Pre-hypertrophic chondrogenic enhancer landscape of limb and axial skeleton development.

Chondrocyte differentiation controls skeleton development and stature. Here we provide a comprehensive map of chondrocyte-specific enhancers and show that they provide a mechanistic framework through which non-coding genetic variants can influence skeletal development and human stature. Working with fetal chondrocytes isolated from mice bearing a Col2a1 fluorescent regulatory sensor, we identify 780 genes and 2'704 putative enhancers specifically active in chondrocytes using a combination of RNA-seq, ATAC-seq and H3K27ac ChIP-seq. Most of these enhancers (74%) show pan-chondrogenic activity, with smaller populations being restricted to limb (18%) or trunk (8%) chondrocytes only. Notably, genetic variations overlapping these enhancers better explain height differences than those overlapping non-chondrogenic enhancers. Finally, targeted deletions of identified enhancers at the Fgfr3, Col2a1, Hhip and, Nkx3-2 loci confirm their role in regulating cognate genes. This enhancer map provides a framework for understanding how genes and non-coding variations influence bone development and diseases.

Cytokine Receptor-like Factor 1 (CRLF1) and Its Role in Osteochondral Repair.

BACKGROUND: Since cytokine receptor-like factor 1 (CRLF1) has been implicated in tissue regeneration, we hypothesized that CRLF1 released by mesenchymal stem cells can promote the repair of osteochondral defects. METHODS: The degree of a femoral osteochondral defect repair in rabbits after intra-articular injections of bone marrow-derived mesenchymal stem cells (BMSCs) that were transduced with empty adeno-associated virus (AAV) or AAV containing CRLF1 was determined by morphological, histological, and micro computer tomography (CT) analyses. The effects of CRLF1 on chondrogenic differentiation of BMSCs or catabolic events of interleukin-1beta-treated chondrocyte cell line TC28a2 were determined by alcian blue staining, gene expression levels of cartilage and catabolic marker genes using real-time PCR analysis, and immunoblot analysis of Smad2/3 and STAT3 signaling. RESULTS: Intra-articular injections of BMSCs overexpressing CRLF1 markedly improved repair of a rabbit femoral osteochondral defect. Overexpression of CRLF1 in BMSCs resulted in the release of a homodimeric CRLF1 complex that stimulated chondrogenic differentiation of BMSCs via enhancing Smad2/3 signaling, whereas the suppression of CRLF1 expression inhibited chondrogenic differentiation. In addition, CRLF1 inhibited catabolic events in TC28a2 cells cultured in an inflammatory environment, while a heterodimeric complex of CRLF1 and cardiotrophin-like Cytokine (CLC) stimulated catabolic events via STAT3 activation. CONCLUSION: A homodimeric CRLF1 complex released by BMSCs enhanced the repair of osteochondral defects via the inhibition of catabolic events in chondrocytes and the stimulation of chondrogenic differentiation of precursor cells.

Extremely low frequency-electromagnetic fields promote chondrogenic differentiation of adipose-derived mesenchymal stem cells through a conventional genetic program.

Progressive cartilage deterioration leads to chronic inflammation and loss of joint function, causing osteoarthritis (OA) and joint disease. Although symptoms vary among individuals, the disease can cause severe pain and permanent disability, and effective therapies are urgently needed. Human Adipose-Derived Stem Cells (ADSCs) may differentiate into chondrocytes and are promising for treating OA. Moreover, recent studies indicate that electromagnetic fields (EMFs) could positively affect the chondrogenic differentiation potential of ADSCs. In this work, we investigated the impact of EMFs with frequencies of 35 Hertz and 58 Hertz, referred to as extremely low frequency-EMFs (ELF-EMFs), on the chondrogenesis of ADSCs, cultured in both monolayer and 3D cell micromasses. ADSC cultures were daily stimulated for 36 min with ELF-EMFs or left unstimulated, and the progression of the differentiation process was evaluated by morphological analysis, extracellular matrix deposition, and gene expression profiling of chondrogenic markers. In both culturing conditions, stimulation with ELF-EMFs did not compromise cell viability but accelerated chondrogenesis by enhancing the secretion and deposition of extracellular matrix components at earlier time points in comparison to unstimulated cells. This study showed that, in an appropriate chondrogenic microenvironment, ELF-EMFs enhance chondrogenic differentiation and may be an important tool for supporting and accelerating the treatment of OA through autologous adipose stem cell therapy.

Sericin promotes chondrogenic proliferation and differentiation via glycolysis and Smad2/3 TGF-ß signaling inductions and alleviates inflammation in three-dimensional models.

Knee osteoarthritis is a chronic joint disease mainly characterized by cartilage degeneration. The treatment is challenging due to the lack of blood vessels and nerve supplies in cartilaginous tissue, causing a prominent limitation of regenerative capacity. Hence, we investigated the cellular promotional and anti-inflammatory effects of sericin, Bombyx mori-derived protein, on three-dimensional chondrogenic ATDC5 cell models. The results revealed that a high concentration of sericin promoted chondrogenic proliferation and differentiation and enhanced matrix production through the increment of glycosaminoglycans, COL2A1, COL X, and ALP expressions. SOX-9 and COL2A1 gene expressions were notably elevated in sericin treatment. The proteomic analysis demonstrated the upregulation of phosphoglycerate mutase 1 and triosephosphate isomerase, a glycolytic enzyme member, reflecting the proliferative enhancement of sericin. The differentiation capacity of sericin was indicated by the increased expressions of procollagen12a1, collagen10a1, rab1A, periostin, galectin-1, and collagen6a3 proteins. Sericin influenced the differentiation capacity via the TGF-ß signaling pathway by upregulating Smad2 and Smad3 while downregulating Smad1, BMP2, and BMP4. Importantly, sericin exhibited an anti-inflammatory effect by reducing IL-1ß, TNF-α, and MMP-1 expressions and accelerating COL2A1 production in the early inflammatory stage. In conclusion, sericin demonstrates potential in promoting chondrogenic proliferation and differentiation, enhancing cartilaginous matrix synthesis through glycolysis and TGF-ß signaling pathways, and exhibiting anti-inflammatory properties.

ß1-Integrin-Mediated Uptake of Chondrocyte Extracellular Vesicles Regulates Chondrocyte Homeostasis.

Osteoarthritis (OA) is the most prevalent age-related degenerative disorder, which severely reduces the quality of life of those affected. Whilst management strategies exist, no cures are currently available. Virtually all joint resident cells generate extracellular vesicles (EVs), and alterations in chondrocyte EVs during OA have previously been reported. Herein, we investigated factors influencing chondrocyte EV release and the functional role that these EVs exhibit. Both 2D and 3D models of culturing C28I/2 chondrocytes were used for generating chondrocyte EVs. We assessed the effect of these EVs on chondrogenic gene expression as well as their uptake by chondrocytes. Collectively, the data demonstrated that chondrocyte EVs are sequestered within the cartilage ECM and that a bi-directional relationship exists between chondrocyte EV release and changes in chondrogenic differentiation. Finally, we demonstrated that the uptake of chondrocyte EVs is at least partially dependent on ß1-integrin. These results indicate that chondrocyte EVs have an autocrine homeostatic role that maintains chondrocyte phenotype. How this role is perturbed under OA conditions remains the subject of future work.

Stem-Cell-Driven Chondrogenesis: Perspectives on Amnion-Derived Cells.

Regenerative medicine harnesses stem cells' capacity to restore damaged tissues and organs. In vitro methods employing specific bioactive molecules, such as growth factors, bio-inductive scaffolds, 3D cultures, co-cultures, and mechanical stimuli, steer stem cells toward the desired differentiation pathways, mimicking their natural development. Chondrogenesis presents a challenge for regenerative medicine. This intricate process involves precise modulation of chondro-related transcription factors and pathways, critical for generating cartilage. Cartilage damage disrupts this process, impeding proper tissue healing due to its unique mechanical and anatomical characteristics. Consequently, the resultant tissue often forms fibrocartilage, which lacks adequate mechanical properties, posing a significant hurdle for effective regeneration. This review comprehensively explores studies showcasing the potential of amniotic mesenchymal stem cells (AMSCs) and amniotic epithelial cells (AECs) in chondrogenic differentiation. These cells exhibit innate characteristics that position them as promising candidates for regenerative medicine. Their capacity to differentiate toward chondrocytes offers a pathway for developing effective regenerative protocols. Understanding and leveraging the innate properties of AMSCs and AECs hold promise in addressing the challenges associated with cartilage repair, potentially offering superior outcomes in tissue regeneration.

Cartilage tissue engineering using decellularized biomatrix hydrogel containing TGF-ß-loaded alginate microspheres in mechanically loaded bioreactor.

Physiochemical tissue inducers and mechanical stimulation are both efficient variables in cartilage tissue fabrication and regeneration. In the presence of biomolecules, decellularized extracellular matrix (ECM) may trigger and enhance stem cell proliferation and differentiation. Here, we investigated the controlled release of transforming growth factor beta (TGF-ß1) as an active mediator of mesenchymal stromal cells (MSCs) in a biocompatible scaffold and mechanical stimulation for cartilage tissue engineering. ECM-derived hydrogel with TGF-ß1-loaded alginate-based microspheres (MSs) was created to promote human MSC chondrogenic development. Ex vivo explants and a complicated multiaxial loading bioreactor replicated the physiological conditions. Hydrogels with/without MSs and TGF-ß1 were highly cytocompatible. MSCs in ECM-derived hydrogel containing TGF-ß1/MSs showed comparable chondrogenic gene expression levels as those hydrogels with TGF-ß1 added in culture media or those without TGF-ß1. However, constructs with TGF-ß1 directly added within the hydrogel had inferior properties under unloaded conditions. The ECM-derived hydrogel group including TGF-ß1/MSs under loading circumstances formed better cartilage matrix in an ex vivo osteochondral defect than control settings. This study demonstrates that controlled local delivery of TGF-ß1 using MSs and mechanical loading is essential for neocartilage formation by MSCs and that further optimization is needed to prevent MSC differentiation towards hypertrophy.

Increased water temperature contributes to a chondrogenesis response in the eyes of spotted wolffish.

Adult vertebrate cartilage is usually quiescent. Some vertebrates possess ocular scleral skeletons composed of cartilage or bone. The morphological characteristics of the spotted wolffish (Anarhichas minor) scleral skeleton have not been described. Here we assessed the scleral skeletons of cultured spotted wolffish, a globally threatened marine species. The healthy spotted wolffish we assessed had scleral skeletons with a low percentage of cells staining for the chondrogenesis marker sex-determining region Y-box (Sox) 9, but harboured a population of intraocular cells that co-express immunoglobulin M (IgM) and Sox9. Scleral skeletons of spotted wolffish with grossly observable eye abnormalities displayed a high degree of perochondrial activation as evidenced by cellular morphology and expression of proliferating cell nuclear antigen (PCNA) and phosphotyrosine. Cells staining for cluster of differentiation (CD) 45 and IgM accumulated around sites of active chondrogenesis, which contained cells that strongly expressed Sox9. The level of scleral chondrogenesis and the numbers of scleral cartilage PCNA positive cells increased with the temperature of the water in which spotted wolffish were cultured. Our results provide new knowledge of differing Sox9 spatial tissue expression patterns during chondrogenesis in normal control and ocular insult paradigms. Our work also provides evidence that spotted wolffish possess an inherent scleral chondrogenesis response that may be sensitive to temperature. This work also advances the fundamental knowledge of teleost ocular skeletal systems.

Gel microspheres enhance the stemness of ADSCs by regulating cell-ECM interaction.

The gel microsphere culture system (GMCS) showed various advantages for mesenchymal stem cell (MSC) expansion and delivery, such as high specific surface area, small and regular shape, extensive adjustability, and biomimetic properties. Although various technologies and materials have been developed to promote the development of gel microspheres, the differences in the biological status of MSCs between the GMCS and the traditional Petri dish culture system (PDCS) are still unknown, hindering gel microspheres from becoming a culture system as widely used as petri dishes. In the previous study, an excellent "all-in-one" GMCS has been established for the expansion of human adipose-derived MSCs (hADSCs), which showed convenient cell culture operation. Here, we performed transcriptome and proteome sequencing on hADSCs cultured on the "all-in-one" GMCS and the PDCS. We found that hADSCs cultured in the GMCS kept in an undifferentiation status with a high stemness index, whose transcriptome profile is closer to the adipose progenitor cells (APCs) in vivo than those cultured in the PDCS. Further, the high stemness status of hADSCs in the GMCS was maintained through regulating cell-ECM interaction. For application, bilayer scaffolds were constructed by osteo- and chondro-differentiation of hADSCs cultured in the GMCS and the PDCS. The effect of osteochondral regeneration of the bilayer scaffolds in the GMCS group was better than that in the PDCS group. This study revealed the high stemness and excellent functionality of MSCs cultured in the GMCS, which promoted the application of gel microspheres in cell culture and tissue regeneration.

Magnetic Seeding of SPIO-BMSCs Into a Biphasic Scaffold Can Promote Tendon-Bone Healing After Rotator Cuff Repair.

BACKGROUND: The tendon-bone interface (TBI) in the rotator cuff has a poor intrinsic capacity for healing, which increases the risk of retear after rotator cuff repair (RCR). However, facilitating regeneration of the TBI still remains a great clinical challenge. Herein, the authors established a novel strategy based on magnetic seeding to enhance the TBI regeneration. HYPOTHESIS: Magnetic seeding bone marrow mesenchymal stem cells labeled with superparamagnetic iron oxide (SPIO-BMSCs) into a biphasic scaffold can promote tendon-bone healing after RCR. STUDY DESIGN: Controlled laboratory study. METHODS: BMSCs were labeled with SPIOs. Prussian blue staining, CCK-8 tests, Western blot, and quantitative reverse transcription polymerase chain reaction (PCR) were used to determine the optimal effect concentration of SPIOs on cell bioactivities and abilities. Then SPIO-BMSCs were magnetically seeded into a biphasic scaffold under a magnetic field. The seeding efficacy was assessed by a scanning electron microscope, and the potential mechanism in chondrogenic differentiation after seeding SPIO-BMSCs into the scaffold was evaluated by Western blot and PCR. Furthermore, the effect of SPIO-BMSC/biphasic scaffold on tendon-bone healing after RCR using a rat model was examined using histological analysis, enzyme-linked immunosorbent assay, and biomechanical evaluation. RESULTS: BMSCs labeled with 100 µg/mL SPIO had no effect on cell bioactivities and the ability of chondrogenic differentiation. SPIO-BMSCs were magnetically seeded into a biphasic scaffold, which offered a high seeding efficacy to enhance chondrogenic differentiation of SPIO-BMSCs via the CDR1as/miR-7/FGF2 pathway for TBI formation in vitro. Furthermore, in vivo application of the biphasic scaffold with magnetically seeded SPIO-BMSCs showed their regenerative potential, indicating that they could significantly accelerate and promote TBI healing with superior biomechanical properties after RCR in a rat rotator cuff tear model. CONCLUSION: Magnetically seeding SPIO-BMSCs into a biphasic scaffold enhanced seeding efficacy to promote cell distribution and condensation. This construct enhanced the chondrogenesis process via the CDR1as/miR-7/FGF2 pathway and further promoted tendon-bone healing after RCR in a rat rotator cuff tear model. CLINICAL RELEVANCE: This study provides an alternative strategy for improving TBI healing after RCR.

Functionally graded hydrogels with opposing biochemical cues for osteochondral tissue engineering.

Osteochondral tissue (OC) repair remains a significant challenge in the field of musculoskeletal tissue engineering. OC tissue displays a gradient structure characterized by variations in both cell types and extracellular matrix components, from cartilage to the subchondral bone. These functional gradients observed in the native tissue have been replicated to engineer OC tissuein vitro. While diverse fabrication methods have been employed to create these microenvironments, emulating the natural gradients and effective regeneration of the tissue continues to present a significant challenge. In this study, we present the design and development of CMC-silk interpenetrating (IPN) hydrogel with opposing dual biochemical gradients similar to native tissue with the aim to regenerate the complete OC unit. The gradients of biochemical cues were generated using an in-house-built extrusion system. Firstly, we fabricated a hydrogel that exhibits a smooth transition of sulfated carboxymethyl cellulose (sCMC) and TGF-ß1 (SCT gradient hydrogel) from the upper to the lower region of the IPN hydrogel to regenerate the cartilage layer. Secondly, a hydrogel with a hydroxyapatite (HAp) gradient (HAp gradient hydrogel) from the lower to the upper region was fabricated to facilitate the regeneration of the subchondral bone layer. Subsequently, we developed a dual biochemical gradient hydrogel with a smooth transition of sCMC + TGF-ß1 and HAp gradients in opposing directions, along with a blend of both biochemical cues in the middle. The results showed that the dual biochemical gradient hydrogels with biochemical cues corresponding to the three zones (i.e. cartilage, interface and bone) of the OC tissue led to differentiation of bone-marrow-derived mesenchymal stem cells to zone-specific lineages, thereby demonstrating their efficacy in directing the fate of progenitor cells. In summary, our study provided a simple and innovative method for incorporating gradients of biochemical cues into hydrogels. The gradients of biochemical cues spatially guided the differentiation of stem cells and facilitated tissue growth, which would eventually lead to the regeneration of the entire OC tissue with a smooth transition from cartilage (soft) to bone (hard) tissues. This promising approach is translatable and has the potential to generate numerous biochemical and biophysical gradients for regeneration of other interface tissues, such as tendon-to-muscle and ligament-to-bone.

Transforming growth factor-ß1-loaded RADA-16 hydrogel scaffold for effective cartilage regeneration.

Cartilage repair remains a major challenge in clinical trials. These current cartilage repair materials can not effectively promote chondrocyte generation, limiting their practical application in cartilage repair. In this work, we develop an implantable scaffold of RADA-16 peptide hydrogel incorporated with TGF-ß1 to provide a microenvironment for stem cell-directed differentiation and chondrocyte adhesion growth. The longest release of growth factor TGF-ß1 release can reach up to 600 h under physiological conditions. TGF-ß1/RADA-16 hydrogel was demonstrated to be a lamellar porous structure. Based on the cell culture with hBMSCs, TGF-ß1/RADA-16 hydrogel showed excellent ability to promote cell proliferation, directed differentiation into chondrocytes, and functional protein secretion. Within 14 days, 80% of hBMSCs were observed to be directed to differentiate into vigorous chondrocytes in the co-culture of TGF-ß1/RADA-16 hydrogels with hBMSCs. Specifically, these newly generated chondrocytes can secrete and accumulate large amounts of collagen II within 28 days, which can effectively promote the formation of cartilage tissue. Finally, the exploration of RADA-16 hydrogel-based scaffolds incorporated with TGF-ß1 bioactive species would further greatly promote the practical clinical trials of cartilage remediation, which might have excellent potential to promote cartilage regeneration in areas of cartilage damage.

PI15, a novel secreted WNT-signaling antagonist, regulates chondrocyte differentiation.

PURPOSE/AIM OF STUDY: During the development of the vertebrate skeleton, the progressive differentiation and maturation of chondrocytes from mesenchymal progenitors is precisely coordinated by multiple secreted factors and signaling pathways. The WNT signaling pathway has been demonstrated to play a major role in chondrogenesis. However, the identification of secreted factors that fine-tune WNT activity has remained elusive. Here, in this study, we have identified PI15 (peptidase inhibitor 15, protease Inhibitor 15, SugarCrisp), a member of the CAP (cysteine rich secretory proteins, antigen 5, and pathogenesis related 1 proteins) protein superfamily, as a novel secreted WNT antagonist dynamically upregulated during chondrocyte differentiation. MATERIALS AND METHODS: ATDC5 cells, C3H10T1/2 micromass cultures and primary chondrocyte cells were used as in vitro models of chondrogenesis. PI15 levels were stably depleted or overexpressed by viral shRNA or expression vectors. Chondrogenesis was evaluated by qPCR gene expression analysis and Alcian blue staining. Protein interactions were determined by coimmunoprecipitation assays. RESULTS AND CONCLUSIONS: shRNA-mediated knockdown of PI15 in ATDC5 cells, C3H10T1/2 cells or primary chondrocytes inhibits chondrogenesis, whereas the overexpression of PI15 strongly enhances chondrogenic potential. Mechanistically, PI15 binds to the LRP6 WNT co-receptor and blocks WNT-induced LRP6 phosphorylation, thus repressing WNT-induced transcriptional activity and alleviating the inhibitory effect of WNT signaling on chondrogenesis. Altogether, our findings suggest that PI15 acts as a key regulator of chondrogenesis and unveils a mechanism through which chondrocyte-derived molecules can modulate WNT activity as differentiation proceeds, thereby creating a positive feedback loop that further drives differentiation.

Bioprinted biomimetic hydrogel matrices guiding stem cell aggregates for enhanced chondrogenesis and cartilage regeneration.

Articular cartilage tissue has limited self-repair capabilities, with damage frequently progressing to irreversible degeneration. Engineered tissues constructed through bioprinting and embedded with stem cell aggregates offer promising therapeutic alternatives. Aggregates of bone marrow mesenchymal stromal cells (BMSCs) demonstrate enhanced and more rapid chondrogenic differentiation than isolated cells, thus facilitating cartilage repair. However, it remains a key challenge to precisely control biochemical microenvironments to regulate cellular adhesion and cohesion within bioprinted matrices simultaneously. Herein, this work reports a bioprintable hydrogel matrix with high cellular adhesion and aggregation properties for cartilage repair. The hydrogel comprises an enhanced cell-adhesive gelatin methacrylate and a cell-cohesive chitosan methacrylate (CHMA), both of which are subjected to photo-initiated crosslinking. By precisely adjusting the CHMA content, the mechanical stability and biochemical cues of the hydrogels are finely tuned to promote cellular aggregation, chondrogenic differentiation and cartilage repair implantation. Multi-layer constructs encapsulated with BMSCs, with high cell viability reaching 91.1%, are bioprinted and photo-crosslinked to support chondrogenic differentiation for 21 days. BMSCs rapidly form aggregates and display efficient chondrogenic differentiation both on the hydrogels and within bioprinted constructs, as evidenced by the upregulated expression of Sox9, Aggrecan and Collagen 2a1 genes, along with high protein levels. Transplantation of these BMSC-laden bioprinted hydrogels into cartilaginous defects demonstrates effective hyaline cartilage repair. Overall, this cell-responsive hydrogel scaffold holds immense promise for applications in cartilage tissue engineering.

Porcine nasal septum cartilage-derived decellularized matrix promotes chondrogenic differentiation of human umbilical mesenchymal stem cells without exogenous growth factors.

BACKGROUND: In the domain of plastic surgery, nasal cartilage regeneration is of significant importance. The extracellular matrix (ECM) from porcine nasal septum cartilage has shown potential for promoting human cartilage regeneration. Nonetheless, the specific biological inductive factors and their pathways in cartilage tissue engineering remain undefined. METHODS: The decellularized matrix derived from porcine nasal septum cartilage (PN-DCM) was prepared using a grinding method. Human umbilical cord mesenchymal stem cells (HuMSCs) were cultured on these PN-DCM scaffolds for 4 weeks without exogenous growth factors to evaluate their chondroinductive potential. Subsequently, proteomic analysis was employed to identify potential biological inductive factors within the PN-DCM scaffolds. RESULTS: Compared to the TGF-ß3-cultured pellet model serving as a positive control, the PN-DCM scaffolds promoted significant deposition of a Safranin-O positive matrix and Type II collagen by HuMSCs. Gene expression profiling revealed upregulation of ACAN, COL2A1, and SOX9. Proteomic analysis identified potential chondroinductive factors in the PN-DCM scaffolds, including CYTL1, CTGF, MGP, ITGB1, BMP7, and GDF5, which influence HuMSC differentiation. CONCLUSION: Our findings have demonstrated that the PN-DCM scaffolds promoted HuMSC differentiation towards a nasal chondrocyte phenotype without the supplementation of exogenous growth factors. This outcome is associated with the chondroinductive factors present within the PN-DCM scaffolds.

The c-Fos/AP-1 inhibitor inhibits sulfur mustard-induced chondrogenesis impairment in zebrafish larvae.

Sulfur mustard (SM, dichlorodiethyl sulfide) is a potent erosive chemical poison that can cause pulmonary lung, skin and eye disease complications in humans. Currently, there is no designated remedy for SM, and its operation's toxicological process remains unidentified. This work employed zebrafish as a model organism to investigate the toxic manifestations and mechanisms of exposure to SM, aiming to offer novel insights for preventing and treating this condition. The results showed that SM caused a decrease in the survival rate of the zebrafish larvae (LC50 = 2.47 mg/L), a reduction in the hatching rate, an increase in the pericardial area, and small head syndrome. However, T-5224 (a selective inhibitor of c-Fos/activator protein) attenuated the reduction in mortality (LC50 = 2.79 mg/L), the reduction in hatching rate, and the worsening of morphological changes. We discovered that SM causes cartilage developmental disorders in zebrafish larvae. The reverse transcription-quantitative polymerase chain reaction found that SM increased the expression of inflammation-related genes (IL-1ß, IL-6, and TNF-α) and significantly increased cartilage development-related gene expression (fosab, mmp9, and atf3). However, the expression of sox9a, sox9b, and Col2a1a was reduced. The protein level detection also found an increase in c-fos protein expression and a significant decrease in COL2A1 expression. However, T-5224,also and mitigated the changes in gene expression, and protein levels caused by SM exposure. The results of this study indicate that SM-induced cartilage development disorders are closely related to the c-Fos/AP-1 pathway in zebrafish.

Electrospun fibers modified with polydopamine for enhancing human mesenchymal stem cell culture.

In this study, we coated electrospun polycaprolactone (PCL) fibers with polydopamine (PDA) to modify their hydrophobicity and fabricated a matrix for culturing mesenchymal stem cells (MSCs). Additionally, we incorporated Arg-Gly-Asp (RGD) peptides into PDA to enhance MSCs culture performance on PCL fibers. PDA and RGD were successfully coated in one step by immersing the electrospun fibers in a coating solution, without requiring an additional surface activation process. The characteristics of functionalized PCL fibers were analyzed by scanning electron microscopy with energy-dispersive x-ray analysis, Fourier transform infrared spectroscopy, water contact angle measurement, and fluorescence measurements using a carboxylic-modified fluorescent microsphere. MSCs cultured on the modified PCL fibers demonstrated enhanced cell adhesion, proliferation, and osteogenic- and chondrogenic differentiation. This study provides insight into potential applications for scaffold fabrication in MSCs-based tissue engineering, wound dressing, implantation, and a deeper understanding of MSCs behaviorin vitro.

BMP signaling maintains auricular chondrocyte identity and prevents microtia development by inhibiting protein kinase A.

Elastic cartilage constitutes a major component of the external ear, which functions to guide sound to the middle and inner ears. Defects in auricle development cause congenital microtia, which affects hearing and appearance in patients. Mutations in several genes have been implicated in microtia development, yet, the pathogenesis of this disorder remains incompletely understood. Here, we show that Prrx1 genetically marks auricular chondrocytes in adult mice. Interestingly, BMP-Smad1/5/9 signaling in chondrocytes is increasingly activated from the proximal to distal segments of the ear, which is associated with a decrease in chondrocyte regenerative activity. Ablation of Bmpr1a in auricular chondrocytes led to chondrocyte atrophy and microtia development at the distal part. Transcriptome analysis revealed that Bmpr1a deficiency caused a switch from the chondrogenic program to the osteogenic program, accompanied by enhanced protein kinase A activation, likely through increased expression of Adcy5/8. Inhibition of PKA blocked chondrocyte-to-osteoblast transformation and microtia development. Moreover, analysis of single-cell RNA-seq of human microtia samples uncovered enriched gene expression in the PKA pathway and chondrocyte-to-osteoblast transformation process. These findings suggest that auricle cartilage is actively maintained by BMP signaling, which maintains chondrocyte identity by suppressing osteogenic differentiation.

Specific lipid magnetic sphere sorted CD146-positive bone marrow mesenchymal stem cells can better promote articular cartilage damage repair.

BACKGROUND: The characteristics and therapeutic potential of subtypes of bone marrow mesenchymal stem cells (BMSCs) are largely unknown. Also, the application of subpopulations of BMSCs in cartilage regeneration remains poorly characterized. The aim of this study was to explore the regenerative capacity of CD146-positive subpopulations of BMSCs for repairing cartilage defects. METHODS: CD146-positive BMSCs (CD146 + BMSCs) were sorted by self-developed CD146-specific lipid magnetic spheres (CD146-LMS). Cell surface markers, viability, and proliferation were evaluated in vitro. CD146 + BMSCs were subjected to in vitro chondrogenic induction and evaluated for chondrogenic properties by detecting mRNA and protein expression. The role of the CD146 subpopulation of BMSCs in cartilage damage repair was assessed by injecting CD146 + BMSCs complexed with sodium alginate gel in the joints of a mouse cartilage defect model. RESULTS: The prepared CD146-LMS had an average particle size of 193.7 ± 5.24 nm, an average potential of 41.9 ± 6.21 mv, and a saturation magnetization intensity of 27.2 Am2/kg, which showed good stability and low cytotoxicity. The sorted CD146 + BMSCs highly expressed stem cell and pericyte markers with good cellular activity and cellular value-added capacity. Cartilage markers Sox9, Collagen II, and Aggrecan were expressed at both protein and mRNA levels in CD146 + BMSCs cells after chondrogenic induction in vitro. In a mouse cartilage injury model, CD146 + BMSCs showed better function in promoting the repair of articular cartilage injury. CONCLUSION: The prepared CD146-LMS was able to sort out CD146 + BMSCs efficiently, and the sorted subpopulation of CD146 + BMSCs had good chondrogenic differentiation potential, which could efficiently promote the repair of articular cartilage injury, suggesting that the sorted CD146 + BMSCs subpopulation is a promising seed cell for cartilage tissue engineering.

Mid-term results of autologous matrix-induced chondrogenesis for large chondral defects in hips with femoroacetabular impingement syndrome.

Aims: The purpose of this study was to evaluate the mid-term outcomes of autologous matrix-induced chondrogenesis (AMIC) for the treatment of larger cartilage lesions and deformity correction in hips suffering from symptomatic femoroacetabular impingement (FAI). Methods: This single-centre study focused on a cohort of 24 patients with cam- or pincer-type FAI, full-thickness femoral or acetabular chondral lesions, or osteochondral lesions ≥ 2 cm2, who underwent surgical hip dislocation for FAI correction in combination with AMIC between March 2009 and February 2016. Baseline data were retrospectively obtained from patient files. Mid-term outcomes were prospectively collected at a follow-up in 2020: cartilage repair tissue quality was evaluated by MRI using the Magnetic Resonance Observation of Cartilage Repair Tissue (MOCART) score. Patient-reported outcome measures (PROMs) included the Oxford Hip Score (OHS) and Core Outcome Measure Index (COMI). Clinical examination included range of motion, impingement tests, and pain. Results: A total of 12 hips from 11 patients were included (ten males, one female, mean age 26.8 years (SD 5.0), mean follow-up 6.2 years (SD 5.2 months)). The mean postoperative MOCART score was 66.3 (SD 16.3). None of the patients required conversion to total hip arthroplasty. Two patients had anterior impingement. External hip rotation was moderately limited in four patients. There was a correlation between MOCART and follow-up time (rs = -0.61; p = 0.035), but not with initial cartilage damage, age, BMI, or imaging time delay before surgery. PROMs improved significantly: OHS from 37.4 to 42.7 (p = 0.014) and COMI from 4.1 to 1.6 (p = 0.025). There was no correlation between MOCART and PROMs. Conclusion: Based on the reported mid-term results, we consider AMIC as an encouraging treatment option for large cartilage lesions of the hip. Nonetheless, the clinical evidence of AMIC in FAI patients remains to be determined, ideally in the context of randomized controlled trials.