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

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

Development and characterization of osteogenic cell sheets in an in vivo model

[Excerpt] Despite some successes in the tissue engineering field its evolution seems to be tampered by limitations such as cell sourcing and the lack of adequate scaffolds to support cell growth and differentiation. The use of stem cells combined with cell sheet engineering technology seems a promising way to overcome these limitations. In this work bone marrow cells were flushed from 3 weeks old Wistar rat femurs and cultured in basal DMEM medium until subconfluence. Cells were then transferred to thermo-responsive dishes (3 x10⁵ cells/dish) and cultured for 3 weeks in osteogenic medium. [...]info:eu-repo/semantics/publishedVersio

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

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

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)

Semipermeable capsules wrapping a multifunctional and self-regulated co-culture microenvironment for osteogenic differentiation

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 â stem cell nicheâ , 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.The authors acknowledge the financial support by the Portuguese Foundation for Science and Technology (FCT) through the Ph.D. (SFRH/BD/69529/2010-Clara R. Correia) and the Post-doc grants (SFRH/BPD/96611/2013- Mariana T. Cerqueira), and the funding of RL3-TECT-NORTE-01-0124-FEDER-000020 for Rogério P. Pirraco. This work was also supported by European Research Council grant agreement ERC-2014-ADG-669858 for project ATLAS