TY - JOUR
AU - FRACS, John E. Greenwood, AM, BSc(Hons), MBChB, MD, DHlthSc, FRCS(Eng), FRCS(Plast),
AB - Abstract The aims were to 1) describe the in vivo studies leading to an optimized model of the biodegradable temporizing matrix (BTM), 2) describe our efforts in effecting closure over this optimized matrix after integration with a cultured composite skin (CCS), and 3) reexamine the ability of the CCS to definitively close fresh wounds (without BTM). Foam scaffolds of biodegradable polyurethane were created to allow in vivo tissue ingrowth or in vitro co-culture. Using the porcine surgical model, multiple BTM optimization studies took place before the BTM-CCS main study was conducted. For the CCS study, optimized sealed 2 mm matrices were implanted into 6-mm deep, 8 × 8 cm wounds (three per pig) and allowed to integrate for 21 days, whereas collected blood and harvested skin tissue were used to prepare autologous composite skins in similar (unsealed) 1 mm matrices. These were then applied at day 21 either over the integrated BTMs or into a freshly created fourth wound. All of the optimized matrices integrated fully, without loss, and were found to resist wound contraction effectively until the composites were ready for application at day 21. The composites demonstrated the ability to generate a bilayer repair with robust epidermis anchored by a basement membrane visible from day 7 after application. The final optimized sealed BTM delaminates easily to produce a clean, temporized wound bed and will be used in the upcoming burn clinical trial. Although the CCS is a magnitude away from human trials, it is still capable of generating a bilayer repair in both BTM-integrated and fresh wounds (onto fat), and with further refinement and optimization of foam structure, seeding densities, and timing, consistent success should be possible. Attempting to reproduce dermal and epidermal functions in deep wounds, where skin graft is not available, is a daunting prospect. In the extensive burn situation, failure to do so can result in death by sepsis or outcomes marred by delayed healing, poor or abnormal scarring, and joint contracture, deformity, and loss of function. With the creation of Integra Dermal Regeneration Template® (Integra Life-Sciences Corp., Plainsboro, NJ) by Burke and Yannas, the dermal matrix strategy for major burn care became possible. The silicone sheet sealing the matrix mimics physiological wound closure,1 whereas the collagen matrix component becomes integrated into the wound to create a stable and robust bed for subsequent grafting, thus improving both function and cosmesis.2,–4 Some issues with the strategy have reduced its potential globally and can be categorized into problems inherent with collagen matrices and problems related to the fact that split-skin graft is still required to effect definitive closure. Collagen-based matrices remain expensive for the end user.5 The biological collagen/glycosaminoglycan structure of the integrating matrix provides no resistance to bacterial infection6 and when combined with burn-induced immune compromise can result in catastrophic material loss and wound infection.7 The dermis is a mesodermal derivative composed predominantly of structural macromolecules. When these structures are lost, their preinjury structure cannot be reestablished. Instead they are repaired, irrespective of the guiding scaffolds that might be used. The basis of the dermal matrix strategy is to compartmentalize this developing dermal scar. Thus, the composition of the compartmentalizing matrix does not necessarily have to be biological. Our technology uses polyurethane chemistry, and the synthetic nature of the scaffolds allows flexibility of design that makes the technology more affordable, scalable, and controlled from a manufacturing perspective. The dermal matrix strategy enables early excision of extensive deep burn and functionally temporizes the wound. This allows time until donor site reharvest is possible while reducing wound contraction. Definitive biological closure is then required. This strategy, however, enables the subsequent split-skin graft required for closure to be much thinner than that usually harvested for closure of an excised burn wound because a “neodermis” has been provided. The split-skin graft allows rapid wound closure with variable wound contraction, dependent largely on its thickness (and thus the volume of dermal component). An intact stratum corneum facilitates a decrease in wound contraction by preventing evaporative water loss and shortening the inflammatory phase of wound healing. Rapid vascularization by inosculation within 48 hours of placement8 allows blood-delivered immune function, which augments the antimicrobial barrier defence provided by the intact epidermis. The split-skin graft is usually a reliable resource, allowing early wound closure in the burn-injured patient. However, in extensive burns, the shortage or complete lack of available donor sites precludes timely repair, whereas failure or retardation of donor site reepithelialization in the septic or compromised patient further complicates wound management. Biological skin substitutes have been engineered in an attempt to mimic the skin graft with the intention of eventual replacement. Historically, they are varied to mirror both dermal and epidermal properties. Tissue-engineered substitutes have become more widely used and reported upon,9 providing greater volumes of evidence to support their use. However, a globally available and affordable substitute that can replace the skin graft in any socioeconomic climate awaits development. In designing and producing materials and substitutes that can realistically replace, or at least reduce our reliance on, the autologous split-skin graft, a number of its advantageous properties have to be examined in detail and incorporated. In previous publications, we have described and illustrated our efforts in this field,10,–15 culminating in a recent proof-of-concept article.15 In this article, we report further design refinements, leading to a much improved and consistent outcome of the two-stage strategy. After optimization of the seal structure and its bonding to the biodegradable temporizing matrix (BTM), an assessment of the outcome produced by our cultured composite skin (CCS) on the integrated BTM was also sought. In addition, we wanted to investigate whether a CCS implanted into a fresh wound would lead to a bilayer repair because this would have implications for significant elective wound closure, such as that after giant hairy nevus excision. METHODS Biodegradable Temporizing Matrix Optimization Studies Groundwork studies investigating the BTM seal were required before the main study could commence. Ten variants of BTM seals consisted of wound generation and implantation, with repeated dressing changes followed by final seal delamination between days 21 and 28 and final wound evaluation. Fifteen pigs were used for the optimization studies. A total of 44 wound sites received BTMs with varying seal properties. The polymer and BTM structure itself were not modified for any of these studies. The BTM consisted of an integrating biodegradable polyurethane foam dermal matrix and a temporary, nonbiodegradable polyurethane seal (or lamina). The seal minimizes evaporative water loss and resists contraction during matrix integration. Delamination refers to the separation of the seal from the BTM. This process is usually planned by the surgeon when the dermal component has integrated fully and prepares the superficial surface of the neodermis for definitive closure. The seal variants ranged in thickness from 50 to 150 μm, some seals were perforated or nonperforated, and a commercial seal was also tested (Table 1). The seal/matrix bonding methods were also evaluated. Table 1. BTM seal optimization studies. View Large Table 1. BTM seal optimization studies. View Large Biodegradable Temporizing Matrix-Cultured Composite Skin Main Study Design Animals. This study was approved by the SA Pathology/CHN Animal Ethics Committee (AEC 56/11). Three large domestic pigs (Sus scrofa) initially weighing 36 kg were acclimatized for 1 week before study commencement. Housing and animal care were provided in accordance with the local Animal Welfare Act and National Health and Medical Research Council Guidelines.16 Sedation, anesthesia and analgesia during surgery, dressing changes, and biopsy procedures were performed as previously described.13,–15 Biodegradable Polyurethane Matrices. The BTM is a white bioabsorbable polyurethane foam, sealed with a transparent nonabsorbable polyurethane film. All BTM (sealed, 2 mm thick) and CCS matrices (unsealed, 1 mm thick) were sterile, dry-packed, and provided by PolyNovo Biomaterials Pty. Ltd (Port Melbourne, Victoria, Australia).15 Day 0: Skin Harvest and Biodegradable Temporizing Matrix Implantation. Four 8 × 8 cm study sites were designed (two on each flank of each animal). Thin split-thickness skin grafts were taken from each animal to provide the autologous components (fibroblasts and keratinocytes) for cell culture and composite construction. Blood (265 mL) from each animal was collected for the preparation of plasma and thrombin to aid in the generation of the composites. One site remained untreated until the day of CCS application; the other three sites were then created to the level of the panniculus adiposus, ensuring that no dermal elements remained. A sealed, 2-mm-thick BTM was then implanted into these deep wounds and held with surgical steel staples. All wounds were then dressed according to our established dressing protocol with Mepitel® (Mölnlycke, Gothenburg, Sweden) and Acticoat® (Smith & Nephew Ltd, Hull, UK) held with Hypafix (BSN Medical, Hamburg, Germany), overdressed with cotton combine also held with Hypafix, all protected by a custom-made pig coat. Wounds were assessed and dressed twice weekly until the CCS was ready for application on day 21. Day 21: Fresh Wound Creation and Cultured Composite Skin Application. The fourth site was excised to the same depth as the other three at day 21 and only received a CCS. At this same time point, the other three sites (where BTM had been implanted at day 0) were delaminated and the superficial surface abraded, ready to also receive a CCS. The composites were carefully applied, trimmed to size (if necessary), and affixed with surgical steel staples. A small Mepitel®, precut to each individual treatment area, was affixed rostrally with staples to minimize shear trauma of the composites. The first dressing change allowed retention of this underdressing. Standard dressing changes followed twice weekly. Cultured Composite Skin Generation (Days 0–21). For CCS manufacture, the established technique for cell isolation of keratinocytes and fibroblasts, plasma gel creation, and cellular seeding were used from previously described methods,15 with some modifications. These included an increase in fibroblast seeding density (6 × 104 cells per cm2) to enable a shorter fibroblast in-matrix culture time before co-culture, isolated cells were used from primary culture rather than passage 1, and the skin graft cell culture dishes (Nalge Nunc International, Rochester, NY) included four inner chambers into which the matrices were precut. The CCS was also ready for implantation on day 21 compared with day 28 in our previous study. One additional CCS was generated per pig for in vitro analysis. Wound Assessment and Analysis. All research animals were anesthetized with isoflurane with O2 for wound evaluation and assessment. Wounds and periwound areas were gently cleaned with sterile saline before evaluation. Following our standard assessment protocol, observations were recorded, photographs taken, and wound areas measured using the Visitrak™ system (Smith & Nephew Ltd, Hull, United Kingdom), with evaporative water loss being assessed by a Vapometer (Delfin Technologies Ltd., Helsinki, Finland).15 Throughout the study period, 4-mm punch biopsy specimens were obtained for histological assessment from central wound locations. Large, full-thickness excision biopsy specimens were collected at necropsy on day 52. All samples were fixed in 10% neutral-buffered formalin, dehydrated, and embedded in paraffin. Sections were cut at 5 μm and stained with standard hematoxylin and eosin and periodic acid-Schiff stains. Some sections were rehydrated for immunohistochemical staining with antibodies for keratin (BovK, 1:500; Dako, Z0622). Immunofluorescence using viability dyes (calcein/ethidium; Sigma, St. Louis, MO) was also performed on biopsy specimens taken from the additional CCS and viewed using the Nikon confocal microscope. To compare the wound area and evaporative water loss between treatments and over time, a linear mixed-effects model was fitted to the data. In the model treatment, time and the interaction between treatment and time were included as categorical predictor variables. Random effects for “pig” and “pig within treatment” were included in the model to account for the dependence in results because of repeated measurements on the same animal (multiple grafts and repeated measures over time). Post hoc analysis was also performed within each treatment group from the original wound size to the final time point to generate P values (Table 1). Post-Biodegradable Temporizing Matrix-Cultured Composite Skin Study: Optimization of Seal Lamination/Bonding A final seal optimization study (aiming to demonstrate easy, quick, one-piece seal delamination) followed the BTM-CCS study. In this study, BTMs with a different seal lamination process were analyzed in two pigs (eight treatment sites). All surgical procedures, dressing changes, and wound measurement were performed as previously described. RESULTS/DISCUSSION Optimization of the Sealed Biodegradable Temporizing Matrix The first groundwork study assessed seal type and thickness. Evidence of tissue ingrowth and matrix integration was clearly visible and seems to occur remarkably early (7–10 days). This may be a major advantage compared with currently available two-stage dermal matrices that can take 2 to 4 weeks.17 The 50- and 100-μm seals were flexible and thus easy to apply compared with the stiffer 150-μm seal. However, these thinner seals displayed some microfracturing. Despite the 50- and 150-μm seals producing the largest wound areas at day 28 (45.9 and 43.2 cm2, respectively; Table 1), the 50-μm seal was omitted from further analysis because of microfragmentation and overgranulation through the resultant fissures. The 150-μm seal was the preferred seal with regard to seal retention. A commercial seal (Tegaderm™; 3M Health Care, St. Paul, MN) was then tested and bonded by two different techniques (labeled commercial a and b in Table 1). Although soft, supple, and elastic, these became loosely detached by day 3 and completely lost by day 7. These were abandoned early because the spontaneous delamination resulted in minimal matrix integration and eventual loss of all foam matrices from all wound sites. In a larger pig study, the first using CCS, BTM seals based on the 150-μm seal resulted in fragmentation and wrinkling that produced a suboptimal wound bed for the application of CCS. Although proof of concept was attained, with composite take being achieved,15 further seal refinements were obviously still required. The next developmental stage was the assessment of whether perforated seals, to allow fluid drainage, would perform better than nonperforated seals in laminae of varying thickness (50 or 150 μm). Neither thickness nor the presence or absence of perforation alone could be demonstrated to affect wound contraction (final wound size compared with initial wound size). Early delamination with unacceptable wound contraction was noted in both animals by day 14 for certain wounds where others maintained the BTM seals and resisted contraction. After unblinding, it was revealed that the manufacturers had used a different method of seal bonding for those matrices, resulting in acceptable wound contraction (Figure 1, seal types highlighted by oval) compared with those allowing unacceptable wound contraction. Figure 1. View largeDownload slide Graphical representation for the optimization of the biodegradable temporizing matrix (BTM) seal. Wound area over time with different BTM seal properties and bonding. Error bars are the SEM. Figure 1. View largeDownload slide Graphical representation for the optimization of the biodegradable temporizing matrix (BTM) seal. Wound area over time with different BTM seal properties and bonding. Error bars are the SEM. Analysis of data from all the seal studies showed that final wound size correlated only with the ability of BTM to retain its seal, with early delamination allowing rapid wound contraction by the end of the study. The post hoc tests also indicated that most wound areas (regardless of seal type) had significantly contracted from their original wound size (P < 0.0001; Table 1). From all of the optimization studies, there was only one seal (thin + holes) that was not significantly different (P = 0.685), ie, there was no difference from starting wound size to end wound size (minimal contraction; Table 1). Figure 1 also demonstrates that the new bonding method used for this seal was the contributing factor for maintaining final wound size, irrespective of seal thickness or perforation. For evaporative water loss comparison, a high reading indicates a seal losing water compared with a lower reading that indicates that the wound is physiologically sealed. The evaporative water loss before delamination was not significantly different among all the different seals. All were, however, higher (Table 1) than normal skin (mean, 7.8 g/m2h), but none displayed the high readings noted from wounds left to heal by secondary intention or those of unsealed wounds (generally >130 g/m2h). This suggests that maintenance of the seal, reducing evaporative water loss and thus resisting wound contraction, is pivotal to the success of the BTM. Thus, for the BTM-CCS main study, a sealed BTM based on the new bonding method was used. Biodegradable Temporizing Matrix-Cultured Composite Skin Main Study Optimized Biodegradable Temporizing Matrix Ready for Cultured Composite Skin Application. The bonding of the polyurethane seal to the matrix reduced early delamination, abolished fragmentation, and restricted tissue ingrowth to the matrix only. Although the seal was intact, the BTM performed its design function of integrating and resisting wound contraction while preparing the wound bed for definitive closure (Figure 2). All BTMs integrated and maintained wound size (representative wound 65 to 62.1 cm2 with a 4.5% reduction in wound size; Figure 2) with acceptable evaporative water loss for 21 days (mean, 40 g/m2h; Table 1). The integrated, sealed BTM was well vascularized, flush with the wound edge, soft and pliable, and clinically as thick as the surrounding skin. The seal was easy to delaminate by gentle teasing with a “Velcro-like” action, leaving vascularized tissue below. The seal showed no signs of spontaneous delamination. During surgical delamination, the superficial surface of the polymer separated (retracting back into tissue), leaving a partially refreshed wound bed, requiring minimal abrasion to refresh the surface. The abraded surface of the delaminated BTM bled gently, ready for CCS application. Figure 2. View largeDownload slide Serial progression of an optimized biodegradable temporizing matrix before seal delamination/removal. A. Day 0, (B) day 3, (C) day 7, (D) day 10, (E) day 14 to (F) day 21 after surgery. Original wound area = 65 cm2; day 21 predelamination wound area = 63 cm2. Figure 2. View largeDownload slide Serial progression of an optimized biodegradable temporizing matrix before seal delamination/removal. A. Day 0, (B) day 3, (C) day 7, (D) day 10, (E) day 14 to (F) day 21 after surgery. Original wound area = 65 cm2; day 21 predelamination wound area = 63 cm2. Cultured Composite Skin Cultured Composite Skin Before Application (In Vitro). The fibroblasts showed typical bipolar morphology at both time points, and the keratinocytes typically demonstrated a cuboidal, honeycomb appearance (Figure 3). Histological sections and immunohistochemical staining demonstrated that the walls of the pores become lined with cells (Figure 3). However, cell distribution was not uniform and appeared to be sporadic, ie, some pores demonstrating few cells. By day 21 of in-matrix culture, the majority of keratinocytes were identified in the deeper levels of the CCS (within the lower 200–300 μm of the 1-mm composite). Figure 3. View largeDownload slide Cultured composite skin in vitro before application. A. Fibroblasts (Fbs) stained green (calcein), filling the polymer pore. B. Co-cultured fibroblasts and keratinocytes (Ks) lining the polymer wall (polymer scaffold stained red). C. Hematoxylin and eosin horizontal section demonstrates a single pore with keratinocytes bordering the polymer edge with central fibroblasts. D. Immunopositive staining (brown) for keratin (BovK). Figure 3. View largeDownload slide Cultured composite skin in vitro before application. A. Fibroblasts (Fbs) stained green (calcein), filling the polymer pore. B. Co-cultured fibroblasts and keratinocytes (Ks) lining the polymer wall (polymer scaffold stained red). C. Hematoxylin and eosin horizontal section demonstrates a single pore with keratinocytes bordering the polymer edge with central fibroblasts. D. Immunopositive staining (brown) for keratin (BovK). Cultured Composite Skin After Application on Biodegradable Temporizing Matrix–Implanted Wounds (In Vivo). Once the CCS had been applied to the temporized wound bed, two phenomena were noted, both of which had been observed in previous studies.15 CCS take: “Take” denotes that both matrix and cellular components are retained as part of the healed tissue. The degree of take varied within BTM-CCS–treated wounds; in general, this was observed early (by day 7 after application; Figure 4Ai–iv). By day 10, most of the polymer was evident in the hyperkeratotic layer external to a well-developed epidermis anchored by a basement membrane (Figure 4Bi–iv). In some sections, polymer microfragments were embedded deep into dermal invaginations of epidermis. By the study end point at day 31 after CCS application (day 52 from original wounding), there were sections of CCS that had been fully incorporated into the wound; however, the majority of the CCS foam had been shed because of epidermal sloughing, but it was left with a robust stratified epithelium with a well-developed basement membrane. Evaporative water loss from this wound at day 31 was relatively low (34.9 g/m2h), suggesting epithelial closure and stratum corneum development (Table 2). CCS as a cellular delivery vehicle (Figure 5Ai–iv): In the first few days after application, it seemed to develop a thin “carapace” over the wound (Figure 5Aiii). This structure could be removed easily at day 10. On removal, we discovered that a fully formed epithelium had developed on the underlying wound surface. This was confirmed with punch biopsies on day 10 that showed a well-developed epithelium and basement membrane (Figure 5Bi–iv). This leads us to surmise that the CCS delivers its cellular content and then persists (like a scab) until encouraged to separate by stratum corneum shedding from the underlying formed epidermis. Therefore, in this situation, the wound was virtually healed 10 days after CCS application. A decline in evaporative water loss readings also indicated wound closure and epithelium thickening over time, with a reading of 20.6 g/m2h on day 14, and by day 31 after application it was 10.1 g/m2h, close to that of normal skin (Table 2). Figure 4. View largeDownload slide Cultured composite skin (CCS) integration and “take” at (A) day 7 and (B) day 10 after application. (i) Central punch biopsy location; (ii) hematoxylin and eosin histology of punch biopsy sites; (iii) high magnification of CCS epithelium; (iv) periodic acid-Schiff section showing basement membrane (BM) development. BTM, biodegradable temporizing matrix. Figure 4. View largeDownload slide Cultured composite skin (CCS) integration and “take” at (A) day 7 and (B) day 10 after application. (i) Central punch biopsy location; (ii) hematoxylin and eosin histology of punch biopsy sites; (iii) high magnification of CCS epithelium; (iv) periodic acid-Schiff section showing basement membrane (BM) development. BTM, biodegradable temporizing matrix. Figure 5. View largeDownload slide Cultured composite skin (CCS) delivery vehicle after application at (A) day 7 and (B) day 10. (i) Central location of punch biopsy sites; (Aii) hematoxylin and eosin histology of punch biopsy sites; (Bii) high magnification of reepithelialization; (iii) high magnification of CCS epithelium; (iv) periodic acid-Schiff section showing basement membrane development (BM). BTM, biodegradable temporizing matrix. Figure 5. View largeDownload slide Cultured composite skin (CCS) delivery vehicle after application at (A) day 7 and (B) day 10. (i) Central location of punch biopsy sites; (Aii) hematoxylin and eosin histology of punch biopsy sites; (Bii) high magnification of reepithelialization; (iii) high magnification of CCS epithelium; (iv) periodic acid-Schiff section showing basement membrane development (BM). BTM, biodegradable temporizing matrix. Cultured Composite Skin Only (No Bio degradable Temporizing Matrix). CCS retention in the fresh wounds (no BTM) was observed in two of the three wounds. Within the first 3 days of application, one CCS treatment area was completely lost because of animal trauma, allowing it to heal by secondary intention. There were no residual polymer remnants. The clinical course appeared different where the CCS was implanted into the fresh wounds created at day 21 (Figure 6). By day 7 after CCS implantation, no clinical composite foam was visible, and the CCS had completely integrated within exuberant vascular tissue filling the wound to the flush point (Figure 6C). The beginning of epithelialization was evident at day 10 with individual islands/foci of epithelium visible (Figure 6D, E). A punch biopsy from one of these foci showed CCS incorporation with developing epithelium and basement membrane formation (Figure 6F–H). The epithelial foci became more complete as time progressed and were visible in all areas of the wound simultaneously, demonstrating that the origin of these epidermal islands was the CCS, not the wound edge. By day 31 after application (day 52, study end point), the CCS scaffold was completely integrated, with the majority disposed in the deep dermis abutting on subcutaneous fat. Clinically, the wound was almost completely reepithelialized (~95%), with a stratified squamous epithelium with continuous basement membrane and rete pegs. The evaporative water loss from this wound at day 31 was also indicative of wound closure (22.7 g/m2h; Table 2). Table 2. Representative evaporative water loss from the BTM-CCS main study View Large Table 2. Representative evaporative water loss from the BTM-CCS main study View Large Figure 6. View largeDownload slide Cultured composite skin (CCS) “take” on a freshly created deep wound at (A) day 21 (0), (B) day 24 (3), (C) day 28 (7), and (D) day 31 (10), (E) enlargement showing foci epithelium deposited from the CCS, (F) hematoxylin and eosin (H&E) histology of punch biopsy sites, (G) H&E (×10) staining of integrated CCS enveloped with epithelium at day 10, and (H) periodic acid-Schiff staining showing basement membrane (BM) development. Figure 6. View largeDownload slide Cultured composite skin (CCS) “take” on a freshly created deep wound at (A) day 21 (0), (B) day 24 (3), (C) day 28 (7), and (D) day 31 (10), (E) enlargement showing foci epithelium deposited from the CCS, (F) hematoxylin and eosin (H&E) histology of punch biopsy sites, (G) H&E (×10) staining of integrated CCS enveloped with epithelium at day 10, and (H) periodic acid-Schiff staining showing basement membrane (BM) development. This was different from the pattern of reepithelialization that progresses from the wound margins following early severe wound contraction in the wound allowed to heal by secondary intention (Figure 7Bi–v). By day 31 after wound generation, this wound had reduced from its original size by 64% compared with a wound that healed through the application of a BTM followed by CCS (with only a 19% reduction by day 52 after original wound creation). Although the healed wound with CCS remained visually pink to study end and clinically might not appear closed on photographs, histologically by day 17, the BTM-CCS had completely reepithelialized, with further epithelial thickening by study end point (day 31 after application; Figure 7Ai–iv). A completely healed wound with color akin to neighboring skin might have been more convincing, but time constraints and housing costs for maintaining pigs for an extended period (beyond the 52-day time point) were not feasible in these studies. Figure 7. View largeDownload slide Wound contraction comparison, starting at day 21 when (A) the cultured composite skin is applied to the integrated biodegradable temporizing matrix and (B) the fresh control wound is created and allowed to heal by secondary intention, (i) day 21 (0); (ii) day 31 (10); (iii) day 38 (17); (iv) day 42 (21); (v) day 52 (31) after wound creation Figure 7. View largeDownload slide Wound contraction comparison, starting at day 21 when (A) the cultured composite skin is applied to the integrated biodegradable temporizing matrix and (B) the fresh control wound is created and allowed to heal by secondary intention, (i) day 21 (0); (ii) day 31 (10); (iii) day 38 (17); (iv) day 42 (21); (v) day 52 (31) after wound creation Previously, we have shown that a suboptimal integrated BTM wound bed will support CCS take.15 Here, we show that the CCS was demonstrably capable of generating a bilayer repair over both the abraded surface of the fully integrated BTM and the fresh wound's fat base. The mode of action of the CCS, ie, take or cellular delivery, seemed to depend on the deposition of the cells within the composite. Regardless, wound closure needs to occur within the first 7 to 10 days after application to ensure that the granulation layer generated between CCS and integrated BTM is kept to a minimum, in turn determining the degree of contraction. Post-Biodegradable Temporizing Matrix-Cultured Composite Skin Study: Seal Lamination/Bonding optimized The pilot human BTM trial (that used the same BTM as the BTM-CCS main study) revealed that unlike the porcine model, the seal tore on delamination and had to be removed in more than one action. This made the removal process unacceptably time-consuming, warranting further seal structure/bond optimization. Therefore, another study was performed to assess a new BTM seal that had been designed to resolve the issues with seal fragmentation on delamination. The new materials were easy to cut and affix and felt slightly more robust than previous iterations. By day 3 after implantation, all matrices were colored by vascular fluid but lacked the solid appearance of tissue ingrowth. The seals showed no tearing at staple sites, fragmentation, or signs of early delamination. By day 7, the BTM color was much darker with obscurity of the polymer foam structure, indicating solidity of the contents and tissue integration. Some wound contraction was evident in the shoulder treatment areas, typically seen in this model. Three treatment sites were removed from analysis because of repeated trauma (at both flank and shoulder areas), causing the seal to delaminate from day 21, such that they could not be included in determining the final mean. By day 28, the largest wound area was 55.3 cm2, a 12% reduction from its original size (63.5 cm2). The mean wound area (n = 5) for the new seals was 64.3 cm2 after creation, and by day 28 it was 53 cm2 (18% contraction). This is lower than those reported in a study that compared a number of commercially available dermal matrices also in a porcine model (Integra® [Integra Life-Sciences, Plainsboro, NJ], 25%; Hyalomatrix® [Anika Therapeutics, Bedford, MA], 24%; Matriderm® [Dr Suwelack Skin & Healthcare, Billerbeck, Germany], 28%; Renoskin® [Groupe Perouse Plastie, Issy Les Moulineaux, France], 34%).18 Although a direct comparison of BTM against other dermal matrices was not performed, our data showed that the wound contraction rate of BTM was comparable with a split-thickness graft applied at the same time point13 and was significantly lower than a secondary intention healing wound (up to 55% reduction in wound area during a 21-day period).13 On delamination at day 28, the seal was removed easily and rapidly in one single piece and action, leaving behind a highly vascularized and clean wound bed (Figure 8A, B). Histological analysis of the punch biopsy specimen before delamination revealed seal/matrix adhesion with no significant development of a scar layer between the seal and the polymer matrix. The BTMs were closely apposed to the fat in the original wound base (Figure 8C). Evaporative water loss from the sealed matrices during the period of the study ranged between 10 and 17 g/m2h (mean, 13.84 g/m2h), with normal skin averaging 4.4 g/m2h. This outperformed the previous best BTM seal (40 g/m2h) and demonstrated that the seals had successfully mimicked wound closure. Furthermore, the results of the evaporative water loss from the optimized sealed BTM were also similar to a split-thickness graft13 and clearly less than a wound allowed to heal by secondary intention or an unsealed matrix,13,–15 demonstrating the suitability of BTM as a dermal substitute. Figure 8. View largeDownload slide Integrated biodegradable temporizing matrix (BTM) and delamination of new seal. A. Seal being delaminated at day 28, (B) highly vascularized “neodermis” before dermabrasion; (C) hematoxylin and eosin section showing seal attachment and minimal ingrowth between the seal and the 2-mm matrix. Figure 8. View largeDownload slide Integrated biodegradable temporizing matrix (BTM) and delamination of new seal. A. Seal being delaminated at day 28, (B) highly vascularized “neodermis” before dermabrasion; (C) hematoxylin and eosin section showing seal attachment and minimal ingrowth between the seal and the 2-mm matrix. CONCLUSIONS The final optimized sealed BTM delaminates more easily (but not spontaneously) and generates a clean, temporized wound bed with negligible contraction and has received ethical committee approval for use in significant burn injuries. Although the cultured composite skins still require substantial development to attain human trials, they were still capable of generating a bilayer repair in both BTM-integrated and fresh wounds (onto fat). Ultimately, a CCS with a stratified epithelium generated before application that takes within days is the most favorable outcome. This would also have implications for elective wound closure procedures because the 1-mm CCS can be premade from a biopsy before surgical excision of a lesion. However, in significant burn injuries, the time to produce the CCS means that alone it is not suitable for deep wound closure, and a two-stage strategy is still required to ensure the best outcome for the patient aesthetically, functionally, and symptomatologically. This study was supported by a grant from BioInnovation SA, a South Australian Government-affiliated grant funding body. The NovoSorb™ biodegradable polyurethane platform is produced by PolyNovo Biomaterials Pty. Ltd. based in Port Melbourne, Victoria, Australia. NovoSkin Pty. Ltd. is a joint venture company established to investigate the role of NovoSorb™ in deep burn wounds. Associate Professor Greenwood owns a 20% share in NovoSkin Pty. Ltd., and the remaining 80% is owned by PolyNovo Pty. Ltd. ACKNOWLEDGMENTS We thank Dr John Finnie, Veterinary Pathologist, SA Pathology, for his help with interpreting the histological findings. General veterinary and animal support was forthcoming from Dr. Tim Kuchel and Dr. Sue Porter of the Preclinical, Imaging and Research Laboratories at South Australian Health and Medical Research Institute. Ms. Loren Matthews expertly delivered animal anesthesia and invaluable hands-on care of our animals. We thank Professor Alison Cowin's group for loan of the Vapometer used in these studies and Tom Sullivan, Statistician within the Data Management & Analysis Centre, Discipline of Public Health, University of Adelaide, for his assistance with the statistical analysis. REFERENCES 1. Greenwood JE, Wagstaff MJ, Mackie IP, Mustoe TA. Silicone action in the open wound: a hypothesis. J Burn Care Res. 2012;33:1–4. 2. Heimbach D, Luterman A, Burke J, et al. Artificial dermis for major burns. 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Split skin graft application over an integrating, biodegradable temporizing polymer matrix: immediate and delayed. J Burn Care Res. 2012;33:7–19. 14. Greenwood JE, Dearman BL. Comparison of a sealed, polymer foam biodegradable temporizing matrix against Integra® dermal regeneration template in a porcine wound model. J Burn Care Res. 2012;33:163–73. 15. Dearman BL, Stefani K, Li A, Greenwood JE. “Take” of a polymer-based autologous cultured composite “skin” on an integrated temporizing dermal matrix: proof of concept. J Burn Care Res. 2013;34:151–60. 16. National Health and Medical Research Council (NHMRC). Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. 20047th ed. Canberra Australian Government National Health and Medical Research Council (NHMRC. 17. Muller M, Gahankari D, Herndon DN.Herndon DN. Chapter 13: operative wound management. Total burn care. 20073rd ed. Philadelphia Sanders Elsevier;:177. 18. 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TI - Optimization of a Polyurethane Dermal Matrix and Experience With a Polymer-Based Cultured Composite Skin
JF - Journal of Burn Care & Research
DO - 10.1097/BCR.0000000000000061
DA - 2014-09-01
UR - https://www.deepdyve.com/lp/oxford-university-press/optimization-of-a-polyurethane-dermal-matrix-and-experience-with-a-0d8g5vs4y3
SP - 437
EP - 448
VL - 35
IS - 5
DP - DeepDyve
ER -