Allogeneic Dermis Research Articles

Skin graft with dermis and appendages generated in vivo by cell competition

Autologous skin grafting is a standard treatment for skin defects such as burns. No artificial skin substitutes are functionally equivalent to autologous skin grafts. The cultured epidermis lacks the dermis and does not engraft deep wounds. Although reconstituted skin, which consists of cultured epidermal cells on a synthetic dermal substitute, can engraft deep wounds, it requires the wound bed to be well-vascularized and lacks skin appendages. In this study, we successfully generate complete skin grafts with pluripotent stem cell-derived epidermis with appendages on p63 knockout embryos’ dermis. Donor pluripotent stem cell-derived keratinocytes encroach the embryos’ dermis by eliminating p63 knockout keratinocytes based on cell-extracellular matrix adhesion mediated cell competition. Although the chimeric skin contains allogenic dermis, it is engraftable as long as autologous grafts. Furthermore, we could generate semi-humanized skin segments by human keratinocytes injection into the amnionic cavity of p63 knockout mice embryos. Niche encroachment opens the possibility of human skin graft production in livestock animals.

(216) Benefit of Closed Wound Drainage System with Negative Pressure in Penile Enhancement Surgery

Abstract Introduction Many patients have had penile enhancement surgery and the surgeon try to reduce postoperative complications including hemorrage, infection, wound dehiscence etc. we want to evaluate the benefits of closed wound drainage system with negative pressure in penile enhancement surgery. Objective we want to evaluate the benefits of closed wound drainage system with negative pressure in penile enhancement surgery. Methods We have experienced 500 penile enhancement surgery. We adopted a pre-pubic minimal incision and dissected penile shaft and pre-pubic space. Inserted graft materials were Allograft dermal grafts which were fixed at the Buck's fascia. And we dissected fundi-form ligaments and sutured transversely pre-pubic dead space with PDS 2.0. In group 1, silastic drain were inserted in 250cases in order to drain postoperative bleeding and prevent inflammation. In group 2, the remain 250 cases adopted closed wound drainage system with negative pressure. Results In group 1, the wound infecton rate was 2.9% and no dermal graft infection was showed. In group 2, the wound infection rate was 0.6% and no dermal graft infection was appeared. Group 1 using closed wound drainage system with negative pressure is more effective than group 2 using silastic drain in order to reduce wound infection rate. Conclusions In penile augmentation surgery using Allogenic dermis, Using closed wound drainage system with negative pressure is safer than using silastic drain as postoperative drainage system. Disclosure No.

AB020. Novel surgical technic for micro penis

Micropenis is an unusually small penis and the common criterion is a dorsal (measured on top) erect penile length of at least 2.5 standard deviations or penis smaller than the mean human penis size and it is occurred in about 0.6% of males. The most common conditions of reduced prenatal androgen production or effect, such as abnormal testicular development (testicular dysgenesis). Testosterone treatment is resumed in adolescence only for boys with hypogonadism. Penile growth is completed at the end of puberty. Testosterone supplement treatment (TST) is resumed in adolescence only for boys with hypogonadism, however, TST no responder, need to surgical treatment. In this presentation, we introduce a novel technique of suspensory ligament incision plus suture fix using 00 Nylon each of proximal tunica albuginea to dermis sub-dermis and insert acellular allogeneic dermis (4×4 cm) under subskin for penile penis length and girth enhancement for treating micro penis.

Development of a new method for the preparation of an acellular allodermis, quality control and cytotoxicity testing.

Demand for use of acellular allodermis is high but commercially appropriate products are not used routinely because of very high price and limited availability. These facts did motivate us to prepare acellular allodermis using a new, simple and less expensive method. We have developed a original method for preparation of acellular allogeneic dermis based on action of a proteolytic enzyme in combination with distilled water. Hypotonic environment in comparison with SDS or Triton ansure no toxicity of the final product. Trials for determination of optimal trypsin concentrations, temperature and time of action were performed. According to our results, the use of 2.5% trypsin/EDTA solution overnight at +4°C was proving to be optimal. The histology confirmed absence of cells in the prepared dermis. No toxicity of final acellular dermis was confirmed by three independent tests (agar diffusion test contact cytotoxicity test and grow curve). The prepared acellular dermis seems to be suitable not only for direct clinical use, but it can be used as a scaffold for cell cultivation as well.

Atomic force microscopy in the production of a biovital skin graft based on human acellular dermal matrix produced in-house and in vitro cultured human fibroblasts.

The most efficient method in III° burn treatment is the use of the autologous split thickness skin grafts that were donated from undamaged body area. The main limitation of this method is lack of suitable donor sites. Tissue engineering is a useful tool to solve this problem. The goal of this study was to find the most efficient way of producing biovital skin substitute based on in house produced acellular dermal matrix ADM and in vitro cultured fibroblasts. Sixty samples of sterilized human allogeneic skin (that came from 10 different donors) were used to examine the influence of decellularizing substances on extracellular matrix and clinical usefulness of the test samples of allogeneic human dermis. Six groups of acellular dermal matrix were studied: ADM-1 control group, ADM-2 research group (24 h incubation in 0.05% trypsin/EDTA solution), ADM-3 research group (24 h incubation in 0.025% trypsin/EDTA solution), ADM-4 research group (24 h incubation in 0.05% trypsin/EDTA solution and 4 h incubation in 0,1% SDS), ADM-5 research group (24 h incubation in 0.025% trypsin/EDTA solution and 4 h incubation in 0,1% SDS), and ADM-6 research group (24 h incubation in 0,1% SDS). Obtained ADMs were examined histochemically and by atomic force microscopy (AFM). ADMs were settled by human fibroblasts. The number of cultured cells and their vitality were measured. The obtained results indicated that the optimal method for production of living skin substitutes is colonization of autologous fibroblasts on the scaffold prepared by the incubation of human allogeneic dermis in 0.05% trypsin/EDTA. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 726-733, 2018.

Own Experience From The Use Of A Substitute Of An Allogeneic Acellular Dermal Matrix Revitalized With In Vitro Cultured Skin Cells In Clinical Practice.

As a result of the removal of cells from human allogeneic dermis, a collagen scaffold is obtained, which can be populated de novo with autologous/allogeneic skin cells and transplanted onto the area of skin loss. The optimal method for production of acellular dermal matrices (ADM) has been selected. Three female patients (a mean age of 54 years) were subjected to the transplantation of either autologous or allogeneic keratinocytes and fibroblasts into the holes of acellular dermal matrix (ADM) mesh graft. The method for burn wound treatment based on the use of a viable dermal-epidermal skin substitute (based on ADM and in vitro cultured fibroblasts and keratinocytes) may be the optimal method of burn treatment.

Adipose tissue regeneration in vivo using micronized acellular allogenic dermis as an injectable scaffold.

Over the past few years, the clinical use of injectable artificial materials in plastic surgery has increased. In addition, autologous lipoimplantation is being performed for volume replacement of soft tissue for aesthetic purposes. In this study, acellular allogenic dermis was utilized as a scaffold for the culturing of preadipocytes, confirming the possibility of three-dimensional proliferation of progenitor cells, the eventual differentiation of stromal cells in adipose tissue into the adipocytes, and the in vivo implantation of such adipocytes to form fat tissue. Preadipocytes, recently called ASCs (adipose tissue-derived stromal/stem cells), were cultured in acellular allogenic dermis, successfully attached to the dermal particles in a three-dimensional structure, and proliferated, differentiated, and eventually formed a cluster. For the in vivo implantation, four groups were formed: the first group was cultured within the dermal scaffold for 24 h before implantation (24-h preconditioned group), the second group was induced for differentiation for 10 days before implantation (10-day preconditioned group), the third group was implanted immediately after cell propagation (nonpreconditioned group), and the control group was implanted with only dermal scaffold. In vivo implanted preadipocytes showed great differentiation into adipocytes within the dermal scaffolds. Also, the 10-day preconditioned group showed a greater volume of fat tissue compared to the 24-h preconditioned group. From these results, we confirmed that after a three-dimensional culture in acellular allogenic dermis, implanted preadipocytes formed a greater amount of fat tissue and that this could be a possible effective method for future soft tissue restoration.

Injectable tissue-engineered soft tissue for tissue augmentation.

Soft tissue augmentation is a process of implanting tissues or materials to treat wrinkles or soft tissue defects in the body. Over the years, various materials have evolved to correct soft tissue defects, including a number of tissues and polymers. Autogenous dermis, autogenous fat, autogenous dermis-fat, allogenic dermis, synthetic implants, and fillers have been widely accepted for soft tissue augmentations. Tissue engineering technology has also been introduced and opened a new venue of opportunities in this field. In particular, a long-lasting filler consisting of hyaluronic acid filler and living human mesenchymal cells called "injectable tissue-engineered soft tissue" has been created and applied clinically, as this strategy has many advantages over conventional methods. Fibroblasts and adipose-derived stromal vascular fraction cells can be clinically used as injectable tissue-engineered soft tissue at present. In this review, information on the soft tissue augmentation method using the injectable tissue-engineered soft tissue is provided.

Acellular porcine skin and allogeneic skin as wound-covering materials for extensive deep burns:a comparative study

Objective To evaluate the advantages of microskin graft using acellular porcine skin for treatment of extensive deep burns by comparing with that using allogeneic skin.Methods A retrospective analysis was conducted on 70 severe burn patients who were treated in the Second Affiliated Hospital of Kunming Medical University during Jan.1999 to Jan.2011.The patients were divided into the acellular porcine skin group and allogeneic skin group,each containing 35 patients.The survival rates of microskin grafts were determined at 4 weeks post-operation.Besides,the rejection of acellular porcine skin and allogeneic skin,changes of body temperature,white blood cell(WBC) count,lymphocyte and serum protein were observed at pre-and post-operation.Results(1) The survival rate was(71.5±6.6)% in acellular porcine skin group and(70.6±7.5)% in allogeneic skin group,with no significant difference found between the two groups(P0.05).(2) Acellular porcine skin group.At 3 days post-operation the acellular porcine skin was still attached to the wound,most of the skin was not discolorated,and small part of the skin became cinnamomeous.The acellular porcine skin was gradually separated from the auto-microskin at 3-4 weeks post operation,and there was small amount of exudates under the acellular porcine skin,which could be drained through a small cut.In the pressed area,there was still a small amount of exudates,but the acellular porcine skin was not dissolved and the microskin grafts survived and became confluent.(3) Allogeneic skin group.The allogeneic epidermal was rejected and was off from the wound at 3-14 days post transplantation,and at 10-30 days after transplantation the allogenic dermis became dry.During 25-60 days after transplantation,the allogenic dermis was completely stripped off,the microskin grafts became confluent,and the wound was healed.(4) The body temperature of the two groups was significantly descended after operation(P0.01).The temperature of acellular porcine skin group was significantly lower than that of allogeneic skin group at 14 day post-operation(P0.01).(5) The WBC count and percentage of neutrophils were also significantly decreased in both two groups after operation(P0.05),and WBC counts in the acellular porcine skin group was significantly lower than those in the allogeneic skin group at 7 and 14 days after operation(P0.05,P0.01).The peripheral blood lymphocytes were also significantly increased after operation(P0.01) and the lymphocytes in the acellular porcine skin group were significantly less than those in the allogeneic skin group at 14 days after operation(P0.01).Blood biochemistry results showed no significant differences in the protein levels between the two groups(P0.05).Conclusion Microskin graft using acellular porcine skin,instead of allogeneic skin,for extensive burn patients can inhibit systematic inflammatory response,improve the nutrition condition,and reduce the using of allogeneic skin.Acellular porcine skin might be a suitable alternative for allogeneic skin.

Autologous Dermal Graft in Breast Reconstruction

The role of allogenic dermis is well established in reconstructive breast surgery, where it acts as a hammock between the detached inferior border of pectoralis major and the inframammary fold. The study reports on the outcome of 19 women (21 breasts as 2 were bilateral) in which autologous dermis was used rather than allogenic material. The autologous dermis was harvested from the abdomen, as a miniabdominoplasty in 15 patients, and from the contralateral breast in 4 patients having simultaneous breast reduction/mastopexy. In all, 15 women underwent immediate reconstruction (7 with tissue expanders, 8 with immediate prosthesis), whereas 4 patients underwent delayed reconstruction (with expanders). Two patients developed infection requiring explantation. There were no abdominal complications after the miniabdominoplasty. The coverage of the prosthesis or tissue expander by the dermal graft was achieved on average in three-quarters of cases (range, just under two-thirds to 100%). The mean follow-up was 17 months (range, 6-36 months). Autologous dermis is a useful alternative to allogenic dermis. It is cheap and readily available. In patients having a contralateral breast reduction, there is no donor site. The risk of complications is not dissimilar to allogenic dermis.

Tissue Thread Grafting

Most double-eyelid operations emphasize the levator insertion into the upper eyelid skin using suture material. For supratarsal fixation, we used autogenous tissue threads from the patients' own temporal fascia, periumbilical tissues, capsulopalpebral fascia, and allogeneic human dermis. We engaged tissue threads using a newly designed Yeop's needle (AILEE Inc., BuSan, South Korea) or modified 5-0 Vicryl suture (Ethicon Inc., Somerville, NJ) material. The suture passed through the whole upper lid in the same pattern used with the conventional nonincisional technique. A total of 78 double-eyelid operations were performed. There were 53 primary cases, 22 secondary cases, and 3 tertiary cases. Tissue threads were maintained for 9.3 days on average. There were no complications regarding tissue threads, including infection or granuloma formation in the upper eyelid or at the donor site. Tissue thread grafts not only offer safe fixation of the palpebral crease, but they also incorporate safely into the eyelid tissues for scarless, natural-looking double-eyelids.

Swine pericardium as dermal substrate for human keratinocyte culture

Synthetic skin analogues or living allogeneic or autologous cells are used as dressings for the care of skin wounds, as well as temporary or permanent substitutes for damaged epithelia. To evaluate if keratinocyte growth on a swine pericardium substrate mimics the natural epithelial layers compared with cultures on allogeneic dermis, which is accepted as having appropriate physical and chemical properties for growth and differentiation. Keratinocytes were cultured on a swine pericardium substrate and allogeneic dermis, either submerged or at the air-liquid interface. At 7, 14 and 21 days postseeding the cultures were evaluated by light microscopy after both haematoxylin and eosin staining and immunohistochemistry. Cell-substrate interactions led to growth, stratification and differentiation of cells, with the definition of epithelial layers. The submerged system showed a continuous growth rise on both composites, but this was more prominent with the swine pericardium substrate. An increase in the number of layers at the air-liquid interface with the dermis composites, in contrast to the submerged cultures, occurred only from days 7 to 14. The pattern of keratinocyte growth on swine pericardium substrate was much better in the submerged than in the air-liquid interface cultures. The results indicate that swine pericardium is a better substrate than allogeneic dermis for keratinocyte cultures in submerged but not in air-liquid interface cultures. Swine pericardium as a substrate opens one more possibility for skin restoration after trauma or burns.

The clinical application of the repair of burn wound with acellular allogenic dermis matrix with split-thickness autologous skin graft

Objective To approach the clinical applieation of burn wound with composite transplanta-tion of acellular dermal matrix with split-thickness autologous skin graft. Methods A clinical observation in 63 patients who had been treated by acellular allogenic dermis matrix with split-thickness autologous skin graft from 2003 in our division. In the peases, 37 cases are burn scars and 26 cases are function site full thickness skin burn wound. THe repair of wound by one time operation only.In this cases, acellular dermal matrix with split-thickness autologous skin grafting are 49, with snip skin grafting are 7, and with mesh skin grafting are 7.Results The composite skin survived after all in 63 examples.Skin grafting and donor site scars proliferation were not obvious after aeerte function rebound satisfaction pigmenatation appearaneed less.Patients feel satisfaction at appoaran ce. Con-clusion The repair of bum wound with cellular allogenic dermis with split-thickness autologous skin graft is a good treatment. Key words: Acellular allogenic dermis matrix; Split-thickness autulogous skin; Composite graft; Wound repair

Hautersatzverfahren in der Verbrennungschirurgie

Patients with extensive deep partial or full thickness burns require early excision of necrotic tissue, however, in many of these cases simultaneous autografting is not possible due to the general condition of the patient. In this instance temporary dressings like allogeneic or xenogeneic skin or foam dressings can be applied to minimize fluid and protein loss. In Europe glycerolized preserved allogeneic skin remains the treatment standard. Dermal replacements are considered to optimize the long-term outcome of split thickness skin grafting. Reduced contracture rates and increased pliability have been reported after additional dermal enhancement with either collagen-glycosaminoglycan matrix, acellular allogeneic dermis or collagen/elastin matrix. True regeneration of the dermis has not yet been observed. However, these materials are suitable for improvement of the wound bed and also the final result after split thickness skin transplantation.

Healing modulation induced by freeze‐dried platelet‐rich plasma and micronized allogenic dermis in a diabetic wound model

The incidence and prevalence of chronic and diabetic wounds are increasing and clinical treatments to tackle these epidemics are still insufficient. In this study, we tested the ability of freeze-dried platelet-rich plasma (PRP) and an allogenic micronized acellular dermal matrix alone and in combination to modulate diabetic wound healing. Therapeutic materials were applied to 1.0 cm(2) excisional wounds on genetically diabetic (db/db) mice. Wound-healing kinetics and new tissue formation were studied at 9 and 21 days posttreatment. Quantitative immunohistochemistry was used to study vascularity and cellular proliferation (days 9 and 21), and collagen deposition was evaluated 21 days postwounding. In vitro, micronized allogenic dermis, when combined with PRP, absorbed nearly 50% of original platelet-derived growth factor, transforming growth factor-beta, vascular endothelial growth factor, and epidermal growth factor from platelets and stimulated fibroblast proliferation. In vivo, micronized dermis increased the formation of vascularized wound tissue by day 9. Freeze-dried PRP alone or in combination with micronized dermis increased wound tissue revascularization and proliferation compared with spontaneous healing. The increase in cell proliferation persisted until day 21 only when freeze-dried PRP was used in combination with micronized dermis. These results indicate that micronized allogenic dermis may be used to provide a dermal matrix to stimulate tissue formation and the combination with PRP may confer additional beneficial growth factors to chronic or diabetic wounds.

Ex vivo produced human conjunctiva and oral mucosa equivalents grown in a serum-free culture system

Purpose We sought to develop full-thickness ex vivo produced human conjunctiva and oral mucosa equivalents using a serum-free culture system without a feeder layer and to compare conjunctiva and oral mucosa equivalents to assess their suitability as graft materials for eyelid reconstruction. Materials and methods Human conjunctival and oral mucosal keratinocytes were cultured, expanded, and seeded onto AlloDerm (LifeCell Corp, Branchburg, NJ), a cadaveric, acellular dermis, to produce ex vivo produced full-thickness mucosa equivalents. Histology of equivalents and their expression of immunoreactive Ki-67, a proliferation marker, and GLUT1, a membrane antigen seen in barrier tissues, were examined at 4, 11, and 18 days after seeding onto AlloDerm. Results Progressive epithelial stratification was observed on day 4, 11, and 18 conjunctiva and oral mucosa equivalents. Ki-67 immunoreactivity progressively increased with cultured time in both types of equivalent, indicating the continued presence of actively proliferating cells. GLUT1 immunoreactivity, concentrated in the basal keratinocytes of stratified epithelia of both types of equivalents, mimicked native tissue and indicated a high glycolytic state of the basal cells. Conclusions Conjunctival and oral mucosal equivalents are similar to native tissue and demonstrate high proliferative and glycolytic states. Due to the similarity to conjunctiva, oral mucosal equivalents may be useful for eyelid reconstruction. Their advantages for surgical reconstruction include 1) ease of obtaining autogenous oral epithelium for expansion in vitro without the possibility of contaminating cellular- or serum-borne biologic agents, 2) growth of intact, confluent epithelia on rigid, transplantable human allogeneic dermis that may be surgically transplanted, and 3) reduced donor site morbidity and surgical time.

Treatment of Infected Burned Wound of Functional Part with Conjunction Skin Transplantation

为功能部位深度烧伤感染创面寻找良好的覆盖材料,挽救功能,减少畸形和疤痕增生.我科于2001年6月至2003年4月间,在大面积烧伤的功能部位深度感染创面进行清创处理后应用异体脱细胞真皮+自体薄皮片复合植皮修复创面共治疗12例29处功能部位,临床应用表明能有效地挽救功能,防止瘢痕增生,预防畸形,移植皮肤生长柔软,有弹性,利于功能部位深度烧伤创面修复,防止瘢痕增生、畸形、功能障碍难题。

Applied tissue engineering in the closure of severe burns and chronic wounds using cultured human autologous keratinocytes in a natural fibrin matrix.

Whereas in severe burns cultured human epithelial cells may well serve as a life saving method, the true value of tissue-engineered skin products in chronic wound care has yet to be clearly defined. Among other well-known clinical problems, the engraftment rate of commercially available multilayered "sheet grafts" has been shown to vary extremely. Adherence of transplanted cells to the wound bed--especially in the presence of potential wound contamination-- is one of the crucial aspects of this technique. Keratinocyte suspensions in a natural fibrin sealant matrix can potentially treat a variety of skin defects. In acute burn wounds, as well as in chronic wounds the clinical application of this type of tissue-engineered skin substitute demonstrates the capacity of cultured human autologous keratinocytes in a fibrin sealant matrix to adhere to wound beds, attach and spread over the wound resulting in reepithelialization of both acute and chronic wounds. In full thickness burns the combination of this new tool with allogenic dermis is a promising option to achieve complete dermal-epidermal reconstitution by means of tissue engineering and guided tissue repair. When transferring this technique into the treatment of chronic wounds we found an optimal preparation of such recipient wound beds to be crucial to the success. The additional application of continuous negative pressure (vacuum therapy) and preliminary chip skin grafting to optimally prepare the recipient site may be helpful tools to achieve such well-prepared and graftable surfaces. Prospective controlled comparative studies should be designed to further assess the clinical efficacy of this technique.

Use of allogenic dermis for radial forearm free flap donor site coverage.

The radial forearm free flap has become the method of choice for reconstruction of head and neck defects following oncologic ablation. Harvesting of a radial forearm free flap leaves a donor site defect. This is most commonly closed with a split-thickness skin graft. Morbidity, most commonly owing to a lack of graft take over the tendons, can be quite high. Recently, an acellular matrix (Alloderm) has been advocated to decrease complications at the radial forearm donor site, as well as obviate taking a split-thickness skin graft from the thigh. Tertiary referral academic centre. Retrospective chart review of 15 patients. Five patients received allogenic dermis, 10 patients received split-thickness skin grafting to the radial forearm donor site. Patients with allogenic dermis took between 12 and 16 weeks to heal completely. Patients undergoing split-thickness skin graft were completely healed within 4 to 6 weeks. Cosmesis was judged to be marginally better in the allogenic dermis group. Allogenic dermis placement had a greater impact on hand function owing to prolonged healing, whereas patients with split-thickness skin graft required wound care at the thigh for a 2- to 3-week period owing to the harvesting of the skin graft. Allogenic dermis may be a viable alternative to split-thickness skin grafting and radial forearm free flap donor sites. Prolonged healing with subsequent increased health care services use needs to be addressed.

Initial experience with a composite autologous skin substitute

Patients with large burns are surviving in increasing numbers, but there remains no durable and reliable permanent skin replacement. After initial favorable small animal experiments, a pilot trial of a composite skin replacement was performed in patients with massive burns. A composite skin replacement (CSR) was developed by culturing autologous keratinocytes on acellular allogenic dermis. This material was engrafted in patients with massive burns and compared to a matched wound covered with split thickness autograft. With human studies committee approval, 12 wounds in 7 patients were grafted with CSR while a matched control wound was covered with split thickness autograft. These 7 children had an average age of 6.4±1.4 yr and burn size of 75.9±5.0% of the body surface. Nine wounds were acute burns and three were reconstructive releases. Successful vascularization at 14 days averaged 45.7±14.2% (range 0–100%) in the study wounds and 98±1% (range 90–100%) in the control sites ( P<0.05). Reduced CSR take seemed to correlate with wound colonization. All children survived. While CSR did not engraft with the reliability of standard autograft, this pilot experience is encouraging in that successful wound closure with this material is possible, if not yet dependable. It is hoped that a more mature epidermal layer may facilitate engraftment, and trials to explore this possibility are in progress.