Permanent Wound Coverage Research Articles

In vivo efficacy proof of concept of a large-size bioprinted dermo-epidermal substitute for permanent wound coverage.

Introduction: An autologous split-thickness skin graft (STSG) is a standard treatment for coverage of full-thickness skin defects. However, this technique has two major drawbacks: the use of general anesthesia for skin harvesting and scar sequelae on the donor site. In order to reduce morbidity associated with STSG harvesting, researchers have developed autologous dermo-epidermal substitutes (DESs) using cell culture, tissue engineering, and, more recently, bioprinting approaches. This study assessed the manufacturing reliability and in vivo efficacy of a large-size good manufacturing practice (GMP)-compatible bio-printed human DES, named Poieskin®, for acute wound healing treatment. Methods: Two batches (40cm2 each) of Poieskin® were produced, and their reliability and homogeneity were assessed using histological scoring. Immunosuppressed mice received either samples of Poieskin® (n = 8) or human STSG (n = 8) immediately after longitudinal acute full-thickness excision of size 1 × 1.5cm, applied on the skeletal muscle plane. The engraftment rate was assessed through standardized photographs on day 16 of the follow-up. Moreover, wound contraction, superficial vascularization, and local inflammation were evaluated via standardized photographs, laser Doppler imaging, and PET imaging, respectively. Histological analysis was finally performed after euthanasia. Results: Histological scoring reached 75% ± 8% and 73% ± 12%, respectively, displaying a robust and homogeneous construct. Engraftment was comparable for both groups: 91.8% (SD = 0.1152) for the Poieskin® group versus 100% (SD = 0) for the human STSG group. We did not record differences in either graft perfusion, PET imaging, or histological scoring on day 16. Conclusion: Poieskin® presents consistent bioengineering manufacturing characteristics to treat full-thickness cutaneous defects as an alternative to STSG in clinical applications. Manufacturing of Poieskin® is reliable and homogeneous, leading to a clinically satisfying rate of graft take compared to the reference human STSG in a mouse model. These results encourage the use of Poieskin® in phase I clinical trials as its manufacturing procedure is compatible with pharmaceutical guidelines.

Mid-Term Follow-Up Study of Children Undergoing Autologous Skin Transplantation for Burns

Deep partial and full-thickness burns require surgical treatment with autologous skin grafts after necrectomy, which is the generally accepted way to achieve permanent wound coverage. This study sought to examine the grafted and donor areas of children who underwent autologous skin transplantation, using two assessment scales to determine the severity of the scarring and the cosmetic outcome during long-term follow-up. At the Surgical Unit of the Department of Paediatrics of the University of Pécs, between 1 January 2015 and 31 December 2019, children who had been admitted consecutively and received autologous skin transplantation were analyzed. Twenty patients met the inclusion criteria in this retrospective cohort study. The authors assessed the results using the Patient and Observer Scar Assessment Scale (POSAS) and the Vancouver Scar Scale (VSS). There was a significant difference in how parents and examiners perceived the children's scars. In the evaluation of the observer scale, the most critical variables for the area of skin grafted were relief and thickness. Besides color, relief was the worst clinical characteristic on the patient scale. However, when medical professionals evaluated the donor site, significantly better results were obtained compared to the transplanted area (average observer scale score: 1.4 and 2.35, p = 0.001; VSS: 0.85 vs. 2.60, p < 0.001), yet it was similar to the graft site in the parents' opinion (Patient Scale: 2.95 and 4.45, p = 0.181).

Use of single-layer artificial dermal template in patients with trauma and burns.

Artificial dermal templates (ADTs), were designed initially to provide extracellular matrix and skin substitute for extensive burn injuries. Use of ADTs in a variety of other indications, has also been described in the literature. In this study, we describe our experience of using ADTs for different indications in burn contractures and wound coverage. In this retrospective study, patients requiring burn scar contracture release, permanent wound coverage for acute traumatic wounds and temporary wound coverage to prepare for complex reconstructions, and where the ADT Pelnac (Gunze Ltd., Japan) was applied, were evaluated. Data regarding patient sex, age, type and location of injury, comorbidities, operations and complications were recorded. A total of 24 patients were included in the study, of whom 12 patients were operated on for burn contractures. ADTs were used with split-thickness skin grafts (STSGs) or Z-plasties in a single-stage procedure. In six patients, ADT and STSGs were used to cover defects with exposed bone or tendon. Of the patients, six had their wounds covered temporarily while they were stabilised for complex reconstructions or were awaiting definitive histopathological results. Revisional surgeries due to graft failures or insufficient contracture releases were required by 12 patients. All temporary wound coverage patients had successful flap reconstructions after stabilisation of their general status, had tumour-free margins in the histopathological examination, and no necrosis or infection was seen on follow-up. In this study, ADTs had positive effects on selected patients, but comprehensive and comparative clinical studies are needed for different indications to choose between these templates.

Wound healing of open fractures: comparison of vaccum assisted dressing versus traditonal dressing

PurposeFor permanent wound coverage of compound fractures, the wound surface should have 100% granulation tissue without any discharge or necrotic slough. Negative pressure wound therapy (NPWT) is a well known modality in the management of open fractures, and post-operative wound problems. This study was conducted to compare the outcome of NPWT and traditional dressing in wound healing of open fractures. Material and methodsA prospective study of 100 cases was conducted to compare the wound healing outcomes of open fracture following vacuum assisted closure (VAC) dressing (50 cases) and traditional wound dressing (50 cases). Patients included in the study underwent initial debridement to remove necrotic slough and tissue when indicated. Foam dressings were used to cover the wounds using aseptic conditions in the VAC group. Dressings were changed daily for patients in traditional dressing group using hydrogen peroxide, normal saline and povidone iodine in a sequential manner and every 3–4 days in the VAC dressing group. Patients were evaluated clinically for appearance of granulation tissue, duration of hospital stay and reduction in wound surface area using appropriate statistical methods. ResultThere was a significant difference between the rate of wound healing in the group NPWT/VAC group compared to the traditional wound dressings group in terms of appearance of granulation tissue, reduction in wound surface area and duration of hospital stay. ConclusionVacuum assisted dressing is more effective than traditional wound dressing in wound healing of open fractures.

A New Form of Artificial Skin to Promote Permanent Wound Coverage: A Preliminary Report.

Although tendon-exposed or bone-exposed wounds can be resurfaced with flaps, such surgeries may not be feasible in patients with poor general or local conditions. Biosynthetic artificial skin is an alternative for critical wound coverage. We designed a new artificial skin bilayer to close difficult wounds permanently. This study compares incorporation and wound contraction between silicone acellular porcine dermis (SAPD) and the Integra graft (Integra Life Sciences Corp., Billerica, Mass) in a rat model. The SAPD was manufactured according to our previously described standard procedures. Integra grafts were obtained commercially. We included 24 male adult Sprague-Dawley rats and divided them into 2 groups. After creating a 3 × 4-cm full-thickness wound on the back, we transplanted the same-sized SAPD and Integra grafts onto the rat wounds. Autologous full-thickness skin (FTS) was grafted onto the acellular porcine dermal matrix (APDM) of the SAPD and the Integra dermal matrix (IDM) 2 weeks later. We measured the wound size and contraction rate of recipient wounds, studied the incorporation of FTS on the dermal matrix, and did pathological examination. Generalized estimating equations were used to assess the data from repeated wound and scar contraction measurements using SAS v9.2. The sizes of wounds of both groups decreased over time. No difference in wound contraction was observed between the SAPD and Integra groups at weeks 2, 4, or 6 after grafting. However, the contraction rates in both groups increased significantly. The pathological examination showed that the FTS was well incorporated in the APDM and IDM. The recipient wounds showed new vessels and cell infiltration in the new matrix, but no severe inflammation. Skin appendages were regenerating in the FTS. There was no rejection sign. Both SAPD and Integra are double-layered artificial skin products. Our results demonstrate that APDM and IDM are good templates and show excellent incorporation with autologous FTS graft. The results also demonstrated gradual wound contraction over time, but the contraction rate was not different between SAPD and Integra 6 weeks after grafting in a rat model.

Treatment of necrotising fasciitis using glycerol-preserved skin allografts for temporary wound coverage

Allografts of human skin have been used for both temporary and permanent wound coverage. They are useful as temporary bridges for the critically ill patient who is not allowed to receive definite wound coverage, or for wound bed preparation before permanent grafting. Glycerol-preserved skin allografts have several benefits including good adherence to the wound bed, water vapour transport, antimicrobial characteristics, low toxicity and antigenicity, ease of application and removal, a long shelf-life, and minimal storage requirements. We achieved lower limb salvage using a glycerol-preserved skin allograft for temporary wound coverage in the treatment of necrotising fasciitis.

L’ingéniérie cutanée pour le traitement des brûlures graves

Severely burned patients need effective and permanent wound coverage. The outcome of massive burn injuries has improved with the use of cultured epithelial autografts (CEA), despite their fragility, frequent failure to take, high cost and long-term tendency to contract. Combining CEA with dermal substitutes provides earlier skin closure and satisfactory functional results. Another promising line of research is skin regeneration with epidermal stem cells, which have the capacity to differentiate into keratinocytes, to promote wound repair, and to regenerate skin appendages. Human mesenchymal stem cells have been evaluated in radiation-induced skin damage.

Coverage of Large Pediatric Wounds With Cultured Epithelial Autografts in Congenital Nevi and Burns: Results and Technique

The use of cultured epithelial autografts (CEA) for the treatment of large burn wounds has gained popularity in recent years. This technique may circumvent the restrictions of limited donor site availability and hasten permanent wound coverage for large TBSA burns. The availability of a large amount of skin from a small donor site with the promise of permanent wound coverage suggests its use in other conditions such as giant congenital nevi (GCN) as well. The risk of malignant transformation of GCN to melanoma although somewhat controversial is significant enough to warrant early excision in childhood. Cultured keratinocytes may provide one-stage coverage of these large wounds, lessening the number of surgeries and the inherent staging problems of tissue expansion or autografting. A retrospective single institution review of was done for 29 children (20 burns and 9 patients with GCN) who underwent coverage of their large surface area wounds with CEA over an 18-year period. Excellent take rates were noted; 76.4% for burn patients and 66% for patients with GCN. Several strategies in preoperative, perioperative, and postoperative care have been standardized and have helped improve outcome. The keys to success with the CEA technique have been aggressive control of wound sepsis, surgical technique, specific use of topical antimicrobials, dressings, and the standardization of nursing and physiotherapy care. Although the cost of CEA is high, the benefits to patient care make this technique an appealing choice for large wound coverage in the pediatric population.

The Skin Allograft Revisited: A Potentially Permanent Wound Coverage Option in the Critically Ill Patient

Pomahac, Bohdan M.D.; Garcia, Jesus A. M.D., Ph.D.; Lazar, Alexander J. M.D., Ph.D.; Tilney, Nicholas M.D.; Orgill, Dennis P. M.D., Ph.D. Author Information

Gene Therapy to Fight Infection in Skin Transplants

Wound closure can be problematic in patients with massive burns covering a large area of the body and in diabetic patients suffering from chronic ulcers. Insufficient donor sites in severely burned patients results in delayed wound closure and increases the likelihood of infection, which is a major cause of burn wound mortality [1]. Complications in patients wi th diabetic foot ulcers, most commonly resulting from infection, lead to as many as 50,000 amputations annually in the USA [2]. Tissue-engineered skin substitutes have been developed to begin to meet the needs of these patient populations [3]. The most basic skin substitutes are synthetic, acellular materials that act primarily as barriers to fluid loss and microbial invasion. Among the most innovative products are those containing cultured skin cells. Commercially available cellular skin substitutes, such as Apligraf ® , contain allogeneic dermal fibroblasts and epidermal keratinocytes, derived from donated neonatal foreskin, combined with a bovine collagen matrix. Apligraf is indicated for use in the treatment of venous leg ulcers and diabetic foot ulcers, increasing the percentage of wounds healed and decreasing the time required for wound closure [4–6]. However, as it is prepared using allogeneic cells, Apligraf and similar products act as biological bandages. Cells within the graft secrete growth factors and other proteins that facilitate wound healing and provide a matrix for cell migration, but the grafted cells do not persist indefinitely. Multiple applications are often required for complete wound healing. For burn patients, epidermal sheet grafts such as Epicel ® can be prepared with patient-derived keratinocytes for permanent wound coverage [7]. These grafts contain only epidermal cells; thus, they are prone to blistering and scar formation [7]. Cultured skin substitutes (CSS) are a bilayered autologous skin substitute currently in Phase II clinical trials. Comprised of cultured autologous keratinocytes, fibroblasts and biopolymers, CSS are used as an adjunctive treatment for coverage of burn wounds of greater than 50% of the body’s surface area, and have also been used to treat chronic wounds [8–10]. Despite the clinical successes observed with skin substitutes, they exhibit anatomical deficiencies, such as delayed vascularization, when compared with native skin autograft, which increase the risk for destruction due to microbial contamination. The use of topical antimicrobials in combination with skin substitutes can enhance engraftment and facilitate wound healing, but may contribute to the emergence of resistant strains of microorganisms. The development of a bioengineered skin substitute with increased innate resistance to microbial contamination could reduce or eliminate the requirement for exogenous antibiotics. Hypothetically, this could be achieved using a cutaneous gene therapy approach. Gene-encoded antimicrobial peptides, including members of the α- and β-defensin families and cathelicidins, have demonstrated antimicrobial activity against a wide variety of pathogens. Significantly, antimicrobial peptides have been shown to act against microorganisms commonly found in burns and chronic wounds, including multiply-resistant strains. As their mechanism of action is distinct from pharmaceutical antibiotic agents, defensins and related peptides represent a novel approach for treatment or prevention of infection. Antimicrobial peptides are effectors of the innate immune system and represent the body’s first line of defense against infection. Various antimicrobial peptides are expressed in neutrophils and in tissues that serve as interfaces with the environment, such as gut and lung epithelia and skin [11,12]. Rather than relying on specific antigen recognition, as in adaptive immunity, mediators of innate immunity take advantage of shared structural and functional characteristics of microorganisms. Antimicrobial peptides are cationic, allowing them to interact with the anionic lipid bilayer of microbial cell membranes through electrostatic interactions [13]. Following adsorption, the peptides disrupt the integrity of the outer cell membrane; irreversible injury of the target cell can result from entry of the antimicrobial peptide and other normally excluded molecules and leakage of essential cell components. Defensin-related cell death has also been linked to interference with protein synthesis and

Management of Exposed Total Knee Prostheses

Exposure of a total knee prosthesis represents a limb-threatening condition and a challenging reconstructive problem, requiring long-term antibiosis, irrigation, and serial debridement to avoid knee arthrodesis or amputation. Although traditional orthopedic surgical doctrine mandates removal of exposed hardware under a dehisced wound, a number of authors have reported salvage of exposed prostheses using local muscle flap coverage. However, the complex three-dimensional geometry of the soft tissue surrounding the knee, as well as the requirement for high sustained local tissue levels of antibiotics to re-sterilize the hardware, suggest that microvascular tissue transfer may be the most advantageous means of permanent wound coverage and may increase both limb salvage and prosthesis salvage rates. The experience using free tissue transfer to reconstruct these difficult three-dimensional wounds was reported.

Experimental model of cultured keratinocytes

The bioengineering research is essential in the development of ideal combination of biomaterials and cultured cells to produce the permanent wound coverage. The experimental model of cultured keratinocytes presents all steps of the culture, since the isolation of the keratinocytes, preparation of the human acellular dermis, preparation of the composite skin graft and their elevation to the air-liquid interface. The research in cultured keratinocytes model advances in two main ways: 1. optimization of the methods in vitro to the skin cells culture and proliferation and 2. developing biomaterials that present similar skin properties.

Immunogenicity of cultured keratinocyte allografts deficient in major histocompatibility complex antigens.

Full-thickness (FT) and cultured keratinocyte (CK) allografts have been used as temporary skin replacements in patients with massive burns, but these grafts are ultimately rejected after restoration of host immunocompetence. Genetic engineering has permitted the creation of knockout (KO) mice deficient in class I or class II major histocompatibility antigens. This study examines the immunogenicity of such grafts to determine if these genetically modified keratinocytes could be used for permanent wound coverage. Host sensitization to alloantigen was assessed by second-set rejection. CBA mice (n = 111) were primed with flank grafts consisting of FT and CK allografts from normal C57BL/6 donors, FT and CK class I KO allografts, FT and CK class II KO allografts, and CK autografts. Three weeks later, hosts were challenged with normal tail allografts and observed for second-set rejection. Median graft survival was analyzed by chi2 and Wilcoxon rank tests. In the second experiment, cytotoxic T lymphocytes (CTLs) were harvested from CBA mice (n = 28) 3 weeks after flank grafting. CTL effectors were tested on radiolabeled targets at various ratios in a 51Cr release assay. Dilution curves of CTL activity were compared by analysis of variance. Hosts primed with CK or FT allografts demonstrated accelerated rejection of second-set tail grafts compared with hosts covered with CBA autografts. CK knockout grafts were less immunogenic than FT knockout skin; class II KO allografts were considerably less immunogenic than class I KO allografts. CTL activity against the knockout CK allografts was negligible compared with that of hosts primed with normal allografts or FT knockout allografts. Although full-thickness knockout skin retains substantial immunogenicity, cultured keratinocytes deficient in class II antigens fail to prime for accelerated second-set rejection and do not elicit a CTL response in the graft recipient. This lack of immunogenicity may permit the indefinite survival of allogeneic knockout keratinocytes in patients requiring massive wound excision and coverage.

Treatment of Pyoderma Gangrenosutn with Cultured Keratinocyte Autografts

Pyoderma gangrenosum is an uncommon chronic skin disease characterized by rapidly enlarging cutaneous ulcers. Immunosuppressive agents, such as corticosteroids, are the mainstay of therapy. We describe a patient with pyoderma gangrenosum treated with cultured keratinocyte autografts for a full-thickness ulcer located on the dorsal and lateral aspects of the foot. After stabilizing the ulcer with intralesional and systemic corticosteroids, the ulcer was debrided and cultured keratinocyte autografts were secured with nylon mesh. An outer dressing of gauze and elastic bandage was used. The patient had > 95% "take" of the grafts and the ulcer was fully healed in less than 1 month. The grafted area preserved the clinical phenotype of the palmar skin from which the original biopsy was taken. Cultured keratinocyte autografts can provide permanent wound coverage for patients on high doses of immunosuppressive medications.

Epidermal culture and grafting. A brief review

In summary, technological advances in culturing epidermis for the purpose of grafting allow this approach in the treatment of cutaneous wounds. Certainly, full- and split-thickness autografts offer immediate availability and permanent wound coverage, but they also involve a large, painful donor site. Cultured epidermal autografts can provide permanent wound coverage, but the delay required for cultivation of confluent sheets of keratinocytes makes them somewhat less desirable. Both allografts and cultured allografts, on the other hand, are available for immediate use, but the possibility of infectious disease transmission may be a concern. In any case, all types of skin grafts function as biological dressings that promote the proliferation of the host's epidermis and, thus, facilitate the ability of the patient's skin to repair itself.

Skin Transplantation

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

Specific unresponsiveness to skin allografts in burns

We have examined the potential to provide long-term or even permanent wound coverage in a mouse model of a 30% total body surface area burn using skin allografts. Treatment of the recipient mouse with rabbit anti-mouse thymocyte serum (ATS) followed by donor bone marrow infusion induces a state of specific unresponsiveness to the skin allograft without the need for chronic immunosuppression. Specifically, a B6AF 1 mouse receives a burn on Day −2 relative to grafting, ATS on Day −1, and Day +2, a skin allograft from a C3H/He mouse on Day 0, and infusion of C3H/He donor bone marrow on Day +6. We studied three groups of burned mice: Group I, allograft control ( n = 5); Group II, allograft plus ATS ( n = 12); and Group III, allograft plus ATS and bone marrow infusion ( n = 15). Mean graft survival was compared using a one-way analysis of variance and a Student-Newman-Keuls post hoc test. There was no statistical difference in animal mortality among any of the three groups, and there was no evidence of infectious morbidity. Mean skin allograft survival was as follows: Group I, 9 days; Group II, 29 days; and Group III, 66 days ( P < 0.05 vs Group I and II). Nine animals in Group III had intact hair bearing grafts at 90 days when the study was terminated. This study suggests the potential use of induced specific unresponsiveness to skin allografts for wound coverage in thermal injury without use of chronic immunosuppression. In our animal study this was accomplished without increased mortality or apparent infectious morbidity.