the artificial dermis and the skin is possible.     

Negative‐Pressure Dressings in the Treatment of Infected Pressure Ulcers

Objective:  Effects of platelet derived wound healing factor (PDWHF)¹⁾ with or without cultured cells on angiogenesis in treatment of defected wounds using artificial dermis (AD) were investigated in rat experimental model.
Methods:  Wistar strain rats were used for this study. The PDWHF was prepared from platelets. The endothelial cells and fibroblasts were prepared from thoracic aorta and back skin respectively. Two sites of 2.5 × 2.5 cm full‐thickness wounds were created on back of the animals. Artificial dermis (TERUDERMIS^®, TERUMO Co, Japan) were grafted to the wounds. Prior to the AD grafts, following 4 groups were established, control group: AD alone (n = 6), PDWHF group: AD treated with PDWHF (0.1 ml/cm²)(n = 6), cultured cells group: disperse of 1 × 10⁵ cultured endothelial cells and fibroblasts between AD and wound (n = 6) and combination group: combination of PDWHF and cultured cells (n = 6). Five days after surgery, degree of angiogenesis was evaluated by gross inspection and histological study. Evans blue perfusions test was performed to evaluate the degree of new capillary formation in the generated dermis quantitatively.
Results:  The combination group showed vascular invasion into AD 5 days after surgery. In this group, the absorbance of the Evans blue extracted from the grafted dermis was highest among the group.
Conclusion:  The result of the present study revealed that treatment with PDWHF combined with cultured endothelial cells and fibroblasts accelerate the wound angiogenesis. This method may be beneficial to shorten the period between the AD grafts and the secondary skin grafting.     

Lando双层人工真皮在手部皮肤缺损中的应用

目的探讨人工真皮在手部皮肤缺损中的临床应用效果。方法2018年6月-2019年6月,共收治20例手部皮肤缺损伴骨或肌腱外露的患者,所有患者均于一期彻底清创后行VSD负压吸引,待创面无明显感染后采用人工真皮覆盖创面,再次行VSD负压吸引,3周后行自体游离植皮手术,观察人工真皮及自体游离植皮成活情况。结果20例人工真皮及自体游离植皮皮片全部成活,术后随访平均5.6(3~12)个月,骨或肌腱外露均得到良好修复,创面愈合后外形良好,皮肤弹性好,无臃肿及明显瘢痕增生,未见明显色素沉着,外观平整。结论手部创面采用人工真皮修复后成活良好,二期中厚皮片植皮可确保手部皮肤外形美观,弹性好,效果满意。     

血管内皮细胞和成纤维细胞混合移植对人工真皮血管化的影响

目的观察同种血管内皮细胞和成纤维细胞移植对人工真皮血管化的促进作用。方法在27只Wistar大鼠背部造成2．5 cm×2．5 cm全层皮肤缺损创面(2处／只),将其分为血管内皮细胞组:将血管内皮细胞混入0．5 ml纤维蛋白胶中,按1．0×105／cm2的密度均匀喷洒于移植床;混合组:将血管内皮细胞和成纤维细胞混入等量纤维蛋白胶后,同前密度喷洒于移植床;对照组:按同样方法喷洒等量纤维蛋白胶。随后各组移植人工真皮,每组9只大鼠18处创面。于移植后5、10 d切取移植的真皮及周围组织行HE、血管内皮生长因子(VEGF)、Masson和墨汁灌注染色,观察新生血管生长情况。于移植后5 d行伊文思蓝灌注,以分光光度计定量检测法测定微血管形成情况。结果移植后5 d,HE、VEGF、Masson和墨汁灌注染色均可见各组移植床有新生血管长入。HE染色见血管内皮细胞组、混合组新生血管数量分别为(14．2±3．6)、(12．1±2．5)条,较对照组[(3．9±1．6)条]明显增多(P<0．05)。移植后10 d,人工真皮内及移植床均有微血管形成,且胶原组织的合成增加。移植后5 d,经伊文思蓝灌注,收集并检测血管内皮细胞组、混合组真皮组织溶出的上清液,吸光度值分别为0．167±0．058、0．155±0．046,均高于对照组的0．066±0．024(P<0．05)。结论同种血管内皮细胞和成纤维细胞移植可促进创面愈合过程中的血管新生,加速人工真皮移植后血管化过程,促进类真皮组织的成熟。     

Observations on the development and clinical use of artificial skin--an attempt to employ regeneration rather than scar formation in wound healing

Artificial skin, a bilaminar membrane, is grafted on an excised wound immediately following injury. This bilayer membrane, made of a dermal and epidermal portion, is populated in place on the wound bed by the patient's own fibroblasts and epidermal cells producing a permanent skin replacement with an anatomically functioning dermis and epidermis. The dermal portion is a porous collagen-chondroitin 6-sulfate fibrous matrix arranged in a three dimensional pattern closely resembling the fiber pattern of normal dermis. A thin silastic covering serves as a temporary epidermis immediately after grafting until the patient's epidermal cells, seeded on the "neodermis", grow into a confluent epidermal replacement. The autogenous "neodermis" is produced as fibroblasts and vessels migrate from the wound bed into the artificial dermal template and, using the artificial fibers as a scaffolding, synthesize new connective tissue in the collagen fiber pattern of normal dermis rather than the pattern of scar while slowly biodegrading the artificial fibers. This replacement dermis functions as normal dermis and not as scar tissue. The patient's epidermal cells seeded on the "neodermis" grow into a confluent normal appearing epidermis and with the neodermis produce a permanent skin composed of normal functioning dermal and epidermal components produced in situ by the patient's own cells. Artificial skin has been successfully used to permanently replace skin destroyed by burn injuries ranging from 10 to over 95% BSA. The long-term functional results in these patients have been excellent and the long term cosmetic results in preliminary studies tend to be superior to autograft. Artificial skin appears to provide a successful physiologic and cosmetic skin replacement in severe burn injury.     

保留变性真皮并移植大张自体皮修复手部深度烧伤的远期疗效观察

目的观察保留变性真皮并移植大张自体皮修复手部深度烧伤的远期疗效。方法对86例患者的152只深度烧伤手经削刮痂保留变性真皮后,行大张自体皮移植术。术后观察疗效并作随访,时间为3个月—3年,对手部皮肤的颜色、弹性、挛缩程度及功能进行评级。结果152只手中外观及功能优良者141只,占92.8%。结论保留变性真皮并移植大张自体皮修复手部深度烧伤,术后外观及功能恢复较好,是修复手部深度烧伤创面的较佳选择。     

In vivo cultured skin composed of two-layer collagen sponges with preconfluent cells.

Although various kinds of cultured skin substitutes have been developed, it takes several weeks to produce them before grafting. In their previous study, the authors succeeded in producing cultured skin easily in a short period of time by layering two collagen sponges. In the current study, to shorten this period even further, they grafted the cell-preconfluent artificial skin immediately after seeding the cells. They used two collagen sponges with different pore sizes and crosslink densities. They seeded 1,000,000 cells per square centimeter of fibroblasts and 1,000,000 cells per square centimeter of keratinocytes on the respective collagen sponges and grafted them on a full-thickness, excised wound on the back of severe combined immunodeficient mice. Two weeks after grafting, epithelium and dermislike tissue were formed. They then decreased the number of keratinocytes and grafted them. Four weeks after grafting, at seeding densities of 50,000 to 1,000,000 cells per square centimeter of keratinocytes, the preconfluent artificial skin took histologically, and human type IV and type VII collagen were stained immunohistochemically. This cell-preconfluent artificial skin composed of two-layer collagen sponges seems promising for widespread clinical use.     

Observations on the development and clinical use of artificial skin —An attempt to employ regeneration rather than scar formation in wound healing—

Artificial skin, a bilaminar membrane, is grafted on an excised wound immediately following injury. This bilayer membrane, made of a dermal and epidermal portion, is populated in place on the wound bed by the patient's own fibroblasts and epidermal cells producing a permanent skin replacement with an anatomically functioning dermis and epidermis. The dermal portion is a porous collagen-chondroitin 6-sulfate fibrous matrix arranged in a three dimensional pattern closely resembling the fiber pattern of normal dermis. A thin silastic covering serves as a temporary epidermis immediately after grafting until the patient's epidermal cells, seeded on the “neodermis”, grow into a confluent epidermal replacement. The autogenous “neodermis” is produced as fibroblasts and vessels migrate from the wound bed into the artificial dermal template and, using the artificial fibers as a scaffolding, synthesize new connective tissue in the collagen fiber pattern of normal dermis rather than the pattern of scar while slowly biodegrading the artificial fibers. This replacement dermis functions as normal dermis and not as scar tissue. The patient's epidermal cells seeded on the “neodermis” grow into a confluent normal appearing epidermis and with the neodermis produce a permanent skin composed of normal functioning dermal and epidermal components producedin situ by the patient's own cells. Artificial skin has been successfully used to permanently replace skin destroyed by burn injures ranging form 10 to over 95% BSA. The long term functional results in these patients have been excellent and the long term cosmetic results in preliminary studies tend to be superior to autograft. Artificial skin appears to provide a successful physiologic and cosmetic skin replacement in severe burn injury. This report is the gist of a paper read by Dr. Naoki Aikawa, a spokesman for Dr. Burke at the 87th Annual Congress of the Japan Surgical Society, Tokyo, Japan, 1987     

成纤维细胞移植促进人工真皮内血管新生的研究

目的观察成纤维细胞与人工真皮共同移植对移植床及真皮海绵内微血管形成、胶原沉积的影响,促进人工真皮海绵内类真皮组织形成,缩短"二期植皮"的间隔时间.方法 Wistar大鼠18只,背部两侧各制作2.5 cm×2.5 cm全层皮肤缺损创面,在移植人工真皮之前分为两组,即成纤维细胞移植组(A组):向创面喷洒混入密度为1.0×105/cm2的同种真皮成纤维细胞的纤维蛋白胶0.5 ml;对照组(B组):只喷洒纤维蛋白胶0.5 ml.于移植后5和10天分别切取移植的真皮及周围组织,进行HE染色、Masson染色、VEGF免疫抗体染色和墨汁灌注染色,观察移植床及真皮海绵内微血管新生及胶原沉积状态;于移植后5天行伊文蓝组织灌注,分光光度计测定真皮海绵内染料含量,定量检测真皮海绵内循环渗透情况.结果移植后5天,新生血管主要集中在真皮下移植床,A组新生血管较B组明显增多,A组(9.64±2.36)个/高倍视野,B组(3.88±1.62)个/高倍视野,差异有统计学意义(P＜0.05);VEGF阳性细胞亦明显增多.移植后10天,移植床及真皮内均有微血管形成,A组血管数量和管径均较B组增加,胶原沉积量也明显增加.A组(46.04±8.90)个/高倍视野,B组(30.08±7.76)个/高倍视野,有统计学意义(P＜0.05).结论同种成纤维细胞移植可明显加快人工真皮内及移植床的血管新生和胶原沉积,加快真皮海绵内类真皮组织的形成.     

皮能快愈敷料在瘢痕整形治疗中的应用

目的:探讨在瘢痕整形中特别是大面积烧伤后形成瘢痕影响功能需行植皮手术整形的新方法。方法:对于深度烧伤后形成瘢痕需行切除植皮改善美观及功能的患者,Ⅰ期行瘢痕切除后人工真皮(皮能快愈敷料即皮耐克)移植术。2～3周后Ⅱ期手术:去除皮耐克硅胶膜行自身刃厚皮片移植。结果:皮片移植均存活,创面外观满意,供皮区轻度色素沉着,无瘢痕增生。结论:皮耐克与自体刃厚皮复合移植在瘢痕整形治疗中具有较好的临床应用价值,值得借鉴推广。     

Autologous skin transplantation: comparison of minced skin to other techniques

BACKGROUND: Skin grafting may be necessary to close nonhealing skin wounds. This report describes a fast and minimally invasive method to produce minced skin suitable for transplantation to skin wounds. The technique was evaluated in an established porcine skin wound healing model and was compared to split-thickness skin grafts and suspensions of cultured and noncultured keratinocytes. MATERIALS AND METHODS: The study included 90 wounds on 3 pigs. Fluid-treated full-thickness skin wounds were grafted with minced skin, split-thickness skin grafts, noncultured keratinocytes, or cultured keratinocytes. Controls received either fluid or dry treatment. The wound healing process was analyzed in histologies collected at Days 8 to 43 postwounding. Wound contraction was quantified by photoplanimetry. RESULTS: Wounds transplanted with minced skin and keratinocyte suspension contained several colonies of keratinocytes in the newly formed granulation tissue. During the healing phase, the colonies progressed upward and reepithelialization was accelerated. Minced skin and split-thickness skin grafts reduced contraction as compared to keratinocyte suspensions and saline controls. Granulation tissue formation was also reduced in split-thickness skin-grafted wounds. CONCLUSIONS: Minced skin grafting accelerates reepithelialization of fluid-treated skin wounds. The technique is faster and less expensive than split-thickness skin grafting and keratinocyte suspension transplantation. Minced skin grafting may have implications for the treatment of chronic wounds.     

Cutometrical measurement confirms the efficacy of the composite skin grafting using allogeneic acellular dermis in burns

Measurement of viscoelasticity of the skin using the device called cutometer seems to be a very precise and promising diagnostic method in different skin pathologies. However, only a very limited number of reports dealing with the use of cutometer in burns is available. The composite skin grafting using an allogeneic acellular dermis covered with a thin epidermal autologous graft is supposed to improve viscoelastic properties of grafted skin in full-thickness burns. Cutometer MPA 580 has been used for examination of healthy skin, healed and maturated autologous split-thickness skin grafts, and in two patiens 6 years after composite skin grafting using the allogeneic acellular dermis + thin autologous epidermal graft in one stage. Typical curves defined by values of R0-R9 parameters are presented. These data document significantly better viscoelastic properties of composite skin grafts than those of conventional split-thickness skin grafts after 6 years. Measurements of viscoelasticity of the skin, using a cutometer, will probably bring useful and precise data for evaluation of the reconstructed skin in burns. Significantly better properties of the composite skin graft were assessed clinically and measured by Cutometer MPA 580 6 years after surgery.     

Infected tibial nonunion: Good results after open cancellous bone grafting in 37 cases

We treated 37 infected tibial shaft nonunions by debridement followed by open autogenous cancellous bone grafting in a 2-stage procedure. Additional surgery was done in 21 fractures including second debridement before bone grafting and/or a second limited bone grafting and/or a split-thickness skin grafting. All fractures healed after an average of 11 (8-16) months. During 2 years follow-up there were no recurrences of the infection. Two cases of early refracture occurred, both healed following new bone grafting.     

Dermal substitutes do well on dura: comparison of split skin grafting +/- artificial dermis for reconstruction of full-thickness calvarial defects

Large, full-thickness calvarial defects present a series of significant reconstructive challenges involving a range of techniques, including local and free flaps. Occasionally these conventional methods may not be possible due to technical, or patient, factors. Artificial dermis is already widely used in burns surgery and is increasing in oncological reconstruction. We believe that artificial dermis coupled with split-thickness skin grafting provides an excellent option for closure of these defects when other techniques are not appropriate.     

The role of fibroblasts in dermal vascularization and remodeling of reconstructed human skin after transplantation onto the nude mouse.

The vascularization and the dermal remodeling of two different types of human skin reconstructed "in vitro" and grafted onto the nude mouse were studied. They were composed of human keratinocytes grown either on a human acellular deepidermized dermis (DED), or on a lattice composed of human fibroblasts embedded in bovine type I collagen, a living dermal equivalent (LDE). At different stages after grafting, the transplants were harvested and processed for an immunohistological study with species-specific and non-species-specific antibodies. At one month after grafting, the two types of grafted dermis contained blood vessels whose vascular basement membranes were labeled with a mouse-specific anti-type IV collagen antibody. With an antibody specific for human type IV collagen, a constant labeling of the vascular basement membrane was only observed in the LDE containing fibroblasts. In the DED, a constant association of the mouse endothelial cells with human type IV collagen was observed at early stages after grafting. At later stages, the human type IV collagen progressively disappeared. On the other hand, the dermal-epidermal junction underneath the human epidermis contained human type IV collagen in the two types of reconstructed skin. Labeling with the species-specific antibodies directed against human or murine type I collagen showed that the ratio murine type I collagen versus human type I collagen increased with time, suggesting that the DED is progressively invaded by mouse fibroblasts that produce the mouse collagen. On the other hand, in the LDE, the preexisting bovine type I collagen became progressively undetectable while both human type I collagen and elastic fibers were deposited by numerous human fibroblasts. Mouse type I collagen was not detected. Altogether, these observations made by grafting human skin reconstructed "in vitro" onto the nude mouse should be interesting for evaluating the usefulness of grafting a dermal substrate together with the epidermal sheet in the treatment of burns.     

The use of artificial dermis for reconstruction of full thickness scalp burn involving the calvaria

The incidence of extensive full thickness scalp burn involving the calvaria is rare and can be very difficult to reconstruct, as the application of local or free tissue transfer is limited. Although wound closure can be achieved with bone debridement and immediate or delayed split-thickness autografting, the result may be problematic due to unstable skin graft surface. The use of artificial dermis that may provide stable thick coverage in the treatment scalp and skull burn has rarely been reported in literature. We encountered two patients who suffered from severe head burns involving the calvarium. Following debridement including the necrotic bone, the artificial dermis (Integra) was used for immediate wound coverage which was 15 cmx10 cm in one case and 5 cmx6 cm in another. Three weeks later, ultra-thin skin grafting was placed on the neodermis. Compared to split-thickness skin graft, this technique provides a thicker coverage for wound closure. Neither skin breakdown nor ulceration was noted in the 1-year follow-up. This paper reports the successful use of artificial dermis for reconstruction of severe scalp burn with calvarial bone involvement.     

Artificial dermis]

An artificial dermis, composed of an inner layer of collagen sponge and an outer silicone layer, was developed by modifying the material reported by Yannas et al. When the artificial dermis is placed on full 14-thickness skin defects, the pores of the inner collagen sponge are infiltrated by fibroblasts and capillaries. This cell-infiltrated membrane is gradually converted into a synthesized connective tissue matrix, similar to true dermis, as the original network of collagen sponge is biodegraded. The secondary skin graft takes easily and postoperative contraction is minimal even if a very thin split-thickness skin graft is used. The requirement for two-stage surgery and long hospitalization remain disadvantages of the material. Consequently, application of the material is limited to cases in which the advantages outweigh the disadvantages. Deep skin defects with partial exposure of bone or muscle appear to be the most suitable indications for the material. Shallow but very wide skin defects also seem to benefit from the application of the material.     

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.     

色素部分缺如性浅表瘢痕的美容性无痕化治疗研究

目的 探讨美容性无痕化治疗色素部分缺如性浅表瘢痕的方法.方法 瘢痕浅层边缘不规则斜坡状部分削除,保留真皮浅层和深层瘢痕组织,创面用刃厚皮片覆盖,医用胶固定皮片.结果 无明显切口缘瘢痕,皮片表面平整,色素均匀,皮肤柔软,局部无明显收缩.结论 色素部分缺如性浅表瘢痕采用浅层部分削除后移植刃厚皮片可作为瘢痕美容性无痕化治疗的一种手段.