(10g) Engineering Cell Transplantation Vehicles for Cardiac Regeneration

Introduction.  Cardiovascular diseases are the main cause of death in developed countries.  Half of the patients diagnosed with heart failure die within three years and suffer from a reduction in their quality of life.  Myocardial infarction, a main cause of heart failure, leads to loss of cardiac tissue and impairment of left ventricular function.  The inadequate current treatments and the lack of donors for heart transplantation make cardiac tissue regeneration the most promising treatment for the patients suffering from severe heart failure.  For this purpose, cell therapy is considered nowadays one of the most optimistic approaches for impaired heart tissue reconstruction [1,2].  Direct injection of cell suspensions has been a focus of research during the last twenty years [3,4].  Even though autologous myoblast transplantation has been performed clinically and contraction of grafted myoblasts have been confirmed [5,6], survival and engraftment of transplanted stem cells is still too low to have a general therapeutic effect.  To overcome this problem, tissue engineering approaches have focused in the development of biodegradable cell-polymer systems as alternatives for a temporary extracellular matrix (ECM).  Some tissues have been engineered into poly(glycolic acid), gelatin, fibrin, alginate or collagen matrices [7,8].  Moreover, injectable scaffolds present additional advantages such as a less invasive methodology, the ability to adopt shape and form of the host environment that alternatively can deliver growth factors or biologically active compounds together with cells [9].  Nevertheless, the encapsulation of cells during polymerization into biodegradable polymer scaffolds attenuate cell-to-cell connections between cardiac myocytes.  These gap junctions are essential for the exchange of small molecules and ions that results in electrically synchronous beating [10].  Alternatively to these technologies, we now propose a novel strategy for cell transplantation that keeps cell-cell interactions while increasing homing and survival of injected cells.  This innovative approach involves the design of a smart cell delivery vehicle that efficiently encapsulates/delivers cells as a response to external stimulus.  By a traditional 2D cell culture, this device provides a biodegradable and injectable platform that keeps cell density and cell-cell connections.

The Technology.  Recently, a cell wrap technology has been developed at Philips Research [11], based on a foldable polymeric material.  The device consists of two polymeric layers coupled by a photopolymerization reaction.  The first one made up of a biodegradable stimuli responsive hydrogel and a second one of a non-responsive network that is optimized for adhesion and viability of cardiomyocytes and/or endothelial cells.  The application of an external stimulus results in the swelling or shrinking of the device, leading to a rolling/unrolling process (Figure 1) that is capable of protecting cells during injection and facilitating the delivery to the infarcted region of the myocardium.

Results and Discussion.  Foldable thermo responsive devices were successfully designed and fabricated based on poly(N-isopropylacrylamide) (PNIPAAm) and poly(ethylene glycol)-Poly(lactide) based block copolymer (PLA-b-PEG-b-PLA).  These microgels are tunable in size and composition and are fabricated by simple photolithographic techniques.

Figure 1.  Schematic depicting of the cell encapsulation procedure using 2D cell seeding techniques (left).  The experimental procedure is structured in four steps: (i) the hydrogels microdevices, called here flakes, are formed on a glass substrate and cells are seeded on top and incubated at 37ºC; (ii) when the substrate is at room temperature for a few minutes the flakes detach from the glass and (iii) curl in a preferred orientation, allowing their injection (iv).  On the right side an optical (a) and fluorescence (b) image of the resulting encapsulated neonatal rat cardiac myocytes.  The hydrogel shown here is 400 μm in diameter and 1.5 mm in legth.  An exposure dose of 2 mW/cm² UV Light at 365 nm wavelength is used for all hydrogel flakes shown here.

As displayed in Figure 1, the use of these materials provides a flat bioactive substrate that facilitates traditional 2D cell culture.  Poly(NIPAAm) is characterized by a lower critical solution temperature (LCST) around 32ºC that affords the swelling and encapsulation of attached cells at room temperature.  The resulting ?cell wrap? protects cells during catheter based delivery to the infarcted region, releasing them in vivo once injected under physiological conditions (over LCST) and exposing the transplanted cells to the native cardiac environment.

Cytotoxicity of the degradation products was tested against neonatal rat cardiomyocytes and human endothelial cells, showing no significant decrease of proliferation compared to control.  Our hydrogels efficiently encapsulated HUVEC, H9C2 cardiomyocytes and neonatal rat primary cardiac cells.  The device environment preserved cell viability and function, showing great survivability and proliferation for more than a month.

Since these substrates provide a temporary ECM to protect cells during injection and engraftment in the native tissue, they have to be cleared out the body.  Our cell vehicles were specially designed to degrade in less than 30 days, displaying diverse degradation profiles in order to adapt them to the clinical needs.  The use of a PEG ? PLA block copolymer as a crosslinker makes these networks susceptible to hydrolysis.  Diacrylate PLA-b-PEG-b-PLA degrades through the isotropic hydrolysis of ester bonds in the PLA block, breaking crosslinks within the gel and removing the degradation products from the network by diffusive transport [12,13].  The regulation of the degradation profile was achieved by using chain transfer agents that control the molecular weight of the polyNIPAAm chains and therefore its diffusion rate from the network.  The degradation of the microdevices was monitored by fluorescence microscopy by using a fluorescent probe covalently attached to the network.  Hydrogels described here reach complete dissolution in a period of 20 to 60 days depending of the composition.

Conclusion.  Smart biomaterials that function as cell carriers can be fabricated through photolithography by using a thermo sensitive polymer.  They provide a mechanically stable substrate for 2D culture and injection in a minimally invasive manner.  Biological studies showed that the materials were not cytotoxic to a range of cell types and that encapsulated primary rat cardiomyocytes remain viable after more than 30 days.  Additionally, the change of the thiol mole fraction in the network provides control over the final degradation product's molecular weight distribution and the weight loss profiles.  These devices are of considerable interest for fundamental materials science investigations and applied studies in stem cell transplantation as well as cardiac tissue regeneration.

Acknowledgements.  This research forms part of the Project P1.04 SMARTCARE of the research program of the BioMedical Materials institute, co-funded by the Dutch Ministry of Economic Affairs.  The financial contribution of the Nederlandse Hartstichting is gratefully acknowledged.

References

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