- Email: [email protected]
Urinary Bladder Matrix Scaffolds Promote Pericardium Repair in a Porcine Model
j o u r n a l o f s u r g i c a l r e s e a r c h m a y 2 0 2 0 ( 2 4 9 ) 2 1 6 e2 2 4
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.JournalofSurgicalResearch.com
Urinary Bladder Matrix Scaffolds Promote Pericardium Repair in a Porcine Model Natalia Amigo, PhD,a Juan Martin Riganti, MD,b,1 Mauricio Ramirez, MD,b Lorenzi Andrea, MD,b Pedro Renda, MD,b Romina Lovera, MD,b Ariel Pascaner, PhD,a Carlos Vigliano, MD, PhD,a,b Damia´n Craiem, PhD,a Thomas W. Gilbert, PhD,c,d Nathaniel T. Remlinger, PhD,c,d and Alejandro Nieponice, MD, PhDa,b,e,* a
Instituto de Medicina Traslacional, Trasplante y Bioingenierı´a (IMeTTyB), Universidad Favaloro-CONICET, Buenos Aires, Argentina b Esophageal Unit, Hospital Universitario Fundacio´n Favaloro, Buenos Aires, Argentina c Department of Bioengineering, University of Pittsburgh, Pittsburgh, Pennsylvania d ACell, Inc, Columbia, Maryland e McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, Pensilvania
article info
abstract
Article history:
Pericardium closure after cardiac surgery is recommended to prevent postoperative ad-
Received 11 September 2019
hesions to the sternum. Synthetic materials have been used as substitutes, with limited
Received in revised form
results because of impaired remodeling and fibrotic tissue formation. Urinary bladder
14 November 2019
matrix (UBM) scaffolds promote constructive remodeling that more closely resemble the
Accepted 27 December 2019
native tissue. The aim of the study is to evaluate the host response to UBM scaffolds in a
Available online xxx
porcine model of partial pericardial resection. Twelve Landrace pigs were subjected to a median sternotomy. A 5 7 cm pericardial defect was created and then closed with a 5 7
Keywords:
cm multilayer UBM patch (UBM group) or left as an open defect (control group). Animals
Urinary bladder matrix
were survived for 8 wk. End points included gross morphology, biomechanical testing,
Pericardium repair
histology with semiquantitative score, and cardiac function. The UBM group showed mild
Regenerative medicine
adhesions, whereas the control group showed fibrosis at the repair site, with robust ad-
Biological scaffold
hesions and injury to the coronary bed. Load at failure (gr) and stiffness (gr/mm) were
Extracellular matrix
lower in the UBM group compared with the native pericardium (199.9 59.2 versus 405.3 99.89 g, P ¼ 0.0536 and 44.23 15.01 versus 146.5 24.38 g/mm, P ¼ 0.0025, respectively). In the UBM group, the histology resembled native pericardial tissue, with neovascularization, neofibroblasts, and little inflammatory signs. In contrast, control group showed fibrotic tissue with mononuclear infiltrates and a lack of organized collagen fibers validated with a histologic score. Both groups had normal ultrasonography results without cardiac motility disorders. In this setting, UBM scaffolds showed appropriate features for pericardial repair, restoring tissue properties that could help reduce postsurgical adhesions and prevent its associated complications. ª 2020 Elsevier Inc. All rights reserved.
* Corresponding author. Esophageal Unit, Hospital Universitario Fundacio´n Favaloro, Av Belgrano 1752, Buenos Aires, Argentina. Tel./fax: þ541143781200x1748. E-mail address: [email protected] (A. Nieponice). 1 Co-first author. 0022-4804/$ e see front matter ª 2020 Elsevier Inc. All rights reserved. https://doi.org/10.1016/j.jss.2019.12.033
amigo et al pericardium repair in a porcine model
Introduction Pericardial repair after cardiac surgery protects the heart from postoperative retrosternal adhesions that can negatively affect the patient’s safety during repeated sternotomy. However, primary repair is not often feasible. Therefore, various synthetic or biological materials have been used as pericardial substitutes. Efficacy of synthetic materials for pericardium repair is debated because of a number of serious disadvantages. Several studies showed that these materials can worsen the normal epicardial reaction causing a significant foreign body reaction and increasing the incidence of cardiac tamponade.1-3 In this context, extracellular matrix (ECM) scaffolds have been emerging as alternatives. ECM scaffolds have been shown to facilitate site-appropriate host tissue remodeling in several preclinical and clinical applications, including repair of skeletal muscle tissue,4,5 soft tissues such as the esophagus,6-8 lower urinary tract,9-12 and vascular tissues,13-15 among others. In several applications, the use of ECM scaffolds has shown improvement in biomechanical properties.15 These considerations are very relevant when working with tissues such as the myocardium or the pericardium, whose normal function depends exclusively on its biomechanical properties and where a rigid scaffold can generate important limitations. Although other biomaterials and other bioabsorbable synthetic materials are currently used worldwide for pericardial repair, their long-term results have been suboptimal, not very encouraging or even contraindicated, with a marked inflammatory response and tissue fibrosis.16,17 The present study aims to evaluate the host response to ECM scaffolds derived from porcine urinary bladder matrix (UBM) in a preclinical model of partial pericardial resection.
Materials and methods A total of 12 Landrace female pigs weighing between 30 and 40 kg were subjected to surgery in sterile conditions, under
217
general anesthesia and mechanical ventilation. A midsternal thoracotomy was performed, and 5 7 cm of pericardium defect was created. Animals were randomly assigned to one of two treatment groups: The UBM group (UBM, n ¼ 6) was repaired with a 5 7 cm of multilayer UBM patch (Fig. 1A) (ACell, Inc, Columbia, MD). The control group (control, n ¼ 6) was left as an open defect of the pericardium. Animals were humanely euthanized at 8 wk. The rationale for this time point was based on previous author’s experience and publications regarding remodeling time of the UBM scaffolds.18-20 Cardiac function was monitored with cardiac ultrasonography before and after implant. At necropsy, after gross morphological evaluation, samples were sent for biomechanical testing and histology. Assessment of macroscopic analysis, biomechanical testing, histologic analysis, and ultrasonography was always performed in a single blinded fashion. All animal procedures were performed in compliance with the 1996 Guide for the Care and Use of Laboratory Animals (NIH 85-23, 1996), with approval and continuous monitoring of the Institutional Committee for the Care and Use of Laboratory Animals (CICUAL) of Institute of Translational Medicine, Transplantation and Bioengineering-Favaloro University (IMeTTyB-UF).
Surgical procedure Each animal was preanesthetized with 20 mg/kg of xylazine and 50 mg/kg of ketamine by intramuscular administration, and anesthesia was maintained by orotracheal intubation (5 mg/kg of isoflurane and intravenous propofol). Briefly, animals were placed in a supine position and prepared for a median sternotomy with standard surgical equipment. A full median sternotomy was performed until the pericardium without opening the pleural cavities. A partial resection of the pericardial sac was performed to create a 5 7 cm defect. Animals randomly assigned to the UBM group were repaired with a 5 7 cm of multilayer UBM patch (Gentrix Surgical Matrix Plus, ACell Inc, MD; Fig. 1) and secured by interrupted nonabsorbable sutures (2-0 Prolene Polypropylene Suture.
Fig. 1 e Progression in the animal, thoracic cavity view, UBM group. (A) Surgery, (B) necropsy with pericardium, and (C) necropsy with pericardium removed. (Color version of figure is available online.)
218
j o u r n a l o f s u r g i c a l r e s e a r c h m a y 2 0 2 0 ( 2 4 9 ) 2 1 6 e2 2 4
Ethicon US LLC, Somerville, NJ) to the remaining pericardium. Pigs in the control group were left with an open pericardial defect. In both groups, the sternotomy was closed without drains using medical grade sternal wires.
Postsurgical care The pigs were recovered from anesthesia, extubated, and monitored in the recovery room until they were resting comfortably in sternal position and were kept in a cubicle specially designed for this kind of animal, where they can stay awake. The pigs were given prophylactic antibiotics consisting of a combination of G penicillin (20.000 UI/kg) and streptomycin sulfate (2 g) via intramuscular administration. After surgery, the pigs received fentanyl (20 mcg/kg intramuscularly) for analgesia as needed and were fed with a high-protein balanced food and water ad libitum. Vital signs and wound care were monitored daily. The weight was checked every 7 d.
elongation (millimeters) were recorded through each test, and a load-elongation curve was plotted. A linear regression was calculated using the linear portion of the curve, from the elbow that indicated end of the collagen recruitment phase until failure.18 Load at failure (gr), elongation at failure (mm), and stiffness (gr/mm) were recorded through each test using GraphPad Prism, version 8.0.2 for Windows, GraphPad Software, La Jolla, CA, www.graphpad.com. The statistical analysis comparison was performed with a onetailed Student t-test, considering P < 0.05 as significant. Results were expressed as mean standard deviation.
Histology Samples of the pericardium tissue were embedded in paraffin, and 5-mm sections were obtained. Sections were processed for staining with hematoxylin and eosin or Masson’s trichrome, and photomicrographs were obtained at 40, 100, 200, and 400, respectively.
Gross morphology Semiquantitative histologic score At time point of 8 wk postsurgery, the animals were humanely euthanized, with an anesthesia overdose with xylazine/ketamine by intramuscular administration and a propofol intravenous bolus. Through a U-shaped thoracic incision, avoiding the previous surgical field, the pericardial area was carefully inspected before tissue harvesting. The presence of fibrotic areas at the repair site was recorded. Particular attention was given to the presence of adhesions to the sternum or between the remodeled tissue and the epicardial surface and to the type of dissection required for separation. After inspection, the remodeled pericardium was harvested and submitted to mechanical testing and histologic processing. Adhesions were categorized using the following score: 1 Mild adhesions/no fibrosisdSoft dissection required for separation. 2 Moderate adhesions/moderate fibrotic layerdCombination of soft and sharp dissection required. 3 Severe adhesions/severe fibrotic layerdUnderlying structures were not identifiable. Therefore, sharp dissection is required.
Histologic evaluation was performed by a certified pathologist blinded to the treatment group, using high-powered light microscopy (40, 100, and 200 magnification) to evaluate the hematoxylin and eosinestained slides. The entire graft-tissue interface (10 nonoverlapping fields per specimen) was evaluated at 100 and 200 using a semiquantitative scoring system graded for inflammation, necrosis, fibrosis and fibrous encapsulation, neovascularization, cellular infiltration and collagen fibers, and global morphology according to an adapted scale (Table 1).21-23 Lower scores on this scale represent more favorable outcomes with respect to greater cellular infiltration and neovascularization with low levels of inflammation and necrosis, fibrosis, and fibrous encapsulation. A composite histologic score was also calculated for each sample by taking the average of the scores in each of the six subcategories mentioned previously. The values obtained were used for statistical analysis between groups using GraphPad Prism, version 8.0.2, with a one-tailed Student ttest, considering P < 0.05 as significant. Results were expressed as mean standard deviation.
Biomechanical testing
Cardiac ultrasonography
To evaluate the remodeling of the UBM patch, biomechanical properties after explantation were compared with those of native pericardium in the same animals. Comparison with the control group was not performed because there was no repair in these animals. Briefly, one strip of tissue from the UBM patch area and native pericardium was obtained using a 10-mm long by 3-mm wide biopsy punch with a dog-bone shape.18 Each test sample was immersed in saline (37 C) bath for tensile testing to failure. The elongation rate was set to 25 mm/s (1.5 mm/min). Stretch was imposed with an accuracy of 10 mm, and strength was measured with a load cell of 10 n (1 kg). Signals of strength and stretching were digitalized at a sampling frequency of 10 Hz with a 12 bit resolution (DVP04ADS module connected to a DVP12SA PLC, Delta Electronics, China). Load (grams) and
Cardiac function was monitored with ultrasound (Sonos 5500; Hewlett Packard, Palo Alto, CA) 24 h preoperatively and 24 h before necropsy. Ejection fraction, motility, and capacity of the cardiac cavities were evaluated in both groups. Index of global systolic function as shortening fraction was calculated as follows: (DDLVDSLV)/DDLV. DDLV is left ventricular diastolic diameter, and DSLV is left ventricular systolic diameter. Both were measured in mode M. Fraction of posterior parietal thickness (PPT%) and fraction of septal parietal thickness (SPT %) are indexes of regional systolic function as walls are measured separately. Both PPT% and SPT% were calculated as follows: ST/DT 100, where ST is systolic thickness and DT is diastolic thickness, at both posterior wall and septum. They were measured in mode M and two-dimensional mode. The values obtained were used for statistical analysis between
Table 1 e Semiquantitative histologic score. Cell type/response
Score 0
Inflammation (PMN cells, lymphocytes, plasma cells, macrophages, FBGCs)
Necrosis
None. No inflammatory cells presents
None
1 Rare (one to five PMNs per HPF). Inflammatory cells present <10% of surface area
Minimal (<10% of surface area)
2
3
4
Mild infiltrate (5-10 PMNs per HPF). Inflammatory cells present in 10%-33% of surface area
Heavy infiltrate (>10 PMNs per HPF). Inflammatory cells present in 33%-75% of surface area
Packed (PMNs too numerous to count). Inflammatory cells present (neutrophils, macrophages, and FBGC) >75% of surface area
Mild (10%-33% of surface area)
Moderate (33%-75% of surface area)
Severe (>75% of surface area)
None. No fibrous encapsulation
Minimal (<10% of surface area). Minimal encapsulation (<10% of periphery)
Mild (10%-33% of surface area). Mild encapsulation (10%-33% of periphery)
Moderate (33%-75% of surface area). Moderate encapsulation (33%-75% of periphery)
Severe (>75% of surface area). Extensive encapsulation (>75% of periphery)
Neovascularization
Marked. Most abundant capillaries present (>20 capillaries/HPF). Vessels penetrate into center of scaffold or resected area
Moderate. Abundant capillaries present (10-20 capillaries/HPF). Vessels penetrate into center of scaffold or resected area
Mild. Many capillaries (5-10 capillaries/HPF). Vessels infiltrate scaffold, but none reach center of scaffold or resected area
Few capillaries (1-5 capillaries/HPF). Vessels present at scaffold periphery, no penetration into scaffold or resected area
None. No capillaries or blood vessels present
Cellular infiltration
Marked (>75% of surface area). Cells penetrate into center of scaffold or resected area
Moderate (33%-75% of surface area). Cells penetrate into center of scaffold or resected area
Mild (10%-33% of surface area). Cells infiltrate scaffold, but none reach center of scaffold or resected area
Minimal (<10% of surface area). Cells contact periphery, no penetration into scaffold or resected area
None. No cells in contact with scaffold or resected area
Collagen fibers and global morphology
Collagen fibers aligned and similar to native tissue. Tissue appears as histologically normal. Highly conserved global morphology
Collagen fibers minimally disorganized. Minimal to mild disorganization of tissue. Minimally altered global morphology (<10% of surface area)
Collagen fibers mildly disorganized. Mild to moderate disorganization of the tissue by dense fibrous connective tissue. Mildly altered global morphology (10%-33% of surface area)
Collagen fibers moderately disorganized. Moderate disorganization of the tissue. Moderately altered global morphology (33%75% of surface area)
PMNs ¼ polymorphonuclear leukocytes; FBGC ¼ foreign body giant cell; HPF ¼ high-powered field (200).
No organized collagen fibers. Disruption of architecture. Severely altered global morphology (>75% of surface area)
amigo et al pericardium repair in a porcine model
Fibrosis and fibrous encapsulation
219
220
j o u r n a l o f s u r g i c a l r e s e a r c h m a y 2 0 2 0 ( 2 4 9 ) 2 1 6 e2 2 4
Fig. 2 e Macroscopic analysis of the pericardium. Thoracic cavity view, necropsy at 8 wk. (A-C) Group reinforced with UBM scaffold. (A) Sternum, (B) pericardium, and (C) patch dissected. (D-F) Control group, open defect without reinforcement mesh. (D and E) Sternum and (F) pericardium dissection. (Color version of figure is available online.)
groups using GraphPad Prism, version 8.0.2. The values obtained from each group were analyzed by paired t-test. Analysis of variance test and Tukey’s multiple comparisons were performed, considering P < 0.05 as significant. Results were expressed as mean standard deviation.
Results Macroscopic evaluation All the animals of the UBM group (Fig. 1B and C; Fig. 2A-C) showed mild and loose type 1 adhesions, with easy and correct dissection of the remodeled tissue from the sternum and between the remodeled tissue and the epicardial surface. No visible or palpable remnants of the UBM material were found. In contrast, all the animals of the control group (Fig. 2D-F) showed strong type 3 adhesions at both the epicardium and sternum. A sharp and acute dissection was required (with scalpel and scissors) to separate the scar tissue generated from the sternum. Epicardial retraction was found, and coronary bed injury was inadvertently caused during the dissection in some cases.
Biomechanical testing Average load at failure (gr) and stiffness (gr/mm) were lower in the UBM group compared with the native pericardium (199.9 59.2 versus 405.3 99.89 g, P ¼ 0.0536 and 44.23 15.01 versus 146.5 24.38 g/mm, P ¼ 0.0025, respectively; Fig. 3).
Control animals were not tested because no deposition of pericardial tissue was observed.
Histology In the UBM group, a well-organized tissue with neovascularization, neofibroblasts, a preserved histoarchitecture, and little signs of inflammation were found. The collagen fibers were observed aligned and similar to native pericardium, with complete resorption of the UBM scaffold (Fig. 4B and E). There was also evidence of scattered mesothelial cells on the surface although they failed to form a continuous layer. In contrast, all control animals showed dense fibrotic tissue with large mononuclear infiltrates, randomly organized collagen fibers, and clear signs of scar tissue (Fig. 4C and F).
Semiquantitative histologic score The results obtained in histologic semiquantitative analysis were consistent in each of the six categories analyzed as well as in the composite score because higher scores on this scale correlate with poorer outcomes. In all subcategories, as well as in the composite score, UBM group recorded significantly lower values compared with the control group (Fig. 5). Briefly, in the inflammation subcategory, the score was significantly lower in the UBM group compared with the control group (0.83 0.69 versus 3.83 0.38, respectively; P < 0.0001, Fig. 5A). In necrosis subcategory, the UBM group recorded significantly lower levels compared with the control
amigo et al pericardium repair in a porcine model
221
Fig. 3 e Biomechanical properties of pericardium reinforcement. (A) Load at failure (gr). (B) Stiffness (gr/mm). Native [ native pericardium tissue. UBM [ remodeled tissue after implantation of the UBM scaffold (**P < 0.01). (Color version of figure is available online.)
group (0.3 0.46 versus 1.80 0.61, respectively; P < 0.0001, Fig. 5B). The fibrosis and fibrous encapsulation subcategory recorded lower values in UBM group compared with the control group (0.5 0.51 versus 3.5 0.63, respectively; P < 0.0001, Fig. 5C). In neovascularization subcategory, lower values were recorded in the UBM group compared with the control group (0.47 0.51 versus 1.83 0.59, respectively; P < 0.0001, Fig. 5D). In the cellular infiltration subcategory, the score was significantly lower in the UBM group compared with the control group (0.2 0.41 versus 1.2 0.56, respectively; P < 0.0001, Fig. 5C). In the collagen fibers and global morphology
subcategories, lower values were found in the UBM group compared with control group (0.13 0.34 versus 3.77 0.43, respectively; P < 0.0001, Fig. 5F). The composite score combined the results obtained in each subcategory and reflected the overall results obtained in the analysis of remodeled tissues in each of the studied groups. The results showed significantly lower values for the UBM group compared with control group (0.41 0.25 versus 2.65 1.17, respectively; P ¼ 0.0005, Fig. 5G). In summary, lower scores on this scale represent better outcomes with respect to cellular infiltration,
Fig. 4 e Histologic analysis. (A and D) Native tissue. (B and E) UBM group: complete resorption of UBM scaffold, angiogenesis, and neovascularization. Fibers of collagen aligned (arrow). (C and F) Control group, open defect: dense fibrous tissue with desalted collagen fibers and global morphology altered (rhombus). Large mononuclear infiltrate (*). H&E stain (A-C: 2003). Masson’s trichrome stain (D and F: 2003; E: 4003). (Color version of figure is available online.)
222
j o u r n a l o f s u r g i c a l r e s e a r c h m a y 2 0 2 0 ( 2 4 9 ) 2 1 6 e2 2 4
Fig. 5 e Semiquantitative histologic score after 8 wk in vivo by subcategory: (A) Inflammation, (B) necrosis, (C) fibrosis and fibrous encapsulation, (D) neovascularization, (E) cellular infiltration, (F) collagen fibers and global morphology; and (G) composite score. Lower scores represent more favorable outcomes with regard to tissue remodeling. UBM group: remodeled tissue after implantation of the UBM scaffold. Control group: remodeled tissue in pericardium open defect, without reinforcement mesh (****P < 0.0001, ***P < 0.001). (Color version of figure is available online.)
neovascularization, arrangement of collagen fibers, and conserved global morphology, together with low levels of inflammation and necrosis found in the UBM group.
results without displaying cardiac motility disorders in any of their walls, as well as in global cardiac motility, preimplant or postimplant, showing no limitation in cardiac function because of the UBM implant.
Ultrasound results No significant differences were observed in the parameters recorded between neither the groups, shortening fraction, fraction of PPT%, and fraction of SPT% nor the preimplant and postimplant measurements (Table 2). Both groups had normal
Discussion This study describes the host response, histologic, and biomechanical properties of pericardial repair with UBM
223
amigo et al pericardium repair in a porcine model
Table 2 e Cardiac ultrasonography of UBM group and control group. Experimental group
Pre-surgery SF%
PPT%
Pre-necropsy SPT%
SF%
PPT%
SPT%
UBM
26.77 7
43.97 11
32.68 10
23.15 5
38.39 13
28.70 17
Control
25.84 10
25.29 10
31.39 16
30.06 10
52.02 24
34.47 7
SF% ¼ shortening fraction; PPT% ¼ fraction of posterior parietal thickness; SPT% ¼ fraction of septal parietal thickness.
scaffolds after partial pericardial resection in an in vivo porcine preclinical model. The UBM group showed complete remodeling with histologic and biomechanical features similar to native tissue. These results are similar to those previously obtained by several authors4-6,9,10,13-15 and show the beneficial use of a biological bioabsorbable pericardial substitute. In contrast, the control group with open pericardial defects had strong postoperative retrosternal adhesions to the heart. Remodeled UBM tissue had similar or weaker mechanical properties than native pericardium. Although it was not tested in this study, unremodeled scaffolds as and synthetic scaffolds are normally stiffer than native tissues. The fact that remodeled UBM showed similar or softer properties in this study as compared with native pericardium is relevant in a setting where myocardial function can be widely affected by external restriction.15 The semiquantitative histologic assessment, and the results obtained in the biomechanical analysis, supported and validated both macroscopic and histologic findings. The present study, in accordance with the previous literature,6-8,10,11,15,18,24,25 provides further evidence about the beneficial use of a pericardial substitute with biological scaffolds. This is the first report about use of UBM to repair a pericardial defect and includes the first complete characterization of pericardial reconstruction with UBM scaffolds in a preclinical porcine model. Other authors have tested anti-inflammatory agents and a reabsorbable polyethylene glycol barrier to limit adhesion formation after cardiac surgery in a pig model26; and the authors conclude that both systemic indomethacin and locally applied polyethylene glycol barrier are suitable methods to markedly reduce pericardial and retrosternal adhesions. However, given the disadvantages of synthetic replacements in general, new bioabsorbable biological structures have recently emerged as safer alternatives. ECM biological scaffolds have been shown to promote constructive remodeling of site-specific tissues of injury in several preclinical and clinical applications, including repair of skeletal muscle tissue,4,5 soft tissues such as the esophagus,6,8 lower urinary tract,9-12 and vascular tissues,13-15 among others. Several studies have shown that UBM scaffolds contribute to a more site-appropriate repair instead of the intrinsic fibrotic response often seen after open surgery. The mechanisms for this homeostatic response are multiple and currently source of intense basic research. The host immune response to UBM tends to include a robust immunomodulatory M2 macrophage phenotype, as opposed to the typical proinflammatory M1 phenotype.24,27-29 We have previously shown the benefits of
UBM-ECM, both in the remodeling of organ-specific tissue like the esophagus where progenitor cells derived from the bone marrow populated the site of esophageal injury24 and in the restoration of biomechanical and histologic properties in the repair of the diaphragmatic hiatus.18,23 Limitations of this study include the lack of a control group with nondegradable materials. Although synthetic pericardial patch devices have been used and described in some occasions, they are rarely used because of the known constraints previously described. Therefore, we considered the current standard of care without pericardial repair as a more relevant control. Other limitations of this include the lack of cardiopulmonary bypass and cardiotomy in the model; therefore, it cannot be considered a full clinical model of open heart surgery. Short-term follow-up of the animals might also be seen as a constraint in this study. Based on previous publications by the authors and others, it has been demonstrated that, by 8 wk, the UBM scaffolds are mostly remodeled with mechanical properties being influenced mainly by remaining tissue that is stable in time. However, tissue remodeling after surgery occurs for up to 1 y. Also, the lack of functional endpoints prevents a more robust conclusion about the clinical impact of the findings.
Conclusion In this preclinical study, UBM scaffolds showed appropriate features for pericardial repair after partial pericardial resection. Remodeled scaffolds can help reduce postoperative adhesions and prevent complications associated with implanted materials without affecting cardiac function. Long-term follow-up and early clinical translation is warranted to further support this evidence.
Acknowledgment Authors’ contributions: N.A. and J.M.R. contributed equally to data collection, analysis and interpretation of the data, and writing of the article. M.R., L.A., P.R., and R.L. contributed to data collection and analysis and interpretation of the data. A.P. and D.C. contributed to analysis and interpretation of the data. C.V. contributed to analysis and interpretation of the data and critical revisions. T.W.G., N.T.R., and A.N. contributed to conceiving and designing the study, critical revisions, and approving the final version of the article. This work was supported by a research grant from ACell, Inc, Columbia, MD to A.N.
224
j o u r n a l o f s u r g i c a l r e s e a r c h m a y 2 0 2 0 ( 2 4 9 ) 2 1 6 e2 2 4
Disclosure 14.
T.W.G. served as Chief Science Officer at ACell, Inc during the completion of the study and writing of the manuscript. A.N. is a member of SAB at ACell, Inc. N.T.R. is currently employed at ACell, Inc. The other authors have no conflicts of interest or financial ties to disclose.
15.
16.
references 17. 1. Vaideeswar P, Mishra P, Nimbalkar M. Infective endocarditis of the Dacron patchda report of 13 cases at autopsy. Cardiovasc Pathol. 2011;20:e169ee175. 2. Henaine R, Roubertie F, Vergnat M, et al. Valve replacement in children: a challenge for a whole life. Arch Cardiovasc Dis. 2012;105:517e528. 3. Sandusky GE, Lantz GC, Badylak SF. Healing comparison of small intestine submucosa and ePTFE grafts in the canine carotid artery. J Surg Res. 1995;58:415e420. 4. Badylak SF, Tullius R, Kokini K, et al. The use of xenogeneic small intestinal submucosa as a biomaterial for Achilles tendon repair in a dog model. J Biomed Mater Res. 1995;29:977e985. 5. Dejardin LM, Amoczky SP, Ewers BJ, et al. Tissue-engineered rotator cuff tendon using porcine small intestine submucosa. Histologic and mechanical evaluation in dogs. Am J Sports Med. 2001;29:175e184. 6. Badylak SF, Meurling S, Chen M, et al. Resorbable bioscaffold for esophageal repair in a dog model. J Pediatr Surg. 2000;35:1097e1103. 7. Nieponice A, McGrath K, Qureshi I, et al. An extracellular matrix scaffold for esophageal stricture prevention after circumferential EMR. Gastrointest Endosc. 2009;69:289e296. 8. Badylak SF, Hoppo T, Nieponice A, et al. Esophageal preservation in five male patients after endoscopic innerlayer circumferential resection in the setting of superficial cancer: a regenerative medicine approach with a biologic scaffold. Tissue Eng Part A. 2011;17:1643e1650. 9. Kropp BP, Eppley B, Prevel CD, et al. Experimental assessment of small intestinal submucosa as a bladder wall substitute. Urology. 1995;46:396e400. 10. Kropp BP, Rippy MK, Badylak SF, et al. Regenerative urinary bladder augmentation using small intestinal submucosa: urodynamic and histopathologic assessment in long-term canine bladder augmentations. J Urol. 1996;155:2097e2104. 11. Sievert KD, Tanagho EA. Organ-specific acellular matrix for reconstruction of the urinary tract. World J Urol. 2000;18:19e25. 12. Yoo JJ, Atala A. Tissue engineering applications in the genitourinary tract system. Yonsei Med J. 2000;41:789e802. 13. Sandusky Jr GE, Badylak SF, Morff RJ, et al. Histologic findings after in vivo placement of small intestine submucosal
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
vascular grafts and saphenous vein grafts in the carotid artery in dogs. Am J Pathol. 1992;140:317e324. Schoder M, Pavcnik D, Uchida BT, et al. Small intestinal submucosa aneurysm sac embolization for endoleak prevention after abdominal aortic aneurysm endografting: a pilot study in sheep. J Vasc Interv Radiol. 2004;15:69e83. Teodori L, Costa A, Marzio R, et al. Native extracellular matrix: a new scaffolding platform for repair of damaged muscle. Front Physiol. 2014;5:218. Woo JS, Fishbein MC, Reemtsen B. Histologic examination of decellularized porcine intestinal submucosa extracellular matrix (CorMatrix) in pediatric congenital heart surgery. Cardiovasc Pathol. 2016;25:12e17. Nezhad ZM, Ponceleta A, Kerchovea L, et al. Small intestinal submucosa extracellular matrix (CorMatrix) in cardiovascular surgery: a systematic review. Interactive Cardiovasc Thorac Surg. 2016;22:839e850. Riganti JM, Ciotola F, Amenabar A, et al. Urinary bladder matrix scaffolds strengthen esophageal hiatus repair. J Surg Res. 2016;204:344e350. Badylak SF, Vorp D, Spievack AR, et al. Esophageal reconstruction with ECM and muscle tissue in a dog model. J Surg Res. 2005;128:87e97. Nieponice A, Ciotola F, Nachman F, et al. Patch esophagoplasty: esophageal reconstruction using biologic scaffolds. Ann Thorac Surg. 2014;97:283e288. Young DA, McGilvray KC, Ehrhart N, et al. Comparison of in vivo remodeling of urinary bladder matrix and acellular dermal matrix in an ovine model. Regen Med. 2018;13:759e773. Jenkins ED, Melman L, Deeken CR, et al. Evaluation of fenestrated and non-fenestrated biologic grafts in a porcine model of mature ventral incisional hernia repair. Hernia. 2010;14:599e610. Amigo N, Zubieta C, Riganti JM, et al. Biomechanical features of reinforced esophageal hiatus repair in a porcine model. J Surg Res. 2019;246:62e72. Badylak SF, Valentin JE, Ravindra AK, et al. Macrophage phenotype as a determinant of biologic scaffold remodeling. Tissue Eng Part A. 2008;14:1835e1842. Young DA, Jackson N, Ronaghan CA, et al. Retrorectus repair of incisional ventral hernia with urinary bladder matrix reinforcement in a long-term porcine model. Regen Med. 2018;13:395e408. Alizzi AM, Summers P, Boon VH, et al. Reduction of postsurgical pericardial adhesions using a pig model. Heart Lung Circ. 2012;21:22e29. Paige JT, Kremer M, Landry J, et al. Modulation of inflammation in wounds of diabetic patients treated with porcine urinary bladder matrix. Regen Med. 2019;14:269e277. Sadtler K, Wolf MT, Ganguly S, et al. Divergent immune responses to synthetic and biological scaffolds. Biomaterials. 2019;192:405e415. Brown BN, Londono R, Tottey S, et al. Macrophage phenotype as a predictor of constructive remodeling following the implantation of biologically derived surgical mesh materials. Acta Biomater. 2012;8:978e987.
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.JournalofSurgicalResearch.com
Urinary Bladder Matrix Scaffolds Promote Pericardium Repair in a Porcine Model Natalia Amigo, PhD,a Juan Martin Riganti, MD,b,1 Mauricio Ramirez, MD,b Lorenzi Andrea, MD,b Pedro Renda, MD,b Romina Lovera, MD,b Ariel Pascaner, PhD,a Carlos Vigliano, MD, PhD,a,b Damia´n Craiem, PhD,a Thomas W. Gilbert, PhD,c,d Nathaniel T. Remlinger, PhD,c,d and Alejandro Nieponice, MD, PhDa,b,e,* a
Instituto de Medicina Traslacional, Trasplante y Bioingenierı´a (IMeTTyB), Universidad Favaloro-CONICET, Buenos Aires, Argentina b Esophageal Unit, Hospital Universitario Fundacio´n Favaloro, Buenos Aires, Argentina c Department of Bioengineering, University of Pittsburgh, Pittsburgh, Pennsylvania d ACell, Inc, Columbia, Maryland e McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, Pensilvania
article info
abstract
Article history:
Pericardium closure after cardiac surgery is recommended to prevent postoperative ad-
Received 11 September 2019
hesions to the sternum. Synthetic materials have been used as substitutes, with limited
Received in revised form
results because of impaired remodeling and fibrotic tissue formation. Urinary bladder
14 November 2019
matrix (UBM) scaffolds promote constructive remodeling that more closely resemble the
Accepted 27 December 2019
native tissue. The aim of the study is to evaluate the host response to UBM scaffolds in a
Available online xxx
porcine model of partial pericardial resection. Twelve Landrace pigs were subjected to a median sternotomy. A 5 7 cm pericardial defect was created and then closed with a 5 7
Keywords:
cm multilayer UBM patch (UBM group) or left as an open defect (control group). Animals
Urinary bladder matrix
were survived for 8 wk. End points included gross morphology, biomechanical testing,
Pericardium repair
histology with semiquantitative score, and cardiac function. The UBM group showed mild
Regenerative medicine
adhesions, whereas the control group showed fibrosis at the repair site, with robust ad-
Biological scaffold
hesions and injury to the coronary bed. Load at failure (gr) and stiffness (gr/mm) were
Extracellular matrix
lower in the UBM group compared with the native pericardium (199.9 59.2 versus 405.3 99.89 g, P ¼ 0.0536 and 44.23 15.01 versus 146.5 24.38 g/mm, P ¼ 0.0025, respectively). In the UBM group, the histology resembled native pericardial tissue, with neovascularization, neofibroblasts, and little inflammatory signs. In contrast, control group showed fibrotic tissue with mononuclear infiltrates and a lack of organized collagen fibers validated with a histologic score. Both groups had normal ultrasonography results without cardiac motility disorders. In this setting, UBM scaffolds showed appropriate features for pericardial repair, restoring tissue properties that could help reduce postsurgical adhesions and prevent its associated complications. ª 2020 Elsevier Inc. All rights reserved.
* Corresponding author. Esophageal Unit, Hospital Universitario Fundacio´n Favaloro, Av Belgrano 1752, Buenos Aires, Argentina. Tel./fax: þ541143781200x1748. E-mail address: [email protected] (A. Nieponice). 1 Co-first author. 0022-4804/$ e see front matter ª 2020 Elsevier Inc. All rights reserved. https://doi.org/10.1016/j.jss.2019.12.033
amigo et al pericardium repair in a porcine model
Introduction Pericardial repair after cardiac surgery protects the heart from postoperative retrosternal adhesions that can negatively affect the patient’s safety during repeated sternotomy. However, primary repair is not often feasible. Therefore, various synthetic or biological materials have been used as pericardial substitutes. Efficacy of synthetic materials for pericardium repair is debated because of a number of serious disadvantages. Several studies showed that these materials can worsen the normal epicardial reaction causing a significant foreign body reaction and increasing the incidence of cardiac tamponade.1-3 In this context, extracellular matrix (ECM) scaffolds have been emerging as alternatives. ECM scaffolds have been shown to facilitate site-appropriate host tissue remodeling in several preclinical and clinical applications, including repair of skeletal muscle tissue,4,5 soft tissues such as the esophagus,6-8 lower urinary tract,9-12 and vascular tissues,13-15 among others. In several applications, the use of ECM scaffolds has shown improvement in biomechanical properties.15 These considerations are very relevant when working with tissues such as the myocardium or the pericardium, whose normal function depends exclusively on its biomechanical properties and where a rigid scaffold can generate important limitations. Although other biomaterials and other bioabsorbable synthetic materials are currently used worldwide for pericardial repair, their long-term results have been suboptimal, not very encouraging or even contraindicated, with a marked inflammatory response and tissue fibrosis.16,17 The present study aims to evaluate the host response to ECM scaffolds derived from porcine urinary bladder matrix (UBM) in a preclinical model of partial pericardial resection.
Materials and methods A total of 12 Landrace female pigs weighing between 30 and 40 kg were subjected to surgery in sterile conditions, under
217
general anesthesia and mechanical ventilation. A midsternal thoracotomy was performed, and 5 7 cm of pericardium defect was created. Animals were randomly assigned to one of two treatment groups: The UBM group (UBM, n ¼ 6) was repaired with a 5 7 cm of multilayer UBM patch (Fig. 1A) (ACell, Inc, Columbia, MD). The control group (control, n ¼ 6) was left as an open defect of the pericardium. Animals were humanely euthanized at 8 wk. The rationale for this time point was based on previous author’s experience and publications regarding remodeling time of the UBM scaffolds.18-20 Cardiac function was monitored with cardiac ultrasonography before and after implant. At necropsy, after gross morphological evaluation, samples were sent for biomechanical testing and histology. Assessment of macroscopic analysis, biomechanical testing, histologic analysis, and ultrasonography was always performed in a single blinded fashion. All animal procedures were performed in compliance with the 1996 Guide for the Care and Use of Laboratory Animals (NIH 85-23, 1996), with approval and continuous monitoring of the Institutional Committee for the Care and Use of Laboratory Animals (CICUAL) of Institute of Translational Medicine, Transplantation and Bioengineering-Favaloro University (IMeTTyB-UF).
Surgical procedure Each animal was preanesthetized with 20 mg/kg of xylazine and 50 mg/kg of ketamine by intramuscular administration, and anesthesia was maintained by orotracheal intubation (5 mg/kg of isoflurane and intravenous propofol). Briefly, animals were placed in a supine position and prepared for a median sternotomy with standard surgical equipment. A full median sternotomy was performed until the pericardium without opening the pleural cavities. A partial resection of the pericardial sac was performed to create a 5 7 cm defect. Animals randomly assigned to the UBM group were repaired with a 5 7 cm of multilayer UBM patch (Gentrix Surgical Matrix Plus, ACell Inc, MD; Fig. 1) and secured by interrupted nonabsorbable sutures (2-0 Prolene Polypropylene Suture.
Fig. 1 e Progression in the animal, thoracic cavity view, UBM group. (A) Surgery, (B) necropsy with pericardium, and (C) necropsy with pericardium removed. (Color version of figure is available online.)
218
j o u r n a l o f s u r g i c a l r e s e a r c h m a y 2 0 2 0 ( 2 4 9 ) 2 1 6 e2 2 4
Ethicon US LLC, Somerville, NJ) to the remaining pericardium. Pigs in the control group were left with an open pericardial defect. In both groups, the sternotomy was closed without drains using medical grade sternal wires.
Postsurgical care The pigs were recovered from anesthesia, extubated, and monitored in the recovery room until they were resting comfortably in sternal position and were kept in a cubicle specially designed for this kind of animal, where they can stay awake. The pigs were given prophylactic antibiotics consisting of a combination of G penicillin (20.000 UI/kg) and streptomycin sulfate (2 g) via intramuscular administration. After surgery, the pigs received fentanyl (20 mcg/kg intramuscularly) for analgesia as needed and were fed with a high-protein balanced food and water ad libitum. Vital signs and wound care were monitored daily. The weight was checked every 7 d.
elongation (millimeters) were recorded through each test, and a load-elongation curve was plotted. A linear regression was calculated using the linear portion of the curve, from the elbow that indicated end of the collagen recruitment phase until failure.18 Load at failure (gr), elongation at failure (mm), and stiffness (gr/mm) were recorded through each test using GraphPad Prism, version 8.0.2 for Windows, GraphPad Software, La Jolla, CA, www.graphpad.com. The statistical analysis comparison was performed with a onetailed Student t-test, considering P < 0.05 as significant. Results were expressed as mean standard deviation.
Histology Samples of the pericardium tissue were embedded in paraffin, and 5-mm sections were obtained. Sections were processed for staining with hematoxylin and eosin or Masson’s trichrome, and photomicrographs were obtained at 40, 100, 200, and 400, respectively.
Gross morphology Semiquantitative histologic score At time point of 8 wk postsurgery, the animals were humanely euthanized, with an anesthesia overdose with xylazine/ketamine by intramuscular administration and a propofol intravenous bolus. Through a U-shaped thoracic incision, avoiding the previous surgical field, the pericardial area was carefully inspected before tissue harvesting. The presence of fibrotic areas at the repair site was recorded. Particular attention was given to the presence of adhesions to the sternum or between the remodeled tissue and the epicardial surface and to the type of dissection required for separation. After inspection, the remodeled pericardium was harvested and submitted to mechanical testing and histologic processing. Adhesions were categorized using the following score: 1 Mild adhesions/no fibrosisdSoft dissection required for separation. 2 Moderate adhesions/moderate fibrotic layerdCombination of soft and sharp dissection required. 3 Severe adhesions/severe fibrotic layerdUnderlying structures were not identifiable. Therefore, sharp dissection is required.
Histologic evaluation was performed by a certified pathologist blinded to the treatment group, using high-powered light microscopy (40, 100, and 200 magnification) to evaluate the hematoxylin and eosinestained slides. The entire graft-tissue interface (10 nonoverlapping fields per specimen) was evaluated at 100 and 200 using a semiquantitative scoring system graded for inflammation, necrosis, fibrosis and fibrous encapsulation, neovascularization, cellular infiltration and collagen fibers, and global morphology according to an adapted scale (Table 1).21-23 Lower scores on this scale represent more favorable outcomes with respect to greater cellular infiltration and neovascularization with low levels of inflammation and necrosis, fibrosis, and fibrous encapsulation. A composite histologic score was also calculated for each sample by taking the average of the scores in each of the six subcategories mentioned previously. The values obtained were used for statistical analysis between groups using GraphPad Prism, version 8.0.2, with a one-tailed Student ttest, considering P < 0.05 as significant. Results were expressed as mean standard deviation.
Biomechanical testing
Cardiac ultrasonography
To evaluate the remodeling of the UBM patch, biomechanical properties after explantation were compared with those of native pericardium in the same animals. Comparison with the control group was not performed because there was no repair in these animals. Briefly, one strip of tissue from the UBM patch area and native pericardium was obtained using a 10-mm long by 3-mm wide biopsy punch with a dog-bone shape.18 Each test sample was immersed in saline (37 C) bath for tensile testing to failure. The elongation rate was set to 25 mm/s (1.5 mm/min). Stretch was imposed with an accuracy of 10 mm, and strength was measured with a load cell of 10 n (1 kg). Signals of strength and stretching were digitalized at a sampling frequency of 10 Hz with a 12 bit resolution (DVP04ADS module connected to a DVP12SA PLC, Delta Electronics, China). Load (grams) and
Cardiac function was monitored with ultrasound (Sonos 5500; Hewlett Packard, Palo Alto, CA) 24 h preoperatively and 24 h before necropsy. Ejection fraction, motility, and capacity of the cardiac cavities were evaluated in both groups. Index of global systolic function as shortening fraction was calculated as follows: (DDLVDSLV)/DDLV. DDLV is left ventricular diastolic diameter, and DSLV is left ventricular systolic diameter. Both were measured in mode M. Fraction of posterior parietal thickness (PPT%) and fraction of septal parietal thickness (SPT %) are indexes of regional systolic function as walls are measured separately. Both PPT% and SPT% were calculated as follows: ST/DT 100, where ST is systolic thickness and DT is diastolic thickness, at both posterior wall and septum. They were measured in mode M and two-dimensional mode. The values obtained were used for statistical analysis between
Table 1 e Semiquantitative histologic score. Cell type/response
Score 0
Inflammation (PMN cells, lymphocytes, plasma cells, macrophages, FBGCs)
Necrosis
None. No inflammatory cells presents
None
1 Rare (one to five PMNs per HPF). Inflammatory cells present <10% of surface area
Minimal (<10% of surface area)
2
3
4
Mild infiltrate (5-10 PMNs per HPF). Inflammatory cells present in 10%-33% of surface area
Heavy infiltrate (>10 PMNs per HPF). Inflammatory cells present in 33%-75% of surface area
Packed (PMNs too numerous to count). Inflammatory cells present (neutrophils, macrophages, and FBGC) >75% of surface area
Mild (10%-33% of surface area)
Moderate (33%-75% of surface area)
Severe (>75% of surface area)
None. No fibrous encapsulation
Minimal (<10% of surface area). Minimal encapsulation (<10% of periphery)
Mild (10%-33% of surface area). Mild encapsulation (10%-33% of periphery)
Moderate (33%-75% of surface area). Moderate encapsulation (33%-75% of periphery)
Severe (>75% of surface area). Extensive encapsulation (>75% of periphery)
Neovascularization
Marked. Most abundant capillaries present (>20 capillaries/HPF). Vessels penetrate into center of scaffold or resected area
Moderate. Abundant capillaries present (10-20 capillaries/HPF). Vessels penetrate into center of scaffold or resected area
Mild. Many capillaries (5-10 capillaries/HPF). Vessels infiltrate scaffold, but none reach center of scaffold or resected area
Few capillaries (1-5 capillaries/HPF). Vessels present at scaffold periphery, no penetration into scaffold or resected area
None. No capillaries or blood vessels present
Cellular infiltration
Marked (>75% of surface area). Cells penetrate into center of scaffold or resected area
Moderate (33%-75% of surface area). Cells penetrate into center of scaffold or resected area
Mild (10%-33% of surface area). Cells infiltrate scaffold, but none reach center of scaffold or resected area
Minimal (<10% of surface area). Cells contact periphery, no penetration into scaffold or resected area
None. No cells in contact with scaffold or resected area
Collagen fibers and global morphology
Collagen fibers aligned and similar to native tissue. Tissue appears as histologically normal. Highly conserved global morphology
Collagen fibers minimally disorganized. Minimal to mild disorganization of tissue. Minimally altered global morphology (<10% of surface area)
Collagen fibers mildly disorganized. Mild to moderate disorganization of the tissue by dense fibrous connective tissue. Mildly altered global morphology (10%-33% of surface area)
Collagen fibers moderately disorganized. Moderate disorganization of the tissue. Moderately altered global morphology (33%75% of surface area)
PMNs ¼ polymorphonuclear leukocytes; FBGC ¼ foreign body giant cell; HPF ¼ high-powered field (200).
No organized collagen fibers. Disruption of architecture. Severely altered global morphology (>75% of surface area)
amigo et al pericardium repair in a porcine model
Fibrosis and fibrous encapsulation
219
220
j o u r n a l o f s u r g i c a l r e s e a r c h m a y 2 0 2 0 ( 2 4 9 ) 2 1 6 e2 2 4
Fig. 2 e Macroscopic analysis of the pericardium. Thoracic cavity view, necropsy at 8 wk. (A-C) Group reinforced with UBM scaffold. (A) Sternum, (B) pericardium, and (C) patch dissected. (D-F) Control group, open defect without reinforcement mesh. (D and E) Sternum and (F) pericardium dissection. (Color version of figure is available online.)
groups using GraphPad Prism, version 8.0.2. The values obtained from each group were analyzed by paired t-test. Analysis of variance test and Tukey’s multiple comparisons were performed, considering P < 0.05 as significant. Results were expressed as mean standard deviation.
Results Macroscopic evaluation All the animals of the UBM group (Fig. 1B and C; Fig. 2A-C) showed mild and loose type 1 adhesions, with easy and correct dissection of the remodeled tissue from the sternum and between the remodeled tissue and the epicardial surface. No visible or palpable remnants of the UBM material were found. In contrast, all the animals of the control group (Fig. 2D-F) showed strong type 3 adhesions at both the epicardium and sternum. A sharp and acute dissection was required (with scalpel and scissors) to separate the scar tissue generated from the sternum. Epicardial retraction was found, and coronary bed injury was inadvertently caused during the dissection in some cases.
Biomechanical testing Average load at failure (gr) and stiffness (gr/mm) were lower in the UBM group compared with the native pericardium (199.9 59.2 versus 405.3 99.89 g, P ¼ 0.0536 and 44.23 15.01 versus 146.5 24.38 g/mm, P ¼ 0.0025, respectively; Fig. 3).
Control animals were not tested because no deposition of pericardial tissue was observed.
Histology In the UBM group, a well-organized tissue with neovascularization, neofibroblasts, a preserved histoarchitecture, and little signs of inflammation were found. The collagen fibers were observed aligned and similar to native pericardium, with complete resorption of the UBM scaffold (Fig. 4B and E). There was also evidence of scattered mesothelial cells on the surface although they failed to form a continuous layer. In contrast, all control animals showed dense fibrotic tissue with large mononuclear infiltrates, randomly organized collagen fibers, and clear signs of scar tissue (Fig. 4C and F).
Semiquantitative histologic score The results obtained in histologic semiquantitative analysis were consistent in each of the six categories analyzed as well as in the composite score because higher scores on this scale correlate with poorer outcomes. In all subcategories, as well as in the composite score, UBM group recorded significantly lower values compared with the control group (Fig. 5). Briefly, in the inflammation subcategory, the score was significantly lower in the UBM group compared with the control group (0.83 0.69 versus 3.83 0.38, respectively; P < 0.0001, Fig. 5A). In necrosis subcategory, the UBM group recorded significantly lower levels compared with the control
amigo et al pericardium repair in a porcine model
221
Fig. 3 e Biomechanical properties of pericardium reinforcement. (A) Load at failure (gr). (B) Stiffness (gr/mm). Native [ native pericardium tissue. UBM [ remodeled tissue after implantation of the UBM scaffold (**P < 0.01). (Color version of figure is available online.)
group (0.3 0.46 versus 1.80 0.61, respectively; P < 0.0001, Fig. 5B). The fibrosis and fibrous encapsulation subcategory recorded lower values in UBM group compared with the control group (0.5 0.51 versus 3.5 0.63, respectively; P < 0.0001, Fig. 5C). In neovascularization subcategory, lower values were recorded in the UBM group compared with the control group (0.47 0.51 versus 1.83 0.59, respectively; P < 0.0001, Fig. 5D). In the cellular infiltration subcategory, the score was significantly lower in the UBM group compared with the control group (0.2 0.41 versus 1.2 0.56, respectively; P < 0.0001, Fig. 5C). In the collagen fibers and global morphology
subcategories, lower values were found in the UBM group compared with control group (0.13 0.34 versus 3.77 0.43, respectively; P < 0.0001, Fig. 5F). The composite score combined the results obtained in each subcategory and reflected the overall results obtained in the analysis of remodeled tissues in each of the studied groups. The results showed significantly lower values for the UBM group compared with control group (0.41 0.25 versus 2.65 1.17, respectively; P ¼ 0.0005, Fig. 5G). In summary, lower scores on this scale represent better outcomes with respect to cellular infiltration,
Fig. 4 e Histologic analysis. (A and D) Native tissue. (B and E) UBM group: complete resorption of UBM scaffold, angiogenesis, and neovascularization. Fibers of collagen aligned (arrow). (C and F) Control group, open defect: dense fibrous tissue with desalted collagen fibers and global morphology altered (rhombus). Large mononuclear infiltrate (*). H&E stain (A-C: 2003). Masson’s trichrome stain (D and F: 2003; E: 4003). (Color version of figure is available online.)
222
j o u r n a l o f s u r g i c a l r e s e a r c h m a y 2 0 2 0 ( 2 4 9 ) 2 1 6 e2 2 4
Fig. 5 e Semiquantitative histologic score after 8 wk in vivo by subcategory: (A) Inflammation, (B) necrosis, (C) fibrosis and fibrous encapsulation, (D) neovascularization, (E) cellular infiltration, (F) collagen fibers and global morphology; and (G) composite score. Lower scores represent more favorable outcomes with regard to tissue remodeling. UBM group: remodeled tissue after implantation of the UBM scaffold. Control group: remodeled tissue in pericardium open defect, without reinforcement mesh (****P < 0.0001, ***P < 0.001). (Color version of figure is available online.)
neovascularization, arrangement of collagen fibers, and conserved global morphology, together with low levels of inflammation and necrosis found in the UBM group.
results without displaying cardiac motility disorders in any of their walls, as well as in global cardiac motility, preimplant or postimplant, showing no limitation in cardiac function because of the UBM implant.
Ultrasound results No significant differences were observed in the parameters recorded between neither the groups, shortening fraction, fraction of PPT%, and fraction of SPT% nor the preimplant and postimplant measurements (Table 2). Both groups had normal
Discussion This study describes the host response, histologic, and biomechanical properties of pericardial repair with UBM
223
amigo et al pericardium repair in a porcine model
Table 2 e Cardiac ultrasonography of UBM group and control group. Experimental group
Pre-surgery SF%
PPT%
Pre-necropsy SPT%
SF%
PPT%
SPT%
UBM
26.77 7
43.97 11
32.68 10
23.15 5
38.39 13
28.70 17
Control
25.84 10
25.29 10
31.39 16
30.06 10
52.02 24
34.47 7
SF% ¼ shortening fraction; PPT% ¼ fraction of posterior parietal thickness; SPT% ¼ fraction of septal parietal thickness.
scaffolds after partial pericardial resection in an in vivo porcine preclinical model. The UBM group showed complete remodeling with histologic and biomechanical features similar to native tissue. These results are similar to those previously obtained by several authors4-6,9,10,13-15 and show the beneficial use of a biological bioabsorbable pericardial substitute. In contrast, the control group with open pericardial defects had strong postoperative retrosternal adhesions to the heart. Remodeled UBM tissue had similar or weaker mechanical properties than native pericardium. Although it was not tested in this study, unremodeled scaffolds as and synthetic scaffolds are normally stiffer than native tissues. The fact that remodeled UBM showed similar or softer properties in this study as compared with native pericardium is relevant in a setting where myocardial function can be widely affected by external restriction.15 The semiquantitative histologic assessment, and the results obtained in the biomechanical analysis, supported and validated both macroscopic and histologic findings. The present study, in accordance with the previous literature,6-8,10,11,15,18,24,25 provides further evidence about the beneficial use of a pericardial substitute with biological scaffolds. This is the first report about use of UBM to repair a pericardial defect and includes the first complete characterization of pericardial reconstruction with UBM scaffolds in a preclinical porcine model. Other authors have tested anti-inflammatory agents and a reabsorbable polyethylene glycol barrier to limit adhesion formation after cardiac surgery in a pig model26; and the authors conclude that both systemic indomethacin and locally applied polyethylene glycol barrier are suitable methods to markedly reduce pericardial and retrosternal adhesions. However, given the disadvantages of synthetic replacements in general, new bioabsorbable biological structures have recently emerged as safer alternatives. ECM biological scaffolds have been shown to promote constructive remodeling of site-specific tissues of injury in several preclinical and clinical applications, including repair of skeletal muscle tissue,4,5 soft tissues such as the esophagus,6,8 lower urinary tract,9-12 and vascular tissues,13-15 among others. Several studies have shown that UBM scaffolds contribute to a more site-appropriate repair instead of the intrinsic fibrotic response often seen after open surgery. The mechanisms for this homeostatic response are multiple and currently source of intense basic research. The host immune response to UBM tends to include a robust immunomodulatory M2 macrophage phenotype, as opposed to the typical proinflammatory M1 phenotype.24,27-29 We have previously shown the benefits of
UBM-ECM, both in the remodeling of organ-specific tissue like the esophagus where progenitor cells derived from the bone marrow populated the site of esophageal injury24 and in the restoration of biomechanical and histologic properties in the repair of the diaphragmatic hiatus.18,23 Limitations of this study include the lack of a control group with nondegradable materials. Although synthetic pericardial patch devices have been used and described in some occasions, they are rarely used because of the known constraints previously described. Therefore, we considered the current standard of care without pericardial repair as a more relevant control. Other limitations of this include the lack of cardiopulmonary bypass and cardiotomy in the model; therefore, it cannot be considered a full clinical model of open heart surgery. Short-term follow-up of the animals might also be seen as a constraint in this study. Based on previous publications by the authors and others, it has been demonstrated that, by 8 wk, the UBM scaffolds are mostly remodeled with mechanical properties being influenced mainly by remaining tissue that is stable in time. However, tissue remodeling after surgery occurs for up to 1 y. Also, the lack of functional endpoints prevents a more robust conclusion about the clinical impact of the findings.
Conclusion In this preclinical study, UBM scaffolds showed appropriate features for pericardial repair after partial pericardial resection. Remodeled scaffolds can help reduce postoperative adhesions and prevent complications associated with implanted materials without affecting cardiac function. Long-term follow-up and early clinical translation is warranted to further support this evidence.
Acknowledgment Authors’ contributions: N.A. and J.M.R. contributed equally to data collection, analysis and interpretation of the data, and writing of the article. M.R., L.A., P.R., and R.L. contributed to data collection and analysis and interpretation of the data. A.P. and D.C. contributed to analysis and interpretation of the data. C.V. contributed to analysis and interpretation of the data and critical revisions. T.W.G., N.T.R., and A.N. contributed to conceiving and designing the study, critical revisions, and approving the final version of the article. This work was supported by a research grant from ACell, Inc, Columbia, MD to A.N.
224
j o u r n a l o f s u r g i c a l r e s e a r c h m a y 2 0 2 0 ( 2 4 9 ) 2 1 6 e2 2 4
Disclosure 14.
T.W.G. served as Chief Science Officer at ACell, Inc during the completion of the study and writing of the manuscript. A.N. is a member of SAB at ACell, Inc. N.T.R. is currently employed at ACell, Inc. The other authors have no conflicts of interest or financial ties to disclose.
15.
16.
references 17. 1. Vaideeswar P, Mishra P, Nimbalkar M. Infective endocarditis of the Dacron patchda report of 13 cases at autopsy. Cardiovasc Pathol. 2011;20:e169ee175. 2. Henaine R, Roubertie F, Vergnat M, et al. Valve replacement in children: a challenge for a whole life. Arch Cardiovasc Dis. 2012;105:517e528. 3. Sandusky GE, Lantz GC, Badylak SF. Healing comparison of small intestine submucosa and ePTFE grafts in the canine carotid artery. J Surg Res. 1995;58:415e420. 4. Badylak SF, Tullius R, Kokini K, et al. The use of xenogeneic small intestinal submucosa as a biomaterial for Achilles tendon repair in a dog model. J Biomed Mater Res. 1995;29:977e985. 5. Dejardin LM, Amoczky SP, Ewers BJ, et al. Tissue-engineered rotator cuff tendon using porcine small intestine submucosa. Histologic and mechanical evaluation in dogs. Am J Sports Med. 2001;29:175e184. 6. Badylak SF, Meurling S, Chen M, et al. Resorbable bioscaffold for esophageal repair in a dog model. J Pediatr Surg. 2000;35:1097e1103. 7. Nieponice A, McGrath K, Qureshi I, et al. An extracellular matrix scaffold for esophageal stricture prevention after circumferential EMR. Gastrointest Endosc. 2009;69:289e296. 8. Badylak SF, Hoppo T, Nieponice A, et al. Esophageal preservation in five male patients after endoscopic innerlayer circumferential resection in the setting of superficial cancer: a regenerative medicine approach with a biologic scaffold. Tissue Eng Part A. 2011;17:1643e1650. 9. Kropp BP, Eppley B, Prevel CD, et al. Experimental assessment of small intestinal submucosa as a bladder wall substitute. Urology. 1995;46:396e400. 10. Kropp BP, Rippy MK, Badylak SF, et al. Regenerative urinary bladder augmentation using small intestinal submucosa: urodynamic and histopathologic assessment in long-term canine bladder augmentations. J Urol. 1996;155:2097e2104. 11. Sievert KD, Tanagho EA. Organ-specific acellular matrix for reconstruction of the urinary tract. World J Urol. 2000;18:19e25. 12. Yoo JJ, Atala A. Tissue engineering applications in the genitourinary tract system. Yonsei Med J. 2000;41:789e802. 13. Sandusky Jr GE, Badylak SF, Morff RJ, et al. Histologic findings after in vivo placement of small intestine submucosal
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
vascular grafts and saphenous vein grafts in the carotid artery in dogs. Am J Pathol. 1992;140:317e324. Schoder M, Pavcnik D, Uchida BT, et al. Small intestinal submucosa aneurysm sac embolization for endoleak prevention after abdominal aortic aneurysm endografting: a pilot study in sheep. J Vasc Interv Radiol. 2004;15:69e83. Teodori L, Costa A, Marzio R, et al. Native extracellular matrix: a new scaffolding platform for repair of damaged muscle. Front Physiol. 2014;5:218. Woo JS, Fishbein MC, Reemtsen B. Histologic examination of decellularized porcine intestinal submucosa extracellular matrix (CorMatrix) in pediatric congenital heart surgery. Cardiovasc Pathol. 2016;25:12e17. Nezhad ZM, Ponceleta A, Kerchovea L, et al. Small intestinal submucosa extracellular matrix (CorMatrix) in cardiovascular surgery: a systematic review. Interactive Cardiovasc Thorac Surg. 2016;22:839e850. Riganti JM, Ciotola F, Amenabar A, et al. Urinary bladder matrix scaffolds strengthen esophageal hiatus repair. J Surg Res. 2016;204:344e350. Badylak SF, Vorp D, Spievack AR, et al. Esophageal reconstruction with ECM and muscle tissue in a dog model. J Surg Res. 2005;128:87e97. Nieponice A, Ciotola F, Nachman F, et al. Patch esophagoplasty: esophageal reconstruction using biologic scaffolds. Ann Thorac Surg. 2014;97:283e288. Young DA, McGilvray KC, Ehrhart N, et al. Comparison of in vivo remodeling of urinary bladder matrix and acellular dermal matrix in an ovine model. Regen Med. 2018;13:759e773. Jenkins ED, Melman L, Deeken CR, et al. Evaluation of fenestrated and non-fenestrated biologic grafts in a porcine model of mature ventral incisional hernia repair. Hernia. 2010;14:599e610. Amigo N, Zubieta C, Riganti JM, et al. Biomechanical features of reinforced esophageal hiatus repair in a porcine model. J Surg Res. 2019;246:62e72. Badylak SF, Valentin JE, Ravindra AK, et al. Macrophage phenotype as a determinant of biologic scaffold remodeling. Tissue Eng Part A. 2008;14:1835e1842. Young DA, Jackson N, Ronaghan CA, et al. Retrorectus repair of incisional ventral hernia with urinary bladder matrix reinforcement in a long-term porcine model. Regen Med. 2018;13:395e408. Alizzi AM, Summers P, Boon VH, et al. Reduction of postsurgical pericardial adhesions using a pig model. Heart Lung Circ. 2012;21:22e29. Paige JT, Kremer M, Landry J, et al. Modulation of inflammation in wounds of diabetic patients treated with porcine urinary bladder matrix. Regen Med. 2019;14:269e277. Sadtler K, Wolf MT, Ganguly S, et al. Divergent immune responses to synthetic and biological scaffolds. Biomaterials. 2019;192:405e415. Brown BN, Londono R, Tottey S, et al. Macrophage phenotype as a predictor of constructive remodeling following the implantation of biologically derived surgical mesh materials. Acta Biomater. 2012;8:978e987.