Elsevier

Biomaterials

Volume 112, January 2017, Pages 264-274
Biomaterials

3D printed complex tissue construct using stem cell-laden decellularized extracellular matrix bioinks for cardiac repair

https://doi.org/10.1016/j.biomaterials.2016.10.026Get rights and content

Abstract

Stem cell therapy is a promising therapeutic method for the treatment of ischemic heart diseases; however, some challenges prohibit the efficacy after cell delivery due to hostile microenvironment of the injured myocardium. 3D printed pre-vascularized stem cell patch can enhance the therapeutic efficacy for cardiac repair through promotion of rapid vascularization after patch transplantation. In this study, stem cell-laden decellularized extracellular matrix bioinks are used in 3D printing of pre-vascularized and functional multi-material structures. The printed structure composed of spatial patterning of dual stem cells improves cell-to-cell interactions and differentiation capability and promotes functionality for tissue regeneration. The developed stem cell patch promoted strong vascularization and tissue matrix formation in vivo. The patterned patch exhibited enhanced cardiac functions, reduced cardiac hypertrophy and fibrosis, increased migration from patch to the infarct area, neo-muscle and capillary formation along with improvements in cardiac functions. Therefore, pre-vascularized stem cell patch provides cardiac niche-like microenvironment, resulting in beneficial effects on cardiac repair.

Introduction

Ischemic cardiovascular diseases are the worldwide major cause of morbidity and mortality [1]. Chronic heart failure usually begins with initial coronary artery blockage and starts progressive cardiomyocyte loss caused by necrotic and apoptotic environments. To heal those coagulative necrosis, cell therapy has emerged as a promising method to achieve functional improvement. The cells are usually transferred via intracoronary delivery method; however, recent meta-analysis of clinical trials showed limited effect on improving cardiac functions because of the death of delivered cells, which resulted in the hostile microenvironment at the injured myocardium [2], [3].

Towards the enhancement of therapeutic efficacy, there have been numerous approaches for promoting high cell survival via delivering of cells with functional biomaterials [4], [5], pro-angiogenic factors [6], [7], and genetically modified cells [8], [9], or using sheet type carriers [10]. In particular, cell delivery through a functionalized patch system (e.g., cell sheet engineering) can be an interesting alternative to achieve the delivery of a larger number of cells at the desired site [11], [12]. These cell patch system have revealed successful results, but low biophysical integration, and absence of organized vascular plexus within the platform remain as key barriers that must be overcome for a high level of functional repair for treating myocardial injury [13].

3D cell printing is a promising technology to produce precisely controlled 3D tissue or organ by mimicking both the outer shape and the inner architecture of native tissues [14]. Currently, the use of decellularized extracellular matrix (dECM) bioinks can recreate more complex biologically and biochemically relevant microenvironment, mimicking tissue specific ECM composition or resident cytokines. It enhances cellular functions, such as survival, maturation, differentiation, and migration, improving therapeutic effects after implanting the 3D printed tissue construct [15], [16]. Seif-Naraghi and colleagues studied the therapeutic effects of injectable heart tissue derived dECM (hdECM) hydrogel via transendocardial injection in small and large animal models. Accordingly, the hdECM hydrogel increased the recruitment of endogenous cardiomyocytes in the infarct area and maintained cardiac function without inducing arrhythmias [3]. Moreover, the resident CPCs migrated toward the infarct from epicardium through triggering the WT1-based migration mechanism [17]. In previous research, we also have examined the use of a bioink composed of hdECM that could intensively mimic the native tissue microenvironment and enhance the stem cell differentiation into the specific lineage [15], [16].

In this study, we developed a 3D pre-vascularized stem cell patch through spatial organization of cardiac progenitor/mesenchymal stem cells using 3D cell printing method. Major features of the prepared cardiac patch are the patterning of dual cells and the use of tissue-specific bioink. They can promote vascularization, prolonged cell survival and tissue remodeling process after transplanting the patch. Moreover, the pre-vascularized stem cell patch provides enhanced therapeutic effects, including decrease of cardiac remodeling and fibrosis, and promotional effects of cardiomyogenesis and neovascularization at the injured myocardium.

Section snippets

Cell isolation and culture

Human c-kit + cardiac progenitor cells (hCPCs) were isolated from human infant-derived heart tissues after surgical procedures by following the protocol previously reported [18]. This protocol was approved by Institutional Review Board of the Pusan National University Hospital of Yangsan, Gyeongsangnam-do, Korea (Approval no. 2012-18). CPCs were cultured in Ham's F12 media supplemented with 10% fetal bovine serum (FBS, Life technologies, Grand Island, NY), 1 × penicillin/streptomycin, 2.5 mU/mL

Fabrication of 3D pre-vascularized stem cell patch

We applied a 3D bioprinting system to manufacture the 3D pre-vascularized stem cell patch (Fig. 1A). Two of the heads were connected to each pneumatic pressure based dispensing system, equipped with a heating unit that enabled dispensing of PCL easily. Each of the other two heads were connected to a robotic micro-extrusion dispensing system for printing cell-laden hydrogel bioink (Fig. 1B). These heads were operating on a 3-axis motorized stage and the movements were controlled with a command

Discussion

In this work, we demonstrated that hdECM bioink provides a beneficial effect on the maturation of CPC compared with that of the collagen bioink; thereby, it is favorable to reconstruct native tissue environment for cardiac repair. In addition, hdECM bioink exhibited improved epicardial activation capability with promotion of angiogenesis and EMT through the WT1-mediated Wnt/β-catenin signaling of epicardium. Taken together, hdECM might potentiate the epicardial-mediated cardiac tissue

Conclusion

A major advantage of the developed platform technique is the spatial patterning of each cell to promote rapid vascularization in a 3D structure and potentiated delivered cells using hdECM as a bioink. The stem cell patch showed therapeutic efficacy through improvement of cardiac function and decrease of negative LV remodeling. We also provided the histological evidences that this 3D pre-vascularized stem cell patch could effectively deliver the stem cells via epicardial delivery route. This

Conflict of interest

The authors confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.

Acknowledgements

This study was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (No. 2010-0018294) (DWC) and Research Fund of Seoul St. Mary's Hospital, The Catholic University of Korea (HJP).

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    J.J and H.-J.P were contributed equally to this work.

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