Elsevier

Acta Biomaterialia

Volume 31, February 2016, Pages 99-113
Acta Biomaterialia

A prosurvival and proangiogenic stem cell delivery system to promote ischemic limb regeneration

https://doi.org/10.1016/j.actbio.2015.12.021Get rights and content

Abstract

Stem cell therapy is one of the most promising strategies to restore blood perfusion and promote muscle regeneration in ischemic limbs. Yet its therapeutic efficacy remains low owing to the inferior cell survival under the low oxygen and nutrient environment of the injured limbs. To increase therapeutic efficacy, high rates of both short- and long-term cell survival are essential, which current approaches do not support. In this work, we hypothesized that a high rate of short-term cell survival can be achieved by introducing a prosurvival environment into the stem cell delivery system to enhance cell survival before vascularization is established; and that a high rate of long-term cell survival can be attained by building a proangiogenic environment in the system to quickly vascularize the limbs. The system was based on a biodegradable and thermosensitive poly(N-Isopropylacrylamide)-based hydrogel, a prosurvival and proangiogenic growth factor bFGF, and bone marrow-derived mesenchymal stem cells (MSCs). bFGF can be continuously released from the system for 4 weeks. The released bFGF significantly improved MSC survival and paracrine effects under low nutrient and oxygen conditions (0% FBS and 1% O2) in vitro. The prosurvival effect of the bFGF on MSCs was resulted from activating cell Kruppel-like factor 4 (KLF4) pathway. When transplanted into the ischemic limbs, the system dramatically improved MSC survival. Some of the engrafted cells were differentiated into skeletal muscle and endothelial cells, respectively. The system also promoted the proliferation of host cells. After only 2 weeks of implantation, tissue blood perfusion was completely recovered; and after 4 weeks, the muscle fiber diameter was restored similarly to that of the normal limbs. These pronounced results demonstrate that the developed stem cell delivery system has a potential for ischemic limb regeneration.

Statement of significance

Stem cell therapy is a promising strategy to restore blood perfusion and promote muscle regeneration in ischemic limbs. Yet its therapeutic efficacy remains low owing to the inferior cell survival under the ischemic environment of the injured limbs. To increase therapeutic efficacy, high rate of cell survival is essential, which current approaches do not support. In this work, we tested the hypothesis that a stem cell delivery system that can continuously release a prosurvival and proangiogenic growth factor will promote high rates of cell survival in the ischemic limbs. The prosurvival effect could augment cell survival before vascularization is established, while the proangiogenic effect could stimulate quick angiogenesis to achieve long-term cell survival. Meanwhile, the differentiation of stem cells into endothelial and myogenic lineages, and cell paracrine effects will enhance vascularization and muscle regeneration.

Section snippets

Introductions

Atherosclerotic peripheral artery disease (PAD) affects more than 27 million people in North America and Europe [1], [2] PAD decreases blood perfusion in the tissues and causes tissue ischemia. Critical limb ischemia (CLI) represents the most severe form of PAD. It is characterized by low blood perfusion, severe tissue ischemia, and degenerated skeletal muscle. Quick restoration of blood perfusion to salvage existing cells and promotion of muscle repair represent the optimal goals for CLI

Materials

All chemicals were purchased from Sigma–Aldrich unless otherwise stated. 2-hydroxyethyl methylmethacrylate (HEMA) was purchased from TCI and passed through an inhibitor remover column to eliminate inhibitor. N-isopropylacrylamide (NIPAAm, Alfa Aesar) was purified by recrystallization for 3 times using hexane. 3,6-Dimethyl-1,4-dioxane-2,5-dione, acryloyl chloride, sodium methoxide and chondroitin sulfate were used as received.

Hydrogel synthesis

The hydrogel was synthesized from NIPAAm, HEMA and a macromer based on

Bioactive bFGF can be gradually released from the hydrogel

The bFGF was able to continuously release from the hydrogel during the 28-day release period. The release exhibited a two-phase profile, i.e., an initial burst release in the first 5 days followed by a slower release until day 28 (Fig. 1A). The bioactivity of the released bFGF was evaluated in terms of its stimulatory effect on fibroblast growth. 1 ng/mL bFGF solution was used as a control as this concentration significantly stimulated fibroblast growth. The cells cultured in the release medium

Discussion

The objective of this work was to develop a stem cell delivery system that augments cell survival under ischemic conditions, thus enhancing ischemic limb regeneration. Ischemic limbs are characterized by a low nutrient and oxygen, and poorly vascularized environment. Inferior cell survival in this environment is one of the key causes that are responsible for the low therapeutic efficacy of stem cell therapy. Augmentation of cell survival under these harsh conditions represents a critical need

Conclusions

In this work, a stem cell delivery system capable of augmenting cell survival in ischemic limbs was created for quick recovery of blood perfusion and enhancing skeletal muscle regeneration. The delivery system was based on an injectable, biodegradable, and thermosensitive hydrogel, bFGF, and MSCs. When tested under low nutrient and oxygen conditions in vitro, the system significantly increased cell survival and paracrine effects. When transplanted in the ischemic limbs, the system not only

Acknowledgments

The assistance of Dr. Jianjie Ma’s group and Dr. Matthew Joseph is greatly appreciated. This work was supported by US National Science Foundation (1006734 and 1160122), American Heart Association (15GRNT25830058 and 13GRNT17150041), US National Institutes for Health (R01HL124122), National Science Foundation of China (81471788), and Institute for Materials Research seed grant at The Ohio State University.

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    These authors contributed equally to this work.

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