Co-cultures and cell sheet engineering as relevant tools to improve the outcome of bone tissue engineering strategies

Taking into consideration the complex biology of bone tissue it is quite clear that the understanding of the cellular interactions that regulate the homeostasis and regeneration of this remarkable tissue is essential for a successful Tissue Engineering strategy. The in vitro study of these cellular interactions relies on co-culture systems, a tremendously useful methodology where two or more cell types are cultured at the same time. Such strategy increases the complexity of typical monoculture systems, allowing the in vitro settings to closely mimic the in vivo environment. Moreover, 2D coculture systems have been extensively used by cell biologists to study cell interactions as an attempt to understand specific cellular mechanisms and signalling pathways. The interaction between osteoblasts/ osteoprogenitor cells and different cell types relevant within the bone Tissue Engineering context, namely mononuclear cells from peripheral blood and umbilical cord blood and fibroblasts, has been addressed in the first part of this thesis. The different co-cultures showed that mononuclear cells from peripheral blood were capable of accelerating the osteogenic differentiation of human bone marrow stromal cells by producing BMP-2. On the other hand, osteoblasts cultured on carrageenan membranes were capable of supporting the culture of endothelial progenitors cells present in the mononuclear fraction of umbilical cord blood that contributed to the in vivo angiogenesis after implantation in an inflammatory setting. Furthermore, fibroblasts, which are key players in the formation of fibrotic tissue after a biomaterial implantation, were shown to decrease the osteogenic activity of osteoblasts through gap junctional communication. A serious limitation of the paradigmatic use of scaffolds for bone Tissue Engineering is the lack of oxygen and nutrient supply to the cells in the core of the engineered construct leading to cell necrosis at the bulk of the constructs. Furthermore, foreign body response to the implanted biomaterial is a frequent reaction of the host and has as a consequence the formation of fibrotic tissue surrounding the implant. The use of cell sheet engineering for bone Tissue Engineering can potentially avoid those shortcomings. One of the explored strategies comprised the production of osteogenic cell sheets using this technology. Its potential for in vivo bone formation was analyzed and the formation of vascularized bone tissue with a marrow was originally demonstrated by implanting a single osteogenic cell sheet in a nude mice model. Furthermore, in order to promote vascularization, co-cultured osteogenic cell sheets with endothelial cells were also created. Endothelial cells, stacked in between osteogenic cell sheets, were proven to contribute to new vessel formation and increased bone formation in vivo This thesis demonstrates that monocytes/macrophages from peripheral blood can accelerate the osteogenic differentiation of osteoprogenitor cells while fibroblasts, have a deleterious effect on the osteogenic phenotype of osteoblasts. In addition, within an inflammatory host reaction, cells from the mononuclear fraction of umbilical cord blood were capable of contributing to new blood vessel formation after co-culture with osteoblasts. Moreover, when using the cell sheet technology to fabricate a bone tissue engineering construct, endothelial cells were also shown to improve in vivo bone formation.Considerando a complexa biologia do tecido ósseo torna-se claro que compreender as interacções celulares que regulam a homeostasia e regeneração deste tecido notável, é de extrema importância numa estratégia de Engenharia de Tecidos. O estudo in vitro destas interacções celulares baseia-se em sistemas de co-cultura, uma metodologia de grande utilidade onde dois ou mais tipos de células diferentes são cultivados simultaneamente e no mesmo espaço. Estes sistemas representam um aumento de complexidade, em relação aos sistemas de monocultura, que permite que as condições in vitro mimetizem melhor o ambiente in vivo. Além disso, sistemas de cultura 2D têm sido muito utilizados por biólogos no estudo de interacções celulares numa tentativa de compreender os mecanismos e vias de sinalização envolvidas. A interacção entre osteoblastos ou células osteoprogenitoras com células mononucleares do sangue periférico e do cordão umbilical, e com fibroblastos para aplicações em Engenharia de Tecido Ósseo foi estudada na primeira parte desta tese. As diferentes co-culturas permitiram mostrar que as células mononucleares do sangue periférico são capazes de acelerar a diferenciação osteogénica de células do estroma de medula óssea humana através da produção de proteína morfogenética do osso 2 (BMP-2). Por outro lado, foi também demonstrado que osteoblastos cultivados em membranas de carragenano suportam a cultura de células progenitoras endoteliais presentes na fracção mononuclear de sangue do cordão umbilical humano e que estas, por sua vez, contribuem para a angiogénese in vivo e em condições inflamatórias. Além disso, foi mostrado que fibroblastos, células-chave na deposição de tecido fibrótico após a implantação de um biomaterial, inibem a actividade osteogénica de osteoblastos através da comunicação por Gap Junctions. A utilização paradigmática de matrizes de suporte para a Engenharia de Tecido Óssea tem como limitação grave a falha de fornecimento de oxigénio e nutrientes às células no interior da matriz construída levando à necrose dessas mesmas células. Além disso, a resposta do hospedeiro à implantação de corpos estranhos, como matrizes tridimensionais, tem como consequência frequente a formação de tecido fibrótico à volta do implante. A aplicação da Engenharia de cell sheets na Engenharia de Tecido ósseo tem a expectativa de ultrapassar estas limitações. Uma das estratégias exploradas teve como base a produção de cell sheets osteogénicas utilizando esta tecnologia. O potencial destas cell sheets para induzir a formação de osso foi analisada in vivo tendo sido demonstrada a formação de osso vascularizado com uma estrutura medular organizada após a implantação de uma única cell sheet num modelo de ratinho nude. Além disso, numa tentativa de promover a vascularização, foram criadas cell sheets osteogénicas co-cultivadas com células endoteliais. Estas, quando cultivadas entre cell sheets osteogénicas sobrepostas, contribuíram para a formação de novos vasos in vivo bem como para o aumento da formação de novo osso. Esta tese demonstra que monócitos/macrófagos do sangue periférico podem acelerar a diferenciação osteogénica de progenitores osteogénicos enquanto os fibroblastos exercem um efeito negativo no fenótipo osteogénico de osteoblastos. Mais ainda, no contexto de uma reacção inflamatória do hospedeiro, ficou comprovado que células da fracção mononuclear do sangue do cordão umbilical participam na formação de novos vasos após co-cultura com osteoblastos. Além disso, as células endoteliais adultas, quando combinadas com cell sheets osteogénicas, promovem a formação de osso in vivo.Fundação para a Ciência e a Tecnologia (FCT) - SFRH / BD / 44893 / 2008

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

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)

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

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

Stromal vascular fraction obtained from subcutaneous adipose tissue: ex-obese and older population as main clinical targets

Introduction Human adipose tissue contains a heterogeneous and synergistic mixture of cells called stromal vascular fraction (SVF) with highly proliferative and angiogenic properties, conferring promising applicability in the field of regenerative medicine. This study aims to investigate if age, body mass index (BMI), history of obesity and massive weight loss, and harvest site are related to SVF cell marker expression. Methods A total of 26 samples of subcutaneous adipose tissue were harvested from patients admitted to the Plastic and Reconstructive department in University Hospital Center of São João, Porto, Portugal, for body contouring surgery. The percentage of cells expressing CD31, CD34, CD45, CD73, CD90, and CD105 was assessed and compared with patient's age, BMI, history of obesity and massive weight loss (ex-obese group), and harvest site. Results In the ex-obese group, a significantly higher number of cells expressing CD90 (P = 0.002) was found. BMI, harvest site, and age appear to have no association with SVF subpopulations

Adipose stem cell-derived osteoblasts sustain the functionality of endothelial progenitors from the mononuclear fraction of umbilical cord blood

Vascularization is the most pressing issue in tissue engineering (TE) since ensuring that engineered constructs are adequately perfused after in vivo transplantation is essential for the construct’s survival. The combination of endothelial cells with current TE strategies seems the most promising approach but doubts persist as to which type of endothelial cells to use. Umbilical cord blood (UCB) cells have been suggested as a possible source of endothelial progenitors. Osteoblasts obtained from human adiposederived stem cells (hASCs) were co-cultured with the mononuclear fraction of human UCB for 7 and 21 days on carrageenan membranes. The expression of vWF and CD31, and the DiI-AcLDL uptake ability allowed detection of the presence of endothelial and monocytic lineages cells in the co-culture for all culture times. In addition, the molecular expression of CD31 and VE-cadherin increased after 21 days of coculture. The functionality of the system was assessed after transplantation in nude mice. Although an inflammatory response developed, blood vessels with cells positive for human CD31 were detected around the membranes. Furthermore, the number of blood vessels in the vicinity of the implants increased when cells from the mononuclear fraction of UCB were present in the transplants compared to transplants with only hASC-derived osteoblasts. These results show how endothelial progenitors present in the mononuclear fraction of UCB can be sustained by hASC-derived osteoblast co-culture and contribute to angiogenesis even in an in vivo setting of inflammatory response.Financial support through the Ph.D. Grant SFRH/BD/44893/2008 to R.P. P. by the Portuguese Foundation for Science and Technology (FCT) and through the European Union NoE EXPERTISSUES (NMP3-CT-2004-500283) is acknowledged