Collagen and non-collagenous proteins molecular crosstalk in the pathophysiology of osteoporosis.

Abstract Collagenous and non-collagenous proteins (NCPs) in the extracellular matrix, as well as the coupling mechanisms between osteoclasts and osteoblasts, work together to ensure normal bone metabolism. Each protein plays one or more critical roles in bone metabolism, sometimes even contradictory, thus affecting the final mechanical, physical and chemical properties of bone tissue. Anomalies in the amount and structure of one or more of these proteins can cause abnormalities in bone formation and resorption, which consequently leads to malformations and defects, such as osteoporosis (OP). The connections between key proteins involved in matrix formation and resorption are far from being elucidated. In this review, we resume knowledge on the crosstalk between collagen type I and selected NCPs (Transforming Growth Factor-β, Insulin-like Growth Factor-1, Decorin, Osteonectin, Osteopontin, Bone Sialoprotein and Osteocalcin) of bone matrix, focusing on their possible involvement and role in OP. The different elements of this network can be pharmacologically targeted or used for the design/development of innovative regenerative strategies to modulate a feedback loop in bone remodelling

Biofabrication and Bone Tissue Regeneration: Cell Source, Approaches, and Challenges

The growing occurrence of bone disorders and the increase in aging population have resulted in the need for more effective therapies to meet this request. Bone tissue engineering strategies, by combining biomaterials, cells, and signaling factors, are seen as alternatives to conventional bone grafts for repairing or rebuilding bone defects. Indeed, skeletal tissue engineering has not yet achieved full translation into clinical practice because of several challenges. Bone biofabrication by additive manufacturing techniques may represent a possible solution, with its intrinsic capability for accuracy, reproducibility, and customization of scaffolds as well as cell and signaling molecule delivery. This review examines the existing research in bone biofabrication and the appropriate cells and factors selection for successful bone regeneration as well as limitations affecting these approaches. Challenges that need to be tackled with the highest priority are the obtainment of appropriate vascularized scaffolds with an accurate spatiotemporal biochemical and mechanical stimuli release, in order to improve osseointegration as well as osteogenesis

Membrane-seeded autologous chondrocytes: cell viability and characterization at surgery

The implantation of chondrocytes, seeded on matrices such as hyaluronic acid or collagen membranes, is a method that is being widely used for the treatment of chondral defects. The aim of the present study was to evaluate the distribution, viability and phenotype expression of the cells seeded on a collagen membrane just at the time of the implantation. Twelve patients who were suffering from articular cartilage lesions were treated by the MACI(®) procedure. The residual part of each membrane was tested by colorimetric assay (MTT) and histochemical and ultrastructural analyses were carried out. In all of the samples a large number of viable cells, quite homogenously distributed, was detected. The cells expressed the markers of the differentiated hyaline chondrocytes. These data reassure in that the MACI procedure provides a suitable engineered tissue for cartilage repair, in line with the clinical evidences emerging in the literature

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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Biofabrication of bundles of poly(lactic acid)-collagen blends mimicking the fascicles of the human Achille tendon

Electrospinning is a promising technique for the production of scaffolds aimed at the regeneration of soft tissues. The aim of this work was to develop electrospun bundles mimicking the architecture and mechanical properties of the fascicles of the human Achille tendon. Two different blends of poly(L-lactic acid) (PLLA) and collagen (Coll) were tested, PLLA/Coll-75/25 and PLLA/Coll-50/50, and compared with bundles of pure PLLA. First, a complete physico-chemical characterization was performed on non-woven mats made of randomly arranged fibers. The presence of collagen in the fibers was assessed by thermogravimetric analysis, differential scanning calorimetry and water contact angle measurements. The collagen release in phosphate buffer solution (PBS) was evaluated for 14 days: results showed that collagen loss was about 50% for PLLA/Coll-75/25 and 70% for PLLA/Coll-50/50. In the bundles, the individual fibers had a diameter of 0.48 ±0.14 μm (PLLA), 0.31 ±0.09 μm (PLLA/Coll-75/25), 0.33 ±0.08 μm (PLLA/Coll-50/50), whereas bundle diameter was in the range 300-500 μm for all samples. Monotonic tensile tests were performed to measure the mechanical properties of PLLA bundles (as-spun) and of PLLA/Coll-75/25 and PLLA/Coll-50/50 bundles (as-spun, and after 48 h, 7 days and 14 days in PBS). The most promising material was the PLLA/Coll-75/25 blend with a Young modulus of 98.6 ±12.4 MPa (as-spun) and 205.1 ±73.0 MPa (after 14 days in PBS). Its failure stress was 14.2 ±0.7 MPa (as-spun) and 6.8 ±0.6 MPa (after 14 days in PBS). Pure PLLA withstood slightly lower stress than the PLLA/Coll-75/25 while PLLA/Coll-50/50 had a brittle behavior. Human-derived tenocytes were used for cellular tests. A good cell adhesion and viability after 14 day culture was observed. This study has therefore demonstrated the feasibility of fabricating electrospun bundles with multiscale structure and mechanical properties similar to the human tendon

The role of the mesenchymal stem cells on breast cancer: friends or foes?

Mesenchymal stem cells (MSCs) have been the subject of an increased interest. Because of their ability to give rise to bone, cartilage, fat and muscle, their role in regenerative medicine has been extensively studied and the fact that they can be recruited at sites of inflammation and tissue repair has prompted their potential use as tissue regenerative cells. Contextually there has been a growing interest in the role of MSCs in cancer progression. The nature of the relationship between MSCs and tumor cells appears dual, with effects pro- as well as anti-tumorigenic. This paradox depends on the source and the degree of differentiation of MSCs and the tumor model used. Moreover, with the large range of cytokines and growth factors they produce, MSCs exert regulatory function on apoptosis, angiogenesis and an immunomodulatory role. Here we evaluate the interaction between MSCs derived from the periprosthetic capsule of mastectomyzed women and the breast cancer cell line MCF-7. Capsular tissue around breast implants is a normal inflammatory reaction versus a foreign body and it is rich of MSCs. To asses how MSCs interact with tumor cells, MCF-7 cells were incubated with medium previously conditioned by MSCs or directly co-coltured with MSCs; subsequently, we evaluated the proliferation and the expression of genes implicated in different pathways (angiogenesis, proliferation, anti-apoptosis, EMT transition). Our results showed that MCF-7 cells cultured together MSCs or using their conditioned medium have a more elevated proliferation rate but tumour cells seem less aggressive, like attested by a reduction of the expression of selected genes. The understanding of the mechanisms that control the interaction between MSCs and tumor cells is still at an early stage but recent literature confirm that MSCs and their progeny are not innocent bystanders in the tumor microenvironment. The study of these interactions is a critical area of future investigation that is needed to better define their role in cancer progression and their potential as therapeutic agents or targets

Loss of nuclear BAP1 protein expression is a marker of poor prognosis in patients with clear cell renal cell carcinoma

BAP1 is a gene situated on chromosome 3p in a region that is deleted in over 90% of Renal Cell Carcinomas (RCCs) (1,2). In the present study we studied BAP1 immunohistochemical expression in a large series of conventional clear cell RCCs (ccRCCs) treated with radical nephrectomy and we assessed the prognostic value of their expression in terms of patients survival at long-term follow-up. 154 consecutive patients with ccRCC were selected from a prospective database and considered for the study purpose; all patients were treated with radical nephrectomy and lymphadenectomy at our Institute of Urology between 1983 and 1985. The features considered in this study were tumor size, grade and stage, vascular and capsular invasion, incidence of metastasis and patient specific survival; all these parameters were correlated with immunohistochemical cytoplasmic and nuclear expression of BPA-1 in tumoral tissue. Median follow-up was 196.18 months (range 5 to 274); median survival was 125.34 months (range 5 to 274 months). We found that nuclear BAP1 expression showed a high frequency of loss in tumoral cells; nuclear BAP1 negative tumors had higher tumor size, higher Fuhrman grade, and higher stage, a greater amount of vascular and capsular invasion and a higher incidence of metastases. We have demonstrated that nuclear BAP1 expression is a marker of prognosis in ccRCC, having an impact on cancer-specific survival. The clinical importance for BAP1 will be realized with the identification and application of targeted therapies and with individualized approaches in the adjuvant and/or metastatic setting

IGF1 and IGF1/VEGF cross-talk on human mesenchymal stromal cells (hMSCs): the role of stem cell sources in bone healing

Repair of skeletal defects remains a considerable biomedical problem. One of the major obstacles of the different tested strategies still remains the vascularization of engineered scaffolds. To this aim we have examined the ability of IGF1, alone or in association with VEGF, to modulate Periosteum Derived Progenitor Cells (PDPCs) (Ferretti et al., 2012) and Skin-Mesenchymal Stromal Cells (S-MSCs) (Orciani and Di Primio, 2013) osteoblastic or endothelial commitment. A selected gene panel for endothelial and osteoblastic differentiation as well as genes that can affect MAPK and PI3K/AKT signalling pathways were investigated. Our results showed a different commitment of PDPCs and S-MSCs under growth factor stimulation: the former are induced towards an osteoblastic differentiation, whilst the latter seem to be brought to an endothelial phenotype. This commitment is also related to a diverse MAPK or PI3K/AKT signalling pathway activation. Our results open intriguing perspective for the development of innovative bone tissue engineering approaches based on associated angiogenesis and osteogenesis. Further investigations are however necessary to gain insight on the real cross-talk between proliferation and differentiation in adult stem cells. The work was supported by Italian FIRB (RBAP10MLK7) and PRIN (2010J8RYS7) project grants

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