Elsevier

Biomaterials

Volume 33, Issue 1, January 2012, Pages 80-90
Biomaterials

Enhancement of mesenchymal stem cell angiogenic capacity and stemness by a biomimetic hydrogel scaffold

https://doi.org/10.1016/j.biomaterials.2011.09.041Get rights and content

Abstract

In this study, we examined the capacity of a biomimetic pullulan–collagen hydrogel to create a functional biomaterial-based stem cell niche for the delivery of mesenchymal stem cells (MSCs) into wounds. Murine bone marrow-derived MSCs were seeded into hydrogels and compared to MSCs grown in standard culture conditions. Hydrogels induced MSC secretion of angiogenic cytokines and expression of transcription factors associated with maintenance of pluripotency and self-renewal (Oct4, Sox2, Klf4) when compared to MSCs grown in standard conditions. An excisonal wound healing model was used to compare the ability of MSC-hydrogel constructs versus MSC injection alone to accelerate wound healing. Injection of MSCs did not significantly improve time to wound closure. In contrast, wounds treated with MSC-seeded hydrogels showed significantly accelerated healing and a return of skin appendages. Bioluminescence imaging and FACS analysis of luciferase+/GFP+ MSCs indicated that stem cells delivered within the hydrogel remained viable longer and demonstrated enhanced engraftment efficiency than those delivered via injection. Engrafted MSCs were found to differentiate into fibroblasts, pericytes and endothelial cells but did not contribute to the epidermis. Wounds treated with MSC-seeded hydrogels demonstrated significantly enhanced angiogenesis, which was associated with increased levels of VEGF and other angiogenic cytokines within the wounds. Our data suggest that biomimetic hydrogels provide a functional niche capable of augmenting MSC regenerative potential and enhancing wound healing.

Introduction

Mesenchymal stem cells (MSCs) have shown beneficial effects in preclinical models of wound healing and in preliminary clinical case reports by accelerating wound closure, improving neovascularization, and reducing scar formation [1], [2], [3], [4], [5], [6]. While the specific mechanism for MSC-mediated improvements in wound healing has not been fully elucidated, a significant role for paracrine interactions in this process has been proposed [7], [8]. The role of MSC differentiation into different cell types within the wound environment has been less well established partially due to the small number of transplanted cells that persist within the wound over time. The lack of significant MSC engraftment at sites of injury is thought to be a major limiting factor of current MSC-based therapeutics [9].

The problem of low MSC engraftment is not unique to wounds and has plagued the therapeutic application of MSCs in numerous models of tissue injury. Despite variable degrees of enhanced tissue repair, MSCs administered systemically have shown long-term engraftment rates of less than 3% in injury models of the pancreas [10], heart [11], kidney [12] and liver [13]. Delivery of MSCs via local injection to cutaneous wound sites has similarly demonstrated low engraftment efficiency [1]. The capacity of MSC-based treatments to exhibit therapeutic efficacy despite such low engraftment efficiency has spurred efforts to improve cell delivery methods in hope of maximizing the regenerative potential of MSCs.

The use of collagen matrices to deliver MSCs in animal models of myocardial infarction has shown promise in augmenting cell engraftment and improving functional endpoints [14], [15]. Similarly, an injectable collagen-based matrix was used to successfully increase the engraftment efficiency of endothelial progenitor cells and upregulate local angiogenesis in a rodent hindlimb ischemia model [16]. These and other tissue engineering-based strategies have suggested that the use of biomaterial-based scaffolds for cell delivery may not only enhance cell viability but can also promote cell proliferation and actively contribute to MSC fate determination [17].

We and others have previously proposed that the ultimate solution to pathologic healing is delivery of multipotent cells in the context of appropriate environmental cues [18], [19]. The use of acellular dermal matrices for stem cell delivery into wounds has shown promise in directing skin-specific regeneration [20]. However, the problems associated with natural biomaterials including cost, availability, seeding and the theoretical risk of disease transmission prompted us to examine synthetic biomaterials that could recapitulate the beneficial structural, mechanical and chemical properties of skin without these problems.

Hydrogels are an ideal physicochemical mimetic of natural extracellular matrix (ECM) as the hygroscopic nature of ECM is one of its key properties [21]. Additionally, a wide array of commercially available hydrogel products are already used for wound dressings, thus potentially accelerating the translation of this technology into the clinic. In this study, we utilize a biomimetic hydrogel scaffold previously developed by our laboratory to deliver MSCs into wounds within a three-dimensional dermal-like microenvironment. We have previously demonstrated that this composite collagen-pullulan hydrogel is non-cytotoxic, recapitulates key features of fetal (scarless) healing, and promotes granulation tissue formation through vascular induction [22]. This cell-biomimetic matrix approach for stem cell delivery may be a promising clinical strategy for maximizing the therapeutic potential of MSCs for cutaneous wound healing.

Section snippets

Animals

All mice were housed in the Stanford University Veterinary Service Center in accordance with NIH and institution-approved animal care guidelines. All procedures were approved by the Stanford Administrative Panel on Laboratory Animal Care.

Bone marrow-derived MSC isolation

Luciferase+/GFP+ bone marrow-derived MSCs were isolated as previously described [23]. Briefly, bone marrow was flushed from the tibias and femurs of 8–12 week old male FVB-luc-eGFP transgenic mice (generous gift of Dr. Christopher Contag) into a suspension of

Hydrogel biocompatibility

To assess the feasibility of our previously developed collagen-pullulan hydrogel system as a delivery vehicle for MSCs, we evaluated cell morphology, viability, proliferation and migratory capacity of MSCs cultured within the hydrogels in vitro. MSCs attached to pullulan–collagen hydrogels and conformed to the scaffold’s three-dimensional topography (Fig. 1A). The majority of cells exhibited a rounded morphology and clustered around the hydrogel pores. MSCs seeded within the hydrogels remained

Discussion

In this study, we utilize a biomimetic hydrogel scaffold to effectively deliver MSCs into cutaneous wounds. Our tissue engineering approach to MSC-based wound therapy enhances the stemness properties of these cells as well as their capacity to secrete cytokines critical to healing. Compared to local MSC injection, delivery of MSCs within a pullulan–collagen hydrogel scaffold accelerates wound healing, improves stem cell survival and promotes angiogenesis.

Previous studies have demonstrated the

Conclusion

These findings demonstrate the capacity of a biomimetic hydrogel system to enhance MSC delivery to cutaneous wounds potentially by preserving cell–matrix interactions, localizing cells within wounds and enhancing stem cell properties. Hydrogel delivery of MSCs significantly enhances MSC viability and engraftment compared to local injection. Biomimetic hydrogels provide a functionalized niche for the in vivo delivery of MSCs which accelerates normal wound healing and promotes neovascularization.

Acknowledgments

This work was supported by a grant to GCG and MTL from the Armed Forces Institute of Regenerative Medicine DOD #W81XWH-08-2-0032, the Hagey Laboratory for Pediatric Regenerative Medicine and the Oak Foundation. We gratefully acknowledge Mrs. Yujin Park for histologic processing, and Mr. Dean Nehama and Ms. Jessica Trang of the Stanford BioADD Center for assistance with hydrogel preparation.

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