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Introduction: Knee osteotomy combined with meniscal allograft transplantation (MAT) showed promising results to treat unicompartmental knee osteoarthritis (OA) secondary to meniscal deficiency and knee malalignment. However, there is still no high-level evidence to demonstrate whether the combination of these two treatments is superior to osteotomy alone., Methods and Analysis: 52 patients with unicompartmental knee OA Kellgren-Lawrence grade ≤3 secondary to meniscal deficiency and knee malalignment (aged 20-60 years) are randomised to undergo knee osteotomy associated with MAT or knee osteotomy alone in a 1:1 ratio. The primary outcome is the International Knee Documentation Committee (IKDC) subjective score at 12 months. The secondary outcomes are the IKDC subjective score at 1, 3 and 6 months, the Western Ontario and McMaster Universities Osteoarthritis Index score, the Knee Injury and Osteoarthritis Outcome Score, the Visual Analogue Scale, the EuroQol Visual Analogue Scale, the Tegner Activity Level Scale, objective parameters (transpatellar circumference, suprapatellar circumference and range of motion), patient expectation on treatment efficacy and patient satisfaction at 1, 3, 6 and 12 months. Radiographs are performed at baseline and at 1-, 6- and 12-month follow-ups and magnetic resonance evaluations at baseline and at 12 months. Biomechanical assessment is performed through gait analysis before surgery and at 12 months, investigating motion data, biomechanical parameters and muscle activation through electromyography. In addition, to detect early joint environment changes and potential MAT effects in protecting the articular surface providing a better knee homeostasis, biological markers of cartilage turnover and inflammation obtained from synovial fluid, serum and urine are evaluated at baseline and at 24 hours, 1-, 3-, 6- and 12-month follow-ups., Ethics and Dissemination: The study protocol has been approved by Emilia Romagna's Ethics Committee (CE-AVEC), Bologna, Italy. Written informed consent is obtained from all participants. Findings of this study will be disseminated through peer-reviewed publications and conference presentations., Protocol Version: March 2023., Trial Registration Number: NCT05840887., Competing Interests: Competing interests: SZ has received institutional support from FidiaFarmaceutici S.p.A., Cartiheal, IGEA Clinical Biophysics, BIOMET and Kensey Nash; grant support from I+ SRL; and royalties from Springer outside the submitted work. The principal investigator and the other authors declare no financial or competing interest., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ Group.)

Advanced tools for the in situ treatment of articular cartilage lesions are attracting a growing interest in both surgery and bioengineering communities. The interest is particularly high concerning the delivery of cell-laden hydrogels. The tools currently available in the state-of-the-art hardly find an effective compromise between treatment accuracy and invasiveness. This paper presents a novel arthroscopic device provided with a bendable tip for the controlled extrusion of cell-laden hydrogels. The device consists of a handheld extruder and a supply unit that allows the extrusion of hydrogels. The extruder is equipped with a disposable, bendable nitinol tip (diameter: 4 mm, length: 92 mm, maximum bending angle: 90°) that guarantees access to hard-to-reach areas of the joint, which are difficult to get to, with conventional arthroscopic instruments. The tip accommodates a biocompatible polymer tube that is directly connected to the cartridge containing the hydrogel, whose plunger is actuated by a volumetric or pneumatic supply unit (both tested, in this study). Three different chondrocyte-laden hydrogels (RGD-modified Vitrogel®, methacrylated gellan gum, and an alginate-gelatine blend) were considered. First, the performance of the device in terms of resolution in hydrogel delivery was assessed, finding values in the range between 4 and 102 µL, with better performance found for the pneumatic supply unit and no significant differences between straight tip and bent tip conditions. Finite element simulations suggested that the shear stresses and pressure levels generated during the extrusion process were compatible with a safe deposition of the hydrogels. Biological analyses confirmed a high chondrocyte viability over a 7-day period after the extrusion of the three cell-laden hydrogel types, with no differences between the two supply units. The arthroscopic device was finally tested ex vivo by nine orthopedic surgeons on human cadaver knees. The device allowed surgeons to easily deliver hydrogels even in hard-to-reach cartilage areas. The outcomes of a questionnaire completed by the surgeons demonstrated a high usability of the device, with an overall preference for the pneumatic supply unit. Our findings provide evidence supporting the future arthroscopic device translation in pre-clinical and clinical scenarios, dealing with osteoarticular treatments., (© 2024. The Author(s).)

While conventional radiography and MRI have a well-established role in the assessment of patients with knee osteoarthritis, ultrasound is considered a complementary and additional tool. Moreover, the actual usefulness of ultrasound is still a matter of debate in knee osteoarthritis assessment. Despite that, ultrasound offers several advantages and interesting aspects for both current clinical practice and future perspectives. Ultrasound is potentially a helpful tool in the detection of anomalies such as cartilage degradation, osteophytes, and synovitis in cases of knee osteoarthritis. Furthermore, local diagnostic and minimally invasive therapeutic operations pertaining to knee osteoarthritis can be safely guided by real-time ultrasound imaging. We are constantly observing a growing knowledge and awareness among radiologists and other physicians, concerning ultrasound imaging. Ultrasound studies can be extremely useful to track the response to various therapies. For this specific aim, tele-ultrasonography may constitute an easy tool aiding precise and repeated follow-up controls. Moreover, raw radio-frequency data from US backscattering signals contain more information than B-mode imaging. This paves the way for quantitative in-depth analyses of cartilage, bone, and other articular structures. Overall, ultrasound technologies and their rapid evolution have the potential to make a difference at both the research and clinical levels. This narrative review article describes the potential of such technologies and their possible future implications.

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., (© 2024 Wiley Periodicals LLC.)

Ewing sarcoma (EWS) is the second most common pediatric bone tumor. The EWS tumor microenvironment is largely recognized as immune-cold, with macrophages being the most abundant immune cells and their presence associated with worse patient prognosis. Expression of CD99 is a hallmark of EWS cells, and its targeting induces inhibition of EWS tumor growth through a poorly understood mechanism. In this study, we analyzed CD99 expression and functions on macrophages and investigated whether the concomitant targeting of CD99 on both tumor and macrophages could explain the inhibitory effect of this approach against EWS. Targeting CD99 on EWS cells downregulated expression of the "don't eat-me" CD47 molecule but increased levels of the "eat-me" phosphatidyl serine and calreticulin molecules on the outer leaflet of the tumor cell membrane, triggering phagocytosis and digestion of EWS cells by macrophages. In addition, CD99 ligation induced reprogramming of undifferentiated M0 macrophages and M2-like macrophages toward the inflammatory M1-like phenotype. These events resulted in the inhibition of EWS tumor growth. Thus, this study reveals what we believe to be a previously unrecognized function of CD99, which engenders a virtuous circle that delivers intrinsic cell death signals to EWS cells, favors tumor cell phagocytosis by macrophages, and promotes the expression of various molecules and cytokines, which are pro-inflammatory and usually associated with tumor regression. This raises the possibility that CD99 may be involved in boosting the antitumor activity of macrophages., (©2023 The Authors; Published by the American Association for Cancer Research.)

The use of piezoelectric nanomaterials combined with ultrasound stimulation is emerging as a promising approach for wirelessly triggering the regeneration of different tissue types. However, it has never been explored for boosting chondrogenesis. Furthermore, the ultrasound stimulation parameters used are often not adequately controlled. In this study, we show that adipose-tissue-derived mesenchymal stromal cells embedded in a nanocomposite hydrogel containing piezoelectric barium titanate nanoparticles and graphene oxide nanoflakes and stimulated with ultrasound waves with precisely controlled parameters (1 MHz and 250 mW/cm 2 , for 5 min once every 2 days for 10 days) dramatically boost chondrogenic cell commitment in vitro . Moreover, fibrotic and catabolic factors are strongly down-modulated: proteomic analyses reveal that such stimulation influences biological processes involved in cytoskeleton and extracellular matrix organization, collagen fibril organization, and metabolic processes. The optimal stimulation regimen also has a considerable anti-inflammatory effect and keeps its ability to boost chondrogenesis in vitro , even in an inflammatory milieu. An analytical model to predict the voltage generated by piezoelectric nanoparticles invested by ultrasound waves is proposed, together with a computational tool that takes into consideration nanoparticle clustering within the cell vacuoles and predicts the electric field streamline distribution in the cell cytoplasm. The proposed nanocomposite hydrogel shows good injectability and adhesion to the cartilage tissue ex vivo , as well as excellent biocompatibility in vivo, according to ISO 10993. Future perspectives will involve preclinical testing of this paradigm for cartilage regeneration.

Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Type-2 Familial Partial Lipodystrophy (FPLD2), a rare lipodystrophy caused by LMNA mutations, is characterized by a loss of subcutaneous fat from the trunk and limbs and excess accumulation of adipose tissue in the neck and face. Several studies have reported that the mineralocorticoid receptor (MR) plays an essential role in adipose tissue differentiation and functionality. We previously showed that brown preadipocytes isolated from a FPLD2 patient's neck aberrantly differentiate towards the white lineage. As this condition may be related to MR activation, we suspected altered MR dynamics in FPLD2. Despite cytoplasmic MR localization in control brown adipocytes, retention of MR was observed in FPLD2 brown adipocyte nuclei. Moreover, overexpression of wild-type or mutated prelamin A caused GFP-MR recruitment to the nuclear envelope in HEK293 cells, while drug-induced prelamin A co-localized with endogenous MR in human preadipocytes. Based on in silico analysis and in situ protein ligation assays, we could suggest an interaction between prelamin A and MR, which appears to be inhibited by mineralocorticoid receptor antagonism. Importantly, the MR antagonist spironolactone redirected FPLD2 preadipocyte differentiation towards the brown lineage, avoiding the formation of enlarged and dysmorphic lipid droplets. Finally, beneficial effects on brown adipose tissue activity were observed in an FPLD2 patient undergoing spironolactone treatment. These findings identify MR as a new lamin A interactor and a new player in lamin A-linked lipodystrophies.

Articular cartilage defects and degenerative diseases are pathological conditions that cause pain and the progressive loss of joint functionalities. The most severe cases are treated through partial or complete joint replacement with prostheses, even if the interest in cartilage regeneration and re-growth methods is steadily increasing. These methods consist of the targeted deposition of biomaterials. Only a few tools have been developed so far for performing these procedures in a minimally invasive way. This work presents an innovative device for the direct deposition of multiple biomaterials in an arthroscopic scenario. The tool is easily handleable and allows the extrusion of three different materials simultaneously. It is also equipped with a flexible tip to reach remote areas of the damaged cartilage. Three channels are arranged coaxially and a spring-based dip-coating approach allows the fabrication and assembly of a bendable polymeric tip. Experimental tests were performed to characterize the tip, showing the ability to bend it up to 90° (using a force of ~ 1.5 N) and to extrude three coaxial biomaterials at the same time with both tip straight and tip fully bent. Rheometric analysis and fluid-dynamic computational simulations were performed to analyze the fluids' behavior; the maximum shear stresses were observed in correspondence to the distal tip and the channel convergence chamber, but with values up to ~ 1.2 kPa, compatible with a safe extrusion of biomaterials, even laden with cells. The cells viability was assessed after the extrusion with Live/Dead assay, confirming the safety of the extrusion procedures. Finally, the tool was tested arthroscopically in a cadaveric knee, demonstrating its ability to deliver the biomaterial in different areas, even ones that are typically hard-to-reach with traditional tools., (© 2022. The Author(s).)

This systematic review is focused on the main characteristics of the hydrogels used for embedding the mesenchymal stromal cells (MSCs) in in vitro/ex vivo studies, in vivo OA models and clinical trials for favoring cartilage regeneration in osteoarthritis (OA). PubMED and Embase databases were used to select the papers that were submitted to a public reference manager Rayyan Systematic Review Screening Software. A total of 42 studies were considered eligible: 25 articles concerned in vitro studies, 2 in vitro and ex vivo ones, 5 in vitro and in vivo ones, 8 in vivo ones and 2 clinical trials. Some in vitro studies evidenced a rheological characterization of the hydrogels and description of the crosslinking methods. Only 37.5% of the studies considered at the same time chondrogenic, fibrotic and hypertrophic markers. Ex vivo studies focused on hydrogel adhesion properties and the modification of MSC-laden hydrogels subjected to compression tests. In vivo studies evidenced the effect of cell-laden hydrogels in OA animal models or defined the chondrogenic potentiality of the cells in subcutaneous implantation models. Clinical studies confirmed the positive impact of these treatments on patients with OA. To speed the translation to the clinical use of cell-laden hydrogels, further studies on hydrogel characteristics, injection modalities, chemo-attractant properties and adhesion strength are needed.

Autophagy is a cellular process that contributes to the maintenance of cell homeostasis through the activation of a specific path, by providing the necessary factors in stressful and physiological situations. Autophagy plays a specific role in chondrocyte differentiation; therefore, we aimed to analyze this process in adipose-derived mesenchymal stromal cells (ASCs) laden in three-dimensional (3D) hydrogel. We analyzed chondrogenic and autophagic markers using molecular biology, immunohistochemistry, and electron microscopy. We demonstrated that ASCs embedded in 3D hydrogel showed an increase expression of typical autophagic markers Beclin 1, LC3, and p62, associated with clear evidence of autophagic vacuoles in the cytoplasm. During ASCs chondrogenic differentiation, we showed that autophagic markers declined their expression and autophagic vesicles were rare, while typical chondrogenic markers collagen type 2, and aggrecan were significantly increased. In line with developmental animal models of cartilage, our data showed that in a 3D hydrogel, ASCs increased their autophagic features. This path is the fundamental prerequisite for the initial phase of differentiation that contributes to fueling the cells with energy and factors necessary for chondrogenic differentiation.

A stable adhesion to the cartilage is a crucial requisite for hydrogels used for cartilage regeneration. Indeed, a weak interface between the tissue and the implanted material may produce a premature detachment and thus the failure of the regeneration processes. Fibrin glue, cellulose nanofibers and catecholamines have been proposed in the state-of-the-art as primers to improve the adhesion. However, no studies focused on a systematic comparison of their performance. This work aims to evaluate the adhesion strength between ex vivo cartilage specimens and polysaccharide hydrogels (gellan gum and methacrylated gellan gum), by applying the mentioned primers as intermediate layer. Results show that the fibrin glue and the cellulose nanofibers improve the adhesion strength, while catecholamines do not guarantee reaching a clinically acceptable value. Stem cells embedded in gellan gum hydrogels reduce the adhesion strength when fibrin glue is used as a primer, being anyhow still sufficient for in vivo applications., (© 2022 The Authors. Macromolecular Bioscience published by Wiley-VCH GmbH.)

Objective: Immune cell evaluation could be useful for clarifying etiopathogenesis, providing a support for formulating the diagnoses of clinically similar joint pathologies or guiding indications for possible therapeutic targets. To contribute to differential diagnosis in joint pathologies we performed an immunophenotypical profile analyzing different immune cells in synovial tissues from patients with rheumatoid arthritis (RA) and osteoarthritis (OA)., Methods: The Krenn and immunologic synovitis (IMSYC) scores, which include the evaluation of T lymphocytes (CD3 positive), B lymphocytes (CD20), endothelial cells (CD31), macrophages (CD68) and proliferating cells (Ki-67 positive) were used to analyze the synovial tissue samples. Moreover, to corroborate immune activation, neutrophils (CD15 positive), NK cells (CD56 positive), plasma cells (CD138 positive), IgG4 and IgG4 secreting-CD138 cells were analyzed using immunohistochemical techniques., Results: We confirmed that all the samples had a high synovitis score according to both the Krenn and IMSYC scores. In both the RA and OA groups, we found similar scores for CD3 (T lymphocytes), CD20 (B lymphocytes), CD31 (endothelial cells), CD56 (NK cells), CD68 (macrophages) CD138 (plasma cells) and IgG4. In contrast, CD15 (neutrophils) was significantly higher in RA compared to OA. Interestingly, IgG4 secreting-CD138 cells were significantly higher in RA than OA, even if CD138 had the same score in both the RA and OA samples., Conclusions: This study found that the scores for different immune cells were similar in both RA and OA synovial tissue with a high synovitis score. CD15 and IgG4 secreting-CD138 were the only immune cells with a higher score in RA compared to OA, suggesting a potential use for discriminating among pathologies with a high synovitis score., (Copyright © 2022 Elsevier GmbH. All rights reserved.)

Articular cartilage is known to have limited intrinsic self-healing capacity when a defect or a degeneration process occurs. Hydrogels represent promising biomaterials for cell encapsulation and injection in cartilage defects by creating an environment that mimics the cartilage extracellular matrix. The aim of this study is the analysis of two different concentrations (1:1 and 1:2) of VitroGel ® (VG) hydrogels without (VG-3D) and with arginine-glycine-aspartic acid (RGD) motifs, (VG-RGD), verifying their ability to support chondrogenic differentiation of encapsulated human adipose mesenchymal stromal cells (hASCs). We analyzed the hydrogel properties in terms of rheometric measurements, cell viability, cytotoxicity, and the expression of chondrogenic markers using gene expression, histology, and immunohistochemical tests. We highlighted a shear-thinning behavior of both hydrogels, which showed good injectability. We demonstrated a good morphology and high viability of hASCs in both hydrogels. VG-RGD 1:2 hydrogels were the most effective, both at the gene and protein levels, to support the expression of the typical chondrogenic markers, including collagen type 2, SOX9, aggrecan, glycosaminoglycan, and cartilage oligomeric matrix protein and to decrease the proliferation marker MKI67 and the fibrotic marker collagen type 1. This study demonstrated that both hydrogels, at different concentrations, and the presence of RGD motifs, significantly contributed to the chondrogenic commitment of the laden hASCs.

Background: Progressive pseudorheumatoid dysplasia (PPRD) is a rare autosomal recessive non-inflammatory skeletal disease with childhood onset and is characterized by a progressive chondropathy in multiple joints, and skeletal abnormalities. To date, the etiopathological relationship between biological modification occurring in PPRD and genetic mutation remains an open issue, partially due to the limited availability of biological samples obtained from PPRD patients for experimental studies., Case Presentation: We describe the clinical features of a PPRD patient and experimental results obtained from the biological characterization of PPRD mesenchymal stromal cells (MSCs) and osteoblasts (OBs) compared to normal cell populations. Phenotypic profile modifications were found in PPRD compared to normal subjects, essentially ascribed to decreased expression of CD146, osteocalcin (OC) and bone sialoprotein in PPRD MSCs and enhanced CD146, OC and collagen type I expression in PPRD OBs. Gene expression of Dickkopf-1, a master inhibitor of WNT signaling, was remarkably increased in PPRD MSCs compared to normal expression range, whereas PPRD OBs essentially exhibited higher OC gene expression levels. PPRD MSCs failed to efficiently differentiate into mature OBs, so showing a greatly impaired osteogenic potential., Conclusions: Since all regenerative processes require stem cell reservoirs, compromised functionality of MSCs may lead to an imbalance in bone homeostasis, suggesting a potential role of MSCs in the pathological mechanisms of PPRD caused by WNT1-inducible signaling pathway protein-3 (WISP3) mutations. In consideration of the lack of compounds with proven efficacy in such a rare disease, these data might contribute to better identify new specific and effective therapeutic approaches., (© 2022. The Author(s).)

Osteoarthritis (OA) is the most prevalent joint disorder, causing pain and disability predominantly in the aging population but also affecting young individuals. Current treatments are limited to use of anti-inflammatory drugs to alleviate symptoms or degenerated joint replacement by a prosthetic implant at the end stage of the disease. We hypothesized that degenerative cartilage defects can be treated using nasal chondrocyte–based tissue-engineered cartilage (N-TEC). We demonstrate that N-TEC maintained cartilaginous properties when exposed in vitro to inflammatory stimuli found in osteoarthritic joints and favorably altered the inflammatory profile of cells from osteoarthritic joints. These effects were at least partially mediated by down-regulation of the WNT (wingless/integrated) signaling pathway through sFRP1 (secreted frizzled-related protein-1). We further report that N-TEC survive and engraft in vivo in ectopic mouse models reproducing a human osteochondral OA tissue environment, as well as in sheep articular cartilage defects that mimic degenerative settings. Last, we tested the safety of autologous N-TEC for the treatment of osteoarthritic cartilage defects in the knees of two patients with advanced OA (Kellgren and Lawrence grades 3 and 4) who were otherwise considered for unicondylar knee arthroplasty. No adverse reactions were recorded, and patients reported reduced pain as well as improved joint function and life quality 14 months after surgery. Together, our findings indicate that N-TEC can directly contribute to cartilage repair in osteoarthritic joints. A suitably powered clinical trial is now required to assess its efficacy in the treatment of patients with OA.

Robotic dispensing-based 3D bioprinting represents one of the most powerful technologies to develop hydrogel-based 3D constructs with enormous potential in the field of regenerative medicine. The optimization of hydrogel printing parameters, proper geometry and internal architecture of the constructs, and good cell viability during the bioprinting process are the essential requirements. In this paper, an analytical model based on the hydrogel rheological properties was developed to predict the extruded filament width in order to maximize the printed structure's fidelity to the design. Viscosity data of two natural hydrogels were imputed to a power-law model to extrapolate the filament width. Further, the model data were validated by monitoring the obtained filament width as the output. Shear stress values occurring during the bioprinting process were also estimated. Human mesenchymal stromal cells (hMSCs) were encapsulated in the silk fibroin-gelatin (G)-based hydrogel, and a 3D bioprinting process was performed to produce cell-laden constructs. Live and dead assay allowed estimating the impact of needle shear stress on cell viability after the bioprinting process. Finally, we tested the potential of hMSCs to undergo chondrogenic differentiation by evaluating the cartilaginous extracellular matrix production through immunohistochemical analyses. Overall, the use of the proposed analytical model enables defining the optimal printing parameters to maximize the fabricated constructs' fidelity to design parameters before the process execution, enabling to achieve more controlled and standardized products than classical trial-and-error approaches in the biofabrication of engineered constructs. Employing modeling systems exploiting the rheological properties of the hydrogels might be a valid tool in the future for guaranteeing high cell viability and for optimizing tissue engineering approaches in regenerative medicine applications.

Selection of feasible hybrid-hydrogels for best chondrogenic differentiation of human mesenchymal stromal cells (hMSCs) represents an important challenge in cartilage regeneration. In this study, three-dimensional hybrid hydrogels obtained by chemical crosslinking of poly (ethylene glycol) diglycidyl ether (PEGDGE), gelatin (G) without or with chitosan (Ch) or dextran (Dx) polysaccharides were developed. The hydrogels, namely G-PEG, G-PEG-Ch and G-PEG-Dx, were prepared with an innovative, versatile and cell-friendly technique that involves two preparation steps specifically chosen to increase the degree of crosslinking and the physical-mechanical stability of the product: a first homogeneous phase reaction followed by directional freezing, freeze-drying and post-curing. Chondrogenic differentiation of human bone marrow mesenchymal stromal cells (hBM-MSC) was tested on these hydrogels to ascertain whether the presence of different polysaccharides could favor the formation of the native cartilage structure. We demonstrated that the hydrogels exhibited an open pore porous morphology with high interconnectivity and the incorporation of Ch and Dx into the G-PEG common backbone determined a slightly reduced stiffness compared to that of G-PEG hydrogels. We demonstrated that G-PEG-Dx showed a significant increase of its anisotropic characteristic and G-PEG-Ch exhibited higher and faster stress relaxation behavior than the other hydrogels. These characteristics were associated to absence of chondrogenic differentiation on G-PEG-Dx scaffold and good chondrogenic differentiation on G-PEG and G-PEG-Ch. Furthermore, G-PEG-Ch induced the minor collagen proteins and the formation of collagen fibrils with a diameter like native cartilage. This study demonstrated that both anisotropic and stress relaxation characteristics of the hybrid hydrogels were important features directly influencing the chondrogenic differentiation potentiality of hBM-MSC., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Scaffolds associated with mesenchymal stem cell (MSC) derivatives, such as extracellular vesicles (EVs), represent interesting carriers for bone regeneration. This systematic review aims to analyze in vitro and in vivo studies that report the effects of EVs combined with scaffolds in bone regeneration. A methodical review of the literature was performed from PubMed and Embase from 2012 to 2020. Sixteen papers were analyzed; of these, one study was in vitro, eleven were in vivo, and four were both in vitro and in vivo studies. This analysis shows a growing interest in this upcoming field, with overall positive results. In vitro results were demonstrated as both an effect on bone mineralization and proangiogenic ability. The interesting in vitro outcomes were confirmed in vivo. Particularly, these studies showed positive effects on bone regeneration and mineralization, activation of the pathway for bone regeneration, induction of vascularization, and modulation of inflammation. However, several aspects remain to be elucidated, such as the concentration of EVs to use in clinic for bone-related applications and the definition of the real advantages.

Human platelet lysate (hPL) is considered a valid substitute to fetal bovine serum (FBS) in the expansion of mesenchymal stromal cells (MSC), and it is commonly produced starting from intermediate side products of whole blood donations. Through freeze-thaw cycles, hPL is highly enriched in chemokines, growth factors, and adhesion and immunologic molecules. Cell therapy protocols, using hPL instead of FBS for the expansion of cells, are approved by regulatory authorities without concerns, and its administration in patients is considered safe. However, published data are fairly difficult to compare, since the production of hPL is highly variable. This study proposes to optimize and standardize the hPL productive process by using instruments, technologies, and quality/safety standards required for blood bank activities and products. The quality and improved selection of the starting material (i.e., the whole blood), together with the improvement of the production process, guarantee a product characterized by higher content and quality of growth factors as well as a reduction in batch-to-batch variability. By increasing the number of freeze/thaw cycles from one (hPL1c) to four (hPL4c), we obtained a favorable effect on the release of growth factors from platelet α granules. Those changes have directly translated into biological effects leading to a decreasing doubling time (DT) of MSC expansion at 7 days (49.41 ± 2.62 vs. 40.61 ± 1.11 h, p < 0.001). Furthermore, mass spectrometry (MS)-based evaluation has shown that the proliferative effects of hPL4c are also combined with a lower batch-to-batch variability (10-15 vs. 21-31%) at the proteomic level. In conclusion, we have considered lot-to-lot hPL variability, and by the strict application of blood bank standards, we have obtained a standardized, reproducible, safe, cheap, and ready-to-use product., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Bianchetti, Chinello, Guindani, Braga, Neva, Verardi, Piovani, Pagani, Lisignoli, Magni, Russo and Almici.)

This paper aims to characterize the wear behavior of hydrogel constructs designed for human articular cartilage replacement. To this purpose, poly (ethylene glycol) diacrylate (PEGDA) 10% w / v and gellan gum (GG) 1.5% w / v were used to reproduce the superior (SUP) cartilage layer and PEGDA 15% w / v and GG 1.5% w / v were used to reproduce the deep (DEEP) cartilage layer, with or without graphene oxide (GO). These materials (SUP and DEEP) were analyzed alone and in combination to mimic the zonal architecture of human articular cartilage. The developed constructs were tested using a four-station displacement control knee joint simulator under bovine calf serum. Roughness and micro-computer tomography (µ-CT) measurements evidenced that the hydrogels with 10% w / v of PEGDA showed a worse behavior both in terms of roughness increase and loss of uniformly distributed density than 15% w / v of PEGDA. The simultaneous presence of GO and 15% w / v PEGDA contributed to keeping the hydrogel construct's characteristics. The Raman spectra of the control samples showed the presence of unreacted C=C bonds in all the hydrogels. The degree of crosslinking increased along the series SUP < DEEP + SUP < DEEP without GO. The Raman spectra of the tested hydrogels showed the loss of diacrylate groups in all the samples, due to the washout of unreacted PEGDA in bovine calf serum aqueous environment. The loss decreased along the series SUP > DEEP + SUP > DEEP, further confirming that the degree of photo-crosslinking of the starting materials plays a key role in determining their wear behavior. μ-CT and Raman spectroscopy proved to be suitable techniques to characterize the structure and composition of hydrogels.

There is a lack ofin vitromodels able to properly represent osteoarthritis (OA) synovial tissue (ST). We aimed to characterize OA ST and to investigate whether a mechanical or enzymatic digestion procedures influence synovial cell functional heterogeneity in vitro. Procedures using mechanical nondigested fragments (NDF), synovial digested fragments (SDF), and filtrated synovial digested cells (SDC) were compared. An immunophenotypic profile was performed to distinguish synovial fibroblasts (CD55, CD73, CD90, CD106), macrophages (CD14, CD68), M1-like (CD80, CD86), and M2-like (CD163, CD206) synovial macrophages. Pro-inflammatory (interleukin 6 IL6), tumor necrosis factor alpha (TNFα), chemokine C-C motif ligand 3 (CCL3/MIP1α), C-X- motif chemokine ligand 10 (CXCL10/IP10) and anti-inflammatory (interleukin 10 (IL10)), transforming growth factor beta 1 (TGFβ1), C-C motif chemokine ligand 18 (CCL18) cytokines were evaluated. CD68 and CD163 markers were higher in NDF and SDF compared to the SDC procedure, while CD80, CD86, and CD206 were higher only in NDF compared to the SDC procedure. Synovial fibroblast markers showed similar percentages. TNFα, CCL3/MIP1α, CXCL10/IP10, and CCL18 were higher in NDF compared to SDC, but not compared to SDF. IL10 and TGFβ1 were higher in NDF than SDC at the molecular level, while IL6 did not show differences among procedures. We demonstrated that NDF isolation procedures better preserved the heterogeneity of specific OA synovial populations (fibroblasts, macrophages), fostering their use for testing new cell therapies or drugs for OA, reducing or avoiding the use of animal models.

Objective: To investigate complement(C) factors(F) and their activation fragments expression in OA joint tissues., Design: Immunohistochemistry and quantitative imaging were performed to analyze C3, C4, and CF (factor) B expression on osteochondral biopsies (43 patients) collected during arthroplasty. Isolated chondrocytes and synoviocytes, cartilage and synovial tissues obtained from surgical specimens of OA patients (15 patients) were cultured with or without IL-1β. Real time PCR for CFB, C3, and C4 was performed. Culture supernatants were analyzed for C3a, C5a, CFBa, and terminal complement complex (TCC) production., Results: In osteochondral biopsies, C factor expression was located in bone marrow, in a few subchondral bone cells and chondrocytes. C3 was the most expressed while factor C4 was the least expressed factor. Gene expression showed that all C factors analyzed were expressed both in chondrocytes and synoviocytes. In chondrocyte cultures and cartilage explants, CFB expression was significantly higher than C3 and C4. Furthermore, CFB, but not C3 and C4 expression was significantly induced by IL-1β. As to C activation factors, C3a was the most produced and CFBa was induced by IL-1β in synovial tissue. TCC production was undetectable in isolated chondrocytes and synoviocytes cell culture supernatants, whereas it was significantly augmented in cartilage explants., Conclusion: C factors were locally produced and activated in OA joint with the contribution of all tissues (cartilage, bone, and synovium). Our results support the involvement of innate immunity in OA and suggest an association between some C alternative pathway component and joint inflammation., (Copyright © 2020 Assirelli, Pulsatelli, Dolzani, Mariani, Lisignoli, Addimanda and Meliconi.)

The objective of this study was to define the effects of osteoarthritic (OA) milieu on good manufactured practice-adipose-derived mesenchymal stromal cells (GMP-ASC) that are commonly utilized in cell therapies. Two different OA milieu: OA synovial fluid (SF) and OA-conditioned medium (CM) from synoviocytes were used to treat GMP-ASC both in normoxia or hypoxia. GMP-ASC were tested for cell migration, proliferation, cytokine receptors expression (CXCR1, CXCR2, CXCR3, CXCR4, CXCR7, CCR1, CCR2, CCR3, CCR5, IL6R), and cytokines (CXCL8/IL8, CXCL10/IP10, CXCL12/SDF-1, CCL2/MCP1, CCL3/MIP1α, CCL4/MIP1β, CCL5/RANTES, IL6) release. Healthy SF was used as controls. We demonstrated that GMP-ASC show an increase in proliferation, migration, and modulation of CXCR1, CXCR3, CCR1, and CCR5 receptors in hypoxic condition. Moreover, GMP-ASC migration increased 15-fold when treated either with OA-SF or OA-CM compared with healthy SF both in normoxia and hypoxia. GMP-ASC treated in both OA milieu showed an increase in CXCR3, CCR3, and IL6R and a decrease in CCR1 and CCR2 receptors. In OA-SF, we detected higher amount of CXCL10/IP10 than in OA-CM, while CCL2/MCP1 and CCL4/MIP1β were higher in OA-CM compared with OA-SF. CXCL10/IP10 was the only chemokine of the OA milieu, which was down-modulated after treatment with GMP-ASC. In conclusion, we demonstrated specific effects of OA milieu on both GMP-ASC proliferation, migration, and cytokine receptor expression that were strictly dependent on the inflammatory and hypoxic environment. The use of characterized OA milieu is crucial to define the therapeutic effect of GMP-ASC and indicates that CXCL10/IP10-CXCR3 axis is partially involved in the GMP-ASC effect on synovial macrophages. © 2019 The Authors. Journal of Orthopaedic Research® published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 38:336-347, 2020., (© 2019 The Authors. Journal of Orthopaedic Research® published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society.)

Hyaluronic acid (HA) is an ideal material for tissue regeneration. The aim of this study was to investigate whether a hyaluronan amide derivative (HAD) can enhance the mineralization of human mesenchymal stem cells (hMSCs). Osteogenically induced hMSCs cultured with or without HAD at different concentrations (0.5 mg/ml or 1 mg/ml) were analyzed for mineral matrix deposition, metabolic activity, cellular proliferation, and the expression of 14 osteogenic genes. Unmodified HA (HYAL) was used as control. We demonstrated that only cells treated daily until day 28 with 0.5 mg/ml HAD, but not with 1 mg/ml of HAD and HYAL, showed a significant induction of mineralization at day 14 compared to the osteogenic control group. HAD at both concentrations tested, significantly decreased the expression of the proliferating marker MKI67 at day 2. By contrast, increased metabolic activity was induced only by HYAL from day 14. HAD at both concentrations significantly down modulated SNAI2, DLX5, RUNX2, COL1A1, and IBSP genes, while significantly up regulated COL15A1. The induction of mineralization of 0.5 mg/ml of HAD at day 14 was significantly dependent on a specific modulation of RUNX2 and COL1A1. Our data demonstrate that only 0.5 mg/ml of HAD, but not HYAL, modulated hMSCs osteogenic differentiation, suggesting that the physicochemical features and concentration of HA products could differently affect osteogenic maturation., (© 2019 Wiley Periodicals, Inc.)

Cartilage tissue engineering remains problematic because no systems are able to induce signals that contribute to native cartilage structure formation. Therefore, we tested the potentiality of gelatin-polyethylene glycol scaffolds containing three different concentrations of chitosan (CH; 0%, 8%, and 16%) on chondrogenic differentiation of human platelet lysate-expanded human bone marrow mesenchymal stromal cells (hBM-MSCs). Typical chondrogenic (SOX9, collagen type 2, and aggrecan), hypertrophic (collagen type 10), and fibrotic (collagen type 1) markers were evaluated at gene and protein level at Days 1, 28, and 48. We demonstrated that 16% CH scaffold had the highest percentage of relaxation with the fastest relaxation rate. In particular, 16% CH scaffold, combined with chondrogenic factor TGFβ3, was more efficient in inducing hBM-MSCs chondrogenic differentiation compared with 0% or 8% scaffolds. Collagen type 2, SOX9, and aggrecan showed the same expression in all scaffolds, whereas collagen types 10 and 1 markers were efficiently down-modulated only in 16% CH. We demonstrated that using human platelet lysate chronically during hBM-MSCs chondrogenic differentiation, the chondrogenic, hypertrophic, and fibrotic markers were significantly decreased. Our data demonstrate that only a high concentration of CH, combined with TGFβ3, creates an environment capable of guiding in vitro hBM-MSCs towards a phenotypically stable chondrogenesis., (© 2019 John Wiley & Sons, Ltd.)

Hydrogen sulfide (H 2 S)-based therapy is a promising therapeutic strategy for several biomedical applications. Following the observation that endogenous and exogenous H 2 S plays a prominent role as a bone anabolic agent, we recently developed a silk fibroin (SF) porous scaffold loaded with GYY4137 (GYY), an H 2 S donor, for applications in bone tissue engineering. Here, we assayed whether the combination of SF with H 2 S 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 H 2 S-releasing SF scaffolds supported cell adhesion, proliferation and viability. Moreover, H 2 S 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 H 2 S-releasing SF scaffolds for bone healing and regeneration applications., (Copyright © 2019. Published by Elsevier B.V.)

Rational design and development of tailorable simple synthesis process remains a centerpiece of investigational efforts toward engineering advanced hydrogels. In this study, a green and scalable synthesis approach is developed to formulate a set of gelatin-based macroporous hybrid hydrogels. This approach consists of four sequential steps starting from liquid-phase pre-crosslinking/grafting, unidirectional freezing, freeze-drying, and finally post-curing process. The chemical crosslinking mainly involves between epoxy groups of functionalized polyethylene glycol and functional groups of gelatin both in liquid and solid state. Importantly, this approach allows to accommodate different polymers, chitosan or hydroxyethyl cellulose, under identical benign condition. Structural and mechanical anisotropy can be tuned by the selection of polymer constituents. Overall, all hydrogels show suitable structural stability, good swellability, high porosity and pore interconnectivity, and maintenance of mechanical integrity during 3-week-long hydrolytic degradation. Under compression, hydrogels exhibit robust mechanical properties with nonlinear elasticity and stress-relaxation behavior and show no sign of mechanical failure under repeated compression at 50% deformation. Biological experiment with human bone marrow mesenchymal stromal cells (hMSCs) reveals that hydrogels are biocompatible, and their physicomechanical properties are suitable to support cells growth, and osteogenic/chondrogenic differentiation, demonstrating their potential application for bone and cartilage regenerative medicine toward clinically relevant endpoints., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Bone marrow and adipose tissue human mesenchymal stem cells were seeded in highly performing 3D gelatin-chitosan hybrid hydrogels of varying chitosan content in the presence of human platelet lysate and evaluated for their proliferation and osteogenic differentiation. Both bone marrow and adipose tissue human mesenchymal stem cells in gelatin-chitosan hybrid hydrogel 1 (chitosan content 8.1%) or gelatin-chitosan hybrid hydrogel 2 (chitosan 14.9%) showed high levels of viability (80%-90%), and their proliferation and osteogenic differentiation was significantly higher with human platelet lysate compared to fetal bovine serum, particularly in gelatin-chitosan hybrid hydrogel 1. Mineralization was detected early, after 21 days of culture, when human platelet lysate was used in the presence of osteogenic stimuli. Proteomic characterization of human platelet lysate highlighted 59 proteins mainly involved in functions related to cell adhesion, cellular repairing mechanisms, and regulation of cell differentiation. In conclusion, the combination of our gelatin-chitosan hybrid hydrogels with hPL represents a promising strategy for bone regenerative medicine using human mesenchymal stem cells., Competing Interests: Declaration of conflicting interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

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., (© 2018 Wiley Periodicals, Inc.)

Scaffold-based bone tissue engineering strategies fail to meet the clinical need to fabricate patient-specific and defect shape-specific, anatomically relevant load-bearing bone constructs. 3D bioprinting strategies are gaining major interest as a potential alternative, but design of a specific bioink is still a major challenge that can modulate key signaling pathways to induce osteogenic differentiation of progenitor cells, as well as offer appropriate microenvironment to augment mineralization. In the present study, we developed silk fibroin protein and gelatin-based conjugated bioink, which showed localized presence and sustained release of calcium. Presence of 2.6 mM Ca 2+ ions within the bioink could further induce enhanced osteogenesis of Bone marrow derived progenitor cells (hMSCs) compared to the bioink without calcium, or same concentration of calcium added to the media, as evidenced by upregulated gene expression of osteogenic markers. This study generated unprecedented mechanistic insights on the role of fibroin-gelatin-CaCl 2 bioink in modulating expression of several proteins which are known to play crucial role in bone regeneration as well as key signaling pathways such as β-catenin, BMP signaling pathway, Parathyroid hormone-dependent signaling pathway, Forkhead box O (FOXO) pathway, and Hippo pathways in hMSC-laden bioprinted constructs.

The perspectives of regenerative medicine are still severely hampered by the host response to biomaterial implantation, despite the robustness of technologies that hold the promise to recover the functionality of damaged organs and tissues. In this scenario, the cellular and molecular events that decide on implant success and tissue regeneration are played at the interface between the foreign body and the host inflammation, determined by innate and adaptive immune responses. To avoid adverse events, rather than the use of inert scaffolds, current state of the art points to the use of immunomodulatory biomaterials and their knowledge-based use to reduce neutrophil activation, and optimize M1 to M2 macrophage polarization, Th1 to Th2 lymphocyte switch, and Treg induction. Despite the fact that the field is still evolving and much remains to be accomplished, recent research breakthroughs have provided a broader insight on the correct choice of biomaterial physicochemical modifications to tune the reaction of the host immune system to implanted biomaterial and to favor integration and healing.

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.

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.

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.

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., (© 2018 The Author(s). Published by S. Karger AG, Basel.)

Mesenchymal stromal cells (MSCs) are key players in the repair or regeneration of the damaged bone tissue. However, heterogeneity exists between MSCs derived from different donors in their bone formation ability both in vitro and in vivo. The identification of markers defining MSCs with different functional phenotypes is fundamental to maximize their clinical potential. In our previous in vivo study, impaired expression in MSCs of cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE), the two key enzymes in the catabolic pathway of homocysteine, was associated to decreased bone formation and to the onset of osteoporosis in mice. Here, we investigated whether osteogenic differentiation of human MSCs (hMSCs) modulates the expression of CBS and CSE. The expression of CBS and CSE was also assessed during chondrogenesis to confirm the specificity of their expression during osteogenesis. hMSCs displayed a heterogeneous mineralizing capacity between donors (70% of the samples mineralized, while 30% did not mineralize). Inducible expression of CBS and CSE was found to be associated with a mineralizing phenotype in hMSCs. In particular, up-regulation of CSE was restricted to hMSCs undergoing mineralization. During chondrogenesis, CBS was significantly up-regulated while CSE expression was not affected. Ex-vivo findings confirmed that mature h-osteoblasts (hOBs) show consistently higher expression of CBS and CSE than hMSCs. Our data provide the first evidence that the expression of CBS and CSE in hMSCs closely correlates with the transition of hMSCs toward the osteoblastic phenotype and that CSE may constitute a novel marker of osteogenic differentiation., (© 2017 Wiley Periodicals, Inc.)

Objective: Assuming that mesenchymal stem cells (MSCs) respond to the osteoarthritic joint environment to exert a chondroprotective effect, we aimed at investigating the molecular response setup by MSCs after priming by osteoarthritic chondrocytes in cocultures., Methods: We used primary human osteoarthritic chondrocytes and adipose stem cells (ASCs) in mono- and cocultures and performed a high-throughput secretome analysis. Among secreted proteins differentially induced in cocultures, we identified thrombospondin-1 (THBS1) as a potential candidate that could be involved in the chondroprotective effect of ASCs., Results: Secretome analysis revealed significant induction of THBS1 in ASCs/chondrocytes cocultures at mRNA and protein levels. We showed that THBS1 was upregulated at late stages of MSC differentiation toward chondrocytes and that recombinant THBS1 (rTHBS1) exerted a prochondrogenic effect on MSC indicating a role of THBS1 during chondrogenesis. However, compared to control ASCs, siTHBS1-transfected ASCs did not decrease the expression of hypertrophic and inflammatory markers in osteoarthritic chondrocytes, suggesting that THBS1 was not involved in the reversion of osteoarthritic phenotype. Nevertheless, downregulation of THBS1 in ASCs reduced their immunosuppressive activity, which was consistent with the anti-inflammatory role of rTHBS1 on T lymphocytes. THBS1 function was then evaluated in the collagenase-induced OA model by comparing siTHBS1-transfected and control ASCs. The protective effect of ASCs evaluated by histological and histomorphological analysis of cartilage and bone was not seen with siTHBS1-transfected ASCs., Conclusion: Our data suggest that THBS1 did not exert a direct protective effect on chondrocytes but might reduce inflammation, subsequently explaining the therapeutic effect of ASCs in OA.

Hydrogen sulfide (H 2 S), 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 H 2 S and blunted gene expression of CSE. However, there are still a lot of unknowns on how H 2 S 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/H 2 S system is in relation with estrogen signaling in bone in a gender specific manner., Competing Interests: CONFLICTS OF INTEREST The authors have no conflicts of interest to declare.

Bisphosphonates (BPs) are the first-line treatment of bone loss resulting from various pathological conditions. Due to their high affinity to bone they have been used to develop conjugates with pro-anabolic or anti-catabolic drugs. We recently demontrated that hydrogen sulfide (H 2 S), promotes osteogenesis and inhibits osteoclast differentiation. Here we developed an innovative molecule, named DM-22, obtained from the combination of alendronate (AL) and the H 2 S-releasing moiety aryl-isothiocyanate. DM-22 and AL were assayed in vitro in the concentration range 1-33 μM for effects on viability and function of human osteoclasts (h-OCs) and mesenchymal stromal cells (h-MSCs) undergoing osteogenic differentiation. Amperometric measures revealed that DM-22 releases H 2 S at a slow rate with a thiol-dependent mechanism. DM-22 significantly inhibited h-OCs differentiation and function, maintaining a residual h-OCs viability even at the high dose of 33 μM. Contrary to AL, in h-MSCs DM-22 did not induce cytotoxicity as revealed by LDH assay, significantly stimulated mineralization as measured by Alizarin Red staining and increased mRNA expression of Collagen I as compared to control cultures. In conclusion, DM-22 is a new BP which inhibits h-OCs function and stimulate osteogenic differentiation of h-MSCs, without cytotoxicity. DM-22 is an ideal candidate for a novel family of osteoanabolic drugs.