Lucília Pereira da Silva

Nanoparticulate bioactive-glass-reinforced gellan-gum hydrogels for bone-tissue engineering

This work presents bioactive-glass-reinforced gellan-gum spongy-like hydrogels (GG-BAG) as novel hydrophilic materials for use as the scaffolding in bone-tissue engineering. The reinforcement with bioactive-glass particles resulted in an improvement to the microstructure and to the mechanical properties of the material. These mechanical properties were found to be dependent on the composition and improved with the amount of bioactive glass; however, values necessary to accommodate biomechanical loading were not achieved in this study. Nevertheless, by incorporating the bioactive-glass particles, the composite material acquired the ability to form an apatite layer when soaked in simulated body fluid. Furthermore, human-adipose-derived stem cells were able to adhere and spread within the gellan-gum, spongy-like hydrogels reinforced with the bioactive glass, and remain viable, which is an important result when considering their use in bone-tissue engineering. Thus, hydrogels based on gellan gum and bioactive glass are promising biomaterials for use either alone or with cells, and with the potential for use in osteogenic differentiation.

Gellan Gum-Hyaluronic Acid Spongy-like Hydrogels and Cells from Adipose Tissue Synergize Promoting Neoskin Vascularization

Currently available substitutes for skin wound healing often result in the formation of nonfunctional neotissue. Thus, urgent care is still needed to promote an effective and complete regeneration. To meet this need, we proposed the assembling of a construct that takes advantage of cell-adhesive gellan gum-hyaluronic acid (GG-HA) spongy-like hydrogels and a powerful cell-machinery obtained from adipose tissue, human adipose stem cells (hASCs), and microvascular endothelial cells (hAMECs). In addition to a cell-adhesive character, GG-HA spongy-like hydrogels overpass limitations of traditional hydrogels, such as reduced physical stability and limited manipulation, due to improved microstructural arrangement characterized by pore wall thickening and increased mean pore size. The proposed constructs combining cellular mediators of the healing process within the spongy-like hydrogels that intend to recapitulate skin matrix aim to promote neoskin vascularization. Stable and off-the-shelf dried GG-HA polymeric networks, rapidly rehydrated at the time of cell seeding then depicting features of both sponges and hydrogels, enabled the natural cell entrapment/encapsulation and attachment supported by cell-polymer interactions. Upon transplantation into mice full-thickness excisional wounds, GG-HA spongy-like hydrogels absorbed the early inflammatory cell infiltrate and led to the formation of a dense granulation tissue. Consequently, spongy-like hydrogel degradation was observed, and progressive wound closure, re-epithelialization, and matrix remodelling was improved in relation to the control condition. More importantly, GG-HA spongy-like hydrogels promoted a superior neovascularization, which was enhanced in the presence of human hAMECs, also found in the formed neovessels. These observations highlight the successful integration of a valuable matrix and prevascularization cues to target angiogenesis/neovascularization in skin full-thickness excisional wounds.

Engineering cell-adhesive gellan gum spongy-like hydrogels for regenerative medicine purposes.

The similarity between the extracellular matrix of soft tissue and hydrogels, characterized by high-water-content viscoelastic polymeric networks, has been sustaining the advancement of hydrogels for tissue engineering and regenerative medicine (TERM) purposes. Current research on hydrogels has focused on introducing cell-adhesive peptides to promote cell adhesion and spreading, a critical applicability limitation. Here we report the development of gellan gum (GG) spongy-like hydrogels with ameliorated mechanical performance and flexibility in relation to hydrogels, using a simple and cost-effective method. Most importantly, these materials allow the entrapment of different cell types representing mesenchymal, epidermal and osteoblastic phenotypes that spread within the three-dimensional microstructure. This effect was associated with microstructural rearrangements characterized by pore wall thickening and pore size augmentation, and lower water content than precursor hydrogels. These properties significantly affected protein adsorption once cell adhesion was inhibited in the absence of serum. Spongy-like hydrogels are not adhesive for endothelial cells; however, this issue was surpassed by a pre-incubation with a cell-adhesive protein, as demonstrated for other substrates but not for traditional hydrogels. The proposed cell-compatible GG-based structures avoid time-consuming and expensive strategies that have been used to include cell-adhesive features in traditional hydrogels. This, associated with their off-the-shelf availability in an intermediary dried state, represents unique and highly relevant features for diverse TERM applications.

Stem Cell-Containing Hyaluronic Acid-Based Spongy Hydrogels for Integrated Diabetic Wound Healing

The detailed pathophysiology of diabetic foot ulcers is yet to be established and improved treatments are still required. We propose a strategy that directs inflammation, neovascularization, and neoinnervation of diabetic wounds. Aiming to potentiate a relevant secretome for nerve regeneration, stem cells were precultured in hyaluronic acid-based spongy hydrogels under neurogenic/standard media before transplantation into diabetic mice full-thickness wounds. Acellular spongy hydrogels and empty wounds were used as controls. Re-epithelialization was attained 4 weeks after transplantation independently of the test groups, whereas a thicker and more differentiated epidermis was observed for the cellular spongy hydrogels. A switch from the inflammatory to the proliferative phase of wound healing was revealed for all the experimental groups 2 weeks after injury, but a significantly higher M2(CD163+)/M1(CD86+) subtype ratio was observed in the neurogenic preconditioned group that also failed to promote neoinnervation. A higher number of intraepidermal nerve fibers were observed for the unconditioned group probably due to a more controlled transition from the inflammatory to the proliferative phase. Overall, stem cell-containing spongy hydrogels represent a promising approach to enhance diabetic wound healing by positively impacting re-epithelialization and by modulating the inflammatory response to promote a successful neoinnervation.

Gellan gum-hydroxyapatite composite spongy-like hydrogels for bone tissue engineering

Osteoinductive biomaterials represent a promising approach to advance bone grafting. Despite promising, the combination of sustained biodegradability, mechanical strength, and biocompatibility in a unique biomaterial that can also support cell performance and bone formation in vivo is demanding. Herein, we developed gellan gum (GG)-hydroxyapatite (HAp) spongy-like hydrogels to mimic the organic (GG) and inorganic (HAp) phases of the bone. HAp was successfully introduced within the GG polymeric networks, as determined by FTIR and XRD, without compromising the thermostability of the biomaterials, as showed by TGA. The developed biomaterials showed sustained degradation, high swelling, pore sizes between 200 and 300 μm, high porosity (>90%) and interconnectivity (<60%) that was inversely proportional to the total polymeric amount and to CaCl2 crosslinker. CaCl2 and HAp reinforced the mechanical properties of the biomaterials from a storage modulus of 40 KPa to 70-80 KPa. This study also showed that HAp and CaCl2 favored the bioactivity and that cells were able to adhere and spread within the biomaterials up to 21 days of culture. Overall, the possibility to tailor spongy-like hydrogels properties by including calcium as a crosslinker and by varying the amount of HAp will further contribute to understand how these features influence bone cells performance in vitro and bone formation in vivo.

Gellan Gum-Hydroxyapatite Composite Hydrogels for Bone Tissue Engineering

Osteoinductive biomaterials represent a promising approach to advance bone grafting. Despite promising, the combination of sustained biodegradability, mechanical strength, and biocompatibility in a unique biomaterial that can also support cell performance and bone formation in vivo is demanding. Herein, we developed gellan gum (GG)-hydroxyapatite (HAp) spongy-like hydrogels to mimic the organic (GG) and inorganic (HAp) phases of the bone. HAp was successfully introduced within the GG polymeric networks, as determined by FTIR and XRD, without compromising the thermostability of the biomaterials, as showed by TGA. The developed biomaterials showed sustained degradation, high swelling, pore sizes between 200 and 300 μm, high porosity (>90%) and interconnectivity (<60%) that was inversely proportional to the total polymeric amount and to CaCl2 crosslinker. CaCl2 and HAp reinforced the mechanical properties of the biomaterials from a storage modulus of 40 KPa to 70-80 KPa. This study also showed that HAp and CaCl2 favored the bioactivity and that cells were able to adhere and spread within the biomaterials up to 21 days of culture. Overall, the possibility to tailor spongy-like hydrogels properties by including calcium as a crosslinker and by varying the amount of HAp will further contribute to understand how these features influence bone cells performance in vitro and bone formation in vivo.

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.

A thermo-/pH-responsive hydrogel (PNIPAM-PDMA-PAA) with diverse nanostructures and gel behaviors as a general drug carrier for drug release

The aim of this research was to develop thermo- and pH-responsive hydrogels based on H-bonds for the sustained release of the small-molecule model drug Methylene Blue (MB). The thermo- or pH-sensitive triblock copolymer based on PNIPAM or PAA was synthesized by sequential RAFT polymerization and hydrolysis. By tuning the temperature or pH, the copolymer was able to form diverse nanostructures, including core–shell micelles, electronegative or uncharged core–shell structures and 3D networks with the hydrophobic forces of PNIPAM or H-bonds between PAA and PNIPAM. Furthermore, the reversible sol–gel transition of the copolymers was modulated by temperature (∼37 °C), pH (∼5) and concentration (∼7 wt%). The drug release potential was evaluated via in vitro injection of copolymer hydrogels into a simulated human body device. The copolymer hydrogels exhibited a sustained drug release behavior and a low instantaneous drug concentration. The MTT studies revealed that there is no noticeable cytotoxicity of NDB or NDA hydrogels against cells. Thus, the tunable drug release efficiency suggests the possibility of PNIPAM- and PAA-based environment responsive hydrogels being applied as drug carrier systems.

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.

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.