Versatile click alginate hydrogels with protease-sensitive domains as cell responsive/instructive 3D microenvironments.

Alginate (ALG) is a widely used biomaterial to create artificial extracellular matrices (ECM) for tissue engineering. Since it does not degrade in the human body, imparting proteolytic sensitivity to ALG hydrogels leverages their properties as ECM-mimics. Herein, we explored the strain-promoted azide-alkyne cycloaddition (SPAAC) as a biocompatible and bio-orthogonal click-chemistry to graft cyclooctyne-modified alginate (ALG-K) with bi-azide-functionalized PVGLIG peptides. These are sensitive to matrix metalloproteinase (MMP) and may act as crosslinkers. The ALG-K-PVGLIG conjugates (50, 125, and 250 µM PVGLIG) were characterized for peptide incorporation, crosslinking ability (double-end grafting), and enzymatic liability. For producing cell-permissive multifunctional 3D matrices for dermal fibroblast culture, oxidized ALG-K was grafted with PVGLIG and with RGD peptides for cell-adhesion. SPAAC reactions were performed immediately before cell-laden hydrogel formation by secondary ionic-crosslinking, considerably reducing the steps and time of preparation. Hydrogels with intermediate PVGLIG concentration (125 µM) presented slightly higher stiffness while promoting extensive cell spreading and higher degree of cell-cell interconnections, likely favored by cell-driven proteolytic remodeling of the network. The hydrogel-embedded cells were able to produce their own pericellular ECM, expressed MMP-2 and 14, and secreted PVGLIG-degrading enzymes. By recapitulating key ECM-like features, these hydrogels provide biologically relevant 3D matrices for soft tissue regeneration.

Microstructured click hydrogels for cell contact guidance in 3D.

The topography of the extracellular matrix (ECM) is a major biophysical regulator of cell behavior. While this has inspired the design of cell-instructive biomaterials, the ability to present topographic cues to cells in a true 3D setting remains challenging, particularly in ECM-like hydrogels made from a single polymer. Herein, we report the design of microstructured alginate hydrogels for injectable cell delivery and show their ability to orchestrate morphogenesis via cellular contact guidance in 3D. Alginate was grafted with hydrophobic cyclooctyne groups (ALG-K), yielding amphiphilic derivatives with self-associative potential and ionic crosslinking ability. This allowed the formation of microstructured ALG-KH hydrogels, triggered by the spontaneous segregation between hydrophobic/hydrophilic regions of the polymer that generated 3D networks with stiffer microdomains within a softer lattice. The azide-reactivity of cyclooctynes also allowed ALG-K functionalization with bioactive peptides via cytocompatible strain-promoted azide-alkyne cycloaddition (SPAAC). Hydrogel-embedded mesenchymal stem cells (MSCs) were able to integrate spatial information and to mechano-sense the 3D topography, which regulated cell shape and stress fiber organization. MSCs clusters initially formed on microstructured regions could then act as seeds for neo-tissue formation, inducing cells to produce their own ECM and self-organize into multicellular structures throughout the hydrogel. By combining 3D topography, click functionalization, and injectability, using a single polymer, ALG-K hydrogels provide a unique cell delivery platform for tissue regeneration.

Engineering injectable vascularized tissues from the bottom-up: Dynamics of in-gel extra-spheroid dermal tissue assembly.

Modular tissue engineering approaches open up exciting perspectives for the biofabrication of vascularized tissues from the bottom-up, using micro-sized units such as spheroids as building blocks. While several techniques for 3D spheroid formation from multiple cell types have been reported, strategies to elicit the extra-spheroid assembly of complex vascularized tissues are still scarce. Here we describe an injectable approach to generate vascularized dermal tissue, as an example application, from spheroids combining fibroblasts and endothelial progenitors (OEC) in a xeno-free (XF) setting. Short-term cultured spheroids (1 day) were selected over mature spheroids (7 days), as they showed significantly higher angiogenic sprouting potential. Embedding spheroids in fibrin was crucial for triggering cell migration into the external milieu, while providing a 3D framework for in-gel extra-spheroid morphogenesis. Migrating fibroblasts proliferated and produced endogenous ECM forming a dense tissue, while OEC self-assembled into stable capillaries with lumen and basal lamina. Massive in vitro interconnection between sprouts from neighbouring spheroids rapidly settled an intricate vascular plexus. Upon injection into the chorioallantoic membrane of chick embryos, fibrin-entrapped pre-vascularized XF spheroids developed into a macrotissue with evident host vessel infiltration. After only 4 days, perfused chimeric capillaries with human cells were present in proximal areas, showing fast and functional inosculation between host and donor vessels. This method for generating dense vascularized tissue from injectable building blocks is clinically relevant and potentially useful for a range of applications.

Engineering a Vascularized 3D Hybrid System to Model Tumor-Stroma Interactions in Breast Cancer.

The stromal microenvironment of breast tumors, namely the vasculature, has a key role in tumor development and metastatic spread. Tumor angiogenesis is a coordinated process, requiring the cooperation of cancer cells, stromal cells, such as fibroblasts and endothelial cells, secreted factors and the extracellular matrix (ECM). In vitro models capable of capturing such complex environment are still scarce, but are pivotal to improve success rates in drug development and screening. To address this challenge, we developed a hybrid alginate-based 3D system, combining hydrogel-embedded mammary epithelial cells (parenchymal compartment) with a porous scaffold co-seeded with fibroblasts and endothelial cells (vascularized stromal compartment). For the stromal compartment, we used porous alginate scaffolds produced by freeze-drying with particle leaching, a simple, low-cost and non-toxic approach that provided storable ready-to-use scaffolds fitting the wells of standard 96-well plates. Co-seeded endothelial cells and fibroblasts were able to adhere to the surface, spread and organize into tubular-like structures. For the parenchymal compartment, a designed alginate gel precursor solution load with mammary epithelial cells was added to the pores of pre-vascularized scaffolds, forming a hydrogel in situ by ionic crosslinking. The 3D hybrid system supports epithelial morphogenesis in organoids/tumoroids and endothelial tubulogenesis, allowing heterotypic cell-cell and cell-ECM interactions, while presenting excellent experimental tractability for whole-mount confocal microscopy, histology and mild cell recovery for down-stream analysis. It thus provides a unique 3D in vitro platform to dissect epithelial-stromal interactions and tumor angiogenesis, which may assist in the development of selective and more effective anticancer therapies.

Effect of surface chemistry on hMSC growth under xeno-free conditions.

Human mesenchymal stem/stromal cells (hMSC) are promising therapeutic agents for regenerative medicine. However, therapeutic doses necessary for clinical application require in vitro expansion, ideally under Xeno-Free (XF) conditions to avoid the use of foetal bovine serum (FBS). We previously reported that hMSCs could be expanded using a pharmaceutical-grade human plasma-derived supplement for cell culture (SCC, Plastem®) combined with bFGF and TGFß1, on fibronectin (Fn)-coated surfaces. hMSCs expansion may also be affected by the chemistry of the culture surface, which modulates protein adsorption at the cell-material interface and, consequently, cell behavior. This work aimed to evaluate the effect of surface chemistry on hMSCs behavior in SCC-based XF media. For that, self-assembled monolayers (SAMs) with hydrophobic (-CH3) and hydrophilic (neutral -OH, positively charged -NH3+ and negatively charged -COO-) groups were used as model surfaces. Under XF conditions, Fn coating showed to be necessary to improve hMSC adhesion (4 h) onto all surfaces, except for OH-SAMs, probably due to a low protein adsorption capacity characteristic of this surface. In terms of cell metabolic activity (5 days) on Fn-coated surfaces, an increase over time under XF conditions was observed in all SAMs except in CH3-SAMs, which can be attributed to strong and irreversible protein adsorption characteristic of hydrophobic surfaces. This trend was also observed under FBS conditions. Nevertheless, none of the surfaces improved hMSC metabolic activity, as compared with tissue-cultured surfaces. Overall, this work describes the role of surface chemistry in XF hMSC expansion.

Coumarin-grafted blue-emitting fluorescent alginate as a potentially valuable tool for biomedical applications.

A novel blue-emitting fluorescent alginate derivative has been successfully synthesised in a simple two-reaction step procedure, using an aqueous conjugation strategy that involved carbodiimide coupling followed by an alkyne-azide "click" reaction. The modified alginate maintained the characteristic ability to form mechanically stable hydrogels by ionic crosslinking. The fluorescent properties of the developed biomaterial were investigated both in solution and hydrogel states, revealing that grafting of the coumarin fluorophore to alginate greatly enhanced its fluorescent properties. Importantly, hydrogels maintained around 80% of their initial fluorescence upon long periods of incubation under physiologic conditions. The fluorescent alginate hydrogels showed to be biocompatible in vitro, supporting the viability, metabolic activity and proliferation of mammary epithelial cells and, more importantly, their morphogenesis into spheroids and polarized acini-like structures. These hydrogels were further applied in the establishment of cell-in-gel microarrays for high-throughput screening of cell behaviour in three-dimensional (3D) matrices, being essential for spotting optimization and analysis. Collectively, our results highlight the potential of coumarin-grafted blue-emitting fluorescent alginate as a valuable tool for biomedical applications where hydrogel tracing is required.

Bottom-up engineering of cell-laden hydrogel microfibrous patch for guided tissue regeneration.

The development of three-dimensional (3D) fibrous networks as platforms for tissue engineering applications has been attracting considerable attention. Opportunely arranged microscaled fibers offer an appealing biomimetic 3D architecture, with an open porous structure and a high surface-to-volume ratio. The present work describes the development of modified-alginate hydrogel microfibers for cell entrapment, using a purpose-designed flow circuit. For microfibers biofabrication, cells were suspended in gel-precursor alginate solution and injected in a closed-loop circuit with circulating cross-linking solution. The flow promoted stretching and solidification of continuous cell-loaded micro-scaled fibers that were collected in a strainer, assembling into a microfibrous patch. The process was optimized to allow obtaining a self-standing cohesive structure. After characterization of the microfibrous patch, the behavior of embedded human mesenchymal stem cells (hMSCs) was evaluated. Microfibers of oxidized alginate modified with integrin-binding ligands provided a suitable 3D cellular microenvironment, supporting hMSCs survival and stimulating the production of endogenous extracellular matrix proteins, such as fibronectin and collagen Type I. Collectively, these features make the proposed microfibrous structures stand out as promising 3D scaffolds for regenerative medicine.

ECM-enriched alginate hydrogels for bioartificial pancreas: an ideal niche to improve insulin secretion and diabetic glucose profile.

INTRODUCTION: The success of a bioartificial pancreas crucially depends on ameliorating encapsulated beta cells survival and function. By mimicking the cellular in vivo niche, the aim of this study was to develop a novel model for beta cells encapsulation capable of establishing an appropriate microenvironment that supports interactions between cells and extracellular matrix (ECM) components. METHODS: ECM components (Arg-Gly-Asp, abbreviated as RGD) were chemically incorporated in alginate hydrogels (alginate-RGD). After encapsulation, INS-1E beta cells outcome was analyzed in vitro and after their implantation in an animal model of diabetes. RESULTS: Our alginate-RGD model demonstrated to be a good in vitro niche for supporting beta cells viability, proliferation, and activity, namely by improving the key feature of insulin secretion. RGD peptides promoted cell-matrix interactions, enhanced endogenous ECM components expression, and favored the assembly of individual cells into multicellular spheroids, an essential configuration for proper beta cell functioning. In vivo, our pivotal model for diabetes treatment exhibited an improved glycemic profile of type 2 diabetic rats, where insulin secreted from encapsulated cells was more efficiently used. CONCLUSIONS: We were able to successfully introduce a novel valuable function in an old ally in biomedical applications, the alginate. The proposed alginate-RGD model stands out as a promising approach to improve beta cells survival and function, increasing the success of this therapeutic strategy, which might greatly improve the quality of life of an increasing number of diabetic patients worldwide.

3D Culture of Mesenchymal Stem Cells in Alginate Hydrogels.

Three-dimensional (3D) cell culture systems have gained increasing interest among the scientific community, as they are more biologically relevant than traditional two-dimensional (2D) monolayer cultures. Alginate hydrogels can be formed under cytocompatibility conditions, being among the most widely used cell-entrapment 3D matrices. They recapitulate key structural features of the natural extracellular matrix and can be bio-functionalized with bioactive moieties, such as peptides, to specifically modulate cell behavior. Moreover, alginate viscoelastic properties can be tuned to match those of different types of native tissues. Ionic alginate hydrogels are transparent, allowing routine monitoring of entrapped cells, and crosslinking can be reverted using chelating agents for easy cell recovery. In this chapter, we describe some key steps to establish and characterize 3D cultures of mesenchymal stem cells using alginate hydrogels.

Biofunctionalized pectin hydrogels as 3D cellular microenvironments.

In situ-forming hydrogels of pectin, a polysaccharide present in the cell wall of higher plants, were prepared using an internal ionotropic gelation strategy based on calcium carbonate/d-glucono-δ-lactone, and explored for the first time as cell delivery vehicles. Since no ultrapure pectins are commercially available yet, a simple and efficient purification method was established, effectively reducing the levels of proteins, polyphenols and endotoxins of the raw pectin. The purified pectin was then functionalized by carbodiimide chemistry with a cell-adhesive peptide (RGD). Its gelation was analyzed by rheometry and optimized. Human mesenchymal stem cells embedded within unmodified and RGD-pectin hydrogels of different viscoelasticities (1.5 and 2.5 wt%) remained viable and metabolically active for up to 14 days. On unmodified pectin hydrogels, cells remained isolated and round-shaped. In contrast, within RGD-pectin hydrogels they elongated, spread, established cell-to-cell contacts, produced extracellular matrix, and migrated outwards the hydrogels. After 7 days of subcutaneous implantation in mice, acellular pectin hydrogels were considerably degraded, particularly the 1.5 wt% hydrogels. Altogether, these findings show the great potential of pectin-based hydrogels, which combine an interesting set of easily tunable properties, including the in vivo degradation profile, for tissue engineering and regenerative medicine.

Correction: Biofunctionalized pectin hydrogels as 3D cellular microenvironments.

Correction for 'Biofunctionalized pectin hydrogels as 3D cellular microenvironments' by Sara C. Neves et al., J. Mater. Chem. B, 2015, 3, 2096-2108.

Injectable alginate hydrogels for cell delivery in tissue engineering.

Alginate hydrogels are extremely versatile and adaptable biomaterials, with great potential for use in biomedical applications. Their extracellular matrix-like features have been key factors for their choice as vehicles for cell delivery strategies aimed at tissue regeneration. A variety of strategies to decorate them with biofunctional moieties and to modulate their biophysical properties have been developed recently, which further allow their tailoring to the desired application. Additionally, their potential use as injectable materials offers several advantages over preformed scaffold-based approaches, namely: easy incorporation of therapeutic agents, such as cells, under mild conditions; minimally invasive local delivery; and high contourability, which is essential for filling in irregular defects. Alginate hydrogels have already been explored as cell delivery systems to enhance regeneration in different tissues and organs. Here, the in vitro and in vivo potential of injectable alginate hydrogels to deliver cells in a targeted fashion is reviewed. In each example, the selected crosslinking approach, the cell type, the target tissue and the main findings of the study are highlighted.

Functionalization of biomaterials with small osteoinductive moieties.

Human mesenchymal stem cells (MSCs) are currently recognized as a powerful cell source for regenerative medicine, notably for their capacity to differentiate into multiple cell types. The combination of MSCs with biomaterials functionalized with instructive cues can be used as a strategy to direct specific lineage commitment, and can thus improve the therapeutic efficacy of these cells. In terms of biomaterial design, one common approach is the functionalization of materials with ligands capable of directly binding to cell receptors and trigger specific differentiation signaling pathways. Other strategies focus on the use of moieties that have an indirect effect, acting, for example, as sequesters of bioactive ligands present in the extracellular milieu that, in turn, will interact with cells. Compared with complex biomolecules, the use of simple compounds, such as chemical moieties and peptides, and other small molecules can be advantageous by leading to less expensive and easily tunable biomaterial formulations. This review describes different strategies that have been used to promote substrate-mediated guidance of osteogenic differentiation of immature osteoblasts, osteoprogenitors and MSCs, through chemically conjugated small moieties, both in two- and three-dimensional set-ups. In each case, the selected moiety, the coupling strategy and the main findings of the study were highlighted. The latest advances and future perspectives in the field are also discussed.

Phenotypic and proliferative modulation of human mesenchymal stem cells via crosstalk with endothelial cells.

The purpose of this work was to investigate if a coculture system of human mesenchymal stem cells (hMSC) with endothelial cells (human umbilical vein endothelial cells, HUVEC) could modulate the phenotype and proliferation of harvested MSCs. In addition to previous investigations on the crosstalk between these two cell types, in the present work different relative cell ratios were analyzed for long, therapeutically relevant, culture periods. Moreover, MSCs osteogenic commitment was assessed in a non-osteogenic medium and in the presence of HUVECs through magnetic cell separation, cell quantification by flow cytometry, morphology by fluorescent microscopy, metabolic activity and gene expression of osteogenic markers. Collectively, the present findings demonstrate that, by coculturing MSCs with HUVECs, there was not only the promotion of osteogenic differentiation (and its enhancement, depending on the relative cell ratios used), but also a significant increase on MSCs proliferation. This augmentation in cell proliferation occurred independently of relative cell ratios, but was favored by higher relative amounts of HUVECs. Taken together, this data suggests that HUVECs not only modulate MSC phenotype but also their proliferation rate. Therefore, a coculture system of MSCs and HUVECs can a have a broad impact on bone tissue engineering approaches.

Injectable in situ crosslinkable RGD-modified alginate matrix for endothelial cells delivery.

Cell-based therapies offer an attractive approach for revascularization and regeneration of tissues. However, and despite the pressing clinical needs for effective revascularization strategies, the successful immobilization of viable vascular cells within 3D matrices has been difficult to achieve. In this paper the in vitro potential of a natural, injectable RGD-alginate hydrogel as an in situ forming matrix to deliver endothelial cells was evaluated. Several techniques were employed to investigate how these microenvironments could influence the behavior of vascular cells, namely their ability to promote the outward migration of viable, proliferative cells, retaining the ability to form a 3D arrangement. Cells within RGD-grafted alginate hydrogel were able to proliferate and maintained 80% of viability for at least 48 h post-immobilization. Additionally, entrapped cells created a 3D organization into cellular networks and, when put in contact with matrigel, cells migrated out of the RGD-matrix. Overall, the obtained results support the idea that the RGD peptides conjugated to alginate provide a 3D environment for endothelial cells adhesion, survival, migration and organization.

Molecularly designed alginate hydrogels susceptible to local proteolysis as three-dimensional cellular microenvironments.

The development of sophisticated three-dimensional (3-D) cell culture microenvironments that recreate some of the complexity of the natural extracellular matrix (ECM) remains a challenging task. Here, the modification of alginate through partial crosslinking with a matrix metalloproteinase (MMP) cleavable peptide (proline-valine-glycine-leucine-isoleucine-glycine, PVGLIG) is described, and its use in the preparation of injectable, in situ crosslinkable hydrogel-like matrices is proposed. PVGLIG-grafted alginates were synthesized by carbodiimide chemistry and characterized. Their biological performance was evaluated by comparing the response of 3-D cultured mesenchymal stem cells (MSCs) to alginate hydrogels containing only cell-adhesion peptides (RGD-alginate) or both peptides (PVGLIG/RGD-alginate). After 1 week, cells remained essentially round within RGD-alginate, while they exhibited an elongated morphology within PVGLIG/RGD-alginate hydrogels, forming cellular networks. This suggests that cells were able to structurally reorganize the matrix, through enzymatic hydrolysis of PVGLIG residues, overcoming biophysical hydrogel resistance. As shown by gelatine-zymography, MSC presented higher activity of MMP-2 when cultured within alginate functionalized with MMP-sensitive peptide, suggesting that the cell's proteolytic phenotype was modulated by the matrix composition. Additionally, PVGLIG/RGD-alginate hydrogels were clearly degraded in cell culture. Taken together, the results demonstrate that the co-incorporation of MMP-labile peptides in cell-adhesive RGD-alginate hydrogels improved their performance as ECM analogues, providing a more dynamic and physiological 3-D cellular microenvironment.

Immobilization of human mesenchymal stem cells within RGD-grafted alginate microspheres and assessment of their angiogenic potential.

In this work, human mesenchymal stem cells (hMSC) immobilized in RGD-coupled alginate microspheres, with a binary composition of high and low molecular weight alginate, were investigated. Cells immobilized within RGD-alginate microspheres (during 21 days) showed metabolic activity, with an overall viability higher than 90%, short cell extensions, and, when induced, they were able to differentiate into the osteogenic lineage. In osteogenic conditions (comparing to basal conditions), immobilized cells presented alkaline phosphatase (ALP) activity and an upregulation of ALP, collagen type I, and Runx 2 expression. Moreover, mineralization was also detected in immobilized cells under osteogenic stimulus. In addition, it was demonstrated for the first time that MSCs immobilized in this 3D matrix were able to enhance the ability of neighboring endothelial cells to form tubelike structures. Overall, these findings represent a step forward in the development of injectable stem cell carriers for bone tissue engineering.

The effect of the co-immobilization of human osteoprogenitors and endothelial cells within alginate microspheres on mineralization in a bone defect.

Bone regeneration seems to be dependant on cell communication between osteogenic and endothelial cells arising from surrounding blood vessels. This study aims to determine whether endothelial cells can regulate the osteogenic potential of osteoprogenitor cells in vitro and in vivo, in a long bone defect, when co-immobilized in alginate microspheres. Alginate is a natural polymer widely used as a biomaterial for cell encapsulation. Human osteoprogenitors (HOP) from bone marrow mesenchymal stem cells were immobilized alone or together with human umbilical vein endothelial cells (HUVEC) inside irradiated, oxidized and RGD-grafted alginate microspheres. Immobilized cells were cultured in dynamic conditions and cell metabolic activity increased during three weeks. The gene expression of alkaline phosphatase and osteocalcin, both specific markers of the osteoblastic phenotype, and mineralization deposits were upregulated in co-immobilized HOPs and HUVECs, comparing to the immobilization of monocultures. VEGF secretion was also increased when HOPs were co-immobilized with HUVECs. Microspheres containing co-cultures were further implanted in a bone defect and bone formation was analysed by muCT and histology at 3 and 6 weeks post-implantation. Mineralization was observed inside and around the implanted microspheres containing the immobilized cells. However, when HOPs were co-immobilized with HUVECs, mineralization significantly increased. These findings demonstrate that co-immobilization of osteogenic and endothelial cells within RGD-grafted alginate microspheres provides a promising strategy for bone tissue engineering.