Elsevier

Biomaterials

Volume 76, January 2016, Pages 187-195
Biomaterials

Early in-situ cellularization of a supramolecular vascular graft is modified by synthetic stromal cell-derived factor-1α derived peptides

https://doi.org/10.1016/j.biomaterials.2015.10.052Get rights and content

Abstract

In an in-situ approach towards tissue engineered cardiovascular replacement grafts, cell-free scaffolds are implanted that engage in endogenous tissue formation. Bioactive molecules can be incorporated into such grafts to facilitate cellular recruitment. Stromal cell derived factor 1α (SDF1α) is a powerful chemoattractant of lymphocytes, monocytes and progenitor cells and plays an important role in cellular signaling and tissue repair. Short SDF1α-peptides derived from its receptor-activating domain are capable of activating the SDF1α–specific receptor CXCR4. Here, we show that SDF1α-derived peptides can be chemically modified with a supramolecular four-fold hydrogen bonding ureido-pyrimidinone (UPy) moiety, that allows for the convenient incorporation of the UPy-SDF1α-derived peptides into a UPy-modified polymer scaffold. We hypothesized that a UPy-modified material bioactivated with these UPy-SDF1α-derived peptides can retain and stimulate circulating cells in an anti-inflammatory, pro-tissue formation signaling environment. First, the early recruitment of human peripheral blood mononuclear cells to the scaffolds was analyzed in vitro in a custom-made mesofluidic device applying physiological pulsatile fluid flow. Preferential adhesion of lymphocytes with reduced expression of inflammatory factors TNFα, MCP1 and lymphocyte activation marker CD25 was found in the bioactivated scaffolds, indicating a reduction in inflammatory signaling. As a proof of concept, in-vivo implantation of the bioactivated scaffolds as rat abdominal aorta interposition grafts showed increased cellularity by CD68+ cells after 7 days. These results indicate that a completely synthetic, cell-free biomaterial can attract and stimulate specific leukocyte populations through supramolecular incorporation of short bioactive SDF1α derived peptides.

Introduction

Replacement of small diameter blood vessels relies mainly on autologous tissue, which is often limited by availability and requires invasive harvesting. Common non-living prostheses for vascular structures have considerable drawbacks such as high risk of occlusion, lack of growth potential, the consequent need for re-operation in pediatric patients, and life-long anti-coagulation therapy [1]. In an in-situ tissue engineering approach, synthetic scaffolds are implanted that provide the necessary mechanical support. Ideally, scaffold material will contain bioactive molecules capable of instructing cells of the recipient to migrate into the graft and stimulate the development of living, growing tissue [2]. Molecules involved in cellular adhesion have largely been the focus to be introduced in synthetic materials [3]. Chemokines play a considerable role in the process of tissue repair by attracting progenitor cells but also by modulating the inflammatory environment. Therefore, immobilization of chemokines on synthetic grafts may simultaneously allow for both specific cell retention and subsequent stimulation of cellular development [2]. Stromal cell derived factor 1 alpha (SDF1α) is a potent chemoattractant of lymphocytes [4], monocytes and progenitor cells but not neutrophils [5]. It is important for the homing of bone-marrow resident stem cells [6], and plays a central role in tissue repair signaling [5]. Importantly, following implantation the systemic response to a cell-free vascular graft material involves the influx of immune cells. The nature and amount of these cells can be influenced by SDF1α [7]. Short SDF1α-peptides that are homologous to the receptor-activating domain of the full protein, have been shown to improve damage repair after local delivery in ischemic tissue [8]. This indicates that short peptide sequences, which are synthetically more accessible compared to full-length proteins, are capable of retaining specific SDF1α activity. Maintaining a stable local gradient of SDF1α and avoiding a burst release of bioactive molecules after implantation has been shown to further improve the retention of progenitor cells under fluid flow conditions [9]. In addition to inducing cellular mobilization as a soluble factor, SDF1a is an important anchoring molecule for progenitor cells in bone marrow stroma [10] as well as a homing beacon bound to the ECM in the vicinity of tissue damage, guiding the migration of cells towards the site of repair [11]. Therefore an approach to anchor the SDF1α protein to a scaffold material may be advantageous for biological signaling at the site of graft implantation.

In this study we apply a synthetic cell-free scaffold based on the supramolecular modification of poly(l-lactic acid caprolactone) (PLLCL) functionalized with quadruple hydrogen bonding ureido-pyrimidinone (UPy) units [12], with SDF1α-derived peptide sequences also modified with these UPy-moieties, in order to facilitate the early cellularization of a vascular graft [2]. The base material consists of PLLCL prepolymers modified with UPy-moieties in the main chain yielding a chain-extended UPy-PLLCL (or CE-UPy-PLLCL) polymer (Fig. 1) [13]. To prevent rapid proteolytic degradation of the SDF1α-derived peptides, we abolished the cleavage sites for the enzymes MMP2 and CD26, which are capable of abrogating the SDF1α signal and are abundant in an inflammatory environment [8], [14]. The second valine in the natural sequence was substituted with a serine residue, leading to two peptide sequences: SKPVSLSYR and SKPVVLSYR, i.e. the proteolytically resistant and non-resistant peptides, respectively (after UPy-modification UPy-SDF1α(R) and UPy-SDF1α(NR), respectively). The material was processed into fibrous scaffolds by electrospinning. Using a previously developed mesofluidic device [15] applying a physiological pulsatile fluid flow of medium-suspended human peripheral blood mononuclear cells (PBMCs) the homing of cells into the scaffolds functionalized with short SDF1α peptides was analyzed. As a proof-of-concept to investigate the in vivo specific recruitment of circulating cells, we implanted electrospun tubular scaffolds in a abdominal aorta interposition graft rat model and analyzed the cellular influx after 24 h and 7 days.

Section snippets

Synthesis of the SDF1α peptides

The synthesis of the investigated peptides is described in the supplemental files (See supplemental Materials & Methods).

Synthesis of CE-UPy-PLLCLa

The CE-UPy-PLLCL polymer was obtained using the same procedure as described for polymer 2 described in Ref. [13] (chain-extended UPy-poly[2-methyl–1,3-propylene adipate) in which the poly[2-methyl–1,3-propylene adipate diol is replaced with poly(l-lactic acid caprolactone) diol (purchased from SyMO-Chem BV) with a Mn of 1 kDa. The CE-UPy-PLLCL polymer was obtained as an

Synthetic SDF1α-derived peptides are biologically active through CXCR4 and are resistant to proteolytic degradation by MMP2

The ability of the SDF1α-derived peptides to induce receptor-mediated migration was analyzed to confirm biological activity though the SDF1α-CXCR4 axis. In a Boyden chamber migration assay both SDF1α(R) and SDF1α(NR) peptides induced significant migration of PBMCs compared to the non-peptide control condition and to a similar degree to full-length SDF1α. Pre-incubation of PBMCs with the CXCR4-specific antagonist AMD-3100 significantly reduced the number of migrating cells (Fig. 2A). This

Discussion

Previous efforts to use biofunctionalized synthetic materials for vascular grafting have mostly focused on ECM derived peptides such as RGD to enhance cellular adhesion [17]. Considering their ability to simultaneously attract and stimulate targeted cell populations chemokines may provide more specific biological activity. We report, for the first time, that the supramolecular bioactivation of a fully synthetic material using short peptides based on the chemokine SDF1α can attract and stimulate

Acknowledgments

This research forms part of the Project P1.01 iValve of the research program of the BioMedical Materials institute, co-funded by the Dutch Ministry of Economic Affairs.

The financial contribution of the Nederlandse Hartstichting is gratefully acknowledged.

Part of this research is funded by the Ministry of Education, Culture and Science (Gravity program 024.001.035), the Netherlands Organisation for Scientific Research (NWO), the European Research Council (FP7/2007–2013), ERC Grant Agreement

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