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Results for “Acellular Dermis”

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PubMed2000

[Use of skin substitutes in treatment of large hypertrophic scars due to burn injury].

The study concerns a clinical experiment in two patients with a dermal substitute based on atelocollagen and hyaluronic acid, and allogeneic acellular dermis. In both cases two-step grafting was performed. At the first step the dermal substitute was implanted into the wound and it was grafted at the second step with thin dermoepidermal autograft. Large hypertrophic scars after burn injury were treated. In both patients the quality of skin cover was significantly improved.

Adult↗

PubMed2002

Transient hyperproliferation of a transgenic human epidermis expressing hepatocyte growth factor.

Hepatocyte growth factor (HGF) is a fibroblast-derived protein that affects the growth, motility, and differentiation of epithelial cells including epidermal keratinocytes. To investigate the role of HGF in cutaneous biology and to explore the possibility of using it in a tissue engineering approach, we used retroviral-mediated gene transfer to introduce the gene encoding human HGF into diploid human keratinocytes. Modified cells synthesized and secreted significant levels of HGF in vitro and the proliferation of keratinocytes expressing HGF was enhanced compared with control unmodified cells. To investigate the effects of HGF in vivo, we grafted modified keratinocytes expressing HGF onto athymic mice using acellular dermis as a substrate. When compared with controls, HGF-expressing keratinocytes formed a hyperproliferative epidermis. The epidermis was thicker, had more cells per length of basement membrane, and had increased numbers of Ki-67-positive proliferating cells, many of which were suprabasal in location. Hyperproliferation subsided and the epidermis was equivalent to controls by 2 weeks, a time frame that coincides with healing of the graft. Transient hyperproliferation may be linked to the loss of factors present in the wound that activate HGF. These data suggest that genetically modified skin substitutes secreting HGF may have applications in wound closure and the promotion of wound healing.

Cell Division↗

PubMed2005

Tissue engineering of skin substitutes.

Cultivated epithelial autografts as multilayered thin sheets represent common standard in clinically applied tissue engineering substitutes, outnumbering all experimental alternatives. However, the unsatisfying short and long term results concerning mechanical stability and scarring demand for alternatives. Our group investigated cultivation and transplantation of cultured autologous keratinocytes as a single cell suspension in a fibrin sealant matrix in athymic mice in combination with allogenic skin grafting. We observed reliable wound reepithialization after a cultivation period of only 2 weeks. Additionally, we could allocate successful combination of a keratinocyte fibrin sealant suspension and acellular dermis in an attempt to regenerate full thickness skin defects in a pig animal model. The potential clinical implication of subconfluently cultured keratinocytes is enhanced by the possibility of co-transplantation with decellularized dermis.

Animals↗

PubMed1997

Differences in dermal analogs influence subsequent pigmentation, epidermal differentiation, basement membrane, and rete ridge formation of transplanted composite skin grafts.

This study evaluated the in vitro and in vivo function of composite skin equivalents based on two different dermal analogs. Keratinocytes derived from the same dark-skinned neonatal foreskins were seeded onto both acellular human dermis and fibroblast-contracted collagen gels. Each type of composite graft readily formed an epithelium in vitro. However, the undulating surface of the acellular dermis acted as a template and organized the seeded keratinocytes into a rete ridge-like pattern, whereas the smooth surface of the fibroblast-contracted collagen gels generated an epithelium with a linear basal layer. Moreover, when acellular dermis was used, the composite grafts demonstrated enhanced melanocyte proliferation. When transplanted to athymic mice, both composite grafts formed a fully differentiated human epidermis, but repigmentation of the grafts when acellular dermis was used was more extensive and only the epidermis on the fibroblast-contracted collagen gels showed signs of hyperproliferation at 6 weeks after grafting. These results demonstrate that the type of dermal analog incorporated into a composite skin graft can influence the subsequent functionality of the skin substitute.

Animals↗

PubMed2004

Fibroblasts improve performance of cultured composite skin substitutes on athymic mice.

BACKGROUND: This study investigated the impact of adding human fibroblasts to a cultured composite skin substitute model of cultured human keratinocytes and acellular human dermis. METHODS: Skin substitutes were prepared by seeding human keratinocytes on the papillary side of acellular dermis with or without seeding fibroblasts on the reticular side. Performance of the grafts was compared both in vitro by histology and in vivo on surgically created full-thickness wounds on athymic mice. Graft size and contraction were measured and immunohistochemical stains were done to reveal vascularization. RESULTS: Skin substitutes with fibroblasts formed thicker epidermis than skin substitutes without fibroblasts. When transplanted onto athymic mice, skin substitutes with fibroblasts maintained their original size with only 2% contraction. In contrast, skin substitutes without fibroblasts showed 29% contraction. Vascular basement membrane specific mouse CD31staining and endothelial cell specific mouse collagen type IV staining revealed vascularization as early as 1 week posttransplant in grafts with fibroblasts, and was significantly higher than grafts without fibroblasts at 2 weeks. CONCLUSIONS: Addition of fibroblasts to keratinocyte based composite skin substitutes improves epidermis formation, enhances vascularization and reduces contraction.

Animals↗

PubMedDate not supplied

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.

Animals↗

PubMed2006

The long-term results of pubovaginal sling surgery using acellular cross-linked porcine dermis in the treatment of urodynamic stress incontinence.

PURPOSE: Acellular cross-linked porcine dermis is a potential substitute for rectus fascia as a sling material with the advantage of decreased morbidity. However, the long-term efficacy is unknown. We compared the 3-year efficacy of PD vs autologous rectus fascia as a sling material for pubovaginal sling surgery in the treatment of urodynamic stress incontinence. MATERIALS AND METHODS: Between July 2000 and December 2001 a total of 101 consecutive, nonrandomized patients with USI underwent a PD (51) or RF (50) sling procedure. Patients were assessed at 6 weeks, and at 3, 6 and 12 months postoperatively. Urodynamic study was repeated in cases of treatment failure. A detailed survey questionnaire was mailed to all patients at least 36 months after surgery and all responders were then retested by telephone interview by a blinded assessor. The primary outcome measure was patient perceived success rate (cured or improved) at least 36 months after PVS. Secondary outcome measures were patient satisfaction 36 months after surgery, durability of success with time and reoperation rate. RESULTS: Complete data were available on 94 patients (48 treated with PD and 46 treated with RF sling). The groups were well matched for age, leak point pressure, prior incontinence surgery and urge symptoms. Pubovaginal sling was successful (cured or improved) in 37 (80.4%) patients treated with RF but in only 26 (54%) patients treated with PD 36 months after surgery (Fisher's exact test p = 0.009; 95% CI 8.03, 44.4). Treatment failure occurred by 9 months after RF and by 24 months after PD sling procedure. Repeat urodynamic study showed USI to be the cause of treatment failure in 18 (37.5%) of 20 patients treated with PD but in only 3 (6.5%) of 8 patients treated with RF. CONCLUSIONS: We have shown that use of the PD sling, although reducing early morbidity, results in a significantly inferior long-term cure rate in comparison to the RF sling. Therefore, acellular cross-linked porcine dermis should not be used as a substitute for rectus fascia.

Adult↗

PubMed1999

Development of autologous human dermal-epidermal composites based on sterilized human allodermis for clinical use.

The aim of this study was to identify a sterilization technique for the preparation of human allodermis which could be used as a dermal component in wound healing and as the dermal base for production of dermal-epidermal composites for one-stage grafting in patients. We report that it is possible to produce dermal-epidermal composites which perform well in vitro and in vivo using a standard ethylene oxide sterilization methodology. Prevention of ethylene oxide-induced damage to the dermis was achieved using gentle dehydration of the skin prior to ethylene oxide sterilization. The issue of whether viable fibroblasts are required for composite production was examined in comparative studies using glycerol vs. ethylene oxide sterilized dermis. Where good collagen IV retention was achieved following preparation of acellular de-epidermized dermis there was no advantage to having fibroblasts present in vitro or in vivo; however, where collagen IV retention was poor or where keratinocytes were initially expanded in culture then there was a significant advantage to introducing fibroblasts to the composites during their preparative 10-day period in vitro. The requirement for fibroblasts became less evident when composites were grafted on to nude mice. In conclusion, we report a protocol for the successful sterilization of human allodermis to achieve an acellular dermis with good retention of collagen IV. This acellular dermis would be appropriate for clinical use as a dermal replacement material. It can also be used for the production of dermal-epidermal composites using autologous keratinocytes (with or without fibroblasts).

Adult↗

PubMed2004

[Preparation of porcine acellular dermal matrix by low concentration of trypsin digestion and repeated freeze-thaw cycles].

OBJECTIVE: To establish a new method for the preparation of porcine acellular dermal matrix. METHODS: The antigenicity of the porcine dermis was weakened by removing epidermal and dermal cells from the porcine skin through the digestion with low-concentration trypsin and repeated freeze-thaw cycles. Split thickness porcine skin was treated with 0.05% trypsin to remove the cells from the epidermis and dermis. Repeated freeze-thaw cycles were employed to further weed out the residual cells within the dermis. The prepared acellular dermis was then examined grossly, as well as histologically, and also by immunohistochemical method. RESULTS: No cell could be identified in the prepared porcine acellular dermal matrix. The integral basement membrane was preserved on the surface of dermal matrix with compact dermal matrix collagen structure. CONCLUSION: Low concentration trypsinization and repeated freeze-thaw cycles seemed to be a simple and effective method for the preparation of xenogeneic acellular dermal matrix.

Animals↗

PubMed2003

[An experimental study of cultured keratinocytes on the acellular xenodermis].

OBJECTIVE: To culture the keratinocytes on the acellular pig dermis and establish a composite skin in vitro. METHODS: Full thickness skin taken from neonatal SD rats of approximate 24-hour-old was cultivated in asepsis condition, which was then separated into epidermal and dermal layers with low-temperature enzyme digestion. The basal lamina cells between the two layers were scraped off and the pure keratinocytes were obtained using gradient density centrifugation. In the experimental group, the keratinocytes were cultured on acellular xenodermis(AX) as the three-dimensional frames. In the control, the keratinocytes were cultured without any trophoblast. The air-liquid interface(ALI) culture continued after the primary culture. Routine histological HE staining and assay of Pancytokeratin and Laminin with strepavidin-biotin-peroxidase complex (SABC) method were used to study the morphology of CK and AX. RESULTS: HE staining demonstrated the formation of more than 4 lays of keratinocytes and basement membrane, with slight keratinization of the cells. Pancytokeratin(+) in immunohistochemical results confirmed that the cultured cells on AX were keratinocytes, not other kinds of cells. Laminin(+) indicated that new procreant basement membrane appeared in CK. CONCLUSION: Keratinocytes cultured on the acellular pig dermis grow well and form basement membrane. This study implies successful construction of composite skin.

Acellular Dermis↗

PubMed2001

Genetic material is present in cadaveric dermis and cadaveric fascia lata.

PURPOSE: We determined whether genetic material is present in the commercially processed cadaveric allografts used in sling surgery. MATERIALS AND METHODS: We evaluated 16 samples from 2 commercial sources of human allograft, including 8 each of freeze-dried gamma irradiated cadaveric fascia lata and acellular cadaveric dermis. Fresh human rectus fascia and sterile saline served as positive and negative controls, respectively. All samples underwent a standard proteinase K/sodium dodecyl sulfate/phenol extraction technique to isolate DNA. Polymerase chain reaction was done to amplify the retrieved DNA material, spectrophotometry to quantify DNA concentration and agarose gel electrophoresis to determine the size of DNA fragments. RESULTS: Of the 16 samples tested from 2 commercial sources of human allograft fascia 14 (87.5%) contained DNA. Mean DNA concentration plus or minus standard error was 258.3 +/- 80.1 and 272.8 +/- 168.8 microg./gm. tissue for cadaveric fascia lata and cadaveric dermis, respectively. Polymerase chain reaction amplified DNA segments of 2,000 bp from 1 of each of the 8 samples of cadaveric fascia lata and cadaveric dermis. CONCLUSIONS: Freeze-dried gamma irradiated cadaveric fascia lata and acellular cadaveric dermis contained intact DNA.

Bioprosthesis↗

PubMed2005

Assessing the long-term retention and permanency of acellular cross-linked porcine dermal collagen as a soft-tissue substitute.

BACKGROUND: Acellular porcine dermis has been used as a soft-tissue substitute in Europe since 1998 and in the United States since 2000. Processing renders the composite acellular, reducing antigenicity and crosslinking the dermal collagen. The purpose of this study was to determine the long-term histologic response of this material to the transfer into a subcutaneous soft-tissue location in an in vivo mouse model. METHODS: A total of 24 mice (ICR strain) underwent an initial procedure transplanting a segment of acellular porcine dermis into a dorsal subcutaneous pocket. The implants were examined for qualitative characteristics including rigidity, migration, inflammation, and healing response. Composites of skin, subcutaneous tissue, implant, and underlying muscle were harvested for histologic evaluation. The specimens were analyzed for inflammatory response, degree, and character of tissue incorporation and degree of implant resorption. RESULTS: One of the 24 animals studied experienced extrusion of the implant. The remaining 23 implants persisted within their respective hosts until the time the animals were killed. The volume and weight of all of the implants remained unchanged from the preoperative to postoperative measurements. Dystrophic calcification and bone formation was seen at 12 months. CONCLUSIONS: The results do not suggest characteristics optimal for implants used in soft-tissue augmentation for the purpose of aesthetics. The porcine dermis would appear from these results to be better suited for situations that require strength and permanence of the implant.

Animals↗

PubMed2005

The application of new biosynthetic artificial skin for long-term temporary wound coverage.

Temporary dressings protect wounds from desiccation and infection. In our previous study, we used meshed acellular porcine dermis (APD) to enhance wound healing and decrease wound contraction; however, the wounds showed meshed scar. In this study, we produced an artificial skin composed of a cross-linked silicon sheet on the surface of APD which we have called silicone acellular porcine dermis (SAPD). This new artificial skin can protect the wound long enough to promote wound healing either by second intention or covered long enough until cultured epithelium autograft (CEA) or autologous skin graft can be harvested for permanent coverage. We delivered 4 cm x 5 cm full-thickness wound on the back of 350 g Sprague-Dawley rats. Thirty-six rats were divided into two groups. Eighteen rats had SAPD and the other 18 were covered with Biobrane. The wounds were first examined 2 weeks after grafting and followed weekly for an additional 4 weeks to evaluate the wound and study pathological changes by using H.E. and Masson's stains. Wound size was calculated by ruler and analyzed by Student's t-test. At the 2-week inspection, both SAPD and Biobrane showed tight adherence to the wound with no change of wound size. Both the SAPD and Biobrane dermal templates were pink. In the Biobrane-covered group, the wounds contracted soon after the tie-over dressing was removed. Its dermal layer is a layer of thin porcine dermal substance, which was promptly digested by tissue hyaluronidase and provides no real dermal template. In the SAPD-covered group however, the wound size was maintained significantly from third to sixth week after grafting (p<0.001). SAPD was designed with thick epidermal silicone and a well-organized porcine dermis so that it incorporates into the recipient wound. Clinically the silicone layer of SAPD dislodged from APD about 6-7 weeks after grafting and was followed by dermal matrix exposure and infection. In pathological examination, much like a human skin graft, new vessels were found in APD about 1 week after grafting with minimal inflammatory cells infiltrated in the graft and wound. Six weeks after grafting, the collagen of APD incorporated into the wound, showing palisade arrangement and no sign of rejection. In the Biobrane group however, the wounds showed severe inflammation, the porcine dermal matrix was digested and disappeared 3 weeks after coverage. In conclusion, SAPD is a thick biosynthetic artificial skin, which protects the rat wound significantly longer than Biobrane and prevents contraction. We expect that using of SAPD for temporary wound coverage will provide enough time to grow autologous-cultured epithelium or to reharvest skin grafts.

Animals↗

PubMedDate not supplied

Debridement of porcine burns with a highly purified, ananain-based cysteine protease preparation.

A novel enzymatic debriding agent was evaluated on experimental full-thickness porcine contact burns. This agent consists of a highly purified, ananain-based, cysteine protease preparation formulated in a hydrophilic cream vehicle. Debridement of full-thickness burns was found to be dependent on several factors including the concentration of enzyme in the vehicle, the duration of treatment, and the hydration status of the burn wound before treatment. With an optimized debridement regimen, burns were consistently debrided of all gelatinized tissue with two 5-hour treatments. Histologic evaluation of the debrided wounds revealed an acellular deeper dermis that was debrided of necrotic cellular debris; however, the collagen matrix of the deeper dermis remained intact. This observation was consistent with a demonstrated in vitro specificity of the ananain-based protease for gelatin over collagen. A direct comparison of debridement efficacy with sutilains ointment, showed the ananain-based, debriding enzyme preparation to provide more rapid debridement of gelatinized tissue. Enzymatically debrided wounds exhibited graft take only after surgical excision of approximately 1 mm of the remaining acellular, avascular dermis. This highly purified enzyme preparation offers the potential for rapid nonsurgical debridement of gelatinized burn tissue, but required additional surgical debridement for graft take in this porcine model.

Animals↗

PubMedDate not supplied

Development of a closed bioreactor system for culture of tissue-engineered skin at an air-liquid interface.

A bioreactor has been developed for the production of tissue-engineered skin at an air-liquid interface for clinical and experimental use. In this closed system, scaffold and bioreactor sterilization, cell seeding, and medium perfusion were all performed with a peristaltic pump. Natural and synthetic dermal substitutes were seeded directly with skin cells without opening the bioreactor and fed either by continuous medium perfusion or by batch-feed. The system was validated by monoculture of human dermal fibroblasts and keratinocytes and the coculture of both cell types in acellular human dermis, Azowipes, electrospun polystyrene, and an electrospun composite of polystyrene and poly-DL-lactide fibers. A comparison was made of culture at an air-liquid interface versus submerged culture and of medium change by continuous perfusion versus batch-feed. Fibroblast and endothelial cells showed greater viability under submerged rather than air-liquid conditions whereas keratinocytes favored culture at an air-liquid interface as did cocultured keratinocytes and fibroblasts. Total cellular viability for reconstructed skin with keratinocytes and fibroblasts was greatest with continuous perfusion rather than batch-feed and with electrospun scaffolds compared with acellular human dermis. The bioreactor could also be easily configured to give replicate small areas for experimental use or one continuous area of construct for clinical use.

Air↗

PubMed2003

Fetal tissue engineering from amniotic fluid.

BACKGROUND: We have recently shown, in an animal model, that amniotic fluid can be a source of cells for fetal tissue engineering. This study was aimed at determining whether fetal tissue constructs could also be engineered from cells normally found in human amniotic fluid. STUDY DESIGN: Cells obtained from the amniotic fluid of pregnant women at 15 to 19 weeks of gestation (n=6) were cultured in Dulbecco's Modified Eagle's medium (Sigma Chemical, St Louis, MO) containing 20% fetal bovine serum and 5 ng/mL basic fibroblast growth factor in a 95% humidified, 5% CO(2) chamber at 37 degrees C. A subpopulation of morphologically distinct cells was then mechanically isolated from the rest and selectively expanded. The lineage of this subpopulation of amniocytes was determined by immunofluorescent staining with antibodies against standard intermediate filaments and surface antigens. Cell proliferation rates were determined by oxidation assay. After cell expansion, colonies of amniocytes were statically and dynamically seeded onto both unwoven, 1-mm-thick polyglycolic acid polymer scaffold and acellular human dermis for 72 hours. The resulting constructs were analyzed by scanning electron microscopy. RESULTS: Amniocytes stained positively for smooth muscle actin, vimentin, cytokeratin 18, and fibroblast surface protein, and negatively for desmin, cluster of differentiation 31, and von Willebrand's factor (Dako, Carpenteria, CA). These findings are consistent with a mesenchymal, fibroblast-myofibroblast cell lineage. Mesenchymal amniocytes could be rapidly expanded in culture, based on results of the proliferation assay. Scanning electron microscopy of amniocyte constructs revealed dense, confluent layers of cells surrounding the polymer matrices and firm cell adhesion to both PGA and Alloderm (Lifecell Corp, Branchburg, NJ) scaffolds. No evidence of cell death was observed. CONCLUSIONS: Subpopulations of fetal mesenchymal cells can be consistently isolated from human amniotic fluid and rapidly expanded in vitro. Human mesenchymal amniocytes attach firmly to both polyglycolic acid polymer and acellular human dermis. The amniotic fluid can be a valuable and practical cell source for fetal tissue engineering.

Amniotic Fluid↗

PubMedDate not supplied

Acellular allodermis in burns surgery: 1-year results of a pilot trial.

We grafted 10 sites on 6 children with limited (< 25%) areas of body surface available for donor harvest with an acellular allogenic dermis and a thin autograft. Matched control sites were grafted with autograft alone. Study site autografts (0.0074 +/- 0.0007 in, median 0.006 in, range 0.006 to 0.012 in) were thinner than control site autografts (0.0102 +/- 0.0008 in, median 0.012 in, range 0.006 to 0.013 in), with a P value of .015. Endopoints were initial engraftment (in percent) as judged by a blinded experienced observer and Vancouver scar scores. The 6 children (3 girls and 3 boys) had an average age of 5.2 +/- 0.9 years (range 2.8 to 10 years) and an average burn size of 68.7% +/- 6.7% total body surface area (range 47% to 85% total body surface area). The 10 study and control sites were treated with 10 separate procedures; 9 of the procedures were reconstructive and 1 was performed for the excision of an acute burn. Successful initial epithelialization was noted at 7 days postburn for 83% +/- 3.4% (range 60% to 95%) at the cryopreserved acellular human dermis sites and 83.3% +/- 4.3% (range 60% to 98%) at the control sites (not significant, P = .96). At a mean follow-up interval of 43.7 +/- 3.6 weeks (median 52, range 26 to 52 weeks), the differences between the study and control sites in the total mean (pigmentation, vascularity, pliability, and height) of the patients' Vancouver scar scores were not different.

Burns↗

PubMed1997

Human acellular dermal matrix as a novel model of malignant epithelial cell invasion.

Cancer invasion and metastasis are associated with matrix degradation. We describe a novel in vivo model of invasion by squamous epithelial neoplastic cells derived from transgenic mice grown on acellular human dermis. Human dermis was subjected to multiple freeze-thaw cycles to render it acellular, maintaining the basement membrane of the former dermal-epidermal junction. Cells representing discrete stages of a multistep transgenic mouse model of epidermal carcinogenesis (neonatal transgenic keratinocytes, moderately/poorly differentiated squamous cell carcinoma, and lymph node metastasis) were seeded onto the basement membrane surface, grown in culture for 4 days, grafted in a subpannicular pocket of athymic mice, and harvested after 3 weeks. Histological analysis demonstrated that neonatal transgenic keratinocytes did not degrade the basement membrane or invade the underlying dermis. In contrast, malignant cells derived from both a moderately differentiated squamous carcinoma and a lymph node metastasis were highly invasive. Immunohistochemical analysis revealed collagenase only in nests of invading malignant cells in contact with the dermal matrix, but not in the tumor mass remaining above the basement membrane, suggesting that this proteinase may be required for stromal invasion. This novel model recapitulates the events seen in malignant invasion: transgenic keratinocytes are unable to penetrate the dermis while cells from a moderately differentiated carcinoma and from lymph node metastasis consistently invade.

Animals↗