Comparison of biopolymer scaffolds for the fabrication of skin substitutes in a porcine wound model.

This study compared three acellular scaffolds as templates for the fabrication of skin substitutes. A collagen-glycosaminoglycan (C-GAG), a biodegradable polyurethane foam (PUR) and a hybrid combination (PUR/C-GAG) were investigated. Scaffolds were prepared for cell inoculation. Fibroblasts and keratinocytes were serially inoculated onto the scaffolds and co-cultured for 14 days before transplantation. Three pigs each received four full-thickness 8 cm × 8 cm surgical wounds, into which a biodegradable temporising matrix (BTM) was implanted. Surface seals were removed after integration (28 days), and three laboratory-generated skin analogues and a control split-thickness skin graft (STSG) were applied for 16 weeks. Punch biopsies confirmed engraftment and re-epithelialisation. Biophysical wound parameters were also measured and analysed. All wounds showed greater than 80% epithelialisation by day 14 post-transplantation. The control STSG displayed 44% contraction over the 16 weeks, and the test scaffolds, C-GAG 64%, Hybrid 66.7% and PUR 67.8%. Immunohistochemistry confirmed positive epidermal keratins and basement membrane components (Integrin alpha-6, collagens IV and VII). Collagen deposition and fibre organisation indicated the degree of fibrosis and scar produced for each graft. All scaffold substitutes re-epithelialised by 4 weeks. The percentage of original wound area for the Hybrid and PUR was significantly different than the STSG and C-GAG, indicating the importance of scaffold retainment within the first 3 months post-transplant. The PUR/C-GAG scaffolds reduced the polymer pore size, assisting cell retention and reducing the contraction of in vitro collagen. Further investigation is required to ensure reproducibility and scale-up feasibility.

Advances in Skin Tissue Bioengineering and the Challenges of Clinical Translation.

Skin tissue bioengineering is an emerging field that brings together interdisciplinary teams to promote successful translation to clinical care. Extensive deep tissue injuries, such as large burns and other major skin loss conditions, are medical indications where bioengineered skin substitutes (that restore both dermal and epidermal tissues) are being studied as alternatives. These may not only reduce mortality but also lessen morbidity to improve quality of life and functional outcome compared with the current standards of care. A common objective of dermal-epidermal therapies is to reduce the time required to accomplish stable closure of wounds with minimal scar in patients with insufficient donor sites for autologous split-thickness skin grafts. However, no commercially-available product has yet fully satisfied this objective. Tissue engineered skin may include cells, biopolymer scaffolds and drugs, and requires regulatory review to demonstrate safety and efficacy. They must be scalable for manufacturing and distribution. The advancement of technology and the introduction of bioreactors and bio-printing for skin tissue engineering may facilitate clinical products' availability. This mini-review elucidates the reasons for the few available commercial skin substitutes. In addition, it provides insights into the challenges faced by surgeons and scientists to develop new therapies and deliver the results of translational research to improve patient care.

Scale-up of a Composite Cultured Skin Using a Novel Bioreactor Device in a Porcine Wound Model.

Extensive deep-burn management with a two-stage strategy can reduce reliance on skin autografts; a biodegradable polyurethane scaffold to actively temporize the wound and later an autologous composite cultured skin (CCS) for definitive closure. The materials fulfilling each stage have undergone in vitro and in vivo pretesting in "small" large animal wounds. For humans, producing multiple, large CCSs requires a specialized bioreactor. This article reports a system used to close large porcine wounds. Three Large White pigs were used, each with two wounds (24.5 cm × 12 cm) into which biodegradable dermal scaffolds were implanted. A sample from discarded tissue allowed isolation/culture of autologous fibroblasts and keratinocytes. CCS production began by presoaking a 1-mm-thick biodegradable polyurethane foam in autologous plasma. In the bioreactor cassette, fibroblasts were seeded into the matrix with thrombin until established, followed by keratinocytes. The CCSs were applied onto integrated dermal scaffolds on day 35, alongside a sheet skin graft (30% of one wound). Serial punch biopsies, trans-epidermal water loss readings (TEWL), and wound measurements indicated epithelialization. During dermal scaffold integration, negligible wound contraction was observed (average 4.5%). After CCS transplantation, the control skin grafts were "taken" by day 11 when visible islands of epithelium were clinically observed on 2/3 CCSs. Closure was confirmed histologically, with complete epithelialization by day 63 post-CCS transplantation (CCS TEWL ~ normal skin average 11.9 g/m2h). Four of six wounds demonstrated closure with robust, stratified epithelium. Generating large pieces of CCS capable of healing large wounds is thus possible using a specialized designed bioreactor.

A Novel Biodegradable Polyurethane Matrix for Auricular Cartilage Repair: An In Vitro and In Vivo Study.

Auricular reconstruction poses a challenge for reconstructive and burns surgeons. Techniques involving cartilage tissue engineering have shown potential in recent years. A biodegradable polyurethane matrix developed for dermal reconstruction offers an alternative to autologous, allogeneic, or xenogeneic biologicals for cartilage reconstruction. This study assesses such a polyurethane matrix for this indication in vivo and in vitro. To evaluate intrinsic cartilage repair, three pigs underwent auricular surgery to create excisional cartilage ± perichondrial defects, measuring 2 × 3 cm in each ear, into which acellular polyurethane matrices were implanted. Biopsies were taken at day 28 for histological assessment. Porcine chondrocytes ± perichondrocytes were cultured and seeded in vitro onto 1 × 1 cm polyurethane scaffolds. The total culture period was 42 days; confocal, histological, and immunohistochemical analyses of scaffold cultures were performed on days 14, 28, and 42. In vivo, the polyurethane matrices integrated with granulation tissue filling all biopsy samples. Minimal neocartilage invasion was observed marginally on some samples. Tissue composition was identical between ears whether perichondrium was left intact, or not. In vitro, the polyurethane matrix was biocompatible with chondrocytes ± perichondrocytes and supported production of extracellular matrix and Type II collagen. No difference was observed between chondrocyte culture alone and chondrocyte/perichondrocyte scaffold coculture. The polyurethane matrix successfully integrated into the auricular defect and was a suitable scaffold in vitro for cartilage tissue engineering, demonstrating its potential application in auricular reconstruction.

Optimization of a polyurethane dermal matrix and experience with a polymer-based cultured composite skin.

The aims were to (1) describe the in vivo studies leading to an optimized model of the biodegradable temporizing matrix (BTM), (2) describe our efforts in effecting closure over this optimized matrix after integration with a cultured composite skin (CCS), and (3) reexamine the ability of the CCS to definitively close fresh wounds (without BTM). Foam scaffolds of biodegradable polyurethane were created to allow in vivo tissue ingrowth or in vitro co-culture. Using the porcine surgical model, multiple BTM optimization studies took place before the BTM-CCS main study was conducted. For the CCS study, optimized sealed 2 mm matrices were implanted into 6-mm deep, 8 × 8 cm wounds (three per pig) and allowed to integrate for 21 days, whereas collected blood and harvested skin tissue were used to prepare autologous composite skins in similar (unsealed) 1 mm matrices. These were then applied at day 21 either over the integrated BTMs or into a freshly created fourth wound. All of the optimized matrices integrated fully, without loss, and were found to resist wound contraction effectively until the composites were ready for application at day 21. The composites demonstrated the ability to generate a bilayer repair with robust epidermis anchored by a basement membrane visible from day 7 after application. The final optimized sealed BTM delaminates easily to produce a clean, temporized wound bed and will be used in the upcoming burn clinical trial. Although the CCS is a magnitude away from human trials, it is still capable of generating a bilayer repair in both BTM-integrated and fresh wounds (onto fat), and with further refinement and optimization of foam structure, seeding densities, and timing, consistent success should be possible.

"Take" of a polymer-based autologous cultured composite "skin" on an integrated temporizing dermal matrix: proof of concept.

This study aimed to investigate the ability of an autologous cultured composite skin (CCS) to close similar biodegradable temporizing matrix (BTM)-integrated wounds, and its effectiveness in healing fresh full-thickness wounds after the failure of cultured epithelial autograft in its two forms (sheets and suspensions) to epithelialize over an integrated polymer BTM. Using a porcine model, autologous split-skin grafts were harvested three of four dorsal 8 × 8 cm treatment sites. These three sites were subsequently converted to full-thickness wounds and BTMs were implanted. The grafts were used to produce autologous CCSs for each pig. These consisted of a 1 mm thick biodegradable polymer foam scaffold into which fibroblasts and keratinocytes harvested from the grafts were cocultured. At Day 28, on each animal, the autologous CCSs were applied to two of the integrated BTMs, an autologous split-skin graft was applied to the third integrated BTM, and one CCS was applied immediately into a fresh, "naked" (no BTM applied) wound. The CCSs were capable of generating a bilayer repair over the naked wound's fat base and BTM-integrated wounds, which consisted of dermal elements and a keratinized stratified squamous epidermis anchored with a basement membrane by day 7. The CCSs behaved in different ways: either as a delivery vehicle allowing similar development of a bilayer repair while the polymer foam was shed from the wound, or generating a bilayer repair with the foam scaffold being retained (composite "take"). These results conclude our porcine program and provide proof of concept that the integrated BTM can be closed with an autologous CCS. Once fully optimized, this may provide robust repair without resorting to the split-skin graft, important in those cases where unburned donor site is unavailable.

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.

Split skin graft application over an integrating, biodegradable temporizing polymer matrix: immediate and delayed.

The objective of this study is to further investigate the NovoSorb™ biodegradable polyurethane in generating dermal scaffolds; to perform a pilot study comparing the previously used spun mat against a recently developed NovoSorb™ foam, ascertaining the optimum structure of the matrix; and to evaluate the successful matrix as an immediate adjunct to split skin grafting and as a temporizing matrix in a prospective six-pig study. A pilot study comparing a previously investigated form of the polymer (spun mat) against a new structural form, a foam, was performed. This was followed by a six-pig study of the foam matrix with three treatment arms-autologous split skin graft alone, polymer foam with immediate engraftment, and polymer foam with delayed engraftment. The foams allowed less wound contraction than the spun mats. The foam structure is less dense (cheaper to produce and having less degradation products). The material remained in situ despite clinical wound infection. Proof of concept was achieved in both treatment modalities in the main study. Split skin graft applied immediately over the polymer foam was able to engraft successfully. The result was "thicker" to pinch and "flush" with the skin surrounding the wound. There was no significant difference in the degree of wound contraction between the graft alone and the polymer plus immediate graft groups. Split skin graft also "took" when applied to the surface of a polymer that had been applied to a wound 11 days earlier, again with a thicker result, flush with the surrounding skin. Split skin grafts alone left a persisting depression. However, a significant degree of wound contraction (compared with the other two groups) was observed in the polymer plus delayed graft group. This has prompted further investigation into "sealing" the polymer foam with a membrane, to prevent evaporative water loss, when the foam is to be used as a biodegradable temporizing matrix. The studies indicate that the NovoSorb™ platform will allow the creation of two inexpensive dermal matrix products; an immediate scaffold to allow a thicker grafting result and a biodegradable temporizing matrix (BTM) for wound integration after burn debridement while donor sites become reharvestable. However, further modification on the BTM structure is necessary to further reduce wound contraction pregrafting.

A comparison of wound area measurement techniques: visitrak versus photography.

OBJECTIVE: To investigate whether a cheap, fast, easy, and widely available photographic method is an accurate alternative to Visitrak when measuring wound area in cases where a non-wound-contact method is desirable. METHODS: The areas of 40 surgically created wounds on porcine models were measured using 2 techniques-Visitrak and photography combined with ImageJ. The wounds were photographed with a ruler included in the photographic frame to allow ImageJ calibration. The images were uploaded to a computer and opened with ImageJ. The wound outline was defined from the photographic image using a digital pad, and the ImageJ software calculated the wound area. The Visitrak method involved a 2-layered transparent Visitrak film placed on the wound and the outline traced onto the film. The top layer containing the tracing was retraced onto the Visitrak digital pad using the Visitrak pen and the software calculated the wound area. RESULTS: The average wound area using the photographic method was 52.264 cm(2) and using Visitrak was 51.703 cm(2). The mean difference in wound area was 0.560 cm(2). Using a 2-tailed paired T test, the T statistic was 1.285 and the value .206, indicating no statistical difference between the two methods. The interclass correlation coefficient was 0.971. CONCLUSIONS: The photographic method is an accurate alternative to Visitrak for measuring wound area, with no statistical difference in wound area measurement demonstrated during this study. The photographic method is a more appropriate technique for clean and uncontaminated wounds, as contact with the wound bed is avoided.

Real-time demonstration of split skin graft inosculation and integra dermal matrix neovascularization using confocal laser scanning microscopy.

OBJECTIVES: During the first 48 hours after placement, an autograft "drinks" nutrients and dissolved oxygen from fluid exuding from the underlying recipient bed ("plasmatic imbibition"). The theory of inosculation (that skin grafts subsequently obtain nourishment via blood vessel "anastomosis" between new vessels invading from the wound bed and existing graft vessels) was hotly debated from the late 19th to mid-20th century. This study aimed to noninvasively observe blood flow in split skin grafts and Integra dermal regeneration matrix to provide further proof of inosculation and to contrast the structure of vascularization in both materials, reflecting mechanism. METHODS: Observations were made both clinically and using confocal microscopy on normal skin, split skin graft, and Integra. The VivaScope allows noninvasive, real-time, in vivo images of tissue to be obtained. RESULTS: Observations of blood flow and tissue architecture in autologous skin graft and Integra suggest that 2 very different processes are occurring in the establishment of circulation in each case. Inosculation provides rapid circulatory return to skin grafts whereas slower neovascularization creates an unusual initial Integra circulation. CONCLUSIONS: The advent of confocal laser microscopy like the VivaScope 1500, together with "virtual" journals such as ePlasty, enables us to provide exciting images and distribute them widely to a "reading" audience. The development of the early Integra vasculature by neovascularization results in a large-vessel, high-volume, rapid flow circulation contrasting markedly from the inosculatory process in skin grafts and the capillary circulation in normal skin and merits further (planned) investigation.

Evaluation of a novel biodegradable polymer for the generation of a dermal matrix.

Dermal skin substitutes can be used to overcome the immediate problem of donor site shortage in the treatment of major skin loss conditions, such as burn injury. In this study, the biocompatibility, safety, and potential of three variants of NovoSorb (a family of novel biodegradable polyurethanes) as dermal scaffolds were determined in a series of in vitro and in vivo systems. All three polymers exhibited minimal cytotoxic effects on human skin cells, allowing keratinocytes, dermal fibroblasts, and microvascular endothelial cells to grow normally in coculture. Subcutaneous implantation of the polymers in rats demonstrated no systemic toxic effects of the materials or their degradation products. The anticipated local foreign body reaction compared favorably with commercially available medical sutures. Assessment of a three-dimensional polymer matrix followed. The success of sequential culturing of dermal fibroblasts and keratinocytes within the matrix indicated that the generation of a cultured skin substitute is achievable. The polymeric matrix also provided a scaffold for the guided formation of a cultured microvasculature. When engrafted onto a surgically created full-thickness sheep wound, the noncellular matrix integrated, healed with an epidermis supported by a basement membrane, and was capable of withstanding wound contraction. The resistance to contraction compared favorably with a commercially available collagen-based dermal matrix (Integra). These results suggest that the NovoSorb matrix could form the basis of an elegant two-stage burn treatment strategy, with an initial noncellular biodegradable temporizing matrix to stabilize the wound bed followed by the application of cultured skin substitute.

Noncultured keratinocyte/melanocyte cosuspension: effect on reepithelialization and repigmentation--a randomized, placebo-controlled study.

Randomized controlled trials in the literature investigating the efficacy of noncultured keratinocyte/melanocyte suspensions are scarce; however, the advocates of such techniques press the value of their application based largely on case studies and anecdote. Caucasian patients with burn hypopigmentation seldom request cosmetic revision making worthwhile clinical trials difficult so that informal case treatments with new therapies generate anecdotal results. A randomized, placebo-controlled trial was carried out to evaluate whether cosuspensions of noncultured skin cells are capable of (1) decreasing the time to reepithelialization and (2) reestablishing pigmentation in vitiligo leukoderma following epidermal/superficial dermal ablation (in the knowledge that a positive result would make the technique likely to be successful in burn hypopigmentation). Vitiligo is common and is socially more debilitating such that suitable trial subjects for new therapies from this pool are more forthcoming. This study demonstrated that suspensions of noncultured keratinocytes and melanocytes do not decrease the time to epithelialization of superficial partial thickness wounds compared with controls. It also suggested that the achievement, quality, and duration of any pigmentation were unpredictable and largely disappointing. Some pigmentation was recorded in placebo-treated areas indicating an effect of the method of epidermal ablation in these patients. These findings have mandated a complete review of the use of these techniques in burn care at the Royal Adelaide Hospital; they have been omitted from surgical protocols where the aim of use was to speed reepithelialization. Their infrequent use in burns hypopigmentation will continue contingent on the successful repigmentation of a test patch.

Nipple-areolar pigmentation: histology and potential for reconstitution in breast reconstruction.

The makeup of nipple-areolar skin, in terms of its melanin and melanocyte content has not previously been established. This histological information is required if pigmentation of the reconstructed nipple-areola is to be successful in post-mastectomy breast reconstruction. We describe examination of 200 parallel sections of nipple-areolar skin of 20 women using histochemical (Masson-Fontana) and immunohistochemical (Mel-5) techniques, evaluated using quantitative image analysis. The amount of melanin present per length of basement membrane was 2.14 times higher in areolar skin than breast skin. The ratio of melanocytes to keratinocytes was 1:9.7 in areolar skin vs. 1:14.7 in breast skin. We also describe a cell culture and skin construct method using autologous human serum without toxic growth promoting additives, which could be used in the clinical setting of nipple-areolar reconstruction.