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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-β1 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. Keywords: Affinity-binding alginate, Bone morphogenic protein 4, Hyaline cartilage, Osteochondral defect, Transforming growth factor-β1

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 β1 (TGF-β1). 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-β1 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.

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.

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.

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-β (TGF-β) 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-β-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-β-bound scaffold indicated that the regulatory function of TGF-β 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.

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.

Tissue regeneration after myocardial infarction (MI) represents a major challenge in cardiovascular therapy, as current clinical approaches are limited in their ability to regenerate damaged myocardium. This chapter presents an overview of two emerging strategies based on the use of biomaterials, as stand-alone therapy or in combination with regeneration signals and cells, for regenerating the infarcted heart. One strategy is cardiac tissue engineering, which creates cardiac patches from functional cells seeded in a biomaterial scaffold, bio-inspired to provide the appropriate interface for cellular interactions. Implementation of perfusion bioreactors and pre-vascularization strategies can nowadays produce thicker cardiac patches that are better integrated into the host, thus advancing the realization of this strategy in the clinics. The second strategy, injection of biomaterials, has shown great promise as a stand-alone therapy. The intracoronary delivery of alginate solution that undergoes gelation only at the infarct in the presence of elevated calcium ions, was shown to increase scar thickness and LV dimensions in acute MI models in rats and pigs and was proven safe in phase I/II clinical studies. To enable the spatio-temporal presentation of regeneration factors, the alginate was modified with sulfate groups to mimic the binding of heparin-binding proteins to heparin/heparan-sulfate. When combining alginate-sulfate with the in-situ formed alginate hydrogel, multiple factor delivery was prolonged in ischemic tissues and enabled regeneration and cardiac repair after MI. The chapter emphasizes the increasing important role of biomaterials in various therapeutic strategies aimed at cardiac regeneration.

The design of focal spatiotemporal delivery systems for multiple regeneration-inducing factors represents a crucial step in the development of effective therapies for osteochondral injuries, osteoarthritis, and other pathologies of cartilage and bone. While endogenous bone regeneration is an established process, cartilage has very limited intrinsic regeneration ability. Thus, cartilage defects progressively affect subchondral bone and alter osteochondral interface homeostasis, leading to pain and disability. Spatiotemporal delivery of osteo- and chondroinductive factors by biomaterial-based systems represents an attractive therapeutic strategy for the currently available clinical therapies that still cannot provide a superior functional replacement for the damaged or lost tissue. This chapter offers an up-to-date review of the acellular biomaterial-based strategies, aimed at simultaneous regeneration of bone and cartilage by the controlled focal delivery of the appropriate factors. It describes the various factors and delivery systems (microspheres, hydrogels, and macroporous scaffolds) developed and tested in animals and presents a novel biomaterial approach, developed by our group, for the affinity binding of TGF-β1 and BMP-4 in two separate layers, which promoted the regeneration of osteochondral interface in rabbits with osteochondral defects.

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.

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-β1), and examine its effects on the chondrogenesis of human mesenchymal stem cells (hMSCs). When attached to matrix via affinity interactions with alginate sulfate, TGF-β1 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-β1 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-β1 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-β1 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-β1 affinity-bound constructs. Our results highlight the fundamental importance of appropriate factor presentation to its biological activity, namely - inducing efficient stem cell differentiation.

Tissue regeneration following myocardial infarction (MI) represents a major challenge in cardiovascular therapy, as current clinical approaches are limited in their ability to regenerate or replace damaged myocardium. The lack of clinically-relevant cell sources, and the growing importance of paracrine effects of cell therapy, mediated by soluble growth factors and cytokines, favors the use of acellular biomaterials for myocardial tissue engineering. While the efficacy of acellular scaffold-based approaches have already been shown, applying the biomaterial in an injectable form represents a more clinically-appealing strategy, where only minimally invasive interventions are required to deliver the biopolymer solution. However, in order to enhance the passive effects mediated by the injected biomaterial on infarct stabilization and mechanical support, and achieve long-term functional improvement and regeneration of the cardiac muscle, the combination with controlled spatio-temporal delivery of bioactive molecules is required. Biomaterial-based growth factor delivery has already been shown to improve therapeutic outcome after MI. Affinity-binding alginate represents an example of such a system. This strategy has promising potential for myocardial repair and regeneration, as it provides mechanical support conferred by in situ hydrogel formation, and can affect multiple processes of myocardial regeneration by controlled delivery of multiple proteins. In conclusion, as the development of novel polymer schemes and approaches continues, the application of biomaterials that can instruct a favorable tissue reconstruction, facilitate self-repair, tissue salvage and regeneration, represents a platform for future modifications and combinations (for instance, with cell therapy). Hopefully, such efforts will have major clinical consequences on the treatment of MI and improve long-term outcome in heart failure patients.

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.

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.

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.