Surfactant-assisted photo-crosslinked silk fibroin sponges: A versatile platform for the design of bone scaffolds.

Critical size bone defects cannot heal without aid and current clinical approaches exhibit some limitations, underling the need for novel solutions. Silk fibroin, derived from silkworms, is widely utilized in tissue engineering and regenerative medicine due to its remarkable properties, making it a promising candidate for bone tissue regeneration in vitro and in vivo. However, the clinical translation of silk-based materials requires refinements in 3D architecture, stability, and biomechanical properties. In earlier research, improved mechanical resistance and stability of chemically crosslinked methacrylate silk fibroin (Sil-Ma) sponges over physically crosslinked counterparts were highlighted. Furthermore, the influence of photo-initiator and surfactant concentrations on silk properties was investigated. However, the characterization of sponges with Sil-Ma solution concentrations above 10 % (w/V) was hindered by production optimization challenges, with only cell viability assessed. This study focuses on the evaluation of methacrylate sponges' suitability as temporal bone tissue regeneration scaffolds. Sil-Ma sponge fabrication at a fixed concentration of 20 % (w/V) was optimized and the impact of photo-initiator (LAP) concentrations and surfactant (Tween 80) presence/absence was studied. Their effects on pore formation, silk secondary structure, mechanical properties, and osteogenic differentiation of hBM-MSCs were investigated. We demonstrated that, by tuning silk sponges' composition, the optimal combination boosted osteogenic gene expression, offering a strategy to tailor biomechanical properties for effective bone regeneration. Utilizing Design of Experiment (DoE), correlations between sponge composition, porosity, and mechanical properties are established, guiding tailored material outcomes. Additionally, correlation matrices elucidate the microstructure's influence on gene expressions, providing insights for personalized approaches in bone tissue regeneration.

Targeting the Inflammatory Hallmarks of Obesity-Associated Osteoarthritis: Towards Nutraceutical-Oriented Preventive and Complementary Therapeutic Strategies Based on n-3 Polyunsaturated Fatty Acids.

Obesity (Ob), which has dramatically increased in the last decade, is one of the main risk factors that contribute to the incidence and progression of osteoarthritis (OA). Targeting the characteristics of obesity-associated osteoarthritis (ObOA) may offer new chances for precision medicine strategies in this patient cohort. First, this review outlines how the medical perspective of ObOA has shifted from a focus on biomechanics to the significant contribution of inflammation, mainly mediated by changes in the adipose tissue metabolism through the release of adipokines and the modification of fatty acid (FA) compositions in joint tissues. Preclinical and clinical studies on n-3 polyunsaturated FAs (PUFAs) are critically reviewed to outline the strengths and weaknesses of n-3 PUFAs' role in alleviating inflammatory, catabolic and painful processes. Emphasis is placed on potential preventive and therapeutic nutritional strategies based on n-3 PUFAs, with a focus on ObOA patients who could specifically benefit from reformulating the dietary composition of FAs towards a protective phenotype. Finally, tissue engineering approaches that involve the delivery of n-3 PUFAs directly into the joint are explored to address the perspectives and current limitations, such as safety and stability issues, for implementing preventive and therapeutic strategies based on dietary compounds in ObOA patients.

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.

Overview of Anti-Inflammatory and Anti-Nociceptive Effects of Polyphenols to Halt Osteoarthritis: From Preclinical Studies to New Clinical Insights.

Knee osteoarthritis (OA) is one of the most multifactorial joint disorders in adults. It is characterized by degenerative and inflammatory processes that are responsible for joint destruction, pain and stiffness. Despite therapeutic advances, the search for alternative strategies to target inflammation and pain is still very challenging. In this regard, there is a growing body of evidence for the role of several bioactive dietary molecules (BDMs) in targeting inflammation and pain, with promising clinical results. BDMs may be valuable non-pharmaceutical solutions to treat and prevent the evolution of early OA to more severe phenotypes, overcoming the side effects of anti-inflammatory drugs. Among BDMs, polyphenols (PPs) are widely studied due to their abundance in several plants, together with their benefits in halting inflammation and pain. Despite their biological relevance, there are still many questionable aspects (biosafety, bioavailability, etc.) that hinder their clinical application. This review highlights the mechanisms of action and biological targets modulated by PPs, summarizes the data on their anti-inflammatory and anti-nociceptive effects in different preclinical in vitro and in vivo models of OA and underlines the gaps in the knowledge. Furthermore, this work reports the preliminary promising results of clinical studies on OA patients treated with PPs and discusses new perspectives to accelerate the translation of PPs treatment into the clinics.

Dietary organosulfur compounds: Emerging players in the regulation of bone homeostasis by plant-derived molecules.

The progressive decline of bone mass and the deterioration of bone microarchitecture are hallmarks of the bone aging. The resulting increase in bone fragility is the leading cause of bone fractures, a major cause of disability. As the frontline pharmacological treatments for osteoporosis suffer from low patients' adherence and occasional side effects, the importance of diet regimens for the prevention of excessive bone fragility has been increasingly recognized. Indeed, certain diet components have been already associated to a reduced fracture risk. Organosulfur compounds are a broad class of molecules containing sulfur. Among them, several molecules of potential therapeutic interest are found in edible plants belonging to the Allium and Brassica botanical genera. Polysulfides derived from Alliaceae and isothiocyanates derived from Brassicaceae hold remarkable nutraceutical potential as anti-inflammatory, antioxidants, vasorelaxant and hypolipemic. Some of these effects are linked to the ability to release the gasotrasmitter hydrogen sulfide (H2S). Recent preclinical studies have investigated the effect of organosulfur compounds in bone wasting and metabolic bone diseases, revealing a strong potential to preserve skeletal health by exerting cytoprotection and stimulating the bone forming activity by osteoblasts and attenuating bone resorption by osteoclasts. This review is intended for revising evidence from preclinical and epidemiological studies on the skeletal effects of organosulfur molecules of dietary origin, with emphasis on the direct regulation of bone cells by plant-derived polysulfides, glucosinolates and isothiocyanates. Moreover, we highlight the potential molecular mechanisms underlying the biological role of these compounds and revise the importance of the so-called 'H2S-system' on the regulation of bone homeostasis.

Optimization of a Monobromobimane (MBB) Derivatization and RP-HPLC-FLD Detection Method for Sulfur Species Measurement in Human Serum after Sulfur Inhalation Treatment.

(1) Background: Hydrogen sulfide (H2S) is a widely recognized gasotransmitter, with key roles in physiological and pathological processes. The accurate quantification of H2S and reactive sulfur species (RSS) may hold important implications for the diagnosis and prognosis of diseases. However, H2S species quantification in biological matrices is still a challenge. Among the sulfide detection methods, monobromobimane (MBB) derivatization coupled with reversed phase high-performance liquid chromatography (RP-HPLC) is one of the most reported. However, it is characterized by a complex preparation and time-consuming process, which may alter the actual H2S level; moreover, a quantitative validation has still not been described. (2) Methods: We developed and validated an improved analytical protocol for the MBB RP-HPLC method. MBB concentration, temperature and sample handling were optimized, and the calibration method was validated using leave-one-out cross-validation and tested in a clinical setting. (3) Results: The method shows high sensitivity and allows the quantification of H2S species, with a limit of detection of 0.5 µM. Finally, it can be successfully applied in measurements of H2S levels in the serum of patients subjected to inhalation with vapors rich in H2S. (4) Conclusions: These data demonstrate that the proposed method is precise and reliable for measuring H2S species in biological matrices and can be used to provide key insights into the etiopathogenesis of several diseases and sulfur-based treatments.

Glucoraphanin Increases Intracellular Hydrogen Sulfide (H2S) Levels and Stimulates Osteogenic Differentiation in Human Mesenchymal Stromal Cell.

Osteopenia and osteoporosis are among the most prevalent consequences of ageing, urging the promotion of healthy nutritional habits as a tool in preventing bone fractures. Glucosinolates (GLSs) are organosulfur compounds considered relatively inert precursors of reactive derivatives isothiocyanates (ITCs). Recent evidence suggests that GLSs may exert biological properties based on their capacity to release hydrogen sulfide (H2S). H2S-donors are known to exert anabolic function on bone cells. Here, we investigated whether a GLS, glucoraphanin (GRA) obtained from Tuscan black kale, promotes osteogenesis in human mesenchymal stromal cells (hMSCs). H2S release in buffer and intracellular H2S levels were detected by amperometric measurements and fluorimetric/cytofluorimetric analyses, respectively. Alizarin red staining assay and real-time PCR were performed to evaluate mineral apposition and mRNA expression of osteogenic genes. Using an in vitro cell culture model, our data demonstrate a sulforaphane (SFN)-independent osteogenic stimulation of GRA in hMSCs, at least partially attributable to H2S release. In particular, GRA upregulated the expression of osteogenic genes and enhanced mineral apposition while increasing intracellular concentrations of H2S. Overall, this study suggests the feasibility of using cruciferous derivatives as natural alternatives to chemical H2S-donors as adjuvant therapies in the treatment of bone-wasting diseases.

Treatment of Human Glioblastoma U251 Cells with Sulforaphane and a Peptide Nucleic Acid (PNA) Targeting miR-15b-5p: Synergistic Effects on Induction of Apoptosis.

Glioblastoma multiforme (GBM) is a lethal malignant tumor accounting for 42% of the tumors of the central nervous system, the median survival being 15 months. At present, no curative treatment is available for GBM and new drugs and therapeutic protocols are urgently needed. In this context, combined therapy appears to be a very interesting approach. The isothiocyanate sulforaphane (SFN) has been previously shown to induce apoptosis and inhibit the growth and invasion of GBM cells. On the other hand, the microRNA miR-15b is involved in invasiveness and proliferation in GBM and its inhibition is associated with the induction of apoptosis. On the basis of these observations, the objective of the present study was to determine whether a combined treatment using SFN and a peptide nucleic acid interfering with miR-15b-5p (PNA-a15b) might be proposed for increasing the pro-apoptotic effects of the single agents. To verify this hypothesis, we have treated GMB U251 cells with SFN alone, PNA-a15b alone or their combination. The cell viability, apoptosis and combination index were, respectively, analyzed by calcein staining, annexin-V and caspase-3/7 assays, and RT-qPCR for genes involved in apoptosis. The efficacy of the PNA-a15b determined the miR-15b-5p content analyzed by RT-qPCR. The results obtained indicate that SFN and PNA-a15b synergistically act in inducing the apoptosis of U251 cells. Therefore, the PNA-a15b might be proposed in a "combo-therapy" associated with SFN. Overall, this study suggests the feasibility of using combined treatments based on PNAs targeting miRNA involved in GBM and nutraceuticals able to stimulate apoptosis.

MicroRNAs miR-584-5p and miR-425-3p Are Up-Regulated in Plasma of Colorectal Cancer (CRC) Patients: Targeting with Inhibitor Peptide Nucleic Acids Is Associated with Induction of Apoptosis in Colon Cancer Cell Lines.

Liquid biopsy has dramatically changed cancer management in the last decade; however, despite the huge number of miRNA signatures available for diagnostic or prognostic purposes, it is still unclear if dysregulated miRNAs in the bloodstream could be used to develop miRNA-based therapeutic approaches. In one author's previous work, nine miRNAs were found to be dysregulated in early-stage colon cancer (CRC) patients by NGS analysis followed by RT-dd-PCR validation. In the present study, the biological effects of the targeting of the most relevant dysregulated miRNAs with anti-miRNA peptide nucleic acids (PNAs) were verified, and their anticancer activity in terms of apoptosis induction was evaluated. Our data demonstrate that targeting bloodstream up-regulated miRNAs using anti-miRNA PNAs leads to the down-regulation of target miRNAs associated with inhibition of the activation of the pro-apoptotic pathway in CRC cellular models. Moreover, very high percentages of apoptotic cells were found when the anti-miRNA PNAs were associated with other pro-apoptotic agents, such as sulforaphane (SFN). The presented data sustain the idea that the targeting of miRNAs up-regulated in the bloodstream with a known role in tumor pathology might be a tool for the design of protocols for anti-tumor therapy based on miRNA-targeting molecules.

Sulforaphane inhibits the expression of interleukin-6 and interleukin-8 induced in bronchial epithelial IB3-1 cells by exposure to the SARS-CoV-2 Spike protein.

BACKGROUND: A key clinical feature of COVID-19 is a deep inflammatory state known as "cytokine storm" and characterized by high expression of several cytokines, chemokines and growth factors, including IL-6 and IL-8. A direct consequence of this inflammatory state in the lungs is the Acute Respiratory Distress Syndrome (ARDS), frequently observed in severe COVID-19 patients. The "cytokine storm" is associated with severe forms of COVID-19 and poor prognosis for COVID-19 patients. Sulforaphane (SFN), one of the main components of Brassica oleraceae L. (Brassicaceae or Cruciferae), is known to possess anti-inflammatory effects in tissues from several organs, among which joints, kidneys and lungs. PURPOSE: The objective of the present study was to determine whether SFN is able to inhibit IL-6 and IL-8, two key molecules involved in the COVID-19 "cytokine storm". METHODS: The effects of SFN were studied in vitro on bronchial epithelial IB3-1 cells exposed to the SARS-CoV-2 Spike protein (S-protein). The anti-inflammatory activity of SFN on IL-6 and IL-8 expression has been evaluated by RT-qPCR and Bio-Plex analysis. RESULTS: In our study SFN inhibits, in cultured IB3-1 bronchial cells, the gene expression of IL-6 and IL-8 induced by the S-protein of SARS-CoV-2. This represents the proof-of-principle that SFN may modulate the release of some key proteins of the COVID-19 "cytokine storm". CONCLUSION: The control of the cytokine storm is one of the major issues in the management of COVID-19 patients. Our study suggests that SFN can be employed in protocols useful to control hyperinflammatory state associated with SARS-CoV-2 infection.

Learning from Monocyte-Macrophage Fusion and Multinucleation: Potential Therapeutic Targets for Osteoporosis and Rheumatoid Arthritis.

Excessive bone resorption by osteoclasts (OCs) covers an essential role in developing bone diseases, such as osteoporosis (OP) and rheumatoid arthritis (RA). Monocytes or macrophages fusion and multinucleation (M-FM) are key processes for generating multinucleated mature cells with essential roles in bone remodelling. Depending on the phenotypic heterogeneity of monocyte/macrophage precursors and the extracellular milieu, two distinct morphological and functional cell types can arise mature OCs and giant cells (GCs). Despite their biological relevance in several physiological and pathological responses, many gaps exist in our understanding of their formation and role in bone, including the molecular determinants of cell fusion and multinucleation. Here, we outline fusogenic molecules during M-FM involved in OCs and GCs formation in healthy conditions and during OP and RA. Moreover, we discuss the impact of the inflammatory milieu on modulating macrophages phenotype and their differentiation towards mature cells. Methodological approach envisaged searches on Scopus, Web of Science Core Collection, and EMBASE databases to select relevant studies on M-FM, osteoclastogenesis, inflammation, OP, and RA. This review intends to give a state-of-the-art description of mechanisms beyond osteoclastogenesis and M-FM, with a focus on OP and RA, and to highlight potential biological therapeutic targets to prevent extreme bone loss.

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.

High Levels of Apoptosis Are Induced in the Human Colon Cancer HT-29 Cell Line by Co-Administration of Sulforaphane and a Peptide Nucleic Acid Targeting miR-15b-5p.

Sulforaphane (SFN) is one of most important dietary constituents of broccoli (Brassica oleracea) and other cruciferous vegetables, which have been reported to exhibit health benefits, including prevention and therapy of cancer, such as colorectal carcinoma (CRC). The objective of this study was to determine whether the anticancer effect of SFN on colon cancer HT-29 cell line could be improved by the combined treatment with molecules inhibiting microRNAs (miRNAs) involved in CRC. As miRNA inhibiting molecules we focused on peptide-nucleic acids (PNAs). As miRNA to be targeted, miR-15b-5p was selected on the basis of several information present in the literature and confirming that miR-15b-5p is overexpressed in colon cancer patients, and that its targeting decreases cell migration and metastasis in colorectal cancer. In this article, we described for the first time the efficacy of targeting miR-15b-5p by using a PNA against miR-15b-5p (R8-PNA-a15b), functionalized with an octoarginine peptide (R8) for maximizing cellular uptake. The miR-15b-5p downregulation in the colon cancer HT-29 cell line was associated with inhibition of in vitro cell growth and activation of the proapoptotic pathway, demonstrated by a sharp increase of late apoptotic cells in HT-29-treated cell populations. A second conclusion of this study is that the R8-PNA-a15b might be proposed in "combo-therapy" associated with SFN. To our knowledge, no report is available in the literature on a combination between SFN and miRNA-targeting molecules. Our data demonstrate that this combined treatment leads to a very high proportion of apoptotic HT-29 cells (over 85%), a value higher than the sum of the values of apoptotic cells obtained after singularly administered regents (either SFN or R8-PNA-a15b).

Hydrogen Sulfide in Bone Tissue Regeneration and Repair: State of the Art and New Perspectives.

The importance of hydrogen sulfide (H2S) in the regulation of multiple physiological functions has been clearly recognized in the over 20 years since it was first identified as a novel gasotransmitter. In bone tissue H2S exerts a cytoprotective effect and promotes bone formation. Just recently, the scientific community has begun to appreciate its role as a therapeutic agent in bone pathologies. Pharmacological administration of H2S achieved encouraging results in preclinical studies in the treatment of systemic bone diseases, such as osteoporosis; however, a local delivery of H2S at sites of bone damage may provide additional opportunities of treatment. Here, we highlight how H2S stimulates multiple signaling pathways involved in various stages of the processes of bone repair. Moreover, we discuss how material science and chemistry have recently developed biomaterials and H2S-donors with improved features, laying the ground for the development of H2S-releasing devices for bone regenerative medicine. This review is intended to give a state-of-the-art description of the pro-regenerative properties of H2S, with a focus on bone tissue, and to discuss the potential of H2S-releasing scaffolds as a support for bone repair.

Hydrogen sulfide-releasing silk fibroin scaffold for bone tissue engineering.

Hydrogen sulfide (H2S)-based therapy is a promising therapeutic strategy for several biomedical applications. Following the observation that endogenous and exogenous H2S plays a prominent role as a bone anabolic agent, we recently developed a silk fibroin (SF) porous scaffold loaded with GYY4137 (GYY), an H2S donor, for applications in bone tissue engineering. Here, we assayed whether the combination of SF with H2S potentiates the osteoconductive properties of SF. Biocompatibility and osteoanabolic activity were assayed in vitro using human bone marrow mesenchymal stromal cells. Cell cultures were performed on a perfusion bioreactor to obtain results closer to the in vivo microenvironment. Cytotoxicity was excluded by lactate dehydrogenase and live/dead assays. Cell colonization and mineral apposition were evaluated by Haematoxylin & Eosin and Von Kossa/Alizarin Red-S stainings respectively. PCR array for human osteogenesis and immunohistochemical analyses were performed to identify pathways and targets involved. Our findings show that H2S-releasing SF scaffolds supported cell adhesion, proliferation and viability. Moreover, H2S activated genes and proteins involved in ossification, osteoblast differentiation, bone mineral metabolism and angiogenesis allowing a high and early mineralization. Based on these properties, we suggest the use of H2S-releasing SF scaffolds for bone healing and regeneration applications.

Effect of microfragmented adipose tissue on osteoarthritic synovial macrophage factors.

Cell-based therapies using adipose-derived mesenchymal stromal cells (ADMSCs) have shown promising results for the treatment of osteoarthritis (OA). In fact, ADMSCs are now indicated as one of the most powerful cell sources through their immunomodulatory and anti-inflammatory activities. Recently, an innovative one-step closed device was developed to obtain microfragmented adipose tissue (MF) to avoid the need for good manufacturing practices for ADMSCs expansion while maintaining their regenerative potential. The aim of this study was to assess the mechanisms of action of MF and ADMSCs from MF (MF-ADMSCs) on an inflammatory cell model of OA synoviocytes. We found that MF produced low levels of inflammatory factors such as interleukin 6 (IL-6), CC-chemokine ligand 5/receptor-activated normal T-cell expressed and secreted (CCL5/RANTES), CC-chemokine ligand 2/monocyte chemoattractant protein-1 (CCL2/MCP-1), and CC-chemokine ligand 3/macrophage inflammatory protein-1α (CCL3/MIP-1α), and a higher level only of CXC-chemokine ligand 8/interleukin 8 compared with MF-ADMSCs. Matrix metalloproteinase 9 (MMP-9) degradative factor but released a lower level of its inhibitor tissue inhibitor of the metalloproteinase (TIMP-1). MF in coculture with synoviocytes significantly induced both the metabolic activity and the release of IL-6. In contrast, MF, not MF-ADMSCs, partially decreased CCL5/RANTES. Moreover, MF reduced the release of both macrophage-specific chemokines (CCL2/MCP-1 and CCL3/MIP-1α) and degradative marker MMP-9. Interestingly, MF increased TIMP-1 (the MMP-9 inhibitor) and down-modulated toll-like receptor (TLR4) receptor and key molecules of NFκB pathways. These data evidenced different effects of MF versus MF-ADMSCs on inflamed synoviocytes. MF reduced typical macrophages markers and its potentiality by switching off macrophages activity was strictly dependent on TLR4 and NFκB signaling.

Clinical and Biological Signature of Osteochondritis Dissecans in a Cross-Sectional Study.

The healing potential of knee osteochondritis dissecans (OCD) focal lesions is not well defined. We performed a cross-sectional study correlating local and systemic biological characteristics with the patients' characteristics. We evaluated both local tissue markers (CD34, CD146, CD166, and tartrate-resistant acid phosphatase (TRAP)) and systemic serum biomarkers (fragments or propeptide of type II collagen: C2C, CTX-II, CPII, and TRAP5b) on histologically scored osteochondral fragments or serum from OCD patients. These biological features were associated with the patients' characteristics (IKDC subjective score, age, and body mass index (BMI)). Histological cartilage tissue score correlated with patients' IKDC and C2C and CPII biomarkers. CPII correlated also with histological bone tissue score. The percentage of CD146 positive cells in cartilage and CD34 positive cells in bone highly correlated with the patient's age and BMI, respectively. The percentage of TRAP in bone was directly correlated with both IKDC and age. Multivariate statistical analysis evidenced that only four parameters significantly predicted IKDC. In conclusion, a complete picture of OCD knee characteristics, defined by local and systemic markers of cartilage and bone remodeling, together with the patients' characteristics, might help to better understand the healing potential of each patient and to target and improve current OCD treatments.

Silk Fibroin Porous Scaffolds Loaded with a Slow-Releasing Hydrogen Sulfide Agent (GYY4137) for Applications of Tissue Engineering.

Hydrogen sulfide (H2S) is a physiological gasotransmitter known to possess a regulatory role in several tissues, including bone. The exogenous administration by injection of solutions of H2S-releasing compounds (e.g., GYY4137) has been previously investigated as a novel therapeutic approach for the treatment of bone diseases. Here, GYY4137 was embedded into fibroin sponges, previously shown to be suitable as scaffolds for bone, thanks to their biocompatibility, scalable porous structure, and biodegradability rate. Fibroin porous scaffolds were produced by solvent casting and the particulate leaching method, and GYY4137 was successively incorporated by using dimethyl sulfoxide (DMSO) as vehicle. The process used to produce GYY4137-loaded scaffolds allowed the incorporation of different controlled amounts of GYY4137 into fibroin matrices. The loading process preserved the properties of the system components in the final products, as assessed by SEM, FT-IR, NMR, and different thermal analyses techniques. Release of H2S from GYY4137 incorporated into the scaffolds was monitored upon incubation in saline solution at physiological pH: H2S-release kinetic was found to be dependent on the amount of GYY4137. To ensure biocompatibility, mouse fibroblasts and human primary bone marrow stromal cells were seeded onto scaffolds, and short-term viability assays were performed. Results showed that the GYY4137-loaded scaffold did not induce cytotoxicity in any of the cell type tested. Our findings demonstrate that embedding an H2S-releasing donor in silk fibroin scaffold is a suitable strategy to achieve a long-lasting release of H2S that preserves cell viability and allows local delivery at sites of tissue injury.

Cartilage and Bone Serum Biomarkers as Novel Tools for Monitoring Knee Osteochondritis Dissecans Treated with Osteochondral Scaffold.

Knee osteochondritis dissecans (OCD) is a focal disease of the joint characterized by modifications of bone and cartilage tissues. Biomimetic osteochondral scaffolds are used to restore these tissues. The aim of this prognostic prospective cohort study was to evaluate serum biomarkers of cartilage (fragments or propeptide of type II collagen: CTXII, C2C, and CPII) and bone (tartrate-resistant acid phosphatase (TRAP) 5b and osteocalcin (OC)) turnover during follow-up of patients treated with an osteochondral scaffold, to identify which were related to healing outcome and clinical score. We found that cartilage (CPII) and bone (OC) synthetic biomarkers were significantly increased during the first-year follow-up, while the respective degradative markers (CTXII, C2C, and TRAP5b) were not modulated. Only CTXII/CPII and C2C/CPII cartilage ratios were significantly modulated, evidencing a higher remodeling of cartilage compared to bone tissue. Cartilage and bone single biomarkers or ratios at one-year follow-up showed values close to or similar to those of healthy subjects. International Knee Documentation Committee (IKDC) score significantly increased from T0 to T2, while the Tegner score did not. Taking into consideration an IKDC score > 70 as clinical success, we found that all OCD cases with both CPII (> 300 pg/ml) and C2C/CPII (<0.35) presented IKDC scores of clinical success. OCD patients treated with an osteochondral scaffold showed an improvement at one-year follow-up, evidenced by both clinical and serum cartilage biomarkers. These data confirmed that cartilage and bone remodeling took place and showed that systemic biomarkers represent a sensitive tool for monitoring OCD patients during the follow-up.

The expression of cystathionine gamma-lyase is regulated by estrogen receptor alpha in human osteoblasts.

Hydrogen sulfide (H2S), generated in the osteoblasts predominantly via cystathionine-γ-lyase (CSE), is bone protective. Previous studies suggested that the onset of bone loss due to estrogen deficiency is associated to decreased levels of H2S and blunted gene expression of CSE. However, there are still a lot of unknowns on how H2S levels influence bone cells function. The present study aims to explore the mechanisms by which estrogen may regulate CSE expression, in particular the role of estrogen receptor alpha (ERα) in human osteoblasts (hOBs). Vertebral lamina derived hOBs were characterized and then assessed for CSE expression by western blot analysis in the presence or absence of ERα overexpression. Bioinformatic analysis, luciferase reporter assay and ChIP assay were performed to investigate ERα recruitment and activity on hCSE gene promoter. Three putative half Estrogen Responsive Elements (EREs) were identified in the hCSE core promoter and were found to participate in the ERα - mediated positive regulation of CSE expression. All osteoblast samples responded to ERα over-expression increasing the levels of CSE protein in a comparable manner. Notably, the ERα recruitment on the regulatory regions of the CSE promoter occurred predominantly in female hOBs than in male hOBs. The obtained results suggest that CSE/H2S system is in relation with estrogen signaling in bone in a gender specific manner.