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Differential efficacy of gels derived from small intestinal submucosa as an injectable biomaterial for myocardial infarct repair
Biomaterials 31 (2010) 7678e7683
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Biomaterials journal homepage: www.elsevier.com/locate/biomaterials
Differential efficacy of gels derived from small intestinal submucosa as an injectable biomaterial for myocardial infarct repair Masaho Okada a, c, Thomas R. Payne a, c, e, f, Hideki Oshima a, c, Nobuo Momoi b, Kimimasa Tobita b, Johnny Huard a, b, c, d, * a
Stem Cell Research Center, Children’s Hospital of Pittsburgh, Pittsburgh, PA, USA Department of Pediatrics, Children’s Hospital of Pittsburgh, Pittsburgh, PA, USA Department of Orthopaedic Surgery, Pittsburgh, PA, USA d Department of Molecular Genetics and Biochemistry, University of Pittsburgh, Pittsburgh, PA, USA e Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA, USA f Cook MyoSite, Pittsburgh, PA, USA b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 20 May 2010 Accepted 28 June 2010 Available online 31 July 2010
Injectable biomaterials have been recently investigated as a therapeutic approach for cardiac repair. Porcine-derived small intestinal submucosa (SIS) material is currently used in the clinic to promote accelerated wound healing for a variety of disorders. In this study, we hypothesized that gels derived from SIS extracellular matrix would be advantageous as an injectable material for cardiac repair. We evaluated 2 forms of SIS gel, types B (SIS-B) and C (SIS-C), for their ability to provide a therapeutic effect when injected directly into ischemic myocardium using a murine model of an acute myocardial infarction. Echocardiography analysis at both 2 and 6 weeks after infarction demonstrated preservation of end-systolic left ventricular geometry and improvement of cardiac contractility in the hearts injected with SIS-B when compared with control hearts injected with saline. However, the SIS-C gel provided no functional efficacy in comparison with control. Histological analysis revealed that SIS-B reduced infarct size and induced angiogenesis relative to control, whereas injection of SIS-C had minimal effect on these histological parameters. Characterization of both gels revealed differential growth factor content with SIS-B exhibiting higher levels of basic fibroblast growth factor than SIS-C, which may explain, at least in part, the differential histological and functional results. This study suggests that SIS gel offers therapeutic potential as an injectable material for the repair of ischemic myocardium. Further understanding of SIS gel characteristics, such as biological and physical properties, that are critical determinants of efficacy would be important for optimization of this biomaterial for cardiac repair. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: Angiogenesis Animal model Bioactivity Cardiac tissue engineering Fibroblast growth factor SIS (small intestine submucosa)
1. Introduction Heart failure remains a significant cause of morbidity and mortality in Western countries [1]. The acute loss of functional myocardium after recovery from a myocardial infarction (MI) results in an abrupt increase in loading conditions within the left ventricle (LV). To compensate, the LV remodels the infarct and remote non-infarcted myocardium through a cascade of biochemical signaling processes that facilitate reparative changes including the dilatation of the left ventricle (LV) [2]. Since these intrinsic mechanisms of repair are limited in fully restoring function, new
* Corresponding author. Department of Orthopaedic Surgery, University of Pittsburgh School of Medicine, 450 Technology Drive, 2 Bridgeside Point, Suite 206, Pittsburgh, PA 15219, USA. Tel.: þ1 412 648 2798; fax: þ1 412 692 7095. E-mail address: [email protected] (J. Huard). 0142-9612/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2010.06.056
therapeutic approaches based on cellular therapy, biomaterials, and tissue engineering are being explored to reduce ventricular dilatation, improve function, and prevent disease progression towards end-stage heart failure. The discovery of small intestinal submucosa (SIS), which is a dense, acellular connective tissue harvested from porcine small intestine, has generated great interest as a naturally derived biomaterial for tissue regeneration and restoration [3]. SIS consists of a sheet of extracellular matrix that is biocompatible, mechanically malleable, resistant to infection, and bioactive. In regard to bioactive properties, SIS promotes proliferation, attachment and migration of various cell types and stimulates angiogenesis through an assortment of matrix-embedded angiogenic factors, including connective tissue growth factor (CTGF), basic fibroblast growth factor (FGF), transforming growth factor b (TGF-b), and fibronectin [4e7]. Researchers have been investigating the use of SIS scaffold
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for the regeneration and repair of various soft tissues and maladies including large vascular grafts, venous valves and leaflets, urinary bladder, dura mater, dermal wounds, fistulas, hernias, esophageal, and rotator cuff [3,8e13]. Of particular interest to this study, prior work has demonstrated a conduciveness of SIS for myocardial tissue ingrowth and vascularization when applied as an atrial prosthesis and a right ventricular full thickness patch [14,15]. In this study, we sought to investigate the use of gels derived from SIS extracellular matrix as an injectable biomaterial for cardiac repair. Two versions of SIS gel were evaluated for intramyocardial injection in a murine model of an acute MI. We postulated that delivery of SIS gel in situ would attenuate adverse LV remodeling and induce accelerated healing of infarct tissue through intrinsic bioactive signals, thereby improving LV function. 2. Materials and methods 2.1. Intramyocardial injection of SIS gel into an acute myocardial infarction The Institutional Animal Care and Use Committee, Children’s Hospital of Pittsburgh approved the animal and surgical procedures performed in this study (Protocols 7/03, 3-04). In conducting the research described in this study, the investigators adhered to the instructions described in the Guide for the Care and Use of Laboratory Animals (NIH Publications 85e23) as promulgated by the Committee of Care and Use of Laboratory Animals of the Institute of Laboratory Sciences, the National Academy of Sciences, and the National Research Council. A total of 26 male non-obese diabetic severe combined immunodeficiency (NOD-SCID) mice (25e30 g body weight, Jackson Laboratory, Bar Harbor, ME) were used for this study. We experienced a 15% mortality rate during the study (n ¼ 4 deaths, n ¼ 26 total injected mice). Two kinds of SIS gel, types B (SIS-B) and C (SIS-C), were provided by Cook Biotech Incorporated (West Lafayette, IN) and were evaluated in this study for cardiac repair. The pH level of SIS gel was adjusted to 7.4e7.8 using sodium hydroxide immediately prior to injection. Twenty micro liters of SIS material were injected directly into the ischemic myocardial zone of NOD-SCID mice immediately after inducing an MI by coronary ligation, as previously described [16]. Basic fibroblast growth factor content was measured by enzyme-linked immunosorbent assay, according to manufacturer’s instructions (R&D Systems, Minneapolis, MN). In control mice, 20 mL of phosphate buffered saline was injected into the same regions of the heart. The investigator performing the coronary ligation injury and injection procedures was blinded to the contents of the injectant. 2.2. Evaluation of cardiac function by echocardiography Two and 6 weeks after surgery, echocardiography was performed by a blinded investigator to assess the heart function of the anesthetized mice, as previously described [16]. End-systolic area (ESA) and end-diastolic area (EDA) were measured from short-axis images of the LV. Fractional area change (FAC), an index of LV contractility, was calculated as FAC (%) ¼ [(EDA ESA) / EDA] 100. 2.3. Histological analysis The mice were sacrificed at 6 weeks after SIS implantation for histological evaluation of the heart tissue. Hearts were flash frozen in 2-methylbutane (SigmaeAldrich, St. Louis, MO) that was pre-cooled in liquid nitrogen. Each specimen was serially cryosectioned at a thickness of 8 mm. 2.4. Measurement of infarct size Masson’s trichrome staining (IMEB, San Marcos, CA), which labels collagen blue and muscle red, was performed to evaluate scar tissue with the treated hearts. As previously described, Image J software (National Institutes of Health) was used to measure the lengths of the entire left ventricular endocardial circumference and the infarcted segment in a total of 5 sections [17]. The infarcted fraction of the left ventricle was calculated from these measurements. 2.5. Capillary density To determine capillary density within the infarct zone after treatment, we performed immunofluorescent staining of CD31, an endothelial cell marker, and analyzed the CD31-positive capillary structures within the infarct and peri-infarct zones at the 6 weeks time-point, as previously described [16]. 2.6. Statistical analysis Data are presented as mean standard error of the mean. Statistical differences in the data were determined by a one-way or two-way analysis of variance (ANOVA).
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The Tukey multiple comparison test was performed for post-hoc analysis when a statistical difference was observed in the ANOVA tests. Statistical significance for all tests was set at a P value < 0.05. Statistical calculations were performed with SigmaPlot software (Systat Inc., San Jose, CA).
3. Results 3.1. Cardiac function We assessed the therapeutic effect of 2 types of injectable SIS gel (SIS-B and SIS-C) for cardiac repair in an immunodeficient mouse model of an acute MI. Two and 6 weeks after transplantation, treatment with either SIS-B or SIS-C gels displayed very little effect on LV EDA relative to control saline injections (Fig. 1A, 2 weeks: SIS-B 8.8 0.2 mm2, n ¼ 9 mice; SIS-C 9.2 0.4, n ¼ 7; control 9.0 0.6, n ¼ 7; 6 weeks: SIS-B 8.4 0.3 mm2, n ¼ 9; SIS-C 9.2 0.5, n ¼ 7; control 8.8 0.2, n ¼ 6). Treatment with SIS-B significantly reduced LV ESA when compared with SIS-C and control saline treatments (Fig. 1B, 2 weeks: SIS-B 5.8 0.1 mm2, SIS-C 7.0 0.4, control 6.7 0.6; 6 weeks: SIS-B 5.7 0.3 mm2, SIS-C 6.9 0.3, control 6.7 0.3; P < 0.05, SIS-B versus SIS-C and control). LV contractility, as determined by FAC values, improved in the SISBetreated hearts when compared with hearts injected with SIS-C and control saline (Fig. 1C, 2 weeks: SIS-B 34.8 1.1%, SIS-C 24.9 2.8, control 25.6 1.9; 6 weeks: SIS-B 31.5 0.8, SIS-C 24.7 1.5, control 24.1 1.9; P < 0.001, SIS-B versus SIS-C and control). Thus, these results indicate that injection of SIS-B more effectively preserved LV systolic geometry and contractility after recovery from an acute MI than did the implantation of either SIS-C or saline. 3.2. Infarct size and fibrosis We examined LV infarct size and scar tissue area using Masson’s Trichrome stain to label collagenous scar tissue (blue) and cardiac muscle (red, Fig. 2AeC). Infarct size was measured as the percentage of endocardial circumferential length that displayed an infarct scar. At 6 weeks after implantation, hearts injected with SISB exhibited smaller infarcts after histological examination than hearts injected with either SIS-C or saline (Fig. 2D, SIS-B 46.4 4.0%, n ¼ 8; SIS-C 65.0 5.0, n ¼ 7; control 63.3 3.3, n ¼ 5; P < 0.05, SIS-B versus SIS-C). To determine the amount of scar tissue, scar area fraction was defined as the ratio of collagenous scar area to the entire cardiac muscle area. We observed a trend of reduced fibrosis in the SISBetreated hearts when compared with hearts injected with SIS-C and saline at the 6 weeks time point (Fig. 2E, SIS-B 0.26 0.03 scar area fraction, SIS-C 0.38 0.08, control 0.37 0.03). Taken together, the infarct size and scar area measurements demonstrate that SIS-B injections more effectively attenuated infarct expansion and fibrosis than either control or SIS-C injections. 3.3. Angiogenesis We measured capillary density within the infarct and periinfarct regions to determine whether the injection of SIS gel induced angiogenesis (Fig. 3AeC). In the infarct zone, hearts injected with SIS-B displayed greater capillary densities when compared with hearts injected with either SIS-C or saline (Fig. 3D, SIS-B 634 37 capillaries/mm2, n ¼ 7; SIS-C 455 33, n ¼ 7; control 503 31, n ¼ 5; P < 0.01, SIS-B versus SIS-C). In the periinfarct region, SIS-B also stimulated greater capillary formation when compared with SIS-C and control (Fig. 3E, SIS-B 640 39 capillaries/mm2, SIS-C 456 47, control 510 28; P < 0.05, SIS-B versus SIS-C).
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2 wks
10
B
6 wks
End-systolic Area (mm2)
End-diastolic Area (mm2)
A 9 8 7 6 5 4
SIS-B
SIS-C
Fractional Area Change (%)
C
Control 2 wks
40 35
2 wks
8
6 wks
7
*
6 5 4 3
SIS-B
SIS-C
Control
6 wks
*
30 25 20 15
SIS-B
SIS-C
Control
Fig. 1. Echocardiographic evaluation of cardiac function. (A) Two and six weeks after transplantation, SIS gel treatment imparted minimal effect on end-diastolic area of the left ventricle (LV). (B) SIS gel type B (SIS-B) significantly reduced LV end-systolic area when compared with SIS gel type C (SIS-C) and control (*P < 0.05, SIS-B versus SIS-C and control). (C) Cardiac contractility, as determined by LV fractional area change values, improved in the hearts injected with SIS-B greater than hearts treated with SIS-C and control saline (*P < 0.001, SIS-B versus SIS-C and control).
3.4. SIS growth factor content Both types of SIS gel consisted of a similar solid concentration: 9.4 mg/mL for SIS-B and 8.2 mg/mL for SIS-C (Table 1). However, SIS-B displayed a 58% higher content of basic fibroblast growth factor (bFGF) than SIS-C (Table 1). 4. Discussion Biomaterials in the injectable form have recently become a vanguard approach in the quest to develop reparative therapeutics for cardiovascular disorders [18]. Alginate, fibrin, matrigel, gelatin, chitosan, self-assembling peptide nanofibers, alpha-cyclodextrin MPEG-PCL-MPEG hydrogel, and the thermosensitive polymer gels Dex-PCL-HEMA/PNIPAAm and poly(NIPAAm-co-AAcco-HEMAPTMC) have been evaluated as an acellular therapy for myocardial infarct repair [19e28]. The fundamental premise of the injectable biomaterial approach for cardiac repair is that the injected agent will form a physical scaffold in situ that will reduce LV wall stresses and stabilize LV remodeling following recovery from an MI. In regards to LV remodeling, our results demonstrate that end-diastolic dimensions were not altered in SIS gel-treated hearts when compared with control saline-treated hearts. However, at endsystole, the dimensions of hearts injected with SIS-B were 15% smaller than hearts injected with control saline. The preservation of end-systolic geometry has also been reported with other injectable biomaterials for cardiac repair and is considered to be an important
predictor of survival after recovery from an MI [21,29]. Preliminary in vitro characterization of mechanical properties demonstrated differential mechanical strength between the 2 polymerized gels. SIS-B exhibited greater compressive modulus and maximum compressive stress than SIS-C, suggesting that SIS-B could more effectively provide physical support for in situ stabilization of LV dimensions than SIS-C (compressive modulus: 1.2e2.5 104 Pa for SIS-B, 0.5e1.0 104 Pa for SIS-C; maximum compressive stress: 6.2e8.2 103 Pa for SIS-B, 1.5e2.5 103 Pa for SIS-C). This disparity in mechanical properties may explain, at least in part, the differential efficacy of SIS-B and SIS-C gels for MI repair. Signaling factors within SIS gel, such as growth factors and cell adhesion motifs, may also be essential for the observed biological and functional effects. In this study, the SIS-B gel displayed a 58% greater content of bFGF than the SIS-C gel. The greater functional and histological effect of SIS-B may also be attributable, at least in part, to its higher growth factor content when compare with SIS-C. The observed attenuation of LV systolic dimensions and infarct expansion in SIS-Betreated hearts could also be due to the preservation of at-risk myocardium along the peri-infarct zone through intrinsic anti-fibrotic, pro-survival, angiogenic, and anti-inflammatory cytokines. Furthermore, SIS has been shown to recruit host c-kit positive cells to the infarct regions, which may aid myocardial regeneration and repair [30]. Others have shown that injectable biopolymers carrying growth factors enhance angiogenesis and LV contractility after recovery from myocardial infarction [23,25,31]. The synergistic effect between growth factors may further enhance the biological effect than a single factor alone. Multiple growth
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B
A
SIS-B
C
SIS-C
D
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Control
80
Infarction Size (%)
70 60 50
*
40 30 20 10 0
SIS-B
SIS-C
Control
Scar Area Fraction
E 0.5 0.4 0.3 0.2 0.1 0.0
SIS-B
SIS-C
Control
Fig. 2. Infarct size and scar area analysis. (AeC) Representative images of Masson’s trichrome stain of infarcted (blue) left ventricle (LV) free wall from each treatment group. Scale bars equal 1 mm. (D) Infarct size analysis by planimetry demonstrated that hearts injected with SIS gel type B (SIS-B) displayed a significant reduction in the percentage of infarcted endocardial circumferences when compared with hearts injected with SIS gel type C (SIS-C) or control saline (*P < 0.05, SIS-B versus SIS-C). (E) Injection of SIS-B also induced a trend towards less LV scar tissue when compared with the implantation of SIS-C and control saline.
factors are known to exist in SIS including CTGF, FGF, and TGF-b, which all have potential to stimulate therapeutic healing in a synergistic manner for cardiac repair [6,7]. In this regard, Hao and colleagues demonstrated that alginate hydrogels loaded with the growth factors VEGF-A and PDGF-BB had a superior functional and angiogenic effect was induced with both growth factors than either factor alone [32]. Collectively, these studies suggest a considerable therapeutic value of growth factors when combined with biomaterials for MI repair and support the hypothesis that intrinsic growth factor content in SIS may play an important and synergistic role in stimulating a beneficial biological response. The ability of injectable biomaterials to promote other biological effects such as host cell adhesion and migration throughout the infarct scar may also enhance the therapeutic response. The extracellular matrix components of SIS include proteoglycans,
glycosaminoglycans, collagens, fibronectin, and laminins [13]. These natural components may promote interaction of host cells with the injected material by presenting multiple cell adhesion, integrin-binding sites. In 2 studies that modified alginate biopolymers with cell adhesion peptides RGD and YIGSR demonstrated that an enhancement of cardiac function was not achieved [33,34]. However, each study presented conflicting results on the effect of the cell adhesion peptides on angiogenesis [33,34]. Additional research is needed to determine whether SIS gel is capable of actively guiding regeneration that leads to superior therapeutic effects when compared with biomaterials lacking bioactive properties. Cellular cardiomyoplasty is intended to be a therapy by which injured myocardium would be replaced by exogenous cells that would create new, functional myocardial tissue. However, various
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A
B
C
SIS-B
SIS-C
Capillaries per mm2
D
Infarct Zone 700
*
600 500 400 300
SIS-B
E Capillaries per mm2
Control
SIS-C
Control
Peri-infarct
*
700 600 500 400 300 200 100 0
SIS-B
SIS-C
Control
Fig. 3. Augmentation of capillary density with SIS gel. (AeC) Representative images of CD31 immunostaining of the peri-infarct regions of hearts injected with SIS gel type B (SIS-B), SIS gel type C (SIS-C), and control saline. Scale bars equal 50 mm. (D) Within the infarct zone, hearts treated with SIS-B displayed significantly greater capillary densities than hearts treated with SIS-C (*P < 0.01, SIS-B versus SIS-C). (E) Within the peri-infarct regions, SIS-B also induced significantly higher capillary densities than SIS-C (*P < 0.05, SIS-B versus SIS-C).
studies have reported that transplanted cells improve function by a mechanical and/or paracrine effect as opposed to de novo myocardial tissue regeneration by implanted cells [35]. Thus, injectable biomaterials could be designed to achieve the same therapeutic outcomes as cell transplantation. Conversely, the ability of cells to aptly sense and respond to the cues of the microenvironment and to secrete paracrine signals in a physiological manner at the site of injury may be major advantage of cell therapy and a limitation of injectable biomaterials. A few research groups have compared injectable, acellular biomaterial therapy to cell transplantation for cardiac repair, including matrigel versus embryonic stem cells, alginate versus neonatal cardiomyocytes, fibrin versus skeletal myoblasts, and chitosan versus embryonic stem cells [21,24,26,36,37]. All of these studies have demonstrated
Table 1 Characterization of injectable SIS gels. Type
Solid concentration (mg/mL)
FGF content (mg/g of dry solid) (mean SD)
SIS-B SIS-C
9.4 8.2
1.47 0.02 0.93 0.11
that injectable biomaterials provided at least an equivalent level of therapeutic benefit to cell transplantation. In our experience with the transplantation of murine skeletal muscle-derived stem cells (MDSCs) and human skeletal musclederived myogenic endothelial cells into the same murine model of acute MI used in this study, we observed a 50e90% improvement in cardiac function with cell therapy when compared with control injections of saline [16,17]. Here, we observed a 31e36% improvement of cardiac function at 2 and 6 weeks with the injection of SISB when compared with the injection of saline. The differences between our inferences and the aforementioned studies may arise from the potency of each cell type for cardiac repair. In particular, our prior work has shown that skeletal myoblasts are inferior to murine MDSCs and human myogenic endothelial cells for cardiac repair [16,17]. In comparison to SIS gel, the functional benefit demonstrated by our experiences with skeletal myoblast transplantation was equal, and sometimes inferior, to the level of therapeutic benefit achieved with injectable SIS gel in this study (i.e., human myoblasts 16e20%, mouse myoblasts 25e50%, and SIS 31e36% improvement relative to control saline injections) [16,17]. A study directly comparing these cell types with injectable biomaterials should be performed to validate these inferences. The use of injectable biomaterials as an acellular therapy would
M. Okada et al. / Biomaterials 31 (2010) 7678e7683
presumably be more conducive than autologous cell therapy in the clinical setting of an acute MI, where an off-the-shelf product could be applied immediately with minimal complexity. Understandably, comparisons between cellular and biomaterial technologies for cardiac repair will continually be assessed as these fields progress. 5. Conclusions Our findings suggest that gel derived from SIS extracellular matrix induces a biologic and therapeutic effect when delivered in situ for the treatment of an acute MI. The evaluation of 2 types of SIS gel demonstrated disparate capacities for cardiac repair, suggesting that the bioactive and physical properties of SIS gel are critical for therapeutic success. Understanding the significance of these characteristics in animal models of disease may help to appropriately design injectable SIS material for tissue repair. Acknowledgements The Donaldson Chair at Children’s Hospital of Pittsburgh and the Henry J. Mankin Chair at the University of Pittsburgh supported this work. We thank James Cummins, Ronald Jankowski, and Michelle Chutka for their assistance in the preparation of this manuscript. We also thank Cook Biotech Incorporated for providing SIS gel material. References [1] Lloyd-Jones D, Adams R, Carnethon M, De Simone G, Ferguson TB, Flegal K, et al. Heart disease and stroke statisticse2009 update: a report from the American Heart Association Statistics Committee and Stroke Statistics Subcommittee. Circulation 2009;119:e21e181. [2] Sutton MG, Sharpe N. Left ventricular remodeling after myocardial infarction: pathophysiology and therapy. Circulation 2000;101:2981e8. [3] Badylak SF. The extracellular matrix as a biologic scaffold material. Biomaterials 2007;28:3587e93. [4] Hodde J, Record R, Tullius R, Badylak S. Fibronectin peptides mediate HMEC adhesion to porcine-derived extracellular matrix. Biomaterials 2002;23:1841e8. [5] Badylak S, Liang A, Record R, Tullius R, Hodde J. Endothelial cell adherence to small intestinal submucosa: an acellular bioscaffold. Biomaterials 1999;20:2257e63. [6] Janis AD, Klingman NV, Berceli SA. Connective tissue growth factor (CTGF) content and bioactivity of small intestinal submucosa (SIS). Wound Repair Regen 2005;13:A4e27. [7] Voytik-Harbin SL, Brightman AO, Kraine MR, Waisner B, Badylak SF. Identification of extractable growth factors from small intestinal submucosa. J Cell Biochem 1997;67:478e91. [8] Badylak SF, Lantz GC, Coffey A, Geddes LA. Small intestinal submucosa as a large diameter vascular graft in the dog. J Surg Res 1989;47:74e80. [9] Cheng EY, Kropp BP. Urologic tissue engineering with small-intestinal submucosa: potential clinical applications. World J Urol 2000;18:26e30. [10] Pavcnik D, Uchida BT, Timmermans HA, Corless CL, O’Hara M, Toyota N, et al. Percutaneous bioprosthetic venous valve: a long-term study in sheep. J Vasc Surg 2002;35:598e602. [11] Nuininga JE, van Moerkerk H, Hanssen A, Hulsbergen CA, Oosterwijk-Wakka J, Oosterwijk E, et al. A rabbit model to tissue engineer the bladder. Biomaterials 2004;25:1657e61. [12] Jankowski R, Pruchnic R, Hiles M, Chancellor MB. Advances toward tissue engineering for the treatment of stress urinary incontinence. Rev Urol 2004;6:51e7. [13] Hodde J. Naturally occurring scaffolds for soft tissue repair and regeneration. Tissue Eng 2002;8:295e308. [14] Badylak S, Obermiller J, Geddes L, Matheny R. Extracellular matrix for myocardial repair. Heart Surg Forum 2003;6:E20e6. [15] Rosen M, Roselli EE, Faber C, Ratliff NB, Ponsky JL, Smedira NG. Small intestinal submucosa intracardiac patch: an experimental study. Surg Innov 2005;12:227e31.
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Contents lists available at ScienceDirect
Biomaterials journal homepage: www.elsevier.com/locate/biomaterials
Differential efficacy of gels derived from small intestinal submucosa as an injectable biomaterial for myocardial infarct repair Masaho Okada a, c, Thomas R. Payne a, c, e, f, Hideki Oshima a, c, Nobuo Momoi b, Kimimasa Tobita b, Johnny Huard a, b, c, d, * a
Stem Cell Research Center, Children’s Hospital of Pittsburgh, Pittsburgh, PA, USA Department of Pediatrics, Children’s Hospital of Pittsburgh, Pittsburgh, PA, USA Department of Orthopaedic Surgery, Pittsburgh, PA, USA d Department of Molecular Genetics and Biochemistry, University of Pittsburgh, Pittsburgh, PA, USA e Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA, USA f Cook MyoSite, Pittsburgh, PA, USA b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 20 May 2010 Accepted 28 June 2010 Available online 31 July 2010
Injectable biomaterials have been recently investigated as a therapeutic approach for cardiac repair. Porcine-derived small intestinal submucosa (SIS) material is currently used in the clinic to promote accelerated wound healing for a variety of disorders. In this study, we hypothesized that gels derived from SIS extracellular matrix would be advantageous as an injectable material for cardiac repair. We evaluated 2 forms of SIS gel, types B (SIS-B) and C (SIS-C), for their ability to provide a therapeutic effect when injected directly into ischemic myocardium using a murine model of an acute myocardial infarction. Echocardiography analysis at both 2 and 6 weeks after infarction demonstrated preservation of end-systolic left ventricular geometry and improvement of cardiac contractility in the hearts injected with SIS-B when compared with control hearts injected with saline. However, the SIS-C gel provided no functional efficacy in comparison with control. Histological analysis revealed that SIS-B reduced infarct size and induced angiogenesis relative to control, whereas injection of SIS-C had minimal effect on these histological parameters. Characterization of both gels revealed differential growth factor content with SIS-B exhibiting higher levels of basic fibroblast growth factor than SIS-C, which may explain, at least in part, the differential histological and functional results. This study suggests that SIS gel offers therapeutic potential as an injectable material for the repair of ischemic myocardium. Further understanding of SIS gel characteristics, such as biological and physical properties, that are critical determinants of efficacy would be important for optimization of this biomaterial for cardiac repair. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: Angiogenesis Animal model Bioactivity Cardiac tissue engineering Fibroblast growth factor SIS (small intestine submucosa)
1. Introduction Heart failure remains a significant cause of morbidity and mortality in Western countries [1]. The acute loss of functional myocardium after recovery from a myocardial infarction (MI) results in an abrupt increase in loading conditions within the left ventricle (LV). To compensate, the LV remodels the infarct and remote non-infarcted myocardium through a cascade of biochemical signaling processes that facilitate reparative changes including the dilatation of the left ventricle (LV) [2]. Since these intrinsic mechanisms of repair are limited in fully restoring function, new
* Corresponding author. Department of Orthopaedic Surgery, University of Pittsburgh School of Medicine, 450 Technology Drive, 2 Bridgeside Point, Suite 206, Pittsburgh, PA 15219, USA. Tel.: þ1 412 648 2798; fax: þ1 412 692 7095. E-mail address: [email protected] (J. Huard). 0142-9612/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2010.06.056
therapeutic approaches based on cellular therapy, biomaterials, and tissue engineering are being explored to reduce ventricular dilatation, improve function, and prevent disease progression towards end-stage heart failure. The discovery of small intestinal submucosa (SIS), which is a dense, acellular connective tissue harvested from porcine small intestine, has generated great interest as a naturally derived biomaterial for tissue regeneration and restoration [3]. SIS consists of a sheet of extracellular matrix that is biocompatible, mechanically malleable, resistant to infection, and bioactive. In regard to bioactive properties, SIS promotes proliferation, attachment and migration of various cell types and stimulates angiogenesis through an assortment of matrix-embedded angiogenic factors, including connective tissue growth factor (CTGF), basic fibroblast growth factor (FGF), transforming growth factor b (TGF-b), and fibronectin [4e7]. Researchers have been investigating the use of SIS scaffold
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for the regeneration and repair of various soft tissues and maladies including large vascular grafts, venous valves and leaflets, urinary bladder, dura mater, dermal wounds, fistulas, hernias, esophageal, and rotator cuff [3,8e13]. Of particular interest to this study, prior work has demonstrated a conduciveness of SIS for myocardial tissue ingrowth and vascularization when applied as an atrial prosthesis and a right ventricular full thickness patch [14,15]. In this study, we sought to investigate the use of gels derived from SIS extracellular matrix as an injectable biomaterial for cardiac repair. Two versions of SIS gel were evaluated for intramyocardial injection in a murine model of an acute MI. We postulated that delivery of SIS gel in situ would attenuate adverse LV remodeling and induce accelerated healing of infarct tissue through intrinsic bioactive signals, thereby improving LV function. 2. Materials and methods 2.1. Intramyocardial injection of SIS gel into an acute myocardial infarction The Institutional Animal Care and Use Committee, Children’s Hospital of Pittsburgh approved the animal and surgical procedures performed in this study (Protocols 7/03, 3-04). In conducting the research described in this study, the investigators adhered to the instructions described in the Guide for the Care and Use of Laboratory Animals (NIH Publications 85e23) as promulgated by the Committee of Care and Use of Laboratory Animals of the Institute of Laboratory Sciences, the National Academy of Sciences, and the National Research Council. A total of 26 male non-obese diabetic severe combined immunodeficiency (NOD-SCID) mice (25e30 g body weight, Jackson Laboratory, Bar Harbor, ME) were used for this study. We experienced a 15% mortality rate during the study (n ¼ 4 deaths, n ¼ 26 total injected mice). Two kinds of SIS gel, types B (SIS-B) and C (SIS-C), were provided by Cook Biotech Incorporated (West Lafayette, IN) and were evaluated in this study for cardiac repair. The pH level of SIS gel was adjusted to 7.4e7.8 using sodium hydroxide immediately prior to injection. Twenty micro liters of SIS material were injected directly into the ischemic myocardial zone of NOD-SCID mice immediately after inducing an MI by coronary ligation, as previously described [16]. Basic fibroblast growth factor content was measured by enzyme-linked immunosorbent assay, according to manufacturer’s instructions (R&D Systems, Minneapolis, MN). In control mice, 20 mL of phosphate buffered saline was injected into the same regions of the heart. The investigator performing the coronary ligation injury and injection procedures was blinded to the contents of the injectant. 2.2. Evaluation of cardiac function by echocardiography Two and 6 weeks after surgery, echocardiography was performed by a blinded investigator to assess the heart function of the anesthetized mice, as previously described [16]. End-systolic area (ESA) and end-diastolic area (EDA) were measured from short-axis images of the LV. Fractional area change (FAC), an index of LV contractility, was calculated as FAC (%) ¼ [(EDA ESA) / EDA] 100. 2.3. Histological analysis The mice were sacrificed at 6 weeks after SIS implantation for histological evaluation of the heart tissue. Hearts were flash frozen in 2-methylbutane (SigmaeAldrich, St. Louis, MO) that was pre-cooled in liquid nitrogen. Each specimen was serially cryosectioned at a thickness of 8 mm. 2.4. Measurement of infarct size Masson’s trichrome staining (IMEB, San Marcos, CA), which labels collagen blue and muscle red, was performed to evaluate scar tissue with the treated hearts. As previously described, Image J software (National Institutes of Health) was used to measure the lengths of the entire left ventricular endocardial circumference and the infarcted segment in a total of 5 sections [17]. The infarcted fraction of the left ventricle was calculated from these measurements. 2.5. Capillary density To determine capillary density within the infarct zone after treatment, we performed immunofluorescent staining of CD31, an endothelial cell marker, and analyzed the CD31-positive capillary structures within the infarct and peri-infarct zones at the 6 weeks time-point, as previously described [16]. 2.6. Statistical analysis Data are presented as mean standard error of the mean. Statistical differences in the data were determined by a one-way or two-way analysis of variance (ANOVA).
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The Tukey multiple comparison test was performed for post-hoc analysis when a statistical difference was observed in the ANOVA tests. Statistical significance for all tests was set at a P value < 0.05. Statistical calculations were performed with SigmaPlot software (Systat Inc., San Jose, CA).
3. Results 3.1. Cardiac function We assessed the therapeutic effect of 2 types of injectable SIS gel (SIS-B and SIS-C) for cardiac repair in an immunodeficient mouse model of an acute MI. Two and 6 weeks after transplantation, treatment with either SIS-B or SIS-C gels displayed very little effect on LV EDA relative to control saline injections (Fig. 1A, 2 weeks: SIS-B 8.8 0.2 mm2, n ¼ 9 mice; SIS-C 9.2 0.4, n ¼ 7; control 9.0 0.6, n ¼ 7; 6 weeks: SIS-B 8.4 0.3 mm2, n ¼ 9; SIS-C 9.2 0.5, n ¼ 7; control 8.8 0.2, n ¼ 6). Treatment with SIS-B significantly reduced LV ESA when compared with SIS-C and control saline treatments (Fig. 1B, 2 weeks: SIS-B 5.8 0.1 mm2, SIS-C 7.0 0.4, control 6.7 0.6; 6 weeks: SIS-B 5.7 0.3 mm2, SIS-C 6.9 0.3, control 6.7 0.3; P < 0.05, SIS-B versus SIS-C and control). LV contractility, as determined by FAC values, improved in the SISBetreated hearts when compared with hearts injected with SIS-C and control saline (Fig. 1C, 2 weeks: SIS-B 34.8 1.1%, SIS-C 24.9 2.8, control 25.6 1.9; 6 weeks: SIS-B 31.5 0.8, SIS-C 24.7 1.5, control 24.1 1.9; P < 0.001, SIS-B versus SIS-C and control). Thus, these results indicate that injection of SIS-B more effectively preserved LV systolic geometry and contractility after recovery from an acute MI than did the implantation of either SIS-C or saline. 3.2. Infarct size and fibrosis We examined LV infarct size and scar tissue area using Masson’s Trichrome stain to label collagenous scar tissue (blue) and cardiac muscle (red, Fig. 2AeC). Infarct size was measured as the percentage of endocardial circumferential length that displayed an infarct scar. At 6 weeks after implantation, hearts injected with SISB exhibited smaller infarcts after histological examination than hearts injected with either SIS-C or saline (Fig. 2D, SIS-B 46.4 4.0%, n ¼ 8; SIS-C 65.0 5.0, n ¼ 7; control 63.3 3.3, n ¼ 5; P < 0.05, SIS-B versus SIS-C). To determine the amount of scar tissue, scar area fraction was defined as the ratio of collagenous scar area to the entire cardiac muscle area. We observed a trend of reduced fibrosis in the SISBetreated hearts when compared with hearts injected with SIS-C and saline at the 6 weeks time point (Fig. 2E, SIS-B 0.26 0.03 scar area fraction, SIS-C 0.38 0.08, control 0.37 0.03). Taken together, the infarct size and scar area measurements demonstrate that SIS-B injections more effectively attenuated infarct expansion and fibrosis than either control or SIS-C injections. 3.3. Angiogenesis We measured capillary density within the infarct and periinfarct regions to determine whether the injection of SIS gel induced angiogenesis (Fig. 3AeC). In the infarct zone, hearts injected with SIS-B displayed greater capillary densities when compared with hearts injected with either SIS-C or saline (Fig. 3D, SIS-B 634 37 capillaries/mm2, n ¼ 7; SIS-C 455 33, n ¼ 7; control 503 31, n ¼ 5; P < 0.01, SIS-B versus SIS-C). In the periinfarct region, SIS-B also stimulated greater capillary formation when compared with SIS-C and control (Fig. 3E, SIS-B 640 39 capillaries/mm2, SIS-C 456 47, control 510 28; P < 0.05, SIS-B versus SIS-C).
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2 wks
10
B
6 wks
End-systolic Area (mm2)
End-diastolic Area (mm2)
A 9 8 7 6 5 4
SIS-B
SIS-C
Fractional Area Change (%)
C
Control 2 wks
40 35
2 wks
8
6 wks
7
*
6 5 4 3
SIS-B
SIS-C
Control
6 wks
*
30 25 20 15
SIS-B
SIS-C
Control
Fig. 1. Echocardiographic evaluation of cardiac function. (A) Two and six weeks after transplantation, SIS gel treatment imparted minimal effect on end-diastolic area of the left ventricle (LV). (B) SIS gel type B (SIS-B) significantly reduced LV end-systolic area when compared with SIS gel type C (SIS-C) and control (*P < 0.05, SIS-B versus SIS-C and control). (C) Cardiac contractility, as determined by LV fractional area change values, improved in the hearts injected with SIS-B greater than hearts treated with SIS-C and control saline (*P < 0.001, SIS-B versus SIS-C and control).
3.4. SIS growth factor content Both types of SIS gel consisted of a similar solid concentration: 9.4 mg/mL for SIS-B and 8.2 mg/mL for SIS-C (Table 1). However, SIS-B displayed a 58% higher content of basic fibroblast growth factor (bFGF) than SIS-C (Table 1). 4. Discussion Biomaterials in the injectable form have recently become a vanguard approach in the quest to develop reparative therapeutics for cardiovascular disorders [18]. Alginate, fibrin, matrigel, gelatin, chitosan, self-assembling peptide nanofibers, alpha-cyclodextrin MPEG-PCL-MPEG hydrogel, and the thermosensitive polymer gels Dex-PCL-HEMA/PNIPAAm and poly(NIPAAm-co-AAcco-HEMAPTMC) have been evaluated as an acellular therapy for myocardial infarct repair [19e28]. The fundamental premise of the injectable biomaterial approach for cardiac repair is that the injected agent will form a physical scaffold in situ that will reduce LV wall stresses and stabilize LV remodeling following recovery from an MI. In regards to LV remodeling, our results demonstrate that end-diastolic dimensions were not altered in SIS gel-treated hearts when compared with control saline-treated hearts. However, at endsystole, the dimensions of hearts injected with SIS-B were 15% smaller than hearts injected with control saline. The preservation of end-systolic geometry has also been reported with other injectable biomaterials for cardiac repair and is considered to be an important
predictor of survival after recovery from an MI [21,29]. Preliminary in vitro characterization of mechanical properties demonstrated differential mechanical strength between the 2 polymerized gels. SIS-B exhibited greater compressive modulus and maximum compressive stress than SIS-C, suggesting that SIS-B could more effectively provide physical support for in situ stabilization of LV dimensions than SIS-C (compressive modulus: 1.2e2.5 104 Pa for SIS-B, 0.5e1.0 104 Pa for SIS-C; maximum compressive stress: 6.2e8.2 103 Pa for SIS-B, 1.5e2.5 103 Pa for SIS-C). This disparity in mechanical properties may explain, at least in part, the differential efficacy of SIS-B and SIS-C gels for MI repair. Signaling factors within SIS gel, such as growth factors and cell adhesion motifs, may also be essential for the observed biological and functional effects. In this study, the SIS-B gel displayed a 58% greater content of bFGF than the SIS-C gel. The greater functional and histological effect of SIS-B may also be attributable, at least in part, to its higher growth factor content when compare with SIS-C. The observed attenuation of LV systolic dimensions and infarct expansion in SIS-Betreated hearts could also be due to the preservation of at-risk myocardium along the peri-infarct zone through intrinsic anti-fibrotic, pro-survival, angiogenic, and anti-inflammatory cytokines. Furthermore, SIS has been shown to recruit host c-kit positive cells to the infarct regions, which may aid myocardial regeneration and repair [30]. Others have shown that injectable biopolymers carrying growth factors enhance angiogenesis and LV contractility after recovery from myocardial infarction [23,25,31]. The synergistic effect between growth factors may further enhance the biological effect than a single factor alone. Multiple growth
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B
A
SIS-B
C
SIS-C
D
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Control
80
Infarction Size (%)
70 60 50
*
40 30 20 10 0
SIS-B
SIS-C
Control
Scar Area Fraction
E 0.5 0.4 0.3 0.2 0.1 0.0
SIS-B
SIS-C
Control
Fig. 2. Infarct size and scar area analysis. (AeC) Representative images of Masson’s trichrome stain of infarcted (blue) left ventricle (LV) free wall from each treatment group. Scale bars equal 1 mm. (D) Infarct size analysis by planimetry demonstrated that hearts injected with SIS gel type B (SIS-B) displayed a significant reduction in the percentage of infarcted endocardial circumferences when compared with hearts injected with SIS gel type C (SIS-C) or control saline (*P < 0.05, SIS-B versus SIS-C). (E) Injection of SIS-B also induced a trend towards less LV scar tissue when compared with the implantation of SIS-C and control saline.
factors are known to exist in SIS including CTGF, FGF, and TGF-b, which all have potential to stimulate therapeutic healing in a synergistic manner for cardiac repair [6,7]. In this regard, Hao and colleagues demonstrated that alginate hydrogels loaded with the growth factors VEGF-A and PDGF-BB had a superior functional and angiogenic effect was induced with both growth factors than either factor alone [32]. Collectively, these studies suggest a considerable therapeutic value of growth factors when combined with biomaterials for MI repair and support the hypothesis that intrinsic growth factor content in SIS may play an important and synergistic role in stimulating a beneficial biological response. The ability of injectable biomaterials to promote other biological effects such as host cell adhesion and migration throughout the infarct scar may also enhance the therapeutic response. The extracellular matrix components of SIS include proteoglycans,
glycosaminoglycans, collagens, fibronectin, and laminins [13]. These natural components may promote interaction of host cells with the injected material by presenting multiple cell adhesion, integrin-binding sites. In 2 studies that modified alginate biopolymers with cell adhesion peptides RGD and YIGSR demonstrated that an enhancement of cardiac function was not achieved [33,34]. However, each study presented conflicting results on the effect of the cell adhesion peptides on angiogenesis [33,34]. Additional research is needed to determine whether SIS gel is capable of actively guiding regeneration that leads to superior therapeutic effects when compared with biomaterials lacking bioactive properties. Cellular cardiomyoplasty is intended to be a therapy by which injured myocardium would be replaced by exogenous cells that would create new, functional myocardial tissue. However, various
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A
B
C
SIS-B
SIS-C
Capillaries per mm2
D
Infarct Zone 700
*
600 500 400 300
SIS-B
E Capillaries per mm2
Control
SIS-C
Control
Peri-infarct
*
700 600 500 400 300 200 100 0
SIS-B
SIS-C
Control
Fig. 3. Augmentation of capillary density with SIS gel. (AeC) Representative images of CD31 immunostaining of the peri-infarct regions of hearts injected with SIS gel type B (SIS-B), SIS gel type C (SIS-C), and control saline. Scale bars equal 50 mm. (D) Within the infarct zone, hearts treated with SIS-B displayed significantly greater capillary densities than hearts treated with SIS-C (*P < 0.01, SIS-B versus SIS-C). (E) Within the peri-infarct regions, SIS-B also induced significantly higher capillary densities than SIS-C (*P < 0.05, SIS-B versus SIS-C).
studies have reported that transplanted cells improve function by a mechanical and/or paracrine effect as opposed to de novo myocardial tissue regeneration by implanted cells [35]. Thus, injectable biomaterials could be designed to achieve the same therapeutic outcomes as cell transplantation. Conversely, the ability of cells to aptly sense and respond to the cues of the microenvironment and to secrete paracrine signals in a physiological manner at the site of injury may be major advantage of cell therapy and a limitation of injectable biomaterials. A few research groups have compared injectable, acellular biomaterial therapy to cell transplantation for cardiac repair, including matrigel versus embryonic stem cells, alginate versus neonatal cardiomyocytes, fibrin versus skeletal myoblasts, and chitosan versus embryonic stem cells [21,24,26,36,37]. All of these studies have demonstrated
Table 1 Characterization of injectable SIS gels. Type
Solid concentration (mg/mL)
FGF content (mg/g of dry solid) (mean SD)
SIS-B SIS-C
9.4 8.2
1.47 0.02 0.93 0.11
that injectable biomaterials provided at least an equivalent level of therapeutic benefit to cell transplantation. In our experience with the transplantation of murine skeletal muscle-derived stem cells (MDSCs) and human skeletal musclederived myogenic endothelial cells into the same murine model of acute MI used in this study, we observed a 50e90% improvement in cardiac function with cell therapy when compared with control injections of saline [16,17]. Here, we observed a 31e36% improvement of cardiac function at 2 and 6 weeks with the injection of SISB when compared with the injection of saline. The differences between our inferences and the aforementioned studies may arise from the potency of each cell type for cardiac repair. In particular, our prior work has shown that skeletal myoblasts are inferior to murine MDSCs and human myogenic endothelial cells for cardiac repair [16,17]. In comparison to SIS gel, the functional benefit demonstrated by our experiences with skeletal myoblast transplantation was equal, and sometimes inferior, to the level of therapeutic benefit achieved with injectable SIS gel in this study (i.e., human myoblasts 16e20%, mouse myoblasts 25e50%, and SIS 31e36% improvement relative to control saline injections) [16,17]. A study directly comparing these cell types with injectable biomaterials should be performed to validate these inferences. The use of injectable biomaterials as an acellular therapy would
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presumably be more conducive than autologous cell therapy in the clinical setting of an acute MI, where an off-the-shelf product could be applied immediately with minimal complexity. Understandably, comparisons between cellular and biomaterial technologies for cardiac repair will continually be assessed as these fields progress. 5. Conclusions Our findings suggest that gel derived from SIS extracellular matrix induces a biologic and therapeutic effect when delivered in situ for the treatment of an acute MI. The evaluation of 2 types of SIS gel demonstrated disparate capacities for cardiac repair, suggesting that the bioactive and physical properties of SIS gel are critical for therapeutic success. Understanding the significance of these characteristics in animal models of disease may help to appropriately design injectable SIS material for tissue repair. Acknowledgements The Donaldson Chair at Children’s Hospital of Pittsburgh and the Henry J. Mankin Chair at the University of Pittsburgh supported this work. We thank James Cummins, Ronald Jankowski, and Michelle Chutka for their assistance in the preparation of this manuscript. We also thank Cook Biotech Incorporated for providing SIS gel material. References [1] Lloyd-Jones D, Adams R, Carnethon M, De Simone G, Ferguson TB, Flegal K, et al. Heart disease and stroke statisticse2009 update: a report from the American Heart Association Statistics Committee and Stroke Statistics Subcommittee. Circulation 2009;119:e21e181. [2] Sutton MG, Sharpe N. Left ventricular remodeling after myocardial infarction: pathophysiology and therapy. Circulation 2000;101:2981e8. [3] Badylak SF. The extracellular matrix as a biologic scaffold material. Biomaterials 2007;28:3587e93. [4] Hodde J, Record R, Tullius R, Badylak S. Fibronectin peptides mediate HMEC adhesion to porcine-derived extracellular matrix. Biomaterials 2002;23:1841e8. [5] Badylak S, Liang A, Record R, Tullius R, Hodde J. Endothelial cell adherence to small intestinal submucosa: an acellular bioscaffold. Biomaterials 1999;20:2257e63. [6] Janis AD, Klingman NV, Berceli SA. Connective tissue growth factor (CTGF) content and bioactivity of small intestinal submucosa (SIS). Wound Repair Regen 2005;13:A4e27. [7] Voytik-Harbin SL, Brightman AO, Kraine MR, Waisner B, Badylak SF. Identification of extractable growth factors from small intestinal submucosa. J Cell Biochem 1997;67:478e91. [8] Badylak SF, Lantz GC, Coffey A, Geddes LA. Small intestinal submucosa as a large diameter vascular graft in the dog. J Surg Res 1989;47:74e80. [9] Cheng EY, Kropp BP. Urologic tissue engineering with small-intestinal submucosa: potential clinical applications. World J Urol 2000;18:26e30. [10] Pavcnik D, Uchida BT, Timmermans HA, Corless CL, O’Hara M, Toyota N, et al. Percutaneous bioprosthetic venous valve: a long-term study in sheep. J Vasc Surg 2002;35:598e602. [11] Nuininga JE, van Moerkerk H, Hanssen A, Hulsbergen CA, Oosterwijk-Wakka J, Oosterwijk E, et al. A rabbit model to tissue engineer the bladder. Biomaterials 2004;25:1657e61. [12] Jankowski R, Pruchnic R, Hiles M, Chancellor MB. Advances toward tissue engineering for the treatment of stress urinary incontinence. Rev Urol 2004;6:51e7. [13] Hodde J. Naturally occurring scaffolds for soft tissue repair and regeneration. Tissue Eng 2002;8:295e308. [14] Badylak S, Obermiller J, Geddes L, Matheny R. Extracellular matrix for myocardial repair. Heart Surg Forum 2003;6:E20e6. [15] Rosen M, Roselli EE, Faber C, Ratliff NB, Ponsky JL, Smedira NG. Small intestinal submucosa intracardiac patch: an experimental study. Surg Innov 2005;12:227e31.
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