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.

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

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

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

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

Collagen type XV and the 'osteogenic status'.

We have previously demonstrated that collagen type XV (ColXV) is a novel bone extracellular matrix (ECM) protein. It is well known that the complex mixture of multiple components present in ECM can help both to maintain stemness or to promote differentiation of stromal cells following change in qualitative characteristics or concentrations. We investigated the possible correlation between ColXV expression and mineral matrix deposition by human mesenchymal stromal cells (hMSCs) with different osteogenic potential and by osteoblasts (hOBs) that are able to grow in culture medium with or without calcium. Analysing the osteogenic process, we have shown that ColXV basal levels are lower in cells less prone to osteo-induction such as hMSCs from Wharton Jelly (hWJMSCs), compared to hMSCs that are prone to osteo-induction such as those from the bone marrow (hBMMSCs). In the group of samples identified as 'mineralized MSCs', during successful osteogenic induction, ColXV protein continued to be detected at substantial levels until early stage of differentiation, but it significantly decreased and then disappeared at the end of culture when the matrix formed was completely calcified. The possibility to grow hOBs in culture medium without calcium corroborated the results obtained with hMSCs demonstrating that calcium deposits organized in a calcified matrix, and not calcium 'per se', negatively affected ColXV expression. As a whole, our data suggest that ColXV may participate in ECM organization in the early-phases of the osteogenic process and that this is a prerequisite to promote the subsequent deposition of mineral matrix.

Distinctive expression pattern of cystathionine-ß-synthase and cystathionine-γ-lyase identifies mesenchymal stromal cells transition to mineralizing osteoblasts.

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-ß-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.

Age-independent effects of hyaluronan amide derivative and growth hormone on human osteoarthritic chondrocytes.

AIM: Increased age is the most prominent risk factor for the initiation and progression of osteoarthritis (OA). The effects of human growth hormone (hGH) combined or not with hyaluronan amide derivative (HAD) were evaluated on human OA chondrocytes, to define their biological action and potentiality in OA treatment. MATERIAL AND METHODS: Cell viability, metabolic activity, gene expression and factors released were tested at different time points on chondrocytes treated with different concentrations of hGH (0.01-10 µg/ml) alone or in combination with HAD (1 mg/ml). RESULTS: We found that OA chondrocytes express GH receptor and that the different doses of hGH tested did not affect cell viability, metabolic activity or the expression of collagen type 2, 1, or 10 nor did it induce the release of IGF-1 or FGF-2. Conversely, hGH treatment increased the expression of hyaluronan receptor CD44. HAD combined with hGH reduced metabolic activity, IL6 release and gene expression, but not the suppressor of cytokine signaling 2 (SOCS2), which was significantly induced and translocated into the nucleus. The parameters analyzed, independently of the treatments used proportionally decreased with increasing age of the patients. CONCLUSIONS: hGH only induced CD44 receptor on OA chondrocytes but did not affect other parameters, such as chondrocytic gene markers or IGF-1 or FGF-2 release. HAD reduced all the effects induced by hGH partially through a significant induction of SOCS2. These data show that GH or HAD treatment does not influence the response of the OA chondrocytes, thus the modulation of cellular response is age-independent.

Novel nano-composite biomimetic biomaterial allows chondrogenic and osteogenic differentiation of bone marrow concentrate derived cells.

In clinical orthopedics suitable materials that induce and restore biological functions together with the right mechanical properties are particularly needed for the regeneration of osteochondral lesions. For this purpose, the ideal scaffold should possess the right properties with respect to degradation, cell binding, cellular uptake, non-immunogenicity, mechanical strength, and flexibility. In addition, it should be easy to handle and serve as a template for chondrocyte and bone cells guiding both cartilage and bone formation. The aim of the present study was to estimate the chondrogenic and osteogenic capability of bone marrow concentrated derived cells seeded onto a novel nano-composite biomimetic material. These properties have been evaluated by means of histological, immunohistochemical and electron microscopy analyses. The data obtained demonstrated that freshly harvested cells obtained from bone marrow were able, once seeded onto the biomaterial, to differentiate either down the chondrogenic and osteogenic pathways as evaluated by the expression and production of specific matrix molecules. These findings support the use, for the repair of osteochondral lesions, of this new nano-composite biomimetic material together with bone marrow derived cells in a "one step" transplantation procedure.

Sodium hydrosulfide inhibits the differentiation of osteoclast progenitor cells via NRF2-dependent mechanism.

Hydrogen sulfide (H2S), which recently emerged as a potent regulator of tissues and organs, is broadly produced in mammalian cells but whether it can regulate bone cell function is still elusive. The main objective of this study was to establish the role of H2S in the regulation of human osteoclast differentiation and function. Sodium hydrosulfide (NaHS), a common H2S-donor, was administered in vitro to CD11b+ human monocytes, the pool of circulating osteoclasts precursors which are critically involved in osteoclast development and function in bone. NaHS dose-dependently decreased human osteoclast differentiation at concentrations which did not induce toxicity. The inhibition of human osteoclast differentiation was associated with a down-regulation in RANKL-dependent intracellular ROS levels in human pre-osteoclasts cells. Furthermore, NaHS up-regulated NRF2 protein expression, its nuclear translocation, and the transcription of the two key downstream antioxidant genes Peroxiredoxin-1 and NAD(P)H dehydrogenase quinone 1, suggesting that NRF2 activation may inhibit human osteoclast differentiation by activating a sustained antioxidant response in osteoclast progenitors; furthermore, NRF2 activators Sulforaphane and Tert-butylhydroquinone inhibited in vitro human osteoclast differentiation. Moreover, silencing NRF2 in human pre-osteoclasts totally abolished NaHS-mediated inhibition of osteoclastogenesis, suggesting that NRF2 is essential to the inhibitory function of NaHS in osteoclast development. Finally, we found that NaHS also downregulated the RANKL/OPG mRNA ratio in human mesenchymal stem cells, the key osteoclast-supporting cells. Our results suggest that NaHS shows a potential therapeutical role in erosive diseases of bone by regulating both direct and indirect mechanisms controlling the differentiation of circulating osteoclasts precursors.

Adipose-derived mesenchymal stem cells exert antiinflammatory effects on chondrocytes and synoviocytes from osteoarthritis patients through prostaglandin E2.

OBJECTIVE: To examine the effect of different sources of good manufacturing practice clinical grade adipose-derived mesenchymal stem cells (AD-MSCs) on inflammatory factors in osteoarthritic (OA) chondrocytes and synoviocytes. METHODS: AD-MSCs from infrapatellar Hoffa fat, subcutaneous (SC) hip fat, and SC abdominal fat were cocultured in Transwells with chondrocytes or synoviocytes. Inflammatory factors (interleukin-1ß [IL-1ß], tumor necrosis factor α, IL-6, CXCL1/growth-related oncogene α, CXCL8/IL-8, CCL2/monocyte chemotactic protein 1, CCL3/macrophage inflammatory protein 1α, and CCL5/RANTES) were evaluated by quantitative reverse transcription-polymerase chain reaction or multiplex bead-based immunoassay. The role of different immunomodulators was analyzed. RESULTS: All the inflammatory factors analyzed were down-modulated at the messenger RNA or protein level independently by all 3 AD-MSC sources or by allogeneic AD-MSCs used in coculture with chondrocytes or synoviocytes. Inflammatory factor down-modulation was observed only when AD-MSCs were cocultured with chondrocytes or synoviocytes that produced high levels of inflammatory factors, but no effect was observed in cells that produced low levels of those factors, thus highlighting a dependence of the AD-MSC effect on existing inflammation. The immunomodulators IL-10, IL-1 receptor antagonist, fibroblast growth factor 2, indoleamine 2,3-dioxygenase 1, and galectin 1 were not involved in AD-MSC effects, whereas the cyclooxygenase 2 (COX-2)/prostaglandin E2 (PGE2 ) pathway exerted a role in the mechanism of antiinflammatory AD-MSC action. CONCLUSION: The antiinflammatory effects of AD-MSCs are probably not dependent on AD-MSC adipose tissue sources and donors but rather on the inflammatory status of OA chondrocytes and synoviocytes. AD-MSCs seem to be able to sense and respond to the local environment. Even though a combination of different molecules may be involved in AD-MSC effects, the COX-2/PGE2 pathway may play a role, suggesting that AD-MSCs may be useful for therapies in osteoarticular diseases.

Engagement of CD99 Activates Distinct Programs in Ewing Sarcoma and Macrophages.

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.

Ultrasound Stimulation of Piezoelectric Nanocomposite Hydrogels Boosts Chondrogenic Differentiation in Vitro, in Both a Normal and Inflammatory Milieu.

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/cm2, 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.

Role of Slug transcription factor in human mesenchymal stem cells.

The pathways that control mesenchymal stem cells (MSCs) differentiation are not well understood, and although some of the involved transcription factors (TFs) have been characterized, the role of others remains unclear. We used human MSCs from tibial plateau (TP) trabecular bone, iliac crest (IC) bone marrow and Wharton's jelly (WJ) umbilical cord demonstrating a variability in their mineral matrix deposition, and in the expression levels of TFs including Runx2, Sox9, Sox5, Sox6, STAT1 and Slug, all involved in the control of osteochondroprogenitors differentiation program. Because we reasoned that the basal expression level of some TFs with crucial role in the control of MSC fate may be correlated with osteogenic potential, we considered the possibility to affect the hMSCs behaviour by using gene silencing approach without exposing cells to induction media. In this study we found that Slug-silenced cells changed in morphology, decreased in their migration ability, increased Sox9 and Sox5 and decreased Sox6 and STAT1 expression. On the contrary, the effect of Slug depletion on Runx2 was influenced by cell type. Interestingly, we demonstrated a direct in vivo regulatory action of Slug by chromatin immunoprecipitation, showing a specific recruitment of this TF in the promoter of Runx2 and Sox9 genes. As a whole, our findings have important potential implication on bone tissue engineering applications, reinforcing the concept that manipulation of specific TF expression levels may elucidate MSC biology and the molecular mechanisms, which promote osteogenic differentiation.

Extracellular calcium chronically induced human osteoblasts effects: specific modulation of osteocalcin and collagen type XV.

Fluctuation in extracellular calcium (Ca(2+)) concentration occurs during bone remodeling. Free ionized Ca(2+) plays a critical role in regulating osteoblast functions. We analyzed the effects of different concentrations of free ionized Ca(2+) (0.5, 1.3, and 2.6 mM) on human osteoblasts and we evaluated osteoblastic phenotype (marker expression and cell morphology) and functions (osteogenic differentiation, cell proliferation, and cell signaling). Our data show human osteoblasts that chronically stimulated with 0.5, 1.3, or 2.6 mM Ca(2+) significantly increase intracellular content of alkaline phosphatase, collagen type I, osteocalcin, and bone sialoprotein, whereas collagen type XV was down-modulated and RUNX2 expression was not affected. We also found a Ca(2+) concentration-dependent increase in osteogenic differentiation and cell proliferation, associated to an increase of signaling protein PLCß1 and p-ERK. Human osteoblast morphology was affected by Ca(2+) as seen by the presence of numerous nucleoli, cells in mitosis, cell junctions, and an increased number of vacuoles. In conclusion, our data show a clear phenotypical and functional effect of extracellular Ca(2+) on human osteoblasts and support the hypothesis of a direct role of this cation in the bone remodeling processes.

Slug contributes to the regulation of CXCL12 expression in human osteoblasts.

CXCL12/CXCR4 chemokine/receptor axis signaling has recently been found to play an important role in the remodeling of bone tissue, but little is known about the molecular mechanisms that are involved. The present study shows that CXCL12 is present at high levels both in human mesenchymal stem cells (hMSCs) and primary osteoblasts (hOBs). When osteogenesis was induced, CXCL12 expression was strictly confined to mineralized nodules. To investigate what mechanisms contribute to the maintenance of a correct expression of CXCL12 in bone cellular context, we analyzed the relationship between CXCL12 and Slug, a transcription factor recently associated with osteoblast maturation. By gene silencing and chromatin immunoprecipitation assay, we showed that both proteins are required for the mineralization process and CXCL12 is transcriptionally and functionally regulated by Slug, which is recruited at specific sites to its gene promoter in vivo. These findings showed for the first time a positive correlation between CXCL12 signaling and Slug activity, thus corroborating the role of these two proteins in bone cellular context and suggesting a new potential target for bone tissue repair and regeneration.

Preliminary study on immune cells in the synovium of end-stage osteoarthritis and rheumatoid arthritis patients: neutrophils and IgG4-secreting plasma cells as differential diagnosis candidates.

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.

Mesenchymal stromal cells from a progressive pseudorheumatoid dysplasia patient show altered osteogenic differentiation.

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.

Primers for the Adhesion of Gellan Gum-Based Hydrogels to the Cartilage: A Comparative Study.

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.

Mesenchymal Stromal Cells Laden in Hydrogels for Osteoarthritis Cartilage Regeneration: A Systematic Review from In Vitro Studies to Clinical Applications.

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 Crucial Path in Chondrogenesis of Adipose-Derived Mesenchymal Stromal Cells Laden in Hydrogel.

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.

RGD-Functionalized Hydrogel Supports the Chondrogenic Commitment of Adipose Mesenchymal Stromal Cells.

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.