River Scholar of China and the Foreign Academicians of the Russian Academy of Engineering and Russian Academy of Science. Her research interests mainly focus on bioeffects and safety evaluation of nanomaterials and environmental pollution analysis and control. Rui L. Reis obtained his Ph.D. and D.Sc. in polymer engineering-biomaterials & tissue engineering from the University of Minho, Portugal. He is vice president for Research and Innovation of UMinho and the director of the 3B's Research Group and ICVS/3B's Associate Laboratory. He is a full professor of Tissue Engineering, Regenerative Medicine and Stem Cells at UMinho and honorary professor in four different Asian Universities. His main area of research is the development of biomaterials from natural origin polymers, and using those in combination with different stem cells for several strategies for tissue engineering and regenerative medicine, applied to distinct human tissues.

Novel highly branched biodegradable macromolecular systems have been developed by grafting carboxymethylchitosan (CMCht) onto low generation poly(amidoamine) (PAMAM) dendrimers. Such structures organize into sphere‐like nanoparticles that are proposed to be used as carriers to deliver bioactive molecules aimed at controlling the behavior of stem cells, namely their proliferation and differentiation. The nanoparticles did not exhibit significant cytotoxicity in the range of concentrations below 1 mg mL−1, and fluorescent probe labeled nanoparticles were found to be internalized with highly efficiency by both human osteoblast‐like cells and rat bone marrow stromal cells, under fluorescence‐activated cell sorting and fluorescence microscopy analyses. Dexamethasone (Dex) has been incorporated into CMCht/PAMAM dendrimer nanoparticles and release rates were determined by high performance liquid chromatography. Moreover, the biochemical data demonstrates that the Dex‐loaded CMCht/PAMAM dendrimer nanoparticles promote the osteogenic differentiation of rat bone marrow stromal cells, in vitro. The nanoparticles exhibit interesting physicochemical and biological properties and have great potential to be used in fundamental cell biology studies as well as in a variety of biomedical applications, including tissue engineering and regenerative medicine.

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A new concept of semipermeable reservoirs containing co-cultures of cells and supporting microparticles is presented, inspired by the multi-phenotypic cellular environment of bone. Based on the deconstruction of the “stem cell niche”, the developed capsules are designed to drive a self-regulated osteogenesis. PLLA microparticles functionalized with collagen I, and a co-culture of adipose stem (ASCs) and endothelial (ECs) cells are immobilized in spherical liquified capsules. The capsules are coated with multilayers of poly(L-lysine), alginate, and chitosan nano-assembled through layer-by-layer. Capsules encapsulating ASCs alone or in a co-culture with ECs are cultured in endothelial medium with or without osteogenic differentiation factors. Results show that osteogenesis is enhanced by the co-encapsulation, which occurs even in the absence of differentiation factors. These findings are supported by an increased ALP activity and matrix mineralization, osteopontin detection, and the up regulation of BMP-2, RUNX2 and BSP. The liquified co-capsules also act as a VEGF and BMP-2 cytokines release system. The proposed liquified capsules might be a valuable injectable self-regulated system for bone regeneration employing highly translational cell sources.

Heterotypic cell interactions are essential for the homeostasis of bone tissue, in particular the widely studied interaction between osteoblasts and osteoclasts. Closely related with osteoclasts are monocytes/macrophages. These have been shown to produce osteogenic factors, e.g. BMP-2, which plays a key role in bone metabolism. However, the mechanisms through which monocytes/macrophages interact with osteoblasts are still elusive. The aim of this work was to assess the influence of human peripheral blood monocytes/macrophages over the early osteogenic differentiation of human bone marrow stromal cells (hBMSCs) in the presence of dexamethasone-supplemented medium. The co-cultures were performed using porous transwells that allowed the interaction between both cell types through the production of paracrine factors. The potential effect of BMP-2 produced by monocytes/macrophages was addressed by adding an anti-BMP-2 antibody to the co-cultures. hBMSCs cultured in the presence of monocytes/macrophages had a higher proliferation rate than hBMSCs monocultures. The quantification of early osteogenic marker alkaline phosphatase (ALP) revealed higher activity of this enzyme in cells in the co-culture throughout the time of culture. Both of these effects were inhibited by adding an anti-BMP-2 antibody to the cultures. Moreover, qRT-PCR for osteocalcin and osteopontin transcripts showed overexpression of both markers. Once again, the effect of monocytes/macrophages over hBMSC osteogenic differentiation was completely inhibited in the co-cultures by blocking BMP-2. The present report confirmed that monocytes/macrophages produce BMP-2, which promotes osteogenic differentiation and proliferation of hBMSCs cumulatively to dexamethasone-supplemented medium. This potentially implies that monocyte/macrophages play a stronger role in bone homeostasis than so far supposed.

Among the wide range of strategies to target skin repair/regeneration, tissue engineering (TE) with stem cells at the forefront, remains as the most promising route. Cell sheet (CS) engineering is herein proposed, taking advantage of particular cell-cell and cell-extracellular matrix (ECM) interactions and subsequent cellular milieu, to create 3D TE constructs to promote full-thickness skin wound regeneration. Human adipose derived stem cells (hASCs) CS were obtained within five days using both thermoresponsive and standard cell culture surfaces. hASCs-based constructs were then built by superimposing three CS and transplanted into full-thickness excisional mice skin wounds with delayed healing. Constructs obtained using thermoresponsive surfaces were more stable than the ones from standard cell culture surfaces due to the natural adhesive character of the respective CS. Both CS-generating strategies lead to prolonged hASCs engraftment, although no transdifferentiation phenomena were observed. Moreover, our findings suggest that the transplanted hASCs might be promoting neotissue vascularization and extensively influencing epidermal morphogenesis, mainly through paracrine actions with the resident cells. The thicker epidermis, with a higher degree of maturation characterized by the presence of rete ridges-like structures, as well as a significant number of hair follicles observed after transplantation of the constructs combining the CS obtained from the thermoresponsive surfaces, reinforced the assumptions of the influence of the transplanted hASCs and the importance of the higher stability of these constructs promoted by cohesive cell-cell and cell-ECM interactions. Overall, this study confirmed the potential of hASCs CS-based constructs to treat full-thickness excisional skin wounds and that their fabrication conditions impact different aspects of skin regeneration, such as neovascularisation, but mainly epidermal morphogenesis.

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.

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.