Objective Calcific aortic valve disease (CAVD) is the leading cause of angina, heart failure, and death from aortic stenosis. However, the molecular mechanisms of its progression, especially the complex disease-related transcriptional regulatory mechanisms, remain to be further elucidated. Methods This study used porcine valvular interstitial cells (PVIC) as a model. We used osteogenic induced medium (OIM) to induce calcium deposition in PVICs to calcify them, followed by basic fibroblast growth factor (bFGF) treatment to inhibit calcium deposition. Transcriptome sequencing was used to study the mRNA expression profile of PVICs and its related transcriptional regulation. We used DaPars to further examine alternative polyadenylation (APA) between different treatment groups. Results We successfully induced calcium deposition of PVICs through OIM. Subsequently, mRNA-seq was used to identify differentially expressed mRNAs for three different treatments: control, OIM-induced and OIM-induced bFGF treatment. Global APA events were identified in the OIM and bFGF treatment groups by bioinformatics analysis. Finally, it was discovered and proven that catalase ( CAT ) is one of the potential targets of bFGF-induced APA regulation. Conclusion We described a global APA change in a calcium deposition model related to CAVD. We revealed that transcriptional regulation of the CAT gene may contribute to bFGF-induced calcium deposition inhibition.

Osteoarthritis (OA) affects approximately 12% of the aging Western population. The sirtuin/forkhead box O (SIRT/FOXO) signaling pathway plays essential roles in various biological processes. Despite it has been demonstrated that ubiquitin-specific protease 3 (USP3) inhibits chondrocyte apoptosis induced by interleukin (IL)-1β, the role of USP3/SIRT3/FOXO3 in the senescence of chondrocytes in OA is unclear. This study initially isolated articular chondrocytes and investigated the role of USP3 in IL-1β-induced senescence of chondrocytes. After USP3 was overexpressed or silenced by lentivirus, expressions of genes and proteins were detected using quantitative polymerase chain reaction and immunoblotting, respectively. Cell cycle analysis was performed using flow cytometry. Reactive oxygen species (ROS) levels and senescence were analyzed. Then, SIRT3 was inhibited or overexpressed to explore the underlying mechanism. We found that overexpression of USP3 hindered IL-1β-mediated cell cycle arrest, ROS generation, and chondrocyte senescence. The inhibition of SIRT3 blocked the protective effect of USP3 on cell senescence, whereas the overexpression of SIRT3 abolished USP3-silencing-induced cell senescence. Furthermore, SIRT3 attenuated cell senescence, probably by deacetylating FOXO3. USP3 upregulated SIRT3 to deacetylate FOXO3 and attenuated IL-1β-induced chondrocyte senescence. This study demonstrated that USP3 probably attenuated IL-1β-mediated chondrocyte senescence by deacetylating FOXO3 via SIRT3.

Articular cartilage (AC) lesions are common but remain a great challenge for researchers and clinicians mainly due to their poor self-healing capability. Decellularized cartilage extracellular matrix (dCECM) derived from natural cartilage, and the incorporated growth factors have been used for potential chondrocyte induction and cartilage tissue regeneration. Moreover, infrapatellar fat pad adipose-derived stem cells (IPFP-ADSCs) have been proven to be an optimal source of seeded cells for AC lesion restoration. Therefore, the therapeutic role of dCECM-incorporated bioink with IPFP-ADSCs loaded in 3D (three-dimensional) scaffolds remain to be explored using an osteochondral defect model. In this work, we successfully extracted IPFP-ADSCs and isolated dCECMs, which were then mixed in a temperature-responsive hydrogel to form a 3D bioink. The 3D bioprinting scaffolds printed with dCECM successfully facilitated chondrogenic differentiation-related growth factor expression in IPFP-ADSCs. Most importantly, implantation of the 3D dCECMs with an IPFP-ADSC-loaded scaffold was capable of providing a favorable cell matrix for stem cell proliferation and promoted chondrogenic differentiation, thereby greatly enhancing successful cartilage repair in rabbit cartilage defects.

Mesenchymal stem cells (MSCs) have emerged as a promising therapeutic method in regenerative medicine. Our previous research adopted a simple nonenzymatic strategy for the preparation of a new type of ready-to-use infrapatellar fat pad (IPFP) cell concentrates. The aim of this study was to compare the therapeutic efficacy of intra-articular (IA) injection of autologous IPFP cell concentrates and allogeneic IPFP-MSCs obtained from these concentrates in a rabbit articular cartilage defect model. IPFP-MSCs sprouting from the IPFP cell concentrates were characterized via flow cytometry as well as based on their potential for differentiation into adipocytes, osteoblasts, and chondrocytes. In the rabbit model, cartilage defects were created on the trochlear groove, followed by treatment with IPFP cell concentrates, IPFP-MSCs, or normal saline IA injection. Distal femur samples were evaluated at 6 and 12 weeks posttreatment via macroscopic observation and histological assessment based on the International Cartilage Repair Society (ICRS) macroscopic scoring system as well as the ICRS visual histological assessment scale. The macroscopic score and histological score were significantly higher in the IPFP-MSC group compared to the IPFP cell concentrate group at 12 weeks. Further, both treatment groups had higher scores compared to the normal saline group. In comparison to the latter, the groups treated with IPFP-MSCs and IPFP cell concentrates showed considerably better cartilage regeneration. Overall, IPFP-MSCs represent an effective therapeutic strategy for stimulating articular cartilage regeneration. Further, due to the simple, cost-effective, nonenzymatic, and safe preparation process, IPFP cell concentrates may represent an effective alternative to stem cell-based therapy in the clinic.

Objective To evaluate the effect of Kartogenin-pretreated exosomes derived from infrapatellar fat pad mesenchymal stem cells on chondrocyte in vitro and articular cartilage regeneration in vivo. Methods Infrapatellar fat pad mesenchymal stem cells (IPFP-MSCs) were isolated from rabbits to harvest exosomes. After identification of mesenchymal stem cells and exosomes, rabbit chondrocytes were divided into three groups for further treatment: the EXO group (chondrocytes treated with exosomes isolated from infrapatellar fat pad mesenchymal stem cells), KGN-EXO group (chondrocytes treated with exosomes isolated from infrapatellar fat pad mesenchymal stem cells pretreated with KGN), and control group. After processing and proliferation, phenotypic changes of chondrocytes were measured. In the in vivo study, 4 groups of rabbits with articular cartilage injury were treated with KGN-EXO, EXO, IPFP-MSCs, and control. Macroscopic evaluation and histological evaluation were made to figure out the different effects of the 4 groups on cartilage regeneration in vivo. Results The proliferation rate of chondrocytes in the EXO or KGN-EXO group was significantly higher than that in the control group (P < 0.05). The qRT-PCR results showed that the expression of Sox-9, Aggrecan, and Col II was the highest in the KGN-EXO group compared with the EXO group and the control group (P < 0.05). The results of Western blot were consistent with the results of qRT-PCR. In vivo, the cartilage defects in the KGN-EXO group showed better gross appearance and improved histological score than those in IPFP-MSC groups, EXO groups, and control groups (P < 0.05). At 12 weeks, the defect site in the KGN-EXO group was almost completely repaired with a flat and smooth surface, while a large amount of hyaline cartilage-like structures and no obvious cracks were observed. Conclusion Our study demonstrates that the exosomes isolated from infrapatellar fat pad mesenchymal stem cells pretreated with KGN have potent ability to induce chondrogenic differentiation of stem cells, effectively promoting the proliferation and the expression of chondrogenic proteins and genes of chondrocytes. The KGN-EXO can also promote the repair of articular cartilage defects more effectively, which can be used as a potential therapeutic method in the future.

BACKGROUND:Tissue engineering is widely applied to treat osteochondral damage in osteoarthritis (OA). However, the superposition of seed cells, material scaffolds, inducing factors, and microenvironmental factors limit their practical application. We intended to develop a novel tissue engineering method for improving the repairment of osteochondral damage and to discuss its effect on repairing osteochondral defects.METHODS:The combined decellularization methods of physics, chemistry and enzymes were used to decellularize rabbit rib cartilage and articular cartilage, and rabbit decellularizated osteochondral composite scaffolds were prepared. The structure and organization of the scaffolds were analyzed. We extracted and identified infrapatellar fat pad stem cells (IPFP-SCs) from healthy rabbits and OA rabbit, which were different in viability, migration, osteogenic and chondrogenic differentiation. Finally, a variety of decellularizated bone cartilage composite scaffolds were loaded with rabbit IPFP-SC for in vitro and in vivo studies.RESULTS:The decellularization effect was strong, and the organic ingredients were lost. The layered scaffold showed lower density, greater porosity, larger pore size and water absorption than the whole scaffold, but the mechanical properties of the two scaffolds were low. IPFP-SCs were successfully extracted, and the migration and cartilage ability of IPFP-SCs in OA group were weak. The decellularized scaffold showed a high biocompatibility. The structure and composition of osteochondral promoted osteogenic differentiation and chondrogenic differentiation of IPFP-SCs. Moreover, the decellularized extracellular matrix loaded with IPFP-SC had the strongest repairing effect.CONCLUSION:The decellularized extracellular matrix loaded with IPFP-SC showed a better repair effect on rabbit osteochondral defects.

Chondral and osteochondral defects caused by trauma or pathological changes, commonly progress into total joint degradation, even resulting in disability.The cartilage restoration is a great challenge because of its avascularity and limited proliferative ability.Additionally, precise diagnosis using non-invasive detection techniques is challenging, which increases problems associated with chondral disease treatment.Methods: To achieve a theranostic goal, we used an integrated strategy that relies on exploiting a multifunctional nanoprobe based on chitosan-modified Fe3O4 nanoparticles, which spontaneously self-assemble with the oppositely charged small molecule growth factor, kartogenin (KGN).This nanoprobe was used to obtain distinctively brighter T2-weighted magnetic resonance (MR) imaging, allowing its use as a positive contrast agent, and could be applied to obtain accurate diagnosis and osteochondral regeneration therapy.Results: This nanoprobe was first investigated using adipose tissue-derived stem cells (ADSCs), and was found to be a novel positive contrast agent that also plays a significant role in stimulating ADSCs differentiation into chondrocytes.This self-assembled probe was not only biocompatible both in vitro and in vivo, contributing to cellular internalization, but was also used to successfully make distinction of normal/damaged tissue in T2-weighted MR imaging.This novel combination was systematically shown to be biosafe via the decrement of apparent MR signals and elimination of ferroferric oxide over a 12-week regeneration period.Conclusion: Here, we established a novel method for osteochondral disease diagnosis and reconstruction.Using the Fe3O4-CS/KGN nanoprobe, it is easy to distinguish the defect position, and it could act as a tool for dynamic observation as well as a stem cell-based therapy for directionally chondral differentiation.

The ring-opening reactions of the furanose forms of the penturonic acids D-arabinuronic acid (1), D-lyxuronic acid (2), D-riburonic acid (3), and D-xyluronic acid (4) in aqueous solution have been studied as a function of temperature and solution pH by 13C saturation-transfer n.m.r. (s.t.-n.m.r.) spectroscopy using 1-13C-substituted compounds. Unidirectional rate constants of ring-opening (kopen) have been determined for the cyclic forms of 1-4 in their protonated (pH 1.5) and ionized (pH 4.5) forms, and have been compared to the k-values measured previously for structurally related furanose sugars. At 50 degrees and pH 1.5, kopen values decrease as follows: alpha-xyluronic acid (2.57 s-1) greater than alpha-riburonic (1.65 s-1) greater than beta-arabinuronic (1.52 s-1) greater than beta-xyluronic (1.09 s-1) greater than beta-riburonic (0.76 s-1) greater than beta-lyxuronic (0.55 s-1) greater than alpha-arabinuronic (0.46 s-1) greater than alpha-lyxuronic (0.40 s-1). At 50 degrees and pH 4.5, this order changes significantly (e.g., beta-arabinuronate is most reactive); in general kopen values for beta anomers appear to be enhanced relative to those for corresponding alpha anomers, suggesting the involvement of intramolecular catalysis in which the carboxylate anion assists in abstracting the hydroxyl proton from O-1. Activation energies of ring-opening, determined for the alpha and beta anomers of 1-4, were found to depend on ring configuration and solution pH.