Generation of 3D melanoma models using an assembloid-based approach.

While 3D tumor models have greatly evolved over the past years, there is still a strong requirement for more biosimilar models which are capable of recapitulating cellular crosstalk within the tumor microenvironment while equally displaying representative levels of tumor aggressiveness and invasion. Herein, we disclose an assembloid melanoma model based on the fusion of individual stromal multicellular spheroids (MCSs). In contrast to more traditional tumor models, we show that it is possible to develop self-organizing, heterotypic melanoma models where tumor cells present stem-cell like features like up-regulated pluripotency master regulators SOX2, POU5F1 and NANOG. Additionally, these assembloids display high levels of invasiveness while embedded in 3D matrices as evidenced by stromal cell promotion of melanoma cell invasion via metalloproteinase production. Furthermore, sensitivity to anticancer drug doxorubicin was demonstrated for the melanoma assembloid model. These findings suggest that melanoma assembloids may play a significant role in the field of 3D cancer models as they more closely mimic the tumor microenvironment when compared to more traditional MCSs, opening the doors to a better understanding of the role of tumor microenvironment in supporting tumor progression. STATEMENT OF SIGNIFICANCE: The development of complex 3D tumor models that better recapitulate the tumor microenvironment is crucial for both an improved comprehension of intercellular crosstalk and for more efficient drug screening. We have herein developed a self-organizing heterotypic assembloid-based melanoma model capable of closely mimicking the tumor microenvironment. Key features recapitulated were the preservation of cancer cell stemness, sensitivity to anti-cancer agents and tumor cell invasion promoted by stromal cells. The approach of pre-establishing distinct stromal domains for subsequent combination into more complex tumor constructs provides a route for developing superior tumor models with a higher degree of similarity to native cancer tissues.

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.

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.

Highly tailorable gellan gum nanoparticles as a platform for the development of T cell activator systems.

BACKGROUND: T cell priming has been shown to be a powerful immunotherapeutic approach for cancer treatment in terms of efficacy and relatively weak side effects. Systems that optimize the stimulation of T cells to improve therapeutic efficacy are therefore in constant demand. A way to achieve this is through artificial antigen presenting cells that are complexes between vehicles and key molecules that target relevant T cell subpopulations, eliciting antigen-specific T cell priming. In such T cell activator systems, the vehicles chosen to deliver and present the key molecules to the targeted cell populations are of extreme importance. In this work, a new platform for the creation of T cell activator systems based on highly tailorable nanoparticles made from the natural polymer gellan gum (GG) was developed and validated. METHODS: GG nanoparticles were produced by a water in oil emulsion procedure, and characterized by dynamic light scattering, high resolution scanning electronic microscopy and water uptake. Their biocompatibility with cultured cells was assessed by a metabolic activity assay. Surface functionalization was performed with anti-CD3/CD28 antibodies via EDC/NHS or NeutrAvidin/Biotin linkage. Functionalized particles were tested for their capacity to stimulate CD4+ T cells and trigger T cell cytotoxic responses. RESULTS: Nanoparticles were approximately 150 nm in size, with a stable structure and no detectable cytotoxicity. Water uptake originated a weight gain of up to 3200%. The functional antibodies did efficiently bind to the nanoparticles, as confirmed by SDS-PAGE, which then targeted the desired CD4+ populations, as confirmed by confocal microscopy. The developed system presented a more sustained T cell activation over time when compared to commercial alternatives. Concurrently, the expression of higher levels of key cytotoxic pathway molecules granzyme B/perforin was induced, suggesting a greater cytotoxic potential for future application in adoptive cancer therapy. CONCLUSIONS: Our results show that GG nanoparticles were successfully used as a highly tailorable T cell activator system platform capable of T cell expansion and re-education.

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.

Effect of squalane-based emulsion on polyphenols skin penetration: Ex vivo skin study.

Polyphenols have gained attractiveness as ingredients in cosmetic formulations as result of their ability to delay the aging process. However, different factors limit their use, including low solubility and poor skin permeability. In this sense, this study describes the potential of squalane to increase the polyphenols ex vivo skin penetration, incorporated into a water-in-oil emulsion. Polyphenols skin permeation followed the Fick's first law and, p-coumaric acid, vitexin, schaftoside and ferulic acid had the higher permeability coefficients (Kp = 6.0-8.0 × 10-3 cm-2 h-1). Addition of squalane to phenolic compounds decreased the permeability coefficients (Kp = 4.1-5.9 × 10-3 cm-2 h-1), indicating that squalane increased the retention of polyphenols in the skin. Gentisic acid, ferulic acid, and p-coumaric acid were the only compounds permeating from water-in-oil emulsion, in the first 8 h of study and, according Krosmeyer-Peppas model, its n value was > 1 indicating a high transport resistance from the formulation and throughout the skin. Results suggest squalane as an efficient vehicle to increase the dermal availability increasing phenolic compounds physiological functions, by enhancing the skin retention time where they should exert antiaging effect.

Vascularization in skin wound healing: where do we stand and where do we go?

Cutaneous healing is a highly complex process that, if altered due to, for example, impaired vascularization, results in chronic wounds or repaired neotissue of poor quality. Significant progress has been achieved in promoting neotissue vascularization during tissue repair/regeneration. In this review, we discuss the strategies that have been explored and how each one of them contributes to regulate vascularization in the context of cutaneous wound healing from two different perspectives - biomaterial-based and a cell-based approaches. Finally, we discuss the implications of these findings on the development of the 'next generation' approaches to target vascularization in wound healing highlighting the importance of going beyond its contribution to regulate vascularization and take into consideration the temporal features of the healing process and of different types of wounds.

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.

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.

Tailoring Gellan Gum Spongy-Like Hydrogels' Microstructure by Controlling Freezing Parameters.

Gellan gum (GG) spongy-like hydrogels have been explored for different tissue engineering (TE) applications owing to their highly attractive hydrogel-like features, and improved mechanical resilience and cell performance. Although the whole process for the preparation of these materials is well-defined, we hypothesized that variations occurring during the freezing step lead to batch-to-batch discrepancies. Aiming to address this issue, two freezing devices were tested, to prepare GG spongy-like hydrogels in a more reproducible way. The cooling and freezing rates, the nucleation time and temperature, and the end freezing time were determined at different freezing temperatures (-20, -80, and -210 °C). The efficacy of the devices was assessed by analyzing the physicochemical, mechanical, and biological properties of different formulations. The cooling rate and freezing rate varied between 0.1 and 128 °C/min, depending on the temperature used and the device. The properties of spongy-like hydrogels prepared with the tested devices showed lower standard deviation in comparison to those prepared with the standard process, due to the slower freezing rate of the hydrogels. However, with this method, mean pore size was significantly lower than that with the standard method. Cell entrapment, adhesion, and viability were not affected as demonstrated with human dermal fibroblasts. This work confirmed that batch-to-batch variations are mostly due to the freezing step and that the tested devices allow fine tuning of the scaffolds' structure and properties.

Injectable pectin hydrogels produced by internal gelation: pH dependence of gelling and rheological properties.

The production of injectable pectin hydrogels by internal gelation with calcium carbonate is proposed. The pH of pectin was increased with NaOH or NaHCO3 to reach physiological values. The determination of the equivalence point provided evidence that the pH can be more precisely modulated with NaHCO3 than with NaOH. Degradation and inability to gel was observed for pectin solutions with pH 5.35 or higher. Therefore, pectin solutions with pH values varying from 3.2 (native pH) to 3.8 were chosen to produce the gels. The increase of the pH for the crosslinked hydrogels, as well as the reduction of the gelling time and their thickening, was dependent upon the amount of calcium carbonate, as confirmed by rheology. Hydrogel extracts were not cytotoxic for L-929 fibroblasts. On the overall, the investigated formulations represent interesting injectable systems providing an adequate microenvironment for cell, drug or bioactive molecules delivery.