Elsevier

Biomaterials

Volume 33, Issue 15, May 2012, Pages 3868-3879
Biomaterials

The in vitro preconditioning of myoblasts to enhance subsequent survival in an in vivo tissue engineering chamber model

https://doi.org/10.1016/j.biomaterials.2012.02.006Get rights and content

Abstract

The effects of in vitro preconditioning protocols on the ultimate survival of myoblasts implanted in an in vivo tissue engineering chamber were examined. In vitro testing: L6 myoblasts were preconditioned by heat (42 °C; 1.5 h); hypoxia (<8% O2; 1.5 h); or nitric oxide donors: S-nitroso-N-acetylpenicillamine (SNAP, 200 μM, 1.5 h) or 1-[N-(2-aminoethyl)-N-(2-aminoethyl)amino]-diazen-1-ium-1,2-diolate (DETA-NONOate, 500 μM, 7 h). Following a rest phase preconditioned cells were exposed to 24 h hypoxia, and demonstrated minimal overall cell loss, whilst controls (not preconditioned, but exposed to 24 h hypoxia) demonstrated a 44% cell loss. Phosphoimmunoblot analysis of pro-survival signaling pathways revealed significant activation of serine threonine kinase Akt with DETA-NONOate (p < 0.01) and heat preconditioning (p < 0.05). DETA-NONOate also activated ERK 1/2 signaling (p < 0.05). In vivo implantation: 100,000 preconditioned (heat, hypoxia, or DETA-NONOate) myoblasts were implanted in SCID mouse tissue engineering chambers. 100,000 (not preconditioned) myoblasts were implanted in control chambers. At 3 weeks, morphometric assessment of surviving myoblasts indicated myoblast percent volume (p = 0.012) and myoblasts/mm2 (p = 0.0005) overall significantly increased in preconditioned myoblast chambers compared to control, with DETA-NONOate-preconditioned myoblasts demonstrating the greatest increase in survival (p = 0.007 and p = 0.001 respectively). DETA-NONOate therefore has potential therapeutic benefits to significantly improve survival of transplanted cells.

Introduction

Tissue engineering offers therapeutic approaches to replace lost or damaged tissue and organs. Recreating an environment that supports and promotes normal tissue growth and homeostatic mechanisms remains a significant challenge. Viable stem/progenitor cells; a scaffold or extra cellular matrix, a functional, growing vascular network and an unencumbered physical space are essential components for successful tissue growth in tissue engineered constructs.

To assure stem/progenitor cell survival after transplantation an adequate oxygen supply, nourishment and the disposal of metabolic waste products are essential [1], [2], [3]. Until a functional vascular network is established in the tissue construct, transplanted cells have to rely on the diffusion of oxygen and nutrients and are susceptible to cell death [4]. Thus, tissue engineering has to allow for vascular networks to form within the matrix material of the new tissue construct. Prior to significant angiogenesis, hypoxic cell death of implanted cells will hamper successful tissue formation [5]. This study therefore seeks to enhance implanted cell survival in a tissue engineering chamber, by increasing the innate survival characteristics of implanted cells by a process termed ‘preconditioning’.

Preconditioning, has largely been studied in whole organs – principally the heart. It is defined as injury – induced tissue protection, acquired through exposure to a sublethal stress, which induces the tissue to be more tolerant of a second subsequent lethal stress. Thus the sublethal stress has a cytoprotective effect in whole organs. First described by Murry et al. [6] in 1986 this phenomenon has reproducibly [7] been shown to decrease infarct size in the ischemic heart. Similar effects, resulting in reduced myocardial infarct size can be induced by a variety of agents that in larger doses may be harmful to the heart, but can initiate a preconditioned state when applied in reduced quantities [8]. Since the work of Murry, preconditioning has been observed to provide whole organ/tissue protection in other tissues including skin and muscle tissue flaps [9], skeletal muscle [10], intestine [11], and kidney [12].

More recently preconditioning of individual cells – whilst in vitro has been used in a limited number of studies to promote subsequent survival of cells in vivo [13], [14], [15], [16]. Preconditioned mesenchymal stem cells (MSCs) have been implanted into infarcted heart tissue, with increased MSC survival. These cells also induced pro-angiogenic effects in the muscle tissue [15].

This study describes cell preconditioning protocols to enhance the survival of myoblasts in the transfer from cell culture to an in vivo environment – a tissue engineering chamber. Preconditioning cells prior to implantation in an in vivo tissue engineering chamber has not been reported previously. The in vivo chamber model has a number of advantages when attempting to assess implanted cell survival as it provides an isolated protected space for implanted cells. Ideally it also has a significant hypoxic environment in the first 7–10 days, but as capillaries grow from the implanted vascular pedicle, hypoxia diminishes and implanted cells that can survive the initial hypoxia, can grow in this supportive niche. A number of different in vitro preconditioning regimes were applied to myoblasts: heat stress, a hypoxic stress, or exposure to nitric oxide (NO) donors – SNAP or DETA-NONOate, and signaling pathways these regimes activate were correlated to preconditioned cell survival (assessed morphometrically) in the in vivo tissue engineering chamber.

Section snippets

Materials and methods

All experiments were performed with the prior approval of the St. Vincent’s Hospital, Melbourne, Animal Ethics Committee, under National Health and Medical Research Council (Australia) guidelines.

Identification of preconditioning regimes which maintain cell survival during 24 h hypoxia

A number of different protocols (Experiment 1–3) for each preconditioning regime (heat, hypoxia or NO donors) were tested in vitro as described in Table 2, which lists the percent survival after the final 24 h hypoxic insult compared to cell survival at the end of the recovery phase. Preconditioning was considered successful in vitro, if cell survival was maintained at or near 100% during the 24 h in vitro hypoxic period (Table 2, and Fig. 1b).

In Experiment 3 all the preconditioned groups

Discussion

The major findings of this study are that although several preconditioning protocols including heat, hypoxia and NO donors maintained cell survival at ∼100% after a subsequent period of 24 h hypoxia in in vitro testing, only one preconditioning protocol, DETA-NONate, maintained a consistent, increased cell survival after three weeks in vivo in a tissue engineering chamber. Notably only DETA-NONate activated two of the major intracellular cell survival signaling pathways ERK and Akt kinases.

Conclusion

We have demonstrated that NO donors when applied to myoblasts in vitro can precondition these cells to significantly enhance their survival to a subsequent prolonged hypoxic insult in an in vivo tissue engineering chamber. The enhanced survival of myoblasts with DETA-NONOate preconditioning was associated with upregulation of the pro-survival factors Akt and ERK in these cells. These results indicate that in vitro preconditioning with DETA-NONOate of cells intended for cell therapy use in

Acknowledgments

This project was supported by a National Health and Medical Research Council (NH&MRC) Project Grant and an NH&MRC Principal Research Fellowship to GJ Dusting. The O’Brien Institute acknowledges the Victorian State Government’s Department of Innovation, Industry and Regional Development’s Operational Infrastructure Support Program.

The authors appreciate the surgical assistance of staff (Sue Mc Kay, Liliana Pepe, Anna Deftereos and Amanda Rixon) from the Experimental and Medical Research Unit, St

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