Chondrogenic Differentiation Of Mesenchymal Stem Cells Research Articles

Silk Fibroin Films with Microscopic Surface Pattern as Promising Cartilage Tissue Engineering Scaffold in Supporting MSCs Amplification and Chondrogenic Differentiation

ABSTRACT Mesenchymal stem cells (MSCs) with multipotential differentiation ability are main cell source for cartilage tissue engineering, but its application is restricted by the difficulty in regulating committed chondrogenic differentiation. Study revealed that certain substrate topographic structure can enhance the chondrogenic differentiation of MSCs. This study prepared silk fibroin (SF) films with gradient microscopic surface patterns based on P280-P7000 type specification sandpapers, and investigated the adhesion, proliferation and chondrogenic differentiation of human adipose-derived stem cells (hADSCs) on the SF films. The results showed that the SF films surface had convex dots with diameter of 3–50 μm, and hADSCs could adhere and proliferate well on the film. Transcriptomics analysis indicated that hADSCs tend to differentiate differently on the SF films, among which hADSCs on P1000 SF films had higher expression of chondrogenic markers Sox-9 and Col-II, with activated yes associated protein (YAP) and restricted F-actin. In vitro co-culture of human OA knee cartilage with hADSCs seeded SF films found satisfactory cartilage protection effect in P1000 group. Overall, this study preliminarily revealed the microscopic surface pattern of SF films may induce chondrogenic differentiation of hADSCs through extracellular matrix mediated Hippo/Yap pathway, and provided a new strategy for cartilage repair.

Gene-activated hyaluronic acid-based cryogels for cartilage tissue engineering

Articular cartilage injuries are very frequent lesions that if left untreated may degenerate into osteoarthritis. Gene transfer to mesenchymal stem cells (MSCs) provides a powerful approach to treat these lesions by promoting their chondrogenic differentiation into the appropriate cartilage phenotype. Non-viral vectors constitute the safest gene transfer tools, as they avoid important concerns of viral systems including immunogenicity and insertional mutagenesis. However, non-viral gene transfer usually led to lower transfection efficiencies when compared with their viral counterparts. Biomaterial-guided gene delivery has emerged as a promising alternative to increase non-viral gene transfer efficiency by achieving sustained delivery of the candidate gene into cellular microenvironment. In the present study, we designed hyaluronic acid-based gene-activated cryogels (HACGs) encapsulating a novel formulation of non-viral vectors based on niosomes (P80PX) to promote MSCs in situ transfection. The developed HACG P80PX systems showed suitable physicochemical properties to promote MSCs in situ transfection with very low cytotoxicity. Incorporation of a plasmid encoding for the transcription factor SOX9 (psox9) into HACG P80PX systems led to an effective MSCs chondrogenic differentiation with reduced expression of fibrocartilage and hypertrophic markers. The capacity of the developed systems to restore cartilage extracellular matrix was further confirmed in an ex vivo model of chondral defect.

Menstrual Blood-Derived Stem Cell Paracrine Factors Possess Stimulatory Effects on Chondrogenesis In Vitro and Diminish the Degradation of Articular Cartilage during Osteoarthritis.

Articular cartilage is an avascular tissue with a limited capacity for self-regeneration, leading the tissue to osteoarthritis (OA). Mesenchymal stem cells (MSCs) are promising for cartilage tissue engineering, as they are capable of differentiating into chondrocyte-like cells and secreting a number of active molecules that are important for cartilage extracellular matrix (ECM) synthesis. The aim of this study was to evaluate the potential of easily accessible menstrual blood-derived MSC (MenSC) paracrine factors in stimulating bone marrow MSC (BMMSCs) chondrogenic differentiation and to investigate their role in protecting cartilage from degradation in vitro. MenSCs and BMMSCs chondrogenic differentiation was induced using four different growth factors: TGF-β3, activin A, BMP-2, and IGF-1. The chondrogenic differentiation of BMMSCs was stimulated in co-cultures with MenSCs and cartilage explants co-cultured with MenSCs for 21 days. The chondrogenic capacity of BMMSCs was analyzed by the secretion of four growth factors and cartilage oligomeric matrix protein, as well as the release and synthesis of cartilage ECM proteins, and chondrogenic gene expression in cartilage explants. Our results suggest that MenSCs stimulate chondrogenic response in BMMSCs by secreting activin A and TGF-β3 and may have protective effects on cartilage tissue ECM by decreasing the release of GAGs, most likely through the modulation of activin A related molecular pathway. In conclusion, paracrine factors secreted by MenSCs may turn out to be a promising therapeutical approach for cartilage tissue protection and repair.

Multifunctional mesoporous polydopamine near-infrared photothermal controlled release of kartogenin for cartilage repair

Improving chondrogenic differentiation while inhibiting hypertrophic differentiation of mesenchymal stem cells (MSCs) is of vital importance to effectively repairing cartilage injury. Accordingly, kartogenin (KGN) and antioxidants, which promote chondrogenic differentiation and inhibit hypertrophic differentiation, respectively, have shown great potential in promoting cartilage repair. However, KGN is poorly soluble in water, hindering efficient intracellular delivery. Near-infrared light (NIR)-responsive mesoporous polydopamine nanoparticles (MPDA) reportedly exert antioxidative effects by eliminating reactive oxygen species (ROS). In this study, we assessed whether KGN loaded in MPDA can accelerate cartilage injury repair in rats. Specifically, a thermosensitive phase-change material (PCM, melting point 39 °C) combined with KGN was loaded into MPDA as a gatekeeper (KGN@MPDA-PCM). The results showed that KGN@MPDA-PCM exhibited excellent photothermal properties under NIR irradiation, which induced PCM melting and consequent KGN release. In combination with antioxidative therapy, NIR-triggered KGN release enabled the nanocomposite to accelerate MSC chondrogenic differentiation and inhibit hypertrophic differentiation. Importantly, negligible damage to primary organs and satisfactory biocompatibility were observed in the rat model. RNA sequencing found that this MPDA-based platform promotes chondrogenic differentiation by promoting fibronectin-1 (Fn1) expression and activating the PI3K/Akt pathway. Collectively, these findings highlight the therapeutic potential of this MPDA-based platform for accelerating cartilage injury repair.

TGF-β1 derived from macrophages contributes to load-induced tendon-bone healing in the murine rotator cuff repair model by promoting chondrogenesis

It has been established that mechanical stimulation benefits tendon-bone (T-B) healing, and macrophage phenotype can be regulated by mechanical cues; moreover, the interaction between macrophages and mesenchymal stem cells (MSCs) plays a fundamental role in tissue repair. This study aimed to investigate the role of macrophage-mediated MSC chondrogenesis in load-induced T-B healing in depth. C57BL/6 mice rotator cuff (RC) repair model was established to explore the effects of mechanical stimulation on macrophage polarization, transforming growth factor (TGF)-β1 generation, and MSC chondrogenesis within T-B enthesis by immunofluorescence and enzyme-linked immunosorbent assay (ELISA). Macrophage depletion was performed by clodronate liposomes, and T-B healing quality was evaluated by histology and biomechanics. In vitro, bone marrow-derived macrophages (BMDMs) were stretched with CELLOAD-300 load system and macrophage polarization was identified by flow cytometry and quantitative real-time polymerase chain reaction (qRT-PCR). MSC chondrogenic differentiation was measured by histochemical analysis and qRT-PCR. ELISA and qRT-PCR were performed to screen the candidate molecules that mediated the pro-chondrogenic function of mechanical stimulated BMDMs. Mechanical stimulation promoted macrophage M2 polarization in vivo and in vitro. The conditioned media from mechanically stimulated BMDMs (MS-CM) enhanced MSC chondrogenic differentiation, and mechanically stimulated BMDMs generated more TGF-β1. Further, neutralizing TGF-β1 in MS-CM can attenuate its pro-chondrogenic effect. In vivo, mechanical stimulation promoted TGF-β1 generation, MSC chondrogenesis, and T-B healing, which were abolished following macrophage depletion. Macrophages subjected to appropriate mechanical stimulation could polarize toward the M2 phenotype and secrete TGF-β1 to promote MSC chondrogenesis, which subsequently augments T-B healing.

Scaffolds containing GAG-mimetic cellulose sulfate promote TGF-β interaction and MSC Chondrogenesis over native GAGs.

Cartilage tissue engineering strategies seek to repair damaged tissue using approaches that include scaffolds containing components of the native extracellular matrix (ECM). Articular cartilage consists of glycosaminoglycans (GAGs) which are known to sequester growth factors. In order to more closely mimic the native ECM, this study evaluated the chondrogenic differentiation of mesenchymal stem cells (MSCs), a promising cell source for cartilage regeneration, on fibrous scaffolds that contained the GAG-mimetic cellulose sulfate. The degree of sulfation was evaluated, examining partially sulfated cellulose (pSC) and fully sulfated cellulose (NaCS). Comparisons were made with scaffolds containing native GAGs (chondroitin sulfate A, chondroitin sulfate C and heparin). Transforming growth factor-beta3 (TGF-β3) sequestration, as measured by rate of association, was higher for sulfated cellulose-containing scaffolds as compared to native GAGs. In addition, TGF-β3 sequestration and retention over time was highest for NaCS-containing scaffolds. Sulfated cellulose-containing scaffolds loaded with TGF-β3 showed enhanced chondrogenesis as indicated by a higher Collagen Type II:I ratio over native GAGs. NaCS-containing scaffolds loaded with TGF-β3 had the highest expression of chondrogenic markers and a reduction of hypertrophic markers in dynamic loading conditions, which more closely mimic in vivo conditions. Studies also demonstrated that TGF-β3 mediated its effect through the Smad2/3 signaling pathway where the specificity of TGF-β receptor (TGF- βRI)-phosphorylated SMAD2/3 was verified with a receptor inhibitor. Therefore, studies demonstrate that scaffolds containing cellulose sulfate enhance TGF-β3-induced MSC chondrogenic differentiation and show promise for promoting cartilage tissue regeneration.

Functional injectable hydrogel with spatiotemporal sequential release for recruitment of endogenous stem cells and in situ cartilage regeneration.

Articular cartilage is refractory to self-healing due to the absence of vascular, nervous, and lymphatic systems, and its repair remains a clinical challenge. Tissue regeneration through in situ recruitment of stem cells via cell-free scaffolds is a promising alternative strategy. Herein, a kind of functional injectable hydrogel system (Col-Apt@KGN MPs), which is a collagen-based and microsphere-embedded cell-free scaffold, was designed to achieve spatiotemporal regulation of endogenous mesenchymal stem cells (MSCs) recruitment and their chondrogenic differentiation by respective release of aptamer 19S (Apt19S) and kartogenin (KGN). In vitro results confirmed that the Col-Apt@KGN MPs hydrogel had sequential release characteristics. Apt19S was rapidly released from the hydrogel within 6 days, while KGN was slowly released for 33 days via the degradation of poly(lactic-co-glycolic acid) (PLGA) microspheres. When cultured with MSCs, the Col-Apt@KGN MPs hydrogel supported the adhesion, proliferation, and chondrogenic differentiation of MSCs. In vivo results indicated that the Col-Apt@KGN MPs hydrogel effectively promoted the recruitment of endogenous MSCs in a rabbit full-thickness cartilage defect model; furthermore, the Col-Apt@KGN MPs hydrogel enhanced the secretion of cartilage specific extracellular matrix and achieved the reconstruction of subchondral bone. This study demonstrates that the Col-Apt@KGN MPs hydrogel possesses great potential in recruitment of endogenous stem cells and cartilage tissue regeneration.

The Role of Extracellular Matrix and Hydrogels in Mesenchymal Stem Cell Chondrogenesis and Cartilage Regeneration.

Diseases associated with articular cartilage disintegration or loss are still therapeutically challenging. The traditional treatment approaches only alleviate the symptoms while potentially causing serious side effects. The limited self-renewal potential of articular cartilage provides opportunities for advanced therapies involving mesenchymal stem cells (MSCs) that are characterized by a remarkable regenerative capacity. The chondrogenic potential of MSCs is known to be regulated by the local environment, including soluble factors and the less discussed extracellular matrix (ECM) components. This review summarizes the process of chondrogenesis, and also the biological properties of the ECM mediated by mechanotransduction as well as canonical and non-canonical signaling. Our focus is also on the influence of the ECM's physical parameters, molecular composition, and chondrogenic factor affinity on the adhesion, survival, and chondrogenic differentiation of MSCs. These basic biological insights are crucial for a more precise fabrication of ECM-mimicking hydrogels to improve cartilage tissue reconstruction. Lastly, we provide an overview of hydrogel classification and characterization. We also include the results from preclinical models combining MSCs with hydrogels for the treatment of cartilage defects, to support clinical application of this construct. Overall, it is believed that the proper combination of MSCs, hydrogels, and chondrogenic factors can lead to complex cartilage regeneration.

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.

The effect of matrix stiffness on the chondrogenic differentiation of mesenchymal stem cells.

Articular cartilage is one of the most important weight-bearing components in human body, thus the chondrogenesis of stem cells is reactive to many intracellular and extracellular mechanical signals. As a unique physical cue, matrix stiffness plays an integral role in commitment of stem cell fate. However, when examining the downstream effects of matrix stiffness, most studies used different soluble factors to assist physical inducing process, which may mask the chondrogenic effects of matrix stiffness. Here we fabricated polyacrylamide (PAAm) hydrogels with gradient stiffness to unravel the role of matrix stiffness in chondrogenic process of mesenchymal stem cells (MSCs), with or without TGF-β3 as induction factor. The results showed that with micromass culture mimicking relatively high cell density in vivo, the chondrogenic differentiation of MSCs can be promoted by soft substrates (about 0.5kPa) independently with assembled cytoskeleton. Further analysis indicated that addition of TGF-β3 generally increased expression level of cartilage-related markers and masked the stiffness-derived expression pattern of hypertrophic markers. These results demonstrate how mechanical cues experienced in developmental context regulate commitment of stem cell fate and have significant impact on the design of tissue regeneration materials.

N-cadherin mimetic hydrogel enhances MSC chondrogenesis through cell metabolism

Mesenchymal stem cells (MSCs) are ideal candidates for tissue engineering and regenerative medicine because of their proliferative capacity and differentiation potential. However, the hypertrophic phenotype occurring in late MSCs chondrogenic differentiation severely limits their clinical translation. While hypertrophy inhibition strategies have been explored, the role of cell metabolism in MSCs chondrogenesis has rarely been studied. In this study, we found that hypertrophy occurred in the late stage of MSCs chondrogenesis with increased fatty acid oxidation (FAO) and decreased glycolysis, as well as cell-cell junctions impairment. Therefore, a N-cadherin mimetic hydrogel was developed to enhance cell-cell junctions via N-cadherin mimetic peptides and high seeding density. The N-cadherin mimetic hydrogel attenuated hypertrophy through regulating glycolysis and FAO. The regulation of cell-cell junctions mechanotransduction on cell metabolism was partly mediated by Hif-1α. In addition, 2D and 3D culture of N-cadherin mimetic hydrogel had similar functions on N-cadherin expression and chondrogenesis in MSCs. Our study is the first to reveal that metabolic remodeling induced hypertrophy during MSCs chondrogenesis, and indicate the effect of N-cadherin mimetic hydrogel on hypertrophy inhibition of MSCs. Statement of significanceThe development of hypertrophy during MSCs chondrogenesis severely limits its clinical translation. Various strategies have been explored to inhibit hypertrophy by chemical and/or mechanical stimulation. However, the role of cell metabolism in MSCs chondrogenesis has rarely been studied. In this study, we developed an RNA sequencing at day 0, 7, and 21 of MSCs chondrogenesis to clarify the mechanisms that mediate hypertrophy. We found that hypertrophy occurred in the late stage of MSCs chondrogenesis with increased FAO and decreased glycolysis, as well as impaired cell-cell junctions. We also found that N-cadherin mimetic hydrogel attenuated hypertrophy and enhanced chondrogenesis through regulating glycolysis and FAO. Our finding provides new insights into the application of MSCs in tissue engineering and regenerative medicine.

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.

RHOA inhibits chondrogenic differentiation of mesenchymal stem cells in adolescent idiopathic scoliosis

ABSTRACT Purpose The etiology of adolescent idiopathic scoliosis (AIS) remains unclear. The chondrogenic differentiation of mesenchymal stem cells (MSCs) is important in AIS, and the Ras homolog gene family member A (RHOA) is associated with chondrogenesis. The purpose of this study was to explore the effect of RHOA on the chondrogenic differentiation of MSCs in AIS. Methods We isolated MSCs from patients with AIS (AIS MSCs) and individuals without AIS (control MSCs). The inhibitor Y27632 was used to inhibit the function of RHOA/ROCK signaling, and plasmid-based overexpression and siRNA-mediated knockdown were used to manipulate RHOA expression. CCK-8 was used to detect cell viability. The phosphorylation levels of LIMK1, MLC2 and cofilin were detected by Western blotting. The mRNA expression of aggrecan, SOX9, and COL2A1 were confirmed using RT-PCR. Immunofluorescence was used to analyze F-actin and collagen II. Alcian blue staining was performed to assess the secretion of glycosaminoglycans (GAGs). Results We found that RHOA was significantly upregulated in AIS MSCs, and the phosphorylation levels of LIMK1, MLC2, and cofilin were increased. The mRNA expressions of aggrecan, SOX9, and COL2A1 were notably reduced in AIS MSCs. However, these effects were abolished by Y27632 treatment and RHOA knockdown in AIS MSCs. In addition, RHOA knockdown in AIS MSCs increased the content of collagen II and GAGs. RHOA overexpression in the control MSCs markedly activated the RHOA/ROCK signaling and decreased the expression of aggrecan, SOX9, and COL2A1, F-actin, and GAGs. Conclusion RHOA regulates the chondrogenic differentiation ability of MSCs in AIS via the RHOA/ROCK signaling pathway and this regulation may involve SOX9.

Soft substrates direct stem cell differentiation into the chondrogenic lineage without the use of growth factors

Mesenchymal stem cells (MSCs) hold great promise for the treatment of cartilage related injuries. However, selectively promoting stem cell differentiation in vivo is still challenging. Chondrogenic differentiation of MSCs usually requires the use of growth factors that lead to the overexpression of hypertrophic markers. In this study, for the first time the effect of stiffness on MSC differentiation has been tested without the use of growth factors. Three-dimensional collagen and alginate scaffolds were developed and characterised. Stiffness significantly affected gene expression and ECM deposition. While, all hydrogels supported chondrogenic differentiation and allowed deposition of collagen type II and aggrecan, the 5.75 kPa hydrogel showed limited production of collagen type I compared to the other two formulations. These findings demonstrated for the first time that stiffness can guide MSCs differentiation without the use of growth factors within a tissue engineering scaffold suitable for the treatment of cartilage defects.

Spatiotemporal regulation of endogenous MSCs using a functional injectable hydrogel system for cartilage regeneration

Hydrogels have been extensively favored as drug and cell carriers for the repair of knee cartilage defects. Recruiting mesenchymal stem cells (MSCs) in situ to the defect region could reduce the risk of contamination during cell delivery, which is a highly promising strategy to enhance cartilage repair. Here, a cell-free cartilage tissue engineering (TE) system was developed by applying an injectable chitosan/silk fibroin hydrogel. The hydrogel system could release first stromal cell-derived factor-1 (SDF-1) and then kartogenin (KGN) in a unique sequential drug release mode, which could spatiotemporally promote the recruitment and chondrogenic differentiation of MSCs. This system showed good performance when formulated with SDF-1 (200 ng/mL) and PLGA microspheres loaded with KGN (10 μΜ). The results showed that the hydrogel had good injectability and a reticular porous structure. The microspheres were distributed uniformly in the hydrogel and permitted the sequential release of SDF-1 and KGN. The results of in vitro experiments showed that the hydrogel system had good cytocompatibility and promoted the migration and differentiation of MSCs into chondrocytes. In vivo experiments on articular cartilage defects in rabbits showed that the cell-free hydrogel system was beneficial for cartilage regeneration. Therefore, the composite hydrogel system shows potential for application in cell-free cartilage TE.

Biofabrication of cell-free dual drug-releasing biomimetic scaffolds for meniscal regeneration

Regenerating the meniscus remains challenging because of its avascular, aneural, and alymphatic nature. Three-dimensional (3D) printing technology provides a promising strategy to fabricate biomimetic meniscal scaffolds with an anisotropic architecture, a proper biomechanical microenvironment, and bioactive components. Herein, 3D printing technology is adopted by coencapsulating chemokines (platelet-derived growth factor-BB, PDGF-BB) and small chondroinductive molecules (kartogenin, KGN) within biomimetic polycaprolactone/hydrogel composite scaffolds. The incorporated PDGF-BB is expected to promote endogenous stem cell homing, and KGN in poly(lactic-co-glycolic) acid microspheres is employed to target the chondrogenesis of resident mesenchymal stem cells (MSCs). First, we chose basic bioinks composed of gelatin methacrylamide and hyaluronic acid methacrylate and then incorporated four concentrations (0%, 0.5%, 1.0%, and 2.0%) of meniscal extracellular matrix into the bioink to systematically study the superiority of these combinations and identify the optimally printable bioink. Next, we investigated the scaffold morphology and drug release profile. The effects of releasing the drugs in a sequentially controlled manner from the composite scaffolds on the fate of MSCs were also evaluated. The biofabricated scaffolds, with and without dual drug loading, were further studied in a rabbit model established with a critical-size medial meniscectomy. We found that meniscal scaffolds containing both drugs had combinational advantages in enhancing cell migration and synergistically promoted MSC chondrogenic differentiation. The dual drug-loaded scaffolds also significantly promoted in vivo neomeniscal regeneration three and six months after implantation in terms of histological and immunological phenotypes. The results presented herein reveal that this 3D-printed dual drug-releasing meniscal scaffold possesses the potential to act as an off-the-shelf product for the clinical treatment of meniscal injury and related joint degenerative diseases.

Nanosecond pulsed electric fields prime mesenchymal stem cells to peptide ghrelin and enhance chondrogenesis and osteochondral defect repair in vivo.

Mesenchymal stem cells (MSCs) are important cell sources in cartilage tissue development and homeostasis, and multiple strategies have been developed to improve MSCs chondrogenic differentiation with an aim of promoting cartilage regeneration. Here we report the effects of combining nanosecond pulsed electric fields (nsPEFs) followed by treatment with ghrelin (a hormone that stimulates release of growth hormone) to regulate chondrogenesis of MSCs. nsPEFs and ghrelin were observed to separately enhance the chondrogenesis of MSCs, and the effects were significantly enhanced when the bioelectric stimulation and hormone were combined, which in turn improved osteochondral tissue repair of these cells within Sprague Dawley rats. We further found that nsPEFs can prime MSCs to be more receptive to subsequent stimuli of differentiation by upregulated Oct4/Nanog and activated JNK signaling pathway. Ghrelin initiated chondrogenic differentiation by activation of ERK1/2 signaling pathway, and RNA-seq results indicated 243 genes were regulated, and JAK-STAT signaling pathway was involved. Interestingly, the sequential order of applying these two stimuli is critical, with nsPEFs pretreatment followed by ghrelin enhanced chondrogenesis of MSCs in vitro and subsequent cartilage regeneration in vivo, but not vice versa. This synergistic prochondrogenic effects provide us new insights and strategies for future cell-based therapies.

Near-Field Direct Write Microfiber-Reinforced Collagen Hydrogel Scaffolds for Articular Cartilage Regeneration

The demand for treating degenerative osteoarthritis is dramatically growing during the past decade due to the increasing aging population. Compared to arthroscopic debridement and injection of lubricating hydrogels, implantation of a cartilage tissue engineering scaffold with a customized architecture and excellent stem cell delivery ability has been deemed as a superior alternative to regenerate defected cartilage tissues. However, relatively low mechanical strength of cell-laden hydrogel limits its potential in treating diseased/defected cartilage or tissues with an anisotropic structure. In this study, a composite cell-laden scaffold composed of three-dimensional (3D) fibrous patterns which was made through near-field electrospinning direct write (NEDW) and mesenchymal stem cell (MSC)-laden collagen I (COL I) hydrogel was fabricated. The hydrogel acted as a favorable microenvironment to load cells and growth factors to facilitate chondrogenic differentiation while the 3D fibrous patterns made of poly(caprolactone)-reinforced hydrogels and provided the composite scaffolds with a precisely designed micro-architecture. Compared to hydrogel scaffold alone, the fabricated composite scaffold had significantly higher mechanical properties. The composite scaffolds were biocompatible and can accelerate the chondrogenic differentiation of MSCs by up-regulating the expression of SOX9, collagen II (COL II) and aggrecan (ACAN), when transforming growth factor (TGF)-[Formula: see text]1 was loaded in COL I hydrogel. However, the morphology of differentiated cells was not identical to that of the natural chondrocytes, suggesting that the modulation of initial MSC morphology is needed to pursue the target phenotype after the chondrogenic differentiation.

Inhibition versus activation of canonical Wnt-signaling, to promote chondrogenic differentiation of Mesenchymal Stem Cells. A review.

Canonical Wnt signaling regulation is essential for controlling stemness and differentiation of mesenchymal stem cells (MSCs). However, the mechanism through which canonical Wnt-dependent MSC lineage commitment leads to chondrogenesis is controversial. Some studies hypothesize that inhibition of canonical Wnt signaling induces MSC chondrogenic differentiation, while others support that the pathway should be activated to achieve MSC chondrogenesis. The purpose of the present review is to analyze data from recent studies to elucidate parameters regarding the role of canonical Wnt signaling in MSC chondrogenic differentiation.