Chondrogenic Differentiation Of Stem Cells Research Articles

Collagen-based hydrogels induce stem cell chondrogenesis and hyaline cartilage regeneration: an in vivo study

Injectable, self-crosslinking collagen-based hydrogels are beneficial for chondrocytes to secrete matrix, positioning them as promising candidates for cartilage tissue engineering. However, previous studies lacked insight into the ability of cell-free collagen-based hydrogels to regenerate hyaline cartilage defect. Therefore, this study aimed to evaluate the potential of collagen-based hydrogels (Col and ColHA) to induce chondrogenic differentiation of stem cells and in situ hyaline cartilage regeneration. Both Col and ColHA hydrogels self-crosslinked in situ and exhibited similar physical properties. In vitro experiments showed they supported the survival, adhesion, spreading, and proliferation of bone marrow stem cells (BMSCs). Moreover, both hydrogels induced ectopic differentiation of BMSCs into chondrocytes when implanted subcutaneously into the back of nude mice. ColHA hydrogel notably enhanced type II collagen secretion. The results of repairing cartilage defects in situ revealed both hydrogels facilitated hyaline cartilage regeneration and maintained cartilage phenotype without exogenous BMSCs. Hydrogels encapsulating BMSCs expedited cartilage repair, and ColHA/BMSC constructs showed better mechanical properties, suggesting their potential for cartilage repair applications. This study implies that collagen-based hydrogels are good candidates for hyaline cartilage regeneration.

Hydroxypropyl chitin-oxidized chondroitin sulfate double-network hydrogel assists microfracture technique to enhance cartilage regeneration

Cartilage has limited self-repair ability, leading to osteoarthritis postinjury and ultimately physical disability in people. In addition, cartilage injury is accompanied by loss of extracellular matrix (ECM) and infiltration of inflammation, which makes cartilage regeneration more difficult. In this study, a hybrid hydrogel of hydroxypropyl chitin and oxidized chondroitin sulfate (HPCH-OCS) with injectable and thermosensitive properties was prepared by a Schiff base reaction. In vitro, the HPCH-OCS hydrogel showed desirable biocompatibility and bioactivity by promoting chondrogenic differentiation of stem cells, maintaining the chondrocyte phenotype, and inhibiting proteins related to cartilage ECM catabolism under inflammatory conditions. In vivo, the HPCH-OCS hydrogel combined with microfracture could effectively repair cartilage defects and promote cartilage regeneration. In conclusion, the HPCH-OCS hydrogel has good biological and mechanical properties, and its combination with microfracture provides an effective strategy for clinical cartilage regeneration.

Engineering sulfated polysaccharides and silk fibroin based injectable IPN hydrogels with stiffening and growth factor presentation abilities for cartilage tissue engineering.

The extracellular matrix (ECM) presents a framework for various biological cues and regulates homeostasis during both developing and mature stages of tissues. During development of cartilage, the ECM plays a critical role in endowing both biophysical and biochemical cues to the progenitor cells. Hence, designing microenvironments that recapitulate these biological cues as provided by the ECM during development may facilitate the engineering of cartilage tissue. In the present study, we fabricated an injectable interpenetrating hydrogel (IPN) system which serves as an artificial ECM and provides chondro-inductive niches for the differentiation of stem cells to chondrocytes. The hydrogel was designed to replicate the gradual stiffening (as a biophysical cue) and the presentation of growth factors (as a biochemical cue) as provided by the natural ECM of the tissue, thus exemplifying a biomimetic approach. This dynamic stiffening was achieved by incorporating silk fibroin, while the growth factor presentation was accomplished using sulfated-carboxymethyl cellulose. Silk fibroin and sulfated-carboxymethyl cellulose (s-CMC) were combined with tyraminated-carboxymethyl cellulose (t-CMC) and crosslinked using HRP/H2O2 to fabricate s-CMC/t-CMC/silk IPN hydrogels. Initially, the fabricated hydrogel imparted a soft microenvironment to promote chondrogenic differentiation, and with time it gradually stiffened to offer mechanical support to the joint. Additionally, the presence of s-CMC conferred the hydrogel with the property of sequestering cationic growth factors such as TGF-β and allowing their prolonged presentation to the cells. More importantly, TGF-β loaded in the developed hydrogel system remained active and induced chondrogenic differentiation of stem cells, resulting in the deposition of cartilage ECM components which was comparable to the hydrogels that were treated with TGF-β provided through media. Overall, the developed hydrogel system acts as a reservoir of the necessary biological cues for cartilage regeneration and simultaneously provides mechanical support for load-bearing tissues such as cartilage.

Regeneration of Rabbit Auricular Cartilage After the Intravenous Stem Cell Injection.

The restoration of auricular cartilage is a major problem of otolaryngology. The low regenerative capacity of cartilage requires alternative approaches such as cell and tissue engineering. Stem cells are one of the ways to repair auricular cartilage damages. The aim of the investigation was the regeneration of an artificial defect of the auricular cartilage of rabbits after the intravenous injection of stem cells. The study was carried out on rabbits. A narrow strip of auricular cartilage was surgically removed. A previously prepared suspension of homologous mesenchymal stem cells (5 million) in 0.5 ml physiological solution was injected into the vein of the opposite ear. Tissue samples from the site of the injury were collected after 1, 2, and 3 months. Histological examinations of the tissues were carried out after staining with fuchsin-eosin, azure II-eosin, and according to Weigert. In addition, the amount of interleukin-6 (IL-6) and the transforming growth factor β1 (TGF-β1) in the blood serum were determined. The main method of healing is the formation of a connective tissue scar. Yret, an increase of the number of fibroblasts and single islands of the newly formed auricular cartilage was found, which indicates the migration of the injected stem cells to the site of the damage and settling there. The intravenous injection of stem cells did not affect the secretion of pro-inflammatory IL-6, but significantly increased the amount of TGF-β1. We assume that regenerative processes were stimulated. Nevertheless, they were aimed at quickly restoring the tissue integrity through the typical stages of scar formation. The restoration of cartilage integrity requires additional regulatory factors which will determine the chondrogenic differentiation of stem cells.

The Properties of Exosomes Derived from Mesenchymal Stem Cells Preconditioned with L-Ascorbic Acid and Cobalt (II) Chloride

Extracellular vesicles including exosomes, are produced by cells for intracellular communication. Preconditioning of parental cells influences exosome properties. The purpose of this study was to examine the effects of L-ascorbic acid (LAA) and cobalt (II) chloride (CoCl2) on human Wharton’s jelly mesenchymal stem cell (hWJ-MSC)-derived exosomes and their ability to promote stem cell differentiation into chondrocytes. The cells were isolated from the umbilical cord and characterized according to the criteria for mesenchymal stem cell. The cells were cultured in a serum-free medium containing LAA and CoCl2. Cell-produced exosomes were isolated and characterized. hWJ-MSCs can grow in serum-free medium containing LAA and CoCl2. Exosomes derived from hWJ-MSCs had a round morphology, particle size within the exosome range, CD 63 expression, and the capacity to be internalized by cells. The production of exosomes by hWJ-MSCs was enhanced by LAA treatment. LAA and CoCl2 promoted stem cell differentiation into chondrocytes, as indicated by the production of collagen type II and glycosaminoglycans. LAA and CoCl2 affect the properties of MSC-derived exosomes. LAA induces cells to produce exosomes in greater quantities, which have the potential to promote chondrogenic differentiation of stem cells.

Viscoelasticity microenvironment constructed by self-crosslinking hyaluronan hybrid hydrogels regulates chondrogenic differentiation of mesenchymal stem cells

Chondrogenic differentiation of stem cells can be regulated by mechanical properties of biomaterials. Here, hybrid hydrogels, composed of sulfhydrylated hyaluronan (HA-SH) with different molecular weight and collagen I (Col Ⅰ), were designed to investigate chondrogenic differentiation of rabbit bone marrow mesenchymal stem cells (rBMSCs). The enhanced molecular weight of HA-SH decreased the mechanical properties of hybrid hydrogels, but improved viscosity and degradation resistance, which could modulate cell spreading and morphological changes, thereby influencing chondrogenic differentiation. Furthermore, 1 MDa HA-SH combined with Col Ⅰ (1M-HAC) with adequate viscoelasticity was beneficial to the circularity and nuclear chromatin condensation of rBMSCs, which induced significant chondrocyte aggregation, increased glycosaminoglycans and collagen Ⅱ stainings, and reduced collagen Ⅹ staining. This was conducive to preserve a hyaline cartilage's phenotype and reducing inflammation and degeneration. Subcutaneous implantation studies revealed that 1M-HAC also facilitated specific matrix secretion, chondrogenic gene expression and potentially anti-inflammatory and anti-degenerative effects.

Facilitation of Osteogenic Differentiation of hASCs on PEDOT:PSS/MXene Composite Sponge with Electrical Stimulation

Innovations in biomedical tissue engineering are on the increase as many researchers look for ways to develop biological scaffolds for cells. Studies have shown that the application of external force and electrical stimulation (ES) promotes stem cell chondrogenic and osteogenic differentiation. Therefore, bioscaffolds sensitive to external stimuli can not only influence the cell behavior with their native physical and biochemical properties but also act as a mediator receiving an outside-in signal to alter the cellular activities under specific conditions. For the first time, the synthetic polymer poly(3,4-ethylene dioxythiophene):polystyrene sulfonate) (PEDOT:PSS) was combined with a two-dimensional, nanoconducting material, titanium carbide (MXene, Ti3C2X3), to prepare three-dimensional (3D) conductive scaffolds. Some studies have shown that MXene has good biocompatibility, osteoinductivity, and bone regeneration activity. The PEDOT:PSS/MXene scaffold was applied to the osteogenic differentiation of hASCs, and ES was used to enhance the osteogenic differentiation. The results showed that the conductive scaffold had low cytotoxicity to hASCs, which could grow and migrate in the 3D scaffold. In addition, osteogenic-specific gene expression significantly differed when the ES was applied. We propose that this PEDOT:PSS/MXene scaffold may serve as a platform for the study of osteogenic differentiation of stem cells with ES and may potentially be ex vivo fabricated as a tissue engineering construct for studying other modes of other ESs such as direct current, capacitive, or inductive coupling in a 3D environment.

Electrosprayed Particles Loaded with Kartogenin as a Potential Osteochondral Repair Implant

The restoration of cartilage damage is a slow and not always successful process. Kartogenin (KGN) has significant potential in this space-it is able to induce the chondrogenic differentiation of stem cells and protect articular chondrocytes. In this work, a series of poly(lactic-co-glycolic acid) (PLGA)-based particles loaded with KGN were successfully electrosprayed. In this family of materials, PLGA was blended with a hydrophilic polymer (either polyethyleneglycol (PEG) or polyvinylpyrrolidone (PVP)) to control the release rate. Spherical particles with sizes in the range of 2.4-4.1 µm were fabricated. They were found to comprise amorphous solid dispersions, with high entrapment efficiencies of >93%. The various blends of polymers had a range of release profiles. The PLGA-KGN particles displayed the slowest release rate, and blending with PVP or PEG led to faster release profiles, with most systems giving a high burst release in the first 24 h. The range of release profiles observed offers the potential to provide a precisely tailored profile via preparing physical mixtures of the materials. The formulations are highly cytocompatible with primary human osteoblasts.

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.

Induced Chondrogenic Differentiation of hESCs by hESC-Derived MSCs Conditioned Medium and Sequential 3D-2D Culture System

Background and Aims: It has been proven that human mesenchymal stem cells (MSCs) conditioned medium (hMSCs-CM) can influence human embryonic stem cells (hESCs) chondrogenic differentiation. In this study, we hypothesized that conditioned medium (CM) from hESCs-derived MSCs in a sequential 3D-2D culture system could facilitate the induction of chondrogenesis in hESCs.
 Materials and Methods: CM was collected from Yazd2 (hESCs; 46, XY) derived MSCs confluent cultures and stored at -20 °C. Yazd4 hESC line (46, XX) was induced for differentiation using EB formation as 3D culture into SD (spontaneously differentiation) and CM groups (differentiation using conditioned medium) for four days. Cell culture continued in a 2D (monolayer) culture system for both groups till day 14. Ultimately, chondrogenic differentiation was assessed by Alcian blue and masson′s trichrome staining at 4 and 14 days of differentiation, and quantitative real-time polymerase chain reaction (PCR) for NANOG, MEOX1, SOX5, SOX6, SOX9, ACAN, COL2A1 and RUNX2 genes for SD and CM groups on days 0 and 4.
 Results: The gene expression profile for chondrogenic genes in the CM group was significantly more than the SD group (p< 0.05). Furthermore, chemical staining assessment illustrated a significant GAG and collagen II difference between the CM and SD groups at days 4 and 14 (p< 0.05).
 Conclusions: Our findings would pave the way for creating an in vitro human chondrogenesis model for further studies in the developmental biology of articular cartilage tissue, which lend itself to cell-based therapy to cure joint diseases such as osteoarthritis.

Simvastatin promotes rat Achilles tendon-bone interface healing by promoting osteogenesis and chondrogenic differentiation of stem cells

To investigate the effect and mechanism of simvastatin on cell components of tendon-bone healing interface. The tendon-bone healing model was established by inserting the end of the Achilles tendon into the tibial tunnel on 24 rats, and simvastatin was used locally at the tendon-bone interface. Healing was evaluated at 8weeks by mechanical testing, micro-CT, and qualitative histology including H&E, Toluidine blue, and immunohistochemical staining. In vitro, bone marrow stromal cells (BMSCs) and tendon-derived mesenchymal stem cells (TDSCs) underwent osteogenic and chondrogenic differentiation respectively by plate co-culture. An analysis was performed on days 7 and 14 of cell differentiation. Biomechanical testing demonstrated a significant increase in maximum stiffness in the simvastatin-treated group. Micro-CT analysis showed that the bone tunnels in the simvastatin group were smaller in diameter and had higher bone density. H&E and Toluidine blue staining demonstrated that tendon-bone healing was significantly greater with better tissue arrangement and more extracellular matrix in the simvastatin-treated group than that in the control group, and immunohistochemical staining showed the expression of VEGF in simvastatin group was significantly higher. Histological staining and RT-PCR confirmed that simvastatin could promote the differentiation of co-cultured BMSCs and TDSCs into osteoblasts and chondroblasts, respectively. The effect of promoting osteogenic differentiation was more tremendous at 14days, while its effect on promoting chondroblast differentiation was more evident on the 7th day of differentiation. In conclusion, local administration of simvastatin can promote the tendon-bone healing by enhancing neovascularization, chondrogenesis, and osteogenesis in different stages of the tendon-bone healing process.

Identifying the key genes regulating mesenchymal stem cells chondrogenic differentiation: an in vitro study

BackgroundMesenchymal stem cells (MSCs) possess the potential to differentiate into chondrocytes, which makes them an ideal source for healing cartilage defects. Here, we seek to identify the essential genes participating in MSCs chondrogenesis.MethodsHuman MSCs were induced for chondrogenesis for 7, 14, and 21 days using a high-density micromass culture system, and RNA was extracted for RNA-seq.ResultsA total of 6247 differentially expressed genes (DEGs) were identified on day 7, and 85 DEGs were identified on day 14. However, no significant DEGs was identified on day 21. The top 30 DEGs at day 7, including COL9A3, COL10A1, and CILP2, are closely related to extracellular matrix organization. While the top 30 DEGs at day 14 revealed that inflammation-related genes were enriched, including CXCL8, TLR2, and CCL20. We also conducted protein–protein interaction (PPI) networks analysis using the search tool for the retrieval of interacting genes (STRING) database and identified key hub genes, including CXCL8, TLR2, CCL20, and MMP3. The transcriptional factors were also analyzed, identifying the top 5 TFs: LEF1, FOXO1, RORA, BHLHE41, and SOX5. We demonstrated one particular TF, RORA, in promoting early MSCs chondrogenesis.ConclusionsTaken together, our results suggested that these DEGs may have a complex effect on MSCs chondrogenesis both synergistically and solitarily.

TGF-β1 and -β3 for Mesenchymal Stem Cells Chondrogenic Differentiation on Poly (Vinyl Alcohol)-Chitosan-Poly (Ethylene Glycol) Scaffold.

Transforming growth factor-beta 1 (TGF-β1) has been reported to promote chondrogenic differentiation and proliferation in the multipotent stromal cell (MSCs), and the transforming growth factor-beta 3 (TGF-β3) tends to be exclusively in promoting cell differentiation alone. The objective of this study was to determine the effect of TGF-β1 and -β3 on the MSCs chondrogenic differentiation on the poly (vinyl alcohol)-chitosan-poly (ethylene glycol) (PVA-NOCC-PEG) scaffold, compared with that of monolayer and pellet cultures. In this study, P2 rabbit bone marrow-derived MSCs were seeded either on the untreated six-well plate (for monolayer culture) or onto the PVA-NOCC-PEG scaffold or cultured as a pellet culture. The cultures were maintained in a chemically defined serum-free medium supplemented with 10 ng/mL of either TGF-β1 or TGF-β3. Cell viability assay, biochemical assay, and real-time polymerase chain reaction were performed to determine the net effect of cell proliferation and chondrogenic differentiation of each of the growth factors. The results showed that the PVA-NOCC-PEG scaffold enhanced MSCs cell proliferation from day 12 to 30 (p < 0.05); however, no significant differences were observed in the cell proliferation between the cultures supplemented with or without TGF-β1 and TGF-β3 (p > 0.05). In terms of chondrogenic differentiation, the PVA-NOCC-PEG scaffold augmented the GAGs secretion in MSCs and the mRNA expression levels of Sox9, Col2a1, Acan, and Comp were elevated (p < 0.05). However, there was no significant difference between both the TGF-β1 and TGF-β3-treated groups (p > 0.05). In conclusion, TGF-β1 and TGF-β3 enhanced the chondrogenic differentiation of MSCs seeded on the PVA-NOCC-PEG scaffold; however, there was no significant difference between the effect of TGF-β1 and TGF-β3. Impact statement Transforming growth factor-beta (TGF-β) superfamily members is a key requirement for the in vitro chondrogenic differentiation of mesenchymal stem cells (MSCs). In this study, the effects of TGF-β1 and -β3 on MSC chondrogenic differentiation and proliferation on a novel three-dimensional scaffold, the poly(vinyl alcohol)-chitosan-poly(ethylene glycol) (PVA-NOCC-PEG) scaffold, was evaluated. In this study, the results showed both TGF-β1 and TGF-β3 can enhance the chondrogenic differentiation of MSCs seeded on the PVA-NOCC-PEG scaffold.

Stepwise Proliferation and Chondrogenic Differentiation of Mesenchymal Stem Cells in Collagen Sponges under Different Microenvironments.

Biomimetic microenvironments are important for controlling stem cell functions. In this study, different microenvironmental conditions were investigated for the stepwise control of proliferation and chondrogenic differentiation of human bone-marrow-derived mesenchymal stem cells (hMSCs). The hMSCs were first cultured in collagen porous sponges and then embedded with or without collagen hydrogels for continual culture under different culture conditions. The different influences of collagen sponges, collagen hydrogels, and induction factors were investigated. The collagen sponges were beneficial for cell proliferation. The collagen sponges also promoted chondrogenic differentiation during culture in chondrogenic medium, which was superior to the effect of collagen sponges embedded with hydrogels without loading of induction factors. However, collagen sponges embedded with collagen hydrogels and loaded with induction factors had the same level of promotive effect on chondrogenic differentiation as collagen sponges during in vitro culture in chondrogenic medium and showed the highest promotive effect during in vivo subcutaneous implantation. The combination of collagen sponges with collagen hydrogels and induction factors could provide a platform for cell proliferation at an early stage and subsequent chondrogenic differentiation at a late stage. The results provide useful information for the chondrogenic differentiation of stem cells and cartilage tissue engineering.

The effect of decellularized cartilage matrix scaffolds combined with endometrial stem cell-derived osteocytes on osteochondral tissue engineering in rats.

Since decellularized tissues may offer the instructive niche for cell differentiation and function, their use as cell culture scaffolds is a promising approach for regenerative medicine. To repair osteochondral tissues, developing a scaffold with biomimetic structural, compositional, and functional characteristics is vital. As a result of their heterogeneous structure, decellularized articular cartilage matrix from allogeneic and xenogeneic sources are considered appropriate scaffolds for cartilage regeneration. We developed a scaffold for osteochondral tissue engineering by decellularizing sheep knee cartilage using a chemical technique. DNA content measurements and histological examinations revealed that this protocol completely removed cells from decellularized cartilage. Furthermore, SEM, MTS assay, and H&E staining revealed that human endometrial stem cells could readily adhere to the decellularized cartilage, and the scaffold was biocompatible for their proliferation. Besides, we discovered that decellularized scaffolds could promote EnSC osteogenic differentiation by increasing bone-specific gene expression. Further, it was found that decellularized scaffolds were inductive for chondrogenic differentiation of stem cells, evidenced by an up-regulation in the expression of the cartilage-specific gene. Also, in vivo study showed the high affinity of acellularized scaffolds for cell adhesion and proliferation led to an improved regeneration of articular lesions in rats after 4weeks. Finally, a perfect scaffold with high fidelity is provided by the developed decellularized cartilage scaffold for the functional reconstruction of osteochondral tissues; these types of scaffolds are helpful in studying how the tissue microenvironment supports osteocytes and chondrocytes differentiation, growth, and function to have a good osteochondral repair effect.

Hypertrophic Effect of Chondrogenic Differentiation Medium Supplemented with BMP-9 and TGFß-3 in Transwell Culture

Mesenchymal stem cells are widely used in the treatment of many diseases, including osteoarthritis, due to their ability to differentiate into cartilage. The high chondrogenic differentiation potential of synovial fluid-derived mesenchymal stem cells increases the importance of these cells in osteoarthritis treatments. Addition of BMP-9 and TGF-ß3 into chondrogenic differentiation medium, increases chondrogenic differentiation and they also cause hypertrophic effects on chondrocytes. In our study, it was aimed to demonstrate the effects of BMP-9 and TGF-ß3 on cell hypertrophy by adding them into the chondrogenic basal medium during in vitro chondrogenic differentiation. In the study, stem cells in passage 5 and chondrocytes in passage 1 were cultured in a transwell co-culture system and six experimental groups were formed. Cell hypertrophy was demonstrated by examining MMP-13 and RUNX-2 gene expressions, in stem cells where chondrogenesis were induced in transwell co-culture. Although the addition of BMP-9 and TGF-ß3 to the chondrogenic medium increased hypertrophic gene expressions in experimental groups compared to control, the results were not statistically significant. The addition of BMP-9 and TGF-ß3, separately or in combination, during the chondrogenic differentiation of stem cells does not cause significant chondrocyte hypertrophy.

Episodic alcohol exposure attenuates mesenchymal stem cell chondrogenic differentiation during bone fracture callus formation.

During bone fracture repair, mesenchymal stem cells (MSC) differentiate into chondrocytes and osteoblasts to form a fracture callus. Our laboratory previously reported that alcohol-exposed rodents with a surgically created tibia fracture display deficient fracture callus formation and diminished signs of endochondral ossification characterized by the absence of chondrocytes and mature hypertrophic chondrocytes, suggesting that alcohol may inhibit MSC differentiation. These findings led to our hypothesis that alcohol exposure inhibits mesenchymal stem cell chondrogenic differentiation within the developing fracture callus. In the present study, we utilized a lineage-tracing approach to determine which stage(s) of chondrogenic differentiation are affected by alcohol exposure. We utilized lineage-specific reporter mice to determine the effects of alcohol on MSC and early and late chondrogenic cell frequencies within the fracture callus. In addition, serially sectioned slides were stained immunofluorescently and immunohistochemically and quantified to determine the effect of alcohol on cell proliferation and apoptosis, respectively, within the fracture callus of alcohol-administered rodents. Alcohol-administered rodents had a reduced fracture callus area at 4, 6, and 9days postfracture. Alcohol had no effect on apoptosis in the fracture callus at any of the examined timepoints. Alcohol-administered rodents had significantly fewer proliferative cells in the fracture callus at 9days postfracture, but no effect on cell proliferation was observed at earlier fracture callus timepoints. Alcohol-administered rodents had reduced Collagen2a1- and Collagen10a1-expressing cells in the developing fracture callus, suggesting that alcohol inhibits both early chondrogenic differentiation and later chondrocyte maturation during fracture callus development. The data suggest that alcohol could affect normal fracture healing through the mitigation of MSC chondrogenic differentiation at the callus site.

MiR-20a-5p facilitates cartilage repair in osteoarthritis via suppressing mitogen-activated protein kinase kinase kinase 2

ABSTRACT Bone marrow mesenchymal stem cell (BMSC) chondrogenic differentiation contributes to the treatment of osteoarthritis (OA). Numerous studies have indicated that microRNAs (miRNAs) regulate the pathogenesis and development of multiple disorders, including OA. Nevertheless, the role of miR-20a-5p in OA remains obscure. Forty male C57BL/6 mice were divided into four groups and were surgically induced OA or underwent sham surgery in the presence or absence of miR-20a-5p. Flow cytometry was implemented to detect surface markers of BMSCs. Reverse transcription quantitative polymerase chain reaction revealed the upregulation of miR-20a-5p during BMSC chondrogenic differentiation. Western blotting displayed that miR-20a-5p inhibition decreased protein levels of cartilage matrix markers but enhanced those of catabolic and hypertrophic chondrocyte markers in BMSCs. Alcian blue staining, hematoxylin‑eosin staining and micro-CT revealed that miR-20a-5p inhibition restrained chondrogenic differentiation and miR-20a-5p overexpression promoted the repair of damaged cartilage and subchondral bone, respectively. Luciferase reporter assay identified that mitogen activated protein kinase kinase kinase 2 (Map3k2) was a target of miR-20a-5p in BMSCs. Moreover, the expression of miR-20a-5p and Map3k2 was negatively correlated in murine cartilage tissues. Knocking down Map3k2 could rescue the suppressive influence of miR-20a-5p inhibition on chondrogenic differentiation of BMSCs. In conclusion, miR-20a-5p facilitates BMSC chondrogenic differentiation and contributes to cartilage repair in OA by suppressing Map3k2.

Atractylodes lancea volatile oils target ADAR2-miR-181a-5p signaling to mesenchymal stem cell chondrogenic differentiation.

Atractylodeslancea Rhizoma (Rhizoma atractylodis [RA]) has long been recommended for the treatment of arthritis in traditional Chinese medicine, but its mechanism of action is still unclear. RA contains a large amount of Atractylodes lancea volatile oils (Atr). In this study, we investigated whether Atr can promote mesenchymal stem cells (MSCs) chondrogenic differentiation. The Atr were extracted from RA by steam distillation method, and the effect of Atr on MSCs was detected by the CCK8 assay. The optimal concentration of Atr for MSCs cultivation was 3μg/ml. The differentially expressed miR-181a-5p was screened by miRNA microarray assay, and its mimics and inhibitors were transfected into MSCs. It was found that the inhibitor of miR-181a-5p could upregulate cartilage-specific genes such as SOX9, COL2A1, and ACAN. Meanwhile, we also found that the expression of gene editing enzyme ADAR2 was significantly increased in the chondrogenic differentiation of MSCs induced by Atr, and the bases of precursor sequence of miR-181a-5p were changed from A to G. After ADAR2 deletion, the expression of cartilage-specific genes was significantly down-regulated and the precursor sequence bases of miR-181a-5p were not changed. Bioinformatics analysis revealed that the predicted target gene of miR-181a-5p was yingyang1 (YY1), and the targeting relationship was verified by dual-luciferase reporter assay. After deleting YY1, the expression of cartilage-specific genes was significantly down-regulated. In conclusion, our study demonstrated that Atr can promote chondrogenic differentiation of MSC through regulation of the ADAR2-miR-181a-5p signaling pathway. This may provide a new insight into the possible mechanism of traditional Chinese medicine (Atr) in treating inflammatory joint diseases.

Three-dimensional culture and chondrogenic differentiation of mesenchymal stem cells in interconnected collagen scaffolds

Interconnected scaffolds are useful for promoting the chondrogenic differentiation of stem cells. Collagen scaffolds with interconnected pore structures were fabricated with poly(lactic acid-co-glycolic acid) (PLGA) sponge templates. The PLGA-templated collagen scaffolds were used to culture human bone marrow-derived mesenchymal stem cells (hMSCs) to investigate their promotive effect on the chondrogenic differentiation of hMSCs. The cells adhered to the scaffolds with a homogeneous distribution and proliferated with culture time. The expression of chondrogenesis-related genes was upregulated, and abundant cartilaginous matrices were detected. After subcutaneous implantation, the PLGA-templated collagen scaffolds further enhanced the production of cartilaginous matrices and the mechanical properties of the implants. The good interconnectivity of the PLGA-templated collagen scaffolds promoted chondrogenic differentiation. In particular, the collagen scaffolds prepared with large pore-bearing PLGA sponge templates showed the highest promotive effect.