Effectiveness of wound healing using the novel collagen dermal substitute INSUREGRAF®

Abstract

Collagen sponges are often used as dermal substitutes in the treatment of burns, trauma, infections, and wounds. Dermal substitutes that can be applied in a one-stage operation are particularly important for dermal regeneration. Some protocols for the production of collagen sponges have been developed, but many issues remain, including low yield, contraction, and expense. In this study, the effectiveness of two skin substitutes was evaluated. Specifically, we compared two thin matrices, i.e., the newly developed INSUREGRAF® 1.2 mm and the widely used Matriderm® 1 mm Single Layer, with respect to their biochemical and mechanical properties, safety, and efficacy. We examined the rate of contractibility and biocompatibility using in vitro and in vivo models. The INSUREGRAF had an interconnected pore structure, which affects cell attachment and proper vascularization. Accordingly, this novel collagen sponge type has the potential to promote skin tissue regeneration and is especially suitable for full-thickness skin defects as a one-stage operation substitute.

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1 Introduction

Tissue engineering is a new technology used to repair or regenerate defects in tissues and organs; it integrates research in many fields, including biology, genetics, material engineering, and medical science.¹ For the regeneration and reconstruction of damaged tissues and organs, an engineered tissue scaffold has to meet several requirements: (1) biocompatibility to prevent rejection responses after implant surgery, (2) a suitable surface chemistry to enhance cell attachment and proliferation, (3) an interconnected pore structure, including apposite pore sizes for cell infiltration (migration) and revascularization, (4) controlled biodegradability to mediate new tissue formation, and (5) sufficient mechanical properties to maintain structure in order to support and retain tissue regeneration immediately after implant placement.^2–8 A regenerative scaffold that meets these conditions can be used in various tissues and organs, such as the heart,⁹ blood vessel,¹⁰ kidney,¹¹ bone,¹² skin,¹³ etc.¹²

The skin functions as a protective barrier against the external environment and prevents water loss. It is damaged by bruises, stab wounds, lacerations, burns, etc. Thermal wounds are the most frequent skin injury. Damage from thermal wounds can be classified into 4 types, epidermal, superficial partial-thickness, deep partial-thickness, and full thickness, according to the depth of the injury. Damaged skin must be reconstructed with skin substitutes to maintain the protective effects.^6,7

Skin substitutes, such as Matriderm® (Germany) and Integra (USA), are used as dressings for skin wounds. These artificial dermises are fabricated by conventional methods, such as freeze drying techniques, using collagen. Skin substitutes have important roles in the treatment of deep dermal and full-thickness wounds of various etiologies. They may allow the construction of a more natural dermis with excellent re-epithelialization characteristics due to the basement membrane. When developing skin substitutes, scaffold composition can be controlled. The ultimate goal is to achieve an ideal skin substitute that provides effective and scar-free wound healing.^14–17

Skin grafting methods using an artificial dermis can be separated into two types, full-thickness skin grafts (FTSG) and split-thickness skin grafts (STSG). The FTSG is generally used for the reconstruction of full-thickness defects, small wounds, and joint areas. The STSG is used for coverage of large-sized defects. When a large-sized skin defect occurs (a defect size of >30% of the total body surface), donor sites can be limited. After skin grafting, the defective skin proceeds to the normal wound healing process.^18,19

Generally, wound healing requires both the restoration of cover by re-epithelialization, and support by the inflow of collagen. Re-epithelialization occurs by the migration and proliferation of keratinocytes from wound edges and by the differentiation of stem cells from the remaining hair follicle bulbs. The inflow of collagen occurs by the influx of growth factors secreted by macrophages, platelets, and fibroblasts, and by fibroblast proliferation and the subsequent synthesis and remodelling of the collagenous dermal matrix. However, in the case of full-thickness acute burn injuries and chronic wounds, these processes, and the natural healing ability of the skin, are severely limited. Thus, new technologies are being developed to improve healing in these conditions.

Collagen is a major component of skin substitutes. It is a key component of all connective tissues and the most abundant protein in mammals. In particular, type I collagen is present in the skin and has the ability to promote cell attachment, cell proliferation, and wound healing.

In this study, a novel collagen sponge (INSUREGRAF®) was compared with a previous collagen sponge (Matriderm®) in vitro and in vivo using a porcine model, with a particular focus on the time period needed for sufficient matrix vascularization to allow epidermal graft take and contraction and on the thickness and architecture of the neodermis. The results of this study support the use of INSUREGRAF® to accelerate wound healing in the skin.

2 Experimental

2.1. Preparation of one-stage operation dermal substitutes (OSDS)

OSDS were fabricated from a purified porcine skin type I collagen (Matrixen-PSC; Bioland, Korea) solution using a freeze-drying method. The collagen solution was solidified by freezing under ultra-low cryogenic conditions. Each component consisted of a rectangular copper mould and an insulation cover, and was directly semi-immersed in liquid nitrogen. The copper mould was maintained in an extremely low-temperature chamber (−196 °C) for 60 min. After solidification, the frozen collagen with ice crystals was stored for at least 5 h in a freezer at −40 °C. Subsequently, the freeze-dried collagen sponges were put in a vacuum oven and maintained in vacuo at room temperature for 2 h to remove trace amounts of water; subsequently, the vacuum was sustained at a temperature of up to 100 °C. After 24 h, it was cooled to room temperature, and the vacuum was purged. In the DHT (dehydrothermal treatment) step, the crosslinked collagen sponge was obtained from the freeze-dried sponge, stabilizing its complex structure and suppressing structural changes. Then, the 1.2 mm-thick collagen sponge was obtained by slicing with a razor blade. The following two OSDS were compared, both of which were used in a one-step procedure.

- Matriderm® (MedSkin Solution Dr Suwelack AG, Billerbeck, Germany) is a 1 mm-thick lyophilized single-laminar matrix of bovine collagen type 1, 3, and 5 with elastin; it is used as a dermal substitute in a one-stage operation.²⁰

- INSUREGRAF® (Bioland) is a 1.2 mm-thick porcine collagen type 1 matrix.

2.2. Scanning electron microscopy

The surface morphology of OSDS with and without cells and cross-sectional images were assessed by scanning electron microscopy (SEM; JSM-5160, JEOL, Tokyo, Japan). The samples were washed with PBS (phosphate-buffered saline) three times and fixed in 10% formalin in PBS (pH 7.4) overnight at 4 °C. The cells were then rinsed in PBS three times, dehydrated in a graded series of alcohol, and subsequently critical point dried. They were then coated with a thin layer of Ag for observation under a scanning electron microscope operated at 10 kV. The INSUREGRAF® pore structure was compared with that of the well-known commercialized product Matriderm® (MedSkin Solutions Dr Suwelack AG).

2.3. Pore size and porosity measurements

OSDS porosity was measured using automatic mercury intrusion porosimetry (PoreMaster®, Quantachrome Instruments, Ltd., Boynton Beach, FL, USA). Three accurately weighed samples were measured (8 mm diameter, 1–1.2 mm triplicate). The average pore diameter of the scaffolds was measured using image analysis software (ImageJ; NIH, Bethesda, MD, USA).

2.4. Tensile strength

The OSDS were cut into pieces of 30 × 10 mm. The tensile strengths of the pieces of OSDS were determined using the LF Plus, NEXYGEN (LLOYD INSTRUMENTS, Bognor Regis, UK) at room temperature with an extension rate of 1 mm min⁻¹ until the specimen ruptured.

2.5. Water uptake test

Samples of the required size were weighed and immersed in PBS (pH 7.4) at room temperature for 5 min. The samples were then removed from the PBS and the unabsorbed solution was removed by aspiration. The wet OSDS were immediately weighed. The water uptake ratio (WUR) was calculated using the following equation:
where W_a is the average weight before absorption and W_b is the average weight after absorption.

2.6. Resistance to collagenase and trypsin

Degradation of collagen fibres in collagenase (type 1; Sigma Aldrich, St. Louis, MO, USA) and trypsin (from the porcine pancreas, Sigma Aldrich) solutions were measured (n = 2 per group) to estimate the extent of cross linking and denaturation, respectively. Solubility in collagenase was measured by incubating a 10 mg sample in 2 mL of 200 units of collagenase and 0.05 M CaCl₂ in a 0.1 M Tris-HCl solution (pH 7.4) at 37 °C for 1 and 2 h. The solubility of trypsin was measured by incubating a 5 mg sample in 2 mL of a 0.1 M ammonium bicarbonate solution (pH 7.5) containing 2000 units of trypsin (from the porcine pancreas, Sigma-Aldrich) at 37 °C for 2 and 24 h.

2.7. Cells proliferation assays

L929 fibroblasts (ATCC cell line CCL 1, NCTC clone 929) were cultured in Dulbecco's modified Eagle's medium and supplemented with 10% foetal bovine serum and antibiotics (100 IU mL⁻¹ penicillin, 0.1 g mL⁻¹ Fungizone). Cells were maintained at 37 °C in a humidified incubator with 5% CO₂ for 24 h, until a monolayer with greater than 80% confluence was obtained. The medium was changed every 2–3 days.

When the cells in the culture flasks became confluent, cell proliferation and morphology were evaluated. Cells (2 × 10⁴ cells per cm²) were seeded on a tissue culture plate (TCP), and divided into the INSUREGRAF® and Matriderm® disc (Ø 8 mm) groups (n = 3). After 4 h, non-adherent cells were removed by medium exchange and/or transfer to a 24-well plate and fresh medium was added to each well. The medium was changed every 2 days until 7 days.

Cell adhesion and proliferation were determined using the Cell Counting Kit-8 (CCK-8) (Dojindo Molecular Technologies, Kumamoto, Japan) based on metabolic activity. At each time point, cultured cells were washed with PBS and transferred to new culture plates. They were incubated with 400 μL of CCK-8 solution, which was mixed at a ratio of 10% in culture medium, for 3 h at 37 °C. The 100 μL reactant was transferred to a 96-well plate and absorbance was measured using a microplate reader (Sunrise™, Tecan, Mannedorf, Switzerland) at 450 nm.

2.8. Histology and immunocytochemistry

To observe the shape and morphology of cells after adhesion to the OSDS, hematoxylin and eosin (H&E) staining and immunocytochemistry were performed. For H&E staining, cells cultured on OSDS were fixed with 3.7% formaldehyde solution in 0.1 M PBS (pH 7.4) for 5 min at room temperature, embedded in paraffin, sectioned (3 μm), and mounted on slides. Serial sections were deparaffinised and stained with H&E. Specimens were then observed by light microscopy (Olympus BX51; Olympus Optical Co., Ltd., Tokyo, Japan).

For immunocytochemistry, sections from cells cultured OSDS were fixed with 3.7% formaldehyde solution in PBS for 5 min at room temperature, followed by extraction with 0.1% Triton X-100 in PBS for 5 min. Alexa Fluor 488 phalloidin and DAPI (Invitrogen, Life Technologies, Carlsbad, CA, USA) were used to stained filamentous actin (F-actin) and nuclei, respectively. Slides were observed using a confocal microscope (Zeiss LSM 510 Meta NLO; Carl Zeiss Microimaging, Thornwood, NY, USA).

2.9. Biocompatibility and efficacy of the OSDS

Miniature pigs were used to confirm the biocompatibility and efficacy of OSDS. For the treatment of full-thickness skin defects, the animals were divided into two groups, a control group (Matriderm®) and an experimental group (INSUREGRAF®), and a one-stage operation surgery was performed.

Four adult (10 month-old) female miniature pigs (CRONEX Co., Ltd., Hwaseong, South Korea) were obtained from a CRONEX closed-barrier facility and were housed in single pens under a controlled environment following Good Laboratory Practice conditions. The animals were allowed two weeks of pre-assessment and acclimatization before the start of the experimental procedures. Food was withheld for at least 6–8 h prior to the administration of anaesthesia. Atropine (0.05 mg kg⁻¹) and a half dose of the combination of 5–25 mg kg⁻¹ tiletamine-zolazepam (Zoletil®, Virbac Animal Health, Carros, France) and 2 mg kg⁻¹ xylazine (Narcosyl®, Merial, Duluth, GA, USA) were given intramuscularly for pre-anaesthesia. General anaesthesia was induced by administering the remaining half dose of Zoletil® and Narcosyl® by intramuscular or intravenous injection. After positioning the animal to ventral recumbency, an appropriately sized (6.5 mm) endotracheal tube was inserted to maintain anaesthesia using a gas anaesthetic machine. Anaesthesia was maintained by isoflurane (Ifran®, Hana Pharm Co., Ltd., Seoul, South Korea) with oxygen, 1 : 1 (5–10 mL kg⁻¹ min). Intravenous hydration with normal saline was maintained through a superficial auricular vein (25 mL h⁻¹). For the final preparation, the animals were shaved in the experimental area and sterilized with 20% povidone solution (Betadine solution).

The in vivo effects of the collagen sponge on wound healing were assayed using a porcine full-thickness skin defect model, as shown in Fig. 7. Each pig was tattooed with a rectangular shape (3 × 6 cm) on the paraspinal space to observe skin contraction during the experimental period. First, dermatome was used to harvest a 0.2 mm split-thickness skin sample, and the harvested skin was soaked in PBS. After skin harvesting, a wound was made by an incision until fascia in order to obtain a full-thickness skin defect. After the bleeding was stopped, a hydrated 3 × 3 cm OSDS was transplanted to the wound using a skin stapler. The harvested skin covered the OSDS to protect the wound site. After operating, the wound was dressed using foam dressing materials (Allevyn®, Smith & Nephew, London, UK). To prevent contamination and OSDS loss, pigs were clothed with meshed pressure garments.

2.10. Statistical analysis

Results are expressed as means ± standard deviation.

3 Results and discussion

3.1. Characteristics of OSDS

We analysed the porous structures of INSUREGRAF® and Matriderm® by SEM. Interconnected micropores were detected throughout the collagen sponges (Fig. 1a and b). The collagen fibres and pores were distributed homogenously, as shown in Fig. 1c and d. The pore structure formed in the vertical direction, and was generally parallel within the collagen sponge using the freeze-drying method.^21,22

Fig. 1 Characterization of OSDS. Morphological comparison obserbation by SEM (a–f). The top surfaces (a, b) and cross-sections (c, d) and high magnification images (e, f) of the OSDS (a, c, e; INSUREGRAF®, b, d, f; Matriderm®) presented pore structure of a unidirectionally solidified and freeze-dried collagen sponge. The pore structure built up from parallel collagen layers connected by single collagen fivers and sizes are very uniform.

The pore distribution and porosity of INSUREGRAF® were similar to those of Matriderm®, which can be mainly adjusted from 20 to 90 μm (Fig. 2b). High porosity values (98.23% and 98.20%) were obtained for INSUREGRAF® and Matriderm®, respectively (Fig. 2c). The average pore diameter of INSUREGRAF® was 38.6 ± 8.9 μm based on SEM images, which was smaller than that of Matriderm® (Fig. 2c). However, the average surface area of INSUREGRAF® was higher than that of Matriderm® (Fig. 2d). For INSUREGRAF®, it was estimated that the tensile strength of the OSDS was similar to that as a result correlated with the thickness of the cross-sectional area, though it was lower than that of Matriderm® (Fig. 2e).

Fig. 2 The average pore size was measured parallel to the c-axis of the plate-like ice crystals, because the behavior of biological cells in collagen matrix depends on the smallest size of pores. The pore size can be adjusted in the range from 20–80 μm (a), pore distribution by porosimeter (b), pore size and porosity (c), surface area (d), and mechanical strength (e).

The porosity and pore size distribution are important determinants of cell proliferation, migration, and nutritional support. An interconnected pore structure can accelerate cell migration, vascularization, nutrient exchange, and the flow of bio-factors.²³ These morphological characteristics influence the properties of OSDS. If pores are too small, cells cannot migrate toward the centre of the construct, limiting the diffusion of nutrients and the removal of waste products. Conversely, if pores are too large, the availability of specific surface area decreases, limiting cell attachment. Cellular activity is influenced by specific integrin–ligand interactions between cells and the surrounding extracellular matrix. Therefore, we inferred that INSUREGRAF® is superior to Matriderm® because although they shared a similar structure, INSUREGRAF® had a larger surface area for the migration and attachment of cells.

3.2. In vitro collagen degradation and trypsin resistance test

The collagen degradation test presents an in vitro measure of the degradation rate for a biological implant. To compare the biodegradation degree of the collagen scaffolds (INSUREGRAF® and Matriderm®), collagenase solution was added, followed by incubation for 2 h at 37 °C. INSUREGRAF® and Matriderm® exhibited thoroughly biodegradation.

A trypsin resistance test was used to evaluate the denaturation of collagen by measuring the degradation degree. The native collagen protein is minimally digested by enzymes due to its stable triple helix, but the denatured collagen protein is easily attacked by trypsin.

As shown in Fig. 3b, Matriderm® was completely degraded after 24 h in trypsin solution at 37 °C. In contrast, INSUREGRAF® maintained its form, without degradation. These results suggest that INSUREGRAF® is composed of high-purity collagen and has favourable biodegradability.

Fig. 3 Degradation behavior of the INSUREGRAF® and Matriderm® in collagenase (a) and trypsin resistance test (b).

3.3. Water uptake ratio of matrix

The goal of OSDS is to replace the dermis, absorb exudate, and provide a humid environment. Accordingly, the WUR of the collagen matrix is very important for wound healing. The water-binding ability of the OSDS could be attributed to their hydrophilicity and the maintenance of their three-dimensional structure. The WURs of INSUREGRAF® and Matriderm® were 2332% and 2492%, respectively (Table 1). According to these results, the ratios of both matrices were very similar. Therefore, the two products are expected to have a similar effect on wound exudate absorption and protection.

Table 1 Water uptake ratio of collagen matrix

Sample	Average Wa	Average Wb	Water uptake ratio (%)
INSUREGRAF	0.0027	0.0660	2332
Matriderm	0.0014	0.0367	2492

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3.4. Proliferation of fibroblasts on the OSDS

To verify the cyto-compatibility of OSDS, L292 fibroblasts were cultured on the OSDS for up to 7 days (Fig. 3a). The growth rate gradually increased in both OSDS groups. However, there was no significant change in the TCP group. This may reflect the limited growth area in the TCP during cell growth.

Additionally, the cell growth rate was higher for INSUREGRAF® than Matriderm®. However, contraction occurred in the two types of OSDS after cell cultivation (Fig. 4b and c). The contraction ratios of INSUREGRAF® and Matriderm® decreased by 48% and 31%, respectively. This difference in contraction can be explained by differences in trypsin resistance and collagen purity. Therefore, the INSUREGRAF® OSDS was expected to show higher cell viability and proliferation than Matriderm®.

Fig. 4 Biocompatibility test. Cell proliferation measured during culture for up to 7 days by CCK-8 (a) and an OSDS contraction graph (b) and images (c) after 7 days.

3.5. Morphological observations

Cell morphology with respect to the size of OSDS after cell cultivation was observed by SEM, confocal scanning laser microscopy, and H&E staining. Based on the SEM images (Fig. 5), cells were detected on the surfaces of both OSDS after 7 days. There was little difference in cell spreading behaviour in INSUREGRAF® (Fig. 5).^24,25

Fig. 5 Morphological observation of fibroblasts cultured on the INSUREGRAF® and Matriderm® by SEM.

The cells formed a pore-like structure. This structure is expected to improve angiogenesis and the transport of nutriment and waste. The cross-section images of INSUREGRAF® and Matriderm® are shown in Fig. 6. Immunocytochemical localization of F-actin in fibroblasts was performed to examine cell attachment and infiltration from the surface to the centre on OSDS at 7 days. Cell infiltration was observed in both OSDS. Therefore, both OSDS provided proper cell growth conditions. However, the thickness of Matriderm® was reduced after 7 days and the cell population was higher on the surface compared to the centre. In contrast, well-distributed cells were observed in INSUREGRAF® from the surface to centre without contraction.

Fig. 6 Morphological observation of fibroblasts cultured on the INSUREGRAF® and Matriderm®. The immunofluorescence images showed distribution of cells. Cell nuclei labeled with DAPI (blue) and cytoskeleton labeled with phalloidin (green). H&E stain also indicated cell distribution and OSDS shape. Cells exhibited a good morphology with no differences between each group both on the external surface.

These results indicated that both OSDS provided an optimal environment for cell growth. However, INSUREGRAF® might improve skin regeneration with more structural stability than existing commercial products, such as Matriderm®.^26–30

3.6. In vivo wound healing

We performed an in vivo study to evaluate the safety and efficacy of both collagen sponges. When comparing skin contraction for INSUREGRAF® and Matriderm® at 4 and 8 weeks, as shown in Fig. 7a, a significant difference appeared. We also observed very rapid degradation and formation of granulation tissue in the Matriderm® wound.

Fig. 7 The effect of wound healing. Scar contraction was observed by macroscopic inspection (a). H&E staining of miniature pig after 4 weeks (c). SMA staining (b, d) images of the wounds of miniature pig on 1 and 4 weeks.

After implantation of the collagen sponge at 1, 4, and 8 weeks, the degree of wound healing was assessed by H&E and smooth muscle actin (SMA) staining. Both implanted OSDS engrafted very well on the paraspinal area of the miniature pig. Additionally, neovascularization in both OSDS was observed in the SMA staining images (Fig. 7b). Based on H&E staining after 4 weeks (Fig. 7c), inflammation and foreign body reactions in the vicinity of the implanted OSDS were not observed. As shown in Fig. 7d, the density of SMA staining of Matriderm® was stronger than that of INSUREGRAF®. Strong SMA staining during the first 2 weeks indicates that angiogenesis is proceeding smoothly, and strong staining after 4 weeks indicates the formation of erythema.

The histological characteristics of the collagen sponges were assessed for each of the implanted substances; an inflammatory response to implanted biomaterials can be characterized by increases in both membrane thickness and the density of cellular infiltration within the collagen sponge.

SMA staining revealed differentiated myofibroblasts of contractile tissue. Matriderm® accelerated fibroblast differentiation and contractile activity.

3.7. Clinical application

To compare the efficiency of INSUREGRAF® and Matriderm®, these two OSDS were applied to the left thigh and leg of a 42% electrical burn patient after debridement of the burn area (Fig. 8). Fig. 8a shows an image of initial burn on the left thigh and leg, which was classified as 2^nd degree. A yellow box in Fig. 8b indicates INSUREGRAF®, while the red box in Fig. 8c indicates Matriderm®. Before STSG, the OSDS were hydrated with PBS for cell proliferation (Fig. 8d). Additionally, 1 : 1 meshed skin was grafted on the OSDS after hydration (Fig. 8e). According to the images obtained after 12 days of STSG, the colour of the wound healing site was similar for INSUREGRAF and Matriderm®. To examine the clinical effectiveness of INSUREGRAF® (Bioland Co.), we transplanted them in patients with severe burns (mean age, 44.7 years; mean total body surface area, 37.7%). In total, 15 joint regions in 10 patients received the INSUREGRAF® graft selectively during the study period between July 2015 and December 2015. The graft results were determined after confirming the take rate of the transplanted skin graft on top of the INSUREGRAF®. Take rates were examined twice, at 7 and 14 days after grafting. Photographs of the skin grafts were evaluated individually by two burn surgeon specialists, and the mean values were recorded. The engraftment rate was 97% after day 7 and 98% after day 14. Accordingly, INSUREGRAF® was successfully developed as an artificial dermis that allows a one-stage operation and exhibited a successful early-stage graft take rate (Table 2).

Fig. 8 Clinical application of INSUREGRAF® on the left thigh and leg of 42% electrical burn patient. (a) Left thigh and leg of initial burn patient, (b) apply a INSUREGRAF® on the burn area (yellow box) after debridement for operation, (c) apply a Matriderm® (red box), (d) hydration of INSUREGRAF® and Matriderm® for skin graft, (e) 1 : 1 auto skin graft, (f) after 12 days of post operation.

Table 2 Patients information and results of operation^a

No	Sex	Age	TBSA (%)	Full thickness burn (%)	No. of operation	Area of used INSUREGRAF	PBD of INSUREGRAF application	Width of INSUREGRAF (cm^2)	Size of mesh	Take rate (%) (POD7)	Take rate (%) (POD14)
a TBSA; total body surface area, PBD; post burn day, POD; post operation day.
1	M	18	40	20	3	Knee, right	12	150	1 : 1	97	99
2	M	46	35	30	3	Knee, right	16	150	1 : 1	97	98
3	M	51	40	40	3	Knee, right	15	300	1 : 1	96	98
						Knee, left	15	150	1 : 1	98	99
4	M	49	40	40	4	Neck	14	100	1 : 1	100	100
5	M	55	15	10	2	Elbow, right	18	150	1 : 1	96	98
6	F	44	57	40	4	Knee right	15	150	1 : 1	99	99
						Knee, left	15	300	1 : 1	99	99
						Hand, both	31	190	Sheet	95	96
						Elbow, right	31	150	1 : 1	96	98
7	M	61	12	7	1	Knee, right	25	150	1 : 1	100	100
8	M	42	60	45	3	Knee, left	14	150	1 : 1	100	100
						Hand, left	14	100	Sheet	96	97
9	MM	34	34	30	3	Knee, right	16	150	1 : 1	96	98
10		47	44	35	3	Hand, Lt.	15	150	Sheet	95	96

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4 Conclusions

A dermal substitute needs to satisfy several conditions, such as (1) biocompatibility, (2) a high surface area to increase cell attachment, and (3) an interconnected pore structure to enable the infiltration of nutrients, growth factors, and hormones. Cell attachment and proliferation are expected to increase in a dermal substitute. After transplantation, the dermal substitute should not cause an inflammatory reaction and should be compatible with various tissues.

The essential goals in the development of a novel dermal substitute are economic feasibility, efficiency, and a sustained healing ability. The freeze-drying method can satisfy these needs. Here, we evaluate a new method using a rapid freeze-drying procedure. Since this method provides a stable interconnected unidirectionally solidified pore structure, the dermal substitutes are expected to induce highly effective cell affinity for medical applications. We compared the collagen sponge graft with other products. The micro-structured collagen sponges significantly stimulated initial cell adhesion, including the expression of adhesive molecules in vitro. Moreover, both collagen sponges can be implanted to full thickness skin defect sites and resulted in successful wound healing. INSUREGRAF® and Matriderm® showed similarly favourable biological behaviours in terms of take, vascularization, and inflammatory responses using a one-step procedure. However, for the one-step procedure using Matriderm®, wound contraction was observed during the healing period.

We speculated that this contraction was associated with a low thickness, low matrix metalloprotease resistance, and rapid early degradation rate in comparison with INSUREGRAF®.

The Matriderm® skin substitute structurally constricted when it was hydrated. These results demonstrate that INSUREGRAF® is more suitable with respect to cell penetration, distribution, and the acceleration of dermis (skin) regeneration compared to Matriderm®.³¹ However, long-term follow-ups and prospective studies are required to prove the superiority of INSUREGRAF® over other materials.

Acknowledgements

This research was supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number: HI15C3000). Also this research supported by Hallym university medical center project (Grant Number: H20011665). This animal study was conducted in accordance with guidelines and approval of the Institutional Animal Care and Use Committees (IACUC) of Hallym University (Hallym-2010-78).

References

1. E. Sachlos and J. T. Czernuszka, Eur. Cells Mater., 2003, 5, 29–39.
2. C. Agrawal and R. B. Ray, J. Biomed. Mater. Res., 2001, 55, 141–150.
3. J. O. Hollinger and K. Leong, Biomaterials, 1996, 17, 187–194.
4. U.-J. Kim, J. Park, H. J. Kim, M. Wada and D. L. Kaplan, Biomaterials, 2005, 26, 2775–2785.
5. Q. Lu, K. Ganesan, D. T. Simionescu and N. R. Vyavahare, Biomaterials, 2004, 25, 5227–5237.
6. R. Papini, BMJ, 2004, 329, 158–160.
7. R. V. Shevchenko, S. L. James and S. E. James, J. R. Soc., Interface, 2010, 7, 229–258.
8. S. Yang, K.-F. Leong, Z. Du and C.-K. Chua, Tissue Eng., 2001, 7, 679–689.
9. M. Boffito, S. Sartori and G. Ciardelli, Polym. Int., 2014, 63, 2–11.
10. C. Vaz, S. Van Tuijl, C. Bouten and F. Baaijens, Acta Biomater., 2005, 1, 575–582.
11. J. J. Song, J. P. Guyette, S. E. Gilpin, G. Gonzalez, J. P. Vacanti and H. C. Ott, Nat. Med., 2013, 19, 646–651.
12. L. Polo-Corrales, M. Latorre-Esteves and J. E. Ramirez-Vick, J. Nanosci. Nanotechnol., 2014, 14, 15.
13. S. Ahn, H. Yoon, G. Kim, Y. Kim, S. Lee and W. Chun, Tissue Eng., Part C, 2009, 16, 813–820.
14. M. Ehrenreich and Z. Ruszczak, Tissue Eng., 2006, 12, 2407–2424.
15. A. Langer and W. Rogowski, BMC Health Serv. Res., 2009, 9, 115.
16. N. Morimoto, N. Kakudo, P. V. Notodihardjo, S. Suzuki and K. Kusumoto, J. Artif. Organs, 2014, 17, 352–357.
17. H. Uchi, A. Igarashi, K. Urabe, T. Koga, J. Nakayama, R. Kawamori, K. Tamaki, H. Hirakata, T. Ohura and M. Furue, Eur. J. Dermatol., 2009, 19, 461–468.
18. J. H. Min, I. S. Yun, D. H. Lew, T. S. Roh and W. J. Lee, Arch. Plast. Surg., 2014, 41, 330–336.
19. K. Sharma, A. Bullock, D. Ralston and S. MacNeil, Burns, 2014, 40, 957–965.
20. V. Cervelli, L. Brinci, D. Spallone, E. Tati, L. Palla, L. Lucarini and B. De Angelis, Int. Wound J., 2011, 8, 400–405.
21. H. Schoof, J. Apel, I. Heschel and G. Rau, J. Biomed. Mater. Res., 2001, 58, 352–357.
22. B. K. Boekema, M. Vlig, L. Olde Damink, E. Middelkoop, L. Eummelen, A. V. Buhren and M. M. Ulrich, J. Mater. Sci.: Mater. Med., 2014, 25, 423–433.
23. C. M. Murphy and F. J. O'Brien, Cell Adhes. Migr., 2010, 4, 377–381.
24. S. Haifei, W. Xingang, W. Shoucheng, M. Zhengwei, Y. Chuangang and H. Chunmao, J. Mech. Behav. Biomed. Mater., 2014, 29, 114–125.
25. C. Philandrianos, L. Andrac-Meyer, S. Mordon, J. M. Feuerstein, F. Sabatier, J. Veran, G. Magalon and D. Casanova, Burns, 2012, 38, 820–829.
26. A. Desmouliere, C. Chaponnier and G. Gabbiani, Wound Repair Regen., 2005, 13, 7–12.
27. B. Hinz, J. Invest. Dermatol., 2007, 127, 526–537.
28. M. P. Horan, N. Quan, S. V. Subramanian, A. R. Strauch, P. K. Gajendrareddy and P. T. Marucha, Brain, Behav., Immun., 2005, 19, 207–216.
29. M. J. Reid, L. J. Currie, S. E. James and J. R. Sharpe, Wound Repair Regen., 2007, 15, 889–896.
30. D. Shin and K. W. Minn, Plast. Reconstr. Surg., 2004, 113, 633–640.
31. O. S. Nachf, W. Suwelack, P. Tewes-Schwarzer and Z. Eckmayer, German Pat., DE 40 28 622 C2, 1990.

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