Co–culture systems of osteoblasts and osteoclasts: Simulating in vitro bone remodeling in regenerative approaches

Bone is an extremely dynamic tissue, undergoing continuous remodeling for its whole lifetime, but its regeneration or augmentation due to bone loss or defects are not always easy to obtain. Bone tissue engineering (BTE) is a promising approach, and its success often relies on a “smart”scaffold, as a sup- port to host and guide bone formation through bone cell precursors. Bone homeostasis is maintained by osteoblasts (OBs) and osteoclasts (OCs) within the basic multicellular unit, in a consecutive cycle of resorption and formation. Therefore, a functional scaffold should allow the best possible OB/OC cooper- ation for bone remodeling, as happens within the bone extracellular matrix in the body. In the present work OB/OC co-culture models, with and without scaffolds, are reviewed. These experimental systems are intended for different targets, including bone remodeling simulation, drug testing and the assessment of biomaterials and 3D scaffolds for BTE. As a consequence, several parameters, such as cell type, cell ratio, culture medium and inducers, culture times and setpoints, assay methods, etc. vary greatly. This review identifies and systematically reports the in vitro methods explored up to now, which, as they al- low cellular communication, more closely resemble bone remodeling and/or the regeneration process in the framework of BTE

Co–culture systems of osteoblasts and osteoclasts: Simulating in vitro bone remodeling in regenerative approaches

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Analysis of multiple protein detection methods in human osteoporotic bone extracellular matrix: From literature to practice

The punctual analysis of bone Extracellular Matrix (ECM) proteins represents a pivotal point for medical research in bone diseases like osteoporosis. Studies in this field, historically done to appreciate bone biology, were mainly conducted on animal samples and, up to today, only a few studies on protein detection in human bone are present. The challenges in bone ECM protein extraction and quantitation protocols are related to both the separation of proteins from the mineral content (i.e. hydroxyapatite) and the difficulty of avoiding protein denaturation during the extraction processes. The aim of the present work was to define appropriate protocol(s) for bone ECM protein extraction that could be applied to investigate both normal and pathological conditions. We compared and optimised some of the most used protocols present in the literature, modifying the protein precipitation method, the buffer used for resuspension and/or the volume of reagent used. Bradford and BCA assays and Western Blotting were used to evaluate the variations in the total protein recovery and the amount of selected proteins (Type I Collagen, TGF-β, IGF-1, Decorin, Osteopontin, Bone Sialoprotein-2 and Osteocalcin). Collectively, we were capable to draw-up two single-extract protocols with optimal recovery and ideal protein content, that can be used for a detailed analysis of ECM proteins in pathological bone samples. Time-consuming multi-extract procedures, optimised in their precipitation methods, are however crucial for a precise detection of specific proteins, like osteocalcin. As the matter of fact, also the demineralization processes, commonly suggested and performed in several protocols, could hinder an accurate protein detection, thus inherently affecting the study of a pathological bone ECM. This study represents a starting point for the definition of appropriate strategies in the study of bone extracellular matrix proteins involved in the onset and maintenance of bone diseases, as well as a tool for the development of customized scaffolds capable to modulate a proper feedback loop in bone remodelling, altered in case of diseases like osteoporosis

Analysis of multiple protein detection methods in human osteoporotic bone extracellular matrix: From literature to practice

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Synovium-derived stromal cell-induced osteoclastogenesis: a potential osteoarthritis trigger

none7noPurpose: To shed light on the idea that mesenchymal stem/stromal cells (MSCs) recruited in synovium (SM) (i.e. Synovium-Derived Stromal Cells, SDSCs) could be involved in Osteoarthritis (OA) pathophysiology. Attention was also paid to a further stromal cell type with a peculiar ultrastructure called telocytes (TCs), whose role is far from clarified. Methods: In the present in vitro study, we compared SDSCs isolated from healthy and OA subjects in terms of phenotype, morphology and differentiation potential as well as in their capability to activate normal Peripheral Blood Mononuclear Cells (PBMCs). Histological, immunohistochemical and ultrastructural analyses were integrated by qRT-PCR and functional resorbing assays. Results: Our data demonstrated that both SDSC populations stimulated the formation of osteoclasts from PBMCs: the osteoclast-like cells generated by healthy-SDSCs via transwell co-cultures were inactive, while OA-derived SDSCs have a much greater effectiveness. Moreover, the presence of TCs was more evident in cultures obtained from OA subjects and suggests a possible involvement of these cells in OA. Conclusions: Osteoclastogenic differentiation capability of PBMCs from OA subjects, also induced by B synoviocytes has been already documented. Here we hypothesized that SDSCs, generally considered for their regenerative potential in cartilage lesions, have also a role in the onset/maintenance of OA. Clinical relevance: Our observations may represent an interesting opportunity for the development of a holistic approach for OA treatment, that considers the multifaceted capability of MSCs in relation to the environment.embargoed_20210517Dicarlo, Manuela; Teti, Gabriella; Cerqueni, Giorgia; Iezzi, Iolanda; Gigante, Antonio; Falconi, Mirella; Mattioli-Belmonte, MonicaDicarlo, Manuela; Teti, Gabriella; Cerqueni, Giorgia; Iezzi, Iolanda; Gigante, Antonio; Falconi, Mirella; Mattioli-Belmonte, Monic

Cobalt chloride supplementation differently affects human mesenchymal stem cells isolated from dental pulp, umbilical cord and adipose tissues in their chondrogenic potential

Articular cartilage is an avascular tissue without innervations, characterized by low cell density and abundant extracellular matrix (ECM). These characteristics leave articular cartilage with very limited capacity of repair and regeneration. Multipotent stem/stromal cells (MSC) are considered promising for cartilage tissue engineering. Stem cells are resided in a special microenvironment known as the stem-cell niche, characterized by the presence of low oxygen concentration. Previous studies have reported that hypoxic conditions could enhance the chondrogenic differentiation of mesenchymal stem cells in the presence of an inductive medium. Cobalt chloride (CoCl2) imitates hypoxia in vitro by preventing hypoxia-inducible factor-alpha (HIF-a) from being destroyed by oxygen. However, the long-term hypoxic culture of stem cells is difficult and requires special attention to avoid cell death due to cobalt treatment. In this study we investigated if CoCl2 affected MSCs isolated from dental pulp, umbilical cord and adipose tissue in their potential to differentiate toward the chondrogenic phenotype. Cells were treated with concentrations of CoCl2 ranging from 50 to 400 uM. Cell proliferation, mRNA expression of stem-cell marker and chondrogenic associated genes were analyzed by RT-PCR and Real-time PCR. The results showed that the CoCl2 supplementation had no effect on the proliferation of all the three type of cells analyzed, while the up-regulation of chondrogenic markers such as aggrecan, sox9, and type II collagen, was dependent on the cellular source. This study shows that hypoxia induced by CoCl2 treatment can differently influence the behavior of MSCs of different sources in their chondrogenic potential. These findings should be taken into consideration in the treatment of cartilage repair and regeneration based on stem cell therapies

Bone and gut microbiota crosstalk: A novel 3D in vitro approach

The present research aimed at shedding light on the interplay between the composition of the human gut microbiota and bone cells

Bone regeneration strategies in the elderly: the role of ageing and replicative senescence in periosteal-derived stem cells

Periosteum contains resident progenitor cells (PDPCs) representing an attractive alternative source of mesenchymal stem cells (MSCs) for skeletal tissue engineering approaches based on cell recruitment (1). Increased in life expectancy point out the necessity for customized strategies to restore bone loss due to trauma and/or disease in elderly. Aim of the present research was the evaluation of the ageing impact on PDPCs isolated from differently aged subjects. Moreover, since long-term culture could lead MSCs to senescence, the effects of culture expansion method on young PDPC through sequential serial passages were examined. Age-related increase of p53 expression and impairment in proliferating capacity were observed; those findings were strictly related to nitric oxide (NO) release. Moreover, qRT-PCR analysis showed a greater expression of genes involved in bone remodelling in elderly donors. As far as replicative in vitro expansion was concerned, we observed that later PDPC passages exhibited the typical “replicative senescence” features (i.e. flattened and enlarged morphology, prolonged population doubling time and increased SA-βgal activity). In these cells, p16 rather than p53 seemed to be involved in senescence processes. Similarly to the elderly, the decrease in proliferating ability of in vitro senescent PDPCs was concomitant with a higher NO production, and the changes in the expression of genes involved in bone resorption and RANKL/OPG ratio were superimposable. Interestingly, the relationship between NO release and ageing could represent a cutting edge “replicative senescence index” as emerged by our System Biology approach. In conclusion, our findings suggest that in vivo cell ageing and in vitro subculturing must be taken into account when testing regenerative tissue strategies that use progenitor cells. Indeed, cells (e.g. MSCs and PDPCs) from the earliest subculture passages could be useful to validate any bone tissue engineering strategies, whilst the later ones could be used to test in vitro scaffolds for regenerative medicine approaches in elderly.This work was supported by grants from MIUR (Project PRIN 2010, MIND-2010J8RYS7)

Aging of periosteal-derived stem cells during expansion: an alternative tool for a customized bone regenerative strategy

Increased in life expectancy points out the necessity for tailored strategies to restore bone loss due to trauma and/or disease in elderly. Moreover, there is a compelling need for improved cell systems to test scaffolds interfacing with an “aging” tissue. For skeletal tissue regeneration, periosteal-derived stem cells (PDPCs) could represent an easily recruited source of Mesenchymal stromal cells (MSCs) [1,2]. This study investigated the effects of long-term in vitro expansion on the stability and function of PDPCs, since extensive culture expansion is usually performed to obtain clinically relevant cell numbers, but its impact on cell behaviour is still unclear. An integrated approach based on flow cytometry, ultrastructural and quantitative Real time PCR (qRT-PCR) analyses was adopted. Senescent cell data were compared with those of cells isolated from differently aged subjects. Both replicative-senescent PDPCs and cells isolated from old donors were permanently blocked in G1 phase of cell cycle, through a pathway that seemed to involve nitric oxide (NO) production and the expression of tumour suppressor proteins p16 or p53, respectively. Changes in the expression of MSC surface markers were detected in PDPCs during subculturing, whilst it was superimposable in young and aged PDPCs. Cytofluorimetric analysis of the physical parameters (i.e. FSC and SSC) showed a trend toward an increase in cell dimension and internal complexity in both populations analysed. This data was consistent with morphological observation that also evidenced similar alterations in mitochondrial shape. In addition, an intense autophagic activity in early passage PDPCs was observed, whilst in the late passages cells had a robust protein synthesis activity that could be related with “senescence-associated secretory phenotype” (SASP). In conclusion, the morphofunctional similarities detected in replicative-senescent and aged PDPCs suggest that their long-term expansion could be a reproducible and useful tool to mimic in vivo ageing

Physicochemical Characterization of Pectin-Gelatin Biomaterial Formulations for 3D Bioprinting.

AbstractDeveloping biomaterial formulations with specific biochemical characteristics and physical properties suitable for bioprinting of 3D scaffolds is a pivotal challenge in tissue engineering. Therefore, the design of novel bioprintable formulations is a continuously evolving research field. In this work, the authors aim at expanding the library of biomaterial inks by blending two natural biopolymers: pectin and gelatin. Cytocompatible formulations are obtained by combining pectin and gelatin at different ratios and using (3‐glycidyloxypropyl)trimethoxysilane (GPTMS) as single crosslinking agent. It is shown that the developed formulations are all suitable for extrusion‐based 3D bioprinting. Self‐supporting scaffolds with a designed macroporosity and micropores in the bioprinted struts are successfully obtained by combining extrusion‐based bioprinting and freeze‐drying. The presence of gelatin in these formulations allows for the modulation of porosity, of water uptake and of scaffold stiffness in respect to pure pectin scaffolds. Results demonstrate that these new biomaterial formulations, processed with this specific approach, are promising candidates for the fabrication of tissue‐like scaffolds for tissue regeneration