Preparation of Porcine Allogenic Acellular Diaphragm Matrix and its Application for Umbilical Hernioplasty in Pig

The herniation of abdominal organ viz. rumen, intestine, omentum etc may result due to defects in abdominal wall as a result of trauma or diseases requires immediate corrections (Sankar et al., 2010). The common complications of surgical reconstruction of abdominal wall defects include incisional hernias with additional complications of adhesions, suture sinus, fistula formation, gangrene etc (Penttinen and Gronroos, 2008). Despite the development of improved surgical techniques, the repair of the abdominal wall defects remains a clinical challenge for the veterinary surgeon and the use of prosthetic materials for surgical management of abdominal wall defect is required when the size of the defect or hernial ring size exceeds 3 cm in diameter (Vilar et al., 2011). More than 80 different types of synthetic mesh have  been used  till date  in the repair of abdominal wall reconstruction; however, although these synthetic meshes present excellent tensile strength and easy to handle, but with potential clinical complications could result following abdominal wall repair includes wound infections, bowel fistula, adhesion, erosion into the abdominal viscera, increased recurrence rate, persistent abdominal pain, mesh extrusion, seroma formation etc (Karrouf et al., 2016). The added advantages of biological mesh over synthetic prosthetic mesh such as minimal adhesion formation, providing better framework for fibroblast proliferation, release of antimicrobial peptides and non-complement fixing antibodies, induction of mild inflammatory response, host cell migration and angiogenesis neovascularization and building of multidirectional fibrous tissue proliferation (Pillay et al., 2002 and Bellows et al., 2007). The preparation of biological mesh involves the removal of cells (Decellularization) from a tissue or an organ leaves the complex mixture of structural and functional proteins that constitute the extracellular matrix (ECM). The goal of decellularization of biological materials is to ameliorate the antigenicity of biological graft by efficiently removing all its cellular and nuclear material while minimizing any adverse effects on the composition, biological activity, and mechanical integrity of the remaining ECM (Gilbert et al., 2006). Use of chemical detergents like sodium deoxycholate solution under physical agitation can be used for complete decellularization of biological tissues like diaphragm, urinary bladder, pericardium etc. In the present study, sodium deoxycholate based decellularized allogenic acellular porcine diaphragm matrix (ADM) was evaluated following application in umbilical hernioplasty in piglets.

The experiment was carried out in the Department of Surgery and Radiology, College of Veterinary Science, Assam Agricultural University, Guwahati, Assam during January 2018 to December 2019. All total eighteen (18) numbers of young piglets of age group 2-3 months of either sex, of weighing 8-14 kg clinically affected with umbilical hernia requiring hernioplasty was selected (Fig 1). The animals were divided randomly into three (3) groups consisting of six animals in each, viz Group A, Group B and Group C. Surgical hernioplasty was carried out with application of ADM in Group A; while hernioplasty was done in Group B animals with application of ADM along with Methylprednisolone @ 1 mg kg^-1 IM at weekly interval for three occasions. Hernioplasty in Group C animals were carried out with application of Autologous Tunica Vaginalis harvested from the same animal after open covered method of castration and was treated as control.



Fresh diaphragm of porcine origin was collected from a local abattoir and immediately preserved in chilled (4°C) sterile 1x phosphate buffer saline (PBS, pH 7.4) containing 0.1% Amikacin (Mikacin, Aristo Pharmaceuticals Private Limited, Mumbai, India) and 0.2025% Ethylene Diamine Tetra Acetic Acid (EDTA). The tendinous portion of each diaphragm was excised and washed thoroughly with sterile 1X PBS to remove all the adherent blood and dirt and subjected to different concentrations of sodium deoxycholate solution (0.5%, 1%, 2% and 4%) at a constant temperature (37^oC) on an orbital shaker (Orbitek, Scigenics Biotech Pvt. Ltd.) under physical agitation at rate of 180 rotations per minute. The sodium deoxycholate solution was changed in every 6 hours and representative samples were collected at interval of 12 hours till 72 hours of detergent treatment and fixed in 10% neutral buffered formalin for histomorphological analysis to confirm decellularization. Following decellularization the resulting porcine ADM was ringed extensively (6 times of 2 h each) with 1x PBS under constant agitation (180 rotations per minute) and temperature (37^oC) on an orbital shaker to remove the residual chemicals and cellular debris. Further the prepared matrices were stored in a sterile 1x PBS solution containing 0.1% Amikacin and 0.1% Sodium Azide at -20^oC until use. The present study was in full compliance with the Institutional Animal Ethics Committee, College of Veterinary Science, Assam Agricultural University, Khanapara, Guwahati, Assam. The decellularization protocol carried out in 2% sodium deoxycholate solution, incubated for duration of 24 h at 37^oC under physical agitation in orbital shaker was found to best in preserving the distinctive, natural, three-dimensional collagen structure within the prepared matrix (Fig 2). Before hernioplasty each piglet was kept off-fed for 12 hours and deprived of water for 4 hours and surgical site was prepared as per standard protocol. The general anaesthesia was produced with atropine sulphate @ 20 µg/kg IM followed by diazepam @ 2 mg/kg IV and kg ketamine hydrochloride@ 10 mg/kg IV. The animal was then positioned in dorsal recumbency and externally the hernial ring was assessed by pressing with thumb (Fig 3). Following application of an elliptical skin incision over the swelling the hernial ring was exposed (Fig 4) and any adhesion remained was removed. In Group A and B hernioplasty was carried out with ADM (Fig 5). The bio-mesh was cut into appropriate size and sutured with abdominal wall in “inlay” fashion using polypropylene suture and skin incision was closed with horizontal mattress suture. In Groups C animals, the male piglets with clinical umbilical hernia were selected; where before surgical reconstruction of hernia autologous tunica vaginalis (Fig 6) was harvested flowing open covered methods of castration and was kept in chilled PBS before implanted in the hernial correction (Fig 7).  In all the animals a course of antibiotic, regular dressing the skin wound with application of antiseptic ointment was followed till 5th day of operation and the external sutures were removed after 7^th post-operative day.

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The clinical parameters viz.  rectal temperature, heart beat and respiration rate and gross evaluation of the surgical wound viz. degree of swelling, degree of exudation, degree of warmth and degree of pain on palpation were recorded as per the methods described by Kumar et al (2002), on 0^th, 3^rd, 5^th, 7^th and 10^th day of post-hernioplasty. The haemato-biochemical examination of whole blood and blood serum viz. Haemoglobin (%), Packed Cell Volume (%), Total Leukocyte Count (103/mm3), Differential Leucocytic Count, Alanine Aminotransferase (ALT- U/L), Creatine Kinase (CK-U/L), Gama-glutamyl transferase (GGT-U/L), Serum Cortisol (ng/ml), Serum Total Protein (g/dl) and Serum Albumin (g/dl)was carried out on 0^th, 3^rd, 5^th, 7^th and 10^th day following surgical hernioplasty. The blood serum was subjected to indirect ELISA as per method described by Mohsina et al., (2014) on 0^th, 5^th, 10^th, 15^th, 20^th and 30^th days of surgical reconstruction.

The histomorphological examination of representative tissue sample collected after shorter duration (12 hours) under physical agitation in sodium deoxycholate of different concentration revealed presence of whole cells with no detectable changes in the architectural fibre pattern; however, the same treated with higher concentration (2.5%) for longer duration (24 to 48 hours) resulted complete decellularization with disintegration of tissue three-dimensional structure. Decellularization protocol run for 24 hours at 37oC in 2% sodium deoxycholate resulted complete removal of cells and preserving the distinctive, natural, three-dimensional collagen structure within the prepared matrix. Gilbert et al., (2006) reported that ionic biological detergent like sodium deoxycholate initiate decellularization with disintegration of cell membrane and productively eliminate cellular residues; which accelerate by mechanical stirring. In contrary to author’s findings Kumar et al., (2013 and 2015) standardized decellularization protocol for small intestine and diaphragm of bubaline origin with application of 2% sodium deoxycholate for a period of 36 and 48 hours at 37^oC under physical agitation respectively. The changes of clinical parameters and gross changes of the surgical wound (Table 1) revealed that the rectal temperature (^oC), heart rate (beat/minute) and respiration rate (rate/minute) were increased significantly on 3^rd day in all the groups, thereafter the values were declining trend towards normalcy at the end of study. The elevated values immediately after surgical reconstruction might be due to surgical trauma. The author’s findings were in accordance with the observations of Singh et al., (2008) in rabbits and Kumar et al., (2015) in pig. There was significant elevation of degree of swelling, exudation, warmth and pain on palpation score of surgical wounds in all the groups on 3^rd post-surgical days followed by reduction towards end; however, there was significant difference of the values between Group A (Fig 8) and Group B on 5^th and 7^th post-surgical days (Fig 9). This might be attributed to the effect of methylprednisolone administration in Group B. Kumar et al., (2013) reported mild to moderate inflammation, oedema and elevation of body physiological parameters following ventral hernioplasty in six (6) goats with application of acellular dermal matrix.



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The changes of haematological parameters (Table 2) showed significant reduction of Hb%, PCV% and Lymphocyte %; while significant increase of TLC, Neutrophil % in all the groups on 3^rd post-surgical day. The changes of Eosinophil %, Basophil % and Monocyte % remained non-significant variation in entire experiment periods. A significant variation of TLC, Neutrophil % and Lymphocyte % between Group A and Group B on 5^th and 7^th day, which might be due attributed to surgical trauma induced local inflammatory response of the host tissue and the result of neutrophilic leucocytosis and use of corticosteroids in Group B post-operatively resulting lyses of lymphoid tissue as well as lyses of circulating lymphocyte. The author’s findings were in accordance with the findings of Tembhurne et al., (2010); Kumar et al., (2016) in goat; Toth et al., (2014) and Singh (2015) in canine.



The changes of biochemical parameters (Table 3) revealed significant elevation of Creatine Kinase (ul^-1), Serum Cortisol (ng ml^-1), Total Serum Protein (gdl^-1), Serum Globulin (gdl^-1) level of in all the groups on 3^rd  day following surgical reconstruction; however there was a significant variation of total serum protein level between Group A and Group B was observed on 5^th day. Tissue traumatisation during surgical intervention, stress, release of acute-phase protein and administration of methylprednisolone in Group B might be attributed to such changes. The values of Alanine Amino Transferase, Gamma-Glutamyle transferase and Serum Albumin were remained non-significant variation during entire experiment period. In agreement with the findings of the present experiment, Munthe-Kass et al., (2018) and Nevill et al., (2010) in dog; Schreiber et al., (1982), Kumar et al., (2015) and Schulz et al., (2007) in piglet and Citil et al., (2004) and Jamma (2004) in cattle and buffaloes recorded similar results .Indirect ELISA (Table 4), absorbance at 492 nm (OD492)revealed negligible antigenicity of autologous Tunica Vaginalis to host tissue (Group C); however minimal extracellular matrix elicits early immunogenic and inflammatory reaction in Group A and Group B, which decreased gradually. The difference of absorbance between Group A and Group B on 15^th and 20^th day of post implantation might be due to immunosuppressive action of methylprednisolone in Group B. Wu et al., (2002) observed that xenogenic acellular dermal matrix produced immunogenic and inflammatory reaction at early stage of implantation which decreased gradually. Coito and Kupiec-Weglinsky (1996) reported that even after remove the cellular components with nuclear debris from biomaterials the extracellular matrix of the acellular tissue itself may elicit some amount of immune response. In corroborated with the author’s findings, Singh et al., (2008) recorded minimal ELISA reaction at 90th day of post implantation of acellular biomaterials of porcine origin in induced abdominal defect in rabbits following initial rise in antibody titre.

Chemical bio-detergent like sodium deoxycholate can effectively be used @ 2% concentration, for 24 hours treatment in orbital shaker for preparation of allogenic acellular diaphragm matrix (ADM) of porcine origin. Clinical, haemato-biochemical and immunological studies revealed that the umbilical hernioplasty with allogenic ADM promises clinical utility. The immune response of host tissue to ADM as revealed by Indirect ELISA was negligible.

The authors are thankful to authority of Assam Agricultural University, Jorhat-13, Assam for providing the required facilities to carry out the experiment.

The authors declared that they have no any conflict of interest.