Cell interactions in bone tissue engineering

Bone fractures, where the innate regenerative bone response is compromised, represent between 4 and 8 hundred thousands of the total fracture cases, just in the United States. Bone tissue engineering (TE) brought the notion that, in cases such as those, it was preferable to boost the healing process of bone tissue instead of just adding artificial parts that could never properly replace the native tissue. However, despite the hype, bone TE so far could not live up to its promises and new bottom-up approaches are needed. The study of the cellular interactions between the cells relevant for bone biology can be of essential importance to that. In living bone, cells are in a context where communication with adjacent cells is almost permanent. Many fundamental works have been addressing these communications nonetheless, in a bone TE approach, the 3D perspective, being part of the microenvironment of a bone cell, is as crucial. Works combining the study of cell-to-cell interactions in a 3D environment are not as many as expected. Therefore, the bone TE field should not only gain knowledge from the field of fundamental Biology but also contribute for further understanding the biology of bone. In this review, a summary of the main works in the field of bone TE, aiming at studying cellular interactions in a 3D environment, and how they contributed towards the development of a functional engineered bone tissue, is presented.The authors acknowledge Marina I. Santos for kindly providing for the micrographs for Figs 2 and 3. Also, financial support through the PhD grant SFRH / BD / 44893 / 2008 to R.P. Pirraco by the Portuguese Foundation for Science and Technology (FCT) is acknowledged

Stem cells in skin wound healing: are we there yet?

Significance: Cutaneous wound healing is a serious problem worldwide that affects patients with various wound types, resulting from burns, traumatic injuries, and diabetes. Despite the wide range of clinically available skin substitutes and the different therapeutic alternatives, delayed healing and scarring are often observed. Recent Advances: Stem cells have arisen as powerful tools to improve skin wound healing, due to features such as effective secretome, self-renewal, low immunogenicity, and differentiation capacity. They represent potentially readily available biological material that can particularly target distinct wound-healing phases. In this context, mesenchymal stem cells have been shown to promote cell migration, angiogenesis, and a possible regenerative rather than fibrotic microenvironment at the wound site, mainly through paracrine signaling with the surrounding cells/tissues. Critical Issues: Despite the current insights, there are still major hurdles to be overcome to achieve effective therapeutic effects. Limited engraftment and survival at the wound site are still major concerns, and alternative approaches to maximize stem cell potential are a major demand. Future Directions: This review emphasizes two main strategies that have been explored in this context. These comprise the exploration of hypoxic conditions to modulate stem cell secretome, and the use of adipose tissue stromal vascular fraction as a source of multiple cells, including stem cells and factors requiring minimal manipulation. Nonetheless, the attainment of these approaches to target successfully skin regeneration will be only evident after a significant number of in vivo works in relevant pre-clinical models.L3–TECT—NORTE-01-0124-FEDER-000020’’ cofinanced by North Portugal Regional Operational Program (ON.2—O Novo Norte), under the National Strategic Reference Framework (NSRF), through the European Regional Development Fund (ERDF), and Portuguese Foundation for Science and Technology (FCT

Modelling the complex nature of the tumor microenvironment: 3D tumor spheroids as an evolving tool

Cancer remains a serious burden in society and while the pace in the development of novel and more effective therapeutics is increasing, testing platforms that faithfully mimic the tumor microenvironment are lacking. With a clear shift from animal models to more complex in vitro 3D systems, spheroids emerge as strong options in this regard. Years of development have allowed spheroid-based models to better reproduce the biomechanical cues that are observed in the tumor-associated extracellular matrix (ECM) and cellular interactions that occur in both a cellâ cell and cell-ECM manner. Here, we summarize some of the key cellular interactions that drive tumor development, progression and invasion, and how successfully are these interactions recapitulated in 3D spheroid models currently in use in the field. We finish by speculating on future advancements in the field and on how these can shape the relevance of spherical 3D models for tumor modelling.Authors would like to acknowledge the fnancial support from the European Research Council through the Starting Grant “CapBed” (ERC-2018-STG-805411) and FCT/MCTES (Fundação para a Ciência e a Tecnologia/ Ministério da Ciência, Tecnologia, e Ensino Superior) through the grant SFRH/BD/119756/2016 (D.B.R.). The authors would additionally like to thank the contributions to this work from the project “TERM RES Hub—Scientifc Infrastructure for Tissue Engineering and Regenerative Medicine”, reference PINFRA/22190/2016 (Norte-01-0145-FEDER-022190), funded by the Portuguese National Science Foundation (FCT) in cooperation with the Northern Portugal Regional Coordination and Development Commission (CCDR-N). Ultimately, we would like to equally acknowledge fnancial support fromhttps://doi.org/10.54499/ UIDB/50026/2020);https://doi.org/10.54499/UIDP/50026/2020) andhttps://doi. org/10.54499/LA/P/0050/2020)

Skin Tissue Models

Skin Tissue Models provides a translational link for biomedical researchers on the interdisciplinary approaches to skin regeneration. As the skin is the largest organ in the body, engineered substitutes have critical medical application to patients with disease and injury - from burn wounds and surgical scars, to vitiligo, psoriasis and even plastic surgery. This volume offers readers preliminary description of the normal structure and function of mammalian skin, exposure to clinical problems and disease, coverage of potential therapeutic molecules and testing, skin substitutes, models as study platforms of skin biology and emerging technologies. The editors have created a table of contents which frames the relevance of skin tissue models for researchers as platforms to study skin biology and therapeutic approaches for different skin diseases, for clinicians as tissue substitutes, and for cosmetic and pharmaceutical industries as alternative test substrates that can replace animal models. Offers descriptions of the normal structure/function of mammalian skin, exposure to clinical problems, and more Presents coverage of skin diseases (cancer, genodermatoses, vitiligo and psoriasis) that extends to clinical requirements and skin diseases in vitro models Addresses legal requirements and ethical concerns in drugs and cosmetics in vitro testing Edited and authored by internationally renowned group of researchers, presenting the broadest coverage possible. © 2018 Elsevier Inc. All rights reserved.(undefined)info:eu-repo/semantics/publishedVersio

Impact of dietary phosphorus on turbot bone mineral density and content

Fish are largely dependent on dietary phosphorus for skeletal development and mineralization. In aquaculture, commercial diets commonly have higher phosphorus concentration than the basal requirements in most fish species to ensure growth and prevent bone mineral disorders. Excessive phosphorus in feeds is harmful for metabolism and results in an increase of wastes in farm effluents, which impact aquatic ecosystems. Previous studies have shown that depletion/excess of dietary phosphorus cause skeletal malformations and reduced/enhanced mineralization in fish. There is scarce information on dietary phosphorus requirements for optimal bone mineralization in species with different types of bone (cellular vs. acellular bone), which is particularly relevant for sustainable aquaculture. Thus, the aim of our study was to analyse the effect of dietary phosphorus concentrations on bone mineralization of turbot, a demersal acellularâ boned fish and valuable aquaculture species. Our results show that the dietary phosphorus concentration did not cause changes to the bone mineral density and the phosphate/calcium concentrations. No apparent skeletal malformations were detected. Additionally, we did not find an altered expression of genes involved in bone mineral metabolism. Taken together, our data show that the phosphorus requirements for optimum growth and bone mineralization in turbot are below those currently used commercially at least for the time period examined: 55â 195 days postfertilization (dpf).Spanish Economy and Competitiveness Ministry project AGL2014-52473R and AGL2017-89648P to JR. PS-B was supported by AGL2014-52473R and AGL2017-89648P project contracts

Interfollicular epidermal stem-like cells for the recreation of the hair follicle epithelial compartment

Background Hair follicle (HF) development and growth are dependent on epithelial-mesenchymal interactions (EMIs). Dermal papilla (DP) cells are recognized as the key inductive mesenchymal player, but the ideal source of receptive keratinocytes for human HF regeneration is yet to be defined. We herein investigated whether human interfollicular epidermal keratinocytes with stem-like features (EpSlKCs), characterized by a α6bri/CD71dimexpression, can replace human hair follicular keratinocytes (HHFKCs) for the recreation of the HF epithelium and respective EMIs. Methods The α6bri/CD71dim cellular fraction was selected from the whole interfollicular keratinocyte population through fluorescence-activated cell sorting and directly compared with follicular keratinocytes in terms of their proliferative capacity and phenotype. The crosstalk with DP cells was studied in an indirect co-culture system, and EpSlKC hair forming capacity tested in a hair reconstitution assay when combined with DP cells. Results EpSlKCs exhibited a phenotypic profile similar to follicular keratinocytes and were capable of increasing DP cell proliferation and, for short co-culture times, the number of alkaline phosphatase-active cells, suggesting an improvement of their inductivity. Moreover, the recreation of immature HFs and sebaceous glands was observed after EpSlKC and DP cell co-grafting in nude mice. Conclusions Our results suggest that EpSlKCs are akin to follicular keratinocytes and can crosstalk with DP cells, contributing to HF morphogenesis in vivo, thus representing an attractive epithelial cell source for hair regeneration strategies.This study was supported by the FCT/MCTES (Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia, e Ensino Superior) through the PD/59/2013, PD/BD/113800/2015 (C.M. Abreu), CEECIND/00695/2017 (M.T. Cerqueira), IF/00347/2015 (R. P. Pirraco), and IF/00945/2014 (A.P. Marques) grants

Hind limb ischemia in type 1 diabetic mice as useful tool to evaluate the neovascularization of tissue engineering constructs

Hind-limb ischemia has been used in type 1 diabetic mice to evaluate treatments for peripheral arterial disease or mechanisms of vascular impairment in diabetes [1]. Vascular deficiency is not only a pathophysiological condition, but also an obvious circumstance in tissue regeneration and in tissue engineering and regenerative medicine (TERM) strategies. We performed a pilot experiment of hind-limb ischemia in streptozotocin(STZ)-induced type 1 diabetic mice to hypothesise whether diabetes influences neovascularization induced by biomaterials. The dependent variables included blood flow and markers of arteriogenesis and angiogenesis. Type 1 diabetes was induced in 8-week-old C57BL/6 mice by an i.p. injection of STZ (50 mg/kg daily for 5 days). Hind-limb ischemia was created under deep anaesthesia and the left femoral artery and vein were isolated, ligated, and excised. The contralateral hind limb served as an internal control within each mouse. Non-diabetic ischaemic mice were used as experiment controls. At the hind-limb ischemia surgical procedure, different types of biomaterials were placed in the blood vessels gap. Blood flow was estimated by Laser Doppler perfusion imager, right after surgery and then weekly. After 28 days of implantation, surrounding muscle was excised and evaluated by histological analysis for arteriogenesis and angiogenesis. The results showed that implanted biomaterials were promote faster restoration of blood flow in the ischemic limbs and improved neovascularization in the diabetic mice. Therefore, we herein demonstrate that the combined model of hind-limb ischemia in type 1 diabetes mice is suitable to evaluate the neovascularization potential of biomaterials and eventually tissue engineering constructs.  Acknowledgments: TCS and RPP acknowledge RL3-TECT-NORTE-01-0124-FEDER-000020, co-financed by ON.2-O Novo Norte; and FCT grants: SFRH/BPD/101952/2014 and SFRH/BPD/101886/2014, respectively. LPS acknowledges FCT grant SFRH/BD/78025/2011

Cell sheet technology-driven re-epithelialization and neovascularization of skin wounds

Skin regeneration remains a challenge, requiring a well-orchestrated interplay of cell–cell and cell–matrix signalling. Cell sheet (CS) engineering, which has the major advantage of allowing the retrieval of the intact cell layers along with their naturally organized extracellular matrix (ECM), has been poorly explored for the purpose of creating skin substitutes and skin regeneration. This work proposes the use of CS technology to engineer cellular constructs based on human keratinocytes (hKC), key players in wound re-epithelialization, dermal fibroblasts (hDFb), responsible for ECM remodelling, and dermal microvascular endothelial cells (hDMEC), part of the dermal vascular network and modulators of angiogenesis. Homotypic and heterotypic three-dimensional (3-D) CS-based constructs were developed simultaneously to target wound re-vascularization and re-epithelialization. After implantation of the constructs in murine full-thickness wounds, human cells were engrafted into the host wound bed and were present in the neotissue formed up to 14 days post-implantation. Different outcomes were obtained by varying the composition and organization of the 3-D constructs. Both hKC and hDMEC significantly contributed to re-epithelialization by promoting rapid wound closure and early epithelial coverage. Moreover, a significant increase in the density of vessels at day 7 and the incorporation of hDMEC in the neoformed vasculature confirmed its role over neotissue vacularization. As a whole, the obtained results confirmed that the proposed 3-D CS-based constructs provided the necessary cell machinery, when in a specific microenvironment, guiding both re-vascularization and re-epithelialization. Although dependent on the nature of the constructs, the results obtained sustain the hypothesis that different CS-based constructs lead to improved skin healing.The authors thank Hospital da Prelada (Porto), in particular Dr. Paulo Costa for lipoaspirate collection, and Skingineering (PTDC/SAU-OSM/099422/2008) for to financial support; a Portuguese Foundation for Science and Technology (FCT) funded project. The research leading to these results has also received funding from the European Union's Seventh Framework Programme (FP7/2007-2013) under grant agreement REGPOT-CT2012-316331-POLARIS

Human mesenchymal stem cells response to multi-doped silicon-strontium calcium phosphate coatings

The search for apatitic calcium phosphate coatings to improve implants osteointegration is, nowadays, preferentially focused in the obtaining of compositions closer to that of the inorganic phase of bone. Silicon and strontium are both present in trace concentrations in natural bone and have been demonstrated, by separate, to significantly improve osteoblastic response on calcium phosphate bioceramics. This work aims the controlled and simultaneous multidoping of carbonated calcium phosphate coatings with both elements, Si and Sr, by pulsed laser deposition technique and the biological response of human mesenchymal stem cells to them. A complete physicochemical characterization has been also performed to analyze the coatings and significant positive effect was obtained at the osteogenic differentiation of cells, confirming the enormous potential of this multi-doping coating approach.Technical staff of CACTI (University of Vigo) is gratefully acknowledged. This work was partially supported by the UE-POCTEP 0330IBEROMARE1P project, UE-INTERREG 2011-1/164MARMED and Ministerio de Ciencia e Innovacion (Project MAT2010-18281). M Lopez-Alvarez thanks funding support from FP7/REGPOT-2012-2013.1 (no 316265, BIOCAPS)