The Biointegration of a Porcine Acellular Dermal Matrix in a Novel Radiated Breast Reconstruction Model.

BACKGROUND: Ideal acellular dermal matrices (ADM) for breast reconstruction exhibit native extracellular matrix (ECM) structure to allow rapid biointegration and appropriate mechanical properties for desired clinical outcomes. In a novel in vivo model of irradiated breast reconstruction, we describe the cellular and vascular ingrowth of Artia, a porcine product chemically prepared to mimic the biomechanics of human ADM, with retained natural ECM structure to encourage cellular ingrowth. METHODS: Utilizing the murine dorsal skinfold model, Artia was implanted into 16 C57bl/6 mice. Eight of the mice received a single dose 35 Gy radiation to the skin, followed by 12 weeks to produce radiation fibrosis and 8 mice served as nonradiated controls. Real-time photoacoustic microscopy of vascular integration and oxygen saturation within the ADM were made over 14 days. At 21 days, vascular ingrowth (CD31), fibroblast scar tissue formation (alpha smooth-muscle actin α-SMA, vimentin), and macrophage function (M2/M1 ratio) were evaluated. Scanning electron microscopy images of Artia were produced to help interpret the potential orientation of cellular and vascular ingrowth. RESULTS: Repeated photoacoustic microscopy imaging demonstrated vascular ingrowth increasing over 14 days, with a commensurate increase in oxygen saturation within both radiated and nonradiated ADM-albeit at an insignificantly lower rate in the radiated group. By day 21, robust CD31 staining was seen that was insignificantly greater in the nonradiated group. Of the fibroblast markers, vimentin expression was significantly greater in the radiated group (P < 0.05). Macrophage lineage phenotype was consistent with remodeling physiology in both radiated and nonradiated groups. Scanning electron microscopy demonstrated transversely organized collagen fibrils with natural porous ECM structure to allow cellular ingrowth. CONCLUSIONS: Artia demonstrates appropriate biointegration, with increased oxygen saturation by 14 days, consistent with the performance of other collagen substrates in this model. Radiation fibrosis resulted in higher vimentin expression yet did not impact macrophage phenotype while only modestly decreasing Artia biointegration suggesting that ADM may have a role in reconstructive efforts in a radiated setting. Taken together with its enhanced biomechanics, this porcine ADM product is well poised to be clinically applicable to breast reconstruction.

Fenestration Improves Acellular Dermal Matrix Biointegration: An Investigation of Revascularization with Photoacoustic Microscopy.

BACKGROUND: Acellular dermal matrices have revolutionized alloplastic breast reconstruction. Furthering our knowledge of their biointegration will allow for improved design of these biomaterials. The ideal acellular dermal matrix for breast reconstruction would provide durable soft-tissue augmentation while undergoing rapid biointegration to promote physiologic elasticity and reduced infectious complications. The inclusion of fenestrations in their design is thought to promote the process of biointegration; however, the mechanisms underlying this theory have not been evaluated. METHODS: Biointegration of standard and fenestrated acellular dermal matrices was assessed with serial photoacoustic microscopic imaging, in a murine dorsal skinfold window chamber model specifically designed to recapitulate the microenvironment of acellular dermal matrix-assisted alloplastic breast reconstruction. Photoacoustic microscopy allows for a serial, real-time, noninvasive assessment of hemoglobin content and oxygen saturation in living tissues, generating high-resolution, three-dimensional maps of the nascent microvasculature within acellular dermal matrices. Confirmatory histologic and immunohistochemical assessments were performed at the terminal time point. RESULTS: Fenestrated acellular dermal matrices demonstrated increased fibroblast and macrophage lineage host cell infiltration, greater mean percentage surface area vascular penetration (21 percent versus 11 percent; p = 0.08), and greater mean oxygen saturation (13.5 percent versus 6.9 percent; p < 0.05) than nonfenestrated matrices by 2 weeks after implantation. By 21 days, host cells had progressed nearly 1 mm within the acellular dermal matrix fenestrations, resulting in significantly more vascularity across the top of the fenestrated matrix (3.8 vessels per high-power field versus 0.07 vessels per high-power field; p < 0.05). CONCLUSIONS: Inclusion of fenestrations in acellular dermal matrices improves the recellularization and revascularization that are crucial to biointegration of these materials. Future studies will investigate the optimal distance between fenestrations.

Evaluating a Variable Porosity Wound Dressing With Anti-Scar Properties in a Porcine Model of Wound Healing.

Introduction: New treatments that promote wound healing while preventing scar formation are needed. One option in topical wound healing is the use of temporary dressings that allow the natural healing process with minimal scar formation. Methods: We evaluated the temporary wound dressings PermeaDerm C, and a PermeaDerm C derivative coated with the anti-scarring agent, salinomycin (PermeaDerm D) in a pig model of wound healing to show the efficacy of these wound dressings in vivo. Results: Porcine fibroblasts grow well in the presence of PermeaDerm C or PermeaDerm A, and salinomycin reduces excessive myofibroblast formation in porcine fibroblasts in vitro. In vivo, wounds treated with PermeaDerm C and PermeaDerm A did not show abnormal or unwanted healing patterns up to 8 weeks post-wound formation. Wounds covered with either PermeaDerm C or PermeaDerm A showed a more mature wound-healing phenotype than the control wounds. Conclusions: PermeaDerm C and PermeaDerm A allowed wound healing, revealing the potential of both PermeaDerm C and PermeaDerm A to promote effective healing while preventing excessive scar formation.

A Novel Small-Animal Model of Irradiated, Implant-Based Breast Reconstruction.

BACKGROUND: There is currently a need for a clinically relevant small-animal model for irradiated, implant-based breast reconstruction. Present models are inadequate in terms of suboptimal location of expander placement and mode of radiation delivery, correlating poorly with the human clinical scenario. The authors hypothesized that by delivering fractionated radiation and placing an expander under the scalp of the animal, they would achieve soft-tissue changes histologically analogous to those seen in human irradiated, implant-based breast reconstruction. METHODS: This study consisted of 11 immunocompetent, hairless rats divided into three groups as follows: untreated control (n = 3), tissue-expanded scalps (n = 4), and fractionated irradiation plus tissue expansion of the scalp (n = 4). At the completion of the experiment for each group, skin tissue samples were analyzed histologically for vascularity, epidermal and dermal thickness, and collagen fiber alignment or scar formation. RESULTS: Expanded rat epidermis was significantly thicker and dermis was more vascular than nonexpanded skin. The authors observed a greater degree of collagen fiber alignment in the expanded group compared with nonexpanded skin. The combination of irradiation and expansion resulted in significant dermal thinning, vascular depletion, and increased scar formation compared with expanded skin alone. CONCLUSIONS: The authors describe a novel small-animal model for irradiated, implant-based breast reconstruction where histologic analysis shows structural changes in the skin consistent with known effects of radiation therapy and expansion in human skin. This model represents a significant improvement from previous ones and, as such, holds the potential to be used to test new therapeutic agents to improve clinical outcomes.

Effects of Collagenase Digestion and Stromal Vascular Fraction Supplementation on Volume Retention of Fat Grafts.

OBJECTIVE: The use of autologous fat as a soft tissue filler has increased over the past decade in both reconstructive and aesthetic surgeries. Enhancement of autologous fat grafts with the addition of the stromal vascular fraction (SVF) has been reported to improve long-term volume retention. Stromal vascular fraction is most commonly isolated using enzymatic digestion, but it is unknown what effect the digestion process has on the adipocytes and SVF cells that comprise the graft. Some clinicians have reported use of enzymatically digested fat grafts to alter the physical properties of the tissue in specialized applications. We have previously reported that increasing collagenase digestion duration adversely affects the viability of adipocytes and SVF cells. Here, we aimed to determine if collagenase digestion of adipocytes before grafting is detrimental to long-term graft retention and if SVF supplementation can abrogate these potential deleterious effects. METHODS AND RESULTS: We used a published xenograft model in which human lipoaspirate was implanted into the scalp of immunocompromised mice to study the effects of collagenase digestion on in vivo graft survival after 12 weeks. We used 4 experimental groups: grafts composed of collagenase-digested and nondigested adipocytes (50-minute digestion) and grafts with and without SVF supplementation. We used microcomputed tomography to serially and noninvasively quantify graft volume, in conjunction with hematoxylin-eosin staining of histological cross-sections of implanted and excised grafts to assess overall tissue viability. We found that adipocytes that were collagenase-digested before implantation had significantly lower retention rates at 12 weeks and poorer tissue health, which was assessed by quantifying the number of intact adipocytes, the number of cystic formations, and by scoring the degree of inflammation and fibrosis. Further, we found that SVF supplementation of the digested grafts improved graft survival, but not to the level observed in undigested grafts. CONCLUSIONS: We conclude that collagenase digestion adversely affects the long-term volume retention of fat grafts, but that graft retention is improved by SVF supplementation. These experimental results can serve as an initial framework to further elucidate the reported efficacy and safety of using collagenase-digested fat grafts and SVF in the clinical setting.

Plasticity of Auricular Cartilage in Response to Hormone Therapy.

INTRODUCTION: Correction of auricular deformities can be accomplished through splinting within the first few weeks of life. This is hypothesized to be due to retained circulating maternal estrogens decreasing the structural density of collagen; however, this has not been fully tested. Cartilage elasticity is dependent on the concentration of the proteoglycan aggregate, and hyaluronic acid, a constituent of proteoglycan aggregate, is increased by estrogens. Nonsurgical correction of these deformities in more developed patients has the potential to change clinical practice and eliminate surgical risks. Previous studies have demonstrated preliminary promise with the use of injectable estrogen to treat auricular deformities. For this study, we have validated an animal model and demonstrated the feasibility of a more therapeutically appropriate topical estrogen treatment in restoring neonatal plasticity of auricular cartilage. METHODS: Ears of 12 New Zealand rabbits were folded and splinted, and assigned an experimental group (estrogen, placebo, and untreated control) (n = 8 ears). Treatment ears received topical estrogen or placebo cream daily for 4 weeks, whereas controls received no treatment. The splints were removed following 2 additional weeks, and photographs were taken to calculate the retained fold angle. Biopsies were also taken for histologic analysis. RESULTS: The 8 control ears showed a statistically increased angle from a folded orientation of 46.6 degrees to return of ear position to a normal upright position of 151.2 degrees by the fourth day after splint removal. Both the estrogen-treated and placebo-treated ears responded to splinting with maintained folding (36.6 degrees and 32.5 degrees, respectively). Auricular cartilage thickness trended toward thicker in ears treated with estrogen, consistent with increased matrix components. CONCLUSIONS: Estrogen and placebo treatment with splinting of ears lead to a significant change to the cartilage configuration, validating the model. The results of this study are very encouraging and provide the foundation for a noninvasive therapeutic approach for correcting auricular deformities. Future work will include a more detailed mechanistic study evaluating the dosing of estrogen and the efficiency of dermal penetration as well as evaluating the long-term outcomes and molecular mechanism-associated cartilaginous responses to estrogen.

Abdominal Wall Allograft: Preclinical Biomechanical Investigation of a Novel Reconstructive Adjunct.

INTRODUCTION: Acellular dermal matrices have revolutionized abdominal wall reconstruction; however, device failure and hernia recurrence remain significant problems. Fascia grafts are a reconstructive adjunct with increased tensile strength compared with acellular dermal matrices; however, clinical use is limited by insufficient donor material and donor site morbidity. To this end, we investigate the biomechanical properties of human abdominal wall allografts (AWAs) consisting of the anterior rectus sheath from xiphoid to pubis. METHODS: After cadaveric procurement of 6 human AWAs, the tissue was divided horizontally and a matched-sample study was performed with specimens randomized to 2 groups: fresh, unprocessed versus processed with gamma irradiation and decellularization. Specimens were evaluated for physical properties, DNA content, tensile strength, and electron microscopy. RESULTS: All AWA donors were male, with a mean age of 55.2 years (range, 35-74 years). Procured AWAs had a mean length of 21.70 ± 1.8 cm, width of 14.30 ± 1.32 cm, and area of 318.50 cm, and processing resulted in a 98.3% reduction in DNA content. Ultimate tensile strength was significantly increased after tissue processing, and after subcutaneous implantation, processed AWA demonstrated 4-fold increased tensile strength compared with unprocessed AWAs. CONCLUSIONS: Acellular AWAs represent a novel reconstructive adjunct for abdominal wall reconstruction with the potential of replacing "like with like" without additional donor site morbidity or antigenicity.

Complement Activation and STAT4 Expression Are Associated with Early Inflammation in Diabetic Wounds.

Diabetic non-healing wounds are a major clinical problem. The mechanisms leading to poor wound healing in diabetes are multifactorial but unresolved inflammation may be a major contributing factor. The complement system (CS) is the most potent inflammatory cascade in humans and contributes to poor wound healing in animal models. Signal transducer and activator of transcription 4 (STAT4) is a transcription factor expressed in immune and adipose cells and contributes to upregulation of some inflammatory chemokines and cytokines. Persistent CS and STAT4 expression in diabetic wounds may thus contribute to chronic inflammation and delayed healing. The purpose of this study was to characterize CS and STAT4 in early diabetic wounds using db/db mice as a diabetic skin wound model. The CS was found to be activated early in the diabetic wounds as demonstrated by increased anaphylatoxin C5a in wound fluid and C3-fragment deposition by immunostaining. These changes were associated with a 76% increase in nucleated cells in the wounds of db/db mice vs. CONTROLS: The novel classical CS inhibitor, Peptide Inhibitor of Complement C1 (PIC1) reduced inflammation when added directly or saturated in an acellular skin scaffold, as reflected by reduced CS components and leukocyte infiltration. A significant increase in expression of STAT4 and the downstream macrophage chemokine CCL2 and its receptor CCR2 were also found in the early wounds of db/db mice compared to non-diabetic controls. These studies provide evidence for two new promising targets to reduce unresolved inflammation and to improve healing of diabetic skin wounds.

Advanced Imaging Techniques for Investigation of Acellular Dermal Matrix Biointegration.

BACKGROUND: Biointegration, a concept involving a dynamic interplay among three processes-inflammation, cellular infiltration, and angiogenesis-is key to understanding the interaction between acellular dermal matrices and the host. The current standard for evaluating acellular dermal matrix biointegration involves histologic analysis at fixed time points; however, the authors' approach uses advanced imaging techniques to serially assess biointegration in real time. METHODS: The authors have adapted two advanced imaging techniques-two-photon microscopy and photoacoustic microscopy-to investigate biointegration in a murine deepithelialized dorsal skin-fold window chamber model, specifically engineered to recapitulate the host microenvironment of acellular dermal matrix-assisted breast reconstruction. Four mice per group were assessed. Two-photon imaging of dual-transgenic mice allows for detection of fluorescently labeled perivascular cells, and macrophage lineage cells. Photoacoustic microscopy noninvasively assesses oxygen and hemoglobin concentration in living tissues, generating high-resolution, three-dimensional mapping of the nascent acellular dermal matrix-associated microvasculature and metabolic consumption of oxygen. These outcomes were corroborated by confirmatory histologic analysis at the terminal time point. RESULTS: The acellular dermal matrix/host interface is characterized by robust inflammation (0 to 3 days), increased oxygen consumption and neoangiogenesis in the matrix border zone (10 to 14 days), and vascular and inflammatory cell penetration into the center of the matrix (>21 days). CONCLUSION: The data broaden the core knowledge of acellular dermal matrix biology, and serve as a potential template for elucidating the key differences among various commercially available and developmental products to guide the reconstructive surgeon to better select a reconstructive adjunct that meets their specific needs.

The influence of absorbable subcuticular staples, continuous subcuticular absorbable suture, and percutaneous metal skin staples on infection in contaminated wounds.

Wound infection is a threatening, troublesome, and costly complication contributing to increased mortality and morbidity. The methods and materials used to close a wound significantly influence the quality of the repair process and the risk of surgical site infection. Six pigs were used to evaluate the influence of four separate skin-closure modalities on the potentiation of infection in contaminated wounds. Full-thickness skin wounds on the abdomen were contaminated with S. aureus and then closed with one of four devices: a novel absorbable staple (InsorbTM) placed in the subcuticular tissue; a braided absorbable suture (VicrylTM); a monofilament absorbable suture (MonocrylTM); percutaneous metal staples. Wound infection was assessed 7 days after closure by clinical signs and quantitative bacterial swabs. InsorbTM staples had significantly lower infection rates than continuous VicrylTM (39% vs. 100%, p=0.002) or MonocrylTM suture (39% vs. 89%, p=0.014). The InsorbTM subcuticular staple and the metal percutaneous skin staple were statistically equivalent in wound infection rate and parameters of inflammation. The combined data for both interrupted staple modalities documented less inflammation compared to the combined data for continuous sutures. These lower levels of inflammatory metrics were statistically significant for edema (p=0.018), gauze exudate observed (p=0.007) and purulent exudate in wound (p<0.0001). In conclusion, InsorbTM staples were shown to be an acceptable choice for the closure of contaminated wounds because they had a significantly lower incidence of wound infection and inflammation when compared to continuous intradermal suture.

Quantitation of the Postoperative Vascular Response in Four Dorsal Bipedicle Flaps in the Rat.

The rodent bipedicle flap has been used in numerous permutations as a model of ischemia and to generate a compromised tissue bed. In this study 24 rats were used to investigate skin perfusion over time along the length of 4 distinct bipedicle flap designs-2 oriented cranial-caudally and 2 transverse. Traditional rectangular flaps were evaluated as well as unique elliptical geometries designed to maximize the area of the resulting ischemic bed. Perfusion was assessed by scanning laser Doppler and intravascular dye penetration. In all models, an initially ischemic bed as measured by Doppler reestablished basal perfusion rates within 2-4 days and went on to produce a hyperemic bed with perfusion levels well above baseline. Reduced perfusion areas visualized by fluorescein dye penetration completely resolved in 55% of flaps within 2 days, and 68% within 4 days. This study underscores the difficulty in developing a representative model of chronic ischemia in young healthy rats as well as their remarkable ability to recover from acute surgical insults.

A revolutionary advance in skin closure compared to current methods.

Six pigs were used to evaluate the influence of three separate modalities on contaminated wounds. Full-thickness skin wounds on the abdomen were contaminated with 10(4) or 10(5) Staphylococcus aureus and then closed with one of three methods. The three closure modalities included (1) a new absorbable staple (Insorb) placed in the subcuticular tissue, (2) a braided Vicryl suture, and (3) percutaneous metal staples. Any foreign body material implanted in tissue increases the risk of infection at that site. Wound closure always involves the use of a foreign body. Historically, sutures have been the primary material used to close tissue. The newer synthetic sutures are significangly more biodegradable and cause less infection than sutures composed of protein, such as silk and catgut. Metal staples are also associated with a low risk of infection. Recently, Incisive Surgical, Inc. (Plymouth, Minnesota) has developed an absorbable polymer staple specifically for subcuticular skin closure. The purpose of this study was to compare the new Insorb staple to both an absorbable polymer suture and a metal staple. Wound infection was assessed 7 days after closure by clinical signs and quantitative bacterial swabs. The results demonstrated that wounds closed with Insorb staples had the lowest incidence (33%) of infection, followed by percutaneous metal staples (44%). All wounds (100%) closed with Vicryl suture became infected. The incidence of wound infection directly correlated with the level of quantitative bacterial count at analysis. The Insorb staple was associated with significantly reduced closure time, less inflammation and infection, and better aesthetic result compared to Vicryl. Compared to metal staples, the Insorb subcuticular staplers demonstrated comparable closure time without the need for later staple removal. In conclusion, the closure of contaminated wounds with the Insorb staples is a superior choice to Vicryl suture because they have a significantly (p = 0.009) lower incidence of infection. The Insorb staple is a revolutionary advance in subcuticular skin stapling.

Octyl 2-cyanoacrylate for repair of peripheral nerve.

The repair of peripheral nerves with sutures is time consuming. The aim of this study was to evaluate the benefits and functional outcome of repairing nerves with octyl 2-cyanoacrylate adhesive. The right peroneal nerve of 64 male, Lewis rats was sectioned and repaired. The rats were randomized into 3 experimental groups: A (n = 27), using only octyl 2-cyanoacrylate; B (n = 27), using 4, 10-0 nylon sutures; and C (n = 10), a sham operation. The recovery of nerve function was quantified through walking-track analyses; group A showed faster return of nerve function than B, especially at 15 days (P < 0.017). Histologic analysis showed a greater axonal regeneration in group A versus group B and no indication of tissue toxicity in group A. No dehiscence occurred during the 6-month study. Use of adhesive shortened the anastomosis time from 12 minutes to 4 minutes. These results indicate that the use of octyl 2-cyanoacrylate adhesive for nerve anastomoses is safe and effective and may have benefits compared with the use of sutures.

Evaluation of an absorbable cyanoacrylate adhesive as a suture line sealant.

BACKGROUND: Previous formulations of cyanoacrylate, though very effective, proved to have too high a tissue reactivity to be used internally. A novel cyanoacrylate compound with less tissue reactivity was recently developed. The objective of this study was to assess this novel cyanoacrylate compound for the use as vascular suture line sealant. MATERIALS AND METHODS: Twelve adult female sheep received a 6 mm PTFE interposition graft in each iliac artery, for a total of 24 grafts. Using oxidized cellulose (Surgicel) as a control, two formulations of a new cyanoacrylate compound (named "compound A" and "compound B") were assessed during this trial. Hemostatic efficiency was measured at the time of operation by the assessment of bleeding time and amount of blood loss. Long-term graft patency was assessed angiographically at 4, 6, and 18 months. Tissue reaction at 2 weeks, 1, 6, and 18 months was assessed grossly by vascular surgeons and microscopically by a blinded pathologist. RESULTS: Average time to hemostasis was 37.6, 50.6, and 219 s in group A, group B, and oxidized cellulose control groups, respectively (P

CAPROSYN*, another major advance in synthetic monofilament absorbable suture.

CAPROSYN* suture is the latest innovation in monofilament synthetic suture. This suture is prepared from POLYGLYTONE*6211, a synthetic polyester composed of glycolide, caprolactone, trimethylene carbonate, and lactide. The purpose of this study was to compare the biomechanical performance of CAPROSYN* suture to that of CHROMIC GUT suture. The biomechanical performance studies included quantitative measurements of wound security, strength loss, mass loss, potentiation of infection, tissue drag, knot security, knot rundown, as well as suture stiffness. Both CAPROSYN* and CHROMIC GUT sutures provided comparable resistance to wound disruption. Prior to implantation, suture loops of CAPROSYN* had a significantly greater mean breaking strength than suture loops of CHROMIC GUT. Three weeks after implantation of these absorbable suture loops, the sutures had no appreciable strength. The rate of loss of suture mass of these two sutures was similar. As expected, CHROMIC GUT sutures potentiated significantly more infection than did the CAPROSYN* sutures. The handling properties of the CAPROSYN* sutures were far superior to those of the CHROMIC GUT sutures. The smooth surface of the CAPROSYN* sutures encountered lower drag forces than did the CHROMIC GUT sutures. Furthermore, it was much easier to reposition the CAPROSYN* knotted sutures than the knotted CHROMIC GUT sutures. In the case of CHROMIC GUT sutures, it was not possible to reposition a two-throw granny knot. These biomechanical performance studies demonstrated the superior performance of synthetic CAPROSYN* sutures compared to CHROMIC GUT sutures and provide compelling evidence of why CAPROSYN* sutures are an excellent alternative to CHROMIC GUT sutures.