RNA Extraction from Cartilage: Issues, Methods, Tips.

The increase in degenerative diseases involving articular cartilage has pushed research to focus on their pathogenesis and treatment, exploiting increasingly complex techniques. Gene expression analyses from tissue are representative of the in vivo situation, but the protocols to be applied to obtain a reliable analysis are not completely cleared through customs. Thus, RNA extraction from fresh samples and specifically from musculoskeletal tissue such as cartilage is still a challenging issue. The aim of the review is to provide an overview of the techniques described in the literature for RNA extraction, highlighting limits and possibilities. The research retrieved 65 papers suitable for the purposes. The results highlighted the great difficulty in comparing the different studies, both for the sources of tissue used and for the techniques employed, as well as the details about protocols. Few papers compared different RNA extraction methods or homogenization techniques; the case study reported by authors about RNA extraction from sheep cartilage has not found an analog in the literature, confirming the existence of a relevant blank on studies about RNA extraction from cartilage tissue. However, the state of the art depicted can be used as a starting point to improve and expand studies on this topic.

The N-Acetyl Phenylalanine Glucosamine Derivative Attenuates the Inflammatory/Catabolic Environment in a Chondrocyte-Synoviocyte Co-Culture System.

Osteoarthritis (OA), the most prevalent degenerative joint disease, still lacks a true disease-modifying therapy. The involvement of the NF-κB pathway and its upstream activating kinases in OA pathogenesis has been recognized for many years. The ability of the N-acetyl phenylalanine glucosamine derivative (NAPA) to increase anabolism and reduce catabolism via inhibition of IKKα kinase has been previously observed in vitro and in vivo. The present study aims to confirm the chondroprotective effects of NAPA in an in vitro model of joint OA established with primary cells, respecting both the crosstalk between chondrocytes and synoviocytes and their phenotypes. This model satisfactorily reproduces some features of the previously investigated DMM model, such as the prominent induction of ADAMTS-5 upon inflammatory stimulation. Both gene and protein expression analysis indicated the ability of NAPA to counteract key cartilage catabolic enzymes (ADAMTS-5) and effectors (MCP-1). Molecular analysis showed the ability of NAPA to reduce IKKα nuclear translocation and H3Ser10 phosphorylation, thus inhibiting IKKα transactivation of NF-κB signalling, a pivotal step in the NF-κB-dependent gene expression of some of its targets. In conclusion, our data confirm that NAPA could truly act as a disease-modifying drug in OA.

Osteochondral tissue cultures: Between limits and sparks, the next step for advanced in vitro models.

The increasing demand for reliable preclinical models and to reduce, refine and, if possible, replace animal studies have brought forth the development of complex tissue cultures in different research areas, including the musculoskeletal field. In this paper, we review the literature within last 10 years on the state of progress for in vitro models of osteochondral tissue cultures, taking into account the clinical relevance of the management and treatment of osteochondral lesions. According to the selected research criteria, 35 works, 27 of which with animal tissues and 8 with human tissues, resulted to be relevant for the purposes of this review. Data analyzed revealed a great heterogeneity among the proposed tissue culture models. The anatomical harvesting sites resulted to be mainly the knee stifle joint, both for animal (prevalently bovines) and human tissues derived from joint replacement surgery, and significant heterogeneity among culture conditions and media were found. To date, very few papers have focused on the set up of a reproducible in vitro model, applicable to a variety of studies, thus suggesting a relevant gap to fill in the development of advanced three-dimensional osteochondral culture models.

Modulation of Alendronate release from a calcium phosphate bone cement: An in vitro osteoblast-osteoclast co-culture study.

In this study, we loaded a biomimetic calcium phosphate bone cement (CPC) with relatively high amounts of a bisphosphonate through the use of Solid Lipid Microparticles (MPs) and investigated bone cells response to the composite cements. 10, 20 and 30% w/w of Alendronate (AL) were successfully introduced into microparticles of Cutina HR and Precirol, which were prepared by means of spray-congealing technique. Addition of AL-loaded MPs to the cement composition provoked a lengthening of the setting and of the hardening processes. However, setting times were still in a range useful for clinical applications, except for the cements at the highest Alendronate content. The composite cements displayed a sustained drug release over time. Cements with the best performances in terms of setting, hardening, mechanical properties and drug release were submitted to in vitro tests using a co-culture model of osteoblast and osteoclast. The results showed that the use of MPs to enrich the cement composition with Alendronate provides materials able to inhibit osteoclast viability and activity, while promoting osteoblast viability and earlier differentiation, indicating that the MPs-cements are good delivery systems for bisphosphonates.

JHDM1B expression regulates ribosome biogenesis and cancer cell growth in a p53 dependent manner.

Tumors characterized by an intense ribosome biogenesis often display a more aggressive behavior. Ribosomal RNA (rRNA) synthesis is controlled at several levels, including the epigenetic regulation of the condensation of chromatin portions containing rRNA genes. JHDM1B (Jumonji C histone demethylase 1B) is a histone demethylase able to regulate the accessibility of rRNA genes. In this study, we aimed to define the contribution of JHDM1B expression to the features of breast cancer, a tumor type whose behavior is related to the rate of ribosome biogenesis. We show that, in breast cancer-derived cell lines, the increase in rRNA transcription that follows JHDM1B knock-down is mirrored by an augmented cell proliferation only in p53 compromised cells, while p53 competent cells undergo cellular senescence and death. The latter effect appears to be mediated by a p38-dependent phosphorylation of p53, inducing the expression of p15(Ink4b) and p21(Waf1). In breast cancers, lower JHDM1B expression correlates with an increased size of specifically stained nucleolar organized regions, a morphological parameter directly related to the rate of ribosome biogenesis and with a poorer prognosis. In addition, in tumors lacking the controller function of p53, a lower expression of JHDM1B is associated with an increased tumor size at diagnosis. Altogether, our data indicate that epigenetic activation of rDNA genes induced by JHDM1B depletion is associated with a p53-dependent growth arrest, but may promote cancer cell growth when p53 is lacking.