Efficacy of Supercritical Fluid Decellularized Porcine Acellular Dermal Matrix in the Post-Repair of Full-Thickness Abdominal Wall Defects in the Rabbit Hernia Model

Abstract

Damage to abdominal wall integrity occurs in accidents, infection and herniation. Repairing the hernia remains to be one of the most recurrent common surgical techniques. Supercritical carbon dioxide (SCCO₂) was used to decellularize porcine skin to manufacture acellular dermal matrix (ADM) for the reparation of full-thickness abdominal wall defects and hernia. The ADM produced by SCCO₂ is chemically equivalent and biocompatible with human skin. The ADM was characterized by hematoxylin and eosin (H&E) staining, 4,6-Diamidino-2-phenylindole, dihydrochloride (DAPI) staining, residual deoxyribonucleic acid (DNA) contents and alpha-galactosidase (α-gal staining), to ensure the complete decellularization of ADM. The ADM mechanical strength was tested following the repair of full-thickness abdominal wall defects (4 × 4 cm) created on the left and right sides in the anterior abdominal wall of New Zealand White rabbits. The ADM produced by SCCO₂ technology revealed complete decellularization, as characterized by H&E, DAPI staining, DNA contents (average of 26.92 ng/mg) and α-gal staining. In addition, ADM exhibited excellent performance in the repair of full-thickness abdominal wall defects. Furthermore, the mechanical strength of the reconstructed abdominal wall after using ADM was significantly (p < 0.05) increased in suture retention strength (30.42 ± 1.23 N), tear strength (63.45 ± 7.64 N and 37.34 ± 11.72 N) and burst strength (153.92 ± 20.39 N) as compared to the suture retention (13.33 ± 5.05 N), tear strength (6.83 ± 0.40 N and 15.27 ± 3.46 N) and burst strength (71.77 ± 18.09 N) when the predicate device materials were concomitantly tested. However, the efficacy in hernia reconstruction of ADM is substantially equivalent to that of predicate material in both macroscopic and microscopic observations. To conclude, ADM manufactured by SCCO₂ technology revealed good biocompatibility and excellent mechanical strength in post-repair of full-thickness abdominal wall defects in the rabbit hernia model.

1. Introduction

Abdominal wall surgery includes primarily techniques and surgical procedures focused to repair hernia defects. Every year, more than 20 million hernia surgeries are carried out worldwide [1]. In the US alone 700,000 inguinal hernia surgeries are performed in one year. The incisional hernias no less often may ascend after a laparotomy, in which between 11 and 20% of laparotomies may progress to an incisional hernia [2]. The tension-free hernia repair technique using biomaterial in a rabbit model is the keystone in the field of surgical procedures intended to repair abdominal wall defects [3,4].

Acellular dermal matrix (ADM) is a biomaterial derived from decellularized animal and human skin and tissues [5]. The ADM’s properties such as structural, mechanical, and biochemical functions are credited to the extracellular matrix (ECM), the main component of ADM [6]. The major element of ECM is collagen I, accountable for hemostasis on the wound bed and for modulating the wound-healing process [7]. In wounds, the role of type I collagen in the wound bed is to avoid the worsening of wounds by attaching to free radicals, proteases, and inflammatory cytokines [8]. The ADM is a collagen-abundant ECM that plays a substantial function in wound healing. Many collagen-based ADM scaffolds are manufactured by the decellularizing animal, and human skin and tissues, such as bovine collagen-derived Integra^®, swine small intestine submucosa-derived Oasis^®, and human placenta-derived Epifix^® [5]. Porcine ADM is an ECM-rich collagen scaffold extensively employed in tissue engineering. It encourages wound healing because of its biodegradable and biocompatible nature [9].

Acellular dermal matrix (ADM) biomaterials can enhance vascular ingrowth and integrate with the host tissues. ADM products possess the ability to support vascular ingrowth and incorporate native tissues. This allows the host immune system to access the repair site, which increases resistance to infection by controlling necrotic debris and bacteria that contribute to the development of chronic wounds [10]. Porcine ADM is an outstanding substitute for human ADM because of its surplus tissue source, cost-effectiveness and structural resemblances to collagen in humans [11]. Porcine ADM possesses immunogenic epitopes which cause host rejection and encapsulation. The chemical cross-linking process is used to decrease the immunogenicity of porcine ADM. The cross-linking process decreases the immunogenicity of the ADM by chemically covering its antigenic epitope, it decreases the scaffold deprivation by matrix metalloprotease and cell infiltration essential for matrix remodeling [12]. The ADM avoids infection by allowing immune cells to contact with necrotic debris and bacteria [10].

Decellularization technology is used to eradicate the antigenicity of the xenogeneic tissue while retaining the components of ECM. Decellularization techniques are classified based on physical, chemical, or enzymatic approaches by adding detergents, such as Triton-X100, sodium dodecyl sulfate (SDS), and sodium deoxycholate [13]. Nevertheless, detergent residues elicit cytotoxicity in vivo and irreversible structural damage to ECM. In addition, decellularization by detergent is laborious [14]. Supercritical carbon dioxide (SCCO₂) extraction technology was an excellent alternative for the decellularization process [15,16]. The SCCO₂ technology can be employed to eradicate fat and cellular components from the fat cells and intracellular, functional cellular proteins while retaining the intact ECM structure without impairment. The SCCO₂ method is efficient in fat reduction and removal from porcine skin [16]. The histological and immunohistochemical (IHC) staining confirmed that there are no alterations and damage to ECM in SCCO₂ decellularization [15]. Carbon dioxide is the most commonly used supercritical fluid due to its suitable critical temperature (31.0 °C) for processing ECM. Carbon dioxide is an easily available ambient gas [16,17]. Moreover, the advantage of SCCO₂ decellularization technology is non-toxic, inexpensive, and eco-friendly [15,18]. The SCCO₂ process has few known disadvantages related to the processing of the tissues, except the fact that the SCCO₂ system is a costly and intricate apparatus working at high pressure.

The protective function, tensile strength and continuity of the skin are due to the presence of type I collagen. Throughout the maturation stage and skin remodeling, it is predicted that type III collagen declines compared to type I collagen to return to the ratio found in normal skin [19,20]. In ADM-treated animals type I collagen revealed a significant elevation in density compared to normal animals. In addition, a rise in total collagen in skin wounds was also observed in the presence of ADM [20].

In the present study, we decellularized the porcine hide employing SCCO₂ technology to produce ADM and examined the biocompatibility and mechanical strength in post-repair of full-thickness abdominal wall defects in the rabbit model.

2. Materials and Methods

2.1. Preparation of Acellular Dermal Matrix

Porcine hide was procured from Tissue Source, LLC (Lafayette, IN, USA). The fat layer of the hide was trimmed off and discarded. The remaining hide part was washed with phosphate-buffered saline (PBS). The hide was delicately sliced to 0.4–2 mm in thickness. The sliced hide was rolled along with polyethene gauze arranged in a tissue holder, which was then placed into a SCCO₂ vessel system (Helix SFE Version R3U, Applied Separations Inc (Allentown, PA, USA), with a cosolvent filled to 10% volume of the vessel with 75% ethanol. The operations of the SCCO₂ system were carried out in dynamic mode at 350 bar and 40 °C for 40 min to decellularize porcine hide to ADM. The flow rate of carbon dioxide is 0.3 liters per minute (LPM). Subsequently washed with acetic acid (1%), sodium hydroxide (0.1 N) and freeze-dried. The ADM is sterilized by γ-irradiation (25 kilo Gray), and the product is marketed as ABCcolla^® Acellular Dermal Matrix in Taiwan.

2.2. Predicate Device

In the present study, the predicate device used is a popular commercial brand, recommended for hernia reconstruction by physicians. This predicate device is approved by US FDA and CE. The biological, chemical, physical and mechanical properties of this commercial brand are comparable to that of the SCCO₂-derived ADM, and therefore we used this predicate device (PRE) to compare in this study.

2.3. Hematoxylin and Eosin Staining

The native porcine hide and ADM were fixed in 4% buffered formaldehyde and paraffin and the sections were carried out using standard routine sectioning, followed by hematoxylin and eosin (H&E) for assessing the decellularization of the porcine hide. The stained native hide and ADM sections were placed on an Olympus bx53 microscope (Olympus, TX, USA) and photomicrographs were documented for assessment.

2.4. DAPI (4,6-Diamidino-2-phenylindole, Dihydrochloride) Staining

The 5 μm thickness of standard routine paraffin-embedded sections was cut, followed by dewaxing in xylene and graded alcohol series were used for rehydration and finally into water. Subsequently, the sections were stained with DAPI and photographs were recorded using a fluorescent microscope.

2.5. DNA Quantification

The native porcine hide and ADMs the standard genomic DNA were extracted employing a kit (Nautia Cat. NO.: NGTZ-S100, Nautiagene, Taipei City, Taiwan). The extracted DNA from the native porcine hide and ADMs DNA was measured employing PicoGreen dsDNa Quantitation Reagent and Kit (P-7589, Thermo Fisher Scientific, Tainan, Taiwan) at 260 nm in a spectrofluorometric microplate reader (BIO-TEK^®Flx800, BioTek Instruments, Inc., Winooski, VT, USA). The residual DNA quantity and fragment size in the native porcine hide and ADM were analyzed by agarose gel electrophoresis.

2.6. Alpha-Gal (α-Gal) Staining

The native porcine hide and ADMs paraffin-embedded sections were carried out as stated in the former section, dewaxed, rehydrated and primary antibody alpha-Gal (α-gal) was added, incubated and developed by employing the standard immunohistochemistry staining Avidin-Biotin Complex (ABC) method, following Wu et al. [21]

2.7. Surgery for the Repair of Full-Thickness Abdominal Wall Defects

In this case, 24 New Zealand rabbits weighing greater than 2.5 kg (Male) were randomly allocated to two groups: the experimental ADM group (n = 12) and the predicate group (PRE) (n = 12), a well-known commercial brand. The animal study protocol was approved by Institutional Animal Care & Use Committee (Master Laboratory Co., Ltd., IACUC: 20T10-10). All animals overnight fasted before surgery, but the water was permitted. The animals were anaesthetized Zoletil and xylazine were injected into the intramuscularly at the dose of 10 mg/kg separately, and then anaesthetized with isoflurane 30 min before surgery. Before the surgery, the fur of the animal’s abdomen was clipped with an electric animal shaver with aseptic techniques.

Using a sterile surgical technique, full-thickness abdominal wall defects with a size of 4 × 4 cm on both sides of the midline were carried out, by which the fascia, the underlying rectus abdominis muscle, and the peritoneum were resected. The experimental ADM (24 experimental ADM, one animal received 2) and PRE (24 predicate device, one animal received 2) (5 × 5 cm) were placed intraabdominal with 1 cm overlap and fixed tension-free to the abdominal wall with eight interrupted 2/0 polypropylene sutures. Next, the abdominal wall fascia was closed with a running 2/0 polypropylene suture. The subcutis and skin were closed with continuous resorbable 2/0 polyglactin sutures. The povidone-iodine was used to disinfect the wound. After surgery, the animals were given antibiotics intramuscularly once a day for seven days. At 2 and 8 weeks after implantation, the animal was sacrificed with humanity. The animal samples were collected for further biomechanical study.

2.8. Mechanical Testing

The mechanical testing was following the in vitro mechanical properties study. The instrument used is the HT 2402EC material testing machine (Hung Ta Instrument Co., Ltd., Taiwan, China) for the tensile strength test. The HT 2402EC capacity is 500 kgf with an accuracy of ±1% and load accuracy of ±0.01%, with a capacity of 0.005–500 mm/min

2.9. Suture Retention Strength Test

The implanted ADM in the abdominal muscle of rabbit specimens were cut into a size of 25 × 50 mm². No #2 suture was used for the retention test, a suture needle was passed through at a distance of 10 mm from the side of the sample and tied the suture with a bowline knot. The suture was passed through a hole in a specimen and fixed on the hook which was attached to the grip of the tensile testing machine.

2.9.1. Tear Strength Test

The tear strength of the implanted ADM in the abdominal muscle of rabbit specimens was cut into a size of 25 × 50 mm², and a 10 mm slit was cut from the midline of the 2.5 cm edge towards the center of the specimen, which is suitable for clamping. Each specimen was clamped in the upper and lower grip of the tensile testing machine. The samples were then stretched at 25.4 mm/min. The test was stopped when the total displacement exceeded 30 mm (the specimen was fully stretched).

2.9.2. Burst Strength Test

The burst strength of the implanted ADM in the abdominal muscle of rabbit specimens was cut to a size of 50 × 50 mm², the surrounding muscle tissue was removed, and the specimens were clamped in the grip. The specimen was fixed into a sample holder, the clamping device could tightly clamp the four sides of the specimen with a diameter of 30 mm in the center tested. A diameter of 25 mm round rod bar fixture was load applied at a rate of 25.4 mm/min on the loading device. The test was stopped when the total displacement exceeded 30 mm or until complete material failure (load = 0 N).

2.9.3. Specimen Tissue Section Preparation and Staining

The raw specimens of each animal (implants on the left or right abdomen) were fixed in 10% buffered neutral formalin solution for 24 h and went through trimming, fixation, dehydration, embedding, and slicing for 3–4 μm slides. The slices were then stained with H&E staining and observed by a veterinary pathologist who was blinded to the experiment and scored by microscope observation.

Semi-quantitative scoring of histopathological lesions according to Shackelford et al. [22], the criteria of the severity grading system for all microscopic lesions were shown as follows: (i) Grade “minimal”—The tissue has undergone less than 10% of the tissue is involved. For hyperplasia, hypoplasia and/or atrophy lesions, the “minimal” grade is given when the affected tissue showed less than 10% increase or decrease in volume. (ii) Grade “mild”—The tissue has undergone 10–39% of the tissue involved. For hyperplasia, hypoplasia and/or atrophy lesions, the “mild” grade is given when the affected tissue has less than 10–39% increase or decrease in volume. (iii) Grade “moderate”—The tissue has undergone 40–79% of the tissue involved. For hyperplasia, hypoplasia and/or atrophy lesions, the “moderate” score is employed when the affected tissue has less than 40–79% increase or decrease in volume. (iv) Grade “marked”—The tissue has undergone 80–100% of the tissue involved. For hyperplasia, hypoplasia and/or atrophy lesions, the “marked” score is given when the affected tissue has experienced 80–100% increase or decrease in volume.

3. Results

3.1. H&E Staining of SCCO₂ Decellularized ADM

In native porcine hide, the nucleus is stained by hematoxylin displaying blue-purple color and eosin binds to the protein of cytoplasm giving pink color (Figure 1i,iii). The H&E staining showed no cells in the SCCO₂ decellularized ADM (Figure 1ii, iv). Therefore, the H&E staining shows ample decellularization of the porcine ADM as compared to normal hide.

3.2. DAPI Staining of SCCO₂ Decellularized ADM

DAPI stains the nucleus of the cell and shows a bright blue color, under a fluorescent microscope. The DAPI staining of the SCCO₂-processed ADM displayed no nucleus demonstrating complete decellularization, whereas nucleus staining was visible in the native porcine hide (Figure 2i, iii). The results indicate that the SCCO₂-decellularization process can successfully remove the cells from the porcine hide (Figure 2ii, iv).

3.3. Residual DNA Content of SCCO₂ Decellularized ADM

The SCCO₂ decellularization of the porcine hide was confirmed by quantifying the content of DNA and agarose gel electrophoresis. For ideal and effective decellularization the criteria are that decellularized tissue must contain < 50 ng of dsDNA per mg of dry tissue. The SCCO₂ decellularized ADMs DNA quantification exhibited an average of 26.92 ng/mg of DNA (Figure 3A), which is below the acceptable level of 50 ng/mg residual DNA for medical implant devices based on Biological Evaluation of Medical Devices—Part 1 (ISO 2018). In addition, the SCCO₂ decellularized ADM by agarose gel electrophoresis showed no DNA band, whereas DNA bands were found in the native porcine hide (Figure 3B).

3.4. Alpha-Gal Staining of SCCO₂ Decellularized ADM

The native porcine hide shows the presence of α-gal indicating live cells. α-gal is a catabolizing enzyme that breakdowns glycoproteins, glycolipids, and polysaccharides. Live cells show positive immunostaining of α -gal in the native porcine hide (Figure 3C). However, SCCO₂ decellularized ADM exhibited negative immunostaining for alpha-gal indicating complete decellularization in the ADM (Figure 3D).

3.5. The Mechanical Strength of SCCO₂ Decellularized ADM

The suture retention strength of SCCO₂ decellularized ADM after implantation was significantly higher (30.42 ± 1.23 N) than that in the PRE control (13.33 ± 5.05 N) at week 2. The suture retention strength of SCCO₂ decellularized ADM and PRE control after implantation were similar at week 8 (30.38 ± 2.31 N and 31.93 ± 3.05 N) (Figure 4A).

In the tear strength tests, the SCCO₂ decellularized ADM (63.45 ± 7.64 N and 37.34 ± 11.72 N) were significantly higher than the PRE control (6.83 ± 0.40 N and 15.27 ± 3.46 N) at week 2 and week 8 (Figure 4B).

In the burst strength test, the SCCO₂ decellularized ADM (153.92 ± 20.39 N) was significantly higher than the PRE control (71.77 ± 18.09 N) at week 2. However, there were no significant differences between the SCCO₂ decellularized ADM (421.50 ± 127.34 N) and the PRE control (276.42 ± 82.67 N) in strength at week 8 (Figure 4C).

3.6. Histological Evaluation of the Abdominal Wall Defect Model

The representative histological photomicrographs 2 weeks after implantation of the SCCO₂ decellularized ADM (Figure 5i,ii) and PRE control (Figure 5iii,iv) were shown in Figure 5. The representative histological photomicrographs 8 weeks after implantation of the SCCO₂ decellularized ADM (Figure 6i,ii) and PRE control (Figure 6iii,iv) were shown in Figure 6.

The histological scores of inflammation in the SCCO₂ decellularized ADM (2.83 ± 0.41 and 3.50 ± 0.55) showed similar levels to those in PRE control (3.17 ± 0.98 and 3.00 ± 0.89) at week 2 and week 8, respectively, indicating no significant difference in the clinical outcome after implantation between the SCCO₂ decellularized ADM and PRE control (Figure 7A,C). The histological scores of fibroblast and vessel formation in the SCCO₂ decellularized ADM and PRE control also showed similar levels at the observation time points. The scores of fibroblast formation in the SCCO₂ decellularized ADM (2.50 ± 0.55 and 2.00 ± 0), and (2.83 ± 0.41 and 2.67 ± 1.03) in PRE control at week 2 and week 8, respectively, indicating no significant changes between the SCCO₂ decellularized ADM and PRE control (Figure 7A,C). The scores of vessel formation in the SCCO₂ decellularized ADM (2.50 ± 0.55 and 2.17 ± 0.41) and (2.50 ± 0.55 and 2.67 ± 0.82) in PRE control at week 2 and week 8, respectively, indicating no significant changes between the SCCO₂ decellularized ADM and PRE control (Figure 7A,C).

The scores of epithelization in the SCCO₂ decellularized ADM (3.33 ± 0.52 and 3.17 ± 0.75), and (3.33 ± 0.98 and 3 ± 1.1) in PRE control at week 2 and week 8, respectively, indicating no significant changes between the SCCO₂ decellularized ADM and PRE control (Figure 7B,D). The scores of PMNL in the SCCO₂ decellularized ADM (3.17 ± 0.98 and 2.83 ± 0.98), and (3.67 ± 0.41 and 3.17 ± 1.17) in PRE control at week 2 and week 8, respectively, indicating no significant changes between the SCCO₂ decellularized ADM and PRE control (Figure 7B,D).

4. Discussion

Today the budding topic of research is the development of various kinds of biomaterial for the restoration of tissue defects in the abdominal wall. For the past 20 years, the development has necessitated alterations to the numerous biomaterials in quest of a prosthetic material displaying ideal performance. However, there is no perfect biomaterial and strategy for tissue repair and regeneration processes so far. The new age of biomaterials has taken into account the features of the host tissue and its biology to ensure that the developed materials with proper characteristics are comparable to the host tissue. Therefore, engineers, biologists and pathologists play a vital role in the product research and development process. Various biomaterials are exposed to in vitro and in vivo biocompatibility studies, and pre-clinical animal performance studies and their mechanical strength before and after implantation are also tested [4]. We have developed and manufactured an innovative regenerative ADM using SCCO₂ technology and examined the biocompatibility and mechanical strength in post-repair of full-thickness abdominal wall defects in the rabbit model.

In the present study, the characterization of ADM was carried out to validate the complete decellularization, competent to be employed as ADM for repairing full-thickness abdominal wall defects. The ADM owns no residual cells as evidenced via H&E and DAPI staining. The SCCO₂ process completely removed the cells as evidenced by H&E staining. In addition, the SCCO₂ process eliminated the nucleus as evidenced by DAPI staining. The residual DNA quantification was found to be negligible and complied with the international standard for regulatory approval. Biomaterials accessible for the treatment of abdominal wall defects are essential to be tested by preclinical animal experimental studies. International Organization for Standardization have developed biocompatibility standards following ISO 10993-6, “Biological evaluation of medical devices “Tests for Local Effects after Implantation,” epitomizes the features that are essential to be taken into consideration when executing implant studies. Among other features, ISO 10993-6 indicates the selection of species and the assessment of biological responses. Though ISO 10993-6 indicates various experimental animals such as rats, mice, and rabbits. Due to its size and easy handling, the rabbit is the animal of choice for implant testing. The New Zealand White rabbit models have offered a shred of outstanding evidence on the behavior of the various mesh implants in the abdominal wall and facilitated the progress of our understanding and predict the consequences that would transpire in human clinical practice [4,23]. Therefore, the current study examines the biocompatibility and mechanical strength in post-repair of full-thickness abdominal wall defects in the rabbit model further studies are needed for extrapolating to humans.

In the present study, ADM was characterized to validate the complete decellularization, α-gal negative in the ADM. A noteworthy difference was detected between the human and porcine dermis. The decellularized porcine ADM owns the immunogenic galactose- α -1,3-galactose epitope, while the galactose epitope was absent in the human dermis. Humans possess a cellular and humoral immune response to this epitope. Its occurrence generates the possibility of immune rejection [24,25]. Porcine ADM is treated to alleviate the galactose-α-1,3-galactose epitope, and this treatment has demonstrated efficacy in evading the immune response during the ADM is implanted in non-human primates. In vitro studies with human fibroblasts displayed higher numbers of human fibroblasts infiltrating deep into human ADM than in porcine ADM, this may be because of the increased density of collagen in the porcine dermis [26]. Therefore, the current study proved that SCCO₂ technology alleviated the galactose-α-1,3-galactose epitope offering biocompatibility and evading the immune response during the ADM implantation.

Hernia repair meshes need to be precisely designed for their uses. The perfect ADM used in the reinforced closure need to offer structural integrity during implantation and an ECM for regeneration. The implanted ADM remodels both mechanically and biologically. Commercially around 14 biologically derived ADMs are available for hernia repair in clinical use [27,28]. Furthermore, the tissue-derived ADMs showed no satisfactory positive responses, in the integration to the host [29]. The current study with the SCCO₂-derived ADM had an excellent integration to the host tissue remodel both mechanically and biologically in the post-repair of full-thickness abdominal wall defects in the rabbit model.

In the case of big incisional hernias, a multifaceted situation of biological and mechanical signaling and remodeling can impact the remodeling of ADM employed for repair. In addition, inflammation is linked to high protease activity, which degrades ADM [30]. ADM-ECM scaffolds implanted in large incisional hernias aid physiologically pertinent to loading and evading repetition, against the occurrence of protease activity. The inflammation and swelling were significantly decreased in gross observations in porcine ADM-reinforcement at 2, 4, and 6 weeks with minimal remodeling at 4 and 6 weeks after the repair [30]. The current study with the SCCO₂-derived ADM is not degraded and no inflammation was observed even after 8 weeks in post-repair of full-thickness abdominal wall defects in the rabbit model.

In Förstemann’s abdominal wall model, the average minimum ultimate tensile strength of the porcine ADM was at least 25 times higher than the average loading in a human abdominal wall [31,32]. The absolute tensile strength of the porcine ADM was lower than the surrounding fascia, which is vital in avoiding hernia reappearance. Comparable results in tensile strength decreased in porcine ADM were observed in primates [29]. Furthermore, the average Young’s modulus of the porcine ADM and fascia at 6 weeks was comparable. Young’s modulus is a measure of the stress required to distort a material. In the abdominal wall defect model, the ADM and abdominal wall interact for a complete closure repair, both the ADM and the peritoneum-fascia complex will work together to counterattack stress and loads in the abdominal wall, totaling the capability of the repair to endure protruding and hernia recurrence in implanting collagen-based ADM in vivo [30]. The current study with the SCCO₂-derived ADM had excellent mechanical strength so that the ADM and abdominal wall interact for a complete closure repair in post-repair of full-thickness abdominal wall defects in the rabbit model.

Implanted porcine ADMs histological evaluation confirmed remodeling into fascia-like tissue, without any adhesions to the internal organs. Moderate revascularization occurs on an average of 2 weeks after implantation of porcine ADM and is increased vascularization in 4 weeks. Cellular repopulation took a similar trend in implanted porcine ADM. In the porcine ADM or peritoneum/fascia complex no inflammation and scarring were observed with the functional repair of a herniated abdominal wall [30]. The synthetic mesh and cross-linked porcine ADM are encapsulated with scar tissue and are not entirely integrated into the host tissue [33,34]. The host resident’s abdominal wall inflammatory cells and the collagen synthesis of fibroblastic cells will respond to the ADM used for hernia repair. The ADM will modulate the integrity and mechanical properties of the repair over time [30]. The present study with the SCCO₂-derived ADM had a minimal infiltration of host cells to ADM for revascularization and to maintain the mechanical strength of the reconstructed abdominal wall in post-repair of full-thickness abdominal wall defects in the rabbit model.

The generally employed bioprosthetic in ventral hernia repair is the human ADM (AlloDerm; Life-Cell Corp., Branchburg, NJ, USA). However, it has a major disadvantage of propensity to stretch after implantation, leading to protruding of the repair site [35,36]. The novel xenogeneic non-crosslinked porcine ADM (Strattice; LifeCell) is a good substitute, due to its negligible bulge rate and the porcine dermis is similar to the human dermis. The temperature regulation of porcine is by the subcutaneous fat instead of hair, which is similar to humans. In addition, similar collagen arrangement and structure between porcine and human dermis, even though porcine collagen is tightly packed and possesses a smaller amount of elastin [26,37]. However, the present study with the SCCO₂-derived ADM had no stretching and protrusion of the repair site was observed that maintain the mechanical strength of the reconstructed abdominal wall in post-repair of full-thickness abdominal wall defects.

In the present study, we produced an innovative porcine ADM by using SCCO₂ extraction technology. Our results proposed that the SCCO₂ method can be successfully used to produce decellularized ADM with excellent mechanical strength, a suitable dermal substitute for the reconstruction of full-thickness abdominal wall defects.