Genetical stability and osteogenic ability of mesenchimal stem cells on demineralized bone matrices

Journal of Osseointegration Volume 7, Issue 1, 1 March 2015, Pages 2-7 Open Access Genetical stability and osteogenic ability of mesenchimal stem cells on demineralized bone matrices (Article) Pozzuoli, A.a, Gardin, C.b, Aldegheri, R.a, Bressan, E.c, Isola, M.d, Calvo-Guirado, J.L.e, Biz, C.a, Arrigoni, P.a, Feroni, L.b, Zavan, B.b a Department of Surgical,Oncological and Gastroenterological Sciences, University of Padua, Padua, Italy b Department of Biomedical Sciences, University of Padua, Padua, Italy c Department of Neurosciences, University of Padua, Padua, Italy d Department of Animal Medicine, Production and Health (MAPS), Italy e Department of General Dentistry, Faculty of Medicine and Dentistry, University of Murcia, Murcia, Spain Hide additional affiliations View references (44) Abstract Aim: Tissue engineering is a rapidly expanding field with regard to the use of biomaterials and stem cells in the orthopedic surgery. Many experimental studies have been done to understand the best characteristics of cells, materials and laboratory methods for safe clinical applications. The aim of this study was to compare the ability of 2 different human demineralized bone matrices (DBMs), the one enriched and the other not enriched with hyaluronic acid, to stimulate in vitro the proliferation and the osteogenic differentiation of human adipose-derived stem cells (ADSCs) seeded onto an osteoconductive scaffold. Materials and Methods: ADSCs were isolated, by enzymatic digestion, from abdominal adipose tissue of 5 patients undergoing cosmetic lipoaspiration surgery. ADSCs were then seeded onto a 3D scaffold in the presence of the two different osteoinductive matrices of human demineralized bone and evaluated for proliferation and osteogenic differentiation. The safety of the methods was verified using array-Comparative Genomic Hybridization (array-CGH). Results: ADSCs were able to differentiate in osteogenic sense. Both DBMs showed the ability to induce osteogenic differentiation of the cells. Conclusion: array-CGH showed no changes at genome level, thus confirming the safety of materials and method

Graphene-based nanomaterials for tissue engineering in the dental field

The world of dentistry is approaching graphene-based nanomaterials as substitutes for tissue engineering. Apart from its exceptional mechanical strength, electrical conductivity and thermal stability, graphene and its derivatives can be functionalized with several bioactive molecules. They can also be incorporated into different scaffolds used in regenerative dentistry, generating nanocomposites with improved characteristics. This review presents the state of the art of graphene-based nanomaterial applications in the dental field. We first discuss the interactions between cells and graphene, summarizing the available in vitro and in vivo studies concerning graphene biocompatibility and cytotoxicity. We then highlight the role of graphene-based nanomaterials in stem cell control, in terms of adhesion, proliferation and differentiation. Particular attention will be given to stem cells of dental origin, such as those isolated from dental pulp, periodontal ligament or dental follicle. The review then discusses the interactions between graphene-based nanomaterials with cells of the immune system; we also focus on the antibacterial activity of graphene nanomaterials. In the last section, we offer our perspectives on the various opportunities facing the use of graphene and its derivatives in associations with titanium dental implants, membranes for bone regeneration, resins, cements and adhesives as well as for tooth-whitening procedure

In vitro and in vivo biological performance of modified gellan gum-based hydrogels for nucleus pulposus tissue engineering

Ionic- (iGG-MA) and photo-crosslinked (phGG-MA) methacrylated gellan gum hydrogels have been proposed as biomaterials for supporting nucleus pulposus (NP) regeneration and/or repair. In this study, the mechanical stability and biocompatibility of these hydrogels have been evaluated in vitro. Human intervertebral disc cells obtained from herniated patients were cultured within both hydrogels, for 1–21 days. Dynamic mechanical analysis and biological characterization (Live/ Dead assay, ATP and DNA quantification, PCR and immunocytochemistry) were performed after specific times of culturing. The in vitro study showed that both cell loading and culturing time do not affect the mechanical properties of hydrogels. In addition, the iGG-MA and phGG-MA hydrogels showed to be effective on supporting cells encapsulation and viability up to 21 days of culturing. In vivo biocompatibility screening was also performed, by subcutaneous implantation of both hydrogels in Lewis rats for the period of 10 and 18 days. Haematoxylin & eosin staining revealed that the hydrogels do not elicit necrosis, calcification or acute inflammatory reaction. The present study demonstrates that the iGG-MA and phGG-MA hydrogels support cells encapsulation and viability, and are well-tolerated, stable and non-cytotoxic in vitro and in vivo, thus possessing promising features for finding application as viable NP substitutes

Mechanical performance and biocompatibility study of methacrylated Gellan gum hydrogels with potential for nucleus pulposus regeneration

Methacrylated gellan gum hydrogels, obtained either by ionic- (iGGMA) and photo-crosslinking (phGG-MA), have been investigated as potential biomaterials for supporting nucleus pulposus (NP) regeneration and/or repair [1,2]. In previous work, some advantages were attributed to GG-MA hydrogels, such as: (i) the possibility to control endothelial cells infiltration and blood vessel ingrowth’s, (ii) tunable and improved mechanical properties, and (iii) in situ gelation, within seconds to few minutes. In this study, the mechanical and biological performance of these hydrogels was firstly evaluated in vitro. Human intervertebral disc (hIVD) cells obtained from herniated patients were cultured within both hydrogels, for 1 up to 21 days. Dynamic mechanical analysis and biological characterization (calcein-AM staining, ATP and DNA quantification and PCR) were performed after specific times of culturing. A biocompatibility study was also performed in vivo, by subcutaneous implantation of acellular iGG-MA and phGG-MA hydrogels in Lewis rats for the period of 10 and 18 days. Tissue response to the hydrogels implantation was determined by histological analysis (haematoxylin-eosin staining). The in vitro study showed that both cell loading and culturing time do not have an effect on the mechanical properties of the hydrogels. Regarding their biological performance, the iGG-MA and phGG-MA hydrogels showed to be effective on supporting hIVD cells encapsulation and viability up to 21 days of culturing. Human IVD cells were homogeneously distributed within the hydrogels and maintained its round-shape morphology during culturing time. The in vivo biocompatibility study showed that iGG-MA and phGG-MA hydrogels do not elicit any deleterious effect, as denoted by the absence of necrosis and calcification, or acute inflammatory reaction. A thin fibrous capsule was observed around the implanted hydrogels. The results presented in this study indicate that the iGG-MA and phGG-MA hydrogels are stable in vitro and in vivo, support hIVD cells encapsulation and viability, and were found to be well-tolerated and non-cytotoxic in vivo, thus being potential candidates for NP regeneration

Transforming growth factor-β/Smad - signalling pathway and conjunctival remodelling in vernal keratoconjunctivitis

Vernal keratoconjunctivitis (VKC) is a chronic ocular allergic inflammation characterized by corneal complications and the formation of giant papillae. Sma- and Mad-related proteins (Smad) modulate extracellular matrix gene expression during wound healing, inflammation and tissue remodelling.Objective To investigate the relationship between allergic inflammation and TGF-β/Smad signalling pathway, expression in VKC patients and in primary cultured conjunctival fibroblasts exposed to mediators found previously over-expressed in VKC.Methods Smad-2, -3, -7, phospho-(p)Smads, TGF-β1 and -β2 were evaluated in the conjunctiva of normal subjects (CT) and VKC patients by immunohistochemistry. The expression of Smads, pro-collagen I (PIP), TGF-β1, -β2, mitogen-activated protein kinase (p38/MAPK), c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK1/2) were also determined in conjunctival fibroblast cultures exposed to histamine, IL-4, -13, TGF-β1, IFN-γ and TNF-α using immunostaining or RT-PCR.Results Immunostaining for Smad-2, -3, pSmad-2, -3, TGF-β1, -β2 and PIP was significantly increased in VKC stroma compared with CT. In conjunctival fibroblast cultures, Smad-3 and PIP were stimulated by histamine, IL-4, -13 and TGF-β1 exposure, while PIP was reduced by IFN-γ, and TNF-α mRNA expression of Smad-3 was increased by histamine, while Smad-7 was reduced by IL-4. In addition, histamine, IL-4 and TNF-α increased JNK and ERK1/2 expression.Conclusion and Clinical Relevance The TGF-β/Smad signalling pathway is over-expressed in VKC tissues and modulated in conjunctival fibroblasts by histamine, IL-4, TGF-β1 and TNF-α. These mechanisms may be involved in fibrillar collagen production, giant papillae formation and tissue remodelling typical of VKC and might provide new therapeutic targets for its treatment. © 2010 Blackwell Publishing Ltd

Hyperbaric oxygen therapy improves the osteogenic and vasculogenic properties of mesenchymal stem cells in the presence of inflammation in vitro

Hyperbaric oxygen (HBO) therapy has been reported to be beneficial for treating many conditions of inflammation-associated bone loss. The aim of this work was to in vitro investigate the effect of HBO in the course of osteogenesis of human Mesenchymal Stem Cells (MSCs) grown in a simulated pro-inflammatory environment. Cells were cultured with osteogenic differentiation factors in the presence or not of the pro-inflammatory cytokine Tumor Necrosis Factor-α (TNF-α), and simultaneously exposed daily for 60 min, and up to 21 days, at 2,4 atmosphere absolute (ATA) and 100% O2. To elucidate osteogenic differentiation-dependent effects, cells were additionally pre-committed prior to treatments. Cell metabolic activity was evaluated by means of the MTT assay and DNA content quantification, whereas osteogenic and vasculogenic differentiation was assessed by quantification of extracellular calcium deposition and gene expression analysis. Metabolic activity and osteogenic properties of cells did not differ between HBO, high pressure (HB) alone, or high oxygen (HO) alone and control if cells were pre-differentiated to the osteogenic lineage. In contrast, when treatments started contextually to the osteogenic differentiation of the cells, a significant reduction in cell metabolic activity first, and in mineral deposition at later time points, were observed in the HBO-treated group. Interestingly, TNF-α supplementation determined a significant improvement in the osteogenic capacity of cells subjected to HBO, which was not observed in TNF-α-treated cells exposed to HB or HO alone. This study suggests that exposure of osteogenic-differentiating MSCs to HBO under in vitro simulated inflammatory conditions enhances differentiation towards the osteogenic phenotype, providing evidence of the potential application of HBO in all those processes requiring bone regeneration

The mechanism of Pseudomonas aeruginosa outer membrane vesicle biogenesis determines their protein composition.

Gram-negative bacteria produce outer membrane vesicles (OMVs) and contain bacterial cargo including nucleic acids and proteins. The proteome of OMVs can be altered by various factors including bacterial growth stage, growth conditions, and environmental factors. However, it is currently unknown if the mechanism of OMV biogenesis can determine their proteome. In this study, we examined whether the mechanisms of OMV biogenesis influenced the production and protein composition of Pseudomonas aeruginosa OMVs. OMVs were isolated from three P. aeruginosa strains that produced OMVs either by budding alone, by explosive cell lysis, or by both budding and explosive cell lysis. We identified that the mechanism of OMV biogenesis dictated OMV quantity. Furthermore, a global proteomic analysis comparing the proteome of OMVs to their parent bacteria showed significant differences in the identification of proteins in bacteria and OMVs. Finally, we determined that the mechanism of OMV biogenesis influenced the protein composition of OMVs, as OMVs released by distinct mechanisms of biogenesis differed significantly from one another in their proteome and functional enrichment analysis. Overall, our findings reveal that the mechanism of OMV biogenesis is a main factor that determines the OMV proteome which may affect their subsequent biological functions

Stem cell-derived small extracellular vesicles embedded into methacrylated hyaluronic acid wound dressings accelerate wound repair in a pressure model of diabetic ulcer

Over the past years, the development of innovative smart wound dressings is revolutionizing wound care management and research. Specifically, in the treatment of diabetic foot wounds, three-dimensional (3D) bioprinted patches may enable personalized medicine therapies. In the present work, a methacrylated hyaluronic acid (MeHA) bioink is employed to manufacture 3D printed patches to deliver small extracellular vesicles (sEVs) obtained from human mesenchymal stem cells (MSC-sEVs). The production of sEVs is maximized culturing MSCs in bioreactor. A series of in vitro analyses are carried out to demonstrate the influence of MSC-sEVs on functions of dermal fibroblasts and endothelial cells, which are the primary functional cells in skin repair process. Results demonstrate that both cell populations are able to internalize MSC-sEVs and that the exposure to sEVs stimulates proliferation and migration. In vivo experiments in a well-established diabetic mouse model of pressure ulcer confirm the regenerative properties of MSC-sEVs. The MeHA patch enhances the effectiveness of sEVs by enabling controlled release of MSC-sEVs over 7 days, which improve wound epithelialization, angiogenesis and innervation. The overall findings highlight that MSC-sEVs loading in 3D printed biomaterials represents a powerful technique, which can improve the translational potential of parental stem cell in terms of regulatory and economic impact

Citrate Mediates Crosstalk between Mitochondria and the Nucleus to Promote Human Mesenchymal Stem Cell In Vitro Osteogenesis

Citrate, generated in the mitochondria, is a key metabolite that might link metabolism with signaling, chromatin structure and transcription to orchestrate mesenchymal stem cells (MSCs) fate determination. Based on a detailed morphological analysis of 3D reconstruction of mitochondria and nuclei in single cells, we identified contact sites between these organelles that drastically increase in volume and number during the early stage of mesenchymal stem cell differentiation. These contact sites create a microdomain that facilitates exchange of signals from mitochondria to the nucleus. Interestingly, we found that the citrate derived from mitochondria is necessary for osteogenic lineage determination. Indeed, inhibition of the citrate transporter system dramatically affected osteogenesis, reduced citrate levels that could be converted in α-ketoglutarate, and consequently affected epigenetic marker H3K9me3 associated with the osteogenesis differentiation process. These findings highlight that mitochondrial metabolites play key regulatory roles in the MSCs differentiation process. Further in-depth investigation is needed to provide novel therapeutic strategies in the field of regenerative medicine