Successful Reconstruction of Full-Thickness Skin Defects in a Swine Model Using Simultaneous Split Thickness Skin Grafting and Composite Collagen Microstructured Dermal Scaffolds

Abstract

Reconstitution of normal skin anatomy after full thickness skin loss may be accomplished using a combination of a dermal regeneration template (DRT) and a split thickness skin graft (STSG). However, because of the relatively low rate of cell infiltration and vascularization of currently available DRTs, reconstruction is almost always performed in a two-step procedure over the course of several weeks, resulting in multiple dressing changes, prolonged immobilization, and increased chance of infection. To mitigate the potential complications of this prolonged process, the collagen-based dermal template DermiSphere^™ was developed and tested in a single-step procedure wherein DermiSphere and STSG were implanted simultaneously. When evaluated in a porcine, full thickness, excisional wound model, DermiSphere successfully supported simultaneous split thickness skin graft take and induced functional neodermal tissue deposition. When compared to a market leading product Integra Bilayer Wound Matrix, which was used in a multistep procedure (STSG placed 14 days after product implantation according to the product IFU), DermiSphere induced a similar moderate and transient inflammatory response that produced similar neodermal tissue maturity, thickness, and vascularity, despite being implanted in a single surgical procedure leading to wound closure 2 weeks earlier. These data suggest that DermiSphere may be implanted in a single-step procedure with an STSG, which would significantly shorten the time course required for the reconstruction of both dermal and epidermal components of skin after full thickness loss.

1. Introduction

Full thickness skin loss greater than a few square centimeters presents a common reconstructive challenge. Because of the relative paucity of donor sites available to provide full thickness skin, these wounds are traditionally reconstructed using split thickness skin grafts (STSG), which reconstitute the epithelial layer along with a relatively minor dermal component. Although this results in a closed wound, dermal tissue does not regenerate, and the lack of a sufficient dermal layer in the grafted skin may result in unstable coverage, or more commonly wound contracture over time that is associated with adverse functional and aesthetic consequences [1,2].

To address the significant clinical inadequacies of closing wounds with STSG that contain only a fraction of the normal dermal thickness, dermal templates were developed to promote the generation of a neodermal layer in situ on a suitable wound. The first commercially available dermal regeneration template (DRT), Integra Bilayer Matrix [3], was intended for use in large total body surface area burns but was quickly deployed by reconstructive surgeons in cases of full thickness skin loss from any cause [4–9], as it had the potential to regenerate dermis, thus obviating the shortcomings of STSG alone.

More than two decades of clinical use have demonstrated that Integra, when successfully engrafted onto the wound bed after full thickness skin loss, can provide improved functional and aesthetic outcomes versus STSG alone [10,11]. However, the structure of the material, derived from fibrillar collagen with a pore size of between 100–200 microns, does not allow for rapid cell infiltration and vascularization, such that the product IFU recommends waiting between 2–4 weeks after placement to ensure sufficient vascularization for it to receive a skin graft [12]. Thus, even under optimal conditions, patients must undergo two separate surgeries, spaced at least 2 weeks apart, to close their wound(s) with a reconstituted dermal and epidermal layer. During this time the wound must remain immobilized and clean, and throughout those first 2–4 weeks until it becomes sufficiently vascularized, the DRT is susceptible to infection [13].

A synchronous or “1-step” procedure in which STSG and DRT are applied simultaneously would overcome these shortcomings and provide significant clinical improvement in the treatment of full thickness skin loss. However, successful single step procedures using Integra over the past two decades have been limited to a handful of case reports because of the protracted time course of vascular infiltration of that specific DRT, which is insufficiently rapid to support a simultaneously engrafted STSG [14–18].

Considering these significant limitations, a novel collagen hydrogel scaffold containing embedded collagen spheres (DermiSphere^™) was developed with a unique microarchitecture that promotes significantly increased rapid cell invasion and vascularization. When evaluated in a rodent full-thickness skin excisional wound model, DermiSphere demonstrated well-developed vascularization throughout the 1.5mm thick matrix after only 7 days [19]. Such rapid vascularization prompted subsequent studies in which DermiSphere was placed simultaneously with a skin graft, resulting in excellent skin graft take and healed wounds with reconstituted dermal and epidermal layers in only one surgical step [19]. Inspired by these promising findings, the 1-step procedure was evaluated in a more clinically relevant porcine full thickness wound model.

2. Materials and Methods:

2.1. Fabrication of composite microsphere scaffolds (DermiSphere)

DermiSphere scaffolds (1.5 mm thickness) were prepared as described in previous work[19]. Briefly, 0.3% telocollagen (Symatese, Chaponost, France) was mixed with 1% atelocollagen (Symatese, Chaponost, France) microspheres at a defined ratio of microsphere : bulk. The mixture was then adjusted to physiologic pH and salt content, injected into molds, and cured into hydrogels for 16 h at 37 °C. Thermoplastic polyurethane (TPU) sheets (18411, SWM, Greenfield, MA) were placed on the hydrogel and the scaffold was terminally sterilized with irradiation (Steri-Tek, USA).

2.2. In vivo swine full thickness skin defect model

2.2.1. Animal studies

An in vivo swine full thickness skin defect model was used to evaluate the performance of the DermiSphere and Integra Bilayer Wound Matrix (Integra, Princeton, NJ). All procedures were done in accordance with Montefiore Medical Center policies and guidelines (per IACUC protocol # 21-02-101). Three female, 7-month-old Yucatan mini swine (>20 kg, Sinclair Bio Resources, Auxvasse, MO) were used for this study. Each pig received 8 full thickness skin wounds on the dorsum (N=8 per experimental group, N=4 on each side of the spine). Three experimental groups were evaluated: 1) DermiSphere/STSG autograft concurrently grafted (DermiSphere), 2) Integra Bilayer Wound Matrix (INT) followed by STSG application 14 days later (according to product IFU), and 3) split thickness skin autograft only (STSG-only) (Figure 1A). Treatments were distributed evenly among all wound sites to control for position of the wound on healing. For each group, a portion of these wounds (N=4) were harvested 7 days post STSG to histologically assess graft “take” (the percent area of transplanted skin that engrafted) and healing. The remaining (N=4) were allowed to heal for 21 days post STSG and were harvested at study termination.

Figure 1:

A) Experimental design. DermiSphere/STSG and STSG-only group followed a one-step procedure, in which grafting of the STSG occurred on the day of wound creation. INT group was utilized according to its IFU in a two-step procedure, where product was allowed to vascularize for 14 days prior to STSG. Wounds were evaluated 7- and 21-days post STSG grafting, and it is important to note that in this study design, INT had an additional 14 days to form neodermal tissue compared to DermiSphere/STSG and STSG-only groups. B) DermiSphere sandwiched between two sheets of thermoplastic polyurethane (TPU) prior to implantation (supplied between TPU sheets).

2.2.2. Surgical Procedure:

Pre-operative procedures:

Prior to surgery, pigs were anesthetized with atropine (0.04 mg/kg), telazol (4.4 mg/kg), propofol (3 mg/kg), and ketamine (2.2 mg/kg) intramuscularly and maintained under isoflurane (1–5%). Analgesia was achieved with buprenorphine (0.02 mg/kg). All wound sites were tattooed for evaluation of wound surface area.

Product grafting:

After tattooing, hair was clipped, and skin was prepped with betadine and ethanol. Using a guide, a 5 cm circle was traced inside the tattoo lines to mark points of incision. Full thickness skin wounds (5 cm diameter) were made using a 10-blade with care to make the wounds as uniform in depth as possible. Following removal of the incised tissue and hemostasis, subsequent steps were completed depending on the group as follows:

STSG-only and DermiSphere/STSG:

Both groups (1) STSG-only and (2) DermiSphere/STSG followed a “1-step”/simultaneous procedure, where (1) STSG autografts were applied directly onto the newly created full thickness wound bed or (2) DermiSphere was first applied onto the wound bed, immediately followed by the STSG (Figure 1A for schematics of the design). STSG (0.016 inch thickness) were harvested using an electric dermatome (Zimmer Biomet, Warsaw, IN) from the lower back of the pig and “pie-crusted” (graft was perforated) using an 11-blade (to prevent fluid accumulation under the graft). STSG donor sites were dressed with Duoderm (Convacec, Reading, United Kingdom). For the STSG-only group: STSG were placed into the wound, trimmed to size, and secured with surgical staples. For the DermiSphere/STSG group: DermiSphere was placed in the wound first, trimmed to size, and then immediately covered with an STSG. Grafts were further secured in place using a tie-over bolster [20]comprised of lubricating jelly and cotton in Xeroform (Covidien, Dublin, Ireland) impregnated with bacitracin (Dynarex, Orangeburg, NY). The bolstered wounds were covered with gauze and an occlusive dressing Tegaderm (3M, Saint Paul, MN). A final layer of fluffy undercast padding (Specialist, BSN Medical, Hamburg, Germany), vet wrap, and a tubular dressing (Curad, Medline, Northfield, IL) followed by a custom fitted jacket (Lomir, Montreal, Canada) was placed to optimize wound cleanliness and minimize the potential for graft shear.

Integra Bilayer Wound Matrix (INT):

INT was used according to its “indication for use” (IFU) label [12]. Immediately after wound creation, the INT bilayer product was “pie-crusted” using 11-blade, placed in the wound, trimmed to size, and secured as described above. Two weeks after initial placement of INT, the top silicone layer was gently removed following manufacturer instructions. An STSG was then applied, secured, and dressed as described above.

Post operative procedures:

Buprenorphine SR (0.2 mg/kg) and Draxxin (2.5 mg/kg) were administered subcutaneously until study termination. Seven days after STSG grafting tie-over bolsters were removed and the grafts were evaluated for take. Wounds were gently cleansed with saline and then redressed with bacitracin impregnated xeroform and covered as described above. Dressing changes were performed three times per week for the remainder of the experiment.

2.2.3. Evaluation of Graft “take” and wound healing/closure

Seven- and 21-days post STSG, graft take was scored (0: 0 – 20% of surface area; 1: 20 – 40 %; 3: 40 – 60%, 4: 60 – 80%; 5: 80 – 100%). Take was grossly assessed by examining the appearance of the graft and gently probing the tissue to ensure it was adherent to the wound bed.

2.2.4. Wound contraction evaluation

Wound contraction was evaluated by imaging wounds 0, 7, 14, and 21 days after STSG implantation. Using the tattoo as a reference, wound area was determined using image J software. Contraction was calculated by the formula:

$$\mathit{\text{CONTRACTION}}=\frac{\mathit{\text{WOUND}}\mathit{\text{AREA}}\mathit{\text{AT}}\mathit{\text{SPECIFIED}}\mathit{\text{TIMEPOINT}}}{\mathit{\text{WOUND}}\mathit{\text{AREA}}\mathit{\text{AT}}\mathit{\text{DAY}}0}\times 100$$

2.2.5. Explant procedure

Seven days after STSG implantation (7 total days for DS and STSG-only groups; and 21 total days for INT), pigs were anesthetized with ketamine induced isoflurane. Strip biopsies (0.5 cm × 6 cm) were taken from N=4 wounds in each experimental group. At 21 days after STSG implantation (21 days for DS and STSG-only, 35 days for INT), the study was terminated and remaining wounds were harvested. After removal of any excess fat, the excised wound specimens were placed in 10% neutral buffered formalin solution (Azer Scientific, Morgantown, PA) for fixation.

2.2.6. Histopathologic evaluation

All samples were processed and dehydrated through a series of alcohol and xylene exposures, followed by embedding in paraffin and staining. All tissue samples were mounted on slides and stained with hematoxylin and eosin (H&E). Digital scans of Hematoxylin/Eosin-stained sections were examined in a blinded fashion by an experienced, independent Board-Certified veterinary pathologist. Tissue response was evaluated based on a predetermined scoring metric adapted from ISO 10993 Part 6 Annex E that evaluates local effects, such as the evolution of the tissue response over time, after implantation of biomaterials intended for use in medical devices [21]. Scores were totaled to determine the relative immune response between treatment days (Table 1). Tissue maturity was scored based on the extent of mature collagen/immature collagen in the wound (Table 2).

Table 1:

ISO 10993 Part 6 Annex E

CELL TYPE/RESPONSE SCORE	0	1	2	3	4
Polymorphonuclear cells (PMNS)	0	Rare, 1–5/hpf	5–10/hpf	Heavy infiltrate	Packed
Lymphocytes	0	Rare, 1–5/hpf	5–10/hpf	Heavy infiltrate	Packed
Plasma cells	0	Rare, 1–5/hpf	5–10/hpf	Heavy infiltrate	Packed
Macrophages	0	Rare, 1–5/hpf	5–10/hpf	Heavy infiltrate	Packed
Giant cells	0	Rare, 1–2/hpf	3–5/hpf	Numerous	Sheets

Table 2:

Tissue maturity scoring rubric

Finding	Score	Description
Tissue Maturity (Wound)
	0	Wound unhealed, devoid of granulation tissue
	1	Wound filled with only immature tissue
	2	Wound filled by immature and mature collagen
	3	Wound mostly filled by mature collagen with some immature tissue
	4	Wound completely filled by mature collagen

2.2.7. Immunohistochemical evaluation

Immunohistochemistry was performed on a Bond III/Rx autostainer (Leica, Germany) with enzyme treatment using standard protocols. Antibodies used were rabbit polyclonal anti-pig CD31 (1:200, ab28364, Abcam, Cambridge, United Kingdom), rabbit polyclonal anti-human alpha-SMA (ab5694, Abcam, Cambridge, United Kingdom), mouse monoclonal anti-human involucrin (sc-21748, Santa Cruz Biotechnology, Dallas, Texas). Bond Polymer Refine Detection (Leica, Germany) was used according to the manufacturer’s protocol. After staining, sections were dehydrated and film coverslipped with a TissueTek-Prisma and Coverslipper (Sakura, USA). Whole slide scanning (40X) was performed on an Aperio AT2 (Leica, Germany).

2.2.8. Dermal thickness

Dermal thickness was defined as the distance from the bottom of the scaffold (when present) up to the top of the dermal layer of the STSG. Five random measurements were taken along the length of each wound and averaged.

2.3. Statistical analysis

Results are presented as mean ± standard deviations unless otherwise specified. Differences between multiple groups were determined using ANOVA tests. Multiple comparisons were conducted using Tukey hypothesis testing using GraphPad Prism software (Dotmatics, Boston, MA). Significance was set at a minimum p-value ≤ 0.05.

3. Results

3.1. Graft take, wound closure/healing, and wound contraction

Seven days after STSG implantation, all groups showed good graft “take” (average graft take scores: 4.32 ± 0.53, 4.88 ± 0.35, 4.75 ± 0.71 for DermiSphere, INT, and STSG-only, respectively, p > 0.5741) (Figure 2). Epidermolysis of the superficial epidermal components of the split-thickness skin grafts (consisting of the superficial epidermal/stratum corneum layers of the STSG) were seen in all groups beginning at day 7, which upon their removal revealed viable, healthy, mostly re-epithelized skin graft, which soon was completely healed. The grafts remained well taken and matured throughout the study, with full re-epithelization observed (grossly) on days 16.00 ± 2.83, 16.25 ± 1.5 and 12.0 ± 0.0 for DermiSphere, INT, and STSG only respectively. At harvest (21 days post-STSG), all groups grossly demonstrated dry, healed wounds with a mature epithelial layer on top (Figure 2). Quantitative evaluation of wound contraction showed no significant contraction (Figure S1) observed in DermiSphere (% area normalized to day 0 at 7d: 101.0 ± 6.7 %, 14d: 97.5 ± 11.4 %, 21d: 99.2 ± 9.0 %, p > 0.9169), INT (% area normalized to day 0 at 7d: 105.8 ± 10.5 %, 14d: 101.7 ± 2.7 %, 21d: 103.8 ± 1.0 %, p > 0.8385), and STSG-only (% area normalized to day 0 at 7d: 98.8 ± 11.3 %, 14d: 108.7 ± 15.1 %, 21d: 113.8 ± 14.8 %, p > 0.2612). No significant differences in contraction were observed between experimental groups (p > 0.1742).

Figure 2:

Wound appearance 7- and 21-days after STSG grafting: (A and D) DermiSphere: applied simultaneously with skin graft (successful STSG take and wound healing observed on day 7 (A) and day 21 (D) post full thickness wound creation); (B and E) Integra Bilayer Wound Matrix: product applied first and STSG grafted 14 days later (successful STSG take and wound healing observed on day 21 (B) and day 35 (E) post full thickness wound creation); (C and F) STSG alone applied directly on the wound bed (successful STSG take and wound healing observed on day 7 (C) and day 21 (F) post full thickness wound creation).

3.2. Histopathological evaluation and scoring

Wounds were excised, processed, and evaluated by an independent board-certified veterinary pathologist following guidance from ISO 10993 Annex E Part 6: Biological Evaluation of Medical Devices - Part 6: Tests for Local Effects After Implantation (See Materials and Methods for scoring details). H&E staining demonstrated viable split-thickness grafts in all groups 7 days after graft placement (Figure 3) with functional blood vessels (as determined by the presence of erythrocytes within vessel lumens) seen throughout both the dermal component of STSG as well as the subjacent dermal template in all three groups. Luminal expression of CD31 further confirmed the presence of blood vessels formed in DermiSphere and INT by day 7 after STSG (Figure 4). As was noted grossly, portions of devitalized superficial epidermis were seen sloughing in all three experimental groups on day 7 post-STSG, but an intact epithelial layer remained below. Involucrin expression was noted within the upper epidermal layer in all groups at 21 days, further substantiating the existence of an intact and fully functional epithelial layer (Figure 5).

Figure 3:

H&E stained histological sections from healing wounds on day 7 (A, B and C) and day 21 (D, E and F) post STSG grafting. DermiSphere (A and D) 7- and 21-days post product/STSG placement; INT (B and E) 7- and 21-days post STSG placement respectively (21- and 35-days post product); STSG only (C and F) 7- and 21-days post STSG placement. Healthy skin grafts seen in all groups on day 7 (denoted by “G”). Luminal structures associated with neovasculature seen in DermiSphere group (denoted by “V”). Newly formed neo-dermal tissue within dermal regeneration templates (denoted by arrows); thin dermal layer seen in STSG only group (dermal/neodermal tissue is defined by the dotted line).

Figure 4:

CD31 expression in healing wounds. Luminal structures expressing CD31 (indicative of new blood vessel formation, denoted by red arrows) were distributed throughout both scaffolds 7- and 21- days (Dermisphere) and 14- and 35- days (INT) after scaffold placement (STSG denoted by “G”).

Figure 5:

Involucrin expression on epidermal layer of wounds 21 days post-STSG grafting. Involucrin expression was present in the upper epithelial layer on all wounds at 21 days post-STSG grafting, indicative of proper maturation and stratification of this layer (denoted by brown staining as indicated by arrows).

Neodermal thickness, defined as the thickness of the newly formed tissue within the dermal templates between the wound bed and the inferior edge of the STSG, demonstrated that both DS and INT generated significantly thicker dermal tissue when compared to STSG-only, both 7 days (DS 2108.3 ± 287.8 mm, INT 1844.5 ± 242.54 mm, STSG-only 866.4 ± 249.7 mm, p < 0.0034) and 21 days after STSG (DS 2856.6 ± 607.9 mm, INT 2106 ± 445.0 mm, STSG-only 1158.6 ± 408.4 mm, p < 0.0053). There were no significant differences in dermal thickness between the DRTs (p > 0.1671) nor any differences within groups between different timepoints (p > 0.2382) within the same group (Figure 6).

Figure 6:

Neodermal thickness (in microns). Significantly thicker neodermal tissue formed by both dermal templates 7 and 21 days after grafting, when compared to STSG only group. Asterisks denote significant difference as assessed by Tukey’s multiple comparison’s test.

Neodermal tissue maturity was assessed semi-quantitatively as part of the histopathological scoring. Maturity, associated with the presence of immature and mature collagen, was scored significantly higher (p < 0.0001) in the dermal templates (DS and INT) compared to the STSG-only group (3.60 ± 0.52, 3.50 ± 0.53, and 2 ± 0 for DS, INT and STSG-only respectively). At day 21 after STSG, all three groups achieved equivalent levels (p > 0.3513) of neodermal tissue maturity according to pathological scoring (Figure 7).

Figure 7:

Assessment of tissue maturity and immune response within wounds. Tissue maturity, associated with the presence of mature and immature collagen were identical for DermiSphere and INT and significantly higher than STSG-only at Day 7. The immune response to DermiSphere scaffolds was quantified by a standard scoring metric outlined in ISO 10993 ISO 10993 part 6, Annex E. Immune response was transient for all groups, with a decrease in response from 7 to 21 days that is typical of the normal wound healing process.

Histopathological evaluation included assessment of the host immune response to the dermal template and/or STSGs (assessment of the presence/number of neutrophils, lymphocytes, macrophages, and giant cells per high-powered 400 × field). In all three groups, the total number of each of these inflammatory cells decreased from 7 to 21 days, suggesting that the inflammatory response was transient and consistent with the typical immune response in a healing wound where there is an elevated immune response on day 7 that largely subsides by day 21 (Figure 7).

Dermal myofibroblasts, as identified by α-SMA expression, were more prevalent in both INT and STSG-only groups, particularly at the interface between the graft and the wound, than in the DS group. Further, in INT and STSG-only the myofibroblasts demonstrated increased linear alignment which was largely absent in the DS wounds. In addition, luminal subendothelial expression of α-SMA was also noted and consistent with the presence of smooth muscle cells and pericytes associated with vascular structures (Figure 8).

Figure 8:

α-SMA expression 21 days post STSG grafting. α-SMA expressing myofibroblasts demonstrated increased number alignment in INT and STSG-only groups, while the DermiSphere group showed fewer myofibroblasts and minimal alignment (aligned, SMA-positive cells denoted with arrows). α-SMA expression was also noted in luminal structures within the sections demonstrating vascularization throughout.

4. Discussion

Skin is the largest organ of the human body and serves as the principal interface between the internal and external environments and as such it is quite vulnerable to damage. The thin outermost layer of skin, the epidermis, is capable of rapid regeneration whereas, the dermis, which is the layer that comprises most of the skin thickness and strength, does not regenerate and if the wound is “full thickness” (involving both epidermal and dermal layers) the dermis is irreversibly lost [22]. The gold standard for definitive treatment of large full thickness (epidermis and dermis) skin defects is a skin autograft, either “split thickness” (STSG) comprised of epidermis with a variable (usually small) amount of dermis or “full thickness” (FTSG) which contains both skin layers in their native proportions [23]. Although ideal for reconstituting all missing layers at their natural thickness, the primary limitation of FTSG is donor site availability [23]. STSG may be harvested from many more anatomic regions, and because they contain only a fraction of dermis, the donor sites may be re-harvested after they heal via re-epithelialization [23]. However, since the amount of dermis reconstituted correlates directly with improved functional and aesthetic outcomes, the major limitation of using STSG for skin resurfacing is the obligatory significant degree of secondary contracture and potential for hypertrophic scar formation that results from incorporating only a small portion of the dermis in the graft. As a result, patients reconstructed using STSG frequently suffer from the effects of the contracted, inextensible, and hypertrophic scars [23].

To overcome these limitations, dermal regeneration templates such as Integra’s Bilayer Wound Matrix have been used to prepare a neodermal layer on the wound bed prior to STSG, resulting in improved aesthetic and functional outcomes [11]. Being cell-free devices, they serve as a scaffold for the infiltration of cells from the wound bed and are transformed into a vascularized neodermis tissue, able to support a split thickness skin graft implanted as a second step surgery 14–28 days later. This 2 to 4-week period is critical for the overall success of the reconstruction and requires full immobilization and maintenance of an immaculately clean wound. While this device has been applied successfully numerous times over the course of more than two decades of use, it is also clear that the relatively slow rate of vascular invasion has major drawbacks including but not limited to: 1) mandatory two separate surgical procedures 2–4-weeks apart [12] 2) non-trivial risk of infection before the scaffold becomes incorporated [13] 3) low efficacy when placed into non-optimal wound beds (e.g. exposed tendon or hardware)[4].

A veritable holy grail of device based full thickness skin reconstructive procedures would be the ability to reconstitute both layers of skin in a single surgical procedure by applying the dermal template and split thickness skin graft simultaneously. This would not only obviate a second surgical procedure and all associated risks and costs, but it would also significantly reduce the time to wound closure by 2–4 weeks and reduce the incidence of infection and other morbidity related to the need for lengthy immobilization.

DermiSphere was developed with a unique microarchitecture that promotes robust cell infiltration and vascularization. A previous study in which DermiSphere and Integra Bilayer wound matrix (INT) were applied with a skin graft in a two-step procedure in a rodent model of full thickness skin loss demonstrated significantly more rapid and robust vascularization in DermiSphere compared to INT with resultant superior graft take (80.7±15.9% vs. 47.9±28.6% respectively) after 7 days [19]. Inspired by these promising results, a simultaneous application of DermiSphere and skin graft in a more clinically relevant swine study was designed. In the current study, DermiSphere was applied simultaneously with an STSG and was evaluated versus an STSG-only control and the INT dermal regeneration template. INT was used according to its IFU, which recommends waiting 14–28 days before removal of the silicone protective layer and placement of STSG [12]. Thus, INT was implanted on the day of wound creation and allowed to vascularize for 14 days as per IFU instructions before the silicone layer was removed and a STSG was applied. For both DermiSphere and STSG-only groups, STSG were applied either directly to DermiSphere over the wound or directly onto the wound (STSG-only) on the day of wound creation. (Figure 1) Because of the design of the experiment where INT needed two surgeries compared to the single stage used for DermiSphere and STSG, a single pig was assigned to each group. While this ensured that only one pig had to undergo multiple procedures, this meant that randomization of wounds was not possible and animal-specific factors could possibly confound the results.

The primary endpoint evaluated was STSG take with all comparisons made defining day 0 at STSG implantation. In this study design, it is important to note that INT had an additional two weeks to form neodermal tissue as opposed to the DermiSphere and STSG-only groups (Figure 1). Evaluation of graft take demonstrated no differences between any group at either 7 or 21 days after STSG application. Sloughing of the superficial epithelial layers of the STSG (epidermolysis) was seen starting at day 7 in all wounds in all three experimental groups, likely due to the use of a thick STSG of 16/1000 inch (vs 8–12/1000 inch in a typical human surgery). Nevertheless, all wounds were fully re-epithelialized by day 16. Good take was expected for the STSG-only group, where grafting occurs on a clean, surgically prepared, vascularized wound bed. It was also anticipated for INT, because in this study this product had two full weeks to become vascularized prior to autografting. Most significantly, DermiSphere was able to support a skin graft despite being implanted simultaneously, suggesting that already by 7 days post wound creation, DermiSphere had become vascularized throughout its 1.5mm thickness fast enough to allow for efficient anastomosis of the graft to neovessels within the underlying DS. These conclusions were further substantiated histologically, where a vast network of new blood vessels was distributed through all three groups, with erythrocytes evident in CD31 expressing luminal structures in both the neodermal tissue and the grafts (Figure 4).

Histologic sections were further evaluated by a blinded, board-certified veterinary pathologist to evaluate the inflammatory response, dermal thickness, and the overall quality of regenerated tissues. A moderate, but expected inflammatory response was seen 7 days after STSG across all groups, as demonstrated by the presence of an inflammatory infiltrate consisting of neutrophils, macrophages, lymphocytes, and giant cells. By 21 days after STSG, inflammation had largely subsided, indicative of normal wound healing within all groups.

At 7- and 21-days post STSG implantation, both DermiSphere and INT had significantly greater dermal thickness than the STSG-only control (Figure 6). This was not surprising and further emphasizes that dermal substitutes can generate thicker dermis than STSG alone, which is associated with better functional and aesthetic results. Most importantly, DermiSphere was able to generate a neodermal thickness at least equivalent to INT but required only one surgical step and 2 fewer weeks of healing time to do so. Semi-quantitative assessment of the neodermal tissue revealed the majority of neotissue was comprised of mature collagen in both DRTs, suggesting that DermiSphere produced dermal tissue at least equivalent to INT (Figure 7), Furthermore, histological analysis in the epidermal portion of the wounds revealed the formation of a functional epithelium at 21 days after STSG for all wounds. Involucrin expression was present in the upper epithelial layers, indicative of proper maturation and stratification of this layer. (Figure 5). Measurement of wound contraction revealed that none of the groups showed any significant signs of wound contraction up to 21 days after STSG implantation (Figure S1). While contraction was expected for the STSG group, assessment was likely performed too early in the healing process to discern any clinically relevant results. On the other hand, processes leading to contraction can be histologically suggested by myofibroblast cells, which are contractile cells rich in alpha-smooth muscle actin (α-SMA) [24]. Analysis of α-SMA expression at 21 days post-STSG revealed aligned myofibroblast cells in both INT and STSG-only groups, suggesting an ongoing or imminent contractile process present in the wound. In contrast, putative dermal myofibroblasts in DermiSphere were significantly fewer in number and did not demonstrate evidence of alignment (Figure 8). Despite the increased presence and alignment of smooth muscle cells in both INT and STSG-only groups compared to DS, all groups demonstrated minimal wound contraction likely because 21 days after grafting is too early to assess contraction and future work will assess these phenomena at later time points.

Taken together, these data demonstrate that DermiSphere can successfully support simultaneous STSG implantation, closing a wound and restoring a significant portion of the dermal thickness in a single surgical procedure. STSG implanted on top of the DermiSphere were well taken and viable, supported by a thick and well-vascularized subjacent neodermal tissue.

5. Conclusions

Presented here is DermiSphere - a microstructured collagen hydrogel scaffold embedded with collagen spheres for dermal tissue regeneration. When implanted in a porcine full-thickness excisional wound, DermiSphere successfully induced functional neodermal tissue deposition and supported simultaneous split thickness skin graft take. Further, DermiSphere was able to form neodermal tissue with a thickness and maturity similar to the market leading scaffold, despite having two fewer weeks to form the tissue and requiring only one surgical step. Although this device holds tremendous promise to change clinical practice by allowing surgeons to use a DRT to reconstitute both layers of skin in only one surgery, clinical evaluation will be necessary to verify such an application.

Supplementary Material

Supporting info

Figure S1: Assessment of wound contraction over time. No significant contraction was observed with no significant differences noted between groups at either timepoint.

List of Abbreviations

DRT

Dermal regeneration template

FTST

Full-thickness skin graft

IFU

Indication for use

INT

Integra Bilayer Wound Matrix

STSG

Split-thickness skin graft