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Hernia (2018) 22:1033–1039 https://doi.org/10.1007/s10029-018-1799-8

ORIGINAL ARTICLE

iBTA-induced bovine Biosheet for repair of abdominal wall defects in a beagle model: proof of concept

Y. Nakayama1,2 · N. Oshima1,3 · E. Tatsumi1 · O. Ichii4 · T. Nishimura5

Received: 27 May 2018 / Accepted: 13 July 2018 / Published online: 18 July 2018 © Springer-Verlag France SAS, part of Springer Nature 2018

AbstractIntroduction We evaluated the usefulness of xeno-Biosheets, an in-body tissue architecture-induced bovine collagenous sheet, as repair materials for abdominal wall defects in a beagle model.Materials and Methods Biosheets were prepared by embedding cylindrical molds into subcutaneous pouches of three Holstein cows for 2–3 months and stored in 70% ethanol. The Biosheets were 0.5 mm thick, cut into 2 cm × 2 cm, and implanted to replace defects of the same size in the abdominal wall of nine beagles. The abdominal wall and Biosheets were harvested and subjected to histological evaluation at 1, 3, and 5 months after implantation (n = 3 each).Results The Biosheet and bovine pericardiac patch (control) were not stressed during the suture operation and did not split, and patches were easily implanted on defective wounds. After implantation, the patch did not fall off and was not perforated, and healing was observed nacroscopically in all cases. During the first month of implantation, accumulation of inflammatory cells was observed along with decomposition around the Biosheet. Decomposition was almost complete after 3 months, and the Biosheet was replaced by autologous collagenous connective tissue without rejection. After 5 months, the abdominal wall muscle elongated from the periphery of the newly formed collagen layer and the peritoneum was formed on the perito-neal cavity surface. Regeneration of almost all layers of the abdominal wall was observed. However, almost all pericardium patches were remained even at 5 months with inflammation.Conclusion Bovine Biosheets requiring no special post-treatment can be useful as off-the-shelf materials for abdominal wall repair.

Keywords Abdominal wall · In-body tissue architecture · Biosheet · Xeno · Repair material

Introduction

Synthetic implants [1–3] or naturally derived tissue matri-ces [4–7] have been used as materials for the repair of large abdominal wall defects in reconstruction and reinforce-ment. Although synthetic materials, such as a polypropyl-ene meshes (Ethicon Inc., NJ) [1] or expanded polytetra-fluoroethylene sheets (Gore & Associates, Inc., DE) [2, 3], have adequate tensile strength to support the abdominal vis-cera, they are associated with unfavorable effects including fibrotic encapsulation, infection, erosion, and mesh extru-sion. Tissue matrices, such as acellular human dermis (Allo-Derm, LifeCell Co., NJ) [4], human dura (SYNTHECELL, DePuySynthes Inc., PA) [5], bovine glutaraldehyde-fixed pericardium (Edwards Lifesciences, CA) [6], and decellular-ized bovine pericardium (Tutomesh, RTI Surgical, FL) [7], have been used because of their biocompatibility. However, problems have been reported in certain materials that have

* Y. Nakayama biovalve@icloud.com

1 Department of Artificial Organs, National Cerebral and Cardiovascular Center Research Institute, 5-7-1, Fujishirodai, Suita, Osaka 565-8565, Japan

2 Biotube Co., Ltd., 3-4-15 Takeshima, Nishiyodogawa, Osaka 555-0011, Japan

3 Veterinary Internal Medicine, Department of Veterinary Medicine, College of Bioresource Sciences, Nihon University, 1866, Kameino, Fujisawa, Kanagawa 252-0880, Japan

4 Laboratory of Anatomy, Department of Basic Veterinary Sciences, Faculty of Veterinary Medicine, Hokkaido University, Kita 18, Nishi 9, Sapporo, Hokkaido 060-0818, Japan

5 Research Faculty of Agriculture, Graduate School of Agriculture, Hokkaido University, Kita 9 Nishi 9, Sapporo, Hokkaido 060-8589, Japan

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exhibited degraded biomechanical properties, i.e., the repair site is weaker than if autologous tissue had been used. As healing and resorption of a biological scaffold occur, loss of tissue support over time may result in relaxation or bulging of the abdominal wall.

We advocate the use of in-body tissue architecture (iBTA), in which implantable collagenous tissues were formed by only embedding molds in subcutaneous pouches [8]. Tubular tissue (Biotube) [9], membranous tissue (Biosheet) [10–12], and three-dimensional valve-shaped tissue (Biovalve) [13] have been fabricated and implanted in several animal models with satisfactory regenerative func-tion. Autologous or allogenic Biosheets have been applied to the cornea [10] as well as to the repair of the diaphragm [11] and esophagus [12]. The targeted tissues could be recon-structed using iBTA-induced tissues as scaffolds within a few months after implantation [8]. However, there are no reports of studies on implantation using iBTA-induced het-erologous tissues. Here, we first performed an implantation study on bovine xeno-Biosheets for the purpose of examin-ing their usefulness as a new restorative material for the repair of dog abdominal wall defects.

Materials and methods

Ethical approval

All animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals, published by the United States National Institutes of Health (NIH Publication No. 85-23, received 1996). The animal experiments were approved by the National Cerebral and Cardiovascular Center Research Institute Committee (No. 17013) and Hokkaido University (No. 16-0110).

Preparation of bovine Biosheets

The mold for Biosheet preparation was assembled by inserting a silicone tube (outer diameter of 16 mm) into a stainless-steel pipe (internal diameter of 18 mm) with many line slits (Fig. 1a). As described previously [14], the molds (n = 6) were surgically embedded in the subcutaneous abdominal pouches of three Holstein livestock cows (body weight of approximately 700 kg) in Hokkaido University under the combination of epidural and infiltration anesthesia (Fig. 1b). After 2–3 months of regular rearing, the molds were harvested with the surrounding connective tissues. The tubular tissues formed in the molds were extracted. Col-lagenous Biosheets (size of 5 cm × 7 cm, thickness of ca. 0.5 mm, Fig. 1e–g) were obtained by cutting the tubular tissues in the longitudinal direction (Fig. 1c). The Biosheets were stored in 70% ethanol solution prior to implantation.

Biosheet implantation

The abdomen, from the xiphoid process to the groin, was surgically prepared in beagle dogs (n = 9). A 10 cm mid-line abdominal incision was made from approximately 2 cm below the xiphoid process to the pubic symphysis. Bilateral skin flaps were raised using blunt dissection of the sub-cutaneous layer. Two standardized 2 cm × 2 cm incisional defects were made at the midline, identified by the linea alba, by excising a full-thickness portion of the abdominal wall, including the peritoneum, abdominal wall muscle, and fascia. Bovine Biosheets and bovine pericardiac sheets (Edwards, Model no. 4700, SN 4294581) as a control were cut in 2 cm × 2 cm (Fig. 1d). After soaking in a saline solu-tion for a minimum of 10 min prior to use, all implants were implanted into abdominal wall defects by a running suture with a 3-0 polypropylene suture (PROLENE, Ethicon) around the perimeter of the implant, attaching the dermis as an overlay to the abdominal wall musculature. Subcutaneous tissues and skin were closed using multi-layer suturing with 3/0 PROLENE. At 1, 3, or 5 months following the repair, three animals per time period were humanely euthanized with a barbiturate overdose. Following euthanasia, lateral skin incisions were made to expose the surgical site without disruption. Evidence of bowel adhesion, hernia formation, and abdominal bulging was macroscopically observed. The extent of vascularization and tissue ingrowth in the implant was noted.

Histological examinations

Samples were fixed with 10% neutral buffered formalin, embedded in paraffin, and sectioned into sections of 3–5 µm in thickness. The specimens were stained with hematoxylin and eosin, Masson’s trichrome stain, and Elastica van Gie-son stain. In the histological examinations, decomposition of implanted materials, inflammation, and formation of abdom-inal wall or abdominal muscle were evaluated in score. The grading in the decomposition was as follows: 0 = no degra-dation; 1 = light; 2 = moderate; 3 = severe; 4 = complete. The grading in the inflammation was as follows: 0 = no inflam-mation; 1 = light; 2 = moderate; 3 = severe. Formation of abdominal wall or abdominal muscle was graded as 0 = no events or 1 = occurrence of events.

Results

Nine beagles underwent patch implantation of bovine Biosheets and bovine pericardium as a control to two defects at the abdominal wall (Fig. 2a). All patch procedures were

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completed without any complications such as anastomotic leak or infection (Fig. 2b). All animals resumed normal appetite, urination, and defecation patterns within 24 h after the operation. Any abnormal abdominal wall defects, hernia formation, or abdominal bulging did not occur during the pre-determined observation period of 1 (n = 3), 3 (n = 3), and 5 months (n = 3) in all cases, including one control, in which severe infection occurred at 5 months after implantation.

Macroscopic observation of the harvested abdominal tis-sues after the first month of implantation showed that the surfaces of both types of biological materials were covered with a similar thin white tissue layer (Fig. 2c, e). Adhesion of bowel tissue on the peritoneal cavity side was minor and could be easily detached. All Biosheet-implanted groups showed evidence of visible angiogenesis toward the grafts at

the peritoneal side (Fig. 2e). There were no signs of inflam-mation, infection, or fibrosis, and both materials were stably embedded in the host subcutaneous tissues. Furthermore, lit-tle seromas or shrinkage of the materials was observed. The healing process continued even after 5 months (Fig. 2d, f). On the peritoneal side, the Biosheet-implanted surface was covered with thicker tissue and it was difficult to recognize the Biosheet.

In the histological examination, during the first month of implantation, degradation of the Biosheets occurred around them and accumulation of numerous inflammatory cells was observed (Fig. 2g; Table 1). At the third month, the Biosheet almost disappeared and was replaced with autologous col-lagen without rejection (Fig. 2h; Table 1). Therefore, inflam-mation associated with Biosheet degradation was minor. In

Fig. 1 Photos of a mold for the preparation of the Biosheet (a) and bovine Biosheet formed in the mold (b). The Biosheet was formed by in-body incubation of the mold embedded in the cow (c). Bovine Biosheet after storage in an ethanol solution (left) and glutaralde-hyde-treated bovine pericardium sheet (right) for implantation (d).

Histological cross-sectional micrographs of the bovine Biosheets (e–g) stained with hematoxylin and eosin (e), Masson’s trichrome (f), and Elastica van Gieson stain (g). Upper side is silicone-contacting surface (e–g)

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Fig. 2 Photo of two incisional defects (2 cm × 2 cm) prepared at the abdominal wall of beagle dogs (a). The bovine Biosheet (B) and the bovine pericardiac sheet (P) were implanted at the respective defect (b). Macroscopic observation of the abdominal wall implanted with the bovine Biosheet (B) and the bovine pericardium sheet (P) view from skin side (c, d) and the peritoneal side (e, f) after 1 (c, e) or

5 months (d, f) of implantation. Histological cross-sectional micro-graphs of the beagle abdominal wall after implantation of the bovine Biosheets (g, h, i) or the bovine pericardiac sheet (j, k, l) for 1 (g, j), 3 (h, k), and 5 months (i, l). All tissues were stained with Masson’s trichrome stain. Lower side is the peritoneal side (g–l)

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addition, almost complete abdominal wall was formed. After 5 months, layered peritoneal regeneration occurred simulta-neously with infiltration of muscle tissue (Fig. 2i; Table 1).

On the other hand, in the control bovine pericardium sheet, accumulation of numerous inflammatory cells around the sheet continued throughout the observation period, and the sheet shape could be confirmed (Fig. 2j, k; Table 1). In one case, severe inflammation occurred at 5 months with fragmentation of the sheet (Fig. 2l).

Discussion

This study showed for the first time the possibility of het-erogeneous iBTA tissues as a biomaterial. So far, Biosheets have been produced using rats, rabbits, dogs, and goats [8]. In Biosheets made from goats, detailed analyses have been performed in terms of materials and mechanics, and a linear relation between their thickness and strength was found [15]. With a thickness of greater than 0.15 mm, the Biosheet had higher breaking strength than that of the human aortic valve leaflet. The Biosheet was cut into the shape of a goat aor-tic leaflet for autologous implantation according to Ozaki’s method [16]. In a 3-month implantation experiment model, the valvular function was maintained without breakage, where reconstruction of the valvular leaflet structure was recognized, and it was proved that the Biosheet had excellent scaffold function as a valve membrane-regenerating mate-rial [17].

Biosheets can also be produced subcutaneously in cows [14]. Tubular tissue Biotubes up to approximately 3 mm in thickness were formed by embedding a mold, made of a stainless-steel pipe and a silicone tube similar to the mold used in this experiment, in cows for 2–3 months. When the obtained Biotubes were opened up, the Biosheet could be obtained as in this study. Biotubes with a thickness of approximately 0.7 mm had a high burst pressure beyond that of the thoracic aorta of beagle dogs and was around the same as that of beagle dog pericardium. Therefore, the

Biosheets obtained in this study were presumed to have simi-lar strength. Indeed, no rupture occurred in all patch implan-tation of Biosheets on the abdominal wall defect.

Polylactic acid, which is one of the most popular biode-gradable polymers, loses strength by hydrolysis [18, 19]. Since the material itself is passively decomposed, material failure can occur if tissue formation is insufficient. In fact, the use of tissue-regenerable polylactic acid-based vascular grafts is limited in low-pressure areas including the pulmo-nary artery [20]. In this study, the shape of the Biosheet remained after the first month of implantation, but almost disappeared at 3 months, where an autologous collagen layer was firmly formed. It was considered that the abdominal wall strength could be maintained owing to the excellent balance between the degradation of the Biosheet and the formation of the autologous collagen membrane.

On the other hand, it was reported that when Biosheets made from rabbits were patch-implanted into diaphragm defects, hernia was present in 21% of cases [11]. The strength of iBTA-induced tissues depends on the size of the animal species. In general, iBTA tissues formed with small animals are weak but those from large animals are strong. Indeed, rabbit Biosheets are soft and their Young’s modulus is less than half that of bovine Biosheets [11, 14]. Therefore, the insufficient strength of the rabbit Biosheets was consid-ered to be the cause of hernia.

Heterogeneous tissues from cows or swine are already widely used clinically [20, 21]. The pericardium of domes-tic animals fixed with glutaraldehyde has long been used as a valve membrane material for biological valves [20] or as a repair material in cardiovascular surgery [21]. Glu-taraldehyde can increase the strength of native tissues by chemical crosslinking. However, residual glutaraldehyde causes inflammation because of its toxicity. In addition, the crosslinking cannot be enzymatically degraded in the living body, so the crosslinked tissues remain as a foreign matter for a long period of time and may cause calcification [22]. In fact, in the bovine pericardiac patch used as a control in this study, inflammation around the pericardium contin-ued from 1 to 5 months of implantation. As a result, the abdominal wall was not reconstructed. On the other hand, the bovine Biosheet was sufficiently robust without glutaral-dehyde treatment and suture with native tissue was possible without cutting.

Decellularization of heterologous tissues has been car-ried out with the aim of tissue regeneration [23]. Although various treatment methods have been developed, it is possi-ble to remove immunogenic cell-derived components while maintaining the strength of the tissues themselves. Decel-lularized tissues have been implanted as blood vessels, heart valves, etc., and reconstruction following implantation has been reported [23]. In this study, all cells were dead because the Biosheets were preserved in an alcohol solution before

Table 1 Summary of efficacy scores in patch implantation models

B Biosheet, P pericardiac patch

Sample Implanta-tion period (month)

Degrada-tion

Inflamma-tion

Abdomi-nal wall formation

Muscle forma-tion

B 1 2.3 1.7 0 0B 3 3 1.3 1 0B 5 4 0 1 0.3P 1 0 2 0 0P 3 0 2.3 0 0P 5 1.3 2.3 0.3 0

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implantation, but the heterogeneous cell-derived compo-nents remained. In the hematoxylin and eosin staining of the Biosheet before implantation, a number of cell nuclei were observed. As a result, severe inflammation occurred around the bovine Biosheet within the first month of implan-tation in beagle dogs. However, inflammation almost con-verged at 3 months after implantation, and the replacement with autologous tissue was smooth without rejection. In this experimental model, the size of the abdominal wall defect was relatively small at 2 cm × 2 cm. Therefore, the size of the Biosheet used for the patch was small. It is necessary to investigate whether it is possible to repair even larger defects in the future.

Conclusion

Bovine Biosheets without any post-treatment could repair abdominal wall defects in beagle dogs. The Biosheets were replaced with native abdominal wall tissues without rejec-tion. The xeno-Biosheets may be useful for regenerable and restorative material for hernia. Since this study indicated the possibility of the xenoimplantation of iBTA-induced tissues, we can expect off-the-shelf applications of iBTA tissues.

Acknowledgements We would like to thank Manami Sone, Tomohiro Mitani, Teppei Ikeda, and Keigo Kosenda for their valuable help.

Compliance with ethical standards

Conflict of interest The authors declare that they have no conflict of interest associated with this work.

Ethical approval The animal experiments were approved by the National Cerebral and Cardiovascular Center Research Institute Com-mittee (No. 17013) and Hokkaido University (No. 16-0110).

Human and animal rights All animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Ani-mals, published by the United States National Institutes of Health (NIH Publication No.85-23, received 1996).

Informed consent For this type of study, formal consent is not required.

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