Articular cartilage regeneration using acellular bioactive affinity-binding alginate hydrogel: A 6-month study in a mini-pig model of osteochondral defects.

BACKGROUND: Despite intensive research, regeneration of articular cartilage largely remains an unresolved medical concern as the clinically available modalities still suffer from long-term inconsistent data, relatively high failure rates and high prices of more promising approaches, such as cell therapy. In the present study, we aimed to evaluate the feasibility and long-term efficacy of a bilayered injectable acellular affinity-binding alginate hydrogel in a large animal model of osteochondral defects. METHODS: The affinity-binding alginate hydrogel is designed for presentation and slow release of chondrogenic and osteogenic inducers (transforming growth factor-ß1 and bone morphogenic protein 4, respectively) in two distinct and separate hydrogel layers. The hydrogel was injected into the osteochondral defects created in the femoral medial condyle in mini-pigs, and various outcomes were evaluated after 6 months. RESULTS: Macroscopical and histological assessment of the defects treated with growth factor affinity-bound hydrogel showed effective reconstruction of articular cartilage layer, with major features of hyaline tissue, such as a glossy surface and cellular organisation, associated with marked deposition of proteoglycans and type II collagen. Microcomputed tomography showed incomplete bone formation in both treatment groups, which was nevertheless augmented by the presence of affinity-bound growth factors. Importantly, the physical nature of the applied hydrogel ensured its shear resistance, seamless integration and topographical matching to the surroundings and opposing articulating surface. CONCLUSIONS: The treatment with acellular injectable growth factor-loaded affinity-binding alginate hydrogel resulted in effective tissue restoration with major hallmarks of hyaline cartilage, shown in large animal model after 6-month follow-up. THE TRANSLATIONAL POTENTIAL OF THIS ARTICLE: This proof-of-concept study in a clinically relevant large animal model showed promising potential of an injectable acellular growth factor-loaded affinity-binding alginate hydrogel for effective repair and regeneration of articular hyaline cartilage, representing a strong candidate for future clinical development.

Live imaging flow bioreactor for the simulation of articular cartilage regeneration after treatment with bioactive hydrogel.

Osteochondral defects (OCDs) are conditions affecting both cartilage and the underlying bone. Since cartilage is not spontaneously regenerated, our group has recently developed a strategy of injecting bioactive alginate hydrogel into the defect for promoting endogenous regeneration of cartilage via presentation of affinity-bound transforming growth factor ß1 (TGF-ß1). As in vivo model systems often provide only limited insights as for the mechanism behind regeneration processes, here we describe a novel flow bioreactor for the in vitro modeling of the OCD microenvironment, designed to promote cell recruitment from the simulated bone marrow compartment into the hydrogel, under physiological flow conditions. Computational fluid dynamics modeling confirmed that the bioreactor operates in a relevant slow-flowing regime. Using a chemotaxis assay, it was shown that TGF-ß1 does not affect human mesenchymal stem cell (hMSC) chemotaxis in 2D culture. Accessible through live imaging, the bioreactor enabled monitoring and discrimination between erosion rates and profiles of different alginate hydrogel compositions, using green fluorescent protein-expressing cells. Mathematical modeling of the erosion front progress kinetics predicted the erosion rate in the bioreactor up to 7 days postoperation. Using quantitative real-time polymerase chain reaction of early chondrogenic markers, the onset of chondrogenic differentiation in hMSCs was detected after 7 days in the bioreactor. In conclusion, the designed bioreactor presents multiple attributes, making it an optimal device for mechanistical studies, serving as an investigational tool for the screening of other biomaterial-based, tissue engineering strategies.

Mechanisms of cellular uptake and endosomal escape of calcium-siRNA nanocomplexes.

Ca2+-siRNA nanocomplexes represent a simple yet an effective platform for siRNA delivery into the cell cytoplasm, with subsequent successful siRNA-induced target gene silencing. Herein, we aimed to elucidate the roles played by calcium ions in siRNA nanocomplex formation, cell uptake, and endosomal escape. We investigated whether the replacement of Ca2+in the nanocomplex by other bivalent cations would affect their cell entry and subsequent gene silencing. Our results indicate that Mg2+ and Ba2+ lead to the formation of nanocomplexes of similar physical features (size=100nm, surface charge ζ=-8mV) as the Ca2+-siRNA nanocomplexes. Yet, these nanocomplexes were not uptaken by the cells to the same extent as those prepared with Ca2+, and siRNA-induced target gene silencing was not obtained. Cell internalization of Ca2+--siRNA nanocomplexes, examined by employing chemical inhibitors to clathrin-, caveolin- and dynamin-mediated endocytosis pathways, indicated the involvement of all mechanisms in the process. Inhibition of endosome acidification by bafilomycin completely abolished the siRNA-mediated silencing by Ca2+-siRNA nanocomplexes. Collectively, our results indicate that Ca2+ promotes cell internalization and rapid endosomal escape, thus leading to the efficient siRNA-induced target gene silencing elicited by the Ca2+-siRNA nanocomplexes.

TGF-ß affinity-bound to a macroporous alginate scaffold generates local and peripheral immunotolerant responses and improves allocell transplantation.

Enhancing vascularization of cell-transplantation devices is necessary for maintaining cell viability and integration within the host, but it also increases the risk of allograft rejection. Here, we investigated the feasibility of generating an immunoregulatory environment in a highly vascularized macroporous alginate scaffold by affinity-binding of the transforming growth factor-ß (TGF-ß) in a manner mimicking its binding to heparan sulfate. Using this device to transplant allofibroblasts under the kidney capsule resulted in the induction of local and peripheral TGF-ß-dependent immunotolerance, characterized by higher frequency of immature dendritic cells and regulatory T cells within the device and by markedly reduced allofibroblast-specific T-cell response in the spleen, thereby increasing the viability of the transplanted cells. Culturing whole splenocytes in the TGF-ß-bound scaffold indicated that the regulatory function of TGF-ß is IL-10-dependent. We thus demonstrate a novel platform for transplantation devices, designed to promote an immunoregulatory microenvironment suitable for cell transplantation and autoimmune regulation. STATEMENT OF SIGNIFICANCE: Allogeneic cell graft transplantation is a potentially optimal treatment for many clinical deficiencies. It is yet challenging to overcome chronic rejection without compromising host immunity to pathogens. We present the features and function of a cell transplantation device designed based on the principle of affinity binding of angiogenic and immunoregulatory factors to extracellular matrix in aim to achieve sustained release of these factors. We show that presentation of these factors in such manner generates the infrastructure for device vascularization and induces profound local allocell-specific tolerance, which then evokes peripheral T-cell tolerance. The tolerance is antigen specific, does not cause immune deficits and may thus serve to improve allocell survival as well as a platform to mitigate pathogenic autoimmunity.

A bridge to silencing: Co-assembling anionic nanoparticles of siRNA and hyaluronan sulfate via calcium ion bridges.

Therapeutic implementation of RNA interference (RNAi) through delivery of short interfering RNA (siRNA) is still facing several critical hurdles, which mostly can be solved through the use of an efficient delivery system. We hereby introduce anionic siRNA nanoparticles (NPs) co-assembled by the electrostatic interactions of the semi-synthetic polysaccharide hyaluronan-sulfate (HAS), with siRNA, mediated by calcium ion bridges. The NPs have an average size of 130nm and a mild (-10mV) negative surface charge. Transmission electron microscopy (TEM) using gold-labeled components and X-ray photoelectron spectroscopy (XPS) demonstrated the spatial organization of siRNA molecules in the particle core, surrounded by a layer of HAS. The anionic NPs efficiently encapsulated siRNA, were stable in physiological-relevant environments and were cytocompatible, not affecting cell viability or homeostasis. Efficient cellular uptake of the anionic siRNA NPs, associated with potent gene silencing (>80%), was observed across multiple cell types, including murine primary peritoneal macrophages and human hepatocellular carcinoma cells. In a clinically-relevant model of acute inflammatory response in IL-6-stimulated human hepatocytes, STAT3 silencing induced by HAS-Ca(2+)-siRNA NPs resulted in marked decrease in the total and activated STAT3 protein levels, as well as in the expression levels of downstream acute phase response genes. Collectively, anionic NPs prove to be an efficient and cytocompatible delivery system for siRNA.

Spontaneous Coassembly of Biologically Active Nanoparticles via Affinity Binding of Heparin-Binding Proteins to Alginate-Sulfate.

Controlled delivery of heparin-binding (HB) proteins represents a challenge in regenerative medicine strategies. Here, we describe the features of novel nanoparticles (NPs), spontaneously coassembled due to affinity interactions between HB proteins and the semisynthetic anionic polysaccharide, alginate-sulfate. The NPs efficiently encapsulated and protected the proteins from proteolysis. Injection of a combination of NPs encapsulating multiple therapeutic growth factors promoted effective and long-term tissue repair in animal models of severe ischemia (murine model of hindlimb ischemia and acute myocardial infarction in rats). This simple yet efficient NP fabrication method is amenable for clinical use.

Alginate biomaterial for the treatment of myocardial infarction: Progress, translational strategies, and clinical outlook: From ocean algae to patient bedside.

Alginate biomaterial is widely utilized for tissue engineering and regeneration due to its biocompatibility, non-thrombogenic nature, mild and physical gelation process, and the resemblance of its hydrogel matrix texture and stiffness to that of the extracellular matrix. In this review, we describe the versatile biomedical applications of alginate, from its use as a supporting cardiac implant in patients after acute myocardial infarction (MI) to its employment as a vehicle for stem cell delivery and for the controlled delivery and presentation of multiple combinations of bioactive molecules and regenerative factors into the heart. Preclinical and first-in-man clinical trials are described in details, showing the therapeutic potential of injectable acellular alginate implants to inhibit the damaging processes after MI, leading to myocardial repair and tissue reconstruction.

Calcium-siRNA nanocomplexes: what reversibility is all about.

Gene silencing using small interfering RNA (siRNA) relies on the critical need for a safe and effective carrier, capable of strong but reversible complexation, siRNA protection, cellular uptake, and cytoplasmatic unloading of its cargo. We hypothesized that a delivery platform based on the eletrostatic interactions of siRNA with calcium ions in solution would fulfill these needs, ultimately leading to effective gene silencing. Physical characterization of the calcium-siRNA complexes, using high resolution microscopy and dynamic light scattering (DLS), showed the formation of stable nanosized complexes ~80nm in diameter, bearing mild (~-7mV) negative surface charge. The complexes were extremely stable in the presence of serum proteins or high concentrations of heparin; they maintained their nanosized features in suspension for days; and effectively protected the siRNA from enzymatic degradation. The Ca-siRNA complexes were disintegrated in the presence of Ca-chelating ion exchange resin, thus proving their reversibility. Excellent cytocompatibility of calcium-siRNA complexes was achieved using physiological calcium ion concentrations. The calcium-siRNA complexes successfully induced a very high (~80%) level of gene silencing in several cell types, at both mRNA and protein levels, associated with efficient cellular uptake. Collectively, our results show that the developed delivery platform based on reversible calcium-siRNA interactions offers a simple and versatile method for enhancing the therapeutic efficiency of siRNA.

Rapid End-Group Modification of Polysaccharides for Biomaterial Applications in Regenerative Medicine.

Polysaccharides have emerged as important functional materials because of their unique properties such as biocompatibility, biodegradability, and availability of reactive sites for chemical modifications to optimize their properties. The overwhelming majority of the methods to modify polysaccharides employ random chemical modifications, which often improve certain properties while compromising others. On the other hand, the employed methods for selective modifications often require excess of coupling partners, long reaction times and are limited in their scope and wide applicability. To circumvent these drawbacks, aniline-catalyzed oxime formation is developed for selective modification of a variety of polysaccharides through their reducing end. Notably, it is found that for efficient oxime formation, different conditions are required depending on the composition of the specific polysaccharide. It is also shown how our strategy can be applied to improve the physical and functional properties of alginate hydrogels, which are widely used in tissue engineering and regenerative medicine applications. While the randomly and selectively modified alginate exhibits similar viscoelastic properties, the latter forms significantly more stable hydrogel and superior cell adhesive and functional properties. Our results show that the developed conjugation reaction is robust and should open new opportunities for preparing polysaccharide-based functional materials with unique properties.

Magnetically actuated alginate scaffold: a novel platform for promoting tissue organization and vascularization.

Among the greatest hurdles hindering the successful implementation of tissue-engineered cardiac patch as a therapeutic strategy for myocardial repair is the know-how to promote its rapid integration into the host. We previously demonstrated that prevascularization of the engineered cardiac patch improves cardiac repair after myocardial infarction (MI); the mature vessel networks were generated by including affinity-bound angiogenic factors in the patch and its transplantation on the blood vessel-enriched omentum. Here, we describe a novel in vitro strategy to promote the formation of capillary-like networks in cell constructs without supplementing with angiogenic factors. Endothelial cells (ECs) were seeded into macroporous alginate scaffolds impregnated with magnetically responsive nanoparticles (MNPs), and after pre-culture for 24 h under standard conditions the constructs were subjected to an alternating magnetic field of 40 Hz for 7 days. The magnetic stimulation per se promoted EC organization into capillary-like structures with no supplementation of angiogenic factors; in the non-stimulated constructs, the cells formed sheets or aggregates. This chapter describes in detail the preparation method of the MNP-impregnated alginate scaffold, the cultivation setup for the cell construct under magnetic field conditions, and the set of analyses performed to characterize the resultant cell constructs.

Three-dimensional perfusion cultivation of human cardiac-derived progenitors facilitates their expansion while maintaining progenitor state.

The therapeutic application of autologous cardiac-derived progenitor cells (CPCs) requires a large cell quantity generated under defined conditions. Herein, we investigated the applicability of a three-dimensional (3D) perfusion cultivation system to facilitate the expansion of CPCs harvested from human heart biopsies and characterized by a relatively high percentage of c-kit(+) cells. The cells were seeded in macroporous alginate scaffolds and after cultivation for 7 days under static conditions, some of the constructs were transferred into a perfusion bioreactor, which was operated for an additional 14 days. A robust and highly reproducible human CPC (hCPC) expansion of more than seven-fold was achieved under the 3D perfusion culture conditions, while under static conditions, the expansion of CPCs was limited only to the first 7 days, after which it leveled-off. On day 21 of perfusion cultivation, the expanded cells exhibited a higher expression level of the progenitor marker c-kit, suggesting that the c-kit-positive CPCs are the main cell population undergoing proliferation. The profile of the spontaneous differentiation in the perfused construct was different from that in the static cultivated constructs; genes typical for cardiac and endothelial cell lineages were more widely expressed in the perfused constructs. By contrast, the differentiation to osteogenic (Von Kossa staining and alkaline phosphatase activity) and adipogenic (Oil Red staining) lineages was reduced in the perfused constructs compared with static cultivated constructs. Collectively, our results indicate that 3D perfusion cultivation mode is an appropriate system for robust expansion of human CPCs while maintaining their progenitor state and differentiation potential into the cardiovascular cell lineages.

Microfabrication of channel arrays promotes vessel-like network formation in cardiac cell construct and vascularization in vivo.

Pre-vascularization is important for the reconstruction of dense and metabolically active myocardial tissue and its integration with the host myocardium after implantation. Herein, we demonstrate that the fabrication of micro-channels in alginate scaffold combined with the presentation of adhesion peptides and an angiogenic growth factor promote vessel-like networks in the construct, both in vitro and in vivo. Using a CO2 laser engraving system, 200 µm diameter channels were formed from top to bottom of the 2 mm thick alginate scaffold, with a channel-to-channel distance of 400 µm. Cells were seeded in a sequential manner onto the scaffolds: first, human umbilical vascular endothelial cells (HUVECs) were seeded and cultured for three days, then neonatal rat cardiomyocytes (CMs) and cardiofibroblasts were added at a final cell ratio of 50:35:15, respectively, and the constructs were cultivated for an additional seven days. A vessel-like network was formed within the cell constructs, wherein HUVECs were organized around the channels in a multilayer manner, while the CMs were located in-between the channels and exhibited the characteristic morphological features of a mature cardiac fiber. Acellular scaffolds with the affinity-bound basic fibroblast growth factor were implanted subcutaneously in mice. Increased cell penetration into the channeled scaffold and greater vessel density were found in comparison with the nonchanneled scaffolds. Our results thus point to the importance of micro-channels as a major structural promoter of vascularization in scaffolds, in conjunction with the sequential preculture of ECs and angiogenic factor presentation.

A multi-shear perfusion bioreactor for investigating shear stress effects in endothelial cell constructs.

Tissue engineering research is increasingly relying on the use of advanced cultivation technologies that provide rigorously-controlled cell microenvironments. Herein, we describe the features of a micro-fabricated Multi-Shear Perfusion Bioreactor (MSPB) designed to deliver up to six different levels of physiologically-relevant shear stresses (1-13 dyne cm(-2)) to six cell constructs simultaneously, during a single run. To attain a homogeneous fluid flow within each construct, flow-distributing nets photo-etched with a set of openings for fluid flow were placed up- and down-stream from each construct. Human umbilical vein endothelial cells (HUVECs) seeded in alginate scaffolds within the MSPB and subjected to three different levels of shear stress for 24 h, responded accordingly by expressing three different levels of the membranal marker Intercellular Adhesion Molecule 1 (ICAM-1) and the phosphorylated endothelial nitric oxide synthetase (eNOS). A longer period of cultivation, 17 d, under two different levels of shear stress resulted in different lengths of cell sprouts within the constructs. Collectively, the HUVEC behaviour within the different constructs confirms the feasibility of using the MSPB system for simultaneously imposing different shear stress levels, and for validating the flow regime in the bioreactor vessel as assessed by the computational fluid dynamic (CFD) model.

Simultaneous regeneration of articular cartilage and subchondral bone induced by spatially presented TGF-beta and BMP-4 in a bilayer affinity binding system.

Subchondral defect repair is a multitask challenge requiring the simultaneous regeneration of cartilage and bone. Herein, we describe the features of a hydrogel system designed to simultaneously induce the endogenous regeneration of hyaline cartilage and subchondral bone. The system was constructed as two layers, spatially presenting the chondroinductive transforming growth factor-ß1 (TGF-ß1) in one layer and the osteoinductive bone morphogenetic protein-4 (BMP-4) in a second layer, via affinity binding to the matrix. Human mesenchymal stem cells seeded in the bilayer system differentiated into chondrocytes and osteoblasts in the respective layers, confirming the spatial presentation and prolonged activity of TGF-ß1 and BMP-4. Administration of the bilayer system with affinity-bound TGF-ß1 and BMP-4 (with no cells) into a subchondral defect in rabbits induced endogenous regeneration of articular cartilage and the subchondral bone underneath within 4weeks. Cartilage extracellular matrix proteoglycans were found in the top layer, with no mineralization, whereas the layer underneath consisted of newly formed woven bone. The results indicate that stem cells migrating into the defect are able to sense the biological cues spatially presented in the hydrogel and respond by differentiation into the appropriate cell lineage. The strategy has a real translational potential for repairing osteochondral defects in humans as it is acellular and can be implanted via a minimally invasive method.

Chondrogenesis of hMSC in affinity-bound TGF-beta scaffolds.

Herein we describe a bio-inspired, affinity binding alginate-sulfate scaffold, designed for the presentation and sustained release of transforming growth factor beta 1 (TGF-ß1), and examine its effects on the chondrogenesis of human mesenchymal stem cells (hMSCs). When attached to matrix via affinity interactions with alginate sulfate, TGF-ß1 loading was significantly greater and its initial release from the scaffold was attenuated compared to its burst release (>90%) from scaffolds lacking alginate-sulfate. The sustained TGF-ß1 release was further supported by the prolonged activation (14 d) of Smad-dependent (Smad2) and Smad-independent (ERK1/2) signaling pathways in the seeded hMSCs. Such presentation of TGF-ß1 led to hMSC chondrogenic differentiation; differentiated chondrocytes with deposited collagen type II were seen within three weeks of in vitro hMSC seeding. By contrast, in scaffolds lacking alginate-sulfate, the effect of TGF-ß1 was short-term and hMSCs could not reach a similar differentiation degree. When hMSC constructs were subcutaneously implanted in nude mice, chondrocytes with deposited type II collagen and aggrecan typical of the articular cartilage were found in the TGF-ß1 affinity-bound constructs. Our results highlight the fundamental importance of appropriate factor presentation to its biological activity, namely - inducing efficient stem cell differentiation.

Bioengineering the infarcted heart by applying bio-inspired materials.

Induction of cardiac muscle regeneration following myocardial infarction (MI) represents a major challenge in cardiovascular therapy, as the current clinical approaches are limited in their ability to regenerate a new muscle tissue and to replace infarcted myocardium. Here, we describe the conception of two strategies based on bio-inspired materials, aimed at myocardial repair after MI. In the first strategy, alginate biomaterial was designed with affinity-binding moieties, enabling the binding of heparin-binding proteins and their controlled presentation and release. The combined features of this unique alginate hydrogel, as a temporary extracellular matrix replacement and a depot for bio-molecules such as insulin-like growth factor-1 and hepatocyte growth factor, led to improvements in cardiac structure and function, as demonstrated by the biomaterial's abilities to thicken the scar and prevent left-ventricular remodeling and dilatation. Endogenous regeneration occurring at the infarct as manifested by the enhanced angiogenesis, cardiomyocyte proliferation, and appearance of cardiac-related stem cells is likely to have contributed to this. In the second strategy, phosphatidylserine (PS)-presenting liposomes were developed to mimic apoptotic cells bodies, specifically their capability of immunomodulating activated macrophages into anti-inflammatory state. In a rat model of acute MI, targeting of PS-presenting liposomes to infarct macrophages after injection via the femoral vein was demonstrated by magnetic resonance imaging. The treatment promoted angiogenesis, the preservation of small scars, and prevention of ventricular dilatation and remodeling. Collectively, the two bio-inspired material-based strategies presented herein represent unique and clinical accessible approaches for myocardial infarct repair.

The promotion of myocardial repair by the sequential delivery of IGF-1 and HGF from an injectable alginate biomaterial in a model of acute myocardial infarction.

Proper spatio-temporal delivery of multiple therapeutic proteins represents a major challenge in therapy strategies aimed at inducing myocardial regeneration after myocardial infarction (MI). We hypothesized that the dual delivery of insulin-like growth factor-1 (IGF-1) and hepatocyte growth factor (HGF) by injectable affinity-binding alginate biomaterial would maximize their therapeutic effects, leading to a more favorable course of tissue restoration after acute MI. A sequential release of IGF-1 followed by HGF was attained from affinity-binding alginate biomaterial, which also protected the proteins from proteolysis (shown by mass spectroscopy). The released factors retained bioactivity, as judged by their capability to activate their respective signaling pathways and to prevent cardiomyocyte apoptosis in vitro. In a rat model of acute MI, an intramyocardial injection of the dual IGF-1/HGF affinity-bound alginate biomaterial preserved scar thickness, attenuated infarct expansion and reduced scar fibrosis after 4 weeks, concomitantly with increased angiogenesis and mature blood vessel formation at the infarct. Furthermore, this treatment prevented cell apoptosis, induced cardiomyocyte cell cycle re-entry and increased the incidence of GATA-4-positive cell clusters. The dual delivery of IGF-1 and HGF from affinity-binding alginate biomaterial represents a useful strategy to treat MI. It showed a marked therapeutic efficacy at various tissue levels, as well as potential to induce endogenous regeneration of cardiac muscle.

Highly efficient osteogenic differentiation of human mesenchymal stem cells by eradication of STAT3 signaling.

Human bone marrow-derived mesenchymal stem cells (hMSCs) are promising candidates for cellular therapy owing to their multipotency to differentiate into several cell lineages. Elucidating the signaling events involved in the response of hMSCs to diverse stimulants affecting their differentiation may considerably promote their clinical use. In this study, we attempted to illuminate the molecular signaling networks involved in bone morphogenetic protein (BMP)-stimulated hMSC osteogenic differentiation. We demonstrate that eradication of signal transducers and activators of transcription (STAT) signaling considerably enhances BMP-induced osteogenic differentiation of hMSCs. BMP 2 and 4 are shown for the first time to activate the Janus-activated kinase (JAK)-STAT pathway in hMSC. Specifically, we reveal that JAK2 mediates STAT3 tyrosine phosphorylation in response to the two BMPs, whereas BMP2- and BMP4-induced STAT3 serine phosphorylation involves two divergent cascades, namely the mTOR and ERK1/2 cascades, respectively. Furthermore, elimination of the STAT3 signaling pathway by the inhibitors, AG490 or STAT3 siRNA, results in the acceleration and augmentation of BMPs-induced osteogenic differentiation, thus proposing a role for JAK-STAT signaling as a negative regulator of this process in MSCs. We believe that the findings presented in this study may be the basis for the development of a useful strategy to better control stem cell fate through intervention in molecular signaling networks. Hopefully, such a strategy will include the development of more efficient and controllable protocols for hMSC differentiation and facilitate their use in regenerative medicine.

Electric field stimulation integrated into perfusion bioreactor for cardiac tissue engineering.

We describe herein the features of a novel cultivation system, combining electrical stimulation with medium perfusion for producing thick, functional cardiac patches. A custom-made electrical stimulator was integrated via inserting two carbon rod electrodes into a perfusion bioreactor, housing multiple neonatal Sprague-Dawley rat cardiac cell constructs between two 96% open-pore-area fixing nets. The stimulator produced adjustable stimulation waveform (i.e., duty cycle, number of stimulating channels, maximum stimulation amplitude, etc.), specially designed for cardiac cell stimulation. The cell constructs were subjected to a homogenous fluid flow regime and electrical stimulation under conditions optimal for cell excitation. The stimulation threshold in the bioreactor was set by first determining its value in a Petri dish under a microscope, and then matching the current density in the two cultivation systems by constructing electric field models. The models were built by Comsol Multiphysics software using the exact three-dimensional geometry of the two cultivation systems. These models illustrate, for the first time, the local electric conditions required for cardiomyocyte field excitation and they confirmed the uniformity of the electrical field around the cell constructs. Bioreactor cultivation for only 4 days under perfusion and continuous electrical stimulus (74.4 mA/cm², 2 ms, bipolar, 1 Hz) promoted cell elongation and striation in the cell constructs and enhanced the expression level of Connexin-43, the gap junction protein responsible for cell-cell coupling. These results thus confirm the validity of the electrical field model in predicting the optimal electrical stimulation in a rather complex cultivation system, a perfusion bioreactor.

The effects of controlled HGF delivery from an affinity-binding alginate biomaterial on angiogenesis and blood perfusion in a hindlimb ischemia model.

Enhancing tissue self-repair through the use of active acellular biomaterials is one of the main goals of regenerative medicine. We now describe the features of an injectable alginate biomaterial designed to affinity-bind heparin-binding proteins and release them at a rate reflected by their association constant to alginate-sulfate. The interactions of hepatocyte growth factor (HGF) with alginate-sulfate resulted in factor protection from proteolysis, as shown by mass spectroscopy analysis after trypsin digestion. When the HGF/alginate-sulfate bioconjugate was incorporated into alginate hydrogel, HGF release was sustained by a factor of 3, as compared to the release rate from non-modified hydrogel. The released factor retained activity, as shown by its induction of ERK1/2 activation and affording cytoprotection in rat neonatal cardiomyocyte cultures. In vivo, an injectable form of the affinity-binding alginate system extended by 10-fold, as compared to a saline-treated group, retention of HGF in myocardial tissue when delivered immediately after myocardial infarction. In a severe murine hindlimb ischemia model, HGF delivery from the affinity-binding system improved tissue blood perfusion and induced mature blood vessel network formation. The therapeutic efficacy of the affinity-binding system, as well as its ease of delivery by injection, provides a proof-of-concept for the potential use of this bioactive biomaterial strategy in cardiovascular repair.