Chondrogenic Expression Research Articles

A xenogenic-free culture medium for cell micro-patterning systems as cell-instructive biomaterials for potential clinical applications

Cell micro-patterning controls cell fate and function and has potential for generating therapeutically usable mesenchymal stromal cell (MSC) populations with precise functions. However, to date, the micro-patterning of human cells in a translational context has been impossible because only ruminant media supplements, e.g. fetal bovine serum (FBS), are established for use with micro-patterns (MPs). Thus, there are currently no good manufacturing practice (GMP)-compliant media available for MPs. This study tested a xenogenic-free human plasma and platelet lysate (hP + PL) medium supplement to determine its compatibility with MPs. Unfiltered hP + PL medium resulted in significant protein deposition, creating a 'carpet-like' layer that rendered MPs ineffective. Filtration (3×/5×) eliminated this effect. Importantly, quantitative comparison using droplet digital PCR revealed that human MSCs in all media types exhibited similar profiles with strong myogenic Calponin 1/Transgelin 2 (TAGLN2) and weaker osteogenic alkaline phosphatase/Runt-related transcription factor 2 marker expression, and much weaker adipogenic (lipoprotein lipase/peroxisome proliferator-activated receptor gamma) and chondrogenic (collagen type II/aggrecan) expression, with profiles being dominated by myogenic markers. Within these similar profiles, an even stronger induction of the myogenic marker TAGLN2 by all hP + PL- compared to FBS-containing media. Overall, this suggested that FBS can be replaced with hP + PL without altering differentiation profiles. However, assessing individual MSC responses to various MP types with defined categories revealed that unfiltered hP + PL medium was unusable. Importantly, FBS- and 3× filtered hP + PL media were comparable in each differentiation category. Summarized, this study recommends 3× filtered hP + PL as a xenogenic-free and potentially GMP-compliant alternative to FBS as a culture medium supplement for micro-patterning cell populations in both basic and translational research that will ensure consistent and reliable MSC micro-patterning for therapeutic use.

Passaged Articular Chondrocytes From the Superficial Zone and Deep Zone Can Regain Zone-Specific Properties After Redifferentiation

Background: Bioengineered cartilage is a developing therapeutic to repair cartilage defects. The matrix must be rich in collagen type II and aggrecan and mechanically competent, withstanding compressive and shearing loads. Biomechanical properties in native articular cartilage depend on the zonal architecture consisting of 3 zones: superficial, middle, and deep. The superficial zone chondrocytes produce lubricating proteoglycan-4, whereas the deep zone chondrocytes produce collagen type X, which allows for integration into the subchondral bone. Zonal and chondrogenic expression is lost after cell number expansion. Current cell-based therapies have limited capacity to regenerate the zonal structure of native cartilage. Hypothesis: Both passaged superficial and deep zone chondrocytes at high density can form bioengineered cartilage that is rich in collagen type II and aggrecan; however, only passaged superficial zone–derived chondrocytes will express superficial zone–specific proteoglycan-4, and only passaged deep zone–derived chondrocytes will express deep zone–specific collagen type X. Study Design: Controlled laboratory study. Methods: Superficial and deep zone chondrocytes were isolated from bovine joints, and zonal subpopulations were separately expanded in 2-dimensional culture. At passage 2, superficial and deep zone chondrocytes were seeded, separately, in scaffold-free 3-dimensional culture within agarose wells and cultured in redifferentiation media. Results: Monolayer expansion resulted in loss of expression for proteoglycan-4 and collagen type X in passaged superficial and deep zone chondrocytes, respectively. By passage 2, superficial and deep zone chondrocytes had similar expression for dedifferentiated molecules collagen type I and tenascin C. Redifferentiation of both superficial and deep zone chondrocytes led to the expression of collagen type II and aggrecan in both passaged chondrocyte populations. However, only redifferentiated deep zone chondrocytes expressed collagen type X, and only redifferentiated superficial zone chondrocytes expressed and secreted proteoglycan-4. Additionally, redifferentiated deep zone chondrocytes produced a thicker and more robust tissue compared with superficial zone chondrocytes. Conclusion: The recapitulation of the primary phenotype from passaged zonal chondrocytes introduces a novel method of functional bioengineering of cartilage that resembles the zone-specific biological properties of native cartilage. Clinical Relevance: The recapitulation of the primary phenotype in zonal chondrocytes could be a possible method to tailor bioengineered cartilage to have zone-specific expression.

Microenvironmental mechanoactivation through Yap/Taz suppresses chondrogenic gene expression.

Chondrocyte phenotype is preserved when cells are round and the actin cytoskeleton is cortical. Conversely, these cells rapidly dedifferentiate in vitro with increased mechanoactive Rho signaling, which increases cell size and causes large actin stress fiber to form. While the effects of Rho on chondrocyte phenotype are well established, the molecular mechanism is not yet fully elucidated. Yap, a transcriptional coregulator, is regulated by Rho in a mechanotransductive manner and can suppress chondrogenesis in vivo. Here, we sought to elucidate the relationship between mechanoactive Rho and Yap on chondrogenic gene expression. We first show that decreasing mechanoactive state through Rho inhibition results in a broad increase in chondrogenic gene expression. Next, we show that Yap and its coregulator Taz are negative regulators of chondrogenic gene expression, and removal of these factors promotes chondrogenesis even in environments that promote cell spreading. Finally, we establish that Yap/Taz is essential for translating Rho-mediated signals to negatively regulate chondrogenic gene expression, and that its removal negates the effects of increased Rho signaling. Together, these data indicate that Rho is a mechanoregulator of chondrogenic differentiation, and that its impact on chondrogenic expression is exerted principally through mechanically induced translocation and activity of Yap and Taz.

STUDY OF SILK STRAIN AND ITS PREPARATION FOR MENISCUS TISSUE ENGINEERING SCAFFOLD

Silk fibroin (SF) has been used as a scaffold for cartilage tissue engineering. Different silkworms strain produced different protein. Also, molecular weight of SF depends on extraction method. We hypothesised that strain of silkworm and method of SF extraction would effect biological properties of SF scaffold. Therefore, cell viability and chondrogenic gene expression of human chondrogenic progenitor cells (HCPCs) treated with SF from 10 silkworm strains and two common SF extraction methods were investigate in this study.Twenty g of 10 strains silk cocoons were separately degummed in 0.02M Na2CO3 solution and dissolved in 100๐C for 30 minutes. Half of them were then dissolved in CaCl2/Ethanol/H2O [1:2:8 molar ratio] at 70±5๐C (method 1) and other half was dissolved in 46% w/v CaCl2 at 105±5๐C (method 2) for 4 hours. HCPCs were cultured in SF added cultured medial according to strain and extraction method. Cell viability at day 1, 3, and 7, were determined. Expression of collagen I, collagen II, and aggrecan at day 7 and 14, was studied. All experiment were done in triplicated samples.Generally, method 1 SF extraction showed higher cell viability in all strains. Cell viability from Nanglai Saraburi, Laung Saraburi and Nangtui strains were higher than those without SF in every time point while Wanasawan and J108 had higher viability at day 1 and decreased by time. Expression in collagen 1, collagen 2 and aggrecan in method 1 are higher at day 7 and day 14. Collagen 1 expression was highest in Nangnoi Srisaket, followed by Laung Saraburi and Nanglai Saraburi in day 7. Nangnoi Srisaket also had highest expression at day 14, followed by Nanglai Saraburi and Laung Saraburi respectively. Nangseaw had highest collagen 2 expression, follow by Laung Saraburi and Nangnoi Srisaket respectively. Higher aggrecan gene expression of Tubtimsiam, Wanasawan, UB 1 and Nangnoi Srisaket was observed at day 7 and increased expression of all strains at day 14.SF extraction using CaCl2/Ethanol/H2O offered better cell viability and chondrogenic expression. Nangseaw, Laung Saraburi and Nangnoi Srisaket strains expressed more chondrogenic phenotype.

In-situ forming injectable GFOGER-conjugated BMSCs-laden hydrogels for osteochondral regeneration

The collagen-mimetic peptide GFOGER possesses the chondrogenic potential and has been used as a cell adhesion peptide or chondrogenic inducer. Here, we prepared an injectable in situ forming composite hydrogel system comprising methoxy polyethylene glycol-b-polycaprolactone (MPEG-PCL) and GFOGER-conjugated PEG-PCL (GFOGER-PEG-PCL) with various GFOGER concentrations based on our recently patented technology. The conjugation of GFOGER to PEG-PCL was confirmed by 1H NMR, and the particle size distribution and rheological properties for the sol-gel transition behavior of the samples with respect to the GFOGER content were evaluated systemically. In vitro experiments using rat bone marrow-derived mesenchymal stem cells (BMSCs) revealed that the GFOGER-PEG-PCL hydrogel significantly enhanced expression of integrins (β1, α2, and α11), increased expression of FAK, and induced downstream signaling of ERK and p38. Overexpression of chondrogenic markers suggested that BMSCs have the potential to differentiate into chondrogenic lineages within GFOGER-PEG-PCL samples. In vivo studies using a rat osteochondral defect model revealed that transplanted BMSCs with GFOGER0.8-PEG-PCL survived at the defect with strong chondrogenic expression after 4 weeks. The stem cell-laden GFOGER0.8-PEG-PCL hydrogel produced remarkable osteochondral regeneration at 8 weeks of transplantation, as determined by histological findings and micro-CT analysis. The histomorphological score in the GFOGER0.8-PEG-PCL + BMSCs group was ~1.7-, 2.6-, and 5.3-fold higher than that in the GFOGER0.8-PEG-PCL, MPEG-PCL, and defect groups, respectively. Taken together, these results provide an important platform for further advanced GFOGER-based stem cell research for osteochondral repair.

Stress deprivation of tendon explants or Tpm3.1 inhibition in tendon cells reduces F-actin to promote a tendinosis-like phenotype.

Actin is a central mediator between mechanical force and cellular phenotype. In tendons, it is speculated that mechanical stress deprivation regulates gene expression by reducing filamentous (F)-actin. However, the mechanisms regulating tenocyte F-actin remain unclear. Tropomyosins (Tpms) are master regulators of F-actin. There are more than 40 Tpm isoforms, each having the unique capability to stabilize F-actin subpopulations. We investigated F-actin polymerization in stress-deprived tendons and tested the hypothesis that stress fiber-associated Tpm(s) stabilize F-actin to regulate cellular phenotype. Stress deprivation of mouse tail tendon down-regulated tenogenic and up-regulated protease (matrix metalloproteinase-3) mRNA levels. Concomitant with mRNA modulation were increases in G/F-actin, confirming reduced F-actin by tendon stress deprivation. To investigate the molecular regulation of F-actin, we identified that tail, Achilles, and plantaris tendons express three isoforms in common: Tpm1.6, 3.1, and 4.2. Tpm3.1 associates with F-actin in native and primary tenocytes. Tpm3.1 inhibition reduces F-actin, leading to decreases in tenogenic expression, increases in chondrogenic expression, and enhancement of protease expression in mouse and human tenocytes. These expression changes by Tpm3.1 inhibition are consistent with tendinosis progression. A further understanding of F-actin regulation in musculoskeletal cells could lead to new therapeutic interventions to prevent alterations in cellular phenotype during disease progression.

Mechanical activation drives tenogenic differentiation of human mesenchymal stem cells in aligned dense collagen hydrogels

Tendons are force transmitting mechanosensitive tissues predominantly comprised of highly aligned collagen type I fibres. In this study, the recently introduced gel aspiration-ejection method was used to rapidly fabricate aligned dense collagen (ADC) hydrogel scaffolds. ADCs provide a biomimetic environment compared to traditional collagen hydrogels that are mechanically unstable and comprised of randomly oriented fibrils. The ADC scaffolds were shown to be anisotropic with comparable stiffness to immature tendons. Furthermore, the application of static and cyclic uniaxial loading, short-term (48 h) and high-strain (20%), resulted in a 3-fold increase in both the ultimate tensile strength and modulus of ADCs. Similar mechanical activation of human mesenchymal stem cell (MSC) seeded ADCs in serum- and growth factor-free medium induced their tenogenic differentiation. Both static and cyclic loading profiles resulted in a greater than 12-fold increase in scleraxis gene expression and either suppressed or maintained osteogenic and chondrogenic expressions. Following the 48 h mechanoactivation period, the MSC-seeded scaffolds were matured by tethering in basal medium without further external mechanical stimulation for 19 days, altogether making up 21 days of culture. Extensive cell-induced matrix remodeling and deposition of collagen types I and III, tenascin-C and tenomodulin were observed, where initial cyclic loading induced significantly higher tenomodulin protein content. Moreover, the initial short-term mechanical stimulation elongated and polarized seeded MSCs, and overall cell alignment was significantly increased in those under static loading. These findings indicate the regenerative potential of the ADC scaffolds for short-term mechanoactivated tenogenic differentiation, which were achieved even in the absence of serum and growth factors that may potentially increase clinical translatability.

Bioprinting of Chondrocyte Stem Cell Co-Cultures for Auricular Cartilage Regeneration.

Advances in 3D bioprinting allows not only controlled deposition of cells or cell-laden hydrogels but also flexibility in creating constructs that match the anatomical features of the patient. This is especially the case for reconstructing the pinna (ear), which is a large feature of the face and made from elastic cartilage that primarily relies on diffusion for nutrient transfer. The selection of cell lines for reconstructing this cartilage becomes a crucial step in clinical translation. Chondrocytes and mesenchymal stem cells are both studied extensively in the area of cartilage regeneration as they are capable of producing cartilage in vitro. However, such monoculture systems involve unfavorable processes and produce cartilage with suboptimal characteristics. Co-cultures of these cell types are known to alleviate these limitations to produce synergically active chondrocytes and cartilage. The current study utilized a 3D bioprinted scaffold made from combined gelatine methacryloyl and methacrylated hyaluronic acid (GelMA/HAMA) to interrogate monocultures and co-cultures of human septal chondrocytes (primary chondrocytes, PCs) and human bone marrow-derived mesenchymal stem cells (BM-hMSCs). This study is also the first to examine co-cultures of healthy human chondrocytes with human BM-hMSCs encapsulated in GelMA/HAMA bioprinted scaffolds. Findings revealed that the combination of MSCs and PCs not only yielded cell proliferation that mimicked MSCs but also produced chondrogenic expressions that mimicked PCs. These findings suggested that co-cultures of BM-hMSCs and healthy septal PCs can be employed to replace monocultures in chondrogenic studies for cartilage regeneration in this model. The opportunity for MSCs used to replace PCs alleviates the requirement of large cartilage biopsies that would otherwise be needed for sufficient cell numbers and therefore can be employed for clinical applications.

Therapeutic application of 3B-PEG injectable hydrogel/Nell-1 composite system to temporomandibular joint osteoarthritis

This study aims to construct a composite system of the tri-block polyethylene glycol injectable hydrogel (3B-PEG IH) and neural epithelial growth factor-like protein 1 (Nell-1), and to analyze its therapeutic effect on temporomandibular joint osteoarthritis (TMJOA). Sol-gel transition temperature was measured via inverting test. The viscoelastic modulus curves was measured by rheometer. Degradation and controlled release profiles of 3B-PEG IH were drawn in vitro. In vivo gel retention and biocompatibility were completed subcutaneously on the back of rats. After primary chondrocytes were extracted and identified, the cell viability in 3B-PEG IH was measured. Evaluation of gene expression in hydrogel was performed by real-time polymerase chain reaction. TMJOA rabbits were established by intra-articular injection of type II collagenase. Six weeks after composite systems being injected, gross morphological score, micro-CT, histological staining and grading were evaluated. The rusults showed that different types of 3B-PEG IH all reached a stable gel state at 37 °C and could support the three-dimensional growth of chondrocytes, but poly(lactide-co-caprolactone)-block-poly(ethyleneglycol)-block-poly(lactide-co-caprolactone) (PLCL-PEG-PLCL) hydrogel had a wider gelation temperature range and better hydrolytic stability for about 4 weeks. Its controlled release curve is closest to the zero-order release kinetics. In vitro, PLCL-PEG-PLCL/Nell-1 could promote the chondrogenic expression and reduce the inflammatory expression. In vivo, TMJOA rabbits were mainly characterized by the disorder of cartilage structure and the destruction of subchondral bone. However, PLCL-PEG-PLCL/Nell-1 could reverse the destruction of the subchondral trabecula, restore the fibrous and proliferative layers of the surface, and reduce the irregular hyperplasia of fibrocartilage layer. In conclusion, by comparing the properties of different 3B-PEG IH, 20 wt% PLCL-PEG-PLCL hydrogel was selected as the most appropriate material. PLCL-PEG-PLCL/Nell-1 composite could reverse osteochondral damage caused by TMJOA, Nfatc1-Runx3 signaling pathway may play a role in it. This study may provide a novel, minimally-invasive therapeutic strategy for the clinical treatment of TMJOA.

MicroRNA-210-3p Promotes Chondrogenic Differentiation and Inhibits Adipogenic Differentiation Correlated with HIF-3α Signalling in Bone Marrow Mesenchymal Stem Cells.

Cartilage injury of the knee joint is very common. Due to the limited self-healing ability of articular cartilage, osteoarthritis is very likely to occur if left untreated. Bone marrow mesenchymal stem cells (BMMSCs) are widely used in the study of cartilage injury due to their low immunity and good amplification ability, but they still have disadvantages, such as heterogeneous undifferentiated cells. MicroRNAs can regulate the chondrogenic differentiation ability of MSCs by inhibiting or promoting mRNA translation and degradation. In this research, we primarily investigated the effect of microRNA-210-3p (miR-210-3p) on chondrogenic and adipogenic differentiation of BMMSCs in vitro. Our results demonstrate that miR-210-3p promoted chondrogenic differentiation and inhibited adipogenic differentiation of rat BMMSCs, which was related to the HIF-3α signalling pathway. Additionally, miR-210-3p promotes mRNA and protein levels of the chondrogenic expression genes COLII and SOX9 and inhibits mRNA and protein levels of the adipogenic expression genes PPARγ and LPL. Thus, miR-210-3p combined with BMMSCs is a candidate for future clinical applications in cartilage regeneration and could represent a promising new therapeutic target for OA.

Human nasal septal chondrocytes (NSCs) preconditioned on NSC-derived matrix improve their chondrogenic potential

BackgroundExtracellular matrix (ECM) has a profound effect on cell behaviors. In this study, we prepare a decellularized human nasal septal chondrocyte (NSC)-derived ECM (CHDM), as a natural (N-CHDM) or soluble form (S-CHDM), and investigate their impact on NSCs differentiation.MethodsN-CHDM, S-CHDM were obtained from NSC. To evaluate function of NSC cultured on each substrate, gene expression using chondrogenic marker, and chondrogenic protein expression were tested. Preconditioned NSCs-loaded scaffolds were transplanted in nude mice for 3 weeks and analyzed.ResultsWhen cultivated on each substrate, NSCs exhibited similar cell spread area but showed distinct morphology on N-CHDM with significantly lower cell circularity. They were highly proliferative on N-CHDM than S-CHDM and tissue culture plastic (TCP), and showed more improved cell differentiation, as assessed via chondrogenic marker (Col2, Sox9, and Aggrecan) expression and immunofluorescence of COL II. We also investigated the effect of NSCs preconditioning on three different 2D substrates while NSCs were isolated from those substrates, subsequently transferred to 3D mesh scaffold, then cultivated them in vitro or transplanted in vivo. The number of cells in the scaffolds was similar to each other at 5 days but cell differentiation was notably better with NSCs preconditioned on N-CHDM, as assessed via real-time q-PCR, Western blot, and immunofluorescence. Moreover, when those NSCs-loaded polymer scaffolds were transplanted subcutaneously in nude mice for 3 weeks and analyzed, the NSCs preconditioned on the N-CHDM showed significantly advanced cell retention in the scaffold, more cells with a chondrocyte lacunae structure, and larger production of cartilage ECM (COL II, glycosaminoglycan).ConclusionsTaken together, a natural form of decellularized ECM, N-CHDM would present an advanced chondrogenic potential over a reformulated ECM (S-CHDM) or TCP substrate, suggesting that N-CHDM may hold more diverse signaling cues, not just limited to ECM component.

Influence of Cellular Microenvironment on Human Articular Chondrocyte Cell Signaling.

Alteration of the cellular microenvironment may influence the intra- and intercellular communication and contribute to cartilage injury and repair. The purpose of this study was to investigate how matrix elasticity/stiffness affects chondrogenic activities, including cell survival, phenotypic expression, and the release of both pro- and anti-inflammatory cytokines. Human articular chondrocytes (HACs) cultured on traditional 2-dimensional (2D) plastic surfaces were compared with those cultured within 3D hydrogel matrices of varying stiffness. Chondrogenic proliferation, differentiation, and the expression of pro- and anti-inflammatory cytokines were evaluated. Both interleukin-1-beta (IL-1β) and human synovial fluid-derived cells (hSFCs) were introduced to study the effects of matrix stiffness on chondrocyte response. Cells demonstrated the most robust chondrogenic differentiation and secreted the least pro-inflammatory cytokines when the matrix stiffness was close to their native microenvironment. The IL-1β effects were attenuated when HACs were co-cultured with hSFCs. Modifying the matrix stiffness to mimic the native cartilage microenvironment not only optimized chondrogenic expression but also was essential for the regulation of physiological homeostasis. This study proposed a new toolkit to study cell-molecule, cell-cell, and cell-matrix influence on cartilage physiology.

Comparison of the Chondrogenic Potential of Mesenchymal Stem Cells Derived from Bone Marrow and Umbilical Cord Blood Intended for Cartilage Tissue Engineering.

Osteoarthritis (OA) remains incurable in humans or horses and mesenchymal stromal/stem cells (MSCs) represent an attractive solution for producing a neocartilage substitute. However, the best MSC source still needs to be identified. This study compared the chondrogenic potential of equine MSCs derived from bone marrow (BM) and umbilical cord blood (UCB), at their undifferentiated status to check if one cell source is better proned, and after chondrogenic-induced differentiation. Chondrogenesis was induced by culture in collagen scaffold with BMP-2 + TGF-ß1 in hypoxia or normoxia. MSCs chondrogenic potential was evaluated using the mRNA and corresponding protein levels for osteogenic, hypertrophic and chondrogenic markers. MSCs characterization demonstrated that BM- and UCB-MSCs differ in proliferation and tripotencies. At undifferentiated status, they also showed differences in their expression of osteogenic, chondrogenic and hypertrophic markers. Upon chondrogenesis induction, both MSCs sources exhibited increased chondrogenic expression and produce an extracellular matrix (ECM) of better quality in hypoxia, although collagen I remained expressed. UCB-MSCs produced higher amounts of collagen II, particularly its IIB isoform, than BM-MSCs, but also collagen I and Htra1, regardless of the oxygen condition. Finally, immunohistochemistry revealed that the BM-MSCs synthesized an ECM of higher quality, regarding the more homogenous distribution of type IIB collagen, compared to UCB-MSCs. Considering collagen I as the major undesirable component in the neo-synthesis of in vitro cartilage, we recommend using BM-MSCs for horse cartilage engineering.

Chondrogenic expression and DNA methylation patterns in prolonged passages of chondrocyte cell lines of the horse

We investigated the activity of chondrogenic markers and variation of methylation patterns in equine cartilaginous cells cultivated in monolayer. The transcriptional and epigenetic effect of the long-term culture of chondrocytes has been evaluated using several passages of chondrocyte cell-lines derived from equine articular cartilage. Using 3 genes as endogenous control we tested the expression of 7 genes important for different stages of chondrocyte differentiation and maturation. CpG islands in RUNX3 locus were inspected for the evaluation of differential methylation state of passaged cell-lines. The general decline of transcript abundance of marker loci was detected in passage 11 which is the sign of dedifferentiation of cultivated chondrocytes in prolonged monolayer culture. Passages 13 and 14 were characterized by the upregulation of a number of genes, possibly due to the heterogeneity of developed cell lines at this stage of the culture. Instead, gradual increase of methylation percent at particular CpG sites of RUNX3 locus was associated with the growing number of passage. This finding led us to the conclusion that epigenetic alterations better describe the stage of cultivated chondrocytes.

High Mobility Group Box 1 Protein in Osteoarthritic Knee Tissue and Chondrogenic Progenitor Cells: An Ex Vivo and In Vitro Study.

In osteoarthritis (OA), a loss of healthy cartilage extracellular matrix (ECM) results in cartilage degeneration. Attracting chondrogenic progenitor cells (CPCs) to injury sites and stimulating them toward chondrogenic expression profiles is a regenerative approach in OA therapy. High mobility group box 1 protein (HMGB1) is associated with chemoattractant and proinflammatory effects in various pathological processes. Here, we investigate the migratory effects of HMGB1 in knee OA and CPCs for the first time. Immunohistochemistry, immunoblotting, and immunocytochemistry were performed to identify HMGB1 and its receptors, receptor for advanced glycation end products (RAGE) and toll-like receptor 4 (TLR4) in OA knee tissue, chondrocytes, and CPCs. In situ hybridization for HMGB1 mRNA was performed in CPCs ex vivo. The chemoattractant effects of HMGB1 on CPCs were analyzed in cell migration assays. HMGB1 expression in OA tissue and OA chondrocytes was higher than in healthy specimens and cells. HMGB1, RAGE, and TLR4 were expressed in CPCs and chondrocytes. In situ hybridization revealed HMGB1 mRNA in CPCs after migration into OA knee tissue, and immunohistochemistry confirmed HMGB1 expression at the protein level. Stimulation via HMGB1 significantly increased the migration of CPCs. Our results show the chemoattractant role of HMGB1 in knee OA. HMGB1 is released by chondrocytes and has migratory effects on CPCs. These effects might be mediated via RAGE and TLR4. The in vitro and ex vivo results of this study need to be confirmed in vivo.

Differential chondro- and osteo-stimulation in three-dimensional porous scaffolds with different topological surfaces provides a design strategy for biphasic osteochondral engineering

Bone/cartilage interfacial tissue engineering needs to satisfy the differential properties and architectures of the osteochondral region. Therefore, biphasic or multiphasic scaffolds that aim to mimic the gradient hierarchy are widely used. Here, we find that two differently structured (topographically) three-dimensional scaffolds, namely, “dense” and “nanofibrous” surfaces, show differential stimulation in osteo- and chondro-responses of cells. While the nanofibrous scaffolds accelerate the osteogenesis of mesenchymal stem cells, the dense scaffolds are better in preserving the phenotypes of chondrocytes. Two types of porous scaffolds, generated by a salt-leaching method combined with a phase-separation process using the poly(lactic acid) composition, had a similar level of porosity (~90%) and pore size (~150 μm). The major difference in the surface nanostructure led to substantial changes in the surface area and water hydrophilicity (nanofibrous ≫ dense); as a result, the nanofibrous scaffolds increased the cell-to-matrix adhesion of mesenchymal stem cells significantly while decreasing the cell-to-cell contracts. Importantly, the chondrocytes, when cultured on nanofibrous scaffolds, were prone to lose their phenotype, including reduced chondrogenic expressions (SOX-9, collagen type II, and Aggrecan) and glycosaminoglycan content, which was ascribed to the enhanced cell–matrix adhesion with reduced cell–cell contacts. On the contrary, the osteogenesis of mesenchymal stem cells was significantly accelerated by the improved cell-to-matrix adhesion, as evidenced in the enhanced osteogenic expressions (RUNX2, bone sialoprotein, and osteopontin) and cellular mineralization. Based on these findings, we consider that the dense scaffold is preferentially used for the chondral-part, whereas the nanofibrous structure is suitable for osteo-part, to provide an optimal biphasic matrix environment for osteochondral tissue engineering.

Shear stresses modulate chondrogenic and osteogenic genes expression in human bone marrow derived stem cells

Background: Mechanical stimuli in the microenvironment may provide cues for differentiation, maintenance of phenotypes, and extracellular matrix homeostasis. This study investigated bone marrow-derived stem cells (BMSCs) response to mechanical stresses in terms of differentiation and the extracellular matrix/matrix metalloproteinase genes expression. Materials & Methods: Human BMSCs were exposed to shear stresses (20 rpm or 50 rpm) for 2, 5, or 10 days with or without osteogenic induction medium. The expression of chondrogenic (type II collagen and aggrecan) and osteogenic (type I collagen, alkaline phosphatase, osteopontin, osteocalcin) phenotypic genes and matrix metalloproteinase genes (aggrecanse, MMP-2, and MMP-9) were analyzed by reverse transcription polymerase chain reaction. In vitro differentiation of BMSCs after shear stress treatment were analyzed by von Kossa staining on monolayer cultures and safarin-O staining on pelleted cells. Results: Shear stress at 20 rpm significantly upregulatedexpression of type II collagen on the 5th day, type I collagen on the 2nd, 5th, and 10th day, osteopontin on the 2nd and 5th day, and osteocalcin on the 5th day as compared to shear stress at 50 rpm. Combination of osteogenic induction medium and shear stress at 20 rpm decreased the mRNA expression levels of chondrogenic genes (type II collagen on all time points and aggrecan on the 5th and 10th day) as compared with shear stress alone. It also decreased the expression of osteogenic genes (type I collagen, alkaline phosphatase, and osteopontin) on the 10th day as compared to osteogenic induction medium. Concomitantly, shear stress upregulated aggrecanase on the 10th day, MMP-2 on the 10th day, and MMP-9 on the 5th and 10th day as compared with osteogenic induction medium. Shear stress increased mineralization of the extracellular matrix as shown by von Kossa staining on the 2nd, 5th, and 10th day. Chondrogenic differentiation by cell pellet analysis found similar amount of proteoglycans but less organized extracellular matrix in shear stress-treated BMSCs as compared to control cells. Discussion: Although shear stress could modulate chondrogenic gene expression as well as osteogenic genes expression in BMSCs, the combination of shear stress inhibited the expression of osteogenic phenotypic genes induced by osteogenic induction medium. Parallel studies revealed that shear stress concomitantly modulate the expression of extracellular matrix genes and the matrix metalloproteinase genes. In vitro differentiation analysis found that shear stress may enhance osteogenesis but interfere with chondrogenesis. Our results may provide data for the design or development of in vitro bioreactor systems to control lineage differentiation of stem cells for tissue engineering. More studies on the mechanotransduction pathways are needed in the future.

Disparate response of articular- and auricular-derived chondrocytes to oxygen tension

ABSTRACTPurpose/Aim: To determine the effect of reduced (5%) oxygen tension on chondrogenesis of auricular-derived chondrocytes. Currently, many cell and tissue culture experiments are performed at 20% oxygen with 5% carbon dioxide. Few cells in the body are subjected to this supra-physiological oxygen tension. Chondrocytes and their mesenchymal progenitors are widely reported to have greater chondrogenic expression when cultured at low, more physiological, oxygen tension (1–7%). Although generally accepted, there is still some controversy, and different culture methods, species, and outcome metrics cloud the field. These results are, however, articular chondrocyte biased and have not been reported for auricular-derived chondrocytes. Materials and Methods: Auricular and articular chondrocytes were isolated from skeletally mature New Zealand White rabbits, expanded in culture and differentiated in high density cultures with serum-free chondrogenic media. Cartilage tissue derived from aggregate cultures or from the tissue engineered sheets were assessed for biomechanical, glycosaminoglycan, collagen, collagen cross-links, and lysyl oxidase activity and expression. Results: Our studies show increased proliferation rates for both auricular and articular chondrocytes at low (5%) O2 versus standard (20%) O2. In our scaffold-free chondrogenic cultures, low O2 was found to increase articular chondrocyte accumulation of glycosaminoglycan, but not cross-linked type II collagen, or total collagen. Conversely, auricular chondrocytes accumulated less glycosaminoglycan, cross-linked type II collagen and total collagen under low oxygen tension. Conclusions: This study highlights the dramatic difference in response to low O2 of chondrocytes isolated from different anatomical sites. Low O2 is beneficial for articular-derived chondrogenesis but detrimental for auricular-derived chondrogenesis.

Effect of Fibroblast Growth Factor 2 on Equine Synovial Fluid Chondroprogenitor Expansion and Chondrogenesis.

Mesenchymal stem cells have been identified in the synovial fluid of several species. This study was conducted to characterize chondroprogenitor (CP) cells in equine synovial fluid (SF) and to determine the effect of fibroblast growth factor 2 (FGF-2) on SF-CP monolayer proliferation and subsequent chondrogenesis. We hypothesized that FGF-2 would stimulate SF-CP proliferation and postexpansion chondrogenesis. SF aspirates were collected from adult equine joints. Colony-forming unit (CFU) assays were performed during primary cultures. At first passage, SF-cells were seeded at low density, with or without FGF-2. Following monolayer expansion and serial immunophenotyping, cells were transferred to chondrogenic pellet cultures. Pellets were analyzed for chondrogenic mRNA expression and cartilage matrix secretion. There was a mean of 59.2 CFU/mL of SF. FGF-2 increased the number of population doublings during two monolayer passages and halved the population doubling times. FGF-2 did not alter the immunophenotype of SF-CPs during monolayer expansion, nor did FGF-2 compromise chondrogenesis. Hypertrophic phenotypic markers were not expressed in control or FGF-2 groups. FGF-2 did prevent the development of a “fibroblastic” cell layer around pellet periphery. FGF-2 significantly accelerates in vitro SF-CP expansion, the major hurdle to clinical application of this cell population, without detrimentally affecting subsequent chondrogenic capacity.

244 CHONDROGENIC PELLET CULTURES FOR CARTILAGE TISSUE ENGINEERING GROW BY DEPOSITION OF MATRIX AND NOT BY CELLULAR PROLIFERATION

Pellet cultures are commonly used to study chondrogenic differentiation in vitro. Our laboratory has demonstrated pellets made with chondrocytes grow in size during culture and produce cartilage matrix, but pellets made with adipose-derived mesenchymal stem cells (ASC) grow only slightly, producing little cartilage matrix. The objective of this study was to determine if differences in chondrocyte and ASC pellet growth result from differences in cell proliferation or in deposition of extracellular matrix. Primary chondrocytes and ASC from adult pigs were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum. To determine baseline proliferation rates in monolayer culture, cells were grown on coverslips in 10 µM bromodeoxyuridine (BrdU) for 24 h and immunostained for BrdU labelling. For pellet cultures, 5 × 105 cells were placed in 15 mL-conical tubes, pelleted by centrifugation in 1.0 mL of chondrogenic base media (CBM: DMEM + 40 µg mL–1 of proline, 50 µM ascorbic acid-2-phosphate, 100 nM dexamethasone, and 1× insulin-transferrin-selenium), and cultured in CBM for 1, and 4 weeks. To detect proliferation in pellets, 1- and 2-week cultured samples were labelled with 10 µM BrdU for 24 h before harvest. Pellets were fixed with 4% paraformaldehyde, embedded, and sectioned on a Leica CM1900 cryostat (Leica Microsystems, Wetzlar, Germany). To assess chondrogenic differentiation and matrix expression, sections were stained for collagen II, keratin sulfate, and chondroitin sulfate. Images were captured and distance between adjacent nuclei in 1- and 4-week pellets were measured using Zeiss imaging software. As expected, cells on coverslips showed BrdU labelling, with higher labelling in ASC cultures indicating faster proliferation (n = 5, 77.3 ± 3.74% chondrocyte v. 92.1 ± 2.88% ASC; α = 0.05; P < 0.0001; Student’s t-test). However, BrdU labelling was not seen in sections from ASC or chondrocyte pellets (n = 5), at either 1 or 2 weeks. Absence of cellular proliferation in pellets was verified by negative staining for the mitotic marker Aurora KinaseB (AurKB). Cartilage matrix staining was strong in chondrocyte pellets at all time points and absent in ASC pellets. Cell nuclei were closely packed in both ASC and chondrocyte pellets at 1 week, but a significant increase in distance between adjacent nuclei with interspersed matrix staining was noted in chondrocyte pellets at 4 weeks (n = 4, 11.88 ± 0.67 µm at 1 week v. 26.85 ± 2.06 µm at 4 weeks; α = 0.05; P < 0.0001; Student’s t-test). As TGFβ3 has been shown to induce chondrogenesis in ASC, ASC pellets were cultured in CBM + 10 ng of TGFβ3 for 1 and 2 weeks (n = 4). The TGFβ3 treatment did not induce cell proliferation in pellets, as sections were negative for BrdU. However, expression of cartilage markers keratan sulfate and chondroitin were noted. Based on our data, neither ASC nor chondrocytes proliferate in pellet culture, and chondrocyte pellet growth is due to extracellular matrix deposition.