Expression Of Chondrogenic Markers Research Articles

Application of human platelet lysate in chondrocyte expansion promotes chondrogenic phenotype and slows senescence progression via BMP–TAK1–p38 pathway

Osteoarthritis (OA) is one of the most common musculoskeletal degenerative. OA treatments are aiming to slow down disease progression; however, lack of cartilage regeneration efficacy. Autologous chondrocyte implantation (ACI) is a promising cartilage-regeneration strategy that uses human articular chondrocytes (HACs) as cellular materials. However, the unreadiness of HACs from prolonged expansion, cellular senescence, and chondrogenic dedifferentiation occurred during conventional expansion, thus, minimizing the clinical efficacy of ACI. We aimed to examine the effects of a human platelet lysate (HPL) as an alternative human-derived HAC medium supplement to overcome the limitations of conventional expansion, and to explain the mechanism underlying the effects of HPL. During passages 2–4 (P2-P4), HPL significantly increased HAC proliferation capacities and upregulated chondrogenic markers. Simultaneously, HPL significantly reduced HAC senescence compared with conventional condition. HACs treated with LDN193189 exhibited a reduction in proliferation capacity and chondrogenic marker expression, whereas the HAC senescence increased slightly. These findings indicated involvement of BMP-2 signaling transduction in the growth-assistive, anti-senescent, and chondrogenic-inductive properties of HPL, which demonstrated its beneficial effects for application as HAC medium supplement to overcome current expansion limitations. Finally, our findings support the roles of platelets in platelet-rich plasma as a promising treatment for patients with OA.

Identifying Fibroblast Growth Factor Receptor 3 as a Mediator of Periosteal Osteochondral Differentiation through the Construction of microRNA-Based Interaction Networks.

Human periosteum-derived progenitor cells (hPDCs) have the ability to differentiate towards both the chondrogenic and osteogenic lineages. This coordinated and complex osteochondrogenic differentiation process permits endochondral ossification and is essential in bone development and repair. We have previously shown that humanised cultures of hPDCs enhance their osteochondrogenic potentials in vitro and in vivo; however, the underlying mechanisms are largely unknown. This study aimed to identify novel regulators of hPDC osteochondrogenic differentiation through the construction of miRNA-mRNA regulatory networks derived from hPDCs cultured in human serum or foetal bovine serum as an alternative in silico strategy to serum characterisation. Sixteen differentially expressed miRNAs (DEMis) were identified in the humanised culture. In silico analysis of the DEMis with TargetScan allowed for the identification of 1503 potential miRNA target genes. Upon comparison with a paired RNAseq dataset, a 4.5% overlap was observed (122 genes). A protein-protein interaction network created with STRING interestingly identified FGFR3 as a key network node, which was further predicted using multiple pathway analyses. Functional analysis revealed that hPDCs with the activating mutation FGFR3N540K displayed increased expressions of chondrogenic gene markers when cultured under chondrogenic conditions in vitro and displayed enhanced endochondral bone formation in vivo. A further histological analysis uncovered known downstream mediators involved in FGFR3 signalling and endochondral ossification to be upregulated in hPDC FGFR3N540K-seeded implants. This combinational approach of miRNA-mRNA-protein network analysis with in vitro and in vivo characterisation has permitted the identification of FGFR3 as a novel mediator of hPDC biology. Furthermore, this miRNA-based workflow may also allow for the identification of drug targets, which may be of relevance in instances of delayed fracture repair.

Silica Replicas Derived from Mammalian Cells as an Innovative Approach to Physically Direct Cell Lineage Decisions of Mesenchymal Stem Cells.

By means of a "live-cell" template strategy, silica replicas displaying the same morphology and topography of the mammalian cells used as templates are fabricated. The replicas are used as substrates to direct the differentiation of mesenchymal stem cells (MSCs) to predefined cell lineages. Upregulation of specific genes shows how the silica replica-based substrates have the ability to induce the molecular characteristics of the mature cell types from which they have been derived from. Thus, MSCs cultured in the presence of silica replicas of human osteoblasts (HObs) differentiate into HObs-like cells, as shown by the upregulation of specific osteogenic genes. Likewise, when MSCs are incubated with silica replicas derived from human chondrocytes, an enhanced expression of chondrogenic markers is observed. Importantly, the effects of the silica replicas are cell type-specific since the incubation of MSCs with HObs silica replicas does not result in upregulation of chondrogenic markers and vice versa. What is more, for both cases, the differentiation rate is enhanced when the silica replicas are used in combination with growth factors, suggesting a potential synergistic effect. These results demonstrate the potential of this innovative method as an efficient and cheap approach with the potential to eliminate, or at least reduce, the use of biochemically soluble compounds in stem cells research.

Evaluation of a synthetic peptide-based bioink (PeptiInk Alpha 1) for in vitro 3D bioprinting of cartilage tissue models

Cartilage pathology in human disease is poorly understood and requires further research. Various attempts have been made to study cartilage pathologies using in vitro human cartilage models as an alternative for preclinical research. Three-dimensional (3D) bioprinting is a technique that has been used to 3D-bioprint cartilage tissue models in vitro using animal-derived materials such as gelatine or hyaluronan, which present challenges in terms of scalability, reproducibility, and ethical concerns. We present an assessment of synthetic self-assembling peptides as bioinks for bioprinted human in vitro cartilage models. Primary human chondrocytes were mixed with PeptiInk Alpha 1, 3D-bioprinted and cultured for 14 days, and compared with 3D chondrocyte pellet controls. Cell viability was assessed through LIVE/DEAD assays and DNA quantification. High cell viability was observed in the PeptiInk culture, while a fast decrease in DNA levels was observed in the 3D pellet control. Histological evaluation using hematoxylin and eosin staining and immunofluorescence labeling for SOX-9, collagen type II, and aggrecan showed a homogeneous cell distribution in the 3D-bioprinted PeptiInks as well as high expression of chondrogenic markers in both control and PeptiInk cultures. mRNA expression levels assessed by - qRT-PCR (quantitative real time-polymerase chain reaction) confirmed chondrogenic cell behavior. These data showed promise in the potential use of PeptiInk Alpha 1 as a bioprintable manufacturing material for human cartilage in vitro models.

Loss of dlx5a/dlx6a Locus Alters Non-Canonical Wnt Signaling and Meckel's Cartilage Morphology.

The dlx genes encode transcription factors that establish a proximal-distal polarity within neural crest cells to bestow a regional identity during craniofacial development. The expression regions of dlx paralogs are overlapping yet distinct within the zebrafish pharyngeal arches and may also be involved in progressive morphologic changes and organization of chondrocytes of the face. However, how each dlx paralog of dlx1a, dlx2a, dlx5a and dlx6a affects craniofacial development is still largely unknown. We report here that the average lengths of the Meckel's, palatoquadrate and ceratohyal cartilages in different dlx mutants were altered. Mutants for dlx5a-/- and dlx5i6-/-, where the entire dlx5a/dlx6a locus was deleted, have the shortest lengths for all three structures at 5 days post fertilization (dpf). This phenotype was also observed in 14 dpf larvae. Loss of dlx5i6 also resulted in increased proliferation of neural crest cells and expression of chondrogenic markers. Additionally, altered expression and function of non-canonical Wnt signaling were observed in these mutants suggesting a novel interaction between dlx5i6 locus and non-canonical Wnt pathway regulating ventral cartilage morphogenesis.

Time-Dependent Anabolic Response of hMSC-Derived Cartilage Grafts to Hydrostatic Pressure

It is generally accepted that the application of hydrostatic pressure (HP) is beneficial for MSC chondrogenesis. There is, however, evidence to suggest that the timing of application might determine its impact on cell fate and tissue development. Furthermore, understanding how the maturity of engineered cartilage affects its response to the application of HP can provide critical insight into determining when such a graft is ready for in vivo implantation into a mechanically loaded environment. In this study, we systematically examined chondrogenic maturation of hMSCs over 35 days in the presence of TGF-β3 in vitro. At specific timepoints, the response of hMSCs to the application of HP following the removal of TGF-β3 was assessed; this partially models conditions such grafts will experience in vivo upon implantation. In free swelling culture, the expression of chondrogenic (COL2A1 and ACAN) and hypertrophic (COL10A1) markers increased with time. At early timepoints, the expression of such markers continued to increase following TGF-β3 withdrawal; however, this was not observed after prolonged periods of chondrogenic priming (35 days). Interestingly, the application of HP was only beneficial after 35 days of chondrogenic priming, where it enhanced sGAG synthesis and improved key chondrogenic gene ratios. It was also found that HP can facilitate a metabolic shift towards oxidative phosphorylation, which can be viewed as a hallmark of successfully differentiating MSCs. These results point to the importance of mechanical loading as a key stimulus for maintaining a chondrogenic phenotype once MSCs are removed from chemically defined culture conditions.

Comparison of multiple synthetic chondroinductive factors in pellet culture against a TGF-β positive control

Despite the advances in surgical and cell therapy regenerative techniques for cartilage repair, the challenge is to overcome an inferior fibrocartilage repair tissue. In vitro, TGF-β1 and TGF-β3 are the primary growth factors employed to induce chondrogenic differentiation. However, the clinical application of native proteins may present challenges regarding stability, cost, or reproducibility. Therefore, there remains an unmet clinical need for the identification of small chondroinductive synthetic molecules. From the literature, two peptides—CM10 and CK2.1—appear to be promising candidates; however, they have not been directly compared to TGF-β with human bone marrow-derived stem cells (hBMSCs). Similarly, two promising compounds—kartogenin and SM04690—have been reported in the literature to exhibit chondroinductive potential in vivo and in vitro; however, kartogenin was not directly compared against TGF-β. In the current study, we evaluated the chondroinductive potential of CM10, CK2.1, kartogenin, and SM04690, and directly compared them to each other and to a TGF-β3 positive control. Following 21 days of culture, none of the evaluated chondrogenic factors, either individually or even in combinations of two, resulted in a higher gene expression of chondrogenic markers as compared to TGF-β3. Additionally, no collagen II gene expression was detected except in the TGF-β3 positive control group. Given that the evaluated factors have confirmed efficacy in the literature, but not in the current study with a positive control, there may be value in the future identification of new chondroinductive factors that are less situation-dependent, with rigorous evaluations of their effect on chondrogenesis using positive controls.

3D Bioprintable Hypoxia-Mimicking PEG-Based Nano Bioink for Cartilage Tissue Engineering

As hypoxia plays a significant role in the formation and maintenance of cartilage tissue, aiming to develop native hypoxia-mimicking tissue engineering scaffolds is an efficient method to treat articular cartilage (AC) defects. Cobalt (Co) is documented for its hypoxic-inducing effects in vitro by stabilizing the hypoxia-inducible factor-1α (HIF-1α), a chief regulator of stem cell fate. Considering this, we developed a novel three-dimensional (3D) bioprintable hypoxia-mimicking nano bioink wherein cobalt nanowires (Co NWs) were incorporated into the poly(ethylene glycol) diacrylate (PEGDA) hydrogel system as a hypoxia-inducing agent and encapsulated with umbilical cord-derived mesenchymal stem cells (UMSCs). In the current study, we investigated the impact of Co NWs on the chondrogenic differentiation of UMSCs in the PEGDA hydrogel system. Herein, the hypoxia-mimicking nano bioink (PEGDA+Co NW) was rheologically optimized to bioprint geometrically stable cartilaginous constructs. The bioprinted 3D constructs were evaluated for their physicochemical characterization, swelling-degradation behavior, mechanical properties, cell proliferation, and the expression of chondrogenic markers by histological, immunofluorescence, and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) methods. The results disclosed that, compared to the control (PEGDA) group, the hypoxia-mimicking nano bioink (PEGDA+Co NW) group outperformed in print fidelity and mechanical properties. Furthermore, live/dead staining, double-stranded DNA (dsDNA) content, and glycosaminoglycans (GAGs) content demonstrated that adding low amounts of Co NWs (<20 ppm) into PEGDA hydrogel system supported UMSC adhesion, proliferation, and differentiation. Histological and immunofluorescence staining of the PEGDA+Co NW bioprinted structures revealed the production of type 2 collagen (COL2) and sulfated GAGs, rendering it a feasible option for cartilage repair. It was further corroborated by a significant upregulation of the hypoxia-mediated chondrogenic and downregulation of the hypertrophic/osteogenic marker expression. In conclusion, the hypoxia-mimicking hydrogel system, including PEGDA and Co2+ ions, synergistically directs the UMSCs toward the chondrocyte lineage without using expensive growth factors and provides an alternative strategy for translational applications in the cartilage tissue engineering field.

Biogenic Carbon Quantum Dots as a Neoteric Inducer in the Game of Directing Chondrogenesis

The journey into the field of stem cell biology has been an endeavor of paramount advancement in biomedicine, establishing new horizons in the avenue of materiobiology. The creative drive of the scientific community focuses on ameliorating the utilization of stem cells, which is currently untapped on a large scale. With similar motivation, we present a nascent strategy of maneuvering biogenic carbon quantum dots (CQDs) to eclipse the toxic hurdles of chemical synthesis of carbon allotropes to serve as a biocompatible trident in stem cell biology employing a three-prong action of stem cell differentiation, imaging, and migration. The derivation of CQDs from garlic peels as a biogenic precursor abets in realizing the optophysical features of CQDs to image mesenchymal stem cells without hampering the biological systems with cytotoxicity. We report the versatility of biogenic CQDs to generate reactive oxygen species (ROS) to robustly influence stem cell migration and concomitantly chondrocyte differentiation from human Wharton's jelly mesenchymal stem cells (hWJ-MSCs). This was orchestrated without the use of chondrogenic induction factors, which was confirmed from the expression of chondrogenic markers (Col II, Col X, ACAN). Even the collagen content of cells incubated with CQDs was quite comparable with that of chondrocyte-induced cells. Thus, we empirically propose garlic peel-derived CQDs as a tangible advancement in stem cell biology from a materiobiological frame of reference to hone significant development in this arena.

TRI-LINEAGE DIFFERENTIATION POTENTIAL OF BMSCS GROWN ON HIPSC-ENGINEERED EXTRACELLULAR MATRIX

Mesenchymal stem cells (MSCs) have the potential to repair and regenerate damaged tissues in response to injury, such as fracture or other tissue injury. Bone marrow and adipose tissue are the major sources of MSCs. Previous studies suggested that the regenerative activity of stem cells can be enhanced by exposure to tissue microenvironments. The aim of our project was to investigate whether extracellular matrix (ECM) engineered from human induced pluripotent stem cells-derived mesenchymal-like progenitors (hiPSCs-MPs) can enhance the regenerative potential of human bone marrow mesenchymal stromal cells (hBMSCs).ECM was engineered from hiPSC-MPs. ECM structure and composition were characterized before and after decellularization using immunofluorescence and biochemical assays. hBMSCs were cultured on the engineered ECM, and differentiated into osteogenic, chondrogenic and adipogenic lineages. Growth and differentiation responses were compared to tissue culture plastic controls.Decellularization of ECM resulted in efficient cell elimination, as observed in our previous studies. Cultivation hBMSCs on the ECM in osteogenic medium significantly increased hBMSC growth, collagen deposition and alkaline phosphatase activity. Furthermore, expression of osteogenic genes and matrix mineralization were significantly higher compared to plastic controls. Chondrogenic micromass culture on the ECM significantly increased cell growth and expression of chondrogenic markers, including glycosaminoglycans and collagen type II. Adipogenic differentiation of hBMSCs on the ECM resulted in significantly increased hBMSC growth, but significantly reduced lipid vacuole deposition compared to plastic controls. Together, our studies suggest that BMSCs differentiation into osteogenic and chondrogenic lineages can be enhanced, whereas adipogenic activity is decreased by the culture on engineered ECM. Contribution of specific matrix components and underlying mechanisms need to be further elucidated.Our studies suggest that the three-lineage differentiation of aged BMSCs can be modulated by culture on hiPSC-engineered ECM. Further studies are aimed at scaling-up to three-dimensional ECM constructs for osteochondral tissue regeneration.

In Vitro Comparison of 2 Clinically Applied Biomaterials for Autologous Chondrocyte Implantation: Injectable Hydrogel Versus Collagen Scaffold.

In autologous chondrocyte implantation (ACI), there is no consensus about used bioscaffolds. The aim of this study was to perform an in vitro comparative analysis of 2 clinically applied biomaterials for cartilage lesion treatment. Monolayer expanded human chondrocytes (n = 6) were embedded in a collagen scaffold (CS) and a hyaluronic acid-based hydrogel (HA). Cells were cultured in chondropermissive medium supplemented with and without interleukin-10 (IL-10) and bone morphogenetic protein-2 (BMP-2). Gene expression of chondrogenic markers (COL1A1, COL2A1, COL10A1, ACAN, SOX9) was detected via quantitative real-time-polymerase chain reaction (RT-qPCR). Biosynthesis of matrix compounds, cell viability, morphology as well as migration from surrounding native bovine cartilage into cell-free scaffolds were analyzed histologically. Adhesion of the material to adjacent cartilage was investigated by a custom-made push-out test. The shift of COL1/2 ratio toward COL2A1 was more pronounced in HA, and cells displayed a more spherical morphology compared with CS. BMP-2 and IL-10 significantly increased COL2A1, SOX9, and ACAN expression, which was paralleled by enhanced staining of glycosaminoglycans (GAGs) and type 2 collagen in histological sections of CS and HA. COL10A1 was not significantly expressed in HA and CS. Better interfacial integration and enhanced cell invasion was observed in CS. Push-out tests using CS showed higher bonding strength to native cartilage. HA-based hydrogel revealed a more chondrocyte-like phenotype but only allowed limited cell invasion, whereas CS were advantageous in terms of cellular invasion and interfacial adhesion. These differences may be clinically relevant when treating cartilaginous or osteochondral defects.

Multiparity and Aging Impact Chondrogenic and Osteogenic Potential at Symphyseal Enthesis: New Insights into Interpubic Joint Remodeling

Pregnancy and childbirth cause adaptations to the birth canal to allow for delivery and fast recovery. To accommodate delivery through the birth canal, the pubic symphysis undergoes changes that lead to the interpubic ligament (IpL) and enthesis formation in primiparous mice. However, successive deliveries influence joint recovery. We aimed to understand tissue morphology and chondrogenic and osteogenic potential at symphyseal enthesis during pregnancy and postpartum in primiparous and multiparous senescent female mice. Morphological and molecular differences were found at the symphyseal enthesis among the study groups. Despite the apparent incapacity to restore cartilage in multiparous senescent animals, the symphyseal enthesis cells are active. However, these cells have reduced expression of chondrogenic and osteogenic markers and are immersed in densely packed collagen fibers contiguous to the persistent IpL. These findings may indicate alterations of key molecules in the progenitor cell population maintenance of the chondrocytic and osteogenic lineages at the symphyseal enthesis in multiparous senescent animals, possibly compromising the mouse joint histoarchitecture recovery. This sheds light on the distention of the birth canal and the pelvic floor that may play a role in pubic symphysis diastasis (PSD) and pelvic organ prolapse (POP), both in orthopedic and urogynecological practice in women.

YTHDF1 Enhances Chondrogenic Differentiation by Activating the Wnt/β-Catenin Signaling Pathway.

Cartilage is derived from the chondrogenic differentiation of stem cells, for which the regulatory mechanism has not been fully elucidated. N6-methyladenosine (m6A) messenger RNA (mRNA) methylation is the most common posttranscriptional modification in eukaryotic mRNAs and is mediated by m6A regulators. However, whether m6A regulators play roles in chondrogenic differentiation is unknown. Herein, we aim to determine the role of a main m6A reader protein, YTH N6-methyladenosine RNA binding protein 1 (YTHDF1), in chondrogenic differentiation regulation. Western blotting (WB) assays found that the expression of YTHDF1 increased during chondrogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs). The results of quantitative polymerase chain reaction, WB, immunohistochemistry, and Alcian blue staining revealed that overexpression of YTHDF1 increased cartilage matrix synthesis and the expression of chondrogenic markers when hBMSCs, ATDC5 cells, or C3H10T1/2 cells were induced to undergo chondrogenesis. Conversely, chondrogenesis was clearly inhibited when YTHDF1 was knocked down in hBMSCs, ATDC5 cells, or C3H10T1/2 cells. Further RNA sequencing and molecular biology experiments found that YTHDF1 activated the Wnt/β-catenin signaling pathway during chondrogenic differentiation. Finally, the effects of overexpression and knockdown of YTHDF1 on chondrogenic differentiation were reversed by inhibiting or activating β-catenin activity. Therefore, we demonstrated that YTDHF1 promoted chondrogenic differentiation through activation of the Wnt/β-catenin signaling pathway.

High-glutathione mesenchymal stem cells isolated using the FreSHtracer probe enhance cartilage regeneration in a rabbit chondral defect model

BackgroundMesenchymal stem cells (MSCs) are a promising cell source for cartilage regeneration. However, the function of MSC can vary according to cell culture conditions, donor age, and heterogeneity of the MSC population, resulting in unregulated MSC quality control. To overcome these limitations, we previously developed a fluorescent real-time thiol tracer (FreSHtracer) that monitors cellular levels of glutathione (GSH), which are known to be closely associated with stem cell function. In this study, we investigated whether using FreSHtracer could selectively separate high-functioning MSCs based on GSH levels and evaluated the chondrogenic potential of MSCs with high GSH levels to repair cartilage defects in vivo.MethodsFlow cytometry was conducted on FreSHtracer-loaded MSCs to select cells according to their GSH levels. To determine the function of FreSHtracer-isolated MSCs, mRNA expression, migration, and CFU assays were conducted. The MSCs underwent chondrogenic differentiation, followed by analysis of chondrogenic-related gene expression. For in vivo assessment, MSCs with different cellular GSH levels or cell culture densities were injected in a rabbit chondral defect model, followed by histological analysis of cartilage-regenerated defect sites.ResultsFreSHtracer successfully isolated MSCs according to GSH levels. MSCs with high cellular GSH levels showed enhanced MSC function, including stem cell marker mRNA expression, migration, CFU, and oxidant resistance. Regardless of the stem cell tissue source, FreSHtracer selectively isolated MSCs with high GSH levels and high functionality. The in vitro chondrogenic potential was the highest in pellets generated by MSCs with high GSH levels, with increased ECM formation and chondrogenic marker expression. Furthermore, the MSCs’ function was dependent on cell culture conditions, with relatively higher cell culture densities resulting in higher GSH levels. In vivo, improved cartilage repair was achieved by articular injection of MSCs with high levels of cellular GSH and MSCs cultured under high-density conditions, as confirmed by Collagen type 2 IHC, Safranin-O staining and O’Driscoll scores showing that more hyaline cartilage was formed on the defects.ConclusionFreSHtracer selectively isolates highly functional MSCs that have enhanced in vitro chondrogenesis and in vivo hyaline cartilage regeneration, which can ultimately overcome the current limitations of MSC therapy.Graphical

Implication of Cellular Senescence in Osteoarthritis: A Study on Equine Synovial Fluid Mesenchymal Stromal Cells.

Osteoarthritis (OA) is described as a chronic degenerative disease characterized by the loss of articular cartilage. Senescence is a natural cellular response to stressors. Beneficial in certain conditions, the accumulation of senescent cells has been implicated in the pathophysiology of many diseases associated with aging. Recently, it has been demonstrated that mesenchymal stem/stromal cells isolated from OA patients contain many senescent cells that inhibit cartilage regeneration. However, the link between cellular senescence in MSCs and OA progression is still debated. In this study, we aim to characterize and compare synovial fluid MSCs (sf-MSCs), isolated from OA joints, with healthy sf-MSCs, investigating the senescence hallmarks and how this state could affect cartilage repair. Sf-MSCs were isolated from tibiotarsal joints of healthy and diseased horses with an established diagnosis of OA with an age ranging from 8 to 14 years. Cells were cultured in vitro and characterized for cell proliferation assay, cell cycle analysis, ROS detection assay, ultrastructure analysis, and the expression of senescent markers. To evaluate the influence of senescence on chondrogenic differentiation, OA sf-MSCs were stimulated in vitro for up to 21 days with chondrogenic factors, and the expression of chondrogenic markers was compared with healthy sf-MSCs. Our findings demonstrated the presence of senescent sf-MSCs in OA joints with impaired chondrogenic differentiation abilities, which could have a potential influence on OA progression.

Scaffolds containing GAG-mimetic cellulose sulfate promote TGF-β interaction and MSC Chondrogenesis over native GAGs.

Cartilage tissue engineering strategies seek to repair damaged tissue using approaches that include scaffolds containing components of the native extracellular matrix (ECM). Articular cartilage consists of glycosaminoglycans (GAGs) which are known to sequester growth factors. In order to more closely mimic the native ECM, this study evaluated the chondrogenic differentiation of mesenchymal stem cells (MSCs), a promising cell source for cartilage regeneration, on fibrous scaffolds that contained the GAG-mimetic cellulose sulfate. The degree of sulfation was evaluated, examining partially sulfated cellulose (pSC) and fully sulfated cellulose (NaCS). Comparisons were made with scaffolds containing native GAGs (chondroitin sulfate A, chondroitin sulfate C and heparin). Transforming growth factor-beta3 (TGF-β3) sequestration, as measured by rate of association, was higher for sulfated cellulose-containing scaffolds as compared to native GAGs. In addition, TGF-β3 sequestration and retention over time was highest for NaCS-containing scaffolds. Sulfated cellulose-containing scaffolds loaded with TGF-β3 showed enhanced chondrogenesis as indicated by a higher Collagen Type II:I ratio over native GAGs. NaCS-containing scaffolds loaded with TGF-β3 had the highest expression of chondrogenic markers and a reduction of hypertrophic markers in dynamic loading conditions, which more closely mimic in vivo conditions. Studies also demonstrated that TGF-β3 mediated its effect through the Smad2/3 signaling pathway where the specificity of TGF-β receptor (TGF- βRI)-phosphorylated SMAD2/3 was verified with a receptor inhibitor. Therefore, studies demonstrate that scaffolds containing cellulose sulfate enhance TGF-β3-induced MSC chondrogenic differentiation and show promise for promoting cartilage tissue regeneration.

Transcriptomic response of bioengineered human cartilage to parabolic flight microgravity is sex-dependent

Spaceflight and simulated spaceflight microgravity induced osteoarthritic-like alterations at the transcriptomic and proteomic levels in the articular and meniscal cartilages of rodents. But little is known about the effect of spaceflight or simulated spaceflight microgravity on the transcriptome of tissue-engineered cartilage developed from human cells. In this study, we investigate the effect of simulated spaceflight microgravity facilitated by parabolic flights on tissue-engineered cartilage developed from in vitro chondrogenesis of human bone marrow mesenchymal stem cells obtained from age-matched female and male donors. The successful induction of cartilage-like tissue was confirmed by the expression of well-demonstrated chondrogenic markers. Our bulk transcriptome data via RNA sequencing demonstrated that parabolic flight altered mostly fundamental biological processes, and the modulation of the transcriptome profile showed sex-dependent differences. The secretome profile analysis revealed that two genes (WNT7B and WNT9A) from the Wnt-signaling pathway, which is implicated in osteoarthritis development, were only up-regulated for female donors. The results of this study showed that the engineered cartilage tissues responded to microgravity in a sex-dependent manner, and the reported data offers a strong foundation to further explore the underlying mechanisms.

Expression of SATB2, RUNX2, and SOX9 and possible osteoblastic and chondroblastic differentiation in chondroblastoma.

Chondroblastoma (CB) is histologically characterized by oval to polygonal-shaped mononuclear neoplastic cells, multinucleated osteoclastic giant cells, and eosinophilic matrix with occasional calcification. Genetically, the majority of CBs harbor H3F3B p.K36M mutation. Despite the historical nomenclature, it has been reported that the matrix of CB is similar to osteoid rather than true cartilage; however, it remains unclear whether neoplastic cells in CB have the potential for osteoblastic differentiation. To clarify this issue, we immunohistochemically examined the expression of osteogenic and chondrogenic markers (SATB2, RUNX2, p63, and SOX9) as well as H3K36M mutant protein in 33 cases of CB. All 33 cases of CB were positive for H3K36M, while SATB2, RUNX2, p63, and SOX9 were expressed in 30/33 (91%), 33/33 (100%), 29/33 (88%), and 31/32 (97%) CB cases, respectively. Our immunohistochemical results suggest that neoplastic cells in CB frequently express both osteogenic and chondrogenic markers and may have an intermediate feature of osteoblastic and chondroblastic nature.

The effect of chondroitin sulfate concentration and matrix stiffness on chondrogenic differentiation of mesenchymal stem cells.

Chondroitin sulfate (CS), a glycosaminoglycan of native cartilage, has shown its potential in promoting chondrogenesis of mesenchymal stem cells (MSCs), whereas the effect of matrix stiffness in a CS-containing 3D environment on chondrogenesis is still poorly understood. Herein, this study aimed at assessing the effect of CS concentration and stiffness of CS-containing hydrogels on the chondrogenesis of MSCs. Hydrogels composed of 6% (w/v) gelatin methacryloyl (GelMA) and three concentrations, i.e., 4%, 6%, or 10% (w/v), of methacrylated chondroitin sulfate (CSMA) were prepared. The hydrogels of each composition were prepared with two stiffness values (33.36 ± 8.25 kPa vs. 8.42 ± 2.83 kPa). Physical characterization showed similar microporous structures among the six groups, higher swelling ratios and faster degradation in the soft hydrogel groups. MSCs were encapsulated in the six groups of hydrogels and they underwent 28-day chondrogenic differentiation. The cell viability in each group on day 1 was similar and most cells exhibited a round shape without spreading. Afterwards, cellular protrusions in soft hydrogels remained filopodium-like from day 14 to day 28, while most protrusions were lamellipodium-like in stiff hydrogels on day 14 and then transformed into a spherical shape on day 28. The expression of chondrogenic markers analysed by real-time qPCR and immunohistochemical staining demonstrated that the optimal CS concentration for chondrogenesis was 6% (w/v) regardless of the stiffness of hydrogels. In addition, with the same CSMA concentration, the trend was observed that the stiff hydrogels supported superior chondrogenesis of MSCs compared to the soft hydrogel. To summarize, this study presents an advancement in the optimization of CSMA concentration and stiffness of hydrogels for chondrogenesis. In the CSMA/GelMA hydrogel, 6% (w/v) CSMA with an initial Young's modulus around 33 kPa was recommended for cartilage tissue engineering.

COG4 mutation in Saul-Wilson syndrome selectively affects secretion of proteins involved in chondrogenesis in chondrocyte-like cells.

Saul-Wilson syndrome is a rare skeletal dysplasia caused by a heterozygous mutation in COG4 (p.G516R). Our previous study showed that this mutation affected glycosylation of proteoglycans and disturbed chondrocyte elongation and intercalation in zebrafish embryos expressing the COG4p.G516R variant. How this mutation causes chondrocyte deficiencies remain unsolved. To analyze a disease-relevant cell type, COG4p.G516R variant was generated by CRISPR knock-in technique in the chondrosarcoma cell line SW1353 to study chondrocyte differentiation and protein secretion. COG4p.G516Rcells display impaired protein trafficking and altered COG complex size, similar to SWS-derived fibroblasts. Both SW1353 and HEK293T cells carrying COG4p.G516R showed very modest, cell-type dependent changes in N-glycans. Using 3D culture methods, we found that cells carrying the COG4p.G516R variant made smaller spheroids and had increased apoptosis, indicating impaired in vitro chondrogenesis. Adding WT cells or their conditioned medium reduced cell death and increased spheroid sizes of COG4p.G516R mutant cells, suggesting a deficiency in secreted matrix components. Mass spectrometry-based secretome analysis showed selectively impaired protein secretion, including MMP13 and IGFBP7 which are involved in chondrogenesis and osteogenesis. We verified reduced expression of chondrogenic differentiation markers, MMP13 and COL10A1 and delayed response to BMP2 in COG4p.G516R mutant cells. Collectively, our results show that the Saul-Wilson syndrome COG4p.G516R variant selectively affects the secretion of multiple proteins, especially in chondrocyte-like cells which could further cause pleiotropic defects including hampering long bone growth in SWS individuals.