The NFATc1/P2X7 receptor relationship in human intervertebral disc cells.

A comprehensive understanding of the molecules that play key roles in the physiological and pathological homeostasis of the human intervertebral disc (IVD) remains challenging, as does the development of new therapeutic treatments. We recently found a positive correlation between IVD degeneration (IDD) and P2X7 receptor (P2X7R) expression increases both in the cytoplasm and in the nucleus. Using immunocytochemistry, reverse transcription PCR (RT-PCR), overexpression, and chromatin immunoprecipitation, we found that NFATc1 and hypoxia-inducible factor-1α (HIF-1α) are critical regulators of P2X7R. Both transcription factors are recruited at the promoter of the P2RX7 gene and involved in its positive and negative regulation, respectively. Furthermore, using the proximity ligation assay, we revealed that P2X7R and NFATc1 form a molecular complex and that P2X7R is closely associated with lamin A/C, a major component of the nuclear lamina. Collectively, our study identifies, for the first time, P2X7R and NFATc1 as markers of IVD degeneration and demonstrates that both NFATc1 and lamin A/C are interaction partners of P2X7R.

Wharton's jelly-derived multifunctional hydrogels: New tools to promote intervertebral disc regeneration in vitro and ex vivo.

The degeneration of intervertebral disc (IVD) is a disease of the entire joint between two vertebrae in the spine caused by loss of extracellular matrix (ECM) integrity, to date with no cure. The various regenerative approaches proposed so far have led to very limited successes. An emerging opportunity arises from the use of decellularized ECM as a scaffolding material that, directly or in combination with other materials, has greatly facilitated the advancement of tissue engineering. Here we focused on the decellularized matrix obtained from human umbilical cord Wharton's jelly (DWJ) which retains several structural and bioactive molecules very similar to those of the IVD ECM. However, being a viscous gel, DWJ has limited ability to retain ordered structural features when considered as architecture scaffold. To overcome this limitation, we produced DWJ-based multifunctional hydrogels, in the form of 3D millicylinders containing different percentages of alginate, a seaweed-derived polysaccharide, and gelatin, denatured collagen, which may impart mechanical integrity to the biologically active DWJ. The developed protocol, based on a freezing step, leads to the consolidation of the entire polymeric dispersion mixture, followed by an ionic gelation step and a freeze-drying process. Finally, a porous, stable, easily storable, and suitable matrix for ex vivo experiments was obtained. The properties of the millicylinders (Wharton's jelly millicylinders [WJMs]) were then tested in culture of degenerated IVD cells isolated from disc tissues of patients undergoing surgical discectomy. We found that WJMs with the highest percentage of DWJ were effective in supporting cell migration, restoration of the IVD phenotype (increased expression of Collagen type 2, aggrecan, Sox9 and FOXO3a), anti-inflammatory action, and stem cell activity of resident progenitor/notochordal cells (increased number of CD24 positive cells). We are confident that the DWJ-based formulations proposed here can provide adequate stimuli to the cells present in the degenerated IVD to restart the anabolic machinery.

Unorthodox localization of P2X7 receptor in subcellular compartments of skeletal system cells.

Identifying the subcellular localization of a protein within a cell is often an essential step in understanding its function. The main objective of this report was to determine the presence of the P2X7 receptor (P2X7R) in healthy human cells of skeletal system, specifically osteoblasts (OBs), chondrocytes (Chs) and intervertebral disc (IVD) cells. This receptor is a member of the ATP-gated ion channel family, known to be a main sensor of extracellular ATP, the prototype of the danger signal released at sites of tissue damage, and a ubiquitous player in inflammation and cancer, including bone and cartilaginous tissues. Despite overwhelming data supporting a role in immune cell responses and tumor growth and progression, a complete picture of the pathophysiological functions of P2X7R, especially when expressed by non-immune cells, is lacking. Here we show that human wild-type P2X7R (P2X7A) was expressed in different samples of human osteoblasts, chondrocytes and intervertebral disc cells. By fluorescence microscopy (LM) and immunogold transmission electron microscopy we localized P2X7R not only in the canonical sites (plasma membrane and cytoplasm), but also in the nucleus of all the 3 cell types, especially IVD cells and OBs. P2X7R mitochondrial immunoreactivity was predominantly detected in OBs and IVD cells, but not in Chs. Evidence of subcellular localization of P2X7R may help to i. understand the participation of P2X7R in as yet unidentified signaling pathways in the joint and bone microenvironment, ii. identify pathologies associated with P2X7R mislocalization and iii. design specific targeted therapies.

Pro­differentiating compounds for human intervertebral disc cells are present in Violina pumpkin leaf extracts.

Intervertebral disc (IVD) degeneration (IDD) is closely associated with inflammation, oxidative stress and loss of the discogenic phenotype, which current therapies are unable to reverse. In the present study, the effects of acetone extract from Violina pumpkin (Cucurbita moschata) leaves on degenerated IVD cells were investigated. IVD cells were isolated from the degenerated disc tissue of patients undergoing spinal surgery and were exposed to acetone extract and three major thin layer chromatography subfractions. The results revealed that, in particular, the cells benefited from exposure to subfraction Fr7, which consisted almost entirely of p­Coumaric acid. Western blot and immunocytochemical analysis showed that Fr7 induced a significant increase in discogenic transcription factors (SOX9 and tricho­rhino­phalangeal syndrome type I protein, zinc finger protein), extracellular matrix components (aggrecan, collagen type II), cellular homeostasis and stress response regulators, such as FOXO3a, nuclear factor erythroid 2­related factor 2, superoxide dismutase 2 and sirtuin 1. Two important markers related to the presence and activity of stem cells, migratory capacity and OCT4 expression, were assessed by scratch assay and western blotting, respectively, and were significantly increased in Fr7­treated cells. Moreover, Fr7 counteracted H2O2­triggered cell damage, preventing increases in the pro­inflammatory and anti­chondrogenic microRNA (miR), miR­221. These findings strengthen the hypothesis that adequate stimuli can support resident cells to repopulate the degenerated IVD and restart the anabolic machinery. Taken together, these data contribute to the discovery of molecules potentially effective in slowing the progression of IDD, a disease for which there is currently no effective treatment. Moreover, the use of part of a plant, the pumpkin leaves, which is usually considered a waste product in the Western world, indicated that it contains substances with potential beneficial effects on human health.

Osteoclast-mediated acidic hydrolysis of thermally gelled curdlan component of the bone scaffolds: Is it possible?

Many biomaterials for bone regeneration have recently been produced using thermally gelled curdlan (1,3-ß-d-glucan) as a binder for bioceramics. As the human organism does not produce enzymes having the ability to degrade curdlan, it is not clear what is the fate of curdlan gel after its implantation in the bone. To clarify this point, in this research osteoclasts were cultured on the curdlan gel to show its degradation by acidic hydrolysis. The studies clearly demonstrated microstructural (AFM and SEM imaging) and chemical changes (Raman spectroscopy) on the curdlan surface caused by osteoclast culture. Moreover, degradation test in a cell-free system using HCl solution (pH = 4.5), mimicking environment in the resorption lacuna, showed great weight loss of the sample, release of glucose, and chemical changes typical of curdlan degradation. Thus, the presented research for the first time provides a strong evidence of osteoclast-mediated acidic hydrolysis of thermally obtained curdlan gel.

Decellularized extracellular matrix-based scaffold and hypoxic priming: A promising combination to improve the phenotype of degenerate intervertebral disc cells.

AIMS: The main cause of low back pain is the intervertebral disc (IVD) degeneration. Designing an effective disc regeneration strategy still remains a major challenge, especially for the lack of effective self-healing capacity. Understanding the properties of IVD cells in the degenerate microenvironment could help to develop in situ regeneration strategies. The objective of the present study was to investigate the ability of degenerate cells to respond to conditions they experience physiologically in their niche in vivo, namely the presence of the hypoxic environment and trophic factors. MAIN METHODS: Degenerate cells from IVD of patients operated for herniated disc were exposed to hypoxic priming and decellularized Wharton's jelly matrix (DWJM) as scaffold and trophic factors source for 48 h in culture. Cell response was evaluated in terms of cell viability, proliferation, cytoskeletal organization, migratory ability and expression of discogenic transcription factors (SOX9, TRPS1), hypoxia-inducible factor 1α (HIF-1α) and longevity transcription factor FOXO3a. The recruitment of HIF-1α at FOXO3a and SOX9 gene promoters was analyzed by Chromatin immunoprecipitation. KEY FINDINGS: Degenerate IVD cells were able to re-acquire the discogenic phenotype, and to re-adapt to hypoxia after exposure to hypoxic priming and DWJM. We demonstrated for the first time that HIF-1α is specifically recruited at the promoter of SOX9 and FOXO3a which are crucial for IVD homeostasis and repair. SIGNIFICANCE: These results open new avenues to engineer IVD by demonstrating that appropriate stimuli are able to dampen the degenerated IVD cell phenotype and to promote anabolic activity in cells which are constitutively characterized by poor reparative capacity.

The P2X7 purinergic receptor in intervertebral disc degeneration.

Mechanisms involved in the development of intervertebral disc (IVD) degeneration are only partially known, thus making the implementation of effective therapies very difficult. In this study, we investigated P2X7 purinergic receptor (P2X7R), NLRP3 inflammasome, and interleukin (IL)-1ß expression in IVD specimens at different stages of disease progression, and during the in vitro dedifferentiation process of the primary cells derived thereof. We found that P2X7R, NLRP3, and IL-1ß expression was higher in the IVD samples at a more advanced stage of degeneration and in the expanded IVD cells in culture which partially recapitulated the in vivo degeneration process. In IVD cells, the P2X7R showed a striking nuclear localization, while NLRP3 was mainly cytoplasmic. Stimulation with the semiselective P2X7R agonist benzoyl ATP together with lipopolysaccharide treatment triggered P2X7R transfer to the cytoplasm and P2X7R/NLRP3 colocalization. Taken together, these findings support pathophysiological evidence that the degenerated disc is a highly inflamed microenvironment and highlight the P2X7R/NLRP3 axis as a suitable therapeutic target. The immunohistochemical analysis and the assessment of subcellular localization revealed a substantial expression of P2X7R also in normal disc tissue. This gives us the opportunity to contribute to the few studies performed in natively expressed human P2X7R so far, and to understand the possible physiological ATP-mediated P2X7R homeostasis signaling. Therefore, collectively, our findings may offer a new perspective and pave the way for the exploration of a role of P2X7R-mediated purinergic signaling in IVD metabolism that goes beyond its involvement in inflammation.

Pro-Osteogenic Properties of Violina pumpkin (Cucurbita moschata) Leaf Extracts: Data from In Vitro Human Primary Cell Cultures.

Traditional medicines rely mainly on use of plant extracts to mitigate or treat a wide range of disorders, including those that affect skeletal homeostasis. In this study, we investigated for the first time the potential pro-osteogenic effects of hexane, acetone and methanol extracts of the leaves of Cucurbita moschata, a very popular pumpkin cultivar in Western countries. We found that in Cucurbita moschata leaves, there are acetone-extractable substances-in particular, fatty acids such as 13-OH-9Z,11E,15E-octadecatrienoic acid (PU-13OH-FA), which is capable of both stimulating the function of human primary osteoblasts, which are responsible for bone formation, and inhibiting the differentiation of human osteoclasts, which are responsible for bone resorption. This dual effect was monitored by analyzing Runx2 expression, deposition of mineralized matrix, ALP activity, TRAP and actin ring staining respectively. This study suggests that bioactive chemicals from Cucurbita moschata leaves are potentially suitable as therapeutics for managing metabolic bone disorders such as osteoporosis and rheumatoid arthritis, and promoting tissue healing and functional recovery after bone fractures. The data we obtained increase knowledge on the biological activities of Cucurbita moschata, and in particular underline the potential benefits of consuming leaves which are a part of the plant currently little considered in the Western world.

Three-Dimensional Co-Culture System of Human Osteoblasts and Osteoclast Precursors from Osteoporotic Patients as an Innovative Model to Study the Role of Nutrients: Focus on Vitamin K2.

Several natural compounds, such as vitamin K2, have been highlighted for their positive effects on bone metabolism. It has been proposed that skeletal disorders, such as osteoporosis, may benefit from vitamin K2-based therapies or its regular intake. However, further studies are needed to better clarify the effects of vitamin K2 in bone disorders. To this aim, we developed in vitro a three-dimensional (3D) cell culture system one step closer to the bone microenvironment based on co-culturing osteoblasts and osteoclasts precursors obtained from bone specimens and peripheral blood of the same osteoporotic patient, respectively. Such a 3-D co-culture system was more informative than the traditional 2-D cell cultures when responsiveness to vitamin K2 was analyzed, paving the way for data interpretation on single patients. Following this approach, the anabolic effects of vitamin K2 on the osteoblast counterpart were found to be correlated with bone turnover markers measured in osteoporotic patients' sera. Overall, our data suggest that co-cultured osteoblasts and osteoclast precursors from the same osteoporotic patient may be suitable to generate an in vitro 3-D experimental model that potentially reflects the individual's bone metabolism and may be useful to predict personal responsiveness to nutraceutical or drug molecules designed to positively affect bone health.

Human osteoclasts/osteoblasts 3D dynamic co­culture system to study the beneficial effects of glucosamine on bone microenvironment.

Glucosamine (GlcN) functions as a building block of the cartilage matrix, and its multifaceted roles in promoting joint health have been extensively investigated. However, the role of GlcN in osteogenesis and bone tissue is poorly understood, mainly due to the lack of adequate experimental models. As a result, the benefit of GlcN application in bone disorders remains controversial. In order to further elucidate the pharmacological relevance and potential therapeutic/nutraceutic efficacy of GlcN, the effect of GlcN treatment was investigated in human primary osteoclasts (hOCs) and osteoblasts (hOBs) that were cultured with two­dimensional (2D) traditional methods or co­cultured in a 3D dynamic system more closely resembling the in vivo bone microenvironment. Under these conditions, osteoclastogenesis was supported by hOBs and sizeable self­assembling aggregates were obtained. The differentiated hOCs were evaluated using tartrate­resistant acid phosphatase assays and osteogenic differentiation was monitored by analyzing mineral matrix deposition via Alizarin Red staining, with expression of specific osteogenic markers determined via reverse transcription­quantitative PCR. It was found that crystalline GlcN sulfate was effective in decreasing osteoclastic cell differentiation and function. hOCs isolated from patients with OA were more sensitive compared with those from healthy donors. Additionally, GlcN exhibited anabolic effects on hOCs both in 2D conventional cell culture and in hOC/hOB 3D dynamic co­culture. The present study demonstrated for the first time the effectiveness of a 3D dynamic co­culture system for characterizing the spectrum of action of GlcN on the bone microenvironment, which may pave the way for more fully determining the potential applications of a compound such as GlcN, which is positioned between pharmaceuticals and nutraceuticals. Based on the present findings, it is hypothesized that GlcN may have potential benefits in the treatment of osteopenic diseases such as osteoporosis, as well as in bone maintenance.

Expression and function of the P2X7 receptor in human osteoblasts: The role of NFATc1 transcription factor.

Bone mineralization is an orchestrated process by which mineral crystals are deposited by osteoblasts; however, the detailed mechanisms remain to be elucidated. The presence of P2X7 receptor (P2X7R) in immature and mature bone cells is well established, but contrasting evidence on its role in osteogenic differentiation and deposition of calcified bone matrix remains. To clarify these controversies in the present study, we investigated P2X7R participation in bone maturation. We demonstrated that the P2X7R is expressed and functional in human primary osteoblasts, and identified in the P2RX7 promoter several binding sites for transcription factors involved in bone mineralization. Of particular interest was the finding that P2X7R expression is enhanced by nuclear factor of activated T cells cytoplasmic 1 (NFATc1) overexpression, and accordingly, NFATc1 is recruited at the P2RX7 gene promoter in SaOS2 osteoblastic-like cells. In conclusion, our data provide further insights into the regulation of P2X7R expression and support the development of drugs targeting this receptor for the therapy of bone diseases.

Extracellular Matrix From Decellularized Wharton's Jelly Improves the Behavior of Cells From Degenerated Intervertebral Disc.

Regenerative therapies for intervertebral disc (IVD) injuries are currently a major challenge that is addressed in different ways by scientists working in this field. Extracellular matrix (ECM) deriving from decellularized non-autologous tissues has been established as a biomaterial with remarkable regenerative capacity and its potential as a therapeutic agent is rising. In the present study, we investigated the potential of decellularized Wharton's jelly matrix (DWJM) from human umbilical cord to act as an ECM-based scaffold for IVD cell culturing. An efficient detergent-enzymatic treatment (DET) was used to produce DWJM maintaining its native microarchitecture. Afterward, immunofluorescence, biochemical assays and electron microscopy analysis showed that DWJM was able to produce sizeable 3D cell aggregates, when combined with human mesenchymal stromal cells isolated from WJ (MSCs) and IVD cells. These latter cells are characterized by the loss of their chondrocyte-like phenotype since they have been isolated from degenerated IVD and in vitro expanded to further de-differentiate. While the effect exerted by DWJM on MSCs was essentially the induction of proliferation, conversely, on IVD cells the DWJM promoted cell differentiation toward a discogenic phenotype. Notably, for the first time, the ability of DWJM to improve the degenerated phenotype of human IVD cells was demonstrated, showing that the mere presence of the matrix maintained the viability of the cells, and positively affected the expression of critical regulators of IVD homeostasis, such as SOX2, SOX9, and TRPS1 transcription factors at specific culture time. Our data are in line with the hypothesis that the strengthening of cell properties in terms of viability and expression of specific proteins at precise times represents an important condition in the perspective of guiding the recovery of cellular functionality and triggering regenerative potential. Currently, there are no definitive surgical or pharmacological treatments for IVD degeneration (IDD) able to restore the disc structure and function. Therefore, the potential of DWJM to revert degenerated IVD cells could be exploited in the next future an ECM-based intradiscal injectable therapeutic.

Reciprocal Regulation of TRPS1 and miR-221 in Intervertebral Disc Cells.

Intervertebral disc (IVD), a moderately moving joint located between the vertebrae, has a limited capacity for self-repair, and treating injured intervertebral discs remains a major challenge. The development of innovative therapies to reverse IVD degeneration relies primarily on the discovery of key molecules that, occupying critical points of regulatory mechanisms, can be proposed as potential intradiscal injectable biological agents. This study aimed to elucidate the underlying mechanism of the reciprocal regulation of two genes differently involved in IVD homeostasis, the miR-221 microRNA and the TRPS1 transcription factor. Human lumbar IVD tissue samples and IVD primary cells were used to specifically evaluate gene expression and perform functional analysis including the luciferase gene reporter assay, chromatin immunoprecipitation, cell transfection with hTRPS1 overexpression vector and antagomiR-221. A high-level expression of TRPS1 was significantly associated with a lower pathological stage, and TRPS1 overexpression strongly decreased miR-221 expression, while increasing the chondrogenic phenotype and markers of antioxidant defense and stemness. Additionally, TRPS1 was able to repress miR-221 expression by associating with its promoter and miR-221 negatively control TRPS1 expression by targeting the TRPS1-3'UTR gene. As a whole, these results suggest that, in IVD cells, a double-negative feedback loop between a potent chondrogenic differentiation suppressor (miR-221) and a regulator of axial skeleton development (TRPS1) exists. Our hypothesis is that the hostile degenerated IVD microenvironment may be counteracted by regenerative/reparative strategies aimed at maintaining or stimulating high levels of TRPS1 expression through inhibition of one of its negative regulators such as miR-221.

SLUG/HIF1-α/miR-221 regulatory circuit in endometrial cancer.

BACKGROUND AND PURPOSE: The pathogenesis of endometrial cancer (EC) involves many regulatory pathways including transcriptional regulatory networks supported by transcription factors and microRNAs only in part known. The aim of this retrospective study was to explore the possible correlation in the EC microenvironment between master regulators of complex phenomena such as steroid responsiveness through estrogen receptor alpha (ERα) and progesterone receptor (PR), epithelial-to-mesenchymal transition (supported by SLUG transcription factor), hypoxia (with hypoxia inducible factor-1 alpha, HIF-1α), and obesity that has been recognized as a EC risk factor. METHODS: Formalin-Fixed Paraffin-Embedded (FFPE) blocks from University of Ferrara Pathology Archive were used and allocated into 2 groups according to their immunohistochemical positivity to ERα and PR, distinguishing the samples with a more benign prognosis (ERα+/PR+) from those with a poorer prognosis (ERα-/PR-). Immunohistochemistry for HIF1-α and SLUG was also performed. Body mass index (BMI) was registered at the time of diagnosis: patients with BMI ≥ 30 kg/m2 were defined obese (OB). Total RNA was isolated for miR-221 analysis. RESULTS: We showed a comparable percentage of HIF1-α and SLUG positive samples in the ERα+/PR+ and ERα-/PR- groups. However, the obesity factor impacted more in the ERα+/PR+ group since the ratio between OB and non-obese (NOB) patients with high expression of HIF1-α and SLUG was higher in ERα+/PR+ than in the ERα-/PR- group. miR-221 levels were significantly higher in the OB than NOB patients, and, also in this case, obesity impacted more in the ERα+/PR+ group. CONCLUSIONS: A molecular circuit of mutual regulation between ERα, PR, HIF1-α, SLUG and miR-221 is feasible in the EC and was firstly suggested by our research. In this interplay miR-221 seems to be in a nodal point of the regulatory system that is particularly strengthened by the metabolic changes in obesity.

Hypoxia Preconditioning of Human MSCs: a Direct Evidence of HIF-1α and Collagen Type XV Correlation.

BACKGROUND/AIMS: Mesenchymal stromal cells (MSCs) hold considerable promise in bone tissue engineering, but their poor survival and potency when in vivo implanted limits their therapeutic potential. For this reason, the study on culture conditions and cellular signals that can influence the potential therapeutic outcomes of MSCs have received considerable attention in recent years. Cell maintenance under hypoxic conditions, in particular for a short period, is beneficial for MSCs, as low O2 tension is similar to that present in the physiologic niche, however the precise mechanism through which hypoxia preconditioning affects these cells remains unclear. METHODS: In order to explore what happens during the first 48 h of hypoxia preconditioning in human MSCs (hMSCs) from bone marrow, the cells were exposed to 1.5% O2 tension in the X3 Hypoxia Hood and Culture Combo - Xvivo System device. The expression modulation of critical genes which could be good markers of increased osteopotency has been investigated by Western blot, immunufluorescence and ELISA. Luciferase reporter assay and Chromatin immunoprecipitation was used to investigate the regulation of the expression of Collagen type XV (ColXV) gene. RESULTS: We identified ColXV as a new low O2 tension sensitive gene, and provided a novel mechanistic evidence that directly HIF-1α (hypoxia-inducible factor-1 alpha) mediates ColXV expression in response to hypoxia, since it was found specifically in vivo recruited at ColXV promoter, in hypoxia-preconditioned hMSCs. This finding, together the evidence that also Runx2, VEGF and FGF-2 expression increased in hypoxia preconditioned hMSCs, is consistent with the possibility that increased ColXV expression in response to hypoxia is mediated by an early network that supports the osteogenic potential of the cells. CONCLUSION: These results add useful information to understand the role of a still little investigated collagen such as ColXV, and identify ColXV as a marker of successful hypoxia preconditioning. As a whole, our data give further evidence that hypoxia preconditioned hMSCs have greater osteopotency than normal hMSCs, and that the effects of hypoxic regulation of hMSCs activities should be considered before they are clinically applied.

Upregulation of the alternative splicing factor NOVA2 in colorectal cancer vasculature.

BACKGROUND: Tumor-specific isoforms generated by alternative splicing (AS) are demonstrated to contribute to tumor progression and can represent potential biomarkers. NOVA2 is an AS factor that in physiological conditions regulates endothelial cells' (ECs) polarity and vessel lumen maturation, likely by mediating AS of apical-basal polarity regulators. However, NOVA2 expression in tumor ECs and its regulation have never been investigated. METHODS: To elucidate this, 40 colorectal cancer patients were enrolled and NOVA2 expression was investigated by immunohistochemistry in samples bearing both the normal mucosa and the tumor tissue. RESULTS: NOVA2 was found expressed in ECs of tumor vasculature and, importantly, it was upregulated in tumor ECs with respect to normal mucosa ECs in all cases (P<0.001). The same samples analyzed by immunohistochemistry for the expression HIF1α, a marker of hypoxia, showed a positive and significant association with NOVA2 levels (P=0.045). Of note, NOVA2 was upregulated by hypoxia also in an in vitro ECs model. CONCLUSION: Our results provide, for the first time, evidence of NOVA2 expression and upregulation in tumor ECs and highlight hypoxia as a potential regulatory factor. These findings open a completely new perspective to study tumor vasculature and to uncover NOVA2 as a potential source of biomarkers and therapeutic targets based on AS isoforms.

MicroRNA-221 silencing attenuates the degenerated phenotype of intervertebral disc cells.

The aim of this study was to investigate the role of an antichondrogenic factor, MIR221 (miR-221), in intervertebral disc degeneration (IDD), and provide basic information for the development of a therapeutic strategy for the disc repair based on specific nucleic acid based drugs, such as miR-221 silencing. We established a relatively quick protocol to minimize artifacts from extended in vitro culture, without selecting the different types of cells from intervertebral disc (IVD) or completely disrupting extracellular matrix (ECM), but by using the whole cell population with a part of resident ECM. During the de-differentiation process miR-221 expression significantly increased. We demonstrated the effectiveness of miR-221 silencing in driving the cells towards chondrogenic lineage. AntagomiR-221 treated cells showed in fact a significant increase of expression of typical chondrogenic markers including COL2A1, ACAN and SOX9, whose loss is associated with IDD. Moreover, antagomiR-221 treatment restored FOXO3 expression and increased TRPS1 expression levels attenuating the severity grade of degeneration, and demonstrating in a context of tissue degeneration and inflammation not investigated before, that FOXO3 is target of miR-221. Data of present study are promising in the definition of new molecules useful as potential intradiscal injectable biological agents.

Immunoelectron microscopic localization of Collagen type XV during human mesenchymal stem cells mineralization.

Purpose/Aim of the study. Collagen type XV (ColXV) was identified, in our previews studies, as a novel component of bone extracellular matrix. The present study aims to investigate ColXV localization during mineralization of osteodifferentiated human mesenchymal stem cells (hMSCs). MATERIAL AND METHODS: hMSCs cultured in osteogenic medium have been analyzed at day 14 and 28 for mineral matrix deposition by alizarin red S staining, ultrastructural analysis and ColXV localization by immunogold electron microscopy. RESULTS: Our data show an intimate association between ColXV and fibrillar components in areas localized far from mineralized nodules. CONCLUSIONS: We have demonstrated the efficacy of ultrastructural analysis, combined with immunocytochemistry, to establish a temporal and spatial localization of ColXV. This data, added to previous evidences, contribute to validate the negative effects of calcium deposits on ColXV expression.