Prevascularized spongy-like hydrogels maintain their angiogenic potential after prolonged hypothermic storage.

The chronic shortage of organs and tissues for transplantation represents a dramatic burden on healthcare systems worldwide. Tissue engineering offers a potential solution to address these shortages, but several challenges remain, with prevascularization being a critical factor for in vivo survival and integration of tissue engineering products. Concurrently, a different challenge hindering the clinical implementation of such products, regards their efficient preservation from the fabrication site to the bedside. Hypothermia has emerged as a potential solution for this issue due to its milder effects on biologic systems in comparison with other cold preservation methodologies. Its impact on prevascularization, however, has not been well studied. In this work, 3D prevascularized constructs were fabricated using adipose-derived stromal vascular fraction cells and preserved at 4 °C using Hypothermosol or basal culture media (α-MEM). Hypothermosol efficiently preserved the structural and cellular integrity of prevascular networks as compared to constructs before preservation. In contrast, the use of α-MEM led to a clear reduction in prevascular structures, with concurrent induction of high levels of apoptosis and autophagy at the cellular level. In vivo evaluation using a chorioallantoic membrane model demonstrated that, in opposition to α-MEM, Hypothermosol preservation retained the angiogenic potential of constructs before preservation by recruiting a similar number of blood vessels from the host and presenting similar integration with host tissue. These results emphasize the need of studying the impact of preservation techniques on key properties of tissue engineering constructs such as prevascularization, in order to validate and streamline their clinical application.

Human adipose-derived mesenchymal stem cells laden in gellan gum spongy-like hydrogels for volumetric muscle loss treatment.

BACKGROUND: volumetric muscle loss (VML) is a traumatic massive loss of muscular tissue which frequently leads to amputation, limb loss, or lifetime disability. The current medical intervention is limited to autologous tissue transfer, which usually leads to non-functional tissue recovery. Tissue engineering holds a huge promise for functional recovery. METHODS: in this work, we evaluated the potential of human adipose-derived mesenchymal stem cells (hASCs) pre-cultured in gellan gum based spongy-like hydrogels (SLHs). RESULTS: in vitro, hASCs were spreading, proliferating, and releasing growth factors and cytokines (i.e. fibroblast growth factor, hepatocyte growth factor, insulin-like growth factor 1, interleukin-6 (IL-6), IL-8, IL-10, vascular endothelial growth factor) important for muscular regeneration. After implantation into a volumetric muscle loss (VML) mouse model, implants were degrading overtime, entirely integrating into the host between 4 and 8 weeks. In both SLH and SLH + hASCs defects, infiltrated cells were observed inside constructs associated with matrix deposition. Also, minimal collagen deposition was marginally observed around the constructs along both time-points. Neovascularization (CD31+vessels) and neoinnervation (ß-III tubulin+bundles) were significantly detected in the SLH + hASCs group, in relation to the SHAM (empty lesion). A higher density ofα-SA+and MYH7+cells were found in the injury site among all different experimental groups, at both time-points, in relation to the SHAM. The levels ofα-SA, MyoD1, and myosin heavy chain proteins were moderately increased in the SLH + hASCs group after 4 weeks, and in the hASCs group after 8 weeks, in relation to the SHAM. CONCLUSIONS: taken together, defects treated with hASCs-laden SLH promoted angiogenesis, neoinnervation, and the expression of myogenic proteins.

Pre-selection of fibroblast subsets prompts prevascularization of tissue engineered skin analogues.

The papillary and reticular dermis harbors phenotypically distinct fibroblasts, whose functions such as maintenance of skin's microvasculature are also distinct. Thus, we hypothesized that pre-selection of the subpopulations of fibroblasts would benefit the generation of skin tissue engineered (TE) constructs, promoting their prevascularization in vitro. We first isolated papillary and reticular fibroblasts using fluorescence-activated cell sorting and studied the effect of their secretome and extracellular matrix (ECM) on human dermal microvascular endothelial cell (hDMEC) organization. Subsequently, we developed a bilayered 3D polymeric structure with distinct layer-associated features to house the subpopulations of fibroblasts, to generate a skin analogue. Both papillary and reticular fibroblasts were able to stimulate capillary-like network formation in a Matrigel assay. However, the secretome of the two subpopulations was substantially different, being enriched in VEGF, IGF-1, and Angio-1 in the case of papillary fibroblasts and in HGF and FGF-2 for the reticular subset. In addition, the fibroblast subpopulations deposited varied levels of ECM proteins, more collagen I and laminin was produced by the reticular subset, but these differences did not impact hDMEC organization. Vessel-like structures with lumens were observed earlier in the 3D skin analogue prepared with the sorted fibroblasts, although ECM deposition was not affected by the cell's pre-selection. Moreover, a more differentiated epidermal layer was obtained in the skin analogue formed by the sorted fibroblasts, confirming that its whole structure was not affected. Overall, we provide evidence that pre-selection of papillary and reticular fibroblasts is relevant for promoting the in vitro prevascularization of skin TE constructs.

Gellan gum-based hydrogels support the recreation of the dermal papilla microenvironment.

The dermal papilla (DP), a specialized compartment within the hair follicle, regulates hair growth. However, human DP cells rapidly lose their inductivity in 2D-culture given the loss of positional and microenvironmental cues. Spheroids have been capable of recreating the 3D intercellular organization of DP cells, however, DP cell-matrix interactions are poorly represented. Considering the specific nature of the DP's extracellular matrix (ECM), we functionalized gellan gum (GG) with collagen IV-(HepIII) or fibronectin-(cRGDfC) derived peptide sequences to generate a 3D environment in which the phenotype and physiological functions of DP cells are restored. We further tuned the stiffness of the microenvironments by varying GG amount. Biomimetic peptides in stiffer hydrogels promoted the adhesion of DP cells, while each peptide and amount of polymer independently influenced the type and quantity of ECM proteins deposited. Furthermore, although peptides did not seem to have an influence, stiffer hydrogels improved the inductive capacity of DP cells after short term culture. Interestingly, independently of the peptide, these hydrogels supported the recapitulation of basic hair morphogenesis-like events when incorporated in an organotypic human skin in vitro model. Our work demonstrates that tailored GG hydrogels support the generation of a microenvironment in which both cell-ECM and cell-cell interactions positively influence DP cells towards the creation of an artificial DP.

Preclinical research studies for treating severe muscular injuries: focus on tissue-engineered strategies.

Severe skeletal muscle injuries are a lifelong trauma with limited medical solutions. Significant progress has been made in developing in vitro surrogates for treating such trauma. However, more attention is needed when translating these approaches to the clinic. In this review, we survey the potential of tissue-engineered surrogates in promoting muscle healing, by critically analyzing data from recent preclinical models. The therapeutic advantages provided by a combination of different biomaterials, cell types, and biochemical mediators are discussed. Current therapies on muscle healing are also summarized, emphasizing their main advantages and drawbacks. We also discuss previous and ongoing clinical trials as well as highlighting future directions for the field.

Spongy-like hydrogels prevascularization with the adipose tissue vascular fraction delays cutaneous wound healing by sustaining inflammatory cell influx.

In vitro prevascularization is one of the most explored approaches to foster engineered tissue vascularization. We previously demonstrated a benefit in tissue neovascularization by using integrin-specific biomaterials prevascularized by stromal vascular fraction (SVF) cells, which triggered vasculogenesis in the absence of extrinsic growth factors. SVF cells are also associated to biological processes important in cutaneous wound healing. Thus, we aimed to investigate whether in vitro construct prevascularization with SVF accelerates the healing cascade by fostering early vascularization vis-à-vis SVF seeding prior to implantation. Prevascularized constructs delayed re-epithelization of full-thickness mice wounds compared to both non-prevascularized and control (no SVF) groups. Our results suggest this delay is due to a persistent inflammation as indicated by a significantly lower M2(CD163+)/M1(CD86+) macrophage subtype ratio. Moreover, a slower transition from the inflammatory to the proliferative phase of the healing was confirmed by reduced extracellular matrix deposition and increased presence of thick collagen fibers from early time-points, suggesting the prevalence of fiber crosslinking in relation to neodeposition. Overall, while prevascularization potentiates inflammatory cell influx, which negatively impacts the cutaneous wound healing cascade, an effective wound healing was guaranteed in non-prevascularized SVF cell-containing spongy-like hydrogels confirming that the SVF can have enhanced efficacy.

Integrin-specific hydrogels for growth factor-free vasculogenesis.

Integrin-binding biomaterials have been extensively evaluated for their capacity to enable de novo formation of capillary-like structures/vessels, ultimately supporting neovascularization in vivo. Yet, the role of integrins as vascular initiators in engineered materials is still not well understood. Here, we show that αvß3 integrin-specific 3D matrices were able to retain PECAM1+ cells from the stromal vascular fraction (SVF) of adipose tissue, triggering vasculogenesis in vitro in the absence of extrinsic growth factors. Our results suggest that αvß3-RGD-driven signaling in the formation of capillary-like structures prevents the activation of the caspase 8 pathway and activates the FAK/paxillin pathway, both responsible for endothelial cells (ECs) survival and migration. We also show that prevascularized αvß3 integrin-specific constructs inosculate with the host vascular system fostering in vivo neovascularization. Overall, this work demonstrates the ability of the biomaterial to trigger vasculogenesis in an integrin-specific manner, by activating essential pathways for EC survival and migration within a self-regulatory growth factor microenvironment. This strategy represents an improvement to current vascularization routes for Tissue Engineering constructs, potentially enhancing their clinical applicability.

Mechanomodulatory biomaterials prospects in scar prevention and treatment.

Scarring is a major clinical issue that affects a considerable number of patients. The associated problems go beyond the loss of skin functionality, as scars bring aesthetic, psychological, and social difficulties. Therefore, new strategies are required to improve the process of healing and minimize scar formation. Research has highlighted the important role of mechanical forces in the process of skin tissue repair and scar formation, in addition to the chemical signalling. A more complete understanding of how engineered biomaterials can modulate these mechanical stimuli and modify the mechanotransduction signals in the wound microenvironment is expected to enable scar tissue reduction. The present review aims to provide an overview of our current understanding of skin biomechanics and mechanobiology underlying wound healing and scar formation, with an emphasis on the development of novel mechanomodulatory wound dressings with the capacity to offload mechanical tension in the wound environment. Furthermore, a broad overview of current challenges and future perspectives of promising mechanomodulatory biomaterials for this application are provided. STATEMENT OF SIGNIFICANCE: Scarring still is one of the biggest challenges in cutaneous wound healing. Beyond the loss of skin functionality, pathological scars, like keloids and hypertrophic, are associated to aesthetic, psychological, and social distress. Nonetheless, the understanding of the pathophysiology behind the formation of those scars remains elusive, which has in fact hindered the development of effective therapeutics. Therefore, in this review we provide an overview of our current understanding of skin biomechanics and mechanobiology underlying wound healing and scar formation, with an emphasis on the development of novel mechanomodulatory wound dressings with the capacity to offload mechanical tension in the wound environment.

3D bioprinting of gellan gum-based hydrogels tethered with laminin-derived peptides for improved cellular behavior.

The treatment of skeletal muscle defects is still a topic of noteworthy concern since surgical intervention is not capable of recovering muscle function. Herein, we propose myoblasts laden in laminin-inspired biofunctionalized gellan gum hydrogels as promising tissue-engineered skeletal muscle surrogates. Gellan gum-based hydrogels were developed by combining native gellan gum (GG) and GG tethered with laminin-derived peptides (CIKVAVS (V), KNRLTIELEVRTC (T) or RKRLQVQLSIRTC (Q)), using different polymer content (0.75%-1.875%). Hydrogels were characterized in terms of compressive modulus, molecules trafficking, and C2C12 adhesion. Hydrogels with higher polymeric content (1.125%-1.875%) showed higher stiffness whereas hydrogels with lower polymer content (0.75%-1.125%) showed higher fluorescein isothiocyanate-dextran molecules diffusion. Cell spreading was achieved regardless of the laminin-derived peptide but preferred in hydrogels with higher polymer content (1.125%-1.875%). Taken together, hydrogels with 1.125% of polymer content were selected for printability analysis. GG-based inks showed a non-newtonian, shear-thinning, and thixotropic behavior suitable for printing. Accordingly, all inks were printable, but inks tethered with T and Q peptides presented some signs of clogging. Cell viability was affected after printing but increased after 7 days of culture. After 7 days, cells were spreading but not showing significant signs of cell-cell communications. Therefore, cell density was increased, thus, myocytes loaded in V-tethered GG-based inks showed higher cell-cell communication, spreading morphology, and alignment 7, 14 days post-printing. Overall, myoblasts laden in laminin-inspired biofunctionalized GG-based hydrogels are a promising skeletal muscle surrogate with the potential to be used as in vitro model or explored for further in vivo applications.

Injectable laminin-biofunctionalized gellan gum hydrogels loaded with myoblasts for skeletal muscle regeneration.

Moderate muscular injuries that exceed muscular tissue's auto-healing capacity are still a topic of noteworthy concern. Tissue engineering appeared as a promising therapeutic strategy capable of overcoming this unmet clinical need. To attain such goal, herein we propose an in situ-crosslinking gellan gum (GG)-based hydrogel tethered with a skeletal muscle-inspired laminin-derived peptide RKRLQVQLSIRTC(Q) and encapsulated with skeletal muscle cells (SMCs). Pre-hydrogel solutions presented decreasing shear viscosity with increasing shear rate and shear stress, and required low forces for extrusion, validating their injectability. The GGDVS hydrogel was functionalized with Q-peptide with 30% of efficiency. C2C12 were able to adhere to the developed hydrogel, remained living and spreading 7 days post-encapsulation. Q-peptide release studies indicated that 25% of the unbound peptide can be released from the hydrogels up to 7 days, dependent on the hydrogel formulation. Treatment of a chemically-induced muscular lesion in mice with an injection of C2C12-laden hydrogels improved myogenesis, primarily promoted by the C2C12. In accordance, a high density of myoblasts (α-SA+ and MYH7+) were localized in tissues treated with the C2C12 (alone or encapsulated in the hydrogel). α-SA protein levels were significantly increased 8 weeks post-treatment with C2C12-laden hydrogels and MHC protein levels were increased in all experimental groups 4 weeks post-treatment, in relation to the SHAM. Neovascularization and neoinnervation was also detected in the defects. Altogether, this study indicates that C2C12-laden hydrogels hold great potential for skeletal muscle regeneration. STATEMENT OF SIGNIFICANCE: We developed an injectable gellan gum-based hydrogel for delivering C2C12 into localized myopathic model. The gellan gum was biofunctinalized with laminin-derived peptide to mimic the native muscular ECM. In addition, hydrogel was physically tuned to mimic the mechanical properties of native tissue. To the best of our knowledge, this formula was used for the first time under the context of skeletal muscle tissue regeneration. The injectability of the developed hydrogel provided non-invasive administration method, combined with a reliable microenvironment that can host C2C12 with nominal inflammation, indicated by the survival and adhesion of encapsulated cells post-injection. The treatment of skeletal muscle defect with the cell-laden hydrogel approach significantly enhanced the regeneration of localized muscular trauma.

Micropatterned gellan gum-based hydrogels tailored with laminin-derived peptides for skeletal muscle tissue engineering.

The efficacy of current therapies for skeletal muscle disorders/injuries are limited urging the need for new treatments. Skeletal muscle tissue engineered platforms represent a promising tool to shed light on the pathophysiology of skeletal muscle disorders/injuries and to investigate the efficacy of new therapies. Herein, we developed a skeletal muscle platform composed of aligned and differentiated myoblasts on micropatterned gellan gum (GG)-based hydrogels tailored with a laminin-derived peptide. To this aim, the binding of murine skeletal muscle cells (C2C12) to different laminin-derived peptides (CIKVAVS (V), KNRLTIELEVRTC (T), and RKRLQVQLSIRTC (Q)) and the binding of laminin-derived peptides to chemically functionalized GG was studied. C2C12-binding to peptide V, T and Q was 10%, 48% and 25%, whereas the peptide tethering to GG was 60%, 40% and 31%, respectively. Peptide-biofunctionalized hydrogels prepared with different polymer content showed different mechanics and peptide exposure at hydrogel surface. Cellular adhesion was detected in all hydrogel formulations, but spreading and differentiation was only promoted in peptide Q-biofunctionalized hydrogels and preferably in stiffer hydrogels. Myoblast alignment was promoted in micropatterned hydrogel surfaces. Overall, the engineered skeletal muscle herein proposed can be further explored as a platform to better understand skeletal muscle disorders/injuries and to screen new therapies.

Micropatterned Silk-Fibroin/Eumelanin Composite Films for Bioelectronic Applications.

There has been growing interest in the use of natural bionanomaterials and nanostructured systems for diverse biomedical applications. Such materials can confer unique functional properties as well as address concerns pertaining to sustainability in production. In this work, we propose the biofabrication of micropatterned silk fibroin/eumelanin composite thin films to be used in electroactive and bioactive applications in bioelectronics and biomedical engineering. Eumelanin is the most common form of melanin, naturally derived from the ink of cuttlefish, having antioxidant and electroactive properties. Another natural biomaterial, the protein silk fibroin, is modified with photoreactive chemical groups, which allows the formation of electroactive eumelanin thin films with different microstructures. The silk fibroin/eumelanin composites are fabricated to obtain thin films as well as electroactive microstructures using UV curing. Here, we report for the first time the preparation, characterization, and physical, electrochemical, and biological properties of these natural silk fibroin/eumelanin composite films. Higher concentrations of eumelanin incorporated into the films exhibit a higher charge storage capacity and good electroactivity even after 100 redox cycles. In addition, the microscale structure and the cellular activity of the fibroin/eumelanin films are assessed for understanding of the biological properties of the composite. The developed micropatterned fibroin/eumelanin films can be applied as natural electroactive substrates for bioapplications (e.g., bioelectronics, sensing, and theranostics) because of their biocompatible properties.

Correction: In vitro vascularization of tissue engineered constructs by non-viral delivery of pro-angiogenic genes.

Correction for 'In vitro vascularization of tissue engineered constructs by non-viral delivery of pro-angiogenic genes' by Helena R. Moreira et al., Biomater. Sci., 2021, DOI: 10.1039/d0bm01560a.

In vitro vascularization of tissue engineered constructs by non-viral delivery of pro-angiogenic genes.

Vascularization is still one of the major challenges in tissue engineering. In the context of tissue regeneration, the formation of capillary-like structures is often triggered by the addition of growth factors which are associated with high cost, bolus release and short half-life. As an alternative to growth factors, we hypothesized that delivering genes-encoding angiogenic growth factors to cells in a scaffold microenvironment would lead to a controlled release of angiogenic proteins promoting vascularization, simultaneously offering structural support for new matrix deposition. Two non-viral vectors, chitosan (Ch) and polyethyleneimine (PEI), were tested to deliver plasmids encoding for vascular endothelial growth factor (pVEGF) and fibroblast growth factor-2 (pFGF2) to human dermal fibroblasts (hDFbs). hDFbs were successfully transfected with both Ch and PEI, without compromising the metabolic activity. Despite low transfection efficiency, superior VEGF and FGF-2 transgene expression was attained when pVEGF was delivered with PEI and when pFGF2 was delivered with Ch, impacting the formation of capillary-like structures by primary human dermal microvascular endothelial cells (hDMECs). Moreover, in a 3D microenvironment, when PEI-pVEGF and Ch-FGF2 were delivered to hDFbs, cells produced functional pro-angiogenic proteins which induced faster formation of capillary-like structures that were retained in vitro for longer time in a Matrigel assay. The dual combination of the plasmids resulted in a downregulation of the production of VEGF and an upregulation of FGF-2. The number of capillary-like segments obtained with this system was inferior to the delivery of plasmids individually but superior to what was observed with the non-transfected cells. This work confirmed that cell-laden scaffolds containing transfected cells offer a novel, selective and alternative approach to impact the vascularization during tissue regeneration. Moreover, this work provides a new platform for pathophysiology studies, models of disease, culture systems and drug screening.

Electric Phenomenon: A Disregarded Tool in Tissue Engineering and Regenerative Medicine.

Tissue engineering and regenerative medicine (TERM) are paving the way to the generation of functional and mature biological tissues that closely emulate cellular, biochemical, and mechanical cues. Electrical fields in the human body modulate myriad biological processes, such as synapses, muscle contraction, hearing, and wound healing, which were disregarded in TERM until recently. To preserve and improve tissue electrophysiology, cells can be loaded in electroactive biomaterials and stimulated with exogenous electrical fields. Here, we review how electrical stimulation and electroactive biomaterials can be used to instruct cells to create more mature and functional tissue-engineered constructs. We also highlight the most recent electroactive engineered tissues developed for TERM.

Lactoferrin-Hydroxyapatite Containing Spongy-Like Hydrogels for Bone Tissue Engineering.

The development of bioactive and cell-responsive materials has fastened the field of bone tissue engineering. Gellan gum (GG) spongy-like hydrogels present high attractive properties for the tissue engineering field, especially due to their wide microarchitecture and tunable mechanical properties, as well as their ability to entrap the responsive cells. Lactoferrin (Lf) and Hydroxyapatite (HAp) are bioactive factors that are known to potentiate faster bone regeneration. Thus, we developed an advanced three-dimensional (3D) biomaterial by integrating these bioactive factors within GG spongy-like hydrogels. Lf-HAp spongy-like hydrogels were characterized in terms of microstructure, water uptake, degradation, and concomitant release of Lf along the time. Human adipose-derived stem cells (hASCs) were seeded and the capacity of these materials to support hASCs in culture for 21 days was assessed. Lf addition within GG spongy-like hydrogels did not change the main features of GG spongy-like hydrogels in terms of porosity, pore size, degradation, and water uptake commitment. Nevertheless, HAp addition promoted an increase of the pore wall thickness (from ~13 to 28 µm) and a decrease on porosity (from ~87% to 64%) and mean pore size (from ~12 to 20 µm), as well as on the degradability and water retention capabilities. A sustained release of Lf was observed for all the formulations up to 30 days. Cell viability assays showed that hASCs were viable during the culture period regarding cell-laden spongy-like hydrogels. Altogether, we demonstrate that GG spongy-like hydrogels containing HAp and Lf in high concentrations gathered favorable 3D bone-like microenvironment with an increased hASCs viability with the presented results.

Hydrogel-Based Strategies to Advance Therapies for Chronic Skin Wounds.

Chronic skin wounds are the leading cause of nontraumatic foot amputations worldwide and present a significant risk of morbidity and mortality due to the lack of efficient therapies. The intrinsic characteristics of hydrogels allow them to benefit cutaneous healing essentially by supporting a moist environment. This property has long been explored in wound management to aid in autolytic debridement. However, chronic wounds require additional therapeutic features that can be provided by a combination of hydrogels with biochemical mediators or cells, promoting faster and better healing. We survey hydrogel-based approaches with potential to improve the healing of chronic wounds by reviewing their effects as observed in preclinical models. Topics covered include strategies to ablate infection and resolve inflammation, the delivery of bioactive agents to accelerate healing, and tissue engineering approaches for skin regeneration. The article concludes by considering the relevance of treating chronic skin wounds using hydrogel-based strategies.

Generation of Gellan Gum-Based Adipose-Like Microtissues.

Adipose tissue is involved in many physiological processes. Therefore, the need for adipose tissue-like analogues either for soft tissue reconstruction or as in vitro testing platforms is undeniable. In this work, we explored the natural features of gellan gum (GG) to recreate injectable stable adipose-like microtissues. GG hydrogel particles with different percentages of polymer (0.5%, 0.75%, 1.25%) were developed and the effect of obtained mechanical properties over the ability of hASCs to differentiate towards the adipogenic lineage was evaluated based on the expression of the early (PPARγ) and late (FABP4) adipogenic markers, and on lipids formation and accumulation. Constructs were cultured in adipogenic induction medium up to 21 days or for six days in induction plus nine days in maintenance media. Overall, no significant differences were observed in terms of hASCs adipogenic differentiation within the range of Young’s moduli between 2.7 and 12.9 kPa. The long-term (up to six weeks) stability of the developed constructs supported its application in soft tissue reconstruction. Moreover, their ability to function as adipose-like microtissue models for drug screening was demonstrated by confirming its sensitivity to TNFα and ROCK inhibitor, respectively involved in the repression and induction of the adipogenic differentiation.

Differentiation of osteoclast precursors on gellan gum-based spongy-like hydrogels for bone tissue engineering.

Bone tissue engineering with cell-scaffold constructs has been attracting a lot of attention, in particular as a tool for the efficient guiding of new tissue formation. However, the majority of the current strategies used to evaluate novel biomaterials focus on osteoblasts and bone formation, while osteoclasts are often overlooked. Consequently, there is limited knowledge on the interaction between osteoclasts and biomaterials. In this study, the ability of spongy-like gellan gum and hydroxyapatite-reinforced gellan gum hydrogels to support osteoclastogenesis was investigated in vitro. First, the spongy-like gellan gum and hydroxyapatite-reinforced gellan gum hydrogels were characterized in terms of microstructure, water uptake and mechanical properties. Then, bone marrow cells isolated from the long bones of mice and cultured in spongy-like hydrogels were treated with 1,25-dihydroxyvitamin D3 to promote osteoclastogenesis. It was shown that the addition of HAp to spongy-like gellan gum hydrogels enables the formation of larger pores and thicker walls, promoting an increase in stiffness. Hydroxyapatite-reinforced spongy-like gellan gum hydrogels support the formation of the aggregates of tartrate-resistant acid phosphatase-stained cells and the expression of genes encoding DC-STAMP and Cathepsin K, suggesting the differentiation of bone marrow cells into pre-osteoclasts. The hydroxyapatite-reinforced spongy-like gellan gum hydrogels developed in this work show promise for future use in bone tissue scaffolding applications.

Gellan Gum Hydrogels with Enzyme-Sensitive Biodegradation and Endothelial Cell Biorecognition Sites.

The survival of a biomaterial or tissue engineered construct is mainly hampered by the deficient microcirculation in its core, and limited nutrients and oxygen availability to the implanted or colonizing host cells. Aiming to address these issues, we herein propose bioresponsive gellan gum (GG) hydrogels that are biodegradable by metalloproteinase 1 (MMP-1) and enable endothelial cells adhesion and proliferation. GG is chemically functionalized with divinyl sulfone (DVS) and then biofunctionalized with thiol cell-adhesive peptides (T1 or C16) to confer GG endothelial cell biorecognition cues. Biodegradable hydrogels are then formed by Michael type addition of GGDVS or/and peptide-functionalized GGDVS with a dithiol peptide crosslinker sensitive to MMP-1. The mechanical properties (6 to 5580 Pa), swelling (17 to 11), MMP-1-driven degradation (up to 70%), and molecules diffusion coefficients of hydrogels are tuned by increasing the polymer amount and crosslinking density. Human umbilical cord vein endothelial cells depict a polarized elongated morphology when encapsulated within T1-containing hydrogels, in contrast to the round morphology observed in C16-containing hydrogels. Cell organization is favored as early as 1 d of cell culture within the T1-modified hydrogels with higher concentration of peptide, while cell proliferation is higher in T1-modified hydrogels with higher modulus. In conclusion, biodegradable and bioresponsive GGDVS hydrogels are promising endothelial cell responsive materials that can be used for vascularization strategies.