Effect of Bacterial Exposure on Acellular Human Dermis in a Rat Ventral Hernia Model

Materials and Methods Lewis rats were randomized to a control and three experimental groups. AHD was placed as an onlay over the intact abdominal wall. Experimental groups ( n = 72) were exposed to Staphilococcus aureus at 1 × 10 4 , 1 × 10 5 , or 1 × 10 6 by direct application; controls ( n = 12) were not exposed. At 5 and 28 d, abdominal walls were explanted and tissue ingrowth assessed via tensiometry measuring energy (E) and max stress (MS) at the AHD–tissue interface. Vascularity, fibroblast migration, and inflammatory cell migration were compared using light microscopy. Results Shear strength reported as energy and max stress were significantly greater at 28 versus 5 d in all experimental groups, remaining unchanged in controls. Plasma cells and histiocytes significantly increased in all groups; macrophages increased in experimental groups only. Vascular ingrowth increased significantly in all groups; fibroblast migration was greater in controls and 1 × 10 6 exposed group only. Conclusions Contamination of AHD results in inflammatory cell influx and a surprising increase in shear strength. Interestingly, shear strength does not increase without contamination. Inflammation stimulates vascular ingrowth, but not equally significant fibroblast migration. Longer survivals are required to determine if energy and max stress of controls increase, and fibroblast migration follows vascular ingrowth. Key Words acellular human dermis bacterial contamination inflammation ingrowth shear strength biologics Introduction Acellular human dermis is described as an effective alternative to prosthetic mesh in contaminated fields [1–3] . First developed in the early 1990s, the acellular dermal matrix is derived from donated cadaver human skin, and is thus classified as a banked human tissue [4] . The most common use for acellular human dermis by general surgeons is to assist with abdominal wall closure when contamination is present. Other uses include reconstruction of soft tissue defects of the head, neck, and oral cavity, breast reconstruction, wound coverage following major burns, pelvic floor reconstruction, and dural repair following brain surgery [5–10] . Placement of acellular human dermis has been found to be safe and effective for use in contaminated fields, but can be associated with complication rates as high as 55%, including graft dehiscence, graft infection, and evisceration [1] . When used as a bridge to repair abdominal wall defects, consistent eventration has been seen over time [11, 12] . Experimental studies are limited regarding the effect of bacterial exposure on tissue ingrowth, and how this affects strength of attachment. Using an experimental model of hernia repair, we attempt to correlate bioprosthetic vascularization, fibroblast migration, and strength of attachment with presence of inflammatory cells in clean and various levels of contaminated fields. Methods A murine model of open ventral hernia repair was used to demonstrate the effects of bacterial contamination on acellular human dermis. Following approval by the Research Review Committee and Institutional Animal Care and Use Committee of the Carolinas Medical Center, adult male Lewis rats were randomized to a control group ( n = 12) and three experimental groups ( n = 72). Animals were anesthetized in an inhalation chamber with isoflurane at a concentration of 0.5% to 3%. Anesthesia was maintained via a nose cone throughout the procedure. A vertical midline incision was made through the skin and subcutaneous tissues, exposing the anterior abdominal wall. Skin flaps were raised circumferentially. Acellular human dermis was placed dermis side down as an onlay (3.5 cm × 3.5 cm) over the intact abdominal wall, and secured along the periphery with six sutures of 3-0 polydioxanone suture (PDS). In the control group, after securing the onlay to the abdominal wall, the skin was closed in two layers with a running subcuticular suture of 4-0 monocryl and skin staples. In the experimental groups, the onlay was exposed to one of three concentrations of Staphylococcus aureus at 1 × 10 4 (n = 24), 1 × 10 5 (n = 24), or 1 × 10 6 (n = 24) by direct application of a 1 mL aliquot to the basement membrane side of the onlay, after being secured to the abdominal wall. Skin was closed over the grafts in an identical fashion to controls. Animals from each of the four groups survived for either 5 or 28 d. The rats were euthanized after receiving anesthesia. The previous incision was opened and skin flaps were raised, exposing the anterior abdominal wall. The entire abdominal wall was harvested intact with the dermal onlay attached. The specimen was processed by sectioning the abdominal wall coronally, leaving equal parts of the onlay on each half. One half was sectioned again, and these specimens were used to ascertain bacterial concentration by quantitative culture and pour plates. The other half was processed for histologic analysis with hematoxylin and eosin stain. The larger specimen remaining was prepared for shear testing by removing all suture material, and disrupting the attachments at the interface of the onlay and abdominal wall over one half the area of the remaining specimen. Tissue adherence was assessed via tensiometry (Instron Mini 44; Canton, MA). Energy (E) and max stress (MS) at the acellular human dermis/abdominal wall interface at pull-off were measured as the onlay and abdominal wall were mechanically distracted. The measure of E represents the external forces required to separate the onlay and the abdominal wall. Max stress was measured using differential variable reluctance transducers (DVRT; MicroStrain, Inc., Williston, VT). A measure of the internal forces holding the onlay to the abdominal wall, MS was considered the primary measure of tissue ingrowth. Light microscopy (×40) was used to compare specimens with regard to percent vascularity, percent fibroblast migration, and number of inflammatory cells per high power field (hpf). Histologic analysis was performed by selecting a 6 mm circumferential area of each slide that included the acellular human dermis–abdominal wall interface, the entire width of the onlay, and the ventral aspect of the onlay. Three consecutive rows of fields that include the most dorsal and ventral aspects of the onlay were examined to account for variations in thickness of the acellular human dermis. All were examined by observers blind to the study groups. Plasma cells, histiocytes, and macrophages were counted separately, these three cell types being the most predominant within the wound. Number of inflammatory cells per hpf was obtained by dividing the visual field into four quadrants; only the upper outer quadrant was scored due to the high numbers of inflammatory cells present in specimens retrieved from contaminated fields. An average number of each cell type per hpf was then determined for each specimen. To measure percent vascularity and percent fibroblast migration, each visual field was divided into four quadrants; when vascular structures or fibroblasts were present, the quadrant was graded as positive so that each visual field can have a score of 0%, 25%, 50%, 75%, or 100%. These percentages were then summed and divided by the number of fields graded to yield a percent vascularity or fibroblast migration for that specimen. Statistically, results were compared using t -test or ANOVA and considered significant when P < 0.05. Results All animals survived the study to procurement. Complications encountered following graft implantation were similar to those seen clinically following hernia repair in an infected field. In the control group, there was one skin dehiscence. Skin dehiscence, hematoma/seroma formation, and abscess formation were seen with varying frequency in all experimental groups ( Table 1 ). Quantitative cultures of acellular human dermis following procurement showed presence of bacteria in both control and experimental groups. Specimens in the control and each experimental group were compared at 5 d versus 28 d. There were no differences in bacterial counts between experimental groups at 5 d or 28 d ( Table 2 ). Bacterial counts at procurement were significantly higher in each of the experimental groups compared with the control group at 5 d ( P = 0.003) and 28 d ( P = 0.014). Bacterial counts were highest in the experimental group exposed to 1 × 10 4 with a mean bacterial count of 1.99 × 10 10 ( Table 2 ). Measures of shear strength at the acellular human dermis-abdominal wall interface increased significantly at 28 d compared with 5 d in all experimental groups, but remained constant in controls ( Table 3 ). This was true for both E and MS. No significant difference between control and experimental groups existed for E at 5 d ( P = 0.427) or 28 d ( P = 0.154), or MS at 5 d ( P = 0.099) or 28 d, ( P = 0.210). Histologic examination of specimens demonstrated significant increases in plasma cells and histiocytes in all groups; macrophages were increased in the experimental groups only ( Table 4 ). Fibroblast migration occurred in unpredictable patterns, increasing significantly at 28 d in controls and specimens exposed to contamination at 1 × 10 6 . Fibroblast ingrowth decreased in specimens exposed at 1 × 10 4 and 1 × 10 5 . Significant increases in vascular ingrowth were seen in both control and experimental groups at 28 d compared with 5 d ( Table 5 ). Discussion Acellular human dermis is used primarily by the general surgeon as an alternative to synthetic mesh in contaminated fields to assist with abdominal wall closure when primary fascial closure is not possible, or to reinforce a primary fascial closure or component separation. Reports of its use in abdominal wall reconstruction demonstrate that it is safe for use in infected fields. Graft dehiscence, infection, evisceration, and hernia recurrence have all been noted as complications following ventral hernia repair with acellular human dermis [1–3] . We, as well as others, have noted a predictable eventration with long-term follow-up when acellular human dermis is used to bridge a hernia defect [11–13] . Few experimental studies of its strength or resistance to infection in a model of hernia repair have been performed. In one study, strength of acellular allograft and expanded polytetrafluoroethylene (ePTFE) were similar when used as a bridge [14] . Others have documented evidence of eventration or stretching in experimental models comparing acellular human dermis with other biologic substrates [15] . Previous work in our laboratory demonstrated acellular human dermis to have similar bacterial counts as other prosthetic and bioprosthetic materials when placed in contaminated fields, demonstrating a similar susceptibility to infection [16] . In our current study, we observed the effects of contamination on an acellular dermal allograft in a model of ventral hernia repair. On a macroscopic level, we observed some of the same complications encountered in the clinically infected field ( Table 5 ). In the control group, the onlay remained grossly unchanged; in experimental groups, obvious abscess and hematoma formation were occasionally seen. Of note, no specimens showed complete degradation. Examining specimens in control and experimental groups postoperatively at 5 d and 28 d demonstrated the effects of contamination on tissue ingrowth and inflammatory cell migration over time in the absence and presence of contamination. By evaluating bacterial counts at harvest, we gained insight into how resistant an allograft was to infection following application of a bacterial load. In our current study, bacterial counts did not significantly change over time following initial inoculation. This persistence of similar bacterial loads at 28 d harvest compared with 5 d harvest demonstrated a propensity for the biologic to harbor bacteria. Use of suture to secure the allograft to the anterior abdominal wall may have contributed to this. The use of PDS, a monofilament suture, was chosen to minimize this confounding variable. Persistently elevated bacterial counts in experimental groups at 28 d demonstrated that any prosthetic, whether of biologic or synthetic origin, is a foreign body until fully vascularized. This study documented the effects of contamination on tissue ingrowth as measured by shear strength at the interface between the allograft and abdominal wall. With regard to shear strength, we measured both energy per unit area and maximum stress at failure at the interface. Energy per unit area is a measure of the external force required to displace the area of overlap between the acellular dermal allograft and abdominal wall. Maximum stress measures the inherent forces of attachment between the biologic and abdominal wall; we consider this to be the primary measure of tissue ingrowth as it is a direct measurement of the strength of attachment inherent to the interface between the acellular human dermis and abdominal wall. Both measures of shear strength increased significantly in the presence of infection, but failed to increase in controls where contamination did not occur. On histologic analysis, plasma cells and histiocytes were seen to increase over time regardless of whether contamination occurred or not. However, the number of macrophages increased exponentially, but only in contaminated fields, mirroring the increases in energy per unit area and maximum stress at failure of the interface. Macrophages are identified as being of primary importance in wound healing, bacterial clearance, and tissue repair, achieving significant numbers within 2 to 4 d of injury [17, 18] . While experimental depletion of neutrophils from areas of injury results in little effect on wound healing, the importance of macrophages in the recruitment and activation of other cells required for wound healing is paramount [17, 18] . The release of TGF-b, vascular endothelial growth factor (VEGF), and other mediators by macrophages play a major role in the angiogenesis and tissue remodeling that follows influx of macrophages [17, 19, 20] . The role of VEGF in wound healing has been well studied. Its up-regulation during early wound healing correlates with maximal capillary growth and is also thought to play a role in collagen deposition and, possibly, epithelialization [21] . Specifically, VEGF-A has been shown to promote the formation of new vessel growth, being especially active in stimulating endothelial cell migration [22] . Measurements of cytokines active in wound healing, such as VEGF and TGF-b, were not performed in this study. Considering the increases in max stress and energy that occurred with macrophage influx into the wound bed seen in experimental groups only, vascular ingrowth stimulated by VEGF may have played a significant role in the increased tissue ingrowth that occurred in contaminated fields. The ability of macrophages to stimulate wound healing and tissue ingrowth was further demonstrated in our model with the concomitant observance of both increased maximum stress at failure and significantly increased numbers of macrophages in contaminated fields. Increases in vascular ingrowth were consistently higher at 28 d for both control and experimental groups. While not statistically significant, greater percent vascularity was seen at 5 and 28 d for experimental groups compared with controls ( Table 5 ). The proliferative phase of wound healing that occurs d 4 through 12 is associated with marked endothelial proliferation. Migration and replication of endothelial cells seen in this phase is influenced by multiple cytokines and growth factors including VEGF [17] . With macrophages representing a major source of VEGF and an abundance of macrophages seen on histologic examination of specimens from experimental groups, it is plausible that the increased shear strength at the interface of acellular human dermis and abdominal wall was due in large part to vascular ingrowth as a result of macrophage influx. Predictable patterns of fibroblast migration were not observed with fibroblasts increasing in both controls and the experimental group exposed to 1 × 10 6 , while decreasing in other experimental groups over time. In our present study, we have demonstrated that bacterial contamination and the ensuing inflammatory reaction produce increased vascularity and accelerated tissue ingrowth. In the case of bioprosthetics, it may be that rapid ingrowth is dependent on the inflammation caused by bacterial contamination. The inflammatory response to infection may stimulate the influx of monocytes and macrophages, and subsequent release of cytokines, needed to begin the process of accelerated vascularization. Conclusion Acellular human dermal allografts are frequently placed in contaminated and infected fields. Few, if any, studies of allograft performance as it relates to tissue attachment or ingrowth in clean versus contaminated fields exist. Bacterial contamination of acellular human dermis results in an influx of inflammatory cells associated with a surprising increased shear strength and tissue attachment. Over time, shear strength is unchanged in the absence of bacterial contamination. Early contamination and the associated inflammatory reaction may initially increase strength of human dermal allografts in infected fields. Inflammation may increase vascular ingrowth, but does not stimulate equally significant fibroblast migration. 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