Scientific Forum. Revascularization of Acellular Human Dermis (Alloderm) in Subcutaneous Implantation

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1 Revascularization of Acellular Human Dermis (Alloderm) in Subcutaneous Implantation Barry L. Eppley, MD Background: Although autogenous soft tissue graft materials, such as fat, fascia, and dermis, are commonly used for subdermal and subcutaneous augmentation, their inherent disadvantages continue to inspire a search for an alternative biomaterial. Objective: This study investigated revascularization after subcutaneous implantation of Alloderm, an allograft derived from acellular human dermis, in a rabbit model. Methods: Alloderm was implanted under the skin in the ears of 8 adult female white rabbits. Harvesting took place after 3, 7, 14, and 28 days. Serial sections were stained and examined under light microscopy to assess vascular ingrowth, as determined by the number of vessels and their location of entry. Results: Postoperatively, minimal vessel ingrowth extending from all sides of the implantation site was visible in the grafts at 3 days. By 7 days, scattered vessels could be seen within the collagen bundles of the grafts. Further vessel ingrowth with nearly complete through-and-through vascularity was evident at 14 days. By 28 days, single-layer grafts were structurally intact, with identifiable vessels throughout the collagen bundles and no volume loss. Conclusions: Acellular human dermis is capable of complete revascularization within weeks after implantation. The orientation of the basement membrane has no impact on vascular ingrowth. The extent of revascularization in more complex grafts awaits further study. Autogenous soft tissue materials, such as fat, fascia, and dermis, have been used for subdermal and subcutaneous augmentation for decades. Graft persistence, mostly for facial augmentation, has been shown to vary depending on implantation techniques and handling of the biomaterials. Despite their biological advantages, all of these autogenous grafts also have inherent disadvantages, which include the need for graft harvesting, the potential for creation of donor scars, and the unpredictability of long-term volume retention. The search continues for a soft tissue graft, such as an offthe-shelf material, that does not require a concomitant donor site but that would biologically integrate into the implantation site without the risks associated with any known synthetic implant. Allograft skin, which is routinely used in burn reconstruction after primary excision, will From the Division of Plastic Surgery, Indiana University School of Medicine, Indianapolis, IN. Accepted for publication June 12, Reprint requests: Barry L. Eppley, MD, Division of Plastic Surgery, Indiana University School of Medicine, 702 Barnhill Drive, #3540, Indianapolis, IN Copyright 2000 by The American Society for Aesthetic Plastic Surgery, Inc /2000/$ /1/ doi: /maj A ESTHETIC S URGERY J OURNAL ~ JULY/AUGUST

2 Figure 1. Toluidine blue stain of a single-layer Alloderm graft with basement membrane down at the third postoperative day (original magnification 4). take as a temporary wound dressing but is ultimately rejected because of foreign major histocompatibility antigens present in the cells of the epidermis and dermis. This is unfortunate, because the collagen matrix of the allograft dermis would be an excellent scaffold for fibrovascular ingrowth and eventual native collagen replacement. Removal of the outer layer of epidermis and the remaining cells of the dermis is one potential approach to the creation of a compact collagenous dermal graft that could be implanted without the risks of foreign body rejection. Alloderm (LifeCell Corporation, Woodlands, TX) has been developed as a dermal/soft tissue replacement in accordance with this approach. It is an allograft that is derived from human cadaveric skin in which the epidermis and all of the cells of the dermis have been removed through a proprietary freeze-dried process. This results in a biomaterial containing components of types IV and VII collagen, elastin, and laminin, which are undamaged within the residual dermal matrix. 1,2 During the past 5 years, this graft material has been extensively evaluated in primary and secondary burn reconstruction (with both autologous split-thickness grafts and cultured epithelial autografts), numerous facial soft tissue augmentation procedures, and intraoral mucosal and gingival replacements. 2-8 Results in selected cases suggest that the graft material is rapidly revascularized and subsequently repopulated by native fibroblasts. However, experimental and clinical biopsies with histologic assessment are currently available only from burn reconstruction results and other topical onlay procedures. 2-4 There are no reports that describe A B Figure 2. Toluidine blue stain of a single-layer Alloderm graft with basement membrane down at the seventh postoperative day. Extensive vessel ingrowth has occurred from all sides of the graft (original magnification 4). A, Graft against the perichondrial surface. B, Graft against the upper dermal surface of the surrounding skin. what initially happens to the graft in a subcutaneous tissue pocket in particular, whether revascularization of the material actually occurs. This information is clinically relevant, because it is important to know whether this type of graft material actually integrates biologically or behaves more like nonvascularized synthetic materials or avascular fibrotic scar tissue. Materials and Methods Implanted biomaterial Prepackaged Alloderm from human donors (size, 1 2 cm; thickness, 30/1000 to 65/1000 in) was obtained from the manufacturer in a dehydrated state. Its origin was human donors. Once the package was opened for use, the material was hydrated in room-temperature normal saline solution for 10 minutes, after which the paper backings to which it was attached were removed. The 292 A ESTHETIC S URGERY J OURNAL ~ JULY/AUGUST 2000 Volume 20, Number 4

3 Figure 3. Toluidine blue stain of a single-layer Alloderm graft with basement membrane up at the 14th postoperative day. Vessel ingrowth has progressed toward the center of the grafts (original magnification 4). material could then be cut and fashioned by scissors to a 5 10-mm flat shape suitable for implantation. Experimental model Female New Zealand white rabbits were selected as the experimental model because the size of their ears makes for a large surface area for implantation in which no other tissue exists between the dermis of the ear skin and the underlying perichondrium. This allows easy postoperative identification of the biomaterial on histologic examination. Given the xenogeneic differences between the human biomaterial and the animal model, however, significant inflammatory infiltrate would be expected to develop eventually. Therefore the study duration was limited to the first postoperative month. Surgical technique Eight rabbits were anesthetized by intramuscular sedation and the ears were shaved and sterilely prepared. Five-mm incisions were made on the dorsal surface of the ears, and a subcutaneous pocket was carefully created with scissors. Two pockets were made in each ear, a total of 4 implantation sites being created in each animal. Alloderm (5 10 mm) was inserted into all 4 of the soft tissue pockets created in each animal, the basement membrane being up in the right ears and down in the left ears. Figure 4. Toluidine blue stain of a single-layer Alloderm graft with basement membrane up at the 28th postoperative day shows maturing vessels in large numbers against the perichondrial surface (original magnification 6.3). The incisions were then closed with interrupted 5-0 black nylon. Intramuscular antibiotics were administered, and the animals were returned to their cages for recovery. Postoperative assessment Harvests were performed at 3, 7, 14, and 28 days after surgery after intramuscular sedation and intracardiac euthanasia. Immediately before the animal was sacrificed, the main dorsal artery in the ear was cannulated with a 23-gauge angiocath and injected with orange-colored silicone microangiographic material until the venous outflow had a similar color. The dorsal ear vein was then ligated and the material allowed to set for 5 minutes, after which the animal was sacrificed. At harvest, the implantation sites, including both sides of the ear skin and cartilage, were removed, placed in 10% formalin for 48 hours, sequentially dehydrated, and embedded in plastic. Serial sections were then cut at 50 µm and colored with a trichrome stain. Examinations were performed under routine light microscopy to assess vascular ingrowth, as measured by the number of vessels and their points of entry into the grafts. Results Wound healing All animals survived the implantation without any infection, wound dehiscence, or complications related to the implantation sites. No signs of inflammation were seen over the implant sites by study completion. 3 days postoperatively Minimal vessel ingrowth was evident at this early time. A very few capillaries could be discerned within the grafts, Revascularization of Acellular Human Dermis (Alloderm) in Subcutaneous Implantation A ESTHETIC S URGERY J OURNAL ~ JULY/AUGUST

4 Discussion Figure 5. Toluidine blue stain of a single-layer Alloderm graft with basement membrane down at the 28th postoperative day shows extensive revascularization of the most central regions (original magnification 6.3). just adjacent to the perichondrium and against the overlying soft tissue. (Figure 1). The vast majority of the grafts remained avascular. 7 days postoperatively Scattered vessels could be seen within the collagen bundles of the grafts that originated from either the underlying perichondrial surface (Figure 2, A) or the overlying soft tissue envelope (Figure 2, B). Penetration of the vessels was limited to the immediate area within 1 mm of the grafts; the more central vessel regions remained avascular. No differences in the presence or obvious number of vessels that penetrated the grafts could be seen, regardless of orientation of the basement membrane. 14 days postoperatively Further vessel ingrowth in the grafts, with progression toward their most central regions, could be seen (Figure 3). Most of the grafts had nearly complete vascularity weaving between the collagen bundles. The source of the vessels appeared to be equally distributed between the overlying soft tissue and the perichondrium. No evidence of inflammatory cellular infiltrate was seen. 28 days postoperatively The single-layer grafts were structurally intact, did not appear to have lost any volume, and had identifiable vessels throughout the differently oriented collagen bundles. The vessels were mature, with clearly identifiable lumens and thickening of the vessel walls (Figure 4). The most central regions of all of the grafts examined showed extensive numbers of vessels (Figure 5). Fibrovascular ingrowth into an implanted biomaterial suggests incorporation and potential replacement with autogenous collagen. Synthetic biomaterials have only a limited capability for fibrovascular encapsulation unless the physical structure of the implant is porous, in which case some intramaterial vascularity may be found. Nevertheless, the synthetic material persists despite the soft tissue response, and it is never replaced by autogenous tissue in the implant. Only collagen and some biodegradable materials have the potential to be revascularized, repopulated by fibroblasts, and ultimately replaced by autogenous collagen. This is the ultimate goal for soft tissue reconstructive materials. Historically, collagen for human implantation was derived from xenogeneic sources, but this was never truly incorporated, being either resorbed quiescently in injectable form, ultimately rejected as a result of foreign body-type reactions, or developed into a stiff avascular geometry of fibrosis. Acellular human dermis (ie, Alloderm) is the first commercially available human collagen material in sheet form that offers the real possibility of a collagen scaffold that can reliably be replaced by native collagen. Extensive experimental and clinical work in burn reconstruction attests to its ability to be incorporated; Alloderm permits a skin graft to be either synchronously or metachronously applied with predictable postoperative take rates. 3-6 This is undoubtedly a consequence of revascularization after implantation; numerous studies indicate that acellular human dermis is completely penetrated by blood vessels (eg, nonmeshed dermal graft application) by the second postoperative week. 9 Alloderm is currently enjoying increasing popularity as a soft tissue augmentation and replacement material, primarily in facial and oral applications. 2,7,8,10 However, it is not known whether complete revascularization of the material occurs when it is placed in a subcutaneous pocket. In addition, experimental and clinical work in burn surgery indicates that it is important to orient the graft material in such a way that the original basement membrane abuts the underlying wound bed. Whether this graft orientation has any implications for subdermal placement is likewise unknown. Conclusion This study demonstrates that single-layer acellular human dermis revascularized fairly quickly; revascularization 294 A ESTHETIC S URGERY J OURNAL ~ JULY/AUGUST 2000 Volume 20, Number 4

5 was essentially complete by the second postoperative week. The orientation of the graft did not change the vessel ingrowth pattern, which occurred equally from all sides. The vessels penetrated the collagen matrix and the basement membrane and did not appear to be preferential for vessel ingrowth. The complete revascularization of Alloderm suggests that it may undergo cell repopulation and eventually be replaced by native collagen. During this replacement process, it is important to ascertain whether any significant volume loss occurs. This is an extremely important clinical question in many subcutaneous soft tissue applications, particularly those involving the face, and the answer is currently unknown. Ongoing animal and postoperative human studies are under way to help clarify these issues and to determine what role Alloderm may have in soft tissue cosmetic augmentation and reconstruction. 10 References 1. Livesey SA, Herndon DN, Hollyoak MA, Atkinson YH, Nag A. Transplanted acellular allograft dermal matrix. Transplantation 1995;60: Achauer BM, VanderKam VM, Celikoz B, Jaconson DG. Augmentation of facial soft tissue defects with Alloderm dermal graft. Ann Plast Surg 1998;41: Wainwright DJ, Madden M, Luterman A, et al. Clinical evaluation of an acellular allograft dermal matrix in full-thickness burns. J Burn Care Rehabil 1996;17: Lattari V, Jones LM, Varcelotti JR, Latenser BA, Sherman HF, Barrette RR. The use of a permanent dermal allograft in full-thickness burns of the hand and foot. J Burn Care Rehabil 1997;18: Rennekampff HO, Kiessig V, Griffey S, Greenleaf G, Hansbrough JF. Acellular human dermis promotes cultured keratinocyte engraftment. J Burn Care Rehabil 1997;18: Sheridan RL, Choucair R, Donelan M, Lydon M, Petras L, Tompkins R. Acellular allodermis in burn surgery: 1 year results of a pilot trial. J Burn Care Rehabil 1998;19: Romo T, Sclafani AP, Falk AN, Toffel PH. A graduated approach to the repair of nasal septal perforations. Plast Reconstr Surg 1999;103: Callan D, Silverstein LH. Use of acellular dermal matrix for increasing keratinized tissue around teeth and implants. Pract Periodontics Aesthetic Dent 1998;10: Reagan BJ, Madden MR, Huo J, Mathwich M, Staiano-Coico L. Analysis of cellular and decellular allogeneic dermal grafts for the treatment of full-thickness wounds in a porcine model. J Trauma Injury Infect Crit Care 1997;43: Rohrich RJ, Reagan BJ, Adams WP Jr, Kenkel JM, Beran SJ. Early results of vermilion lip augmentation using acellular allogeneic dermis: an adjunct in facial rejuvenation. Plast Reconstr Surg 2000;105: Full-text access to Aesthetic Surgery Journal Online is now available to all print subscribers. To activate your individual online subscription, please point your browser to follow the prompts to activate online access here, and follow the instructions. To activate your account, you will need your subscriber account number, which you can find on your mailing label (note: your subscriber account number may contain 6 to 10 digits). See the example below in which the subscriber account number has been circled: This is your subscription account number Sample mailing label * * * * * * * * * * * * * * *3-DIGIT 001 SJ P1 FEB00 J070 C: U 05/00 Q:1 J.H. DOE, MD 531 MAIN ST CENTER CITY, NY Personal subscriptions to Aesthetic Surgery Journal Online are for individual use only and may not be transferred. Use of Aesthetic Surgery Journal Online is subject to agreement to the terms and conditions indicated online. Revascularization of Acellular Human Dermis (Alloderm) in Subcutaneous Implantation A ESTHETIC S URGERY J OURNAL ~ JULY/AUGUST