Hydrogels as a Platform for Stem Cell Delivery to the Heart

Abstract

Stem cell therapy offers great promise to repair the injured or failing heart. The outcomes of clinical trials to date, however, have shown that the actual benefit realized falls far short of the promise. A number of factors may explain why that is the case, but poor stem cell retention and engraftment in the hostile environment of the injured heart would seem to be a major factor. Improving stem cell retention and longevity once delivered would seem a logical means to enhance their reparative function. One way to accomplish this goal may be injectable hydrogels, which would serve to fix stem cells in place while providing a sheltering environment. Hydrogels also provide a means to allow for the paracrine factors produced by encapsulated stem cells to diffuse into the injured myocardium. Alternatively, hydrogels themselves can be used for the sustained delivery of reparative factors. Here we discuss chitosan-based hydrogels.

The idea that stem cells, in addition to forming new tissue, can be employed as a drug depot for the sustained release of therapeutic biomolecules or paracrine factors in an injured tissue is a new and exciting direction for pharmacology.¹ Two challenges that need to be overcome for this to be a reality are recreating a 3-D environment that resembles natural extracellular matrices and providing protection against hostile extrinsic factors, while permitting the free passage of the therapeutic biomolecules. In addition, any manipulation should not preclude the possible engraftment or differentiation of the stem cells. Hydrogels are being commonly employed in this endeavor, in bone, cartilage, and skeletal bioengineering, as well as corneal, wound, or ulcer repair. However, very little research in this regard has involved the heart. A PubMed search (January 14, 2010) using the terms “hydrogel AND stem cell AND (heart OR cardiac)” found only 23 publications, most of which reported on the favorable effects of hydrogels on the in vitro growth, differentiation, or regulation of embedded cardiac myocytes, myofibroblasts, endothelial progenitor cells, or HL-1 cardiomyocytes (Fig. 1). Nearly half of the publications appeared in the last year. Overall only 5 dealt with the utility of this approach for repairing the injured heart with stem cells.^2–6 These studies report enhanced survivability of stem cells embedded in hydrogels in the infarcted heart along with enhanced improvements in cardiac function and remodeling (wall thinning, new vessel formation and scar reduction).

Figure 1.

Published articles dealing with both hydrogels and stem cells in the context of the heart. A PubMed search was performed (1/14/10) using “hydrogel AND stem cell AND (heart OR cardiac)”. Results are shown from the oldest publication in 1989 to the present. For those intervening years not shown, no publications were found.

Both natural (e.g., collagen, gelatin, and chitosan) and synthetic polymers can be used to generate thermosensitive solutions that transform into hydrogels when injected in the body. Such gels are particularly useful in tissue engineering because they are thermosensitive at body temperature, meaning they undergo gelation at 37°C.^7,8 One example of this type of thermosensitive hydrogel consists of a mixture of the polysaccharide chitosan and β-glycerophosphate (β-GP).⁷ Biodegradability of the chitosan-GP hydrogel can be fine tuned by modifying the polysaccharide structure. For example, gels formed using highly deacetylated chitosan (≥ 90%), are essentially non-biodegradable; those made from chitosan that is 75–90% deacetylated, e.g., Protasan UP CL 113 (NovaMatrix), are biodegradable, but can be long-lived in vivo when chemical cross-links are generated in the matrix.^9,10 Another attractive feature of chitosan-based hydrogels for in vivo work is their biocompatibility. The bulking agent, hydroxyethyl-cellulose, helps protect cells during gel formation and, if it is of commercial grade quality rather than being ultrapure, provides the crosslinking agent glyoxal (Fig. 2).⁹ This hydrogel platform, which is easily constructed and manipulated, gels at body temperature, allowing for cardiac delivery of stem cells via intramyocardial injection or a Cricket^™ micro-infusion catheter. Once formed, the hydrogels are stable as can be assessed by their constant weight over several days in culture (Fig. 3). Examples of such gels, cast at a relatively large volume (0.75 mL) compared to the size that would be placed into the heart are pictured in Figure 4. As can be seen from the pink-tinted border arising from the diffusion of phenol red in the culture medium into the hydrogel, the aqueous phase of these hydrogels “communicates” with the surrounding milieu. The gels are exudative in that they can be designed to slowly release certain factors over time, such as the angiogenic factor vascular endothelial growth factor (VEGF; Fig. 5).

Figure 2.

Schematic showing one protocol for forming a chitosan-based hydrogel. A highly deacetylated and high-purity chitosan (Protasan) is used. A 1 hour wait is needed after mixing Protasan with β-glycerophosphate (β-GP) to allow microbubbles time to dissipate. The size of the boxes depicting the gel components are proportional to the volume ratios that are used.

Figure 3.

Hydrogel shrinkage assay. After gelation at 37°C, Protasan-based hydrogels were washed 3 times for 5 minutes each with culture medium and weighed (0 Min). Hydrogels were incubated at 37°C in 2 mL of culture medium and reweighed after 80 minutes. The hydrogels were then incubated overnight in 2 mL of culture medium. To study gel shrinkage, the weight of the hydrogel was measured after carefully removing the medium. Afterwards new medium was added. The findings indicate that the gels once formed are stable in culture following an initial shrinkage due to the tightening of associations among the neutralized Protasan polymer following efflux of excess β-GP (as previously shown by Hoemann et al.⁹). Values shown are mean ± SEM of 3 independent experiments each performed on 4 replicates.

Figure 4.

Protasan-based hydrogels plated in a 6-well cell culture plate and incubated overnight at 37°C. Those in the upper row were not incubated with cell culture medium; those in the lower row were and show uptake of phenol red, as indicated by the pink tint around the periphery.

Figure 5.

Time course of VEGF release from Protasan-based hydrogels. VEGF₁₆₅ (1.25 ng/mL; Millipore) was added to the hydroxyethyl-cellulose prior to adding it to the Protasan – β-glycerophosphate mixture. Once the hydrogels formed, the medium was sampled at 1, 2, 4, 6, and 20 hours (Day 1). New medium was added, and samples taken at 1, 2, 4, and 6 hours (Day 2). VEGF in the medium was assayed by ELISA and expressed as a percentage of the initial VEGF hydrogel content. Values are mean ± SD for 4 replicates. A single experiment was performed.

Others have reported VEGF diffusion from other types of hydrogels such as those made with alginate or hyaluronan;^11,12 the chitosan-hydrogel would seem to have slower release kinetics compared to the latter. The Hubbell lab has also designed a presolidified hydrogel that has a plasmin-dependent cleavage site between the VEGF and matrix.¹³ Other implantable hydrogels have been developed for angiogenic factor release;¹¹ however, injectable in situ-solidifying gels have the advantage of minimal invasiveness which is important when you need to avoid disrupting tissue function for example, in already damaged cardiac tissue. Interestingly, bone marrow-derived mesenchymal stem cells were recently reported to constitutively release high quantities of VEGF in monolayer culture,^14,15 which opens the possibility that in situ VEGF release could be also achieved through delivery of hydrogel-encapsulated stem cells. But VEGF delivery alone may be ineffective in cardiac repair as suggested by the negative outcome on myocardial perfusion, exercise treadmill time, and anginal symptoms in the NOGA angiogenesis Revascularization Therapy: assessment by RadioNuclide imaging (NORTHERN) trial, in which patients with refractory Canadian Cardiovascular Society (CCS) class 3 or 4 anginal symptoms received either VEGF plasmid DNA or placebo.¹⁶ A recent animal study would indicate that combined stem cell, chemokine, and angiogenic growth factor gene therapy may offer an effective approach.¹⁷ Using a rat model, it was observed that intramyocardial injection one week post-MI of mesenchymal stem cells transduced with an adenovirus expressing stromal cell-derived factor-1 (SDF-1), a chemotactic factor for stem cell recruitment, as well as VEGF, reduced infarct size and fibrosis, increased vascular density, left ventricle wall thickness, and left ventricular function assessed one month later.

Incontrovertible evidence shows that the heart, like many other organs and tissues, possesses niches that harbor stem cells.¹⁸ These stem cells may not only be endogenous to the heart, but could be from the circulation and bone marrow-derived.^19,20 For the latter, niches may serve as “way stations” or temporary repositories. For both types of stem cells, the niches likely serve to protect and acclimatize the stem cells to the heart environment through paracrine means, as well as direct communication with mature heart cells or possibly nurse cells via connexins.¹⁸ As knowledge of these niches advances, hydrogel design can be specifically optimized to provide a better nurturing environment for stem cells in the heart.

Summary

The successful design of a protective scaffold for stem cells when placed into diseased tissues will have a major impact on their use in all areas of regenerative medicine affecting millions of individuals. Moreover, demonstrating that the harboring environment can be manipulated so as to regulate stem cell behavior and improve their reparative functions, without any genetic manipulation and the consequent oncogenic concerns, will open up a new area of pharmaceutical research and lead to a broader therapeutic use of stem cells.