Growth factor delivery systems have been designed from a variety of materials – both inert and bioactive. These systems are diverse and numerous, taking the form of injectable microspheres [61
], implantable patches [38
], and in situ
gelling scaffolds [3
]. In vivo
, growth factor delivery is achieved via sequestration in the native extracellular matrix. Through binding to sulfated glycosaminoglycans linked to ECM proteins, growth factors can be immobilized for presentation or stored for future release [64
]. When designing tissue engineering approaches that mimic this system, important design parameters include providing for prolonged presentation and release of the incorporated growth factors, as well as a fibrous, porous template that allows for cellular infiltration and remodeling. A wide array of materials have previously been investigated for encapsulation of angiogenic factors for therapeutic delivery [29
], including synthetic materials such as poly(lactide-co-glycolide) (PLG) [65
] and poly-NiPAam co-polymers [66
], as well as natural materials such as alginate [67
], fibrin [26
], collagen [70
], gelatin [71
], and chitosan [72
], among others. Often, a material that has been demonstrated to be a useful scaffold for a certain tissue engineering application will be modified to deliver growth factors in order to enhance the therapeutic effect. Common modifications include crosslinking the material or covalently attaching growth factors [38
] or growth factor-binding molecules such as heparin [75
]. Previous work has shown that when angiogenic growth factors are immobilized on a scaffold, angiogenic potential is enhanced [37
]. Unfortunately, the modifications used to increase growth factor activity or stability may change the chemistry of many natural biopolymers and therefore change their activity in vivo
]. ECM-derived hydrogels processed from decellularized tissue have shown great promise as scaffolds for tissue repair, but their potential as growth factory delivery systems had not been previously explored. These matrix hydrogels have been demonstrated to retain components of the native ECM, including proteins, glycoproteins, and glycosaminoglycans [6
]; implying the possibility of employing the same method for sequestration and delivery observed in the native tissue microenvironment. With this work, the natural incorporation of a heparin binding growth factor, bFGF, in a pericardial matrix hydrogel was explored.
Confirmation of the sulfated GAG content in the PPM gel was performed by comparing the FTIR spectra obtained for PPM and collagen and identifying a sulfate peak and a sugar peak (), similar to previous work comparing proteoglycans and collagen in cartilage [55
]. A Blyscan sulfated glycosaminoglycan assay allowed for quantification of the sGAG content, which was comparable to matrix hydrogels derived from myocardial and adipose tissue [7
]. While bFGF diffused readily out of the collagen gels over 5 days in vitro,
significant release of bFGF from the PPM gels was only observed after the addition of NaCl (), indicating that the growth factor was bound to the matrix through electrostatic means, presumably through the sGAGs in the material. A portion of bFGF did diffuse out of the PPM gel over 5 days, indicating that some of the growth factor was simply encapsulated in the material instead of bound. Upon the addition of collagenase, additional bFGF was removed as the collagen structure was degraded; however, the remaining components of the gel, which were visible in an intact 3D structure, retained a significant amount of growth factor that was dissociated upon the further addition of NaCl. These in vitro
results with enhanced retention were further validated by the increased retention in vivo
, quantified five days post-injection (). This data, taken in conjunction with the similar mechanical and structural properties of the two materials, indicates that the growth factors are likely associating with the extant binding domains in the decellularized ECM hydrogel.
Using biomaterials for immobilization and prolonged release of angiogenic factors has been shown to enhance their activity; in this study, injection of bFGF in pericardial matrix was the only treatment group to significantly increase acute neovascularization compared to controls (). Additionally, a significantly greater density of large-diameter vessels (25–50 μm) were identified in the infarcts in these animals (), indicating a maturation of the vasculature. These results may be due to the extended presence of bFGF in the injected PPM or an increase in stability or activity due to immobilization on the PPM scaffold. There may also be synergistic effects with the addition of bFGF to the matrix hydrogel. Inflammation is known to influence neovascularization, and the inclusion of bFGF [77
] and GAGs [79
] as well as differences between allogeneic and xenogeneic material sources could influence this process. We therefore obtained a semi-quantitative score of the inflammation in each group. While an increase in the degree of inflammation was observed with injection of all groups with a growth factor (bFGF alone, bFGF in collagen, and bFGF in pericardial matrix) when compared to the saline injection, there was no significant difference in the inflammatory response of either material alone (pericardial matrix or collagen) when compared to saline or each other. Thus, while an increase in inflammation may be one of the mechanisms by angiogenesis is enhanced [80
], since the inflammatory response was not significantly different among the growth factor groups, it is likely not the major contributing factor in the increased arteriole density observed when bFGF is delivered with the pericardial matrix.
It is important to note that while other studies have shown extant growth factor content (bFGF and VEGF) in decellularized matrix materials after processing, the quantity is on the order of picograms per milligram of dry weight ECM [81
]. While we previously did not identify bFGF in the PPM using mass spectrometry [8
], any potential extant bFGF content should be masked by the much larger quantity added to the injection. Additionally, the control pericardial matrix injection did not show a significant increase in arteriole density compared to collagen or saline, indicating the material alone does not enhance neovascularization over other injections at this time point.
Given the potential for hemangioma formation [82
], where vascular tissue proliferates without being connected to the host vasculature, it was important to determine if using the pericardial matrix hydrogel to deliver bFGF would result in anastomosis of the blood vessels formed in the infarct region. This was done by perfusing red fluorescent carboxylated microspheres retrograde before euthanasia. Red fluorescence in the vessels formed in the infarct region for all bFGF groups indicated that the vessels were connected to the host vasculature (), supporting the feasibility of the pericardial matrix as a delivery system for bFGF. Importantly, even though the high concentration of bFGF injected did not cause hemangioma formation, it would still be necessary to optimize the loading dose of growth factor for any application. In previous studies utilizing bFGF for treatment post-MI, the administered dose varied greatly, from 30 mg injected with 3 mg of heparin sulfate in a canine model [85
], 110 mg daily intracoronary bolus injection also in a canine model [86
], or a total of 5 ug incorporated in heparin-sepharose-alginate beads in a porcine model [61
]. Further investigation with ECM-derived hydrogels will be necessary to determine the maximum loading capacity for a heparin-binding growth factor. This value will vary for matrix hydrogels derived via different methods and from different tissues as it will depend on the concentrations of growth factor-binding motifs in the processed materials.
Injectable matrix hydrogels derived from different tissue sources have been previously explored as therapies for the prevention of heart failure post-myocardial infarction [11
]. Intramyocardial injection of hydrogels derived from porcine myocardium and small-intestinal submucosa has been demonstrated to preserve ejection fraction post-MI while saline-injected control animals continue to decline [13
]. While the exact mechanism behind this success has not yet been elucidated, it may be that the fibrous microstructure provides a template for cell infiltration and vascularization [87
] or that degradation may promote cell in-growth and tissue remodeling [11
]. ECM-derived hydrogels are of special clinical interest as they are deliverable via catheter and therefore could potentially be administered via minimally invasive methods [13
]. While long-term studies with this growth factor delivery system will be necessary to evaluate its effect on cardiac function and remodeling post-MI, with this work, we have established proof-of-concept for using an ECM-derived hydrogel to enhance retention and delivery of a heparin-binding growth factor.