Sulfurous thermal waters stimulate the osteogenic differentiation of human mesenchymal stromal cells - An in vitro study.

Strategies aimed at delaying the onset of bone tissue degeneration and the resulting skeletal fragility are key to decrease the risk of bone fracture correlated to ageing. The therapeutic properties of sulfurous thermal waters (STWs), rich in hydrogen sulfide (H2S), have been claimed for centuries. However, the direct regulation of bone cells by STWs has not been investigated yet. Here we aimed at analyzing the effect of STWs on cultured human mesenchymal stromal cells (hMSCs) derived from bone tissue. Two concentrations of STWs from 2 health spa centers in Italy (here named STW-1 and STW-2) containing, respectively, high and moderate quantities of H2S, were added to the culture media. Cytotoxicity and osteogenic differentiation were evaluated. We provided first evidence that treatment of hMSCs with STWs results in a sharp increase in intracellular H2S content, coherent with the different concentrations of H2S, thereby reveling that STWs-released H2S is internalized by cells. STWs treatment significantly induced osteogenic differentiation of hMSCs. In particular, mineral apposition was increased with a similar pattern by the two STWs, while mRNA expression of osteogenic markers (BSP, OC, RUNX-2, OPN) was differently affected. Only STW-2 induced a significant, dose-dependent increase in these gene expression. These findings support the rationale for the use of STWs as a complementary treatment of bone wasting diseases

Engineered Cartilage Maturation Regulates Cytokine Production and Interleukin-1β Response

Background: Because the injured joint has an actively inflammatory environment, the survival and repair potential of cartilage grafts may be influenced by inflammatory processes. Understanding the interactions of those processes with the graft may lead to concepts for pharmacologic or surgical solutions allowing improved cartilage repair. Questions/purposes: We asked whether the maturation level of cartilaginous tissues generated in vitro by expanded human articular chondrocytes (HACs) modulate (1) the spontaneous production of cytokines and (2) the response to interleukin (IL)-1β. Methods: Twelve pellets/donor prepared with monolayer-expanded HACs (n=6 donors) were evaluated at six different culture times for mRNA expression (n=72) and spontaneous baseline release of monocyte chemoattractant protein (MCP)-1, IL-8, and transforming growth factor (TGF)-β1 (n=72). We cultured 24 pellets/donor from each of four donors for 1 or 14days (defined as immature and mature, respectively) and exposed the pellets to IL-1β for 3days. MCP-1, IL-8, TGF-β1, and metalloprotease (MMP)-1 and MMP-13 were quantified in pellets and culture supernatants. Results: By increasing culture time, the spontaneous release of IL-8 and MCP-1 decreased (12.0- and 5.5-fold, respectively), whereas that of TGF-β1 increased (5.4-fold). As compared with immature pellets, mature pellets responded to IL-1β by releasing lower amounts of MMP-1 (2.9-fold) and MMP-13 (1.7-fold) and increased levels of IL-8, MCP-1, and TGF-β1 (1.5-, 5.0-, and 7.5-fold, respectively). IL-8 and MCP-1 promptly returned to baseline on withdrawal of IL-1β. Conclusions: Our observations suggest more mature cartilaginous tissues are more resistant to IL-1β exposure and can activate chemokines required to initiate tissue repair processes. Clinical Relevance: The implantation of more mature cartilaginous tissues might provide superior graft survival and improve/accelerate cartilage repai

Physicochemical and biological properties of natural and synthetic C-22 and C-23 hydroxylated bile acids.

In order to define the effect of a side chain hydroxy group on bile acid (BA) physicochemical and biological properties, 23-hydroxylated bile acids were synthesized following a new efficient route involving the alpha-oxygenation of silylalkenes. 22-Hydroxylated bile acids were also studied. The synthesized bile acids included R and S epimers of 3 alpha,7 alpha,23-trihydroxy-5 beta-cholan-24-oic acid (23R epimer: phocaecholic acid), 3 alpha,12 alpha,23-trihydroxy-5 beta-cholan-24-oic (23R epimer: bitocholic acid), and 3 alpha,7 beta,23-trihydroxy-5 beta-cholan-24-oic acid. A 3 alpha,7 alpha,22-trihydroxy-5 beta-cholan-24-oic acid (haemulcholic acid) was also studied. The presence of a hydroxy group on the side chain slightly modified the physicochemical behavior in aqueous solution with respect to common BA: the critical micellar concentration (CMC) and the hydrophilicity were similar to naturally occurring trihydroxy BA such as cholic acid. The pKa value was lowered by 1.5 units with respect to common BA, being 3.8 for all the C-23 hydroxy BA. C-22 had a higher pKa (4.2) as a result of the increased distance of the hydroxy group from the carboxy group. When the C-23 hydroxylated BA were intravenously administered to bile fistula rats, they were efficiently recovered in bile (more than 80% unmodified) while the corresponding analogs, lacking the 23- hydroxy group, were almost completely glycine- or taurine-conjugated. On the other hand, the C-22 hydroxylated BA were extensively conjugated with taurine and less than 40% of the administered dose was secreted without being conjugated. In the presence of intestinal bacteria, they were mostly metabolized to the corresponding 7-dehydroxylated compound similar to common BA with the exception of bitocholic acid which was relatively stable. The presence of a hydroxy group at the C-23 position increased the acidity of the BA and this accounted for poor absorption within the biliary tree and efficient biliary secretion without the need for conjugation. 3 alpha,7 beta-23 R/S trihydroxy-5 beta-cholan-24-oic acids could improve the efficiency of ursodeoxycholic acid (UDCA) for gallstone dissolution or cholestatic syndrome therapy, as it is relatively hydrophilic and efficiently secreted into bile without altering the glycine and taurine hepatic pool

Glucosamine affects intracellular signalling through inhibition of mitogen-activated protein kinase phosphorylation in human chondrocytes

The aim of this study was to determine the effects of glucosamine on matrix metalloprotease (MMP) production, on mitogen-activated protein kinase (MAPK) phosphorylation, and on activator protein (AP)-1 transcription factor activation in human chondrocytes. The human immortalized cell line lbpva55 and healthy human chondrocytes (obtained from healthy donors) were subjected to challenge with 10 ng/ml IL-1β after pretreatment with 2.5 or 10 mmol/l glucosamine. MMP mRNA expression levels were evaluated using quantitative real-time PCR, and MMP protein production levels were evaluated in the culture supernatant using ELISA. MAPK phosphorylation was evaluated using Western blotting. AP-1 transcription factor activation was evaluated by measuring AP-1 DNA-binding activity. After IL-1β stimulation, levels of MMP-1, MMP-3 and MMP-13 production were markedly increased. Treatment with 2.5 and 10 mmol/l glucosamine reduced expression of these metalloproteases. MMP expression is regulated by transcription factors such as the AP-1 complex, which is activated by phosphorylated MAPKs. IL-1β stimulated phosphorylation of c-jun amino-terminal kinase, p38 MAPK and extracellular signal-regulated kinase-1/2. Glucosamine inhibited c-jun amino-terminal kinase and p38 phosphorylation, and consequently c-jun binding activity. These findings demonstrate, for the first time, that glucosamine inhibits IL-1β-stimulated MMP production in human chondrocytes by affecting MAPK phosphorylation

Methacrylated Silk Fibroin Additive Manufacturing of Shape Memory Constructs with Possible Application in Bone Regeneration

: Methacrylated silk (Sil-MA) is a chemically modified silk fibroin specifically designed to be crosslinkable under UV light, which makes this material applicable in additive manufacturing techniques and allows the prototyping and development of patient-specific 2D or 3D constructs. In this study, we produced a thin grid structure based on crosslinked Sil-MA that can be withdrawn and ejected and that can recover its shape after rehydration. A complete chemical and physical characterization of Sil-MA was first conducted. Additionally, we tested Sil-MA biocompatibility according to the International Standard Organization protocols (ISO 10993) ensuring the possibility of using it in future trials. Sil-MA was also tested to verify its ability to support osteogenesis. Overall, Sil-MA was shown to be biocompatible and osteoconductive. Finally, two different additive manufacturing technologies, a Digital Light Processing (DLP) UV projector and a pneumatic extrusion technique, were used to develop a Sil-MA grid construct. A proof-of-concept of its shape-memory property was provided. Together, our data support the hypothesis that Sil-MA grid constructs can be injectable and applicable in bone regeneration applications

In Vitro Synovial Membrane 3D Model Developed by Volumetric Extrusion Bioprinting

Background: Synovial tissue plays a fundamental role in inflammatory processes. Therefore, understanding the mechanisms regulating healthy and diseased synovium functions, as in rheumatic diseases, is crucial to discovering more effective therapies to minimize or prevent pathological progress. The present study aimed at developing a bioartificial synovial tissue as an in vitro model for drug screening or personalized medicine applications using 3D bioprinting technology. (2) Methods: The volumetric extrusion technique has been used to fabricate cell-laden scaffolds. Gelatin Methacryloyl (GelMA), widely applied in regenerative medicine and tissue engineering, was selected as a bioink and combined with an immortalized cell line of fibroblast-like synoviocytes (K4IM). (3) Results: Three different GelMA formulations, 7.5–10–12.5% w/v, were tested for the fabrication of the scaffold with the desired morphology and internal architecture. GelMA 10% w/v was chosen and combined with K4IM cells to fabricate scaffolds that showed high cell viability and negligible cytotoxicity for up to 14 days tested by Live & Dead and lactate dehydrogenase assays. (4) Conclusions: We successfully 3D bioprinted synoviocytes-laden scaffolds as a proof-of-concept (PoC) towards the fabrication of a 3D synovial membrane model suitable for in vitro studies. However, further research is needed to reproduce the complexity of the synovial microenvironment to better mimic the physiological condition

Prospective double-blind randomised controlled trial protocol comparing bone marrow aspirate concentrate intra-articular injection combined with subchondral injection versus intra-articular injection alone for the treatment of symptomatic knee osteoarthritis

Introduction: Subchondral and intra-articular injections of bone marrow aspirate concentrate (BMAC) showed promising results for knee osteoarthritis (OA) patients. To date, there is no evidence to demonstrate whether the combination of these treatments provides higher benefits than the intra-articular injection alone. Methods and analysis: Eighty-six patients with symptomatic knee OA (aged between 40 and 70 years) are randomised to BMAC intra-articular injection combined with subchondral BMAC injection or BMAC intra-articular injection alone in a ratio of 1:1. The primary outcome is the total Western Ontario and McMaster Universities Osteoarthritis Index, the secondary outcomes are the International Knee Documentation Committee Subjective and Objective Knee Evaluation Form, the Tegner activity scale, the EuroQol-Visual Analogue Scale, and the health questionnaire European Quality of Life Five Dimension score. Additional CT and MRI evaluations are performed at the baseline assessment and at the final 12-month follow-up. The hypothesis is that the combined injections provide higher knee pain and function improvement compared with BMAC intra-articular injection alone. The primary analysis follows an intention to treat principle. Ethics and dissemination: The study protocol has been approved by the Emilia Wide Area Ethical Committee of the Emilia-Romagna Region (CE-AVEC), Bologna, Italy. Written informed consent is obtained from all the participants. Findings of this study will be disseminated through peer-reviewed publications and conference presentations. Protocol version: Version 1 (14 May 2018). Trial registration number: NCT03876795

Effective Label-Free Sorting of Multipotent Mesenchymal Stem Cells from Clinical Bone Marrow Samples

Mesenchymal stem cells (MSC) make up less than 1% of the bone marrow (BM). Several methods are used for their isolation such as gradient separation or centrifugation, but these methodologies are not direct and, thus, plastic adherence outgrowth or magnetic/fluorescent-activated sorting is required. To overcome this limitation, we investigated the use of a new separative technology to isolate MSCs from BM; it label-free separates cells based solely on their physical characteristics, preserving their native physical properties, and allows real-time visualization of cells. BM obtained from patients operated for osteochondral defects was directly concentrated in the operatory room and then analyzed using the new technology. Based on cell live-imaging and the sample profile, it was possible to highlight three fractions (F1, F2, F3), and the collected cells were evaluated in terms of their morphology, phenotype, CFU-F, and differentiation potential. Multipotent MSCs were found in F1: higher CFU-F activity and differentiation potential towards mesenchymal lineages compared to the other fractions. In addition, the technology depletes dead cells, removing unwanted red blood cells and non-progenitor stromal cells from the biological sample. This new technology provides an effective method to separate MSCs from fresh BM, maintaining their native characteristics and avoiding cell manipulation. This allows selective cell identification with a potential impact on regenerative medicine approaches in the orthopedic field and clinical applications

Small Extracellular Vesicles from Inflamed Adipose Derived Stromal Cells Enhance the NF-κB-Dependent Inflammatory/Catabolic Environment of Osteoarthritis

The last decade has seen exponentially growing efforts to exploit the effects of adipose derived stromal cells (ADSC) in the treatment of a wide range of chronic degenerative diseases, including osteoarthritis (OA), the most prevalent joint disorder. In the perspective of developing a cell-free advanced therapy medicinal product, a focus has been recently addressed to the ADSC secretome that lends itself to an allogeneic use and can be further dissected for the selective purification of small extracellular vesicles (sEVs). sEVs can act as "biological drug carriers" to transfer information that mirror the pathophysiology of the providing cells. This is important in the clinical perspective where many OA patients are also affected by the metabolic syndrome (MetS). ADSC from MetS OA patients are dysfunctional and "inflammatory" primed within the adipose tissue. To mimic this condition, we exposed ADSC to IL-1 beta, and then we investigated the effects of the isolated sEVs on chondrocytes and synoviocytes, either cultured separately or in co-culture, to tease out the effects of these "IL-1 beta primed sEVs" on gene and protein expression of major inflammatory and catabolic OA markers. In comparison with sEVs isolated from unstimulated ADSC, the IL-1 beta primed sEVs were able to propagate NF-kappa B activation in bystander joint cells. The effects were more prominent on synoviocytes, possibly because of a higher expression of binding molecules such as CD44. These findings call upon a careful characterization of the "inflammatory fingerprint" of ADSC to avoid the transfer of an unwanted message as well as the development of in vitro "preconditioning" strategies able to rescue the antiinflammatory/anticatabolic potential of ADSC-derived sEVs