Tissue Response to Bovine Fetal Collagen Extracellular Matrix in Full ...

AJCP / Original Article

Tissue Response to Bovine Fetal Collagen Extracellular Matrix in Full-Thickness Skin Wounds James S. A. Neill, MD,1 and William C. Lineaweaver, MD2 From 1Ameripath, Flowood, Mississippi, and 2Joseph M. Still Burn and Reconstructive Center. Brandon, MS. Key Words: Dermal substitute; Histology; Dermatopathology; Wound healing; Bovine fetal collagen DOI: 10.1309/AJCPMF3B9XJAKXKM

ABSTRACT Objectives: To present the findings of the biological response and assimilation of bovine fetal collagen (BFC) as observed in biopsy specimens taken after implant. Methods: The biopsy specimens were from 9 patients with full-thickness skin wounds who received the BFC biomaterial in the first stage of a 2-stage reconstruction. Biopsy specimens were taken at the second stage of skin grafting from the wound margins. Results: The response to the BFC included neovascularization and infiltration of the collagen matrix with fibroblasts. The acellular matrix had the tinctural properties of devitalized tissue, which may be mistaken for coagulative necrosis if one is unaware of the biomaterial implant. Conclusions: The characteristics of BFC histology should be recognized by pathologists involved in patients treated for reconstruction and wound care.

In extensive burns and other full-thickness skin wounds, replacement of skin is a major clinical challenge. Myriad skin dermal substitutes have been used in the treatment of a variety of full-thickness skin wounds such as diabetic ulcers, venous stasis ulcers, burns, surgical wounds, and Mohs surgery for malignant tumors.1,2 Extracellular matrix biomaterials have been made from bovine, porcine, and equine sources and have origins from pericardium, small intestine submucosa, and dermis.3 Sometimes these devices are referred to as scaffolds. Tissue responses to such biomaterials have included the classic foreign body encapsulation, reabsorption, and adaption/ incorporation.4,5 Processing the biomaterial using techniques such as crosslinking and removal of lipids, cells, and glycosaminoglycans has improved and altered the host tissue response. The objective of this review is to report the host tissue response to uncrosslinked bovine fetal collagen (BFC) in full-thickness skin wounds.

Materials and Methods This report is a single-center retrospective review of biopsy samples from patients who had received implants of a bioactive and regenerative extracellular matrix for treatment of full-thickness skin wounds of various etiologies and sites ❚Table 1❚. The extracellular matrix was manufactured by TEI Biosciences (Boston, MA) and marketed under the trade name of Primatrix. Primatrix is an uncrosslinked collagen biomaterial derived from fetal bovine dermis. The BFC has been processed to remove cellular elements, lipids, carbohydrates, and noncollagenous proteins. The collagen that remains is primarily type I and type III in composition, and the fibrils are

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© American Society for Clinical Pathology

DOI: 10.1309/AJCPMF3B9XJAKXKM

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❚Table 1❚ Clinical Data Patient Age, No. Sex y Site Type of Wound

Age of Wound at Excision Associated Disease

1 M 49 Abdominal wall hernia Open abdominal wall, exposed 2 wk Abdominal wall cellulitis, necrosis, sepsis, complicated by infection intestine and peritonitis 2 M 51 Left arm Burn with hypertrophic scarring Many years None and contracture 3 F 29 Left foot Traumatic ulcer wound with Ulcer 3 mo Type 2 diabetes mellitus, peripheral vascular peripheral vascular disease disease, and stroke 4 M 53 Right leg Nonhealing wound 1 y Peripheral vascular disease Right lower leg Nonhealing wound 2 mo Peripheral vascular disease 5 F 27 Neck Burn with hypertrophic scarring 1 y Skin biopsy specimen at debridement and contracture showed cellulitis and microabscesses Right arm Burn with hypertrophic scarring 18 mo Elbow contracture and immobility and contracture 6 M 27 Left chest wall area Chemical burn with scarring 4 mo None and contractures 7 M 35 Chest Burn with scarring and bleeding 1 y Liver failure and coagulopathy 8 M 14 Left heel Scarring and contracture Years Epidermolysis bullosa 9 M 52 Right leg Nonhealing wound 7 mo Traumatic injury

arranged in its native architecture. The resulting material is a sheet of sterile porous BFC without viable cells. Each patient underwent a 2-stage repair procedure. Initially, the chronic wounds were excised to the peripheral margins and entire thickness, leaving viable soft, pliable tissue, fascia, or muscle in the wound bed. The BFC was applied on this surface. The biomaterial was soaked in saline before application and attached to the wound with sutures. In the second stage, a split-thickness skin graft was performed 11 to 27 days following the application of the BFC. The skin graft was laid directly on the BFC implant. Biopsy specimens were obtained from the edge of the healing wound, including both native skin and the BFC graft.

Orientation was preserved by marking the edge of the biopsy specimen toward the native skin with a suture. Longitudinal sections were taken of the tissue fixed in 10% buffered neutral formaldehyde and processed for routine histology with hematoxylin and eosin sections.

Results Nine patients (Table 1) under went 14 biopsies that were available for review ❚Table 2❚. The most frequent BFC graft age at biopsy was around 2 weeks (7 biopsy specimens), and second in frequency was 27 days (3 biopsy specimens). The

❚Table 2❚ Biopsy Specimen Summaries Biopsy Size of BFC Specimen ID Site Graft, cm

Age of BFC Implant at Biopsy Histology

1A Abdominal wall hernia 1,050 cm2 9 mo Fibroblast proliferation 2A Left arm 18 × 6, 8 × 3 15 d Fibroblast/vessels granulation tissue 3A Left foot 6 × 7 15 d Sparse fibroblast/vascular ingrowth 3B Left foot 11 × 8 23 d Fibroblast proliferation in the graft matrix 4A Right leg 10 × 10 13 d Sparse fibroblasts/columns of granulation tissue 4B Right lower leg 9 × 8 11 d Sparse fibroblast granulation at base 5A Neck 13 × 7 13 d Sparse fibroblasts/vascular ingrowth 5B Right arm 10 × 5 18 d Fibroblast between graft collagen bundles and granulation tissue 6A Left chest wall area 18 × 4 13 d Sparse fibroblast columns of granulation tissue 6B Left arm 12 × 6 27 d Fibroblast proliferation between graft collagen bundles/granulation tissue 7A Chest 32 × 12 14 d Granulation tissue with residual collagen fibers 7B Chest 45 × 13 20 d Exuberant granulation tissue 8 Left heel 21 × 22 27 d Fibroblast proliferation between graft collagen bundles and foreign body reaction 9 Right leg 11 × 7 27 d Fibroblast proliferation between graft collagen bundles and columns of granulation tissue BFC, bovine fetal collagen.


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age range extended from 11 days to 9 months. These early biopsy specimens most frequently showed fibroblast proliferation, neovascularization in the BFC implant, and columns of granulation tissue in various pattern combinations. In biopsy specimens 3A, 4B, and 6A, columns of granulation tissue alternated with columns of BFC ❚Image 1❚. The granulation tissue extended vertically to the surface of the graft area as a result of the mesh pattern in the BFC application on

the surface. In the intervening BFC matrix, the spindle/cell inflammatory infiltrate was sparse. In biopsy specimens 2A and 5A, the granulation tissue reaction was largely absent, with focal fibroblastic proliferation and ingrowth of the vessel seen in the graft. Reepithelialization of the surface had already occurred at the edge. Loose collections of lymphocytes were seen in the superficial dermis. In biopsy specimen 4B, granulation tissue and ingrowth of the vessels were seen at the base of the graft ❚Image 2❚. As the biopsy (specimens 3B, 6B, 8, and 9) age increased (range, 20-27 days), the fibroblasts became denser in the extracellular matrix. The granulation tissue in some of these biopsy specimens was less pronounced, except in biopsy specimen 7B. The collagen bundles of the BFC graft had a more eosinophilic tincture than that of the native collagen in many cases (Image 2). The BFC was devoid of cells and vessels at the time of implantation, but neovascularization was present in the biopsy specimens and advanced from the interface edge of the BFC implant with or without adjacent granulation tissue ❚Image 3❚. The vessel endothelium in the graft matrix was easily demonstrated by endothelial cell markers such as CD34. Vessel distribution was highest at the base of the graft or adjacent to the columns of granulation tissue within the graft matrix. Noticeably absent from 13 of 14 biopsy specimens was dense inflammatory reactions within the BFC matrix apart from the adjacent granulation tissue ❚Image 4❚. Small cells consistent with lymphocytes could be seen in the BFC, and neutrophils were sparse to absent.

❚Image 2❚ Bovine fetal collagen matrix at day 11 is seen overlying granulation tissue and fibroblast proliferation (biopsy specimen 4B).

❚Image 3❚ The interface between the bovine fetal collagen and the host tissue shows prominent ingrowth of vessels and fibroblasts on day 11 (biopsy specimen 4B).

❚Image 1❚ Alternating columns of granulation tissue and bovine fetal collagen extending to the graft surface are shown. The collagen is seen as eosinophilic fibers as compared with the cellular inflammatory tissue (biopsy specimen 6A).

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❚Image 4❚ Typical eosinophilia characterizes bovine fetal collagen in the biopsy sections. In young grafts, this resembles coagulative necrosis. At the edge, sparse fibroblasts and neovascularization are seen advancing into the collagen fibers (biopsy specimen 4A).

❚Image 5❚ The oldest bovine fetal collagen graft was 9 months in patient 1. The orientation of the collagen has a basket weave pattern similar to that seen in the native dermis (biopsy specimen 1A).

In the 9-month-old graft (biopsy specimen 1A), the fibrosis in the graft dermis ❚Image 5❚ was more pronounced, but clinically, the graft area was supple and scar contracture was not seen. The split-thickness skin graft showed stable, reticulated adherence to the underlying implant layer. Biopsy specimen 6B, taken on day 27 after the BFC graft, showed a pronounced granulation tissue reaction and resorption of BFC. The site was selected from the graft bed for biopsy because of the clinical impression of tissue necrosis and the surgeon believed the area required debridement. The excised tissue accounted for only 1.2% of the area of the implant bed. Resorption of BFC was seen in the exuberant granulation reaction. Biopsy specimen 7B, with BFC resorption, exuberant granulation tissue, and acute inflammation, was from a patient who had liver failure with coagulopathies. These factors directly complicated the management of the implant graft. Unlike other sites, acute inflammation was seen in the granulation tissue of this patient.

This review provides a small glimpse of the tissue response to this novel and promising therapy.1,2,10 The patients in this review all had developed chronic wounds characterized by a dysfunction in wound healing. The types of wounds undergoing implantation of BFC included exuberant and impaired wound-healing response. The tissue response to BFC extracellular matrix implantation produced a spectrum of changes ranging from a process of adaption and incorporation, formation of granulation tissue within the implanted material, and apparent resorption of BFC collagen in 1 patient. One biopsy specimen provided a late look (9 months) at the graft bed and showed prominent remodeling with fibroblasts, a natural sequence in wound healing. In the model of wound healing that highlights inflammation, tissue deposition, and remodeling phases, it is important to note that the phases are overlapping and interdependent.6 The BFC primarily modulates this sequence with a therapy to alter the tissue deposition and remodeling phases. This study demonstrates the tissue response in primarily the histologic patterns that may be observed from a sequence of biopsy specimens after the wound preparation surgery and grafting of the BFC extracellular matrix. Several observations may be made: (1) a granulation reaction of some sort was present in most of the early biopsy specimens, (2) cellular infiltrates populated the BFC matrix loosely in all biopsy specimens, (3) neovascularization took place from the edge of the BFC graft with or without granulation tissue present, (4) exuberant granulation tissue was

Discussion The dynamics of skin wound healing involves inflammation, tissue formation, and tissue remodeling.6 How healing is altered by the use of dermal substitutes such as BFC has not been systematically studied in human patients.7 The use of dermal substitutes has been tested in several clinical series.8,9 © American Society for Clinical Pathology

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accompanied by resorption of the BFC matrix, (5) aging of the graft site showed increased fibroblasts and less granulation tissue, and (6) remodeling of the BFC graft appeared similar to other healing wounds. One limitation in the review was that the biopsy specimens were taken randomly from the edge of the wound and therefore only sample a small fraction of the total graft area. The representative nature of the findings cannot be fully ascertained in these circumstances. In cutaneous wounds, tissue formation usually involves the production of granulation tissue, epithelialization, and subsequent remolding over an extended period of weeks to months. Likewise, in some of the biopsy specimens, the granulation tissue coexisted alongside the BFC matrix. The lack of intense inflammatory reactions in the BFC biomaterial suggests that it is well tolerated by the host tissue. It may be suggested from these findings that by mechanisms not yet clear, the material helps modulate a healing response in chronic wounds that otherwise are prone to dysfunction because of host and wound factors.1,9 The BFC is a porous material that readily absorbs blood and allows migration of inflammatory cells, characteristics that may support the formation of granulation tissue. Address correspondence to Dr Neill: Ameripath, 1033 N. Flowood Dr, Flowood, MS 39232; e-mail: [email protected].

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