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In the last few years, additive manufacturing (AM) has been gaining great interest in the fabrication of complex structures for soft-to-hard tissues regeneration, with tailored porosity, and boosted structural, mechanical, and biological properties. 3D printing is one of the most known AM techniques in the field of biofabrication of tissues and organs. This technique opened up opportunities over the conventional ones, with the capability of creating replicable, customized, and functional structures that can ultimately promote effectively different tissues regeneration. The uppermost component of 3D printing is the bioink, i.e. a mixture of biomaterials that can also been laden with different cell types, and bioactive molecules. Important factors of the fabrication process include printing fidelity, stability, time, shear-thinning properties, mechanical strength and elasticity, as well as cell encapsulation and cell-compatible conditions. Collagen-based materials have been recognized as a promising choice to accomplish an ideal mimetic bioink for regeneration of several tissues with high cell-activating properties. This review presents the state-of-art of the current achievements on 3D printing using collagen-based materials for hard tissue engineering, particularly on the development of scaffolds for bone and cartilage repair/regeneration. The ultimate aim is to shed light on the requirements to successfully print collagen-based inks and the most relevant properties exhibited by the so fabricated scaffolds. In this regard, the adequate bioprinting parameters are addressed, as well as the main materials properties, namely physicochemical and mechanical properties, cell compatibility and commercial availability, covering hydrogels, microcarriers and decellularized matrix components. Furthermore, the fabrication of these bioinks with and without cells used in inkjet printing, laser-assisted printing, and direct in writing technologies are also overviewed. Finally, some future perspectives of novel bioinks are given.

Background Mesenchymal stem cell therapy for osteoarthritis (OA) has been widely investigated, but the mechanisms are still unclear. Exosomes that serve as carriers of genetic information have been implicated in many diseases and are known to participate in many physiological processes. Here, we investigate the therapeutic potential of exosomes from human embryonic stem cell-induced mesenchymal stem cells (ESC-MSCs) in alleviating osteoarthritis (OA). Methods Exosomes were harvested from conditioned culture media of ESC-MSCs by a sequential centrifugation process. Primary mouse chondrocytes treated with interleukin 1 beta (IL-1β) were used as an in vitro model to evaluate the effects of the conditioned medium with or without exosomes and titrated doses of isolated exosomes for 48 hours, prior to immunocytochemistry or western blot analysis. Destabilization of the medial meniscus (DMM) surgery was performed on the knee joints of C57BL/6 J mice as an OA model. This was followed by intra-articular injection of either ESC-MSCs or their exosomes. Cartilage destruction and matrix degradation were evaluated with histological staining and OARSI scores at the post-surgery 8 weeks. Results We found that intra-articular injection of ESC-MSCs alleviated cartilage destruction and matrix degradation in the DMM model. Further in vitro studies illustrated that this effect was exerted through ESC-MSC-derived exosomes. These exosomes maintained the chondrocyte phenotype by increasing collagen type II synthesis and decreasing ADAMTS5 expression in the presence of IL-1β. Immunocytochemistry revealed colocalization of the exosomes and collagen type II-positive chondrocytes. Subsequent intra-articular injection of exosomes derived from ESC-MSCs successfully impeded cartilage destruction in the DMM model. Conclusions The exosomes from ESC-MSCs exert a beneficial therapeutic effect on OA by balancing the synthesis and degradation of chondrocyte extracellular matrix (ECM), which in turn provides a new target for OA drug and drug-delivery system development.

Background The mechanisms underpinning the regenerative capabilities of mesenchymal stem cells (MSC) were originally thought to reside in their ability to recognise damaged tissue and to differentiate into specific cell types that would replace defective cells. However, recent work has shown that molecules produced by MSCs (secretome), particularly those packaged in extracellular vesicles (EVs), rather than the cells themselves are responsible for tissue repair. Methods Here we have produced a secretome from adipose-derived mesenchymal stem cells (ADSC) that is free of exogenous molecules by incubation within a saline solution. Various in vitro models were used to evaluate the effects of the secretome on cellular processes that promote tissue regeneration. A cardiotoxin-induced skeletal muscle injury model was used to test the regenerative effects of the whole secretome or isolated extracellular vesicle fraction in vivo. This was followed by bioinformatic analysis of the components of the protein and miRNA content of the secretome and finally compared to a secretome generated from a secondary stem cell source. Results Here we have demonstrated that the secretome from adipose-derived mesenchymal stem cells shows robust effects on cellular processes that promote tissue regeneration. Furthermore, we show that the whole ADSC secretome is capable of enhancing the rate of skeletal muscle regeneration following acute damage. We assessed the efficacy of the total secretome compared with the extracellular vesicle fraction on a number of assays that inform on tissue regeneration and demonstrate that both fractions affect different aspects of the process in vitro and in vivo. Our in vitro, in vivo , and bioinformatic results show that factors that promote regeneration are distributed both within extracellular vesicles and the soluble fraction of the secretome. Conclusions Taken together, our study implies that extracellular vesicles and soluble molecules within ADSC secretome act in a synergistic manner to promote muscle generation.

Background Angiogenesis, as an endogenous repair mechanism, plays crucial roles in wound healing and tissue regeneration. However, this process is impaired in the elderly due to aging-related vascular endothelial dysfunction. This study was aimed to explore the pro-angiogenic effects of exosomes from human embryonic stem cells (ESC-Exos) in aged mice of pressure-induced ulcer model and the underlying mechanism. Methods Pressure ulcer wounds were created on the back of d -galactose-induced aging mice. ESC-Exos were locally applied onto the wound beds, with PBS as control. The effects of ESC-Exos on wound healing were analyzed by measuring wound closure rates, histological and immunofluorescence analyses. Then, the anti-aging effect of ESC-Exos on vascular endothelial cells was tested in an in vitro d -galactose-induced HUVEC senescence model. Results ESC-Exos could accelerate wound closure and enhance angiogenesis, and the senescence of vascular endothelial cells was significantly ameliorated after ESC-Exos treatment. In vitro, ESC-Exos could rejuvenate the senescence of endothelial cells and recover compromised proliferation, migratory capacity, and tube formation. This recovery was Nrf2-activation-dependent, since cotreatment with Nrf2 inhibitor Brusatol could abolish the rejuvenative effects of ESC-Exos. Further study revealed that miR-200a was highly enriched in ESC-Exos and played a crucial role in ESC-Exos-mediated rejuvenation through downregulating Keap1, which negatively regulates Nrf2 expression. Conclusions ESC-Exos ameliorate endothelial senescence by activating Nrf2 and recover aging-related angiogenic dysfunction, thereby accelerating wound healing in aged mice. ESC-Exos might be a natural nano-biomaterial for aging-related diseases therapy.

Background Primary mesenchymal stem cells (MSCs) are fraught with aging-related shortfalls. Human-induced pluripotent stem cell (iPSC)-derived MSCs (iMSCs) have been shown to be a useful clinically relevant source of MSCs that circumvent these aging-associated drawbacks. To date, the extent of the retention of aging-hallmarks in iMSCs differentiated from iPSCs derived from elderly donors remains unclear. Methods Fetal femur-derived MSCs (fMSCs) and adult bone marrow MSCs (aMSCs) were isolated, corresponding iPSCs were generated, and iMSCs were differentiated from fMSC-iPSCs, from aMSC-iPSCs, and from human embryonic stem cells (ESCs) H1. In addition, typical MSC characterization such as cell surface marker expression, differentiation capacity, secretome profile, and trancriptome analysis were conducted for the three distinct iMSC preparations—fMSC-iMSCs, aMSC-iMSCs, and ESC-iMSCs. To verify these results, previously published data sets were used, and also, additional aMSCs and iMSCs were analyzed. Results fMSCs and aMSCs both express the typical MSC cell surface markers and can be differentiated into osteogenic, adipogenic, and chondrogenic lineages in vitro . However, the transcriptome analysis revealed overlapping and distinct gene expression patterns and showed that fMSCs express more genes in common with ESCs than with aMSCs. fMSC-iMSCs, aMSC-iMSCs, and ESC-iMSCs met the criteria set out for MSCs. Dendrogram analyses confirmed that the transcriptomes of all iMSCs clustered together with the parental MSCs and separated from the MSC-iPSCs and ESCs. iMSCs irrespective of donor age and cell type acquired a rejuvenation-associated gene signature, specifically, the expression of INHBE , DNMT3B , POU5F1P1 , CDKN1C , and GCNT2 which are also expressed in pluripotent stem cells (iPSCs and ESC) but not in the parental aMSCs. iMSCs expressed more genes in common with fMSCs than with aMSCs. Independent real-time PCR comparing aMSCs, fMSCs, and iMSCs confirmed the differential expression of the rejuvenation ( COX7A , EZA2 , and TMEM119 ) and aging ( CXADR and IGSF3 ) signatures. Importantly, in terms of regenerative medicine, iMSCs acquired a secretome (e.g., angiogenin, DKK-1, IL-8, PDGF-AA, osteopontin, SERPINE1, and VEGF) similar to that of fMSCs and aMSCs, thus highlighting their ability to act via paracrine signaling. Conclusions iMSCs irrespective of donor age and cell source acquire a rejuvenation gene signature. The iMSC concept could allow circumventing the drawbacks associated with the use of adult MSCs und thus provide a promising tool for use in various clinical settings in the future.

Background Low-intensity pulsed ultrasound (LIPUS) can induce mesenchymal stem cell (MSC) differentiation, although the mechanism of its potential effects on chondrogenic differentiation is unknown. Since autophagy is known to regulate the differentiation of MSCs, the aim of our study was to determine whether LIPUS induced chondrogenesis via autophagy regulation. Methods MSCs were isolated from the rat bone marrow, cultured in either standard or chondrogenic medium, and stimulated with 3 MHz of LIPUS given in 20% on–off cycles, with or without prior addition of an autophagy inhibitor or agonist. Chondrogenesis was evaluated on the basis of aggrecan (AGG) organization and the amount of type II collagen (COL2) and the mRNA expression of AGG, COL2, and SRY-related high mobility group-box gene 9 (SOX9) genes. Results LIPUS promoted the chondrogenic differentiation of MSCs, as shown by the changes in the extracellular matrix (ECM) proteins and upregulation of chondrogenic genes, and these effects were respectively augmented and inhibited by the autophagy inhibitor and agonist. Conclusions Taken together, these results indicate that LIPUS promotes MSC chondrogenesis by inhibiting autophagy.

Background Mesenchymal stem cells (MSCs) produced for clinical purposes rely on culture media containing fetal bovine serum (FBS) which is xenogeneic and has the potential to significantly alter the MSC phenotype, rendering these cells immunogenic. As a result of bovine-derived exogenous proteins expressed on the cell surface, MSCs may be recognized by the host immune system as non-self and be rejected. Platelet lysate (PL) may obviate some of these concerns and shows promising results in human medicine as a possible alternative to FBS. Our goal was to evaluate the use of equine platelet lysate (ePL) pooled from donor horses in place of FBS to culture equine MSCs. We hypothesized that ePL, produced following apheresis, will function as the sole media supplement to accelerate the expansion of equine bone marrow-derived MSCs without altering their phenotype and their immunomodulatory capacity. Methods Platelet concentrate was obtained via plateletpheresis and ePL were produced via freeze-thaw and centrifugation cycles. Population doublings (PD) and doubling time (DT) of bone marrow-derived MSCs ( n = 3) cultured with FBS or ePL media were calculated. Cell viability, immunophenotypic analysis, and trilineage differentiation capacity of MSCs were assessed accordingly. To assess the ability of MSCs to modulate inflammatory responses, E. coli lipopolysaccharide (LPS)-stimulated monocytes were cocultured with MSCs cultured in the two different media formulations, and cell culture supernatants were assayed for the production of tumor necrosis factor (TNF)-α. Results Our results showed that MSCs cultured in ePL media exhibited similar proliferation rates (PD and DT) compared with those cultured in FBS at individual time points. MSCs cultured in ePL showed a statistically significant increased viability following a single washing step, expressed similar levels of MSC markers compared to FBS, and were able to differentiate towards the three lineages. Finally, MSCs cultured in ePL efficiently suppressed the release of TNF-α when exposed to LPS-stimulated monocytes similar to those cultured in FBS. Conclusion ePL has the potential to be used for the expansion of MSCs before clinical application, avoiding the concerns associated with the use of FBS.

BackgroundMedical management of the severe musculocutaneous radiation syndrome involves surgical intervention with debridement of necrotic tissue. Even when skin excision is replaced by specific plastic surgery, treatment of the muscle radiation injury nonetheless remains difficult, for it involves a massive muscle defect in an unpredictable environment, subject to inflammatory waves weeks to months after irradiation, which delay healing and predispose the patient to the development of fibrous scar tissue. In this study, we investigated the long-term effect of local injections of bone marrow-derived mesenchymal stromal cells (BM-MSCs), combined with plastic surgery, to treat muscle necrosis in a large animal model.MethodsThree months after irradiation to the rump, minipigs were treated by excision of necrotic muscle tissue, vascularized flap surgery, and four injections with or without local autologous BM-MSCs, performed weekly. The quality of the muscle wound healing was examined 1year post-surgery.ResultsThe skeletal muscle surgery without MSC treatment led to permanent deposition of collagen 1 and 3, decreased myofiber diameter, failed muscle fiber regeneration, a reduced number of capillaries, and the accumulation of high calcium and fat. In animals treated by surgery and MSC injections, these indicators were substantially better and demonstrated established regeneration. MSC therapy acts at several levels by stimulating growth factors such as VEGF, which is involved in angiogenesis and satellite cell pool maintenance, and creating a macrophage M1/M2 balance.ConclusionThus, cell therapy using BM-MSCs is an effective and safe way to improve recovery of irradiation-induced skeletal muscle damage without signs of long-term degeneration.

Titanium (Ti) is a material frequently used in orthopedic applications, due to its good mechanical properties and high corrosion resistance. However, formation of a non-adherent fibrous tissue between material and bone drastically could affect the osseointegration process and, therefore, the mechanical stability of the implant. Modifications of topography and configuration of the tissue/material interface is one of the mechanisms to improve that process by manipulating parameters such as morphology and roughness. There are different techniques that can be used to modify the titanium surface; plasma electrolytic oxidation (PEO) is one of those alternatives, which consists of obtaining porous anodic coatings by controlling parameters such as voltage, current, anodizing solution and time of the reaction. From all of the above factors, and based on previous studies that demonstrated that bone cells sense substrates features to grow new tissue, in this work commercially pure Ti (c.p Ti) and Ti6Al4V alloy samples were modified at their surface by PEO in different anodizing solutions composed of H2SO4 and H3PO4 mixtures. Treated surfaces were characterized and used as platforms to grow osteoblasts; subsequently, cell behavior parameters like adhesion, proliferation and differentiation were also studied. Although the results showed no significant differences in proliferation, differentiation and cell biological activity, overall results showed an important influence of topography of the modified surfaces compared with polished untreated surfaces. Finally, this study offers an alternative protocol to modify surfaces of Ti and their alloys in a controlled and reproducible way in which biocompatibility of the material is not compromised and osseointegration would be improved.

Background Dental pulp represents an easily accessible autologous source of adult stem cells. A subset of these cells, named dental pulp pluripotent-like stem cells (DPPSC), shows high plasticity and can undergo multiple population doublings, making DPPSC an appealing tool for tissue repair or maintenance. Methods DPPSC were harvested from the dental pulp of third molars extracted from young patients. Growth factors released by DPPSC were analysed using antibody arrays. Cells were cultured in specific differentiation media and their endothelial, smooth and skeletal muscle differentiation potential was evaluated. The therapeutic potential of DPPSC was tested in a wound healing mouse model and in two genetic mouse models of muscular dystrophy ( Scid/mdx and Sgcb-null Rag2-null γc-null ). Results DPPSC secreted several growth factors involved in angiogenesis and extracellular matrix deposition and improved vascularisation in all three murine models. Moreover, DPPSC stimulated re-epithelialisation and ameliorated collagen deposition and organisation in healing wounds. In dystrophic mice, DPPSC engrafted in the skeletal muscle of both dystrophic murine models and showed integration in muscular fibres and vessels. In addition, DPPSC treatment resulted in reduced fibrosis and collagen content, larger cross-sectional area of type II fast-glycolytic fibres and infiltration of higher numbers of proangiogenic CD206 + macrophages. Conclusions Overall, DPPSC represent a potential source of stem cells to enhance the wound healing process and slow down dystrophic muscle degeneration.