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Several studies have shown that nanosilicate-reinforced scaffolds are suitable for bone regeneration. However, hydrogels are inherently too soft for load-bearing bone defects of critical sizes, and hard scaffolds typically do not provide a suitable three-dimensional (3D) microenvironment for cells to thrive, grow, and differentiate naturally. In this study, we bypass these long-standing challenges by fabricating a cell-free multi-level implant consisting of a porous and hard bone-like framework capable of providing load-bearing support and a softer native-like phase that has been reinforced with nanosilicates. The system was tested with rat bone marrow mesenchymal stem cells in vitro and as a cell-free system in a critical-sized rat bone defect. Overall, our combinatorial and multi-level implant design displayed remarkable osteoconductivity in vitro without differentiation factors, expressing significant levels of osteogenic markers compared to unmodified groups. Moreover, after 8 weeks of implantation, histological and immunohistochemical assays indicated that the cell-free scaffolds enhanced bone repair up to approximately 84% following a near-complete defect healing. Overall, our results suggest that the proposed nanosilicate bioceramic implant could herald a new age in the field of orthopedics.

Bacterial colonization of acute and chronic wounds is often associated with delayed wound healing and prolonged hospitalization. The rise of multi-drug resistant bacteria and the poor biocompatibility of topical antimicrobials warrant safe and effective antimicrobials. Antimicrobial agents that target microbial membranes without interfering with the mammalian cell proliferation and migration hold great promise in the treatment of traumatic wounds. This article reports the utility of superhydrophilic electrospun gelatin nanofiber dressings (NFDs) containing a broad-spectrum antimicrobial polymer, ε-polylysine (εPL), crosslinked by polydopamine (pDA) for treating second-degree burns. In a porcine model of partial thickness burns, NFDs promoted wound closure and reduced hypertrophic scarring compared to untreated burns. Analysis of NFDs in contact with the burns indicated that the dressings trap early colonizers and elicit bactericidal activity, thus creating a sterile wound bed for fibroblasts migration and re-epithelialization. In support of these observations, in porcine models of Pseudomonas aeruginosa and Staphylococcus aureus colonized partial thickness burns, NFDs decreased bacterial bioburden and promoted wound closure and re-epithelialization. NFDs displayed superior clinical outcome than standard-of-care silver dressings. The excellent biocompatibility and antimicrobial efficacy of the newly developed dressings in pre-clinical models demonstrate its potential for clinical use to manage infected wounds without compromising tissue regeneration.

The innate graded structural and compositional profile of musculoskeletal tissue interfaces is disrupted and replaced by fibrotic tissue in the context of disease and degeneration. Tissue engineering strategies focused on the restoration of the transitional complexity found in those junctions present special relevance for regenerative medicine. Herein, we developed a gelatin-based multiphasic hydrogel system, where sections with distinct composition and microstructure were integrated in a single unit. In each phase, hydroxyapatite particles or cellulose nanocrystals (CNC) were incorporated into an enzymatically cross-linked gelatin network to mimic bone or tendon tissue, respectively. Stiffer hydrogels were produced with the incorporation of mineralized particles, and magnetic alignment of CNC resulted in anisotropic structure formation. The evaluation of the biological commitment with human adipose-derived stem cells toward the tendon-to-bone interface revealed an aligned cell growth and higher synthesis and deposition of tenascin in the anisotropic phase, while the activity of the secreted alkaline phosphatase and the expression of osteopontin were induced in the mineralized phase. These results highlight the potential versatility offered by gelatin-transglutaminase enzyme tandem for the development of strategies that mimic the graded, composite, and complex intersections of the connective tissues.

The emergence of nontoxic, eco-friendly, and biocompatible polymers derived from natural sources has added a new and exciting dimension to the development of low-cost and scalable biomaterials for tissue engineering applications. Here, we have developed a mechanically strong and durable hydrogel composed of an eco-friendly biopolymer that exists within the cell walls of fruits and plants. Its trade name is pectin, and it bears many similarities with natural polysaccharides in the native extracellular matrix. Specifically, we have employed a new pathway to transform pectin into a ultraviolet (UV)-cross-linkable pectin methacrylate (PEMA) polymer. To endow this hydrogel matrix with cell differentiation and cell spreading properties, we have also incorporated thiolated gelatin into the system. Notably, we were able to fine-tune the compressive modulus of this hydrogel in the range ∼0.5 to ∼24 kPa: advantageously, our results demonstrated that the hydrogels can support growth and viability for a wide range of three-dimensionally (3D) encapsulated cells that include muscle progenitor (C2C12), neural progenitor (PC12), and human mesenchymal stem cells (hMSCs). Our results also indicate that PEMA-gelatin-encapsulated hMSCs can facilitate the formation of bonelike apatite after 5 weeks in culture. Finally, we have demonstrated that PEMA-gelatin can yield micropatterned cell-laden 3D constructs through UV light-assisted lithography. The simplicity, scalability, processability, tunability, bioactivity, and low-cost features of this new hydrogel system highlight its potential as a stem cell carrier that is capable of bridging the gap between clinic and laboratory.

Islet transplantation has shown to be a successful alternative in type 1 diabetes treatment, but donor scarcity precludes its worldwide clinical translation. Stem cells are an unlimited source that could circumvent the lack of donors if complete differentiation into insulin-producing cells (IPCs) could be accomplished. We have performed the differentiation of mesenchymal stem cells (MSCs) from different sources into IPCs within three-dimensional (3D) alginate matrixes. We quantified an increased insulin release at the final stage of differentiation compared to undifferentiated MSCs, which is more pronounced in IPCs differentiated from pancreatic-derived MSCs tissues. Moreover, the addition of hyaluronic acid (HA) in alginate microcapsules enhanced, even more, the insulin release from the final IPCs, independent of the MSC source. We can conclude that MSCs can be differentiated into IPCs within alginate microcapsules, enhancing insulin release when HA is present in the 3D alginate matrixes.

Despite the promise of hydrogel-based stem cell therapies in orthopedics, a significant need still exists for the development of injectable microenvironments capable of utilizing the  regenerative potential of donor cells. Indeed, the quest for biomaterials that can direct stem cells into bone without the need of external factors has been the "Holy Grail" in orthopedic stem cell therapy for decades. To address this challenge, we have utilized a combinatorial approach to screen over 63 nanoengineered hydrogels made from alginate, hyaluronic acid, and two-dimensional nanoclays. Out of these combinations, we have identified a biomaterial that can promote osteogenesis in the absence of well-established differentiation factors such as bone morphogenetic protein 2 (BMP2) or dexamethasone. Notably, in our "hit" formulations we observed a 36-fold increase in alkaline phosphate (ALP) activity and a 11-fold increase in the formation of mineralized matrix, compared to the control hydrogel. This induced osteogenesis was further supported by X-ray diffraction, scanning electron microscopy, Fourier transform infrared spectroscopy, and energy-dispersive X-ray spectroscopy. Additionally, the Montmorillonite-reinforced hydrogels exhibited high osteointegration as evident from the relatively stronger adhesion to the bone explants as compared to the control. Overall, our results demonstrate the capability of combinatorial and nanoengineered biomaterials to induce bone regeneration through osteoinduction of stem cells in a natural and differentiation-factor-free environment.

The potential clinical application of alginate cell microencapsulation has advanced enormously during the past decade. However, the 3D environment created by alginate beads does not mimic the natural extracellular matrix surrounding cells in vivo, responsible of cell survival and functionality. As one of the most frequent macromolecules present in the extracellular matrix is hyaluronic acid, we have formed hybrid beads with alginate and hyaluronic acid recreating a closer in vivo cell environment. Our results show that 1% alginate-0.25% hyaluronic acid microcapsules retain 1.5% alginate physicochemical properties. Moreover, mesenchymal stem cells encapsulated in these hybrid beads show enhanced viability therapeutic protein release and mesenchymal stem cells' potential to differentiate into chondrogenic lineage. Although future studies with additional proteins need to be done in order to approach even more the extracellular matrix features, we have shown that hyaluronic acid protects alginate encapsulated mesenchymal stem cells by providing a niche-like environment and remaining them competent as a sustainable drug delivery system.

The beneficial effect of combining alginate hydrogel with graphene oxide (GO) on microencapsulated C 2 C 12 -myoblast viability has recently been described. However, the commercially available GO lacks homogeneity in size, this parameter being of high relevance for the cell fate in two-dimensional studies. In three-dimensional applications the capacity of this material for binding different kinds of proteins can result in the reduction of de novo released protein that can effectively reach the vicinity of the microcapsules. Undoubtedly, this could be an important hurdle in its clinical use when combined with alginate-PLL microcapsules. Here, we demonstrate that the homogenization of GO nanoparticles is not a mandatory preparation step in order to get the best of this material upon cell microencapsulation. In fact, when the superficial area of these particles is increased, higher amounts of the therapeutic protein erythropoietin (EPO) are adsorbed on their surface. On the other hand, we have been able to improve even more the favorable effects of this graphene derivative on microencapsulated cell viability by forming a protein biocorona. These proteins block the potential binding sites of EPO and, therefore, enhance the amount of therapeutic drug that is released. Finally, we prove that these hybrid alginate-protein-coated GO-microcapsules are functional in vivo.

The combination of mesenchymal stem cells (MSCs) and biomimetic matrices for cell-based therapies has led to enormous advances, including the field of cell microencapsulation technology. In the present work, we have evaluated the potential of genetically modified MSCs from mice bone marrow, D1-MSCs, immobilized in alginate microcapsules with different RGD (Arg-Gly-Asp) densities. Results demonstrated that the microcapsules represent a suitable platform for D1-MSC encapsulation since cell immobilization into alginate matrices does not affect their main characteristics. The in vitro study showed a higher activity of D1-MSCs when they are immobilized in RGD-modified alginate microcapsules, obtaining the highest therapeutic factor secretion with low and intermediate densities of the bioactive molecule. In addition, the inclusion of RGD increased the differentiation potential of immobilized cells upon specific induction. However, subcutaneous implantation did not induce differentiation of D1-MSCs toward any lineage remaining at an undifferentiated state in vivo.

Cell encapsulation technology holds promise for the sustained and controlled delivery of different therapeutic proteins. Alginate-poly-L-lysine-alginate (APA) microcapsules represent one of the most widely studied alginate-polycation microcapsules. On the basis of this technology, two types of hydrogel-based scaffolds have been developed and analyzed with the aim of improving the retention and the retrieval of erythropoietin (Epo) secreting cell-loaded microcapsules in the tissue where they are implanted. Furthermore, these hydrogels may help to reduce the post-transplant inflammation and pericapsular fibrotic overgrowth typically observed around capsules. The hydrogel-based scaffolds could be administered as implantable forms (preformed scaffolds) or injectable forms (in situ formed scaffolds). The in vitro studies confirmed the correct adaptation of the enclosed cells to the scaffolds in terms of viability and protein expression. The posterior implantation of the cell-loaded capsules containing hydrogel-based scaffolds in mice revealed that the hematocrit levels were maintained up to 80% for at least 2 months. The histological analysis of the explanted microcapsules performed at that point showed that pericapsular overgrowth was reduced when cell-loaded microcapsules were enclosed in the hydrogels scaffolds. Incorporating microencapsulated cells within hydrogel-based scaffolds may help to improve their administration protocol and retention while reducing post-transplantation inflammation.

The in vitro and in vivo characterization of cell-loaded immobilization devices is an important challenge in cell encapsulation technology for the long-term efficacy of this approach. In the present paper, alginate-poly-l-lysine-alginate (APA) microcapsules containing erythropoietin (Epo)-secreting C2C12 myoblasts have been elaborated, characterized, and tested both in vitro and in vivo. High mechanical and chemical resistance of the elaborated microcapsules was observed. Moreover, the in vitro cultured encapsulated cells released 81.9 +/- 8.2 mIU/mL/24 h (by 100 cell-loaded microcapsules) by day 7, reaching the highest peak at day 21 (161.7 +/- 0.9 mIU/mL/24 h). High and constant hematocrit levels were maintained over 120 days after a single subcutaneous administration of microcapsules and lacking immunosuppressive protocols. No major host reaction was observed. On the basis of the results obtained in our study, cell encapsulation technology might be considered a suitable therapeutic strategy for the long-term delivery of biologically active products, such as Epo.

Biocompatibility of biomaterials and biomaterial-based medical devices is a critical issue for the long-term function on multiple therapeutic systems. In the past few years, there has been an increasing interest in producing more biocompatible biomaterials and in developing novel assays to analyze the quality of the products. In this study, a battery of in vitro techniques to assess the biocompatibility of alginates with different compositions and purities and alginate-based microcapsules is presented. Study of the protein and polyphenol content of the alginates revealed clear differences between the nonpurified and the purified alginates. A similar behavior was observed when the mitogenic activity and the tumor necrosis factor-alphasecretion induced by the alginates were assessed. Interestingly, when the latter two techniques were adapted to evaluate the different alginate microcapsules, a correlation with the results obtained for the alginate samples was observed. These results reinforce the idea of using the full battery of assays here reported to screen alginates and alginate-based microcapsules before implantation.