Chondrogenesis Of hMSCs Research Articles

An assessment of the response of human MSCs to hydrostatic pressure in environments supportive of differential chondrogenesis

Mechanical stimulation can modulate the chondrogenic differentiation of stem/progenitor cells and potentially benefit tissue engineering (TE) of functional articular cartilage (AC). Mechanical cues like hydrostatic pressure (HP) are often applied to cell-laden scaffolds, with little optimization of other key parameters (e.g. cell density, biomaterial properties) known to effect lineage commitment. In this study, we first sought to establish cell seeding densities and fibrin concentrations supportive of robust chondrogenesis of human mesenchymal stem cells (hMSCs). High cell densities (15*106 cells/ml) were more supportive of sGAG deposition on a per cell basis, while collagen deposition was higher at lower seeding densities (5*106 cells/ml). Employment of lower fibrin (2.5 %) concentration hydrogels supported more robust chondrogenesis of hMSCs, with higher collagen type II and lower collagen type X deposition compared to 5 % hydrogels. The application of HP to hMSCs maintained in identified chondro-inductive culture conditions had little effect on overall levels of cartilage-specific matrix production. However, if hMSCs were first temporally primed with TGF-β3 before its withdrawal, they responded to HP by increased sGAG production. The response to HP in higher cell density cultures was also associated with a metabolic shift towards glycolysis, which has been linked with a mature chondrocyte-like phenotype. These results suggest that mechanical stimulation may not be necessary to engineer functional AC grafts using hMSCs if other culture conditions have been optimised. However, such bioreactor systems can potentially be employed to better understand how engineered tissues respond to mechanical loading in vivo once removed from in vitro culture environments.

3D bioprinting of cartilaginous templates for large bone defect healing.

Damaged or diseased bone can be treated using autografts or a range of different bone grafting biomaterials, however limitations with such approaches has motivated increased interest in developmentally inspired bone tissue engineering (BTE) strategies that seek to recapitulate the process of endochondral ossification (EO) as a means of regenerating critically sized defects. The clinical translation of such strategies will require the engineering of scaled-up, geometrically defined hypertrophic cartilage grafts that can be rapidly vascularised and remodelled into bone in mechanically challenging defect environments. The goal of this study was to 3D bioprint mechanically reinforced cartilaginous templates and to assess their capacity to regenerate critically sized femoral bone defects. Human mesenchymal stem/stromal cells (hMSCs) were incorporated into fibrin based bioinks and bioprinted into polycaprolactone (PCL) frameworks to produce mechanically reinforced constructs. Chondrogenic priming of such hMSC laden constructs was required to support robust vascularisation and graft mineralisation in vivo following their subcutaneous implantation into nude mice. With a view towards maximising their potential to support endochondral bone regeneration, we next explored different in vitro culture regimes to produce chondrogenic and early hypertrophic engineered grafts. Following their implantation into femoral bone defects within transiently immunosuppressed rats, such bioprinted constructs were rapidly remodelled into bone in vivo, with early hypertrophic constructs supporting higher levels of vascularisation and bone formation compared to the chondrogenic constructs. Such early hypertrophic bioprinted constructs also supported higher levels of vascularisation and spatially distinct patterns of new formation compared to BMP-2 loaded collagen scaffolds (here used as a positive control). In conclusion, this study demonstrates that fibrin based bioinks support chondrogenesis of hMSCs in vitro, which enables the bioprinting of mechanically reinforced hypertrophic cartilaginous templates capable of supporting large bone defect regeneration. These results support the use of 3D bioprinting as a strategy to scale-up the engineering of developmentally inspired templates for BTE. STATEMENT OF SIGNIFICANCE: Despite the promise of developmentally inspired tissue engineering strategies for bone regeneration, there are still challenges that need to be addressed to enable clinical translation. This work reports the development and assessment (in vitro and in vivo) of a 3D bioprinting strategy to engineer mechanically-reinforced cartilaginous templates for large bone defect regeneration using human MSCs. Using distinct in vitro priming protocols, it was possible to generate cartilage grafts with altered phenotypes. More hypertrophic grafts, engineered in vitro using TGF-β3 and BMP-2, supported higher levels of blood vessel infiltration and accelerated bone regeneration in vivo. This study also identifies some of the advantages and disadvantages of such endochondral bone TE strategies over the direct delivery of BMP-2 from collagen-based scaffolds.

Pre-culture of human mesenchymal stromal cells in spheroids facilitates chondrogenesis at a low total cell count upon embedding in biomaterials to generate cartilage microtissues

Material-assisted cartilage tissue engineering has limited application in cartilage treatment due to hypertrophic tissue formation and high cell counts required. This study aimed at investigating the potential of human mesenchymal stromal cell (hMSC) spheroids embedded in biomaterials to study the effect of biomaterial composition on cell differentiation. Pre-cultured (3 days, chondrogenic differentiation media) spheroids (250 cells/spheroid) were embedded in tyramine-modified hyaluronic acid (THA) and collagen type I (Col) composite hydrogels (four combinations of THA (12.5 vs 16.7 mg/ml) and Col (2.5 vs 1.7 mg/ml) content) at a cell density of 5 × 106 cells/ml (2 × 104 spheroids/ml). Macropellets derived from single hMSCs (2.5 × 105 cells, ScMP) or hMSC spheroids (2.5 × 105 cells, 103 spheroids, SpMP) served as control. hMSC differentiation was analyzed using glycosaminoglycan (GAG) quantification, gene expression analysis and (immuno-)histology. Embedding of hMSC spheroids in THA-Col induced chondrogenic differentiation marked by upregulation of aggrecan (ACAN) and COL2A1, and the production of GAGs . Lower THA led to more pronounced chondrogenic phenotype compared to higher THA content. Col content had no significant influence on hMSC chondrogenesis. Pellet cultures showed an upregulation in chondrogenic-associated genes and production of GAGs with less upregulation of hypertrophic-associated genes in SpMP culture compared to ScMP group. This study presents hMSC pre-culture in spheroids as promising approach to study chondrogenic differentiation after biomaterial encapsulation at low total cell count (5 × 106/ml) without compromising chondrogenic matrix production. This approach can be applied to assemble microtissues in biomaterials to generate large cartilage construct. Statement of significanceIn vitro studies investigating the chondrogenic potential of biomaterials are limited due to the low cell-cell contact of encapsulated single cells. Here, we introduce the use of pre-cultured hMSC spheroids to study chondrogenesis upon encapsulation in a biomaterial. The use of spheroids takes advantage of the high cell-cell contact within each spheroid being critical in the early chondrogenesis of hMSCs. At a low seeding density of 5·106 cells/ml (2 × 104 spheroids/ml) we demonstrated hMSC chondrogenesis and cartilaginous matrix deposition. Our results indicate that the pre-culture might have a beneficial effect on hypertrophic gene expression without compromising chondrogenic differentiation. This approach has shown potential to assemble microtissues (here spheroids) in biomaterials to generate large cartilage constructs and to study the effect of biomaterial composition on cell alignment and migration.

Biomaterial-mediated presentation of wnt5a mimetic ligands enhances chondrogenesis and metabolism of stem cells by activating non-canonical Wnt signaling

The presentation of development-relevant bioactive cues by biomaterial scaffolds is essential to the guided differentiation of seeded human mesenchymal stem cells (hMSCs) and subsequent tissue regeneration. Wnt5a is a critical non-canonical Wnt signaling ligand and plays a key role in the development of musculoskeletal tissues including cartilage. Herein we investigate the efficacy of biofunctionalizing the hyaluronic acid hydrogel with a synthetic Wnt5a mimetic ligand (Foxy5 peptide) to promote the chondrogenesis of hMSCs and the potential underlying molecular mechanism. Our findings show that the conjugation of Foxy5 peptide in the hydrogels activates non-canonical Wnt signaling of encapsulated hMSCs via the upregulation expression of PLCE1, CaMKII-β, and downstream NFATc1, leading to enhanced expression of chondrogenic markers such as SOX9. The decoration of Foxy5 peptide also promotes the metabolic activities of encapsulated hMSCs as evidenced by upregulated gene expression of mitochondrial complex components and glucose metabolism biomarkers, leading to enhanced ATP biosynthesis. Furthermore, the conjugation of Foxy5 peptide activates the non-canonical Wnt, PI3K-PDK-AKT and IKK/NF-κB signaling pathways, thereby inhibiting the hypertrophy of the chondrogenically induced hMSCs in the hydrogels under both in vitro and in vivo conditions. This enhanced chondrogenesis and attenuated hypertrophy of hMSCs by the biomaterial-mediated bioactive cue presentation facilitates the potential clinical translation of hMSCs for cartilage regeneration. Our work provides valuable guidance to the rational design of bio-inductive scaffolds for various applications in regenerative medicine.

Precision 3D printed meniscus scaffolds to facilitate hMSCs proliferation and chondrogenic differentiation for tissue regeneration

BackgroundThe poor regenerative capability and structural complexity make the reconstruction of meniscus particularly challenging in clinic. 3D printing of polymer scaffolds holds the promise of precisely constructing complex tissue architecture, however the resultant scaffolds usually lack of sufficient bioactivity to effectively generate new tissue.ResultsHerein, 3D printing-based strategy via the cryo-printing technology was employed to fabricate customized polyurethane (PU) porous scaffolds that mimic native meniscus. In order to enhance scaffold bioactivity for human mesenchymal stem cells (hMSCs) culture, scaffold surface modification through the physical absorption of collagen I and fibronectin (FN) were investigated by cell live/dead staining and cell viability assays. The results indicated that coating with fibronectin outperformed coating with collagen I in promoting multiple-aspect stem cell functions, and fibronectin favors long-term culture required for chondrogenesis on scaffolds. In situ chondrogenic differentiation of hMSCs resulted in a time-dependent upregulation of SOX9 and extracellular matrix (ECM) assessed by qRT-PCR analysis, and enhanced deposition of collagen II and aggrecan confirmed by immunostaining and western blot analysis. Gene expression data also revealed 3D porous scaffolds coupled with surface functionalization greatly facilitated chondrogenesis of hMSCs. In addition, the subcutaneous implantation of 3D porous PU scaffolds on SD rats did not induce local inflammation and integrated well with surrounding tissues, suggesting good in vivo biocompatibility.ConclusionsOverall, this study presents an approach to fabricate biocompatible meniscus constructs that not only recapitulate the architecture and mechanical property of native meniscus, but also have desired bioactivity for hMSCs culture and cartilage regeneration. The generated 3D meniscus-mimicking scaffolds incorporated with hMSCs offer great promise in tissue engineering strategies for meniscus regeneration.Graphical

Recent Advances in Promoting Chondrogenesis of Human Mesenchymal Stem Cells Using Physically and Chemically Modified Hydrogels

Tissue engineering strategies for regenerating damaged cartilage using hydrogels have garnered significant attention due to the limited self-healing capacity of damaged cartilage tissue and the restrictions of current medical treatment methods. In particular, using human mesenchymal stem cells (hMSCs) as the cell source has shown the potential to differentiate along a chondrogenic lineage. Hydrogels, whether made of synthetic polymers, natural polymers, or combinations, are widely explored as scaffolding materials mimicking the natural cartilage environment. Based on the understanding of the importance of surface nanotopographies and mechanical stiffness, hydrogels have been presented in various forms and tested for the differentiation of hMSCs. The primary focus of this review is to provide a summary of recent advances in physically and chemically modified hydrogels promoting the chondrogenesis of hMSCs. Advances in micromachining have helped in forming surfaces with the required roughness or an array of micropillars of defined architecture. Hydrogels have been combined with various stimulants such as small peptides, growth factors, and many modified matrix elements. Creating anisotropic hydrogels mimicking the extracellular matrix of cartilage has also been reported. These studies show promising results and identify a niche for in-vitro differentiation of chondrocytes from hMSCs.

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.

Bi-functional nanoparticle-stabilized hydrogel colloidosomes as both extracellular matrix and bioactive factor delivery vehicle.

Maintaining both cell-cell and cell-extracellular matrix (ECM) interactions is often a critical component of three-dimensional (3D) tissue regeneration. In high-density cell condensation systems, lack of appropriate cell-ECM interactions can result in limited and/or slow cell differentiation and tissue formation. To address these problems, a colloidosome microsphere system that is composed of a gelatin hydrogel core and a porous nanoparticle shell is developed. The colloidosome microsphere functions as an ECM and morphogen carrier for the induction of cartilage formation of high-density human mesenchymal stem cell (hMSC) in 3D cultures. With the protection of the nanoparticle shell, the colloidosome microspheres can be readily suspended in aqueous solution without clumping, thus incorporated homogeneously within high-density cell condensations. The gelatin-based colloidosome microspheres stimulate chondrogenesis of hMSCs and degrade rapidly to facilitate ECM remodeling for new tissue formation. When loaded with human transforming growth factor-β1, a potent chondrogenic morphogen, the colloidosomes serve as a bioactive factor delivery vehicle as well. The dual functionality of the colloidosomes as an ECM and a growth factor carrier effectively supports the chondrogenic differentiation of high-density hMSC condensations. These capabilities render the colloidosomes a promising platform system amenable to large-scale production of high-density 3D tissue culture constructs.

Senolytic activity of small molecular polyphenols from olive restores chondrocyte redifferentiation and promotes a pro-regenerative environment in osteoarthritis.

Articular cartilage and synovial tissue from patients with osteoarthritis (OA) show an overactivity of connexin43 (Cx43) and accumulation of senescent cells associated with disrupted tissue regeneration and disease progression. The aim of this study was to determine the effect of oleuropein on Cx43 and cellular senescence for tissue engineering and regenerative medicine strategies for OA treatment. Oleuropein regulates Cx43 promoter activity and enhances the propensity of hMSCs to differentiate into chondrocytes and bone cells, reducing adipogenesis. This small molecule reduce Cx43 levels and decrease Twist-1 activity in osteoarthritic chondrocytes (OACs), leading to redifferentiation, restoring the synthesis of cartilage ECM components (Col2A1 and proteoglycans), and reducing the inflammatory and catabolic factors mediated by NF-kB (IL-1ß, IL-6, COX-2 and MMP-3), in addition to lowering cellular senescence in OACs, synovial and bone cells. Our in vitro results demonstrate the use of olive-derived polyphenols, such as oleuropein, as potentially effective therapeutic agents to improve chondrogenesis of hMSCs, to induce chondrocyte re-differentiation in OACs and clearing out senescent cells in joint tissues in order to prevent or stop the progression of the disease.

Sodium Hyaluronate Supplemented Culture Media as a New hMSC Chondrogenic Differentiation Media-Model for in vitro/ex vivo Screening of Potential Cartilage Repair Therapies.

Surgical strategies to treat articular cartilage injury such as microfracture, expose human bone marrow stem cells (hMSCs) to synovial fluid and its components. High molecular weight hyaluronan (hMwt HA) is one of the most abundant bioactive macromolecules of healthy synovial fluid (hSF) and it plays an important role in the protection of opposing articular cartilage surfaces within the synovial joint. Although hMwt HA has been extensively used to attempt the engineering of the cartilage tissue, its effect as media supplement has not been established. Indeed, current media are often simple in their composition and doesn’t recapitulate the rheological and biological features of hSF. In addition, critical in vivo molecules that can potentially change the chondrogenic behavior of hBMSCs to make the in vitro results more predictive of the real in vivo outcome, are lacking. In order to be one step closer to the in vivo physiology of hSF, a new culture media supplemented with physiological level of hMwt HA was developed and the effect of the hMwt HA on the chondrogenesis of hMSCs that would be present in a traumatic defect after marrow stimulation techniques, was investigated. hBMSC-seeded fibrin-polyurethane constructs were cultured in a serum free chondropermissive control medium (HA- TGFβ-). This medium was further supplemented with 10 ng/mL TGFβ1 (HA- TGFβ+) or 2 mg/ml hMwt HA 1.8 MDa (HA+ TGFβ-) or both (HA+ TGFβ+). Alternatively, 1 MDa HA was mixed with the fibrin at 0.2 mg/ml (HASc TGFβ+). The effect of hMwt HA on hMSC differentiation was investigated at the gene expression level by RT-qPCR and total DNA, sulfated glycosaminoglycans and Safranin O staining were evaluated. Addition of hMwt HA to the culture media, significantly increased the synthesis of sulfated glycosaminoglycans, especially in the early days of chondrogenesis, and reduced the upregulation of the hypertrophic cartilage marker collagen X. hMwt HA added inside the fibrin gel(HASc TGF+) led to the best matrix deposition. hMwt HA can be one key medium component in a more reliable in vitro/ex vivo system to reduce in vitro artifacts, enable more accurate pre-screening of potential cartilage repair therapies and reduce the need for animal studies.

MiR-892b Inhibits Hypertrophy by Targeting KLF10 in the Chondrogenesis of Mesenchymal Stem Cells

We investigated the functional role of miR-892b as a novel inhibitor of chondrocyte hypertrophy during TGF-β-mediated chondrogenesis of human mesenchymal stem cells (hMSCs). The expression of miR-892b during TGF-β-mediated chondrogenesis of hMSCs and the effects of miR-892b overexpression on chondrogenic and hypertrophic marker genes in the chondrogenesis of hMSCs were investigated. Targets of miR-892b were identified and verified by overexpression of synthetic miRNA mimics and luciferase assays. Cross-talk between Kruppel-like factor 10 (KLF10) and Indian hedgehog (Ihh) was investigated using KLF10 knockdown (KD). miR-892b enhanced chondrogenic makers and suppressed hypertrophy in hMSC chondrogenesis, mimicking parathyroid hormone-related peptide (PTHrP). KLF10, a transcription factor and miR-892b target, directly regulated Ihh promoter activity. Like miR-892b, KLF10 KD enhanced hMSC chondrogenesis and inhibited hypertrophy. Our findings suggest a key role of miR-892b in targeting the KLF10-Ihh axis as a regulator of hypertrophy in TGF-β-mediated chondrogenesis of hMSCs and provide a novel strategy for preventing hypertrophy in chondrogenesis from MSCs.

Biocomposites composed of natural rubber latex and cartilage tissue derived from human mesenchymal stem cells

Natural rubber latex (NRL) is mainly used for traditional industrial products, but currently, its target application is continuously expanding into tissue engineering. Here, we have presented the chondrogenesis ability of NRL nanoparticles against human mesenchymal stem cells (hMSCs). The effective gene expression of in vitro chondrogenesis of hMSCs with administration of NRL was confirmed as revealed by the chondrogenic gene expression analysis. We have successfully for the first time fabricated the cartilage/NRL biocomposites via hMSC spheroid formation, where the administration of NRL nanoparticles exhibited the mechanically stable structure with hard surface structure in the spheroid.

Expression of microRNA-892B in human mesenchymal stem cells promotes cartilage regeneration in athymic nude rat osteochondral defect model

Purpose: Articular cartilage (AC) is poorly supplied by blood vessels, nerves, and the lymphatic system. Therefore, AC has a very limited capacity that can be repaired after an injury. Although several therapeutic measures have been developed to promote the healing of AC, no widely accepted treatment has been established. But transfection of a cocktail of genes into cells has recently been attractive to stem cell differentiation. Here, based on our previous in vitro studies, we confirm in vivo effectiveness of miR-892b for cartilage regeneration in the rat osteochondral defect model in vivo. Our strategy modulating miR-892b gene can suggest a new strategy for effective cartilage regeneration in MSC mediated tissue engineering fields. Methods: The knee joints of the male arthymic nude rat were exposed by a medial parapatellar incision, and the trochlear groove was severed due to the lateral obstruction of the patella. A 1.5 mm outer diameter trephine drill was employed to create osteochondral defects (1.5 × 3.0 mm) in the trochlear groove of the femur. The osteochondral defects was managed using one of the following methods : filling with HCF only (Group 1, n = 3), filling with TGF-â1-loaded HCF (Group 2, n = 3); and filling with lenti-control/hMSC-loaded HCF (Group 3, n = 3); and filling with lenti-control/hMSC and TGF-â1-loaded HCF (Group 4, n = 3); and filling with lenti-miR-892b/hMSC and TGF-â1-loaded HCF (Group 5, n = 3). An equal volume of thrombin solution was simultaneously injected with HCF in all groups. Each group consisted of 3 rats. After 8 weeks, the rats were sacrificed. Results: After genomic DNA PCR, a sequence correspond to pri-miR-892b was cloned into NheI-EcoRI sites of pCDH-copGFP lentiviral plasmid (Fig. 1a). As shown in Fig. 1b, lentiviral control and lenti-miR-892b transduced hMSCs were dually labeled with a far-red fluorescent dye (CellVeu) for in vivo visualization. After 8 weeks of post-surgery, this far-red fluorescent signals were only detected osteochondral defects of CellVeu labeled hMSCs transplanted groups (Fig. 1c). The ICRS macroscopic score was highest in group 5 (lenti-miR-892b-transduced hMSCs and TGF-β1: 11.0), Group 5 had significantly higher score than the other groups (Fig. 1d). Histological assessment showed that group 5 defects had good healing and covered with hyaline cartilage-like tissue. Although group 4 defects had complete healing with hyaline cartilage-like tissue, remodeling status in subchondral bone of the defects was irregular and incomplete. Defects of the rest group were mostly filled with fibrous tissue (Fig. 1f). According to the Wakitani Histological Grading Scale, group 5 was significantly lower than other group in surface, cell distribution, and subchondral bone (Fig. 1e). Conclusions: In conclusion, we previously found that miR-892b can enhance in vitro chondrogenesis of hMSCs. Here, we additionally evaluated its in vivo efficacy using rat osteochondral defect model. The macroscopic assessment of the regenerated cartilages demonstrated that the defect had become firm and smooth after 8 weeks in Groups 4, 5 while a visible defect was present in Group 1, 2 and 3. However, the reconstitution of subchondral bone was better in Group 5 than in Groups 4. The histological score was significantly better in Group 5 than in the other groups. These in vivo results confirmed that the expression of miR-892b in hMSC under TGF-β1 stinuli might promote the healing of osteochondral defects.

Donor Variation and Optimization of Human Mesenchymal Stem Cell Chondrogenesis in Hyaluronic Acid.

Mesenchymal stem cells (MSCs) are an attractive cell type for cartilage repair that can undergo chondrogenesis in a variety of three-dimensional (3D) scaffolds. Hyaluronic acid (HA) hydrogels provide a biologically relevant interface for cell encapsulation. While previous studies have shown that MSC-laden HA constructs can mature in vitro to match native mechanical properties using cells from animal sources, clinical application will depend on the successful translation of these findings to human cells. Though numerous studies have investigated chondrogenesis of human MSC (hMSC)-laden constructs, their functional outcomes were quite inferior to those using animal sources, and donor-specific responses to 3D HA hydrogels have not been fully investigated. To that end, hMSCs were derived from seven donors, and their ability to undergo chondrogenesis in pellet culture and HA hydrogels was evaluated. Given the initial observation of overt cell aggregation and/or gel contraction for some donors, the impact of variation in cell and HA macromer concentration on functional outcomes during chondrogenesis was evaluated using one young/healthy donor. The findings show marked differences in functional chondrogenesis of hMSCs in 3D HA hydrogels based on donor. Increasing cell density resulted in increased mechanical properties, but also promoted construct contraction. Increasing the macromer density generally stabilized construct dimensions and increased extracellular matrix production, but limited the distribution of formed matrix at the center of the construct and reduced mechanical properties. Collectively, these findings suggest that the use of hMSCs may require tuning of cell density and gel mechanics on a donor-by-donor basis to provide for the most robust tissue formation for clinical application.

The Effects of the WNT-Signaling Modulators BIO and PKF118-310 on the Chondrogenic Differentiation of Human Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) are multipotent cells, mainly from bone marrow, and an ideal source of cells in bone and cartilage tissue engineering. A study of the chondrogenic differentiation of MSCs is of particular interest for MSCs-based cartilage regeneration. In this study, we aimed to optimize the conditions for the chrondogenic differentiation of MSCs by regulating WNT signaling using the small molecule WNT inhibitor PKF118-310 and activator BIO. Human mesenchymal stem cells (hMSCs) were isolated from bone marrow aspirates and cultured in hMSCs proliferation medium. Pellet culture was subsequently established for three-dimensional chondrogenic differentiation of 5 weeks. WNT signaling was increased by the small molecule glycogen synthase kinase-3 inhibitor 6-bromoindirubin-3-oxim (BIO) and decreased by the WNT inhibitor PKF118-310 (PKF). The effects of BIO and PKF on the chondrogenesis of hMSCs was examined by real-time PCR, histological methods, and ELISA. We found that activation of canonical WNT-signaling by BIO significantly downregulated the expression of cartilage-specific genes SOX9, COL2A1, and ACAN, and matrix metalloproteinase genes MMP1/3/9/13, but increased ADAMTS 4/5. Inhibition of WNT signaling by PKF increased the expression of SOX9, COL2A1, ACAN, and MMP9, but decreased MMP13 and ADAMTS4/5. In addition, a high level of WNT signaling induced the expression of hypertrophic markers COL10A1, ALPL, and RUNX2, the dedifferentiation marker COL1A1, and glycolysis genes GULT1 and PGK1. Deposition of glycosaminoglycan (GAG) and collagen type II in the pellet matrix was significantly lost in the BIO-treated group and increased in the PKF-treated group. The protein level of COL10A1 was also highly induced in the BIO group. Interestingly, BIO decreased the number of apoptotic cells while PKF significantly induced apoptosis during chondrogenesis. The natural WNT antagonist DKK1 and the protein level of MMP1 in the pellet culture medium were decreased after PKF treatment. All of these chondrogenic effects appeared to be mediated through the canonical WNT signaling pathway, since the target gene Axin2 and other WNT members, such as TCF4 and β-catenin, were upregulated by BIO and downregulated by PKF, respectively, and BIO induced nuclear translocation of β-catenin while PKF inhibited β-catenin translocation into the nucleus. We concluded that addition of BIO to a chondrogenic medium of hMSCs resulted in a loss of cartilage formation, while PKF induced chondrogenic differentiation and cartilage matrix deposition and inhibited hypertrophic differentiation. However, BIO promoted cell survival by inhibiting apoptosis while PKF induced cell apoptosis. This result indicates that either an overexpression or overinhibition of WNT signaling to some extent causes harmful effects on chondrogenic differentiation. Cartilage tissue engineering could benefit from the adjustment of the critical level of WNT signaling during chondrogenesis of hMSC.

Shockwaves Inhibit Chondrogenic Differentiation of Human Mesenchymal Stem Cells in Association with Adenosine and A2B Receptors

Extracorporeal shockwave therapy (ESWT) has emerged as the important choice for the treatment of many orthopedic disorders. Our previous mechanistic studies suggest that ESWT promoted osteogenesis of human mesenchymal stem cells (hMSCs) through mechanisms that involve adenosine 5′-triphosphate (ATP) release. In this study, we investigated the effect of ESWT on chondrogenesis of hMSCs. We demonstrate that ESWT treatment caused a significant release of adenosine from hMSCs; ESWT treatment increased the levels of A2B receptor (A2BR) in hMSCs under 3-D culture conditions. ESWT, exogenous adenosine and specialized A2BR agonist suppressed hMSC chondrogenic differentiation through downregulating the expressions of aggrecan (ACAN), Collagen Type I alpha 2(COL1A2), Collagen Type II alpha 1(COL2A1), Sex-Determining Region YBox 9 (SOX9) and Sex-Determining Region YBox 6 (SOX6). Selective A2BR antagonists induced chondrogenic differentiation of hMSCs. This study indicated that shockwave therapy inhibits hMSC chondrogenic differentiation through or partially through regulation of adenosine release and activation of A2B receptor under 3-D culture conditions.

Self-assembled N-cadherin mimetic peptide hydrogels promote the chondrogenesis of mesenchymal stem cells through inhibition of canonical Wnt/β-catenin signaling.

N-cadherin, a transmembrane protein and major component of adherens junction, mediates cell-cell interactions and intracellular signaling that are important to the regulation of cell behaviors and organ development. Previous studies have identified mimetic peptides that possess similar bioactivity as that of N-cadherin, which promotes chondrogenesis of human mesenchymal stem cells (hMSCs); however, the molecular mechanism remains unknown. In this study, we combined the N-cadherin mimetic peptide (HAVDI) with the self-assembling KLD-12 peptide: the resultant peptide is capable of self-assembling into hydrogels functionalized with N-cadherin peptide in phosphate-buffered saline (PBS) at 37°C. Encapsulation of hMSCs in these hydrogels showed enhanced expression of chondrogenic marker genes and deposition of cartilage specific extracellular matrix rich in proteoglycan and Type II Collagen compared to control hydrogels, with a scrambled-sequence peptide after 14 days of chondrogenic culture. Furthermore, western blot showed a significantly higher expression of active glycogen synthase kinase-3β (GSK-3β), which phosphorylates β-catenin and facilitates ubiquitin-mediated degradation, as well as a lower expression of β-catenin and LEF1 in the N-cadherin peptide hydrogels versus controls. Immunofluorescence staining revealed significantly less nuclear localization of β-catenin in N-cadherin mimetic peptide hydrogels. Our findings suggest that N-cadherin peptide hydrogels suppress canonical Wnt signaling in hMSCs by reducing β-catenin nuclear translocation and the associated transcriptional activity of β-catenin/LEF-1/TCF complex, thereby enhancing the chondrogenesis of hMSCs. Our biomimetic self-assembled peptide hydrogels can serve as a tailorable and versatile three-dimensional culture platform to investigate the effect of biofunctionalization on stem cell behavior.

Sulfated hyaluronic acid hydrogels with retarded degradation and enhanced growth factor retention promote hMSC chondrogenesis and articular cartilage integrity with reduced hypertrophy.

In this paper, we conjugated sulfate groups to hyaluronic acid (HA) and demonstrated the slow degradation and growth factor delivery of sulfated HA. Furthermore, the in vitro and in vivo culture of hMSCs laden HA hydrogels proved that the sulfation of HA hydrogels not only promotes the chondrogenesis of hMSCs but also suppresses hypertrophic differentiation of the chondrogenically induced hMSCs. The animal OA model study showed that the injected sulfated HA hydrogels significantly reduced the cartilage abrasion and hypertrophy in the animal OA joints. We believe that this study will provide important insights into the design and optimization of the HA-based hydrogels as the scaffold materials for cartilage regeneration and OA treatment in clinical setting.

Construction of PLGA Nanoparticles Coated with Polycistronic SOX5, SOX6, and SOX9 Genes for Chondrogenesis of Human Mesenchymal Stem Cells.

Transfection of a cocktail of genes into cells has recently attracted attraction in stem cell differentiation. However, it is not easy to control the transfection rate of each gene. To control and regulate gene delivery into human mesenchymal stem cells (hMSCs), we employed multicistronic genes coupled with a nonviral gene carrier system for stem cell differentiation. Three genes, SOX5, SOX6, and SOX9, were successfully fabricated in a single plasmid. This multicistronic plasmid was complexed with the polycationic polymer polyethylenimine, and poly(lactic-co-glycolic) acid (PLGA) nanoparticles were coated with this complex. The uptake of PLGA nanoparticles complexed with the multicistronic plasmid was tested first. Thereafter, transfection of SOX5, SOX6, and SOX9 was evaluated, which increased the potential for chondrogenesis of hMSCs. The expression of specific genes triggered by transfection of SOX5, SOX6, and SOX9 was tested by RT-PCR and real-time qPCR. Furthermore, specific proteins related to chondrocytes were investigated by a glycosaminoglycan/DNA assay, Western blotting, histological analyses, and immunofluorescence staining. These methods demonstrated that chondrogenesis of hMSCs treated with PLGA nanoparticles carrying this multicistronic genes was better than that of hMSCs treated with other carriers. Furthermore, the multicistronic genes complexed with PLGA nanoparticles were more simple than that of each single gene complexation with PLGA nanoparticles. Multicistronic genes showed more chondrogenic differentiation than each single gene transfection methods.

Stem cell differentiation-related protein-loaded PLGA microspheres as a novel platform micro-typed scaffold for chondrogenesis

During cell differentiation for tissue regeneration, several factors, including growth factors and proteins, influence cascades in stem cells such as embryonic stem cells and mesenchymal stem cells (MSCs). In this study, transforming growth factor (TGF)-β3 and SOX9, which is an important protein in chondrocytes, were used to generate mature chondrocytes from human MSCs (hMSCs). For safe and effective delivery of bioactive molecules into hMSCs, biodegradable poly-(d,l-lactide-co-glycolide) (PLGA) microspheres (MSs) were coated with TGF-β3 and loaded with SOX9. Instead of SOX9 protein, release of the model protein FITC-bovine serum albumin (BSA) from PLGA MS was evaluated in vitro and in vivo by confocal laser microscopy and Kodak imaging. The bioactivities of TGF-β3 and SOX9 were evaluated by assessing α-helical formation using circular dichroism. PLGA MS loaded with FITC-BSA easily entered hMSCs without causing cytotoxicity. To confirm that internalization of PLGA MSs harboring TGF-β3 and SOX9 induced chondrogenesis of hMSCs, we performed several molecular analyses. By analysis, the specific marker gene expression levels in hMSCs adhered onto PLGA MSs coated with TGF-β3 and loaded with SOX9 were more than 3–5 times that of the control group both in vitro and in vivo. This result revealed that PLGA MS uptake and subsequent release of SOX9 induced chondrogenesis of hMSCs was enhanced by coating PLGA MSs with TGF-β3.