Chondrogenic Microenvironment Research Articles

Extremely low frequency–electromagnetic fields promote chondrogenic differentiation of adipose-derived mesenchymal stem cells through a conventional genetic program

Progressive cartilage deterioration leads to chronic inflammation and loss of joint function, causing osteoarthritis (OA) and joint disease. Although symptoms vary among individuals, the disease can cause severe pain and permanent disability, and effective therapies are urgently needed. Human Adipose-Derived Stem Cells (ADSCs) may differentiate into chondrocytes and are promising for treating OA. Moreover, recent studies indicate that electromagnetic fields (EMFs) could positively affect the chondrogenic differentiation potential of ADSCs. In this work, we investigated the impact of EMFs with frequencies of 35 Hertz and 58 Hertz, referred to as extremely low frequency-EMFs (ELF–EMFs), on the chondrogenesis of ADSCs, cultured in both monolayer and 3D cell micromasses. ADSC cultures were daily stimulated for 36 min with ELF–EMFs or left unstimulated, and the progression of the differentiation process was evaluated by morphological analysis, extracellular matrix deposition, and gene expression profiling of chondrogenic markers. In both culturing conditions, stimulation with ELF–EMFs did not compromise cell viability but accelerated chondrogenesis by enhancing the secretion and deposition of extracellular matrix components at earlier time points in comparison to unstimulated cells. This study showed that, in an appropriate chondrogenic microenvironment, ELF–EMFs enhance chondrogenic differentiation and may be an important tool for supporting and accelerating the treatment of OA through autologous adipose stem cell therapy.

A Hybrid Scaffold Induces Chondrogenic Differentiation and Enhances In Vivo Cartilage Regeneration.

Extensively researched tissue engineering strategies involve incorporating cells into suitable biomaterials, offering promising alternatives to boost tissue repair. In this study, a hybrid scaffold, Gel-DCM, which integrates a photoreactive gelatin-hyaluronic acid hydrogel (Gel) with an oriented porous decellularized cartilage matrix (DCM), was designed to facilitate chondrogenic differentiation and cartilage repair. The Gel-DCM exhibited excellent biocompatibility in vitro, promoting favorable survival and growth of human adipose-derived stem cells (hADSCs) and articular chondrocytes (hACs). Gene expression analysis indicated that the hACs expanded within the Gel-DCM exhibited enhanced chondrogenic phenotype. In addition, Gel-DCM promoted chondrogenesis of hADSCs without the supplementation of exogenous growth factors. Following this, invivo experiments were conducted where empty Gel-DCM or Gel-DCM loaded with hACs/hADSCs were used and implanted to repair osteochondral defects in a rat model. In the control group, no implants were delivered to the injury site. Interestingly, macroscopic, histological, and microcomputed tomography scanning results revealed superior cartilage restoration and subchondral bone reconstruction in the empty Gel-DCM group compared with the control group. Moreover, both hACs-loaded and hADSCs-loaded Gel-DCM implants exhibited superior repair of hyaline cartilage and successful reconstruction of subchondral bone, whereas defects in the control groups were predominantly filled with fibrous tissue. These observations suggest that the Gel-DCM can provide an appropriate three-dimensional chondrogenic microenvironment, and its combination with reparative cell sources, ACs or ADSCs, holds great potential for facilitating cartilage regeneration.

Tendon stem/progenitor cells are promising reparative cell sources for multiple musculoskeletal injuries of concomitant articular cartilage lesions associated with ligament injuries

BackgroundTrauma-related articular cartilage lesions usually occur in conjunction with ligament injuries. Torn ligaments are frequently reconstructed with tendon autograft and has been proven to achieve satisfactory clinical outcomes. However, treatments for the concomitant articular cartilage lesions are still very insufficient. The current study was aimed to evaluate whether stem cells derived from tendon tissue can be considered as an alternative reparative cell source for cartilage repair.MethodsPrimary human tendon stem/progenitor cells (hTSPCs) were isolated from 4 male patients (32 ± 8 years) who underwent ACL reconstruction surgery with autologous semitendinosus and gracilis tendons. The excessive tendon tissue after graft preparation was processed for primary cell isolation with an enzyme digestion protocol. Decellularization cartilage matrix (DCM) was used to provide a chondrogenic microenvironment for hTSPCs. Cell viability, cell morphology on the DCM, as well as their chondrogenic differentiation were evaluated.ResultsDAPI staining and DNA quantitative analysis (61.47 μg per mg dry weight before and 2.64 μg/mg after decellularization) showed that most of the cells in the cartilage lacuna were removed after decellularization process. Whilst, the basic structure of the cartilage tissue was preserved and the main ECM components, collagen type II and sGAG were retained after decellularization, which were revealed by DMMB assay and histology. Live/dead staining and proliferative assay demonstrated that DCM supported attachment, survival and proliferation of hTSPCs with an excellent biocompatibility. Furthermore, gene expression analysis indicated that chondrogenic differentiation of hTSPC was induced by the DCM microenvironment, with upregulation of chondrogenesis-related marker genes, COL 2 and SOX9, without the use of exogenous growth factors.ConclusionDCM supported hTSPCs attachment and proliferation with high biocompatibility. Moreover, TSPCs underwent a distinct chondrogenesis after the induction of a chondrogenic microenvironment provided by DCM. These results indicated that TSPCs are promising reparative cell sources for promoting cartilage repair. Particularly, in the cohort that articular cartilage lesions occur in conjunction with ligament injuries, autologous TSPCs can be isolated from a portion of the tendon autograph harvested for ligaments reconstruction. In future clinical practice, combined ligament reconstruction with TSPCs- based therapy for articular cartilage repair can to be considered to achieve superior repair of these associated injuries, in which autologous TSPCs can be isolated from a portion of the tendon autograph harvested for ligaments reconstruction.

Injectable hypoxia-preconditioned cartilage progenitor cells-laden GelMA microspheres system for enhanced osteoarthritis treatment.

Osteoarthritis (OA) is the most common age-related degenerative joint disease mainly characterized by the destruction of articular cartilage. Owing to its native avascular property, intrinsic repair of articular cartilage is very limited. Thus, a chondrogenic microenvironment in the joint is essential to the preservation of healthy chondrocytes and OA treatment. Recently, cartilage progenitor cells (CPCs)-based therapy is emerging as a promising strategy to repair degenerated and damaged articular cartilage. In this study, injectable hypoxia-preconditioned three-dimensional (3D) cultured CPCs-laden gelatin methacryloyl (GelMA) microspheres (CGMs) were constructed and characterized. Compared to normoxia-pretreated 3D CPCs and two-dimensional (2D) cultured CPCs, hypoxia-preconditioned 3D cultured CPCs exhibited enhanced cartilage extracellular matrix (ECM) secretion and greater chondrogenic ability. In addition, hypoxia-preconditioned 3D cultured CPCs more effectively maintained cartilage matrix metabolism balance and attenuated articular cartilage degeneration in subacute and chronic rat OA models. Mechanistically, our results demonstrated hypoxia-preconditioned 3D cultured CPCs exerted chondro-protective effects by inhibiting inflammation and oxidative stress via NRF2/HO-1 pathway in vitro and in vivo. Together, through the 3D culture of CPCs using GelMA microspheres (GMs) under hypoxia environment, our results proposed an efficient articular cartilage regeneration strategy for OA treatment and could provide inspiration for other stem cells-based therapies.

Porous scaffolds with enzyme-responsive Kartogenin release for recruiting stem cells and promoting cartilage regeneration

In situ cartilage regeneration with endogenous stem/progenitor cells (ESPCs) and bioactive scaffolds is attractive in cartilage regeneration. Nevertheless, the optimization of ESPCs recruitment and the establishment of chondrogenic microenvironment are still a huge challenge for better therapeutic outcomes. Herein, a in situ cartilage tissue engineering porous scaffold with enzyme-responsive kartogenin (KGN) release is prepared to not only recruit ESPCs via chemoattractant, but also promote their chondrogenesis. Polyethylene glycol-modified Kartogenin (PEGKGN) and polycaprolactone (PCL) are grafted on poly(L-glutamic acid) (PLGA) to endow the porous scaffolds with good mechanical property, capillarity and enzyme-responsiveness. The scaffolds show a fast absorption of protein solution through capillarity, indicating its capacity of rapid fill with bone marrow blood at the early stage after implantation for the maximum absorption and retention of ESPCs. The release dose of PEGKGN is found to be accelerated by matrix metalloproteinase-2 (MMP-2). And the high-dose PEGKGN can stimulate the migration of bone marrow mesenchyml stem cells (BMSCs) obviously, and the low-dose PEGKGN can promote the chondrogenesis of BMSCs in vitro. The in vivo data demonstrates that the bioactive porous scaffolds enhance in situ cartilage regeneration. The neo-tissues show close structures and components to the healthy cartilages. Thus, the in situ cartilage tissue engineering porous scaffolds with enzyme-responsive KGN release can recruit ESPCs to defects and promote the chondrogenesis effectively, which is a potential treatment for in situ cartilage regeneration.

Perichondrium-inspired permeable nanofibrous tube well promoting differentiation of hiPSC-derived pellet toward hyaline-like cartilage pellet

The pellet formation has been regarded as a golden standard for in vitro chondrogenic differentiation. However, a spatially inhomogeneous chondrogenic microenvironment around a pellet resulted from the use of a traditional impermeable narrow tube, such as the conical tube, undermines the differentiation performance and therapeutic potential of differentiated cartilage pellet in defective articular cartilage treatment. To address this drawback, a perichondrium-inspired permeable nanofibrous tube (PINaT) well with a nanofibrous wall permeable to gas and soluble molecules is proposed. The PINaT well was fabricated with a micro deep drawing process where a flat thin nanofibrous membrane was transformed to a 3.5 mm deep tube well with a ∼50 µm thick nanofibrous wall. Similar to in vivo perichondrium, the PINaT well was found to allow oxygen and growth factor diffusion required for chondrogenic differentiation across the entire nanofibrous wall. Analyses of gene expressions (COL2A1, COL10A1, ACAN, and SOX9), proteins (type II and X collagen), and glycosaminoglycans contents were conducted to assess the differentiation performance and clinical efficacy of differentiated cartilage pellet. The regulated spatially homogeneous chondrogenic microenvironment around the human induced pluripotent stem cell-derived pellet (3 × 105 cells per pellet) in the PINaT well remarkably improved the quality of the differentiated pellet toward a more hyaline-like cartilage pellet. Furthermore, an accelerated chondrogenic differentiation process of the pellet produced by the PINaT well was achieved for 14 days, demonstrating a hyaline cartilage-specific marker similar to the control pellet differentiated for 20 days. Finally, the enhanced clinical efficacy of the hyaline-like cartilage pellet was confirmed using an osteochondral defect rat model, with the repaired tissue resembling hyaline cartilage rather than fibrous cartilage after 8 weeks of regeneration.

Solubilized Cartilage ECM Facilitates the Recruitment and Chondrogenesis of Endogenous BMSCs in Collagen Scaffolds for Enhancing Microfracture Treatment

Articular cartilage has very poor intrinsic healing ability and its repair remains a significant clinical challenge. To promote neocartilage regeneration, we fabricated two collagen (Col) scaffolds functionalized with a porcine decellularized extracellular matrix (dECM) in the forms of particle and solution named pE-Col and sE-Col, respectively. Their differences were systematically compared, including the biochemical compositions, scaffold properties, cell-material interactions, and in situ cartilage regeneration. While it is demonstrated that both forms of dECM could enhance the cell recruitment, proliferation, and chondrogenesis of bone marrow stem cells (BMSCs) in vitro, better performance was seen in the sE-Col group, which could quickly provide a more favorable chondrogenic microenvironment for endogenous BMSCs. The superiority of sE-Col was also proved by our in vivo study, which showed that the sE-Col scaffold achieved better structural hyaline-like neocartilage formation and subchondral bone repair compared to the pE-Col scaffold, according to the gross morphology, biological assessment, and micro-CT imaging analysis. Together, this study suggests that the sE-Col scaffold holds great potential in developing the one-step microfracture-based strategy for cartilage repair and also reminds us that despite dECM being a promising biomaterial in tissue engineering, the optimization of the proper processing methodology would be a crucial consideration in the future design of dECM-based scaffolds in articular cartilage regeneration.

Repopulation of decellularised articular cartilage by laser-based matrix engraving.

BackgroundIn spite of advances in the treatment of cartilage defects using cell and scaffold-based therapeutic strategies, the long-term outcome is still not satisfying since clinical scores decline years after treatment. Scaffold materials currently used in clinical settings have shown limitations in providing suitable biomechanical properties and an authentic and protective environment for regenerative cells. To tackle this problem, we developed a scaffold material based on decellularised human articular cartilage.MethodsHuman articular cartilage matrix was engraved using a CO2 laser and treated for decellularisation and glycosaminoglycan removal. Characterisation of the resulting scaffold was performed via mechanical testing, DNA and GAG quantification and in vitro cultivation with adipose-derived stromal cells (ASC). Cell vitality, adhesion and chondrogenic differentiation were assessed. An ectopic, unloaded mouse model was used for the assessment of the in vivo performance of the scaffold in combination with ASC and human as well as bovine chondrocytes. The novel scaffold was compared to a commercial collagen type I/III scaffold.FindingsCrossed line engravings of the matrix allowed for a most regular and ubiquitous distribution of cells and chemical as well as enzymatic matrix treatment was performed to increase cell adhesion. The biomechanical characteristics of this novel scaffold that we term CartiScaff were found to be superior to those of commercially available materials. Neo-tissue was integrated excellently into the scaffold matrix and new collagen fibres were guided by the laser incisions towards a vertical alignment, a typical feature of native cartilage important for nutrition and biomechanics. In an ectopic, unloaded in vivo model, chondrocytes and mesenchymal stromal cells differentiated within the incisions despite the lack of growth factors and load, indicating a strong chondrogenic microenvironment within the scaffold incisions. Cells, most noticeably bone marrow-derived cells, were able to repopulate the empty chondrocyte lacunae inside the scaffold matrix.InterpretationDue to the better load-bearing, its chondrogenic effect and the ability to guide matrix-deposition, CartiScaff is a promising biomaterial to accelerate rehabilitation and to improve long term clinical success of cartilage defect treatment.FundingAustrian Research Promotion Agency FFG (“CartiScaff” #842455), Lorenz Böhler Fonds (16/13), City of Vienna Competence Team Project Signaltissue (MA23, #18-08)

3D Printed Cartilage-Like Tissue Constructs with Spatially Controlled Mechanical Properties.

Developing biomimetic cartilaginous tissues that support locomotion while maintaining chondrogenic behavior is a major challenge in the tissue engineering field. Specifically, while locomotive forces demand tissues with strong mechanical properties, chondrogenesis requires a soft microenvironment. To address this challenge, 3D cartilage-like tissue is bioprinted using two biomaterials with different mechanical properties: a hard biomaterial to reflect the macromechanical properties of native cartilage, and a soft biomaterial to create a chondrogenic microenvironment. To this end, a hard biomaterial (MPa order compressive modulus) composed of an interpenetrating polymer network (IPN) of polyethylene glycol (PEG) and alginate hydrogel is developed as an extracellular matrix (ECM) with self-healing properties, but low diffusive capacity. Within this bath supplemented with thrombin, fibrinogen containing human mesenchymal stem cell (hMSC) spheroids is bioprinted forming fibrin, as the soft biomaterial (kPa order compressive modulus) to simulate cartilage's pericellular matrix and allow a fast diffusion of nutrients. The bioprinted hMSC spheroids improve viability and chondrogenic-like behavior without adversely affecting the macromechanical properties of the tissue. Therefore, the ability to print locally soft and cell stimulating microenvironments inside of a mechanically robust hydrogel is demonstrated, thereby uncoupling the micro- and macromechanical properties of the 3D printed tissues such as cartilage.

Co-culture of bone marrow stromal cells and chondrocytes in vivo for the repair of the goat condylar cartilage defects.

This study explored the feasibility of inducing the differentiation of BMSCs into chondrocytes through co-culture with chondrocytes in hydrogel constructs (Pluronic F-127 gel) in vivo for the repair of goat mandibular condylar cartilage defects. Chondrocytes and BMSCs were isolated from goat auricular cartilage and bone marrow, respectively, and were mixed at a ratio of 3:7. BMSCs were labelled with green fluorescence protein (GFP) using a retrovirus vector for tracing. Mixed cells were re-suspended in 30% Pluronic F-127 at a concentration of 5×107 cells/ml to form a gel-cell complex. The gel-cell complex was implanted into the temporomandibular joint condylar articular cartilage defects. The whole temporomandibular joint and adjacent tissues were harvested at 4, 8, and 12 weeks after surgery, and gross observation, histology and collagen II expression were evaluated. In the co-culture group, cartilage-like tissues were formed, and abundant type II collagen could be detected by immunohistochemistry in the condylar cartilage defects. Confocal microscopy revealed that implanted GFP-labelled BMSCs were embedded in cartilage-like tissues. The co-culture system described herein provides a chondrogenic microenvironment to induce the chondrogenic differentiation of BMSCs in vivo without any additional cellular factors.

Angiogenic Potential of Human Bone Marrow-Derived Mesenchymal Stem Cells in Chondrocyte Brick-Enriched Constructs Promoted Stable Regeneration of Craniofacial Cartilage

Craniofacial deformities caused by congenital defects or trauma remain challenges for clinicians, whereas current surgical interventions present limited therapeutic outcomes. Injection of bone marrow‐derived mesenchymal stem cells (BMSCs) into the defect is highly desirable because such a procedure is microinvasive and grafts are more flexible to fill the lesions. However, preventing hypertrophic transition and morphological contraction remain significant challenges. We have developed an “all host derived” cell transplantation system composed of chondrocyte brick (CB)‐enriched platelet‐rich plasma (P) gel and BMSCs (B). Without exogenous biomaterials or growth factors, such grafts regenerate cartilage efficiently and present great clinical promise. In immunodeficient mice, we compared performance of BMSCs and BMSCs lacking angiogenic potential in CB‐B‐P constructs and followed the cartilage maturation process by histology, immunostaining, micro‐computed tomography, and protein analysis. We determined that angiogenesis occurred quickly inside rudimentary cartilage derived from CB‐B‐P constructs after implantation, which improved tissue survival, tissue growth, and production of chondrogenic signals from chondrocytes. In contrast, silencing angiogenic potential of BMSCs led to poor chondrogenesis accompanied by necrosis. Chondrocyte bricks merged rapidly with angiogenesis, which constituted an enclosed chondrogenic niche and effectively inhibited runt‐related transcription factor‐2‐dependent hypertrophic transition of BMSCs as well as endochondral ossification; progressive chondrogenic differentiation of BMSCs resulted in vascularization regression, thus favoring persistent chondrogenesis and effectively augmenting nasal cartilage. In conclusion, these findings provided a novel, efficient approach to regenerating cartilage tissues in vivo. Chondrocyte bricks mixed with P provide transient vascularization and a persistently chondrogenic microenvironment for BMSCs; this provides a mini‐invasive approach for craniofacial cartilage reconstruction. Stem Cells Translational Medicine 2017;6:601–612

GFOGER-Modified MMP-Sensitive Polyethylene Glycol Hydrogels Induce Chondrogenic Differentiation of Human Mesenchymal Stem Cells

The cellular microenvironment plays a crucial role in directing proliferation and differentiation of stem cells. Cells interact with their microenvironment via integrins that recognize certain peptide sequences of extracellular matrix proteins. This receptor-ligand binding has profound impact on cell fate. Interactions of human bone marrow mesenchymal stem cells (hMSCs) with the triple helical collagen mimetic, GPC(GPP)5-GFOGER-(GPP)5GPC-NH2, and the fibronectin adhesion peptide, RGD, were studied in degradable or nondegradable polyethylene glycol (PEG) gels formed by Michael-type addition chemistry. Proliferation, cytoskeletal morphology, and chondrogenic differentiation of encapsulated hMSCs were evaluated. The hMSCs adopted a highly spread morphology within the GFOGER-modified gels, whereas RGD induced a star-like spreading of the cells. hMSCs within GFOGER-modified degradable gels had a high proliferation rate compared with cells in peptide-free gels (p=0.017). Gene expression of type II collagen was highest in GFOGER-modified degradable gels after 21 days. Peptide incorporation increased GAG production in degradable gels after 7 and 21 days and GFOGER-modified degradable hydrogels had on average the highest GAG content, a finding that was confirmed by Alcian blue staining. In conclusion, the GFOGER peptide enhances proliferation in degradable PEG gels and provides a better chondrogenic microenvironment compared with the RGD peptide.

High-Density Cell Systems Incorporating Polymer Microspheres as Microenvironmental Regulators in Engineered Cartilage Tissues

To address the significant clinical need for tissue-engineered therapies for the repair and regeneration of articular cartilage, many systems have recently been developed using bioactive polymer microspheres as regulators of the chondrogenic microenvironment within high-density cell cultures. In this review, we highlight various densely cellular systems utilizing polymer microspheres as three-dimensional (3D) structural elements within developing engineered cartilage tissue, carriers for cell expansion and delivery, vehicles for spatiotemporally controlled growth factor delivery, and directors of cell behavior via regulation of cell-biomaterial interactions. The diverse systems described herein represent a shift from the more traditional tissue engineering approach of combining cells and growth factors within a biomaterial scaffold, to the design of modular systems that rely on the assembly of cells and bioactive polymer microspheres as building blocks to guide the creation of articular cartilage. Cell-based assembly of 3D microsphere-incorporated structures represents a promising avenue for the future of tissue engineering.

Electromagnetic fields enhance chondrogenesis of human adipose-derived stem cells in a chondrogenic microenvironment in vitro

We tested the hypothesis that electromagnetic field (EMF) stimulation enhances chondrogenesis in human adipose-derived stem cells (ADSCs) in a chondrogenic microenvironment. A two-dimensional hyaluronan (HA)-coated well (2D-HA) and a three-dimensional pellet culture system (3D-pellet) were used as chondrogenic microenvironments. The ADSCs were cultured in 2D-HA or 3D-pellet, and then treated with clinical-use pulse electromagnetic field (PEMF) or the innovative single-pulse electromagnetic field (SPEMF) stimulation. The cytotoxicity, cell viability, and chondrogenic and osteogenic differentiations were analyzed after PEMF or SPEMF treatment. The modules of PEMF and SPEMF stimulations used in this study did not cause cytotoxicity or alter cell viability in ADSCs. Both PEMF and SPEMF enhanced the chondrogenic gene expression (SOX-9, collagen type II, and aggrecan) of ADSCs cultured in 2D-HA and 3D-pellet. The expressions of bone matrix genes (osteocalcin and collagen type I) of ADSCs were not changed after SPEMF treatment in 2D-HA and 3D-pellet; however, they were enhanced by PEMF treatment. Both PEMF and SPEMF increased the cartilaginous matrix (sulfated glycosaminoglycan) deposition of ADSCs. However, PEMF treatment also increased mineralization of ADSCs, but SPEMF treatment did not. Both PEMF and SPEMF enhanced chondrogenic differentiation of ADSCs cultured in a chondrogenic microenvironment. SPEMF treatment enhanced ADSC chondrogenesis, but not osteogenesis, when the cells were cultured in a chondrogenic microenvironment. However, PEMF enhanced both osteogenesis and chondrogenesis under the same conditions. Thus the combination of a chondrogenic microenvironment with SPEMF stimulation can promote chondrogenic differentiation of ADSCs and may be applicable to articular cartilage tissue engineering.

Spatiotemporal Regulation of Chondrogenic Differentiation with Controlled Delivery of Transforming Growth Factor-β1 from Gelatin Microspheres in Mesenchymal Stem Cell Aggregates

The precise spatial and temporal presentation of growth factors is critical for cartilage development, during which tightly controlled patterns of signals direct cell behavior and differentiation. Recently, chondrogenic culture of human mesenchymal stem cells (hMSCs) has been improved through the addition of polymer microspheres capable of releasing growth factors directly to cells within cellular aggregates, eliminating the need for culture in transforming growth factor-β1 (TGF-β1)-containing medium. However, the influence of specific patterns of spatiotemporal growth factor presentation on chondrogenesis within microsphere-incorporated cell systems is unclear. In this study, we examined the effects of altering the chondrogenic microenvironment within hMSC aggregates through varying microsphere amount, growth factor concentration per microsphere, and polymer degradation time. Cartilage formation was evaluated in terms of DNA, glycosaminoglycan, and type II collagen in hMSCs from three donors. Chondrogenesis equivalent to or greater than that of aggregates cultured in medium containing TGF-β1 was achieved in some conditions, with varied differentiation based on the specific conditions of microsphere incorporation. A more spatially distributed delivery of TGF-β1 from a larger mass of fast-degrading microspheres improved differentiation by comparison with delivery from a smaller mass of microspheres with a higher TGF-β1 concentration per microsphere, although the total amount of growth factor per aggregate was the same. Results also indicated that the rate and degree of chondrogenesis varied on a donor-to-donor basis. Overall, this study elucidates the effects of varied conditions of TGF-β1-loaded microsphere incorporation on hMSC chondrogenesis, demonstrating that both spatiotemporal growth factor presentation and donor variability influence chondrogenic differentiation within microsphere-incorporated cellular constructs.

Chondrogenesis by Co-culture of Adipose-Derived Stromal Cells and Chondrocytes In Vitro

Adipose-derived stromal cells (ADSCs) could be induced to differentiate into chondrocytes in the presence of cellular factors. In this study, we explored the feasibility of inducing the differentiation of ADSCs into chondrocytes in the presence of chondrocytes. Human ADSCs and porcine auricular chondrocytes were expanded in vitro and then were mixed at the ratio of 7:3. 5.0 x× 107 mixed cells were seeded onto a polyglycolic acid/polylactic acid scaffold as co-culture group. Chondrocytes and ADSCs with the same cell number were seeded onto the scaffold as positive control group and negative control group. A total of 1.5 x× 107 chondrocytes were seeded as low-concentration chondrocyte group. After culturing for 8 weeks, gross observation, wet weight, histology, glycosaminoglycan quantification, and collagen II expression were evaluated. Cells in all groups well adhered to the scaffold and could secrete extracellular matrices. In the co-culture group and positive control group, cell–scaffold constructs could maintain the original size and shape during the culture. At the 8th week, cartilage-like tissues were formed, and abundant type II collagen could be detected by immunohistochemistry and reverse transcription-polymerase chain reaction in co-culture and positive control groups. Wet weights and glycosaminoglycan contents of tissues in co-culture group were approximately onefold of those in the negative control group. In the negative control group, constructs shrunk gradually without mature cartilage lacuna formation. In low-concentration chondrocyte group, constructs also shrunk obviously with small amount of cartilage formation. Chondrocytes can provide chondrogenic microenvironment to induce chondrogenic differentiation of ADSCs and thus promote the chondrogenesis of ADSCs in vitro.

Preliminary study of in vitro chondrogenesis by co-culture of chondrocytes and adipose-derived stromal cells

To explore the feasibility of in vitro chondrogenesis by co-culture of chondrocytes and adipose-derived stromal cells (ADSCs) so as to confirm the hypothesis that chondrocytes can provide chondrogenic microenvironment to induce chondrogenic differentiation of ADSCs. Human ADSCs and porcine auricular chondrocytes were in vitro expanded respectively and then were mixed at the ratio of 7:3 (ADSCs: chondrocytes). 200 microl mixed cells (5.0 x 10(7)/ml) were seeded onto a polyglycolic acid/polylactic acid (PGA/PLA) scaffold, 8 mm in diameter and 2 mm in thickness, as co-culture group. Chondrocytes and ADSCs with the same cell number were seeded respectively onto the scaffold as positive control group and negative control group. 200 microl chondrocytes (1.5 x 10(7)/ml) were seeded as low concentration chondrocyte group. There were 6 specimens in each group. All specimens were harvested after in vitro culture for 8 weeks in DMEM plus 10% FBS. Gross observation, histology, immunohistochemistry, wet weight measurement and glycosaminoglycan (GAG) quantification were used to evaluate the results. Multiple-sample t-test statistics analysis was done to compare the difference of wet weight and glycosaminoglycan(GAG) content between the groups. Cells in all groups had fine adhesion to the scaffold and could secrete extracellular matrix. In co-culture group and positive control group, cell-scaffold constructs could maintain the original size and shape during in vitro culture. At 8 weeks, cartilage-like tissue formed in gross appearance and histological features, and abundant type II collagen could be detected by immunohistochemistry. Wet weight and glycosaminoglycan(GAG) content of co-culture group were respectively (174 +/- 12) mg and (7.6 +/- 0.4) mg. There were respectively 75% (P < 0.01) and 79% (P<0.01) of those of positive control group. In negative control group, however, constructs shrunk gradually without mature cartilage lacuna in histology. In low concentration chondrocyte group, constructs also shrunk obviously with small amount of cartilage formation at the edge area of the construct, and wet weight was (85 +/- 5) mg, which was 37% (P<0.01) of that of positive control group. Chondrocytes can provide chondrogenic microenvironment to induce chondrogenic differentiation of ADSCs and thus promote the in vitro chondrogenesis of ADSCs.

Coculture between periosteal explants and articular chondrocytes induces expression of TGF- 1 and collagen I

Repair of focal articular cartilage lesions is usually performed by employing cell-based therapeutic strategies such as autologous chondrocyte implantation (ACI). The aim of this study was to determine whether periosteum exerts pro-chondrogenic effects on the transplanted cells beyond its biomechanical role in ACI. Micromass pellets of human articular chondrocytes were cocultured for up to 28 days with human periosteal explants either with physical contact or separated by a membrane allowing paracrine interactions only. Quantitative reverse transcription (RT)-PCR, ELISA, immunohistochemistry and collagen isolation were used to analyse the expression and secretion of TGF-beta1, collagens I and II and chondrogenic differentiation markers such as MIA (CD-RAP) and aggrecan. TGF-beta1 gene expression was induced significantly in paracrine cocultures in periosteum, whereas it was repressed in physical contact cocultures. However, a higher TGF-beta1 secretion rate was observed in physical contact cocultures compared with periosteal monocultures. The expression of COL2A1, melanoma inhibitory activity (cartilage-derived retinoic acid-sensitive protein) [MIA (CD-RAP)] and aggrecan was mainly unaffected by culture conditions, whereas COL1A1 gene expression was increased in periosteal paracrine cocultures. Collagen I staining was induced in micromass pellets from paracrine cocultures, whereas it was repressed in chondrocytes from physical contact cocultures. We found evidence for a bidirectional regulating system with paracrine signalling pathways between periosteum and articular chondrocytes. Stimulation of TGF-beta1 and COL1A1 gene expression in periosteal paracrine cocultures and the increased release of TGF-beta1 protein in physical contact conditions indicate an anabolic, and not merely chondrogenic micro-environment in this in vitro model for periosteal-based ACI.

Effects of Chondrogenic Microenvironment on Construction of Cartilage Tissues Using Marrow Stromal Cells In Vitro

To investigate whether it is feasible to use the chondrogenic microenvironment provided by cartilage cells to construct cartilage tissues in vitro with bone marrow stromal cells (BMSC). We isolated and cultured BMSC and cartilage cells from Sprague Dawley rats (SD rats). The supernatant of cartilage culture was used as inducing solution to cause differentiation of BMSC from the second generation of cells cultured in vitro. Cells were examined seven days later, using immunohistochemistry to determine the expression of collagen specific to type II cartilage. RT-PCR was used to detect the expression of type II collagen and aggrecan mRNA. BMSC and cartilage cells were isolated from SD rats and cultured in vitro. The BMSC and cartilage cells in culture were mixed evenly in an 8:2 ratio and inoculated into a polyglycolic acid/polylactic acid (PGA/PLA) scaffold to a final concentration of 5.0x10(7) cells/ml. PGA/PLA preparations with pure cartilage cells or pure BMSC served as the positive and negative controls, respectively. The control group of low-concentration cartilage cells consisted of PGA/PLA preparations containing cartilage cells at 20% of the above mentioned concentration (1.0x10(7) cells/ml). Samples were collected eight weeks later, at which time general observations, wet weight, and glycosaminoglycan (GAG) levels were determined, and histological and immunohistochemical examinations were performed. Immunohistochemistry showed the induction of BMSC type II collagen, and RT-PCR indicated the expression of type II collagen and aggrecan mRNA. In the mixed-cell group and the positive control group, pure mature cartilage cells were produced after eight weeks of culture in vitro, and the size and shape of the scaffold were maintained throughout the culture period. The two groups gave rise to newly generated cartilage cells essentially identical in appearance and histological properties. The immunohistochemical results showed that the cartilage cells of both groups expressed abundant cartilage-specific type II collagen. The average wet weight and GAG content were more than 70% of the values in the positive control group. Only an extremely small amount of immature cartilage tissue formed in local regions in the BMSC-only sample, and the scaffold was obviously shrunken and deformed. Although the wet weight of newly generated cartilage tissue in the low-concentration cartilage cell sample reached 30% of the value of the positive control group, the scaffold was obviously shrunken and deformed. Only regional and discontinuous cartilage tissues were formed, and the amount of newly generated cartilage was obviously less than in the co-culture and positive control groups. Cartilage cells can provide a microenvironment for cartilage formation to some extent, and also effectively induce BMSC to differentiate into cartilage cells and form tissue-engineered cartilage in vitro.

Cartilaginous ECM component-modification of the micro-bead culture system for chondrogenic differentiation of mesenchymal stem cells

In this study a 3-D alginate microbead platform was coated with cartilaginous extracellular matrix (ECM) components to emulate chondrogenic microenvironment in vivo for the differentiation of bone marrow-derived mesenchymal stem cells (BMSCs). BMSCs were seeded onto the microbead surface and the effect of the modified microbead on BMSC adhesion, proliferation and chondrogenic differentiation was studied, and compared to chondrogenesis in conventional pellet culture. Our results indicated that microbead system promoted BMSC proliferation and protein deposition resulting in the formation of bigger aggregates compared to conventional pellet culture. Analysis of the aggregates indicated that chondroitin sulfate (CS)- and Col2-coated microbeads enhanced the chondrogenic differentiation of hBMSCs, with increasing formation of glycosaminoglycan (GAG) and collagen II deposition in histology, immunohistochemistry and real time PCR analysis. In addition, Col2-coated microbeads resulted in hypertrophic maturation of the differentiated chondrocytes, similar to conventional pellet culture, while CS-coated microbeads were able to retain the pre-hypertrophy state of the differentiated cells. Our result suggested that provision of suitable cartilaginous microenvironment in a 3-D system can promote the chondrogenic differentiation of BMSC and influence the phenotype of resulting chondrocytes. Our microbead system provides an easy method of processing a 3-D alginate system that allows the possibility of scaling up chondrogenic pellet production for clinical application, while the modifiable microbeads also provide an adjustable 3-D platform for the study of co-interaction of ECM and differentiation factors during the stem cell differentiation.