Chondrogenesis Of Mesenchymal Stem Cells Research Articles

Synergistic effects of Indian hedgehog and sonic hedgehog on chondrogenesis during cartilage repair.

Sonic hedgehog (Shh) and Indian hedgehog (Ihh) have been shown to control the induction of early cartilaginous differentiation. However, it is unclear whether Ihh and Shh exert synergistic effects on chondrogenesis during articular cartilage repair. Herein, we investigate the effects of chondrogenesis of bone-derived mesenchymal stem cells (BMSCs) following co-transfection with Shh and Ihh via adenoviral vectors in vitro and in vivo. A rotary cell culture system (RCCS) and Cytodex 3 microcarriers were used to create a stereoscopic dynamic environment for cell culture. In the RCCS environment, BMSCs co-transfected with Ihh and Shh displayed stronger chondrogenic differentiation and chondrogenesis than BMSCs transfected with Ihh or Shh alone, and exhibited higher expression levels of Sox 9, ACAN and collagen II, stronger toluidine blue and collagen II immunohistochemical staining. After transplanted into the osteochondral defect at 8weeks, Ihh/Shh co-transfected BMSCs showed a significantly better cartilage repair than BMSCs transfected with Ihh or Shh alone. Ihh and Shh have synergistic effects on the induction of chondrogenic differentiation and chondrogenesis under a microgravity environment, and help to repair damaged cartilage and reverse subchondral defects during the early stages.

Electrospun fibers enhanced the paracrine signaling of mesenchymal stem cells for cartilage regeneration

BackgroundSecretome profiles of mesenchymal stem cells (MSCs) are reflective of their local microenvironments. These biologically active factors exert an impact on the surrounding cells, eliciting regenerative responses that create an opportunity for exploiting MSCs towards a cell-free therapy for cartilage regeneration. The conventional method of culturing MSCs on a tissue culture plate (TCP) does not provide the physiological microenvironment for optimum secretome production. In this study, we explored the potential of electrospun fiber sheets with specific orientation in influencing the MSC secretome production and its therapeutic value in repairing cartilage.MethodsConditioned media (CM) were generated from MSCs cultured either on TCP or electrospun fiber sheets of distinct aligned or random fiber orientation. The paracrine potential of CM in affecting chondrogenic differentiation, migration, proliferation, inflammatory modulation, and survival of MSCs and chondrocytes was assessed. The involvement of FAK and ERK mechanotransduction pathways in modulating MSC secretome were also investigated.ResultsWe showed that conditioned media of MSCs cultured on electrospun fiber sheets compared to that generated from TCP have improved secretome yield and profile, which enhanced the migration and proliferation of MSCs and chondrocytes, promoted MSC chondrogenesis, mitigated inflammation in both MSCs and chondrocytes, as well as protected chondrocytes from apoptosis. Amongst the fiber sheet-generated CM, aligned fiber-generated CM (ACM) was better at promoting cell proliferation and augmenting MSC chondrogenesis, while randomly oriented fiber-generated CM (RCM) was more efficient in mitigating the inflammation assault. FAK and ERK signalings were shown to participate in the modulation of MSC morphology and its secretome production.ConclusionsThis study demonstrates topographical-dependent MSC paracrine activities and the potential of employing electrospun fiber sheets to improve the MSC secretome for cartilage regeneration.

Glucose Availability Affects Extracellular Matrix Synthesis During Chondrogenesis In Vitro.

Understanding in vitro chondrogenesis of human mesenchymal stem cells (hMSCs) is important as it holds great promise for cartilage tissue engineering and other applications. The current technology produces the end tissue quality that is highly variable and dependent on culture conditions. We investigated the effect of nutrient availability on hMSC chondrogenesis in a static aggregate culture system by varying the medium-change frequency together with starting glucose levels. Glucose uptake and lactate secretion profiles were obtained to monitor the metabolism change during hMSC chondrogenesis with different culture conditions. Higher medium-change frequency led to increases in cumulative glucose uptake for all starting glucose levels. Furthermore, increase in glucose uptake by aggregates led to increased end tissue glycosaminoglycan (GAG) and hydroxyproline (HYP) content. The results suggest that increased glucose availability either through increased medium-change frequency or higher initial glucose levels lead to improved chondrogenesis. Also, cumulative glucose uptake and lactate secretion were found to correlate well with GAG and HYP content, indicating both molecules are promising biomarkers for noninvasive assessment of hMSC chondrogenesis. Collectively, our results can be used to design optimal culture conditions and develop dynamic assessment strategies for cartilage tissue engineering applications. Impact statement In this study, we investigated how culture conditions, medium-change frequency and glucose levels, affect chondrogenesis of human mesenchymal stem cells in an aggregate culture model. Doubling the medium-change frequency significantly increased the biochemical quality of the resultant tissue aggregates, as measured by their glycosaminoglycan and hydroxyproline content. We attribute this to increased glucose uptake through the glycolysis pathway, as secretion of lactate, a key endpoint product of the glycolysis pathway, increased concurrently. These findings can be used to design optimal culture conditions for tissue engineering and regenerative medicine applications.

Linc-ROR promotes mesenchymal stem cells chondrogenesis and cartilage formation via regulating SOX9 expression

The present study is to characterize the role of long intergenic non-coding RNA, regulator of reprogramming (linc-ROR) in bone marrow mesenchymal stem cell (BMSCs) chondrogenesis, cartilage formation and OA development. Linc-ROR expression pattern in articular cartilage tissue sample from OA patients were studied by real-time PCR. Linc-ROR lentivirus mediated BMSCs were constructed. In vitro micromass cultured BMSCs chondrogenesis or in vivo MeHA hydrogel encapsulated BMSCs cartilage formation activity were studied. Linc-ROR associating miRNAs which repressed SOX9 expression were characterized by luciferase assay, real-time PCR and Western blot. Linc-ROR was co-transfected with miRNAs into BMSCs to study its rescue effect on SOX9 expression and chondrogenesis activity. Linc-ROR was down-regulated in articular cartilage tissue from OA patients and was positively correlated with the expression level of SOX9 (R2=0.43). Linc-ROR expression was upregulated during BMSCs chondrogenesis. Linc-ROR ectopic expression significantly promoted in vitro BMSCs chondrogenesis and in vivo cartilage formation activities as revealed by safranin O, alcian blue and COL II staining. The mRNA expression level of chondrogenesis markers including COL II, SOX9 and ACAN were increased, and the hypertrophy markers MMP13 and COL X were decreased upon linc-ROR overexpression in BMSCs. Linc-ROR functioned as a miRNA sponge for miR-138 and miR-145. Both miR-138 and miR-145 suppressed BMSCs chondrogenesis activity and SOX9 expression, while co-expression of linc-ROR displayed a rescuing effect. Taken together, linc-ROR modulated BMSCs chondrogenesis differentiation and cartilage formation by acting as a competing endogenous RNA for miR-138 and miR-145 and activating SOX9 expression. Linc-ROR could be considered as a new diagnostic and therapeutic target for OA treatment.

Combination of collagen scaffold with doxycycline for the treatment of cartilage and subchondral bone defects

The repair of cartilage and bone tissue post arthritis or injury in the knee joint is a challenge to the orthopedics. Both doxycycline and collagen scaffolds had been shown with positive influences on the repair of the tissue. Doxycycline inhibits catabolism related enzymes. Collagens provide tissue repair materials. In this study, the influence of doxycycline and jellyfish collagen (JFC) scaffold to the cells in vitro and tissue repair in vivo was studied. Doxycycline was found with a broad inhibition of the matrix metallopeptidases. It also had an enhancement to the progenitor chondrocytes but suppressed the maturation of chondrocytes. JFC (major type II collagen) was seen had improved both type I and type II collagen production during chondrogenesis of mesenchymal stem cells in vitro. Both doxycycline and JFC enhanced the bone formation and had synergistic effects in a late-stage during the bone repair process. However, the cartilage repair was found with no significant influence after the implantations. Overall, the control of metabolism by doxycycline and providing stimulation to collagen production by the implantation of JFC only contribute to bone growth but had little influence on cartilage repair. This indicates the growth of cartilage tissue needs more delicate regulation. The subchondral bone repair, even with some chondrocytes and proteoglycan deposition in the extracellular matrix could not convert to a repair with mature hyaluronic cartilage tissue.

Downregulation of MicroRNA-495 Alleviates IL-1β Responses among Chondrocytes by Preventing SOX9 Reduction.

PurposeOur previous work demonstrated that miRNA-495 targets SOX9 to inhibit chondrogenesis of mesenchymal stem cells. In this study, we aimed to investigate whether miRNA-495-mediated SOX9 regulation could be a novel therapeutic target for osteoarthritis (OA) using an in vitro cell culture model.Materials and MethodsAn in vitro model mimicking the OA environment was established using TC28a2 normal human chondrocyte cells. Interleukin-1β (IL-1β, 10 ng/mL) was utilized to induce inflammation-related changes in TC28a2 cells. Safranin O staining and glycosaminoglycan assay were used to detect changes in proteoglycans among TC28a2 cells. Expression levels of COX-2, ADAMTS5, MMP13, SOX9, CCL4, and COL2A1 were examined by qRT-PCR and/or Western blotting. Immunohistochemistry was performed to detect SOX9 and CCL4 proteins in human cartilage tissues obtained from patients with OA.ResultsmiRNA-495 was upregulated in IL-1β-treated TC28a2 cells and chondrocytes from damaged cartilage tissues of patients with OA. Anti-miR-495 abolished the effect of IL-1β in TC28a2 cells and rescued the protein levels of SOX9 and COL2A1, which were reduced by IL-1β. SOX9 was downregulated in the damaged cartilage tissues of patients with OA, and knockdown of SOX9 abolished the effect of anti-miR-495 on IL-1β-treated TC28a2 cells.ConclusionWe demonstrated that inhibition of miRNA-495 alleviates IL-1β-induced inflammatory responses in chondrocytes by rescuing SOX9 expression. Accordingly, miRNA-495 could be a potential novel target for OA therapy, and the application of anti-miR-495 to chondrocytes could be a therapeutic strategy for treating OA.

Chondrocyte secretome enriched microparticles encapsulated with the chondrocyte membrane to facilitate the chondrogenesis of BMSCs and reduce hypertrophy.

Co-culture of chondrocytes and mesenchymal stem cells (MSCs) represents an effective way to stimulate the chondrogenesis of MSCs and reduce hypertrophy, but the limited donor site supply and the requirement of two-stage operations are among the major barriers of using autologous chondrocytes in clinical settings. With recent evidence indicating that the chondrogenic effects of the above co-culture mainly lied on the paracrine secretion, and that cell membranes also played crucial roles during the chondrocyte-MSC interaction, we fabricated a multifunctional design of "artificial chondrocytes", which consist of chondrocyte secretome enriched PLGA microparticles with the encapsulation of chondrocytes' membrane fragments. The artificial chondrocytes had shown a similar diameter and surface electrical charge to natural chondrocytes, with the preserved key chondrocyte membrane surface proteins and sustainedly released chondrogenic cytokines from the chondrocyte secretome to extend their effects in vivo. Consequently, the co-culture studies of artificial chondrocytes and bone marrow MSCs had shown the beneficial effects from both chondrocyte secretome and membrane fragments, which also synergistically facilitated the cell proliferation, chondrogenic gene expression, cartilaginous matrix production, and reduced phenotypic hypertrophy in vitro and in vivo. Together, this study has successfully developed the proof-of-concept design of "artificial chondrocytes", which could potentially conquer many major barriers of using natural chondrocytes and provided a novel synthetic-cell approach to current therapeutical strategies towards the functional regeneration of articular cartilage.

Which Culture System Is better for Chondrogenesis of Adipose-Derived Stem Cells ; Pellet or Micromass?

Background and Aims: The current study was conducted to compare the expression levels of collagen type Π and X during chondrogenesis of human adipose-derived mesenchymal stem cells (hADMSCs) pellet and micromass cultures. Materials and Methods: Extracted hADMSCs were cultured until three passages and then transferred to pellet and micromass cultures in the experimental groups of day 7 and day14. For pellet and micromass cultures, aliquots of 5×105 cells/ml were centrifuged and respectively cultured in the conical tubes and droplets (12.5 µl) of the 24-well plates containing chondrogenic medium. Realtime-polymerase chain reaction technique was performed for gene expression levels. Results: Increased expression of collagen type Π was shown in micromass day14 compared to micromass day 7, pellet day 14 (p<0.01) and pellet day 7 (p<0.001). Also, an increased expression of collagen type Π was seen in micromass day 7 and pellet day 14 compared to pellet day 7 (p< 0.05). Expression of collagen type X increased in pellet day 14 compared to micromass on days 7 and 14 (p<0.001, p<0.01) and pellet day14 compared to pellet day7 (p< 0.05). An increased expression of collagen type X was shown in pellet day 7 compared to micromass on days 7 and 14 (p<0.05). Conclusions: According to the results, higher expression of collagen type Π and lower expression of collagen type X in micromass cultures that are prepared by cell suspension play a better role during cellular condensation that leads to the formation of large nodules exhibiting cartilage-like morphology, suggests a higher efficiency for micromass cultures.

MiR-335-5P contributes to human osteoarthritis by targeting HBP1.

MicroRNA (miR)-335-5P has the ability to regulate chondrogenic differentiation and promote chondrogenesis in mouse mesenchymal stem cells. It is also abnormally elevated in human osteoarthritic chondrocytes. However, the biological function of miR-335-5P in osteoarthritis (OA) is not well understood. The present study investigated the mechanism of miR-335-5P in the pathogenesis of OA. To investigate the effect of miR-335-5P on the pathogenesis of OA in vitro, a miR-335-5P mimic and inhibitor were transfected into chondrocytes. Cell Counting kit-8 assay and flow cytometry were used to observe the effects of miR-335-5P on chondrocyte apoptosis and the expression of cartilage-specific genes, such as aggrecan, collagen II, matrix metalloproteinase 13 and collagen X, were detected by reverse transcription-quantitative PCR and western blot analysis. Moreover, the current study assessed whether HMG-box transcription factor 1 (HBP1) is a novel target of miR-335-5P with dual luciferase reporter assays. Finally, a rescue experiment was used to prove the regulation between miR-335-5P and HBP1. The results revealed that HBP1 was a novel target of miR-335-5P, and that miR-335-5P mediated the apoptosis of chondrocytes and changes in cartilage-specific genes via targeting HBP1. Overall, the present study revealed that miR-335-5P mediated the development of OA by targeting the HBP1 gene and promoting chondrocyte apoptosis. These data suggested that miR-335-5P may be used to develop novel early-stage diagnostic and therapeutic strategies for OA.

Aqueous extract of Arctium lappa L. root (burdock) enhances chondrogenesis in human bone marrow-derived mesenchymal stem cells

BackgroundArctium lappa L. root (burdock root) has long been recommended for the treatment of different diseases in traditional Chinese medicine. Burdock root possesses anti-oxidative, anti-inflammatory, anti-cancer, and anti-microbial activities. The aim of the study was to elucidate whether aqueous extract of burdock root regulates mesenchymal stem cell proliferation and differentiation.MethodsHuman bone marrow-derived mesenchymal stem cells in 2D high density culture and in 3D micromass pellets were treated with chondrogenic induction medium and chondral basal medium in the absence or presence of aqueous extract of burdock root. The chondrogenic differentiation was accessed by staining glucosaminoglycans, immunostaining SOX9 and type II collagen and immuonblotting of SOX9, aggrecan and type II collagen.ResultsTreatment of aqueous extract of burdock root increased the cell proliferation of hMSCs. It did not have significant effect on osteogenic and adipogenic differentiation, but significantly enhanced chondrogenic induction medium-induced chondrogenesis. The increment was dose dependent, as examined by staining glucosaminoglycans, SOX9, and type II collagen and immunobloting of SOX9, aggrecan and type II collagen in 2D and 3D cultures. In the presence of supplemental materials, burdock root aqueous extract showed equivalent chondrogenic induction capability to that of TGF-β.ConclusionsThe results demonstrate that aqueous extract of Arctium lappa L. root promotes chondrogenic medium-induced chondrogenic differentiation. The aqueous extract of burdock root can even be used alone to stimulate chondrogenic differentiation. The study suggests that the aqueous extract of burdock root can be used as an alternative strategy for treatment purposes.

Fabrication of cartilage/natural rubber latex biocomposites derived from human mesenchymal stem cells in hypoxia

To broaden the knowledge regarding regenerative medicine in natural rubber latex (NRL) nanoparticles, we have examined the chondrogenesis of human mesenchymal stem cells (hMSCs) with NRL nanoparticles under hypoxia condition. We have successfully fabricated the cartilage/NRL biocomposites via hMSCs spheroid under hypoxic condition, where the administration of NRL nanoparticles exhibits the suppression of the spheroid contraction due to the cellular proliferation of hMSCs. It has been found that the NRL nanoparticles acted as a main component, which provides surface heterogeneity of the spheroid, leading to the mechanically stable structure with higher modulus in comparison with the control as revealed by atomic force microscopy microindentation. Under hypoxia condition, the effective gene expression of in vitro chondrogenesis of hMSCs with administration of NRL was confirmed as revealed by the chondrogenic gene expression analysis compared with that under normoxia condition.

Exosome-mediated delivery of kartogenin for chondrogenesis of synovial fluid-derived mesenchymal stem cells and cartilage regeneration

Transplantation of synovial fluid-derived mesenchymal stem cells (SF-MSCs) is a viable therapy for cartilage degeneration of osteoarthritis (OA). But controlling chondrogenic differentiation of the transplanted SF-MSCs in the joints remains a challenge. Kartogenin (KGN) is a small molecule that has been discovered to induce differentiation of SF-MSCs to chondrocytes both in vitro and in vivo. The clinical application of KGN however is limited by its low water solubility. KGN forms precipitates in the cell, resulting in low effective concentration and thus limiting its chondrogesis-promoting activity. Here we report that targeted delivery of KGN to SF-MSCs by engineered exosomes leads to even dispersion of KGN in the cytosol, increases its effective concentration in the cell, and strongly promotes the chondrogenesis of SF-MSCs in vitro and in vivo. Fusing an MSC-binding peptide E7 with the exosomal membrane protein Lamp 2b yields exosomes with E7 peptide displayed on the surface (E7-Exo) that has SF-MSC targeting capability. KGN delivered by E7-Exo efficiently enters SF-MSCs and induces higher degree of cartilage differentiation than KGN alone or KGN delivered by exosomes without E7. Co-administration of SF-MSCs with E7-Exo/KGN in the knee joints via intra-articular injection also shows more pronounced therapeutic effects in a rat OA model than KGN alone or KGN delivered by exosomes without E7. Altogether, transplantation of SF-MSCs with in situ chondrogenesis enabled by E7-Exo delivered KGN holds promise towards as an advanced stem cell therapy for OA.

Validation of the 1,4-butanediol thermoplastic polyurethane as a novel material for 3D bioprinting applications.

Tissue engineering (TE) seeks to fabricate implants that mimic the mechanical strength, structure, and composition of native tissues. Cartilage TE requires the development of functional personalized implants with cartilage‐like mechanical properties capable of sustaining high load‐bearing environments to integrate into the surrounding tissue of the cartilage defect. In this study, we evaluated the novel 1,4‐butanediol thermoplastic polyurethane elastomer (b‐TPUe) derivative filament as a 3D bioprinting material with application in cartilage TE. The mechanical behavior of b‐TPUe in terms of friction and elasticity were examined and compared with human articular cartilage, PCL, and PLA. Moreover, infrapatellar fat pad‐derived human mesenchymal stem cells (MSCs) were bioprinted together with scaffolds. in vitro cytotoxicity, proliferative potential, cell viability, and chondrogenic differentiation were analyzed by Alamar blue assay, SEM, confocal microscopy, and RT‐qPCR. Moreover, in vivo biocompatibility and host integration were analyzed. b‐TPUe demonstrated a much closer compression and shear behavior to native cartilage than PCL and PLA, as well as closer tribological properties to cartilage. Moreover, b‐TPUe bioprinted scaffolds were able to maintain proper proliferative potential, cell viability, and supported MSCs chondrogenesis. Finally, in vivo studies revealed no toxic effects 21 days after scaffolds implantation, extracellular matrix deposition and integration within the surrounding tissue. This is the first study that validates the biocompatibility of b‐TPUe for 3D bioprinting. Our findings indicate that this biomaterial can be exploited for the automated biofabrication of artificial tissues with tailorable mechanical properties including the great potential for cartilage TE applications.

Directionalities of magnetic fields and topographic scaffolds synergise to enhance MSC chondrogenesis

Mesenchymal stem cell (MSC) chondrogenesis is modulated by diverse biophysical cues. We have previously shown that brief, low-amplitude pulsed electromagnetic fields (PEMFs) differentially enhance MSC chondrogenesis in scaffold-free pellet cultures versus conventional tissue culture plastic (TCP), indicating an interplay between magnetism and micromechanical environment. Here, we examined the influence of PEMF directionality over the chondrogenic differentiation of MSCs laden on electrospun fibrous scaffolds of either random (RND) or aligned (ALN) orientations. Correlating MSCs’ chondrogenic outcome to pFAK activation and YAP localisation, MSCs on the RND scaffolds experienced the least amount of resting mechanical stress and underwent greatest chondrogenic differentiation in response to brief PEMF exposure (10 min at 1 mT) perpendicular to the dominant plane of the scaffolds (Z-directed). By contrast, in MSC-impregnated RND scaffolds, greatest mitochondrial respiration resulted from X-directed PEMF exposure (parallel to the scaffold plane), and was associated with curtailed chondrogenesis. MSCs on TCP or the ALN scaffolds exhibited greater resting mechanical stress and accordingly, were unresponsive, or negatively responsive, to PEMF exposure from all directions. The efficacy of PEMF-induced MSC chondrogenesis is hence regulated in a multifaceted manner involving focal adhesion dynamics, as well as mitochondrial responses, culminating in a final cellular response. The combined contributions of micromechanical environment and magnetic field orientation hence will need to be considered when designing magnetic exposure paradigms.

Directionalities of Magnetic Fields and Topographic Scaffolds Synergise to Enhance MSC Chondrogenesis

Mesenchymal stem cell (MSC)-based chondrogenesis is modulated by diverse biophysical cues. We have previously shown that brief, low-amplitude pulsed electromagnetic fields (PEMFs) differentially enhance MSC chondrogenesis in scaffold-free pellet cultures versus conventional tissue culture plastic (TCP), indicating an interplay between magnetism and micromechanical environment. Here, we examined the influence of PEMF directionality over the chondrogenic differentiation of MSCs laden onto electrospun fibrous scaffolds of either random (RND) or aligned (ALN) orientations. Interactions were detected between magnetic field orientation with that of the cells that combined with micromechanical environment at the level of mitochondrial respiration. Based on pFAK staining and YAP localisation, MSCs on the RND scaffolds were shown to be experiencing the least amount of resting mechanical stress and were found to undergo robust chondrogenic differentiation in response to PEMF exposure perpendicular to the dominant plane of the scaffolds (Z-directed) for only ten minutes at an amplitude of 1 mT. By contrast, MSCs plated onto TCP or the ALN scaffolds gave indications of greater resting mechanical stress accordingly, were unresponsive, or responded negatively, to Z-directed PEMFs. Such interactions agree with a mitohormetic mechanism of cumulative interaction between magnetic fields and the mechanical environment. Moreover, PEMFs demonstrated a strong interaction with MSC alignment, whereby Y-directed magnetic fields applied parallel to the long-axis of MSCs on Y-ALN scaffolds exhibited reduced chondrogenesis. These results indicate that diverse biophysical parameters interact with magnetic fields to culminate in a final cell response that will need to be taken into consideration when designing an effective magnetic exposure paradigm.

In situ ornamenting poly(ε-caprolactone) electrospun fibers with different fiber diameters using chondrocyte-derived extracellular matrix for chondrogenesis of mesenchymal stem cells

Biomimetic instructive tissue engineering scaffolds are critical for achieving successful tissue regeneration. In the present study, we developed a novel scaffold via ornamenting poly(ε-caprolactone) (PCL) electrospun fibers with a chondrocyte-derived extracellular matrix (ECM)-coating, which was applied for chondrogenesis of mesenchymal stem cells (MSCs). PCL fibrous films with different fiber diameters (1282±121 nm, 549±61 nm and 285±38 nm) were first prepared via electrospinning. Rabbit articular chondrocytes (rACs) were cultured on PCL fibrous scaffolds, followed by a decellularization treatment to generate decellularized ECM (dECM)-coated PCL scaffolds (dECM/PCL). Rabbit bone marrow-derived MSCs (rMSCs) were then seeded onto these scaffolds and adhesion, proliferation and chondrogenic differentiation were evaluated. dECM/PCL scaffolds displayed distinct surface microstructural features with varying fiber diameters and fibrous mesh-like ECM with more developed collagen fibers was observed on nanofibers. On dECM/PCL scaffolds, rMSCs tended to spread more at 24 h post-seeding and proliferated better within 7 d compared to those on uncoated PCL scaffolds. Based on analysis of gene expression, rMSCs underwent the best chondrogenic differentiation on dECM/PCL scaffolds of 549-nm fibers. Collectively, such dECM/PCL composite scaffolds are very promising for cartilage tissue regeneration.

Chondrogenesis of cocultures of mesenchymal stem cells and articular chondrocytes in poly(l-lysine)-loaded hydrogels

This work investigated the effect of poly(l-lysine) (PLL) molecular weight and concentration on chondrogenesis of cocultures of mesenchymal stem cells (MSCs) and articular chondrocytes (ACs) in PLL-loaded hydrogels. An injectable dual-network hydrogel composed of a poly(N-isopropylacrylamide)-based synthetic thermogelling macromer and a chondroitin sulfate-based biological network was leveraged as a model to deliver PLL and encapsulate the two cell populations. Incorporation of PLL into the hydrogel did not affect the hydrogel's swelling properties and degradation characteristics, nor the viability of encapsulated cells. Coculture groups demonstrated higher type II collagen expression compared to the MSC monoculture group. Expression of hypertrophic phenotype was also limited in the coculture groups. Histological analysis indicated that the ratio of MSCs to ACs was an accurate predictor of the degree of long-term chondrogenesis, while the presence of PLL was shown to have a more substantial short-term effect. Altogether, this study demonstrates that coculturing MSCs with ACs can greatly enhance the chondrogenicity of the overall cell population and offers a platform to further elucidate the short- and long-term effect of polycationic factors on the chondrogenesis of MSC and AC cocultures.

MiR-520d-5p modulates chondrogenesis and chondrocyte metabolism through targeting HDAC1.

MicroRNAs (miRNAs) play an essential role in the chondrogenesis and the progression of osteoarthritis (OA). This study aimed to determine miRNAs associated with chondrogenesis of human mesenchymal stem cells (hMSCs) and chondrocyte metabolism. MiRNAs were screened in hMSCs during chondrogenesis by RNA-seq and qRT-PCR. MiRNA expression was determined in primary human chondrocytes (PHCs), and degraded cartilage samples. MiRNA mimics and inhibitors were transfected to cells to determine the effect of miRNA. Bioinformatic analysis and luciferase reporter assays were applied to determine the target gene of miRNA. The results demonstrated that miR-520d-5p was increased in hMSCs chondrogenesis. The overexpression and knockdown of miR-520d-5p promoted and inhibited chondrogenesis, and regulated chondrocyte metabolism. Histone deacetylase 1 (HDAC1) was decreased in hMSCs chondrogenesis, and HDAC1 was a targeting gene of miR-520d-5p. CI994, HDAC1 inhibitor, elevated cartilage-specific gene expressions and promoted hMSCs chondrogenesis. In IL-1β-treated PHCs, CI994 promoted AGGRECAN expression and suppressed MMP-13 expression, abolishing the effect of IL-1β on PHCs. Taken together, these results suggest that miR-520d-5p promotes hMSCs chondrogenesis and regulates chondrocyte metabolism through targeting HDAC1. This study provides novel understanding of the molecular mechanism of OA progression.

Amygdalin Promotes Fracture Healing through TGF-β/Smad Signaling in Mesenchymal Stem Cells.

Chondrogenesis and subsequent osteogenesis of mesenchymal stem cells (MSCs) and angiogenesis at injured sites are crucial for bone fracture healing. Amygdalin, a cyanogenic glycoside compound derived from bitter apricot kernel, has been reported to inhibit IL-1β-induced chondrocyte degeneration and to stimulate blood circulation, suggesting a promising role of amygdalin in fracture healing. In this study, tibial fractures in C57BL/6 mice were treated with amygdalin. Fracture calluses were then harvested and subjected to radiographic, histological, and biomechanical testing, as well as angiography and gene expression analyses to evaluate fracture healing. The results showed that amygdalin treatment promoted bone fracture healing. Further experiments using MSC-specific transforming growth factor- (TGF-) β receptor 2 conditional knockout (KO) mice (Tgfbr2Gli1-Cre) and C3H10 T1/2 murine mesenchymal progenitor cells showed that this effect was mediated through TGF-β/Smad signaling. We conclude that amygdalin could be used as an alternative treatment for bone fractures.

Assessment and Comparison of the Efficacy of Methotrexate, Prednisolone, Adalimumab, and Tocilizumab on Multipotency of Mesenchymal Stem Cells.

Mesenchymal stem cell (MSC)–based articular regeneration might be beneficial for both protecting and rebuilding cartilaginous tissues in the management of rheumatoid arthritis. However, it is unclear how current immunosuppressive strategies influence the multipotency of MSCs. The present study was undertaken to profile the direct effectiveness of major antirheumatic drugs including methotrexate, prednisolone, adalimumab, and tocilizumab on the multipotency of MSCs, with a special focus on chondrogenesis. The inhibitory effects of methotrexate on adipogenesis, osteogenesis, and chondrogenesis were observed to occur in a dose-dependent manner in an in vitro differentiation system. Prednisolone enhanced adipogenesis, but reduced alkaline phosphatase activity in osteoprogenitors and suppressed the formation of chondrospheroids. Adalimumab suppressed alkaline phosphatase activity, while tocilizumab diminished osteogenesis and chondrogenesis of MSCs in vitro. Chondrogenesis of antirheumatic drug-treated MSCs was also evaluated in vivo using a scaffolded spheroid-engrafted murine model. The biologics examined appeared to be relatively safe for cartilaginous formation, but methotrexate and prednisolone exhibited opposing influences on chondrogenesis. Taken together, these results reveal the direct efficacy of major antirheumatic agents on the multipotency of MSCs. Therefore, our findings suggest that optimization of medication protocols is further required for therapeutic approaches involving cartilaginous tissue engineering.