A Retrospective Clinical Review of Extracellular Matrices for Tissue Reconstruction: Equine Pericardium as a Biological Covering

Complex wounds frequently undergo surgical excision and grafting in an attempt to optimize the wound environment and to facilitate wound closure. Individuals who have not responded to conventional non-surgical therapies and dressings may not be good candidates for autologous grafting. A fully flexible, cross-linked, acellular equine pericardium biological xenograft was used to address difficult-to-treat and recalcitrant wounds of the lower extremity. Twenty-four complex wounds of varying etiology, including diabetic, venous, trauma, vasculitic, and post-surgical wounds underwent surgical debridement and xenograft application. The individual results were reviewed in a retrospective study over an 18-month period. The duration of the wounds ranged between 3 months to 2 years. The average time to wound closure was 5.96 weeks. The median time to closure was 6 weeks. No significant adverse events were noted. The data review suggests the use of equine pericardium as a xenograft and biological cover may significantly benefit patients with difficult-to-heal wounds. Additional animal and clinical studies are in progress to help understand the mechanism of action of the xenograft in the clinical environment.

A variety of treatment modalities are available for the treatment of difficult to treat wounds.¹ Topical dressings, ointments, drugs, devices, and other external products offer varying benefits depending on the nature of the wound environment. Regardless of modality, creating an environment that will optimize the wound environment by facilitating cellular activity may be of significant benefit in promoting wound closure.²

Biological matrices may consist of allogenic, xenogenic, or chemical constructs that act as temporary matrices that are eventually replaced by host tissue. While not containing living cells or antigenic materials, these products provide a structure for cell migration and activity that is absent in many full-thickness defects.

This retrospective clinical review examined results from 24 complex wounds treated with an acellular equine pericardium on patients with known barriers to tissue repair and wound closure. The primary purpose of the review was to determine potential benefits of this particular xenograft to assist in the development of future studies that would support continued use and help explain the mechanism of action leading to positive results and wound closure in wounds otherwise recalcitrant to other therapies, since little data are currently available on the clinical and scientific basis of acellular mechanisms of action in the clinical setting. Similarities and differences with other acellular materials are discussed and contrasted.

The data reviewed include wounds of varying etiology: diabetic, venous, trauma, post-surgical, vasculitic, and autoimmune. The primary consideration of this retrospective study was not with wound etiology, rather it was with the overall physical presentation of the wound, previous attempts at wound closure, failure to attain wound closure, and host as well as wound response to the xenograft. Secondary factors reviewed included reduction of pain, decreased wound dressings and daily treatments, and overall acceptance by the patient. The patients reviewed were all treated over an 18-month period with an initial application of non-fenestrated equine pericardium biomatrix (Unite® Biomatrix, Synovis Life Technologies, St. Paul, MN). The compilation of 24 patients is intended to provide a broad overview of possible applications.

Equine pericardium biomatrix. The equine pericardium biomatrix ([EPB], Unite Biomatrix) product line is used as a wound dressing for a variety of chronic wounds. These products are equine pericardia based, which go through a decellularization process followed by a novel stabilization (crosslinking) process and sterilization methods with EDC (a non-toxic, water-soluble chemical). This results in a cross-linked tissue that is biocompatible, pliable, has a natural feel and texture, and highly resistant to proteolytic enzyme degradation. The final product retains the native structural attributes of collagen³ (Figure 1).

Regardless of wound etiology, once an ulcer develops and does not proceed to heal, the chronic wound environment takes on its own unique characteristics, which include excessive proteases, increased cellular senescence, and increased bacterial bioburden.⁴ At the cellular level, evidence suggests that a higher level of matrix metalloproteinase (MMPs) production by fibroblasts is one factor that contributes to delayed healing associated with advanced age.⁵ Similarly, the absence of appropriate vascularization is a known problem in most chronic wounds, and cells from an ischemic or hypoxic skin are known to have higher levels of MMPs combined with unstable collagen production resulting in continued degradation and consequently, skin ulcers.⁶ Some of the noncollagen degrading proteases are also known to impair anchoring of the remodeling cells entering chronic wounds.⁷ Thus, a matrix such as EPB, which is resistant to premature degradation, can provide a better environment to progress healing.

No currently completed randomized or controlled Level 1 trials on EPB exist. Level 1 trials with other xenografts are also nonexistent. This retrospective review supports potential benefits of xenograft use for chronic wounds and encourages studies through well-designed, controlled clinical trials.

Twenty-four patients were treated over an 18-month period and their results were reviewed. The etiology, location, and time to closure for each wound were recorded (Table 1). In all cases, the wounds were debrided in the operating theater by excisional debridement prior to application of a xenograft, which facilitated removal of nonviable tissue, any bacterial biofilm present on the wound surface, and senescent cells. Intraoperative tissue cultures were taken and gram stains were requested. All patients were determined to be free of clinical signs of infection and were later placed on appropriate antibiotics if any pathogens were found to be present subsequent to an intraoperative wound culture; although, this did not preclude the presence of significant levels of bacteria in the wound bed prior to debridement. Patients were required to be medically cleared for surgery prior to being scheduled for excisional debridement, including adequate vascular flow needed for wound closure (determined by noninvasive vascular lab tests), adequate albumin levels (> 3.5) and no additional contraindications to surgery as determined by the patient’s primary care provider. Since the EPB dressing is approved for use in the treatment of chronic wounds, the decision for surgery and application of xenograft was based on the patient’s past treatment history, medical status, and expected benefit from the procedure.

All patients underwent preoperative preparation of their lower extremity adhering to standard operating room procedures. Wounds were excised utilizing sharp excisional and hydrosurgical debridement (Versajet®, Smith and Nephew, Fort Lauderdale, FL) to prepare the wound bed prior to xenograft application. Once hemostasis was obtained, the wound bed was examined to determine whether the application of other biological materials was required. In one case where viable tendon was exposed, a silicone-free, collagen-glycosaminoglycan based product (Integra™, Integra LifeSciences, Plainsboro, NJ) was placed in the wound base. In another case, a cavity whose depth was probed and determined not to be a fistula, an injectable collagen-glycosaminoglycan (Integra) was used. This was then covered with the EPB. In both cases, the collagen-glycosaminoglycan was applied in an attempt to expedite cell migration into defects where contact between the xenograft and the underlying tissue was not possible. The collagen-glycosaminoglycan matrix is designed to act as a temporary matrix that allows cell integration and eventual replacement by host cells. The decision to use the combination of products in two of the total number of patients was based on wound evaluation and clinical judgment at the time of surgery. Periwound tissue status helped determine which retention materials to use. Staples were used in the majority of cases. The exception was in cases of fragile periwound tissue where tincture of benzoin followed by Steri-Strip™ (3M Healthcare, St. Paul, MN) application to secure the product to intact skin. The xenograft was then covered with a single layer of Adaptic, bolstered gauze, Kerlix™ (Covidien, Mansfield, MA), and when edema or venous disease was present or when deemed necessary, with compression wraps. All dressings were left intact for 3 days to 1 week (± 1 day). At the first postoperative visit (week 1 for outpatients and day 3 for inpatients), all dressings were removed and the underlying xenograft was examined to determine retention over the wound, drainage, or any indication of clinical infection. Staples were removed at the postoperative week 1 visit, although the Steri-Strips were left intact for an additional week. Dressings were changed weekly until the xenograft detached itself and the underlying wound had closed. Dressing changes after week 1 consisted of a single layer of petrolatum impregnated gauze covered with Kerlix. Compression was applied if edema or venous disease was present.

Twenty-four wounds from 24 patients (19 men and 5 women) were retrospectively reviewed. The average age at the day of surgery was 58.3 ± 15.16 years (range 23 to 86). The duration of wounds prior to surgery ranged between 3 months and 2 years. Thirteen wounds were diabetic foot ulcers, 5 ulcers were a direct result of trauma that involved the leg and foot, 3 venous leg ulcers, 2 with mixed etiology (VLU/vasculitic and VLU/lymphedema), and 1 vasculitic ulcer.

The average time to wound closure was 5.96 ± 2.1 weeks (range 2 to 9). The median time to wound closure was 6 weeks.

Acellular products have been used extensively for the treatment of burns and chronic wounds. Its primary use in burn wounds is as a temporary matrix to promote granulation that prepares the wound for subsequent grafting.⁸

The use of acellular products for the treatment of chronic wounds is a recent development, although extensive literature is available on the use of acellular products (cadaver, porcine and equine) in the medical literature.^12–15 While cadaver skin is readily available for clinical use, human tissue still carries the risk of viral transmission, particularly hepatitis and HIV. 

The term extracellular matrix (ECM) as applied to wounds is commonly associated with the use of topically-applied, biological materials. The term ECM implies the introduction of a scaffold that assists with cellular activity leading to cellular migration and modulation, granulation, and eventual wound closure. To maintain a scaffold that allows cellular infiltration, an ECM must remain in the wound bed for a sufficient amount of time to promote ongoing activities and processes that will eventually lead to tissue repair. A material that is rapidly denatured in the wound bed in a matter of days cannot be expected to act as a durable scaffold.

A recent article by Winters et al¹⁶ discussed the use of an allograft for chronic wounds; however, there was no direct correlation between use of the product and complete wound closure as a result of allograft application alone. The study is particularly misleading since the majority of patients seemed to be treated with multiple modalities to attain would closure, making it difficult to determine the value of the allograft. In the present study, wound closure was defined as complete epithelialization and no discharge. The difference in outcomes using the allograft in the Winters study and the information presented in this review is significant, as our data only reviewed patients whose wounds attained complete closure without use of additional modalities—with the exception of the patient who required a final 3-week treatment with a topical dressing for the remaining superficial wound after the xenograft was removed (Figure 5).¹⁶ Additionally, the ulcers in the Winters et al¹⁶ study were classified using the UT Wound Classification, which was designed for diabetic foot ulcers, even though the study ulcers presented (foot, ankle, and lower extremity) did not all appear to be diabetic, based on presentation and location. The wounds in the present study were all full-thickness, regardless of etiology.

The patients included in this retrospective review presented with full-thickness wounds that had been either unresponsive to previous attempts at conservative treatment modalities, which included alginate, collagen, combination alginate/collagen, foam, topical ointments and medications, compression wraps, application of growth factors, cellular skin constructs, or split- thickness skin grafts, or were considered at high risk for deterioration if the wound did not close. All wounds had been open for a minimum of 3 months with a range of 3 months to 2 years. All wounds attained complete closure in less time than would be anticipated for a wound of similar etiology based on anticipated times to closure published in the literature. Veves et al¹⁷ reported a 65-day median time to wound closure in the Apligraf® (Organogenesis, Canton, MA) group versus 90 days in the saline moistened gauze group in the treatment of diabetic foot ulcers, while Winters et al¹⁶ reported a mean and median time to wound closure of 13.8 weeks and 11 weeks, respectively, in the treatment of diabetic lower extremity wounds with an allograft.¹⁶ Mostow et al¹⁸ reported a 55% healing rate of VLU (> 1 month duration) by 12 weeks using small intestine submucosa (SIS) and compression compared with 34% in the compression-only group.¹⁸ Falanga and Sabolinski¹⁹ reported 47% healing rate VLU (> 1 year duration) at 6 months using graftskin (Apligraf) and compression compared with 19% in the compression-only group. This retrospective review is not a randomized or controlled trial and was not designed to be compared with such studies. However, the observations on healing times suggest significantly improved time to healing rates with use of this xenograft, warranting more extensive and controlled studies. Figures 2, 3, 4, and 5 illustrate wounds of varying etiology before and after treatment.

Mechanism of action
Currently, the mechanism of action of stabilized equine pericardium in assisting with wound closure has not been well defined or studied. Scientific assumptions on its clinical benefits are based on available animal data.^9–11 While the mechanism of action of equine pericardium is better understood when used as a true implant for tendon repair, the role in chronic wounds remains unclear. When not used as an implant, the material does not appear to act as a true, fully incorporated matrix, as it is eventually displaced from the wound surface with the exception of deep cavities where cellular invasion and replacement with host cells and tissue eventually occurs.

Clinical and animal studies investigating the cellular and chemical activity in chronic wounds treated with equine pericardium are warranted to better understand the mechanism of action when applying this xenograft as a biological cover in either the surgical or nonsurgical setting on full-thickness wounds. Hypothetically, a biological dressing, which consists primarily of protein could bind harmful proteases, facilitate and promote optimal cell and cytokine activity, and expedite wound closure.²⁰ This hypothesis needs to be supported by well designed and controlled clinical trials.

Twenty-four patients treated with xenograph over an 18-month period were selected for a retrospective review of the use of an equine derived xenograft for difficult-to-heal wounds. Significant adverse events were not noted for any of the patients. The wounds attained complete closure without complications or difficulties. The data reviewed suggest that the use of equine pericardium as a temporary biological cover and scaffold may be of significant benefit in the surgical treatment of chronic wounds. Additional animal and prospective multinational randomized controlled clinical studies are underway to hopefully provide a better understanding of the mechanism of action and role of this product in the treatment of difficult-to-heal wounds.

Affiliation: University of California San Diego, Department of Surgery/Division of Trauma 

Address correspondence to:
Gerit Mulder, DPM, MS, FAPWCA
UCSD Medical Center
200 West Arbor Drive First Floor, Suite 4
San Diego, CA 92103
Phone: 303-733-4181
Email: gmulder@ucsd.edu