Staff member

JTD Keywords: clinical-trials, collagen i, discovery, mcf-7 cells, phenotype, progression, spheroids, translation, tumor microenvironment, Extracellular-matrix

JTD Keywords: angiogenesis, bioactive materials, bone regeneration, bone-formation, calcium-phosphate, extracellular calcium, in-vitro, interstitial flow, ion release, microfluidic model, signalling gradient, substitutes, vascularization, vegf, Mesenchymal stem-cells, Tissue engineering

In situ tissue engineering strategies are a promising approach to activate the endogenous regenerative potential of the cardiac tissue helping the heart to heal itself after an injury. However, the current use of complex reprogramming vectors for the activation of reparative pathways challenges the easy translation of these therapies into the clinic. Here, we evaluated the response of mouse neonatal and human induced pluripotent stem cell-derived cardiomyocytes to the presence of exogenous lactate, thus mimicking the metabolic environment of the fetal heart. An increase in cardiomyocyte cell cycle activity was observed in the presence of lactate, as determined through Ki67 and Aurora-B kinase. Gene expression and RNA-sequencing data revealed that cardiomyocytes incubated with lactate showed upregulation of BMP10, LIN28 or TCIM in tandem with downregulation of GRIK1 or DGKK among others. Lactate also demonstrated a capability to modulate the production of inflammatory cytokines on cardiac fibroblasts, reducing the production of Fas, Fraktalkine or IL-12p40, while stimulating IL-13 and SDF1a. In addition, the generation of a lactate-rich environment improved ex vivo neonatal heart culture, by affecting the contractile activity and sarcomeric structures and inhibiting epicardial cell spreading. Our results also suggested a common link between the effect of lactate and the activation of hypoxia signaling pathways. These findings support a novel use of lactate in cardiac tissue engineering, modulating the metabolic environment of the heart and thus paving the way to the development of lactate-releasing platforms for in situ cardiac regeneration.Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.

JTD Keywords: cardiac regeneration, cardiac tissue engineering, cell cycle, failure, growth, heart regeneration, induced pluripotent stem cells, ischemia, lactate, metabolic environment, metabolism, mouse, proliferation, repair, Bone morphogenetic protein-10, Cardiac tissue engineering, Cardiomyocytes, Cell cycle, Induced pluripotent stem cells, Lactate, Metabolic environment

The tumor extracellular matrix (ECM) plays a vital role in tumor progression and drug resistance. Previous studies have shown that breast tissue-derived matrices could be an important biomaterial to recreate the complexity of the tumor ECM. We have developed a method for decellularizing and delipidating a porcine breast tissue (TDM) compatible with hydrogel formation. The addition of gelatin methacrylamide and alginate allows this TDM to be bioprinted by itself with good printability, shape fidelity, and cytocompatibility. Furthermore, this bioink has been tuned to more closely recreate the breast tumor by incorporating collagen type I (Col1). Breast cancer cells (BCCs) proliferate in both TDM bioinks forming cell clusters and spheroids. The addition of Col1 improves the printability of the bioink as well as increases BCC proliferation and reduces doxorubicin sensitivity due to a downregulation of HSP90. TDM bioinks also allow a precise three-dimensional printing of scaffolds containing BCCs and stromal cells and could be used to fabricate artificial tumors. Taken together, we have proven that these novel bioinks are good candidates for biofabricating breast cancer models.

JTD Keywords: 3d in vitro cancer model, Bioink, Bioprinting, Breast tissue, Crosstalk, Decellularization, Extracellular-matrix, Growth, Hydrogels, In-vitro, Inhibition, Mechanical-properties, Metastasis, Proliferation

The use of cell-derived matrices (CDM) is a promising alternative to decellular-ized tissues/organs consisting of a complex assembly of macro-molecules. 3D cell-cultured PLA microparticles combined withmacromolecular crowding ( MMC) effect, hold a great potential todevelop bioactive materials for disease modeling applications. Wepropose CDMs as colorectal tumor models for personalizedmedicinebymimickingtissuemicroenvironmentproper-ties1.CDMs are produced by seeding human mesenchymal stemcells on fibronectin-coated PLA microparticles2 and cultured inpresence of MMCs. Obtained CDMs are biochemically charac-terized by immunostaining and mass spectrometry; gene expres-sion by qRT-PCR and RNA-sequencing; and mechanicalproperties by atomic force microscopy. Decellularized and parti-cle-free CDMs are recellularized with colorectal cancer cells andcancer associated fibroblasts (CAFs)3 to further characterize cell-cell interactions, gene expression and CDM-remodeling potential.Obtained CDMs are compared with human colorectal tumorsamples from patients.MMC effect enhances protein deposition inCDMs. Collagen types I, III and fibronectin are CDMs’ mainconstituents, resembling colon tumor extracellular matrix. Decel-lularized and particle-free CDMs were recellularized with coloncancer cells and CAFs. Cancer CDMs’ characterization is takingplace to develop an in vitro tumor model to understand cancerpromoting mechanisms, develop patient-specific drug screeningplatforms and to identify potential therapeutic targets.CDMscomposition and the tunable matrix stiffness provides reproducibletissue microenvironment. By repopulating CDMs with cancer andstroma cells, we pretend to mimic native tissue structure andproperties to obtain a promising platform for in vitro tumor modelgeneration.Authors thank MINECO ( MAT2015-68906-R) andRTI2018-096320-B-C21 for funding

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Introduction: Chronic wounds represent a major burden in humansociety, with high costs associated with extensive care treatments.Research has focussed on the development of new wound healingdevices. However, a device that enables fast-effective closure, lowcost, and scalability is still missing.Ions such as calcium and zinc are essential for skin homeostasis.Calcium regulates a plethora of skin vital functions. We have shownthat calcium releasing platforms such as calcium phosphate nano-particles (NPs) stimulate in vitro and in vivo wound healing [1,2]. Onthe other hand, zinc deficiencies are associated with impaired woundhealing. zinc’s antimicrobial properties make this ion a potentialsubstitute for antibiotics.This work aims to develop different ion releasing platforms basedon nanocomposites for local and sustained ion release at the woundsite.Methods: NPs incorporating Zn2+and Ca2+were synthesized bydouble emulsification, nanoprecipitation and sol-gel synthesis. Ionrelease was measured by colorimetric methods. NPs were encapsu-lated in 3D printed gelatine scaffolds.Results: NPs were synthesized with diameters*300 nm andnarrow size dispersity. Both ions were successfully incorporated intothe carrier NPs. Scaffolds showed good particle distribution. Therelease of zinc ions showed enhanced antibacterial activity.Discussion and Conclusion: Ion releasing platforms were suc-cessfully produced. Their composition, size, and ion release profilesindicate their potential use in soft tissue applications such as woundhealing therapies.

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INTRODUCTION: Lactate, an important metabolite during car-diogenesis and cardiac development, has been recently shown topromote cardiomyocyte proliferation and reprogramming towards adedifferentiated stem cell-like state.1 Non-myocytic cardiac fibro-blasts (CFBs) have a key role during cardiac remodeling aftermyocardial infarction, where they become activated and releaseextracellular matrix proteins. Excessive extracellular matrix depo-sition or myofibroblast activation is detrimental to cardiac repair.The aim of this work is to elucidate the effect of lactate on CFBsfor a complete interpretation of its potential proregenerative cap-abilities. Then, we developed bioactive lactate-releasing scaffoldsfor cardiac regeneration.METHODS: CFBs metabolic activity and proliferation were as-sessed. The inflammatory and fibrotic responses to exogenous lactatewere investigated in terms of cytokine and collagen production,migration assays and myofibroblast differentiation. Then, polylactic-acid(PLA) scaffolds were fabricated by electrospinning and phy-siochemically characterized (SEM, DSC, lactate release,.).RESULTS: Our results indicate that lactate does not affect fi-broblast proliferation, migration, collagen production, or activationand significantly reduces the expression of detrimental cytokines forcardiac repair. Furthermore, our PLA scaffolds are amorphous,biodegradable, nanofibrous materials that release lactate in a sus-tained manner. They do not affect CFB viability and promote cellattachment.DISCUSSION: Altogether, this study further supports the pro-spective use of lactate as a bioactive signal in new endogenouscardiac regeneration therapies. Thus, PLA patches that releaselactate as a major product from its breakdown can then be used as anABSTRACTSS-271effective source of lactate for alleviating the aftermath of myo-cardial infarction.This project was funded by ’’LaCaixa’’ Foundation(LCF/BQ/DR19/11740025) and MINECO/FEDER(RTI2018-096320-B-C21).

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JTD Keywords: Breast cancer, Hydrogels, Spheroids

INTRODUCTION: Mesenchymal stem cells ( MSCs) regulatecell processes by secreting growth factors, cytokines, chemokines,hormones and extracellular vesicles, known as secretome. This se-cretome has successfully exhibited therapeutic properties.1 Althoughsecretome can be directly administered in the target site, its fastclearance is still challenging. Therefore, new efforts have been madeto develop delivery platforms,1 that sustain secretome release andincrease its retention time.The aim of this work is to encapsulate secretome into nano-particles (NPs) for achieving a sustained release and to evaluate theirpotential in wound healing.EXPERIMENTAL METHODS: Secretome was obtained byculturing human umbilical cord MSCs under hypoxic conditions.Protein content was determined using a protein array. Secretome wasencapsulated in poly(lactic-co-glycolic) (PLGA) NPs by doubleemulsification. NPs size and zeta potential were measured using aZetasizer. The loading content and release was evaluated using amicroBCA. The secretome integrity was also assessed. NPs cellularcytocompatibility was studied using human dermal fibroblasts andkeratinocytes.RESULTS: The most expressed bioactive molecules detected inthe secretome were TIMP-2, TIMP-1, IL-6, IL-8, RANTES. NPsencapsulate between 7.2-13.5mg/mg NPs, having sizes of 300-400 nm and zeta potentials below -20 mV. NPs were biocompatibleand sustained the release for 7 days.DISCUSSION AND CONCLUSIONS: The secretome composi-tion evidenced its potential in wound healing. Secretome was suc-cessfully encapsulated in the NPs, showing a sustained release. Ourresults indicate that these nanoplatforms are promising woundhealing agents.

JTD Keywords: Msc, Nanoparticle, Secretome

Introduction: Metallic meshes filled with different bone grafts areone of the common treatments for maxillary bone defects. Due totheir mechanical properties, they can maintain the stability of thegraft during the regeneration process. Nevertheless, a second surgeryis needed to remove the mesh, with all the side effects that entails.Also, when autologous bone graft is used, the possibility of sideeffects associated to bone harvest like pain and morbidity increase.The main objective of this industrial project is to develop patientspecific bioactive and biodegradable implants to substitute currenttechniques.Methods: Different bioactive calcium phosphate-based glass mi-croparticles ( MPs) were dispersed into polycaprolactone (PCL).Then, the mixtures obtained were 3D printed by fused deposition.Scaffolds were tested with human mesenchymal stem cells (hMSCs)and human gingival fibroblasts (hGFIB). Moreover, production ofproteins such us alkaline phosphatase and vascular endothelialgrowth factor have also been quantified.Results: Parallel patterned scaffolds with interconnected macroporosity and homogeneous MPs dispersion were obtained. No cy-totoxicity was detected by LDH assay. High biocompatibility wasachieved regarding metabolic activity for both cell types. Printabilityof clinical cases with complex geometries have been achieved.Printed implants prototypes show a proper structure stability.Discussion & Conclusions: This platform holds a promising po-tential to develop safe bioactive and biodegradable patient specificimplants, with good biocompatible properties and response to hGFibpreventing future implant exposure.Acknowledgements: This industrial project is being developed incollaboration with AVINENT Implant System and supported by‘‘Doctorat Industrial’’ grant (2017DI076) from AGAUR.

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Most morphogenetic and pathological processes are driven by cells responding to the surrounding matrix, such as its composition, architecture, and mechanical properties. Despite increasing evidence for the role of extracellular matrix (ECM) in tissue and disease development, many in vitro substitutes still fail to effectively mimic the native microenvironment. We established a novel method to produce macroscale (>1 cm) mesenchymal cell-derived matrices (CDMs) aimed to mimic the fibrotic tumor microenvironment surrounding epithelial cancer cells. CDMs are produced by human adipose mesenchymal stem cells cultured in sacrificial 3D scaffold templates of fibronectin-coated poly-lactic acid microcarriers (MCs) in the presence of macromolecular crowders. We showed that decellularized CDMs closely mimic the fibrillar protein composition, architecture, and mechanical properties of human fibrotic ECM from cancer masses. CDMs had highly reproducible composition made of collagen types I and III and fibronectin ECM with tunable mechanical properties. Moreover, decellularized and MC-free CDMs were successfully repopulated with cancer cells throughout their 3D structure, and following chemotherapeutic treatment, cancer cells showed greater doxorubicin resistance compared to 3D culture in collagen hydrogels. Collectively, these results support the use of CDMs as a reproducible and tunable tool for developing 3D in vitro cancer models.

JTD Keywords: 3d cell-derived matrices, adipose mesenchymal stem cells, collagen matrix, colorectal adenocarcinoma, cytotoxicity assay, deposition, expansion, extracellular microenvironment, extracellular-matrix, fibronectin, growth, macromolecular crowders, microcarriers, scaffolds, tissue, 3d cell-derived matrices, Adipose mesenchymal stem cells, Cytotoxicity assay, Extracellular microenvironment, Macromolecular crowders, Mesenchymal stem-cells, Microcarriers

The creation of cardiac tissue models for preclinical testing is still a non-solved problem in drug discovery, due to the limitations related to thein vitroreplication of cardiac tissue complexity. Among these limitations, the difficulty of mimicking the functional properties of the myocardium due to the immaturity of the used cells hampers the obtention of reliable results that could be translated into human patients.In vivomodels are the current gold standard to test new treatments, although it is widely acknowledged that the used animals are unable to fully recapitulate human physiology, which often leads to failures during clinical trials. In the present work, we present a microfluidic platform that aims to provide a range of signaling cues to immature cardiac cells to drive them towards an adult phenotype. The device combines topographical electrospun nanofibers with electrical stimulation in a microfabricated system. We validated our platform using a co-culture of neonatal mouse cardiomyocytes and cardiac fibroblasts, showing that it allows us to control the degree of anisotropy of the cardiac tissue inside the microdevice in a cost-effective way. Moreover, a 3D computational model of the electrical field was created and validated to demonstrate that our platform is able to closely match the distribution obtained with the gold standard (planar electrode technology) using inexpensive rod-shaped biocompatible stainless-steel electrodes. The functionality of the electrical stimulation was shown to induce a higher expression of the tight junction protein Cx-43, as well as the upregulation of several key genes involved in conductive and structural cardiac properties. These results validate our platform as a powerful tool for the tissue engineering community due to its low cost, high imaging compatibility, versatility, and high-throughput configuration capabilities.

JTD Keywords: bioreactor, cardiac tissue engineering, cardiomyocytes, electrospinning, fabrication, fibroblasts, heart-on-a-chip, heart-tissue, in vitro models, myocardium, orientation, platform, scaffolds, Cardiac tissue engineering, Electrospinning, Field stimulation, Heart-on-a-chip, In vitro models, Microphysiological system

Objective: Wound healing is a complex process that involves the interaction between different cell types and bioactive factors. Impaired wound healing is characterized by a loss in synchronization of these interactions, resulting in nonhealing chronic wounds. Chronic wounds are a socioeconomic burden, one of the most prominent clinical manifestations of diabetes, however, they lack satisfactory treatment options. The objective of this study was to develop polymeric composites that deliver ions having wound healing properties and evaluate its performance using a pressure ulcer model in diabetic mice. Approach: To develop a polymeric composite wound dressing containing ion-releasing nanoparticles for chronic wound healing. This composite was chemically and physically characterized and evaluated using a pressure ulcer wound model in diabetic (db/db) mice to explore their potential as novel wound dressing. Results: This dressing exhibits a controlled ion release and a goodin vitrobioactivity. The polymeric composite dressing treatment stimulates angiogenesis, collagen synthesis, granulation tissue formation, and accelerates wound closure of ischemic wounds created in diabetic mice. In addition, the performance of the newly designed composite is remarkably better than a commercially available dressing frequently used for the treatment of low-exuding chronic wounds. Innovation: The developed nanoplatforms are cell- and growth factor free and control the host microenvironment resulting in enhanced wound healing. These nanoplatforms are available by cost-effective synthesis with a defined composition, offering an additional advantage in potential clinical application. Conclusion: Based on the obtained results, these polymeric composites offer an optimum approach for chronic wound healing without adding cells or external biological factors.

JTD Keywords: angiogenesis, bioactive dressings, chronic wounds, Angiogenesis, Bioactive dressings, Bioactive glass, Bioglass, Cells, Chronic wounds, Diabetes, Endothelial growth-factor, Expression, Hydrogel, Induction

Significance: The incidence of chronic wounds is increasing due to our aging population and the augment of people afflicted with diabetes. With the extended knowledge on the biological mechanisms underlying these diseases, there is a novel influx of medical technologies into the conventional wound care market. Recent Advances: Several nanotechnologies have been developed demonstrating unique characteristics that address specific problems related to wound repair mechanisms. In this review, we focus on the most recently developed nanotechnology-based therapeutic agents and evaluate the efficacy of each treatment in in vivo diabetic models of chronic wound healing. Critical Issues: Despite the development of potential biomaterials and nanotechnology-based applications for wound healing, this scientific knowledge is not translated into an increase of commercially available wound healing products containing nanomaterials. Future Directions: Further studies are critical to provide insights into how scientific evidences from nanotechnology-based therapies can be applied in the clinical setting.

JTD Keywords: chronic, diabetes, liposomes, nanofibers, nanoparticles, Chronic, Chronic wound, Diabetes, Diabetic wound, Diabetic-rats, Dressings, Drug mechanism, Extracellular-matrix, Growth-factor, Human, In-vitro, Liposome, Liposomes, Mesenchymal stem-cells, Metal nanoparticle, Nanofiber, Nanofibers, Nanofibrous scaffolds, Nanoparticles, Nanotechnology, Nonhuman, Polyester, Polymer, Polysaccharide, Priority journal, Protein, Review, Self assembled protein nanoparticle, Silk fibroin, Skin wounds, Wound healing, Wound healing promoting agent

Tissue engineering and regenerative medicine approaches use biomaterials in combination with cells to regenerate lost functions of tissues and organs to prevent organ transplantation. However, most of the current strategies fail in mimicking the tissue's extracellular matrix properties. In order to mimic native tissue conditions, we developed cell-derived matrix (CDM) microtissues (MT). Our methodology uses poly-lactic acid (PLA) and Cultispher(R) S microcarriers' (MCs') as scaffold templates, which are seeded with rat bone marrow mesenchymal stem cells (rBM-MSCs). The scaffold template allows cells to generate an extracellular matrix, which is then extracted for downstream use. The newly formed CDM provides cells with a complex physical (MT architecture) and biochemical (deposited ECM proteins) environment, also showing spontaneous angiogenic potential. Our results suggest that MTs generated from the combination of these two MCs (mixed MTs) are excellent candidates for tissue vascularization. Overall, this study provides a methodology for in-house fabrication of microtissues with angiogenic potential for downstream use in various tissue regenerative strategies.

JTD Keywords: angiogenesis, cell-derived matrix, cultispher® s, microtissue, poly-lactic acid microcarriers, Angiogenesis, Cell-derived matrix, Cultispher (r) s, Microtissue, Poly-lactic acid microcarriers, Rat bone marrow mesenchymal stem cells

© 2020 Elsevier B.V. We examine different approaches for the controlled release of L-lactate, which is a signaling molecule that participates in tissue remodeling and regeneration, such as cardiac and muscle tissue. Robust, flexible, and self-supported 3-layers films made of two spin-coated poly(lactic acid) (PLA) layers separated by an electropolymerized poly(3,4-ethylenedioxythiophene) (PEDOT) layer, are used as loading and delivery systems. Films with outer layers prepared using homochiral PLA and with nanoperforations of diameter 146 ± 70 experience more bulk erosion, which also contributes to the release of L-lactic acid, than those obtained using heterochiral PLA and with nanoperforations of diameter 66 ± 24. Moreover, the release of L-lactic acid as degradation product is accelerated by applying biphasic electrical pulses. The four approaches used for loading extra L-lactate in the 3-layered films were: incorporation of L-lactate at the intermediate PEDOT layer as primary dopant agent using (1) organic or (2) basic water solutions as reaction media; (3) substitution at the PEDOT layer of the ClO4− dopant by L-lactate using de-doping and re-doping processes; and (4) loading of L-lactate at the outer PLA layers during the spin-coating process. Electrical stimuli were applied considering biphasic voltage pulses and constant voltages (both negative and positive). Results indicate that the approach used to load the L-lactate has a very significant influence in the release regulation process, affecting the concentration of released L-lactate up to two orders of magnitude. Among the tested approaches, the one based on the utilization of the outer layers for loading, approach (4), can be proposed for situations requiring prolonged and sustained L-lactate release over time. The biocompatibility and suitability of the engineered films for cardiac tissue engineering has also been confirmed using cardiac cells.

JTD Keywords: biphasic voltage pulse, cardiac tissue regeneration, cardiomyocytes proliferation, conducting polymer, nanoperforated films, sustained delivery, Biphasic voltage pulse, Cardiac tissue regeneration, Cardiomyocytes proliferation, Conducting polymer, Nanoperforated films, Sustained delivery

© 2021 Elsevier B.V. Thymidine kinase expressing human adipose mesenchymal stem cells (TK-hAMSCs) in combination with ganciclovir (GCV) are an effective platform for antitumor bystander therapy in mice models. However, this strategy requires multiple TK-hAMSCs administrations and a substantial number of cells. Therefore, for clinical translation, it is necessary to find a biocompatible scaffold providing TK-hAMSCs retention in the implantation site against their rapid wash-out. We have developed a microtissue (MT) composed by TKhAMSCs and a scaffold made of polylactic acid microparticles and cell-derived extracellular matrix deposited by hAMSCs. The efficacy of these MTs as vehicles for TK-hAMSCs/GCV bystander therapy was evaluated in a rodent model of human prostate cancer. Subcutaneously implanted MTs were integrated in the surrounding tissue, allowing neovascularization and maintenance of TK-hAMSCs viability. Furthermore, MTs implanted beside tumors allowed TK-hAMSCs migration towards tumor cells and, after GCV administration, inhibited tumor growth. These results indicate that TK-hAMSCs-MTs are promising cell reservoirs for clinical use of therapeutic MSCs in bystander therapies.

JTD Keywords: adipose mesenchymal stem cells, bioluminescence, bystander therapy, cancer, Adipose mesenchymal stem cells, Bioluminescence, Bystander therapy, Cancer, Self-assembled cell-based microtissues

© 2020 Elsevier B.V. In the current work, our purpose was based on the assessment of bioactive chitosan (CS)/Poly(ethylene glycol) diacrylate (PEGDA) based scaffolds ability to stimulate in vitro angiogenesis process. The bioactivation of the scaffolds was accomplished by using organic (BMP-2 peptide) and inorganic (hydroxyapatite nanoparticles) cues. In particular, the properties of the materials in terms of biological response promotion on human umbilical vein endothelial cells (HUVECs) were studied by using in vitro angiogenesis tests based on cell growth and proliferation. Furthermore, our interest was to examine the scaffolds capability to modulate two important steps involved in angiogenesis process: migration and tube formation of cells. Our data underlined that bioactive signals on CS/PEGDA scaffolds surface induce a desirable effect on angiogenic response concerning angiogenic marker expression (CD-31) and endothelial tissue formation (tube formation). Taken together, the results emphasized the concept that bioactive CS/PEGDA scaffolds may be novel implants for stimulating neovascularization of tissue-engineered constructs in regenerative medicine field.

JTD Keywords: angiogenesis, bmp-2 peptide, chitosan/pegda based scaffolds, human umbilical vein endothelial cells huvecs, Angiogenesis, Bmp-2 peptide, Chitosan/pegda based scaffolds, Human umbilical vein endothelial cells huvecs, Osteogenesis

Degenerative cartilage pathologies are nowadays a major problem for the world population. Factors such as age, genetics or obesity can predispose people to suffer from articular cartilage degeneration, which involves severe pain, loss of mobility and consequently, a loss of quality of life. Current strategies in medicine are focused on the partial or total replacement of affected joints, physiotherapy and analgesics that do not address the underlying pathology. In an attempt to find an alternative therapy to restore or repair articular cartilage functions, the use of bioengineered tissues is proposed. In this study we present a three-dimensional (3D) bioengineered platform combining a 3D printed polycaprolactone (PCL) macrostructure with RAD16-I, a soft nanofibrous self-assembling peptide, as a suitable microenvironment for human mesenchymal stem cells’ (hMSC) proliferation and differentiation into chondrocytes. This 3D bioengineered platform allows for long-term hMSC culture resulting in chondrogenic differentiation and has mechanical properties resembling native articular cartilage. These promising results suggest that this approach could be potentially used in articular cartilage repair and regeneration.

JTD Keywords: 3D printing, Chondrogenic differentiation, Polycaprolactone, RAD16-I self-assembling peptide

Regenerative medicine and disease models have evolved in recent years from two to three dimensions, providing in vitro constructs that are more similar to in vivo tissues. By mimicking native tissues, cell-derived matrices (CDMs) have emerged as new modifiable extracellular matrices for a variety of tissues, allowing researchers to study basic cellular processes in tissue-like structures, test tissue regeneration approaches, and model disease development. In this review, different fabrication techniques and characterization methods of CDMs are presented and examples of their application in cell behavior studies, tissue regeneration, and disease models are provided. In addition, future guidelines and perspectives in the field of CDMs are discussed.

JTD Keywords: 3D models, Biomaterials, Cell-derived matrices, Extracellular matrix, Personalized therapies

The introduction of 3D bioprinting to fabricate living constructs with tailored architecture has provided a new paradigm for biofabrication, with the potential to overcome several drawbacks of conventional scaffold-based tissue regeneration strategies. Hydrogel-based materials are suitable candidates regarding cell biocompatibility but often display poor mechanical properties. Self-assembling peptides are a promising source of biomaterials to be used as 3D scaffolds based on their similarity to extracellular matrices (structurally and mechanically). In this study, an advanced bioink for biofabrication is presented based on the optimization of a RAD16-I-based biomaterial. The strategy followed to build 3D predefined structures by 3D printing is based on an enhancement of bioink viscosity by adding methylcellulose (MC) to a RAD16-I solution. The resultant constructs display high shape fidelity and stability and embedded human mesenchymal stem cells present high viability after 7 days of culture. Moreover, cells are also able to differentiate to the adipogenic lineage, suggesting the suitability of this novel biomaterial for soft tissue engineering applications.

JTD Keywords: 3D bioprinting, Biofabrication, Bioinks, Self-assembling peptides, Tissue engineering

Electroactive and biocompatible fibrous scaffolds have been prepared and characterized using polyaniline (PAni) doped with dodecylbenzenesulfonic acid (DBSA) combined with poly(lactic acid) (PLA) and PLA/poly(ethylene glycol) (PEG) mixtures. The composition of simple and core–shell fibers, which have been obtained by both uniaxial and coaxial electrospinning, respectively, has been corroborated by Fourier-transform infrared and micro-Raman spectroscopies. Morphological studies suggest that the incorporation of PEG enhances the packing of PLA and PAni chains, allowing the regulation of the thickness of the fibers. PAni and PEG affect the thermal and electrical properties of the fibers, both decreasing the glass transition temperature and increasing the electrical conductivity. Interestingly, the incorporation of PEG improves the PAni-containing paths associated with the conduction properties. Although dose response curves evidence the high cytotoxicity of PAni/DBSA, cell adhesion and cell proliferation studies on PLA/PAni fibers show a reduction of such harmful effects as the conducting polymer is mainly retained inside the fibers through favorable PAni···PLA interactions. The incorporation of PEG into uniaxial fibers resulted in an increment of the cell mortality, which has been attributed to its rapid dissolution into the culture medium and the consequent enhancement of PAni release. In opposition, the delivery of PAni decreases and, therefore, the biocompatibility of the fibers increases when a shell coating the PAni-containing system is incorporated through coaxial electrospinning. Finally, morphological and functional studies using cardiac cells indicated that these fibrous scaffolds are suitable for cardiac tissue engineering applications.

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Skin wound healing aims to repair and restore tissue through a multistage process that involves different cells and signalling molecules that regulate the cellular response and the dynamic remodelling of the extracellular matrix. Nowadays, several therapies that combine biomolecule signals (growth factors and cytokines) and cells are being proposed. However, a lack of reliable evidence of their efficacy, together with associated issues such as high costs, a lack of standardization, no scalable processes, and storage and regulatory issues, are hampering their application. In situ tissue regeneration appears to be a feasible strategy that uses the body's own capacity for regeneration by mobilizing host endogenous stem cells or tissue-specific progenitor cells to the wound site to promote repair and regeneration. The aim is to engineer instructive systems to regulate the spatio-temporal delivery of proper signalling based on the biological mechanisms of the different events that occur in the host microenvironment. This review describes the current state of the different signal cues used in wound healing and skin regeneration, and their combination with biomaterial supports to create instructive microenvironments for wound healing.

JTD Keywords: Instructive biomaterials, Skin regeneration, Wound healing, Signalling release, In situ tissue engineering

The use of human mesenchymal stromal cells (hMSC) for treating diseased tissues with poor vascularization has received significant attention, but low cell survival has hampered its translation to the clinic. Bioglasses and glass-ceramics have also been suggested as therapeutic agents for stimulating angiogenesis in soft tissues, but these effects need further evaluation in vivo. In this study, calcium-releasing particles and hMSC were combined within a hydrogel to examine their vasculogenic potential in vitro and in vivo. The particles provided sustained calcium release and showed proangiogenic stimulation in a chorioallantoic membrane (CAM) assay. The number of hMSC encapsulated in a degradable RGD-functionalized PEG hydrogel containing particles remained constant over time and IGF-1 release was increased. When implanted in the epidydimal fat pad of immunocompromised mice, this composite material improved cell survival and stimulated vessel formation and maturation. Thus, the combination of hMSC and calcium-releasing glass-ceramics represents a new strategy to achieve vessel stabilization, a key factor in the revascularization of ischemic tissues. Statement of Significance: Increasing blood vessel formation in diseased tissues with poor vascularization is a current clinical challenge. Cell therapy using human mesenchymal stem cells has received considerable interest, but low cell survival has hampered its translation to the clinic. Bioglasses and glass-ceramics have been explored as therapeutic agents for stimulating angiogenesis in soft tissues, but these effects need further evaluation in vivo. By incorporating both human mesenchymal stem cells and glass-ceramic particles in an implantable hydrogel, this study provides insights into the vasculogenic potential in soft tissues of the combined strategies. Enhancement of vessel formation and maturation supports further investigation of this strategy.

JTD Keywords: Calcium, Glass-ceramic particles, Vascularization, hMSC, Hydrogel

Extracellular calcium has been proved to influence the healing process of injuries and could be used as a novel therapy for skin wound healing. However, a better understanding of its effect, together with a system to obtain a controlled release is needed. In this study, we examined whether the ionic dissolution of the calcium–phosphate-based ormoglass nanoparticles coded SG5 may produce a similar stimulating effect as extracellular calcium (from CaCl2) on rat dermal fibroblast in vitro. Cells were cultured in the presence of medium containing different calcium concentrations, normally ranging from 0.1 to 3.5 mM Ca2+. A concentration of 3.5 mM of CaCl2 increased metabolic activity, in vitro wound closure, matrix metalloproteinases (MMP) activity, collagen synthesis and cytokine expression, and reduced cell contraction capacity. Interestingly, the levels of migration and contraction capacity measured followed a dose-dependent behavior. In addition, media conditioned with SG5 stimulated the same activities as media conditioned with CaCl2, but undesired effects in chronic wound healing such as inflammatory factor expression and MMP activity were reduced compared to the equivalent CaCl2 concentration. In summary, calcium-releasing particles such as SG5 are potential biological-free biostimulators to be applied in dressings for chronic wound healing.

JTD Keywords: Nanomaterials, Cell signaling, Skin wound healing

This chapter will review the interactions between calcium phosphate (CaP) glasses and different cell types. These glasses are less established in the biomaterials field than silicate-based glasses, but phosphate glasses generate interest owing to their higher solubility. CaP glasses have been less studied than silicate-based glasses, possibly due to the commercialization of Hench's Bioglass that allowed many laboratory groups to use them for different studies, including cell culture studies, without having to prepare them in-house. Studies on CaP glasses focused on compositional modification in order to elicit different properties to enhance biodegradability and bioactivity, two main properties for the application of these glasses. These properties have opened the application of these glasses and have enhanced the effect on cells allowing exploration of the bioactivity of ions released by these exceptionally interesting biomaterials.

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In this work we have evaluated the capacity of bone morphogenetic protein-2 (BMP-2) and fibrin-binding platelet-derived growth factor-BB (PDGF-BB) to support cell growth and induce bone regeneration using two different imaging technologies to improve the understanding of structural and organizational processes participating in tissue repair. Human mesenchymal stem cells from adipose tissue (hAMSCs) expressing two luciferase genes, one under the control of the cytomegalovirus (CMV) promoter and the other under the control of a tissue-specific promoter (osteocalcin or platelet endothelial cell adhesion molecule), were seeded in fibrin matrices containing BMP-2 and fibrin-binding PDGF-BB, and further implanted intramuscularly or in a mouse calvarial defect. Then, cell growth and bone regeneration were monitored by bioluminescence imaging (BLI) to analyze the evolution of target gene expression, indicative of cell differentiation towards the osteoblastic and endothelial lineages. Non-invasive imaging was supplemented with micro-computed tomography (μCT) to evaluate bone regeneration and high-resolution μCT of vascular casts. Results from BLI showed hAMSC growth during the first week in all cases, followed by a rapid decrease in cell number; as well as an increment of osteocalcin but not PECAM-1 expression 3 weeks after implantation. Results from μCT show that the delivery of BMP-2 and PDGF-BB by fibrin induced the formation of more bone and improves vascularization, resulting in more abundant and thicker vessels, in comparison with controls. Although the inclusion of hAMSCs in the fibrin matrices made no significant difference in any of these parameters, there was a significant increment in the connectivity of the vascular network in defects treated with hAMSCs.

JTD Keywords: Angiogenesis, Bioluminescence imaging, Bone regeneration, Fibrin, Mesenchymal stem cell

Each bone of the skeleton constantly undergoes modeling during life to help it to adapt to changing biomechanical forces as well as remodeling to remove old bone and replace it with new, mechanically stronger bone to help preserve bone strength. Bone remodeling involves the removal of mineralized bone by osteoclasts, followed by the formation of bone matrix through the osteoblasts that subsequently become mineralized. All these assets make bone a suitable model for regeneration. Bone tissue can be grossly divided into inorganic mineral material (mostly HA), and organic material from cells and the extracellular matrix. This chapter outlines some of the bone diseases such as osteoporosis and Paget's disease. Bone can be considered as a biphasic composite material, with two phases: one the mineral and the other collagen. This combination confers better mechanical properties on the tissue than each component itself.

JTD Keywords: Bone biology, Bone cells, Bone diseases, Bone extracellular matrix, Bone mechanics, Bone remodeling, Bone tissue regeneration, Skeleton

Cerebral Palsy (CP) is the most frequent disability affecting children. Although the effects of CP are diverse this chapter focuses on the impaired motor control of children suffering from spastic diplegia, particularly in the lower limb. The chapter collects the most relevant techniques that are used or might be useful to overcome the current limitations existing in the diagnosis and rehabilitation of CP. Special emphasis is placed on the role that emerging technologies can play in this field. Knowing in advance the type and site of brain injury could assist the clinician in selecting the appropriate therapy. In this context, neuroimaging techniques are being recommended as an evaluation tool in children with CP; we describe a variety of imaging technologies such as Magnetic Resonance Imaging (MRI), Diffusion Tensor Imaging (DTI), etc. But creating new knowledge in itself is not enough; there must be a transfer from progress through research to advances in the clinical field. The classic therapeutic approach of CP thus hampers the optimal rehabilitation of the targeted component. Traditional therapies may be optimized if complemented with treatments. We try to collect a wide range of emerging technologies and provide some criteria to select the adequate technology based on the characteristics of the neurological injury. For example, exoskeleton based over-ground gait training is suggested to be more effective than treadmill-based gait training. So, we suggest a new point of view combining different technologies in order to provide the foundations of a rational design of the individual rehabilitation strategy.

JTD Keywords: Cerebral palsy, Robotics, Neurostimulation, Neuroimaging, Myoelectric signals

Background: All living organisms are made of individual and identifiable cells, whose number, together with their size and type, ultimately defines the structure and functions of an organism. While the total cell number of lower organisms is often known, it has not yet been defined in higher organisms. In particular, the reported total cell number of a human being ranges between 1012 and 1016 and it is widely mentioned without a proper reference. Aim: To study and discuss the theoretical issue of the total number of cells that compose the standard human adult organism. Subjects and methods: A systematic calculation of the total cell number of the whole human body and of the single organs was carried out using bibliographical and/or mathematical approaches. Results: A current estimation of human total cell number calculated for a variety of organs and cell types is presented. These partial data correspond to a total number of 3.72×1013. Conclusions: Knowing the total cell number of the human body as well as of individual organs is important from a cultural, biological, medical and comparative modelling point of view. The presented cell count could be a starting point for a common effort to complete the total calculation.

JTD Keywords: Cell size, Human cell number, Organ, Theoretical issue, Total cell count