Three-dimensional Pellet Culture Research Articles

Non-additive effect of the DNA methylation inhibitor, 5-Aza-dC, and glass as a culture surface on osteogenic differentiation

The clinical need for bone regenerative solutions is expanding with increasing life expectancy and escalating incidence of accidents. Several strategies are being investigated to enhance the osteogenic differentiation of stem cells. We previously reported two different approaches for this purpose, in monolayer and three-dimensional cell culture. The first approach was based on pretreating cells with 5-Aza-dC, a DNA methylation inhibitor, before the applying the differentiation media. The second approach was based on culturing cells on a glass surface during differentiation. In this study, we investigated the potential effect of combining both methods. Our results suggested that both approaches were associated with decreasing global DNA methylation levels. Cells cultured as a monolayer on glass surface showed enhancement in alkaline phosphatase activity at day 10, while 5-Aza-dC pretreatment enhanced the activity at day 5, irrespective of the culture surface. In three-dimensional pellet culture, 5-Aza-dC pretreatment enhanced osteogenesis through Runx-2 and TGF-β1 upregulation while the glass surface induced Osterix.Furthermore, pellets cultured on glass showed upregulation of a group of miRNAs, including pro-osteogenesis miR- 20a and miR -148b and anti-osteogenesis miR -125b, miR -31, miR -138, and miR -133a. Interestingly, 5-Aza-dC was not associated with a change of miRNAs in cells cultured on tissue culture plastic but reverted the upregulated miRNAs on the glass to the basal level. This study confirms the two approaches for enhancing osteogenic differentiation and contradicts their combination.

An Improved Diagnostic Tool to Predict Cartilage Formation in an Osteoarthritic Joint Environment.

Osteoarthritis (OA) is characterized by progressive articular cartilage loss. Due to the chondrogenic potential of human mesenchymal stromal cells (MSCs), MSC-based therapies are promising treatment strategies for cartilage loss. However, the local joint microenvironment has a great impact on the success of cartilage formation by MSCs. There are great interpatient differences in this local joint environment, therefore, the result of MSC therapies is uncertain. We previously developed gene promoter-based reporter assays as a novel tool to predict the effect of a patient's OA joint microenvironment on the success of MSC-based cartilage formation. In this study, we describe an improved version of this molecular tool with increased prediction accuracy. For this, we generated 14 stable cell lines using transcription factor (TF)-binding elements (AP1, ARE, CRE, GRE, ISRE, NFAT5, NFκB, PPRE, SBE, SIE, SOX9, SRE, SRF, and TCF/LEF) to drive luciferase reporter gene expression, and evaluated the cell lines for their responsiveness to an osteoarthritic microenvironment by stimulation with OA synovium-conditioned medium (OAs-cm; n = 31). To determine the effect of this OA microenvironment on MSC-based cartilage formation, MSCs were stimulated with OAs-cm while cultured in a three-dimensional pellet culture model. Pellets were assessed histologically and sulfated glycosaminoglycan production was quantified as a measure of cartilage formation. Six TF reporters correlated significantly with the effect of OAs-cm on cartilage formation. We validated the predictive value of these TF reporters with an independent cohort of OAs-cm (n = 22) and compared the prediction accuracy between our previous and the current new tool. Furthermore, we investigated which combination of reporters could predict the effect of the OA microenvironment on cartilage repair with the highest accuracy. A combination between the TF (NFκB) and the promoter-based (IL6) reporter proved to reach a more accurate prediction compared with the tools separately. These developments are an important step toward a diagnostic tool that can be used for personalized cartilage repair strategies for OA patients. Impact Statement We demonstrate the improvement of a novel diagnostic tool to predict if an osteoarthritis joint microenvironment is permissive for cartilage repair or not. The enhanced prediction accuracy is of great importance for the development of a diagnostic tool that can determine the success of mesenchymal stromal cell-based cartilage repair strategies.

Folic acid pretreatment and its sustained delivery for chondrogenic differentiation of MSCs

Dietary uptake of folic acid (FA) improves cartilage regeneration. In this work, we discovered that three days of FA treatment is highly effective for promoting chondrogenic differentiation of tonsil-derived mesenchymal stem cells (TMSCs). In a three-dimensional pellet culture, the levels of typical chondrogenic biomarkers, sulfated glycosaminoglycan, proteoglycan, type II collagen (COL II), SRY box transcription factor 9 (SOX 9), cartilage oligomeric matrix protein (COMP), and aggrecan (ACAN) increased significantly in proportion to FA concentration up to 30 μM. At the mRNA expression level, COL II, SOX 9, COMP, and ACAN increased 3.6–6.0-fold with FA treatment at 30 μM compared with the control system that did not receive FA treatment, and the levels with FA treatment were 1.6–2.5 times greater than those in the kartogenin-treated positive control system. FA treatment did not increase type I collagen α1 (COL I α1), an osteogenic biomarker which is a concern with most chondrogenic promoters. At the high FA concentration of 100 μM, significant decreases in chondrogenic biomarkers were observed, which might be related to DNA methylation. A thermogel system incorporating TMSCs and FA provided sustained release of FA over several days, similar to the FA treatment. The thermogel system confirmed the efficacy of FA in promoting chondrogenic promotion of TMSCs. The increased nuclear translocation of core-binding factor β subunit (CBFβ) and the runt-related transcription factor 1 (RUNX1) expression after FA treatment, together with molecular docking studies, suggest that the chondrogenic enhancement mechanism of FA is mediated by CBFβ and RUNX1.

Endochondral Bone Tissue Engineering Using Human Induced Pluripotent Stem Cells

The use of induced pluripotent stem cells (iPSCs) shows potential in bone regenerative strategies. In this study, we investigated whether implantation of chondrogenically differentiated iPSC-derived mesenchymal stem cells (iMSCs) can lead to successful bone regeneration in nude mice with bone defects. Two human iPSC clones (201B7 and 454E2) were used. After generating iMSCs, chondrogenic differentiation was achieved by three-dimensional pellet culture. Thereafter, a 2-mm defect was created in the radius of nude mice, and chondrogenically differentiated iMSC pellets were transplanted in the defect. Microcomputed tomography imaging was performed 8 weeks posttransplantation to assess bone regeneration. All (100%) radii in the 201B7 cell-derived pellet transplantation group and 7 of 10 (70%) radii in the 454E2 cell-derived pellet transplantation group showed bone union. In contrast, 2 of 11 radii (18%) in the control group showed bone union. Thus, the experimental groups showed significantly higher bone union rates than the control group (p < 0.05). Histological analysis 2 weeks postimplantation in the experimental groups revealed hypertrophic chondrocytes within grafted iMSC pellets and the formation of woven bone around them. This hypertrophic chondrocyte transitioning to newly formed bone suggests that the cartilaginous template can trigger endochondral bone ossification (ECO). Four weeks postimplantation, the cartilage template was reduced in size; newly formed woven bone was predominant in the defect site. New vessels were surrounded by a matrix of woven bone, and hypertrophic chondrocytes transitioning to newly formed bone indicated the progression of ECO. Eight weeks postimplantation, the pellets were completely resorbed and replaced by bone; complete bone union was observed. Dense mature bone developed with evidence of lamellar-like bone formation. Collectively, our results suggest that using iMSC-based cartilage grafts recapitulating the morphogenetic process of ECO in the context of embryonic skeletogenesis is a promising strategy for repairing large bone defects. Impact statement We investigated whether implantation of chondrogenically differentiated iPSC-derived mesenchymal stem cells (iMSCs) could lead to the successful regeneration of bone defects in vivo. We implanted two different clones of human induced pluripotent stem cells into a radial bone defect model. Eleven of 11 (100%) and 7 of 10 (70%) radii in the 201B7 and 454E2 cell-derived pellet transplantation groups, respectively, showed bone union, which were significantly higher than those in the control group [only 2 of 11 radii (18%)]. Overall, our results support the use of iMSC-based cartilage grafts recapitulating the morphogenetic process of endochondral bone ossification for repairing large bone defects.

Prediction of the Effect of the Osteoarthritic Joint Microenvironment on Cartilage Repair.

Osteoarthritis (OA) is characterized by progressive articular cartilage loss. Human mesenchymal stromal cells (MSCs) can be used for cartilage repair therapies based on their potential to differentiate into chondrocytes. However, the joint microenvironment is a major determinant of the success of MSC-based cartilage formation. Currently, there is no tool that is able to predict the effect of a patient's OA joint microenvironment on MSC-based cartilage formation. Our goal was to develop a molecular tool that can predict this effect before the start of cartilage repair therapies. Six different promoter reporters (hIL6, hIL8, hADAMTS5, hWISP1, hMMP13, and hADAM28) were generated and evaluated in an immortalized human articular chondrocyte for their responsiveness to an osteoarthritic microenvironment by stimulation with OA synovium-conditioned medium (OAs-cm) obtained from 32 different knee OA patients. To study the effect of this OA microenvironment on MSC-based cartilage formation, MSCs were cultured in a three-dimensional pellet culture model, while stimulated with OAs-cm. Cartilage formation was assessed histologically and by quantifying sulfated glycosaminoglycan (sGAG) production. We confirmed that OAs-cm of different patients had significantly different effects on sGAG production. In addition, significant correlations were obtained between the effect of the OAs-cm on cartilage formation and promoter reporter outcome. Furthermore, we validated the predictive value of measuring two promoter reporters with an independent cohort of OAs-cm and the effect of 87.5% of the OAs-cm on MSC-based cartilage formation could be predicted. Together, we developed a novel tool to predict the effect of the OA joint microenvironment on MSC-based cartilage formation. This is an important first step toward personalized cartilage repair strategies for OA patients. Impact statement We describe the development of a novel molecular tool to predict if an osteoarthritis joint microenvironment is permissive for cartilage repair or not. Such a tool is of great importance in determining the success of mesenchymal stromal cell-based cartilage repair strategies.

A promoter-based bioassay that predicts the effect of the osteoarthritic microenvironment on cartilage formation

Purpose: Recent developments have demonstrated that intrinsic cartilage repair is possible in certain osteoarthritis (OA) patients. A growing body of literature suggests an influence of joint microenvironment on intrinsic cartilage repair via affecting chondrogenic capacity of stem cells; i.e. this environment can be permissive or non-permissive for repair. A method able to discriminate between patients with permissive or non-permissive articular joint microenvironments would be of great use to predict the outcome of cartilage repair therapies. We postulate this can be best done with a bioassay, as in a bioassay different cellular signals are integrated and multiple reporters can be measured simultaneously. Therefore, our goal is to develop a rapid bioassay that will be able to discriminate between permissive and non-permissive joint conditions. Methods: To construct our bioassay, promoter sequences of four different genes were isolated from human gDNA using PCR; i.e. IL6 (-600 till +78), IL8 (-572 till +100 bp), ADAMTS5 (-1432 till +37) and WISP1 (-833 till +50) and cloned in the pNL1.2 vector (Promega) which expressed the unstable Nano luciferase. Promoter reporter constructs were efficiently introduced by transient transfection with lipofectamine in H11 chondrocytes. Transfected cells were stimulated for 24 hours with OA-synovium conditioned medium (OAs-cm), as a substitute for OA synovial fluid, from sixteen individual patients and luciferase activity was measured on a luminometer. OAs-cm was obtained by culturing synovium from end-stage OA patients for 24 hours. Fold change was calculated relative to control stimulation. These results hereof were correlated (Pearson) with the ability of these OAs-cm to inhibit sGAG formation during chondrogenesis. This was tested by chondrogenic differentiation of human fetal bone-marrow derived MSCs in an established three-dimensional pellet culture model. Pellets were cultured for 14 days in serum-free chondrogenic medium (without dexamethasone). After 10 days of differentiation, MSCs were exposed to the OAs-cm from the same sixteen individual donors. Cartilage formation was determined at day 14 by measuring sulfated glycosaminoglycan (sGAG) content. Results: Stimulation of the hIL6, hIL8 and hADAMTS5 promoter reporters with OAs-cm form sixteen different patients resulted in an up-regulation of promoter activity compared to control stimulation, whereas stimulation of the hWISP1 promoter reporter resulted in a down-regulation of the promoter activity compared to control. Interestingly, large heterogeneity was observed in the response of the promoter reporters between the OAs-cm from distinct patients with hIL6 (FC 33.9 ± 32.3), hIL8 (FC 32.2 ± 27.7), hADAMTS5 (FC 3.7 ± 2.4) and hWISP1 (FC 0.4 ± 0.24). Furthermore, individual OAs-cm reduced sGAG content of differentiating MSCs by a range between 3% and 65%. Significant correlations were found between the fold change of the promoter activity and the percentage of inhibition on chondrogenesis. We observed a positive correlation with hIL6 (p=0.01 and r=0.67), hIL8 (p=0.01 and r=0.66) and with hADAMTS5 (p=0.01 and r=0.72), whereas a negative correlation was seen with hWISP1 (p=0.002 and r=-0.78). To validate if this correlation will predict the effect of other OAs-cm, sixteen newly collected OAs-cm were tested and eleven out of sixteen OAs-cm were within the 95% prediction interval. Conclusions: The current study demonstrates that our reporter assays are able to predict the effect of the joint microenvironment (OAs-cm) on cartilage formation as measured by sGAG production. Significant correlations were observed between the fold change of these promoter reporters and the inhibition of chondrogenic capacity of hMSCs, with positive correlations for hIL6, hIL8 and hADAMTS5 and a negative correlation with hWISP1. Our promoter constructs appear to be able to predict repair capacity of stem cells and could be of use for a personalized treatment strategy.

Evidence that TD-198946 enhances the chondrogenic potential of human synovium-derived stem cells through the NOTCH3 signaling pathway.

Human synovium-derived stem cells (hSSCs) are an attractive source of cells for cartilage repair. At present, the quality of tissue and techniques used for cartilage regeneration have scope for improvement. A small compound, TD-198946, was reported to enhance chondrogenic induction from hSSCs; however, other applications of TD-198946, such as priming the cell potential of hSSCs, remain unknown. Our study aimed to examine the effect of TD-198946 pretreatment on hSSCs. HSSCs were cultured with or without TD-198946 for 7 days during expansion culture and then converted into a three-dimensional pellet culture supplemented with bone morphogenetic protein-2 (BMP2) and/or transforming growth factor beta-3 (TGFβ3). Chondrogenesis in cultures was assessed based on the GAG content, histology, and expression levels of chondrogenic marker genes. Cell pellets derived from TD-198946-pretreated hSSCs showed enhanced chondrogenic potential when chondrogenesis was induced by both BMP2 and TGFβ3. Moreover, cartilaginous tissue was efficiently generated from TD-198946-pretreated hSSCs using a combination of BMP2 and TGFβ3. Microarray analysis revealed that NOTCH pathway-related genes and their target genes were significantly upregulated in TD-198946-treated hSSCs, although TD-198946 alone did not upregulate chondrogenesis related markers. The administration of the NOTCH signal inhibitor diminished the effect of TD-198946. Thus, TD-198946 enhances the chondrogenic potential of hSSCs via the NOTCH3 signaling pathway. This study is the first to demonstrate the gradual activation of NOTCH3 signaling during chondrogenesis in hSSCs. The priming of NOTCH3 using TD-198946 provides a novel insight regarding the regulation of the differentiation of hSSCs into chondrocytes.

A comprehensive analysis of human dental pulp cell spheroids in a three-dimensional pellet culture system

ObjectiveThree-dimensional (3D) cell culture methods are of high importance to studies of biological processes. This is particularly the case with spheroid cultures, which create 3D cell aggregates without the use of exogenous materials. Compared to conventional monolayer cultures, cellular spheroid cultures have been demonstrated to improve multilineage potential and extracellular matrix production. To address this issue in depth, we present a more comprehensive analysis of 3D human dental pulp cell (hDPC) spheroids. DesignhDPC spheroids were fabricated by the pellet culture method and were cultured without adding any reagent to induce differentiation. The gene-expression profiles of the 3D and two-dimensional (2D) cultured hDPCs were compared by complementary DNA microarray analysis. Odontoblastic and osteoblastic differentiation marker gene expression was evaluated by quantitative real-time PCR (RT-qPCR). Hematoxylin-eosin (HE) staining and transmission electron microscopy (TEM) were applied to examine the morphology of hDPC spheroids and extracellular matrix components. ResultsCompared with 2D monolayer culture, microarray analysis identified 405 genes and 279 genes with twofold or greater differential expression after 3 days and 28 days of 3D culture, respectively. In 3D hDPC spheroids, gene ontology analysis revealed upregulation of extracellular matrix-related genes and downregulation of cell growth-related genes. RT-qPCR analysis showed higher expression levels of osteocalcin, dentin sialophosphoprotein, and alkaline phosphatase. TEM revealed the morphological characteristics of the fibrillar collagen-rich matrix and cell-cell interactions. ConclusionsThe present findings provide clues to understanding the mechanisms of pellet-cultured hDPCs and contribute to future research in the comparative studies of different 3D culture methods.

Impact of expansion and redifferentiation under hypothermia on chondrogenic capacity of cultured human septal chondrocytes

A critical limitation in the cultivation of cartilage for tissue engineering is the dedifferentiation in chondrocytes, mainly during in vitro amplification. Despite many previous studies investigating the influence of various conditions, no data exist concerning the effects of hypothermia. Our aim has been to influence chondrocyte dedifferentiation in vitro by hypothermic conditions. Chondrocytes were isolated from cartilage biopsies and seeded in monolayer and in three-dimensional pellet-cultures. Each cell culture was either performed at 32.2°C or 37°C during amplification. Additionally, the influence of the redifferentiation of chondrocytes in three-dimensional cell culture was examined at 32.2°C and 37°C after amplification at 32.2°C or 37°C. An 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay was used to measure cell proliferation in monolayer, whereas the polymerase chain reaction and immunohistochemical and histological staining were used in three-dimensional pellet-cultures. Real-time polymerase chain reaction was employed to measure the relative expression of the target genes collagen II, collagen I, aggrecan and versican. Ratios were estimated between collagen II/collagen I and aggrecan/versican to evaluate differentiation. A higher value of these ratios indicated an advantageous status of differentiation. In monolayer, hypothermia at 32.2°C slowed down the proliferation rate of chondrocytes significantly, being up to two times lower at 32.2°C compared with culture at 37°C. Simultaneously, hypothermia in monolayer decelerated dedifferentiation. The ratio of aggrecan/versican was significantly higher at 32.2°C compared with that at 37°C. In three-dimensional pellet-culture, the chondrocytes redifferentiated at 32.2°C and at 37°C, and this process is more distinct at 37°C than at 32.2°C. Similar results were obtained for the ratios of collagen II/collagen I and aggrecan/versican and were supported by immunochemical and histological staining. Thus, hypothermic conditions for chondrocytes are mainly advantageous in monolayer culture. In three-dimensional pellet-culture, redifferentiation predominates at 37°C compared with at 32.2°C. In particular, the results from the monolayer cultures show potential in the avoidance of dedifferentiation.

Acute stimulation of mesenchymal stem cells with cigarette smoke extract affects their migration, differentiation, and paracrine potential.

Mesenchymal stem cells (MSCs) are known to play a key role in tissue regeneration, while smoking cigarettes is described to impair it. This work focuses on the effect cigarette smoke extract (CSE) has on the migration, differentiation, and paracrine potential of human adipose derived MSCs (AdMSCs). To mimic native conditions in vitro, AdMSCs were cultured in either monolayer or three-dimensional pellet cultures. While constant exposure to high concentrations of CSE had lethal effects on AdMSCs, lower concentrations of CSE impaired cell migration when compared to control conditions. The secretion of key interleukins was downregulated when CSE was exposed to the cells at low concentrations. Moreover, in this work AdMSCs were exposed to CSE while simultaneously being induced to differentiate into adipocytes, osteoblasts, and chondrocytes to determine the effect of CSE on the cells potential to differentiate. While adipogenic differentiation showed no significant variation, AdMSCs exposed to osteogenic and chondrogenic supplements showed both early and late genetic level variation when acutely exposed to low concentrations of CSE. Our results indicate that even a small amount of cigarette smoke can have detrimental effects on the regenerative potential of MSCs.

Fetal Cartilage-Derived Cells Have Stem Cell Properties and Are a Highly Potent Cell Source for Cartilage Regeneration.

Current strategies for cartilage cell therapy are mostly based on the use of autologous chondrocytes or mesenchymal stem cells (MSCs). However, these cells have limitations of a small number of cells available and of low chondrogenic ability, respectively. Many studies now suggest that fetal stem cells are more plastic than adult stem cells and can therefore more efficiently differentiate into target tissues. However, the characteristics and the potential of progenitor cells from fetal tissue remain poorly defined. In this study, we examined cells from human fetal cartilage at 12 weeks after gestation in comparison with bone marrow-derived MSCs or cartilage chondrocytes from young donors (8-25 years old). The fetal cartilage-derived progenitor cells (FCPCs) showed higher yields by approximately 24 times than that of chondrocytes from young cartilage. The morphology of the FCPCs was polygonal at passage 0, being similar to that of the young chondrocytes, but it changed later at passage 5, assuming a fibroblastic shape more akin to that of MSCs. As the passages advanced, the FCPCs showed a much greater proliferation ability than the young chondrocytes and MSCs, with the doubling times ranging from 2∼4 days until passage 15. The surface marker profile of the FCPCs at passage 2 was quite similar to that of the MSCs, showing high expressions of CD29, CD90, CD105, and Stro-1. When compared to the young chondrocytes, the FCPCs showed much less staining of SA-β-gal, a senescence indicator, at passage 10 and no decrease in SOX9 expression until passage 5. They also showed a much greater chondrogenic potential than the young chondrocytes and the MSCs in a three-dimensional pellet culture in vitro and in polyglycolic acid (PGA) scaffolds in vivo. In addition, they could differentiate into adipogenic and osteogenic lineages as efficiently as MSCs in vitro. These results suggest that FCPCs have stem cell properties to some extent and that they are more active in terms of proliferation and chondrogenic differentiation than young chondrocytes or MSCs.

The Potential Application of Pulsed Ultrasound on Bone Defect Repair via Developmental Engineering: An In Vitro Study.

Repairing bone defect by recapitulation of endochondral bone formation, known as developmental engineering, has been a promising approach in bone tissue engineering. The critical issue in this area is how to effectively construct the hypertrophic cartilaginous template in vitro and enhance in vivo endochondral ossification process after implantation. Pulsed ultrasound stimulation has been widely used in the clinic for accelerating bone healing in fractures and nonunions. The aim of this study was to investigate whether ultrasound (US) could accelerate in vitro chondrogenesis and the hypertrophic process in certain microenvironments. Rat bone marrow mesenchymal stem cells were chondrogenic or hypertrophic differentiated in a three-dimensional pellet culture system with different media, and treated with different intensities of US. US exposure promoted chondrogenic differentiation of stem cells and inhibited their transition into the hypertrophic stage in a chondrogenic-friendly microenvironment. US significantly advanced hypertrophic differentiation of bone marrow stem cell pellets in hypertrophic medium after chondrogenesis. Our data indicated that pulsed US promoted in vitro chondrogenic and hypertrophic differentiation of stem cell pellets in specific culture conditions. The present study proves the potential application of US in the in vitro stage of "developmental engineering" for bone development and repair.

Pulsed electromagnetic field may accelerate in vitro endochondral ossification.

Recapitulation of embryonic endochondral bone formation is a promising alternative approach to bone tissue engineering. However, the time-consuming process is one of the reasons the approach is unpractical. Here, we aimed at accelerating the in vitro endochondral ossification process of tissue engineering by using a pulsed electromagnetic field (PEMF). The rat bone marrow-derived stem cells were chondrogenic or hypertrophic differentiated in a three-dimensional pellet culture system, and treated with different intensities of PEMF (1, 2, and 5 mT with modulation frequency 750 Hz, carrier frequency 75 Hz and a duty ratio of 0.8, 3 h/day for 4 weeks). The effects of PEMF on hypertrophy and endochondral ossification were assessed by safranin O staining, immunohistochemistry, and quantitative real-time polymerase chain reaction. The results suggest that PEMF at 1, 2, and 5 mT may inhibit the maintenance of the cartilaginous phenotype and increase cartilage-specific extracellular matrix degradation in the late stage of chondrogenic differentiation. In addition, among the three different intensities, only PEMF at 1 mT directed the differentiation of chondrogenic-induced stem cell pellets to the hypertrophic stage and promoted osteogenic differentiation. Our findings provide the feasibility to optimize the process of in vitro endochondral ossification with PEMF stimulation.

Nanocomposite Scaffold for Chondrocyte Growth and Cartilage Tissue Engineering: Effects of Carbon Nanotube Surface Functionalization

The goal of this study was to assess the long-term biocompatibility of single-wall carbon nanotubes (SWNTs) for tissue engineering of articular cartilage. We hypothesized that SWNT nanocomposite scaffolds in cartilage tissue engineering can provide an improved molecular-sized substrate for stimulation of chondrocyte growth, as well as structural reinforcement of the scaffold's mechanical properties. The effect of SWNT surface functionalization (-COOH or -PEG) on chondrocyte viability and biochemical matrix deposition was examined in two-dimensional cultures, in three-dimensional (3D) pellet cultures, and in a 3D nanocomposite scaffold consisting of hydrogels+SWNTs. Outcome measures included cell viability, histological and SEM evaluation, GAG biochemical content, compressive and tensile biomechanical properties, and gene expression quantification, including extracellular matrix (ECM) markers aggrecan (Agc), collagen-1 (Col1a1), collagen-2 (Col2a1), collagen-10 (Col10a1), surface adhesion proteins fibronectin (Fn), CD44 antigen (CD44), and tumor marker (Tp53). Our findings indicate that chondrocytes tolerate functionalized SWNTs well, with minimal toxicity of cells in 3D culture systems (pellet and nanocomposite constructs). Both SWNT-PEG and SWNT-COOH groups increased the GAG content in nanocomposites relative to control. The compressive biomechanical properties of cell-laden SWNT-COOH nanocomposites were significantly elevated relative to control. Increases in the tensile modulus and ultimate stress were observed, indicative of a tensile reinforcement of the nanocomposite scaffolds. Surface coating of SWNTs with -COOH also resulted in increased Col2a1 and Fn gene expression throughout the culture in nanocomposite constructs, indicative of increased chondrocyte metabolic activity. In contrast, surface coating of SWNTs with a neutral -PEG moiety had no significant effect on Col2a1 or Fn gene expression, suggesting that the charged nature of the -COOH surface functionalization may promote ECM expression in this culture system. The results of this study indicate that SWNTs exhibit a unique potential for cartilage tissue engineering, where functionalization with bioactive molecules may provide an improved substrate for stimulation of cellular growth and repair.

Human Bone Marrow-Derived Mesenchymal Stem Cells Display Enhanced Clonogenicity but Impaired Differentiation With Hypoxic Preconditioning

Stem cells are promising candidate cells for regenerative applications because they possess high proliferative capacity and the potential to differentiate into other cell types. Mesenchymal stem cells (MSCs) are easily sourced but do not retain their proliferative and multilineage differentiative capabilities after prolonged ex vivo propagation. We investigated the use of hypoxia as a preconditioning agent and in differentiating cultures to enhance MSC function. Culture in 5% ambient O(2) consistently enhanced clonogenic potential of primary MSCs from all donors tested. We determined that enhanced clonogenicity was attributable to increased proliferation, increased vascular endothelial growth factor secretion, and increased matrix turnover. Hypoxia did not impact the incidence of cell death. Application of hypoxia to osteogenic cultures resulted in enhanced total mineral deposition, although this effect was detected only in MSCs preconditioned in normoxic conditions. Osteogenesis-associated genes were upregulated in hypoxia, and alkaline phosphatase activity was enhanced. Adipogenic differentiation was inhibited by exposure to hypoxia during differentiation. Chondrogenesis in three-dimensional pellet cultures was inhibited by preconditioning with hypoxia. However, in cultures expanded under normoxia, hypoxia applied during subsequent pellet culture enhanced chondrogenesis. Whereas hypoxic preconditioning appears to be an excellent way to expand a highly clonogenic progenitor pool, our findings suggest that it may blunt the differentiation potential of MSCs, compromising their utility for regenerative tissue engineering. Exposure to hypoxia during differentiation (post-normoxic expansion), however, appears to result in a greater quantity of functional osteoblasts and chondrocytes and ultimately a larger quantity of high-quality differentiated tissue.

Bioengineered Cartilage in a Scaffold‐Free Method by Human Cartilage‐Derived Progenitor Cells: A Comparison With Human Adipose‐Derived Mesenchymal Stromal Cells

The objective of our study was to investigate chondrogenesis potential of human adipose-derived mesenchymal stromal cells (MSCs), using as a positive control a human source of cartilage-derived progenitor cells (PCs). This source of PCs was recently described by our group and dwells on the surface of nasoseptal cartilage. Histological analysis using Safranin O staining and immunofluorescence for actin filaments and collagen type II was performed on three-dimensional (3D) pellet cultures. Cartilage PCs and adipose MSCs showed similarities in monolayer culture related to cell morphology and proliferation. Our 3D pellet cultures substantially reduced the actin stress and after 21 days under chondrogenic medium, we observed an increase in the pellet diameter for cartilage PCs (7.4%) and adipose MSCs (21.2%). Adipose-derived MSCs responded to chondrogenic stimulus, as seen by positive areas for collagen type II, but they were not able to recreate a mature extracellular matrix. Using semi-quantitative analysis, we observed a majority of Safranin O areas rising from blue (no stain) to orange (moderate staining) and no changes in fibroblastic morphology (P < 0.0001). For cartilage PCs, chondrogenic induction is responsible for morphological changes and a high percentage of matrix area/number of cells (P ≤ 0.0001), evaluated by computerized histomorphometry. Morphological analyses reveal that adipose-derived MSCs were not able to recreate a bioengineered cartilage. The cost of culture was reduced, as the cartilage PCs under growth-factor free medium exhibit a high score for cartilage formation compared with the induced adipose mesenchymal stromal cells (P = 0.0021). Using a pellet 3D culture, our cartilage PCs were able to produce a cartilage tissue in vitro, leading to the future development of bioengineered products.

Serum-free media for articular chondrocytes in vitro expansion

Background In vitro chondrocyte expansion is a major challenge in cell-based therapy for human articular cartilage repair. Classical culture conditions usually use animal serum as a medium supplement, which raises a number of undesirable questions. In the present study, two kinds of defined, serum-free media were developed to expand chondrocytes in monolayer culture for the purpose of cartilage tissue engineering. Methods Bovine chondrocytes were expanded in serum-free media supplemented with fibroblast growth factor-2 and platelet-derived growth factor or fibroblast growth factor-2 and insulin-like growth factor. Expansion culture in a conventional 10% fetal bovine serum (FBS) medium served as control. Fibronectin coating was used to help cell adhesion in serum-free medium. Next, in vitro three-dimensional pellet culture was used to evaluate the chondrocyte capacity. Cell pellets were expanded in different media to re-express the differentiated phenotype (re-differentiation) and to form cartilaginous tissue. The pellets were assessed by glycosaminoglycans contents, collagen II, collagen I and collagen X immunohistological staining. Results Chondrocytes cultured in serum-free media showed no proliferation difference than cells grown with 10% FBS medium. In addition, chondrocytes expanded in both serum-free media expressed more differentiated phenotypes at the end of monolayer culture, as indicated by higher gene expression ratios of collagen type II to collagen type I. Pellets derived from chondrocytes cultured in both serum-free media displayed comparable chondrogenic capacities to pellets from cells expanded in 10% FBS medium. Conclusion These findings provide alternative culture approaches for chondrocytes in vitro expansion, which may benefit the clinical use of autologous chondrocytes implantation.

Chondrogenic capacity and alterations in hyaluronan synthesis of cultured human osteoarthritic chondrocytes

During osteoarthritis there is a disruption and loss of the extracellular matrix of joint cartilage, composed primarily of type II collagen, aggrecan and hyaluronan. In young patients, autologous chondrocyte implantation can be used to repair cartilage defects. However, for more elderly patients with osteoarthritis, such a repair approach is contraindicated because the procedure requires a large expansion of autologous chondrocytes in vitro leading a rapid, perhaps irreversible, loss of the chondrocyte phenotype. This study investigates whether osteoarthritic chondrocytes obtained from older patients can be expanded in vitro and moreover, induced to re-activate their chondrocyte phenotype. A decrease in chondrocyte phenotype markers, collagen II, aggrecan and SOX9 mRNA was observed with successive expansion of cells in monolayer culture. However, chondrogenic induction in three-dimensional pellet culture successfully rescued the expression of all three marker genes to native levels, even with 4th passage cells–cells representing an approximate 625-fold expansion in cell number. This data supports the use of osteoarthritic cells for autologous implantation repair. In addition, another set of gene products were explored as useful markers of the chondrocyte phenotype. Differentiated primary chondrocytes exhibited a common pattern of hyaluronan synthase isoforms that changed upon cell expansion in vitro and, reverted back to the original pattern following pellet culture. Moreover, the change in isoform pattern correlated with changes in the molecular size of synthesized hyaluronan.

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.

Matrix formation is enhanced in co-cultures of human meniscus cells with bone marrow stromal cells

The ultimate aim of this study was to assess the feasibility of using human bone marrow stromal cells (BMSCs) to supplement meniscus cells for meniscus tissue engineering and regeneration. Human menisci were harvested from three patients undergoing total knee replacements. Meniscus cells were released from the menisci after collagenase treatment. BMSCs were harvested from the iliac crest of three patients and were expanded in culture until passage 2. Primary meniscus cells and BMSCs were co-cultured in vitro in three-dimensional (3D) pellet culture at three different cell-cell ratios for 3 weeks under normal (21% O2 ) or low (3% O2 ) oxygen tension in the presence of serum-free chondrogenic medium. Pure BMSCs and pure meniscus cell pellets served as control groups. The tissue generated was assessed biochemically, histochemically and by quantitative RT-PCR. Co-cultures of primary meniscus cells and BMSCs resulted in tissue with increased (1.3-1.7-fold) deposition of proteoglycan (GAG) extracellular matrix (ECM) relative to tissues derived from BMSCs or meniscus cells alone under 21% O2 . GAG matrix formation was also enhanced (1.3-1.6-fold) under 3% O2 culture conditions. Alcian blue staining of generated tissue confirmed increased deposition of GAG-rich matrix. mRNA expression of type I collagen (COL1A2), type II collagen (COL2A1) and aggrecan were upregulated in co-cultured pellets. However, SOX9 and HIF-1α mRNA expression were not significantly modulated by co-culture. Co-culture of primary meniscus cells with BMSCs resulted in increased ECM formation. Co-delivery of meniscus cells and BMSCs can, in principle, be used in tissue engineering and regenerative medicine strategies to repair meniscus defects.