Sunday, April 1, 2012

ADM treatment of venous stasis ulcers

April 1, 2012

Here is a preliminary study describing the results of a new method of treating a venous stasis leg ulcers.  This report shows the outcome in one patient whose ulcer was implanted with acellular dermal matrix after which the treated leg was dressed with pressure dressings for a period of 92 days.  The results were quite encouraging.  I wish we had been able to study more patients.

ADM treatment of venous stasis ulcers




Use of Acellular Dermal Matrix in the Treatment of Venous Stasis Ulcers:

A Preliminary Report

 by

Mark D. Hoffman, MD, Michael D. Tharp, MD, Robert J. Walter, PhD 1

 
Department of Dermatology, Rush University Medical Center, Chicago, IL 60612

1  Department of Surgery, Cook County Hospital, Chicago, IL  60612

 
Address all correspondence to:
Robert J. Walter, Ph.D.
Department of Surgery
Hektoen Institute for Medical Research
625 South Wood Street
Chicago, IL 60612
Telephone: (312) 633-7237
FAX: (312) 732-3102

Running Title: ADM Treating Venous Stasis Ulcers

Keywords: dermis, acellular matrix, venous stasis ulcer, decubitis ulcer, wound healing, skin wounds

Abbreviations:
ADM, acellular dermal matrix


ABSTRACT

     Chronic leg ulcers are common, but often intractable wounds which impose significant burdens on patients and society alike.  These wounds negatively impact the quality of life circumscribing the range of activities and frequently causing discomfort   Standard compression therapy has only limited effectiveness, but recently success has been reported using skin substitutes in the treatment of leg ulcers.  Skin substitutes are products composed of biologically derived materials such as collagen, matrix proteins, complex sugars, and occasionally living cells which are applied to wounds as dressings.  The relative contributions of the matrix materials and cells comprising the skin substitute remain, however, undefined.  Our study will examine the efficacy of one type of skin substitute, acellular dermal matrix (ADM), in treating leg ulcers.  ADM is a native collagen meshwork derived from human skin that has been treated to remove all cells and cell debris.  As with other skin substitutes, it is applied as a dressing, and serves as a cushion and replacement for skin dermis lost from the wound.  Comparisons will be made with conventional compression stocking treatment.  Additionally, some subsets of patients will receive implants of living cells derived from a small biopsy of their own skin either used alone or in combination with ADM.  We expect this investigation to increase the understanding of how skin substitutes function therapeutically and to provide an improved method for treating leg ulcers.  Here we report the results of treatment with ADM of a venous stasis leg ulcer in one patient.


INTRODUCTION

     Ulcers of the lower extremities are extremely common in the United States (>600,000 cases per year) and account for significant morbidity and loss of productivity (1) with more than 2 million workdays lost in the US annually due to these persistent wounds.  In addition, these wounds frequently have a deleterious emotional impact on affected patients.  The majority of chronic leg ulcers result from venous disease leading to ambulatory venous hypertension.  These wounds, referred to as "venous ulcers", are generally about 2-5 centimeters in diameter and are usually located over the medial malleolus.  Improper venous functioning may occur as a result of damage to the veins themselves, as a sequela of deep venous thrombosis or superficial thrombophlebitis, or may reflect dysfunction of the surrounding neuromuscular tissues that normally propel blood through these vascular conduits.  Conventional therapy is often arduous and time-consuming for patients, and healing frequently progresses slowly.  

     The most common therapy utilized in the treatment of venous ulcers involves compression or support stockings, but healing is achieved in only approximately 50% of patients despite 4-6 months of such treatment (2,3).  Therapeutic approaches involving medical (pentoxifylline) (4), or surgical (vein repair) (5) strategies have provided mixed results and small split-thickness skin grafts or autologous plugs have been used with some success (6,7).  More recently, certain artificial skin products (Dermagraft, Apligraf) have been applied to ulcers, including those related to venous insufficiency, to augment their healing, with generally favorable results (2,8,9).  These skin substitutes are composed of biologically derived materials, which may include collagen gels or living cells such as keratinocytes or fibroblasts.  They are considered biological dressings and do not become permanently incorporated into the wound.  These materials are thought to duplicate properties of the skin vital to healing including the production and release of growth factors and cytokines.

     The factors pertinent to the healing of venous stasis ulcers are manifold, and may include the influences of  structural proteins and other macromolecules, cellular elements, or the growth factors they elaborate.  Recent studies have demonstrated accelerated ulcer healing with the use of artificial skin products as noted above.  However, the mechanism of this effect is not at all clear, and may involve the infrastructural and space-occupying properties of the matrices used rather than the cellular components or growth factors  purported to be acting.  This question has not been addressed previously but will be examined more closely in the study proposed here.

     In addition, Dermagraft (Advanced Tissue Sciences) and Apligraft (Novartis) contain allogeneic epidermal and dermal cellular components.  These foreign materials evoke an immune reaction in the wound and the effect of this host inflammatory response on wound healing is unclear, but such inflammation may act to undermine healing.  Our study will examine the impact of autologous epidermal and dermal cellular populations (from "mini-implants") on the healing response when introduced into venous ulcers in various contexts.  In addition, ADM appears to be virtually non-immunogenic when it is implanted within the same species as that from which it was derived so it does not induce an inflammatory response in the wound. (10,11).

     Our objective in this study was to preliminarily determine the efficacy of acellular human dermal matrix (ADM) and autologous skin "mini-implants" in the treatment of venous stasis ulcers of the leg.  We intended for this investigation to increase our understanding of the therapeutic functions of skin substitutes and to provide an improved method for treating leg ulcers.  


METHODS AND EXPERIMENTAL DESIGN

MATERIALS
     Acellular Dermal Matrix (ADM) is a thin sheet of human tissue, approximately 1/100 of an inch thick, which is composed mainly of native collagen and other extracellular matrix components such as laminin and keratan sulfate (12).  ADM is treated with enzymes and detergent to remove cells and cell debris.  ADM was derived from donors unrelated to study participants.  We used ADM as a biological cushion to permanently replace lost dermis in venous stasis ulcers.  Cadaver skin was obtained from the Skin Bank at the University of Cincinnati Shriner’s Burn Center which is certified by the American Association of Tissue Banking.  This tissue has been thoroughly screened for human fungal, bacterial, and viral pathogens and has been certified to be negative for these pathogens.  ADM was prepared by treating 0.012" thick pieces of cadaver skin with Dispase (2.5 U/ml, 24 h @ 4°C) followed by 0.5% Triton X-100 detergent (24 h @ 25°C) as described previously (10).  We have reported the composition of such ADM analyzed by immunocytochemistry in conjunction with light microscopy (12).  We have shown greatly improved healing of experimental full-thickness wounds utilizing ADM with overlying autologous split-thickness skin grafts in rats (10,11).  In addition, we have used ADM successfully in 5 human burn victims, with no adverse events attributable to this material and with evidence of improved healing (13).   A commercially available cadaveric acellular dermal matrix (Alloderm) has also been employed in burn patients and is described in the literature (14,15).  Alloderm, however, has not been studied in venous ulcers.  Its preparation and characteristics are different from those of ADM, thus allaying concerns regarding patent or proprietary constraints.

     As discussed above, small split-thickness skin grafts or autologous plugs have also been used with some success in the treatment of venous ulcers (6,7).  We have used a variation on this using "mini-implants", tissue from a finely-minced autologous 4 mm skin punch biopsy, placed either directly onto the surface of the ulcer or on top of the ADM in the ulcer and in both cases held in place by the usual wound dressings and a compression stocking.  Keratinocytes and fibroblasts in the resulting "mini-implants" should readily migrate into and onto the wound surface or ADM and rapidly populate these regions.  These cells may then serve as a source of a variety of biological factors that can augment wound healing.

STUDY DESIGN
     The study is a prospective, controlled, parallel-group, comparative trial.  There will be 4 groups of 20 patients studied, with patients receiving:
     1)  Standard compression therapy only,
     2)  Autologous "mini-implants" combined with standard compression therapy,
     3)  ADM placed onto ulcerated wounds combined with standard compression therapy,
     4)  ADM and autologous "mini-implants" combined with standard compression therapy.
     For analysis of safety and efficacy, the end points of this study will be prospectively set at 6 months, mirroring the parameters of the study by Falanga et al. (2)  Patients will be entered into the study after informed consent is obtained.  Patients who qualify for participation in the study will be assigned to one of the above four treatment groups according to computer generated randomization schedules.  The present preliminary report shows results from one patient.

STUDY POPULATION
     Eventually, there will be a total of 80 patients enrolled in this study and the clinical aspects will be conducted entirely at Rush.  Eligible patients aged 18 to 85 years will be offered the opportunity to enroll in this study, and be randomly assigned in the outpatient setting to a treatment group.  Leg ulcers (3 to 6 cm in diameter) will be due to venous insufficiency as determined by the criteria discussed in Section G (below) and inclusion and exclusion criteria set as described therein.

TREATMENT PROTOCOL AND FOLLOW-UP
     The ulcer sites of control patients will be dressed with a standard compression dressing only.  Such a dressing will be comprised of a non-adherent primary dressing (Tegapore, 3M Health Care, St. Paul MN), gauze bolster, zinc oxide impregnated paste bandage (Unna boot), and a self-adherent elastic wrap (Coban, 3M Health Care, St. Paul, MN). In the standard compression plus mini-implant group, patients will have a 4 mm skin punch biopsy removed from the buttock or thigh.  The tissue will be finely minced, and placed upon the wound bed, after which the standard compression dressing described above will be applied.  Patients assigned to the ADM treatment group will have ADM placed directly on the ulcer, with the standard compression dressing applied over the ADM to immobilize it and keep it in place.  Patients assigned to the ADM plus mini-implant group will have ADM  first  placed upon the wound, after which minced tissue derived from the 4 mm skin punch biopsy will be placed upon the ADM, over which the standard compression dressing will then be applied.

     When ADM is to be used, it will applied only once, at the start of treatment. The Coban elastic wrap, when in place, will extend from the metatarsal heads to the infrapatellar notch and will be secured at midstretch tension with at least 50% overlap.  Compression therapy will be reapplied weekly for the first 4 weeks.  Dressings will need to remain in place and kept dry until changed during follow-up visits. After 4 weeks or on complete healing (defined as full epithelialization of the wound and no drainage from the site), patients will use graded elastic stockings as a means of compression (Fast Fit, Beiersdorf-Jobst, Charlotte, NC) for the remainder of the study.  These graded elastic stockings will be removed during bathing and while sleeping.

     The overall design of this study is such as to allow ready comparison with the data presented by Falanga et al. who used multiple applications of Apligraf to treat venous ulcers of the leg (2).

STUDY EVALUATIONS
     Demographic characteristics of each treatment group will be tabulated and compared.  Parameters will include gender, race, age, ulcer area at baseline, and ulcer duration.  All patients will be evaluated weekly for the first 4 weeks; thereafter, examinations will be performed at weeks 12 and 24.  At each visit, ulcer status will be recorded by photographs and wound tracings.  Ulcer size will be determined by computerized planimetry of surface tracings made with plastic films.  The primary efficacy end points will include incidence of complete healing by 6 months and the time required for complete healing to occur.  Other efficacy end points to be evaluated will include incidence and time to 50% and 75% wound closure by 6 months.  Safety will be evaluated by several parameters, including spontaneous reports of adverse events at each visit, and direct questioning of patients regarding their experiences with the treatment.

ANALYSIS OF DATA
     Statistical calculations will employ SAS software (SAS Inc, Cary, NC). Comparisons between treatment groups for demographics and baseline ulcer evaluations will use Fisher’s exact test or X2 analysis.  Analysis of frequency of wound closure will be determined by Fisher’s exact test (2-tailed), and time to wound closure computed using a survival analysis by the Kaplan-Meier life-table method.  Statistical significance of the differences in time to wound closure by treatments will be tested using the log-rank test.  The individual and combined effects of pre-existing wound size and duration will be subjected to Cox proportional hazards regression analysis.

RESULTS

     One patient was studied here using the protocol described for group 3, i.e., ADM implantation with mini-implants of the patients own dermis.  The appearance of the leg ulcer at each visit is shown in the photographs below (Figure 1).  The wound was implanted with ADM and mini-implants and the patient’s progress followed for 13 weeks.  The wound,which was initially dry, assumed a moister appearance after implantation.  The wound margins began to close as time progressed and the wound size decreased.  In addition, the wound appeared to re-epithelialize from the wound margins to a large extent.  Epithelialization was not complete by the end of the observation period however.   

     Wound area was calculated from these photos and is shown in Figure 2.  The wound area decreased by 72% by the end of the 13 week followup period.


DISCUSSION

                 Chronic leg ulcers are common, but often intractable wounds which are usually treated with compression therapy, but recent success has been reported using skin substitutes.  The healing seen over the 13 week period studied here suggests that ADM implantation may be an effective method of treating these long-standing, hard-to-heal venous stasis ulcers.  The followup period n this patient needs to be extended for at least 6 months to see if the improvement is lasting and to determine if complete healing occurs.  Of course, additional patients must be treated and the remaining study groups must be examined to make any firm conclusions regarding possibilities for treatment of chronic venous stasis ulcers using ADM.


BIBLIOGRAPHY

  1.  Phillips TJ, et al. A study of the impact of leg ulcers on quality of life:  Financial, social, and psychologic. J Amer Acad Dermatol. 1994;31:49-153.
  2.  Falanga V, Margolis D, Alvarez O, Auletta M, Maggiacomo F, Altman M et al. Rapid healing of venous ulcers and lack of clinical rejection with an allogeneic cultured human skin equivalent. Arch Dermatol. 1998;134:293-300.
  3.  Falanga V, Sabolinski M. A bilayered living skin construct (Apligraf) accelerates complete closure of hard to heal venous ulcers. Wound Repair Regen. 1999;7:201-207.
  4.  Samlaska C, et al. Pentoxifylline. J Amer Acad Dermatol. 1994;30:603-621.
  5.  Darke S, et al. Venos ulceration and saphenous ligation. Eur J Vasc Surg. 1992;6:4-9.
  6.  Ceilley R, et al. Pinch grafting for chronic ulcers on lower extremities. J Dermatol Surg Oncol. 1977;3:303-309.
  7.  Kirsner RS, Falanga V, Kerdel FA, Eaglstein WH. Split-thicknesss grafting of leg ulcers. Dermatol Surg. 1995;21:701-703.
  8.  Gentzkow G, Totoritis M, Iwasaki S, Allman R, Page J, Alvarez O et al. Healing diabetic foot ulcers with a cultured human dermis-final data. Wound Repair Regen. 1997;5:A100-A100.
  9.  Gentzkow G, Pollak R, Kroeker R, Jensen J, Edington H, Totoritis M et al. Healing diabetic foot ulcers with Dermagraft, a cultured human dermis. Wound Repair Regen. 1997;5:A257-A257.
  10.  Takami Y, Matsuda T, Yoshitake M, Hanumadass M, Walter RJ. Dispase/detergent treated dermal matrix as a dermal substitute. Burns. 1996;22:182-190.
  11.  Walter RJ, Jennings LJ, Matsuda T, Reyes HM, Hanumadass M. Dispase/Triton treated acellular dermal matrix as a dermal substitute in rats. Curr Surg. 1997;54:371-374.
  12.  Walter RJ, Matsuda T, Reyes HM, Walter JM, Hanumadass M. Characterization of acellular dermal matrices (ADMs) prepared by two different methods. Burns. 1998;24:104-113.
  13.  Hanumadass M, Srivastava A, Botts J, Jennings LJ, Walter RJ. Dispase/ detergent treated allogenic acellular dermal matrix (ADM) as a dermal substitute in full-thickness burn wounds. Proc 10th Cong Intl Soc Burn Injur. 1998;7:37A-7A.
  14.  Wainwright DJ. Use of an acellular allograft dermal matrix (AlloDerm) in the management of full-thickness burns. Burns. 1995;21:243-248.
  15.  Livesey SA, Herndon DN, Hollyoak MA, Atkinson YH, Nag A. Transplanted acellular allograft dermal matrix.  Potential as a template for the reconstruction of viable dermis. Transplantation. 1995;60:1-9.
  16.  Sabolinski M, Alvarez O, Auletta M, Mulder G, Parenteau NL. Cultured skin as a 'smart material' for healing wounds:  Experience in venous ulcers. Biomaterials. 1996;17:311-320.



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