Expression Of Chondrogenic Markers Research Articles

AdipoRon promotes diabetic fracture repair through endochondral ossification-based bone repair by enhancing survival and differentiation of chondrocytes

Diabetic bone defects may exhibit impaired endochondral ossification (ECO) leading to delayed bone repair. AdipoRon, a receptor agonist of adiponectin polymers, can ameliorate diabetes and related complications, as well as overcome the disadvantages of the unstable structure of artificial adiponectin polymers. Here, the effects of AdipoRon on the survival and differentiation of chondrocytes in a diabetic environment were explored focusing on related mechanisms in gene and protein levels. In vivo, AdipoRon was applied to diet-induced-obesity (DIO) mice, a model of obesity and type 2 diabetes, with femoral fracture. Sequential histological evaluations and micro-CT were examined for further verification. We found that AdipoRon could ameliorate cell viability, apoptosis, and reactive oxygen species (ROS) production and promote mRNA expression of chondrogenic markers and cartilaginous matrix production of ATDC5 cells in high glucose medium via activating ERK1/2 pathway. Additionally, DIO mice with intragastric AdipoRon administration had more neocartilage and accelerated new bone formation. These data suggest that AdipoRon could stimulate bone regeneration via ECO in diabetes.

Additive manufacturing of an elastic poly(ester)urethane for cartilage tissue engineering

Although a growing knowledge on the field of tissue engineering of articular cartilage exists, reconstruction or in-vitro growth of functional hyaline tissue still represents an unmet challenge. Despite the simplicity of the tissue in terms of cell population and absence of innervation and vascularization, the outstanding mechanical properties of articular cartilage, which are the result of the specificity of its extra cellular matrix (ECM), are difficult to mimic. Most importantly, controlling the differentiation state or phenotype of chondrocytes, which are responsible of the deposition of this specialized ECM, represents a milestone in the regeneration of native articular cartilage. In this study, we fabricated fused deposition modelled (FDM) scaffolds with different pore sizes and architectures from an elastic and biodegradable poly(ester)urethane (PEU) with mechanical properties that can be modulated by design, and that ranged the elasticity of articular cartilage. Cell culture in additive manufactured 3D scaffolds exceeded the chondrogenic potential of the gold-standard pellet culture. In-vitro cell culture studies demonstrated the intrinsic potential of elastic (PEU) to drive the re-differentiation of de-differentiated chondrocytes when cultured in-vitro, in differentiation or basal media, better than pellet cultures. The formation of neo-tissue was assessed as a high deposition of GAGs and fibrillar collagen II, and a high expression of typical chondrogenic markers. Moreover, the collagen II / collagen I ratio commonly used to evaluate the differentiation state of chondrocytes (ratio > 1 being chondrocytes and, ratio < 0 being de-differentiated chondrocytes) was higher than 5. Statement of significanceTissue engineering of articular cartilage requires material scaffolds capable of driving the deposition of a coherent and specific ECM representative of articular cartilage. Materials explored so far account for low mechanical properties (hydrogels), or are too stiff to mimic the elasticity of the native tissue (traditional polyesters). Here, we fabricated 3D fibrous scaffolds via FDM with a biodegradable poly(ester)urethane. The compressive Young`s modulus and elastic limit of the scaffolds can be tuned by designed, mimicking those of the native tissue. The designed scaffolds showed an intrinsic potential to drive the formation of a GAG and collagen II rich ECM, and to drive a stable chondrogenic cell phenotype.

Comparison of the Chondrogenic Potential of Mesenchymal Stem Cells Derived from Bone Marrow and Umbilical Cord Blood Intended for Cartilage Tissue Engineering.

Osteoarthritis (OA) remains incurable in humans or horses and mesenchymal stromal/stem cells (MSCs) represent an attractive solution for producing a neocartilage substitute. However, the best MSC source still needs to be identified. This study compared the chondrogenic potential of equine MSCs derived from bone marrow (BM) and umbilical cord blood (UCB), at their undifferentiated status to check if one cell source is better proned, and after chondrogenic-induced differentiation. Chondrogenesis was induced by culture in collagen scaffold with BMP-2 + TGF-ß1 in hypoxia or normoxia. MSCs chondrogenic potential was evaluated using the mRNA and corresponding protein levels for osteogenic, hypertrophic and chondrogenic markers. MSCs characterization demonstrated that BM- and UCB-MSCs differ in proliferation and tripotencies. At undifferentiated status, they also showed differences in their expression of osteogenic, chondrogenic and hypertrophic markers. Upon chondrogenesis induction, both MSCs sources exhibited increased chondrogenic expression and produce an extracellular matrix (ECM) of better quality in hypoxia, although collagen I remained expressed. UCB-MSCs produced higher amounts of collagen II, particularly its IIB isoform, than BM-MSCs, but also collagen I and Htra1, regardless of the oxygen condition. Finally, immunohistochemistry revealed that the BM-MSCs synthesized an ECM of higher quality, regarding the more homogenous distribution of type IIB collagen, compared to UCB-MSCs. Considering collagen I as the major undesirable component in the neo-synthesis of in vitro cartilage, we recommend using BM-MSCs for horse cartilage engineering.

Spatially Regulated Multiphenotypic Differentiation of Stem Cells in 3D via Engineered Mechanical Gradient.

Within the osteochondral interface, cellular and extracellular matrix gradients provide a biomechanical and biochemical niche for homeostatic tissue functions. Postnatal joint loading is critical for the development of such tissue gradients, leading to the formation of functional osteochondral tissues composed of superficial, middle, and deep zones of cartilage, and underlying subchondral bone, in a depth-dependent manner. In this regard, a novel, variable core-shell electrospinning strategy was employed to generate spatially controlled strain gradients within three-dimensional scaffolds under dynamic compressive loading, enabling the local strain-magnitude dependent, multiphenotypic stem cell differentiation. Human mesenchymal stem cells (hMSCs) were cultured in electrospun scaffolds with a linear or biphasic mechanical gradient, which was computationally engineered and experimentally validated. The cell/scaffold constructs were subjected to various magnitudes of dynamic compressive strains in a scaffold depth-dependent manner at a frequency of 1 Hz for 2 h daily for up to 42 days in osteogenic media. Spatially upregulated gene expression of chondrogenic markers (ACAN, COL2A1, PRG4) and glycosaminoglycan deposition was observed in the areas of greater compressive strains. In contrast, osteogenic markers (COL1A1, SPARC, RUNX2) and calcium deposition were downregulated in response to high local compressive strains. Dynamic mechanical analysis showed the maintenance of the engineered mechanical gradients only under dynamic culture conditions, confirming the potent role of biomechanical gradients in developing and maintaining a tissue gradient. These results demonstrate that multiphenotypic differentiation of hMSCs can be controlled by regulating local mechanical microenvironments, providing a novel strategy to recapitulate the gradient structure in osteochondral tissues for successful regeneration of damaged joints in vivo and facile development of interfacial tissue models in vitro.

Transmission and regulation of biochemical stimulus via a nanoshell directly adsorbed on the cell membrane to enhance chondrogenic differentiation of mesenchymal stem cell.

A nanoscale artificial extracellular matrix (nanoshell) formed by layer-by-layer adsorption can enhance and modulate the function of stem cells by transferring biochemical stimulus to the cell directly. Here, the nanoshell composed of fibronectin (FN) and chondroitin sulfate (CS) is demonstrated to promote chondrogenic differentiation of mesenchymal stem cells (MSCs). The multilayer structure of nanoshell is formed by repeating self-assembly of FN and CS, and its thickness can be controlled through the number of layers. The expression of chondrogenic markers in MSCs coated with the FN/CS nanoshell was increased as the number of bilayers in the nanoshell increased until four, but when it exceeds five bilayers, the effect began to decrease. Finally, the MSCs coated with optimized four bilayers of FN/CS nanoshell have high chondrogenic differentiation efficiency and showed the potential to increase formation of cartilage tissue when it is transplanted into mouse kidney. So, the precise regulation of stem cell fate at single cell level can be possible through the cellular surface modification by self-assembled polymeric film.

Impact of Hydrogel Stiffness on Differentiation of Human Adipose-Derived Stem Cell Microspheroids.

Hydrogels represent an attractive material platform for realization of three-dimensional (3D) tissue-engineered constructs, as they have tunable mechanical properties, are compatible with different types of cells, and resemble elements found in natural extracellular matrices. So far, numerous hydrogel-cartilage/bone tissue engineering (TE)-related studies were performed by utilizing a single cell encapsulation approach. Although multicellular spheroid cultures exhibit advantageous properties for cartilage or bone TE, the chondrogenic or osteogenic differentiation potential of stem cell microspheroids within hydrogels has not been investigated much. This study explores, for the first time, how stiffness of gelatin-based hydrogels (having a storage modulus of 538, 3584, or 7263 Pa) affects proliferation and differentiation of microspheroids formed from telomerase-immortalized human adipose-derived stem cells (hASC/hTERT). Confocal microscopy indicates that all tested hydrogels supported cell viability during their 3- to 5-week culture period in the control, chondrogenic, or osteogenic medium. Although in the softer hydrogels cells from neighboring microspheroids started outgrowing and interconnecting within a few days, their protrusion was slower or limited in stiffer hydrogels or those cultured in chondrogenic medium, respectively. High expressions of chondrogenic markers (SOX9, ACAN, COL2A1), detected in all tested hydrogels, proved that the chondrogenic differentiation of hASC/hTERT microspheroids was very successful, especially in the two softer hydrogels, where superior cartilage-specific properties were confirmed by Alcian blue staining. These chondrogenically induced samples also expressed COL10A1, a marker of chondrocyte hypertrophy. Interestingly, the hydrogel itself (with no differentiation medium) showed a slight chondrogenic induction. Regardless of the hydrogel stiffness, in the samples stimulated with osteogenic medium, the expression of selected markers RUNX2, BGLAP, ALPL, and COL1A1 was not conclusive. Nevertheless, the von Kossa staining confirmed the presence of calcium deposits in osteogenically stimulated samples in the two softer hydrogels, suggesting that these also favor osteogenesis. This observation was also confirmed by Alizarin red quantification assay, with which higher amounts of calcium were detected in the osteogenically induced hydrogels than in their controls. The presented data indicate that the encapsulation of adipose-derived stem cell microspheroids in gelatin-based hydrogels show promising potential for future applications in cartilage or bone TE.Impact StatementOsteochondral defects represent one of the leading causes of disability in the world. Although numerous tissue engineering (TE) approaches have shown success in cartilage and bone tissue regeneration, achieving native-like characteristics of these tissues remains challenging. This study demonstrates that in the presence of a corresponding differentiation medium, gelatin-based hydrogels support moderate osteogenic and excellent chondrogenic differentiation of photo-encapsulated human adipose-derived stem cell microspheroids, the extent of which depends on hydrogel stiffness. Because photosensitive hydrogels are a convenient material platform for creating stiffness gradients in three dimensions, the presented microspheroid-hydrogel encapsulation strategy holds promise for future strategies of cartilage or bone TE.

Effects of Conditioned Medium From Osteoarthritic Cartilage Fragments on Donor-Matched Infrapatellar Fat Pad–Derived Mesenchymal Stromal Cells

Background: Mesenchymal stromal cell (MSC)–based therapies have emerged as a promising strategy for osteoarthritis (OA) treatment. In particular, infrapatellar fat pad (IPFP)–derived MSCs have become a good option to treat knee OA. Purpose: To investigate the influence of the local microenvironment of the knee joint, especially OA cartilage, on the bioactivities of injected/implanted IPFP MSCs. Study Design: Controlled laboratory study. Methods: Conditioned medium (CM) derived from OA cartilage fragments was collected and characterized. Donor-matched IPFP MSCs were treated with control medium (Dulbecco’s modified Eagle medium (DMEM)/F-12 or chondrogenic medium), control medium + CM, or CM alone; and a series of behaviors including the viability, migration, chondrogenic and hypertrophic differentiation, and catabolic activity of IPFP MSCs were evaluated among groups. Results: There were 14 cytokines detected in CM. CM treatment improved the viability of IPFP MSCs. CM hindered the migration of IPFP MSCs. In chondrogenic differentiation, the presence of CM increased the expression of chondrogenic markers but also enhanced the state of hypertrophy and catabolism. Conclusion: OA cartilage–secreted factors could induce chondrogenic differentiation but also resulted in negative effects including the weakened migration, increased hypertrophy, and catabolism of IPFP MSCs in vitro. Clinical Relevance: These findings provide an insight on the fate of IPFP MSCs after intra-articular injections.

The poly (l-lactid-co-glycolide; PLGA) fiber component of brushite-forming calcium phosphate cement induces the osteogenic differentiation of human adipose tissue-derived stem cells

A brushite-forming calcium phosphate cement (CPC) was mechanically stabilized by addition of poly (l-lactid-co-glycolide; PLGA) fibers (≤10% w/w). It proved highly biocompatible and its fiber component enhanced bone formation in a sheep lumbar vertebroplasty model. However, possible effects on the osteogenic differentiation of resident mesenchymal stem cells (MSCs) remained unexplored. The present study used a novel approach, simultaneously analyzing the influence of a solid CPC scaffold and its relatively low PLGA proportion (a mimicry of natural bone) on osteogenic, chondrogenic, and adipogenic differentiation, as well as the pluripotency of human adipose tissue-derived mesenchymal stem cells (hASCs). hASCs were cultured on CPC discs with/without PLGA fibers (5% and 10%) in the absence of osteogenic medium for 3, 7, and 14 d. Gene expression of osteogenic markers (Runx2, osterix, alkaline phosphatase, collagen I, osteonectin, osteopontin, osteocalcin), chondrogenic markers (collagen II, Sox9, aggrecan), adipogenic markers (PPARG, Leptin, and FABP4), and pluripotency markers (Nanog, Tert, Rex) was analyzed by RT-PCR. The ability of hASCs to synthesize alkaline phosphatase was also evaluated. Cell number and viability were determined by fluorescein diacetate/propidium iodide staining. Compared to pure CPC, cultivation of hASCs on fiber-reinforced CPC transiently induced the gene expression of Runx2 and osterix (day 3), and long-lastingly augmented the expression of alkaline phosphatase (and its enzyme activity), collagen I, and osteonectin (until day 14). In contrast, augmented expression of all chondrogenic, adipogenic, and pluripotency markers was limited to day 3, followed by significant downregulation. Cultivation of hASCs on fiber-reinforced CPC reduced the cell number, but not the proportion of viable cells (viability > 95%). The PLGA component of fiber-reinforced, brushite-forming CPC supports long-lasting osteogenic differentiation of hASCs, whereas chondrogenesis, adipogenesis, and pluripotency are initially augmented, but subsequently suppressed. In view of parallel animal results, PLGA fibers may represent an interesting clinical target for future improvement of CPC- based bone regeneration.

Structure-Function Relationships of the Temporomandibular Joint in Response to Altered Loading.

To elucidate the effects of decreased occlusal loading (DOL), with or without reloading (RL), on the structure and bite force function of the mandibular condylar fibrocartilage in skeletally mature male mice. At 13 weeks old, 30 wild type (WT) male mice were subjected to: (1) 6 weeks normal loading (NL); (2) 6 weeks DOL; or (3) 4 weeks DOL + 2 weeks RL. Histomorphometry, cell metabolic activity, gene expression of chondrogenic markers, and bite force tests were performed. DOL resulted in a significant increase in apoptosis (P < .0001) and significant decreases in fibrocartilage thickness (P < .05) and hypertrophic chondrocyte markers indian hedgehog and collagen type X (P < .05). A corresponding decrease in bite force was also observed (P < .05). RL treatment resulted in a return to values comparable to NL of chondrogenic maturation markers (P > .10), apoptosis (P > .999), and bite force (P > .90), but not in mandibular condylar fibrocartilage thickness (P > .05). DOL in skeletally mature mice induces mandibular condylar fibrocartilage atrophy at the hypertrophic cell layer with a corresponding decrease in bite force.

Development, characterization and research of efficacy on in vitro cell culture of glucosamine carrying hyaluronic acid nanoparticles

Intra-articular hyaluronic acid (HA) and oral glucosamine (GA) are frequently used to decrease pain and increase function in osteoarthritis patients. Injectable chondroitin sulfate and HA improve effectiveness however a formulation that combines GA and HA is currently not available. Aims of this study were to synthesize and characterize N-acetyl-d-GA immobilized HA based nanoparticles [GA-PEG@nano(HA)] to increase extracellular matrix production and to decrease OA markers of healthy and OA chondrocytes. Mean size of synthesized GA-PEG@nano(HA) was 175 nm and 20% of GA released from these composites during 21 days. Released GA reduced the proliferation of chondrosarcoma cells. Chondrogenic marker expression of bone marrow mesenchymal stem cells decreased slightly but not significantly. osteoarthritis and extracellular matrix markers of healthy and osteoarthritic chondrocytes at seven days in culture remained unchanged. In conclusion, GA-PEG@nano(HA) composites had limited effects on mesenchymal stem cells, healthy and osteoarthritic chondrocytes.

3D bioprinted hydrogel model incorporating β-tricalcium phosphate for calcified cartilage tissue engineering

One promising strategy to reconstruct osteochondral defects relies on 3D bioprinted three-zonal structures comprised of hyaline cartilage, calcified cartilage, and subchondral bone. So far, several studies have pursued the regeneration of either hyaline cartilage or bone in vitro while—despite its key role in the osteochondral region—only few of them have targeted the calcified layer. In this work, we present a 3D biomimetic hydrogel scaffold containing β-tricalcium phosphate (TCP) for engineering calcified cartilage through a co-axial needle system implemented in extrusion-based bioprinting process. After a thorough bioink optimization, we showed that 0.5% w/v TCP is the optimal concentration forming stable scaffolds with high shape fidelity and endowed with biological properties relevant for the development of calcified cartilage. In particular, we investigate the effect induced by ceramic nano-particles over the differentiation capacity of bioprinted bone marrow-derived human mesenchymal stem cells in hydrogel scaffolds cultured up to 21 d in chondrogenic media. To confirm the potential of the presented approach to generate a functional in vitro model of calcified cartilage tissue, we evaluated quantitatively gene expression of relevant chondrogenic (COL1, COL2, COL10A1, ACAN) and osteogenic (ALPL, BGLAP) gene markers by means of RT-qPCR and qualitatively by means of fluorescence immunocytochemistry.

Human umbilical cord-derived scaffolds for cartilage tissue engineering.

Since articular cartilage is an avascular tissue, it has limited self-regeneration capacity after damage. Current methods for human bone marrow-derived mesenchymal stem cell (hBM-MSC) differentiation into cartilage result in tissues with a lower quality as compared to native articular cartilage. Decellularized biological scaffolds have the potential to provide appropriate signals, in order to support cellular retention, migration, proliferation, and differentiation. Given the high amount of collagen, hyaluronic acid (HA), and glycosaminoglycan (GAG) in umbilical cord, this tissue can be considered as an abundant natural biomaterial for tissue engineering applications. Human umbilical cord-derived scaffolds were prepared, and the chondrogenic induction of hBM-MSCs loaded onto the scaffolds was investigated. Gelatin-based scaffolds as a commercial material was used as a control. The results show that hBM-MSCs in tissue-derived scaffolds have an increased expression of chondrogenic markers compared with gelatin, whereas there are no significant differences between the expression of hypertrophic and osteogenic markers between tissue and gelatin scaffolds. In conclusion, it is confirmed that umbilical cord-derived scaffolds are able to provide a native environment for the cells and can promote cartilage differentiation. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1793-1802, 2019.

Efficacy of pulsed electromagnetic fields and electromagnetic fields tuned to the ion cyclotron resonance frequency of Ca2+ on chondrogenic differentiation.

Previous studies provide strong evidence for the therapeutic effect of electromagnetic fields (EMFs) on different tissues including cartilage. Diverse exposure parameters applied in scientific reports and the unknown interacting mechanism of EMF with biological systems make EMF studies challenging. In 1985, Liboff proposed that when magnetic fields are tuned to the cyclotron resonance frequencies of critical ions, the motion of ions through cell membranes is enhanced, and thus biological effects appear. Such exposure system consists of a weak alternating magnetic field (B1 ) in the presence of a static magnetic field (B0 ) and depends on the relationship between the magnitudes of B0 and B1 and the angular frequency Ω. The purpose of the present study is to determine the chondrogenic potential of EMF with regards to pulsed EMF (PEMF) and the ion cyclotron resonance (ICR) theory. We used different stimulating systems to generate EMFs in which cells are either stimulated with ubiquitous PEMF parameters, frequently reported, or parameters tuned to satisfy the ICR for Ca2+ (including negative and positive control groups). Chondrogenesis was analysed after 3weeks of treatment. Cell stimulation under the ICR condition showed positive results in the context of glycosaminoglycans and type II collagen synthesis. In contrast, the other electromagnetically stimulated groups showed no changes compared with the control groups. Furthermore, gene expression assays revealed an increase in the expression of chondrogenic markers (COL2A1, SOX9, and ACAN) in the ICR group. These results suggest that the Ca2+ ICR condition can be an effective factor in inducing chondrogenesis.

Exostosin-1 activity enhances the strength of wnt signaling during chondrogenic differentiation

Purpose: The exostosin-1 (EXT1) gene encodes a glycosyltransferase that is required for heparan sulfate (HS) chain elongation, which is a key step in HS-proteoglycan biosynthesis. Importantly, HS chains serve as binding partners for key signaling proteins such as hedgehogs and BMPs, affecting their distribution, activity and target selection, and thus influencing their roles in joint development and growth. The Wnt signaling pathway has emerged as a critical regulator of skeletal cell differentiation processes. However, how EXT1 activity and subsequent HS levels affect the activation of the Wnt signaling during endochondral ossification remains poorly understood. In this study, we aimed to define the function of EXT1 and HS-proteoglycan levels in determining the strength of Wnt signaling during chondrogenesis. Methods: Ext1 was stably knocked down or overexpressed in ATDC5 chondrogenic cells cultured as micromasses. HS content was determined using ELISA. Gene expression of chondrogenic markers (Col2a1, Acan andSox9) and direct Wnt target genes (Axin2 andLef1) was measured by RT q-PCR, at day 1, 7 and 14 of the chondrogenic differentiation process. Total proteoglycan content was evaluated by Alcian blue staining and DMMB assay. Activation of canonical Wnt signaling was assessed by Western blot of active β-catenin protein compared to total β-catenin levels, and by TOP/FOP luciferase activity assay. To evaluate whether Wnt signaling reciprocally regulates Ext1 expression, wild-type ATDC5 micromasses were treated with Wnt activators (recombinant WNT3A protein and CHIR99021) and a pharmacological Wnt antagonist (XAV939). Results: Ext1 was efficiently knocked down and overexpressed in ATDC5 cells during the whole chondrogenic differentiation process. Ext1 knockdown cells, which exhibited reduced HS content, showed increased expression of chondrogenic markers Col2a and Acan compared to control cells, and this paralleled an increase in total proteoglycan content. These results indicate that Ext1 knockdown facilitates the chondrogenic differentiation process. Next, Ext1 knockdown led to a reduced active Wnt signaling, as it resulted in a lower gene expression of Axin2, lower ratio of active β-catenin/totalβ-catenin protein levels, and lower luciferase activity compared to control cells. Contrarily, Ext1 overexpressing cells, which had a higher HS content, showed decreased chondrogenic differentiation potential and enhanced Wnt signaling activation compared to corresponding control cells. Interestingly, activation of Wnt signaling led to a downregulation of Ext1 expression in a concentration-dependent manner, and a specific Wnt antagonist upregulated Ext1 expression. Conclusions: Changes in EXT1 function, which has an impact on HS-proteoglycan content, affect the chondrogenic differentiation potential of precursor cells, and these effects are in part attributable to modifications in the strength of the Wnt signaling pathway. Simultaneously, Wnt signaling controls the expression of Ext1. Collectively, these results suggest the existence of a regulatory loop between Ext1 and the Wnt signaling during chondrogenesis. These insights may also have implications in osteoarthritis, since excessive Wnt signaling in articular cartilage is strongly linked to disease development and progression.

A 300 μm Organotypic Bone Slice Culture Model for Temporal Investigation of Endochondral Osteogenesis.

Translational studies to elucidate the response of immature bone to biologic and physical stimuli have been held back by the lack of a viable long-term functional bone explant model. This study attempts to bridge this gap between cell culture and animal model studies. In this study, we describe a methodology to derive a 300 μm organotypic femur slice comprising physiological zones (epiphysis and meta-diaphysis) essential for endochondral bone development. The unique capability of slice culture model incorporating enhanced nutrient access to distinct bone tissue components associated with linear bone growth facilitates the investigation of the orchestrated cellular transition of chondrogenic and osteogenic cells involved in endochondral bone development in an ex vivo setup. Bone slices of 300 μm were prepared from 4-day-old postnatal rats and were viable in culture up to 21 days. On days 7 and 15, an increase in chondrogenic and osteogenic modulations was confirmed in epiphysis, metaphysis, and diaphysis. An increase in osteocytes, osteoblasts, and hypertrophic cells were found at these time points, as well as a noticeable increased expression of chondrogenic and osteogenic markers (collagen II, Runx2, and osteocalcin) confirmed endochondral progression. Osteoclast-mediated bone resorption was demonstrated on day 15 by tartrate-resistant acid phosphatase staining. Attenuated total reflection infrared spectroscopic analyses, furthermore, confirmed a time-dependent increase in phosphate levels, bone minerals, and hydroxyapatite for 15 days. Our establishment of a bone slice culture model closely mimicking the in vivo cellular transitions and endochondral microenvironment of a mineralizing bone provides a vital new tool for the elucidation of cellular and endochondral mechanisms of bone development, maturation, and growth plate modulations. The presented model has the potential to be utilized in implementation of preclinical, toxicological, and therapeutic investigations.

Stromal cell-derived factor-1α promotes recruitment and differentiation of nucleus pulposus-derived stem cells.

BACKGROUNDIntervertebral disc (IVD) degeneration is a condition characterized by a reduction in the water and extracellular matrix content of the nucleus pulposus (NP) and is considered as one of the dominating contributing factors to low back pain. Recent evidence suggests that stromal cell-derived factor 1α (SDF-1α) and its receptor C-X-C chemokine receptor type 4 (CXCR4) direct the migration of stem cells associated with injury repair in different musculoskeletal tissues.AIMTo investigate the effects of SDF-1α on recruitment and chondrogenic differentiation of nucleus pulposus-derived stem cells (NPSCs).METHODSWe performed real-time RT-PCR and enzyme-linked immunosorbent assay to examine the expression of SDF-1α in nucleus pulposus cells after treatment with pro-inflammatory cytokines in vitro. An animal model of IVD degeneration was established using annular fibrosus puncture in rat coccygeal discs. Tissue samples were collected from normal control and degeneration groups. Differences in the expression of SDF-1α between the normal and degenerative IVDs were analyzed by immunohistochemistry. The migration capacity of NPSCs induced by SDF-1α was evaluated using wound healing and transwell migration assays. To determine the effect of SDF-1α on chondrogenic differentiation of NPSCs, we conducted cell micromass culture and examined the expression levels of Sox-9, aggrecan, and collagen II. Moreover, the roles of SDF-1/CXCR4 axis in the migration and chondrogenesis differentiation of NPSCs were analyzed by immunofluorescence, immunoblotting, and real-time RT-PCR.RESULTSSDF-1α was significantly upregulated in the native IVD cells cultured in vitro with pro-inflammatory cytokines, such as interleukin-1β and tumor necrosis factor-α, mimicking the degenerative settings. Immunohistochemical staining showed that the level of SDF-1α was also significantly higher in the degenerative group than in the normal group. SDF-1α enhanced the migration capacity of NPSCs in a dose-dependent manner. In addition, SDF-1α induced chondrogenic differentiation of NPSCs, as evidenced by the increased expression of chondrogenic markers using histological and immunoblotting analyses. Real-time RT-PCR, immunoblotting, and immunofluorescence showed that SDF-1α not only increased CXCR4 expression but also stimulated translocation of CXCR4 from the cytoplasm to membrane, accompanied by cytoskeletal rearrangement. Furthermore, blocking CXCR4 with AMD3100 effectively suppressed the SDF-1α-induced migration and differentiation capacities of NPSCs.CONCLUSIONThese findings demonstrate that SDF-1α has the potential to enhance recruitment and chondrogenic differentiation of NPSCs via SDF-1/CXCR4 chemotaxis signals that contribute to IVD regeneration.

Osteochondritis Dissecans (OCD)-Derived Chondrocytes Display Increased Senescence, Oxidative Stress, Chaperone-Mediated Autophagy and, in Co-Culture with Adipose-Derived Stem Cells (ASCs), Enhanced Expression of MMP-13.

Osteochondritis dissecans (OCD) in equids, especially in sport horses, has become a growing issue as it contributes to the occurrence of lameness. Thus the aim of this study was to investigate the cytophysiological properties of OCD chondrocytes including expression of chondrogenic genes, apoptosis, mitochondria dynamics and autophagy. Horse chondrocytes were isolated from healthy (HE) and OCD cartilages. Properties of cells were evaluated using multiple assays e.g., polymerase chain reaction (PCR), immunofluorescence, Western blot. OCD chondrocytes were characterized by increased apoptosis and senescence. Expression of chondrogenic genes (vimentin, aggrecan) was decreased while mRNA levels of matrix metalloproteinase 13 significantly upregulated in comparison to HE cells. Moreover, OCD cells displayed increased mitochondrial fusion while fission events were diminished. Interestingly, chaperone mediated autophagy was triggered in those cells and it predominated over macroautophagy. Furthermore, co-culture of LPS-treated chondrocytes with adipose-derived stem cells (ASC) decreased p62/sequestosome 1 (SQSTM) and increases MMP-13 expression in OCD cells. Our results suggest that OCD affected horse chondrocytes are characterized by senescent phenotype due to endoplasmic reticulum stress and mitochondria dynamics deterioration. Expression of chondrogenic markers is decreased in those cells while expression of chaperone mediated autophagy (CMA)-related genes increased. Increased malfunctioning of cells leads to loss of their functionality and capacity to maintain tissue homeostasis.

Platelet-rich plasma promotes the regeneration of cartilage engineered by mesenchymal stem cells and collagen hydrogel via the TGF-β/SMAD signaling pathway.

The tissue engineering technique using mesenchymal stem cells (MSCs) and scaffolds is promising. Transforming growth factor-β1 (TGF-β1) is generally accepted as an chondrogenic agent, but immunorejection and unexpected side effects, such as tumorigenesis and heterogeneity, limit its clinical application. Autogenous platelet-rich plasma (PRP), marked by low immunogenicity, easy accessibility, and low-cost, may be favorable for cartilage regeneration. In our study, the effect of PRP on engineered cartilage constructed by MSCs and collagen hydrogel in vitro and in vivo was investigated and compared with TGF-β1. The results showed that PRP promoted cell proliferation and gene and protein expressions of chondrogenic markers via the TGF-β/SMAD signaling pathway. Meanwhile, it suppressed the expression of collagen type I, a marker of fibrocartilage. Furthermore, PRP accelerated cartilage regeneration on defects with engineered cartilage, advantageous over TGF-β1, as evaluated by histological analysis and immunohistochemical staining. Our work demonstrates that autogenous PRP may substitute TGF-β1 as a potent and reliable chondrogenic inducer for therapy of cartilage defect.

A Subpopulation of Synovial Fibroblasts Leads to Osteochondrogenesis in a Mouse Model of Chronic Inflammatory Rheumatoid Arthritis.

Specific major histocompatibility complex (MHC) class II genes result in a high susceptibility to rheumatoid arthritis (RA), with co-stimulatory molecules working together with MHC class II during the progression of the disease. To elucidate the involvement of the B7.1 co-stimulatory molecule in RA, we analyzed the phenotype of B7.1 transgenic (named D1BC) mice and the sequential differentiation of synovial fibroblasts (SFs) by studying the expression of chondrogenic and osteogenic lineage markers together with lineage tracing experiment using B7.1 transgene in vivo. The B7.1 transgene was driven by a collagen type II (CII) promoter and enhancer in the D1BC mouse. A low-dose of bovine CII (bCII) was used to induce chronic articular inflammation with interstitial pneumonitis. Joint damage was analyzed by histopathological examination and computed tomography. B7.1 was expressed in articular cartilage and SFs of D1BC mice. Chronic inflammatory arthritis in the bCII-D1BC mouse shared common features with those found in patients with RA, such as pannus formation, bone destruction, osteoporosis, and joint ankylosis. A subpopulation of SFs (Runx2 +, Sox9 +, Col10a1 +, Osx+, and CX-) in the pannus was classified as osteochondrogenic lineage rather than mesenchymal stromal lineage. These cells underwent differentiation into osteogenic lineage via hypertrophic chondrocytes at the end of the chronic phase. The ectopic expression of B7.1 in chondrocytes and SFs leads to an increased susceptibility to chronic inflammatory arthritis and subsequent new bone formation, reminiscent of ankylosis. The regulation of cartilage remodeling in pannus tissue is an important consideration in the treatment of RA.

MicroRNA-218 promotes early chondrogenesis of mesenchymal stem cells and inhibits later chondrocyte maturation

BackgroundMicroRNAs (miRNAs) reportedly participate in the mesenchymal stem cell (MSC) chondrogenic differentiation regulation. We objected to examine how miR-218 regulate chondrogenic differentiation of synovium-derived MSCs (SDSCs) and the maturation of RCJ3.1C5.1 chondrocytes. SDSCs were sourced from the knee joint synovium of New Zealand white rabbits, and their multilineage differentiation potentials were examined. The level of miR-218 was measured during SDSC chondrogenic differentiation, together with determination of SDSCs chondrogenic markers and RCJ3.1C5.1 chondrocytes maturation markers expression level after transfection of miR-218 mimics/inhibitor.ResultsmiR-218 expression was notably upregulated in early chondrogenesis but mostly ceased during the maturation phases of chondrogenic differentiation in SDSCs. The transfection of miR-218 mimics notably enhanced SDSCs chondrocytes differentiation, as evidenced by augmented expressions of chondrogenic markers (SOX9, COL2A1, ACAN, GAG, and COMP) in terms of mRNA and protein level, and the inhibition of miR-218 yielded opposite resutls. Additionally, miR-218 overexpression substantially suppressed the expression of osteogenic markers (ALP, BSP, COL1A1, OCN and OPN) during the early stage of chondrogenesis while increasing that of chondrogenic markers (SOX9, COL2A1, ACAN, GAG and COMP). However, miR-218 mimics notably suppressed maturation markers (CMP, COL10A1, MMP-13 and VEGF) expression in RCJ3.1C5.18 chondrocytes, and the miR-218 inhibitor promoted the expression of these maturation markers. We proposed miR-218 plays a regulatory role on 15-hydroxyprostaglandin dehydrogenase (HPGD), which plays a key role in chondrogenic differentiation, and this finding indicates that miR-218 directly regulates HPGD expression in SDSCs.ConclusionOur study suggests that miR-218 contributes to early chondrogenesis while suppressing later chondrocyte maturation. The miR-218-HPGD pathway offers us a perspective into how SDSCs differentiate into chondrogenic cells.