Anna F. Falabella

Tissue-Engineered Skin

Tissue-engineered skin is a significant advance in the field of wound healing and was developed due to limitations associated with the use of autografts. These limitations include the creation of a donor site which is at risk of developing pain, scarring, infection and/or slow healing.A number of products are commercially available and many others are in development. Cultured epidermal autografts can provide permanent coverage of large area from a skin biopsy. However, 3 weeks are needed for graft cultivation. Cultured epidermal allografts are available immediately and no biopsy is necessary. They can be cryopreserved and banked, but are not currently commercially available.A nonliving allogeneic acellular dermal matrix with intact basement membrane complex (Alloderm®) is immunologically inert. It prepares the wound bed for grafting allowing improved cultured allograft ‘take’ and provides an intact basement membrane. A nonliving extracellular matrix of collagen and chondroitin-6-sulfate with silicone backing (Integra®) serves to generate neodermis.A collagen and glycosaminoglycan dermal matrix inoculated with autologous fibroblasts and keratinocytes has been investigated but is not commercially available. It requires 3 to 4 weeks for cultivation. Dermagraft® consists of living allogeneic dermal fibroblasts grown on degradable scaffold. It has good resistance to tearing. An extracellular matrix generated by allogeneic human dermal fibroblasts (TransCyte™) serves as a matrix for neodermis generation.Apligraf® is a living allogeneic bilayered construct containing keratinocytes, fibroblasts and bovine type I collagen. It can be used on an outpatient basis and avoids the need for a donor site wound. Another living skin equivalent, composite cultured skin (OrCel™), consists of allogeneic fibroblasts and keratinocytes seeded on opposite sides of bilayered matrix of bovine collagen. There are limited clinical data available for this product, but large clinical trials are ongoing. Limited data are also available for 2 types of dressing material derived from pigs: porcine small intestinal submucosa acellular collagen matrix (Oasis™) and an acellular xenogeneic collagen matrix (E-Z-Derm™). Both products have a long shelf life.Other novel skin substitutes are being investigated. The potential risks and benefits of using tissue-engineered skin need to be further evaluated in clinical trials but it is obvious that they offer a new option for the treatment of wounds.

Dermal fibroblasts from venous ulcers are unresponsive to the action of transforming growth factor-β 11

Failure to reepithelialize is the major clinical problem in venous ulcers. It is not clear whether the problem resides with keratinocytes or with inadequate and improper formation of extracellular matrix. In this study, we characterized the biosynthetic activity and response to transforming growth factor-beta 1 (TGF-beta) of dermal fibroblast cultures isolated from biopsies of venous ulcers and from normal thigh skin (controls) of seven patients. We found that baseline 3H-proline incorporation was similar in fibroblasts from venous ulcers and control skin (p= 0.1716). No difference was detected by ELISA between ulcer and control fibroblasts in the synthesis of total TGF-beta (p = 0.2309), and the mRNA levels for alpha 1(I) procollagen and TGF-beta were comparable in both groups. However, TGF-beta (0.1-5 ng/ml) enhanced collagen protein synthesis by more than 60% and in a dose-dependent manner (r = 0.997) in control fibroblast cultures, while failing to stimulate collagen production by venous ulcer fibroblasts (p = 0.0001). This unresponsiveness to TGF-beta was associated with up to a fourfold decrease in TGF-beta Type II receptors. We conclude that fibroblasts from the edge of non-healing venous ulcers are unresponsive to the action of TGF-beta, and that this blunted response may cause faulty deposition of the extracellular matrix needed for reepithelialization and wound healing.

The safety and efficacy of a proteolytic ointment in the treatment of chronic ulcers of the lower extremity.

BACKGROUND Elase is a widely used ointment consisting of a combination of 2 proteolytic enzymes, fibrinolysin and desoxyribonuclease (DNAse). It is said to promote debridement of necrotic and purulent debris from skin ulcers. OBJECTIVE Our purpose was to assess the efficacy and safety of this ointment and its components in the treatment of chronic ulcers of the lower extremity. METHODS This was a double-blind, randomized, prospective study of 84 patients with leg ulcers exhibiting necrotic and purulent debris, who were treated for 21 days with twice-daily applications of the ointment, fibrinolysin, DNAse, or who received the ointment vehicle (placebo). We assessed 6 efficacy features: ulcer size, purulent exudate, necrotic tissue, erythema, pain, and overall condition of the lesion at days 8, 15, and 21 after initiation of treatment. We also assessed the frequency of adverse effects. RESULTS All treatments produced some improvement in the efficacy parameters and overall condition of the ulcers by week 3, but no statistically significant difference was found when compared with placebo. No serious adverse effects were noted. A later retrospective reanalysis of the data found a statistically significant reduction of purulent exudate only at days 3 and 7 of treatment in the group treated with the complete ointment, but not in the other features. CONCLUSION The proteolytic ointment provides no long-term clinical benefit in reducing purulent exudate, pain, erythema, necrotic tissue, or overall condition of chronic leg ulcers when compared with either of its two components or placebo.

Un‐cross‐linked fibrin substrates inhibit keratinocyte spreading and replication: Correction with fibronectin and factor XIII cross‐linking

Wound repair is characterized by the presence of a fibrin‐rich matrix, but the effect of fibrin on re‐epithelialization remains unclear. In this study, we determined the effects of different fibrin matrices on cultured human neonatal keratinocytes. Using purified fibrinogen and fibrin gels generated by the enzymatic action of thrombin, batroxobin (it leads to retention of fibrinopeptide B), or Agkistrodon contortrix thrombin‐like enzyme (ACTE; it leads to retention of fibrinopeptide A), we determined the effect of each of these matrices on keratinocyte morphology, attachment, spreading, and replication as compared to tissue culture plastic. Morphologically, keratinocytes seeded on fibrin surfaces were more rounded and formed three‐dimensional structures. Specific cell attachment, as measured at either 37°C or 4°C, was not altered on the different fibrin substrates (P > .05) but was increased on fibrinogen and factor XIII cross‐linked fibrin (P < .01). However, keratinocytes seeded on fibrin, regardless of the presence or absence of fibrinopeptides A or B, showed a marked decrease (up to 71%) in cell numbers by days 5 (P = .0357) and 10 (P = .0114). Keratinocyte spreading was decreased by 78.8% (P = .0006), 80.3% (P = .0001), and 89.2% (P = .0001) on thrombin‐, batroxobin‐, and ACTE‐generated fibrin, respectively, but not on fibrinogen‐coated dishes. However, either the addition of fibronectin or cross‐linking of fibrin with factor XIII allowed full keratinocyte spreading to occur (P = .0002 and P = .0013, respectively). We conclude that fibrin inhibits keratinocyte spreading in the absence of other matrix or plasma proteins or cross‐linking by factor XIII. J. Cell. Physiol. 174:58–65, 1998.

Tissue‐Engineered Skin in the Healing of Wound Stumps from Limb Amputations Secondary to Purpura Fulminans

Abstract: Currently wound treatment options of amputation stumps due to purpura fulminans include healing by secondary intention from wound debridement, split‐thickness skin grafting, tissue and muscle flaps, plantar skin free transfer, skin expansion, artificial skin, and hyperbaric oxygen therapy. We saw a 6‐month‐old girl with purpura fulminans as a complication of meningococcemia. She developed necrosis of the distal extremities resulting in bilateral amputation of the lower limbs. Shortly thereafter the leg stumps also became necrosed and she underwent unsuccessful split‐thickness grafts of lower limb ulcers. The patients difficult‐to‐heal wounds made her an excellent candidate for treatment with tissue‐engineered skin. At 10 months of age, this was applied to her previously nonhealing wounds. The tissue‐engineered skin induced rapid healing of the patients chronic amputation stump ulcers and provided her with substantial pain relief. In conclusion, tissue‐engineered skin appears to be a potential beneficial treatment for chronic wounds in children with nonhealing amputation stumps.

Durometer Measurements of Skin Induration in Venous Disease

BACKGROUND The degree of skin induration (lipodermatosclerosis) around venous ulcers has prognostic significance; however, objective measurements are needed to assess the induration. The durometer, an engineering instrument used to measure the hardness of metals and plastic, has recently been adapted to assess skin induration. OBJECTIVE The purpose of this study was to measure skin induration and its relationship to skin ulceration by the use of a durometer, and to determine the influence of edema, if any, on durometer measurements. METHODS The degree of skin induration on the medial leg was determined in six sequential, nonselected patients with lipodermatosclerosis and leg ulcers, and in five normal volunteers by using a blinded observers clinical score (0 = normal to 3 = maximal induration) and a hand‐held Type 0 durometer. In addition, durometer readings in 14 patients with edema and eight control subjects were taken on the tibia, the dorsum of the foot, and behind the ankle. RESULTS Durometer readings in patients with leg ulcers and lipodermatosclerosis diminished as one measured from the superior edge of the ulcer to the knee (r = 0.925). The higher the clinical skin score the higher were the durometer readings (P = 0.0062). The presence of edema did not influence durometer measurements. CONCLUSION The durometer is an effective and reliable instrument for measuring the degree of skin induration in venous ulceration and its readings are not affected by edema. Ulcers occur in skin most affected by lipodermatosclerosis.

USE OF SKIN SUBSTITUTES IN DERMATOLOGY

The use of skin substitutes to treat acute and chronic wounds should continue to increase as newer products are developed. The goals for the future are to eliminate the need for autografting, eliminate the risk for transmission of disease, improve the shelf life and simplify storage requirements, improve cosmetic outcomes, and reduce cost. A number of researchers are also looking to incorporate gene therapy into skin substitutes.

Un-cross-linked fibrin substrates inhibit keratinocyte spreading and replication: Correction with fibronectin and factor XIII cross-linking

Wound repair is characterized by the presence of a fibrin-rich matrix, but the effect of fibrin on re-epithelialization remains unclear. In this study, we determined the effects of different fibrin matrices on cultured human neonatal keratinocytes. Using purified fibrinogen and fibrin gels generated by the enzymatic action of thrombin, batroxobin (it leads to retention of fibrinopeptide B), or Agkistrodon contortrix thrombin-like enzyme (ACTE; it leads to retention of fibrinopeptide A), we determined the effect of each of these matrices on keratinocyte morphology, attachment, spreading, and replication as compared to tissue culture plastic. Morphologically, keratinocytes seeded on fibrin surfaces were more rounded and formed three-dimensional structures. Specific cell attachment, as measured at either 37 degrees C or 4 degrees C, was not altered on the different fibrin substrates (P > .05) but was increased on fibrinogen and factor XIII cross-linked fibrin (P < .01). However, keratinocytes seeded on fibrin, regardless of the presence or absence of fibrinopeptides A or B, showed a marked decrease (up to 71%) in cell numbers by days 5 (P = .0357) and 10 (P = .0114). Keratinocyte spreading was decreased by 78.8% (P = .0006), 80.3% (P = .0001), and 89.2% (P = .0001) on thrombin-, batroxobin-, and ACTE-generated fibrin, respectively, but not on fibrinogen-coated dishes. However, either the addition of fibronectin or cross-linking of fibrin with factor XIII allowed full keratinocyte spreading to occur (P = .0002 and P = .0013, respectively). We conclude that fibrin inhibits keratinocyte spreading in the absence of other matrix or plasma proteins or cross-linking by factor XIII.