3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer cartilage tissue offer a promising solution to repair articular cartilage. To select the optimal cell source for tissue repair, it is important to develop an appropriate culture platform to systematically examine the biological and biomechanical differences in the tissue-engineered cartilage by different cell sources. Here we applied a three-dimensional (3D) biomimetic hydrogel culture platform to systematically examine cartilage regeneration potential of juvenile, adult, and osteoarthritic (OA) chondrocytes. The 3D biomimetic hydrogel consisted of synthetic component poly(ethylene glycol) and bioactive component chondroitin sulfate, which provides a physiologically relevant microenvironment for in vitro culture of chondrocytes. In addition, the scaffold may be potentially used for cell delivery for cartilage repair in vivo. Cartilage tissue engineered in the scaffold can be evaluated using quantitative gene expression, immunofluorescence staining, biochemical assays, and mechanical testing. Utilizing these outcomes, we were able to characterize the differential regenerative potential of chondrocytes of varying age, both at the gene expression level and in the biochemical and biomechanical properties of the engineered cartilage tissue. The 3D culture model could be applied to investigate the molecular and functional differences among chondrocytes and progenitor cells from different stages of normal or aberrant development.     

Computational Modeling to Predict the Temporal Regulation of Chondrocyte Metabolism in Response to Various Dynamic Compression Regimens

Based on previously published experimental work, computational models were developed to simulate the effect of different dynamic compression regimens on the activity of chondrocytes seeded in agarose constructs. In particular, the balance between proliferation and matrix synthesis can be adjusted by applying different intervals of continuous or intermittent mechanical compression. A phenomenological compartment based-modeling approach was used as first model. A more mechanistic cell cycle model was used as the second model. The compartment-based modeling approach was found to be useful in representing a balance between proliferation and proteoglycan synthesis, when the effect of a certain stimulation protocol is known. In order to predict the response to different intervals of mechanical stimulation, however, a more mechanistic cell cycle-based approach is required. The cell cycle model supports an important role of the onset of loading. In addition, an inhibitory effect of further loading is required, which is more likely to be related to cell cycle progression velocity than to a decreased probability of commitment to the cell cycle. The mechanisms behind this inhibitory effect and the computational implementation, however, require further investigation.     

Regenerative Potential of Blood-Derived Products in 3D Osteoarthritic Chondrocyte Culture System

Intra-articular injection of different types of blood-derived products is gaining popularity and clinical importance in the treatment of degenerative cartilage disorders such as osteoarthritis. The regenerative potential of two types of platelet-rich plasma (PRP), prepared in the presence of EDTA (EPRP) and citrate (CPRP) and an alternative blood product-hyperacute serum (hypACT) was evaluated using a 3D osteoarthritic chondrocyte pellet model by assessing the metabolic cell activity, cartilage-related gene expression and extracellular matrix deposition within the pellets. Chondrocyte viability was determined by XTT assay and it revealed no significant difference in metabolic activity of OA chondrocyte pellets after supplementation with different blood products. Nevertheless, the selection of blood products influenced the cartilage-related genes expression, ECM morphology and the tissue quality of pellets. Both PRP types had a different biological effect depending upon concentration and even though CPRP is widely used in clinics our assessment did not reveal good results in gene expression either tissue quality. HypACT supplementation resulted in superior cartilage-related genes expression together with tissue quality and seemed to be the most stable product since no remarkable changes were observed between the two different concentrations. All in all, for successful regenerative therapy, possible molecular mechanisms induced by blood-derived products should be always carefully investigated and adapted to the specific medical indications.     

A Comparative Study of Fibroblast Behaviors under Cyclic Stress Stimulus and Static Culture on 3D Patterned Matrix

Mechanical stress and patterned surface of the scaffolds has been recognized as a crucial factor in determining cell functionality and tissue development, which in turn can direct the cell responses. In this study, fibroblasts M-3T3 in three-dimensional (3D) honeycomb patterning Chitosan/Poly(L-Lactic Acid) (CS/PLLA) composites was stimulated by a 15% sinusoidal (1 Hz) strain applied by a biodynamic test instrument. The effects of mechanical stimulus on the cell proliferation and basic Fibroblast Growth Factor (bFGF) secretion were studied in comparison to the non-strain groups and blank control. Results show that fibroblasts are able to sense the mechanical stimulation and respond, resulting in a time dependent increase of bFGF secretion and promoting cell proliferation. Moreover, the cells seeded in the scaffolds showed a higher cell proliferation and bFGF secretion. These findings support the hypothesis that suitable mechanical stimulus has positive effect on fibroblasts, and such a 3D honeycomb patterned scaffold may play a positive role in regulating cell behaviors in vitro.     

Epigenetic Regulation of Chondrocyte Catabolism and Anabolism in Osteoarthritis

Osteoarthritis (OA) is one of the most prevalent forms of joint disorder, associated with a tremendous socioeconomic burden worldwide. Various non-genetic and lifestyle-related factors such as aging and obesity have been recognized as major risk factors for OA, underscoring the potential role for epigenetic regulation in the pathogenesis of the disease. OA-associated epigenetic aberrations have been noted at the level of DNA methylation and histone modification in chondrocytes. These epigenetic regulations are implicated in driving an imbalance between the expression of catabolic and anabolic factors, leading eventually to osteoarthritic cartilage destruction. Cellular senescence and metabolic abnormalities driven by OA-associated risk factors appear to accompany epigenetic drifts in chondrocytes. Notably, molecular events associated with metabolic disorders influence epigenetic regulation in chondrocytes, supporting the notion that OA is a metabolic disease. Here, we review accumulating evidence supporting a role for epigenetics in the regulation of cartilage homeostasis and OA pathogenesis.     

Dynamic 3D Cell Rearrangements Guided by a Fibronectin Matrix Underlie Somitogenesis

Somites are transient segments formed in a rostro-caudal progression during vertebrate development. In chick embryos, segmentation of a new pair of somites occurs every 90 minutes and involves a mesenchyme-to-epithelium transition of cells from the presomitic mesoderm. Little is known about the cellular rearrangements involved, and, although it is known that the fibronectin extracellular matrix is required, its actual role remains elusive. Using 3D and 4D imaging of somite formation we discovered that somitogenesis consists of a complex choreography of individual cell movements. Epithelialization starts medially with the formation of a transient epithelium of cuboidal cells, followed by cell elongation and reorganization into a pseudostratified epithelium of spindle-shaped epitheloid cells. Mesenchymal cells are then recruited to this medial epithelium through accretion, a phenomenon that spreads to all sides, except the lateral side of the forming somite, which epithelializes by cell elongation and intercalation. Surprisingly, an important contribution to the somite epithelium also comes from the continuous egression of mesenchymal cells from the core into the epithelium via its apical side. Inhibition of fibronectin matrix assembly first slows down the rate, and then halts somite formation, without affecting pseudopodial activity or cell body movements. Rather, cell elongation, centripetal alignment, N-cadherin polarization and egression are impaired, showing that the fibronectin matrix plays a role in polarizing and guiding the exploratory behavior of somitic cells. To our knowledge, this is the first 4D in vivo recording of a full mesenchyme-to-epithelium transition. This approach brought new insights into this event and highlighted the importance of the extracellular matrix as a guiding cue during morphogenesis.     

3D水凝胶压应力模型建立及压应力对成骨细胞的影响

目的:通过建立3D水凝胶细胞模型,采用不同强度、频率、时间对MC3T3-E1细胞进行机械加压干预,进而探讨促进成骨细胞生长的适宜压应力方案。方法:设计不同强度、频率、时间的压应力梯度方案对成骨细胞MC3T3-E1进行干预。加压干预结束后即刻收集细胞,对样本中的ATF4、ALP、Runx2、Osteocalcin、RANKL和RANK mRNA进行定量检测。结果:经统计学分析后发现不同强度和频率对ALP(P0.05)、Runx2(P0.01)交互作用显著。此外,4h加压Runx2的表达量比12h加压的高(P0.05);4h加压RANKL的表达量比12h加压的低(P0.05)。结论:确定了压力强度和频率后,对3D细胞水凝胶模型施加一段时间的压应力发现,以1%强度、0.5Hz频率、4h的压应力干预方案能够促进成骨细胞系的生长。     

Dynamic Hubs Show Competitive and Static Hubs Non-Competitive Regulation of Their Interaction Partners

Date hub proteins have 1 or 2 interaction interfaces but many interaction partners. This raises the question of whether all partner proteins compete for the interaction interface of the hub or if the cell carefully regulates aspects of this process? Here, we have used real-time rendering of protein interaction networks to analyse the interactions of all the 1 or 2 interface hubs of Saccharomyces cerevisiae during the cell cycle. By integrating previously determined structural and gene expression data, and visually hiding the nodes (proteins) and their edges (interactions) during their troughs of expression, we predict when interactions of hubs and their partners are likely to exist. This revealed that 20 out of all 36 one- or two- interface hubs in the yeast interactome fell within two main groups. The first was dynamic hubs with static partners, which can be considered as ‘competitive hubs’. Their interaction partners will compete for the interaction interface of the hub and the success of any interaction will be dictated by the kinetics of interaction (abundance and affinity) and subcellular localisation. The second was static hubs with dynamic partners, which we term ‘non-competitive hubs’. Regulatory mechanisms are finely tuned to lessen the presence and/or effects of competition between the interaction partners of the hub. It is possible that these regulatory processes may also be used by the cell for the regulation of other, non-cell cycle processes.     

Oxidative Stress Inhibits Insulin-like Growth Factor-I Induction of Chondrocyte Proteoglycan Synthesis through Differential Regulation of Phosphatidylinositol 3-Kinase-Akt and MEK-ERK MAPK Signaling Pathways

The ability of insulin-like growth factor I (IGF-I) to stimulate cartilage matrix synthesis is reduced in aged and osteoarthritic cartilage. Aging and osteoarthritis are associated with an increase in reactive oxygen species, which we hypothesized would interfere with normal IGF-I signaling. We compared IGF-I signaling in normal and osteoarthritic human articular chondrocytes and investigated the effects of oxidative stress induced by tert-butylhydroperoxide (tBHP). In normal human chondrocytes, IGF-I initiated a strong and sustained phosphorylation of IRS-1 (Tyr-612) and Akt (Ser-473) and transient ERK phosphorylation. In contrast, in osteoarthritic chondrocytes, which possessed elevated basal IRS-1 (Ser-312) and ERK phosphorylation, IGF-I failed to stimulate IRS-1 (Tyr-612) or Akt phosphorylation. In normal human chondrocytes, tBHP triggered strong IRS-1 (Ser-312 and Ser-616) and ERK phosphorylation and inhibited IGF-I-induced IRS-1 (Tyr-612) and Akt phosphorylation. Lentivirus-mediated overexpression of constitutively active (CA) Akt significantly enhanced proteoglycan synthesis, whereas both dominant negative Akt and CA MEK inhibited proteoglycan synthesis. CA Akt also promoted type II collagen and Sox9 expression, whereas tBHP treatment and CA MEK inhibited aggrecan, collagen II, and Sox9 mRNA expression. In osteoarthritic chondrocytes, the antioxidants Mn(III) tetrakis(4-benzoic acid)porphyrin and N-acetylcysteine increased the ratio of Akt to ERK phosphorylation and promoted IGF-I-mediated proteoglycan synthesis. Chemical inhibition of ERK significantly enhanced IGF-I phosphorylation of Akt and alleviated tBHP inhibition of Akt phosphorylation. These results demonstrate opposing roles for phosphatidylinositol 3-kinase-Akt and MEK-ERK in cartilage matrix synthesis and suggest that elevated levels of reactive oxygen species cause chondrocyte IGF-I resistance by altering the balance of Akt to ERK activity.     

Effect of Matrix on Cardiomyocyte Viscoelastic Properties in 2D Culture

Cardiomyocyte phenotype changes significantly in 2D culture systems depending on the substrate composition and organization. Given the variety of substrates that are used both for basic cardiac cell culture studies and for regenerative medicine applications, there is a critical need to understand how the different matrices influence cardiac cell mechanics. In the current study, the mechanical properties of neonatal rat cardiomyocytes cultured in a subconfluent layer upon aligned and unaligned collagen and fibronectin matrices were assessed over a two week period using atomic force microscopy. The elastic modulus was estimated by fitting the Hertz model to force curve data and the percent relaxation was determined from stress relaxation curves. The Quasilinear Viscoelastic (QLV) and Standard Linear Solid (SLS) models were fit to the stress relaxation data. Cardiomyocyte cellular mechanical properties were found to be highly dependent on matrix composition and organization as well as time in culture. It was observed that the cells stiffened and relaxed less over the first 3 to 5 days in culture before reaching a plateau in their mechanical properties. After day 5, cells on aligned matrices were stiffer than cells on unaligned matrices and cells on fibronectin matrices were stiffer than cells on collagen matrices. No such significant trends in percent relaxation measurements were observed but the QLV model fit the data very well. These results were correlated with observed changes in cellular structure associated with culture on the different substrates and analyzed for cell-to-cell variability.     

Human Lung Cancer Cells Grown in an Ex Vivo 3D Lung Model Produce Matrix Metalloproteinases Not Produced in 2D Culture

We compared the growth of human lung cancer cells in an ex vivo three-dimensional (3D) lung model and 2D culture to determine which better mimics lung cancer growth in patients. A549 cells were grown in an ex vivo 3D lung model and in 2D culture for 15 days. We measured the size and formation of tumor nodules and counted the cells after 15 days. We also stained the tissue/cells for Ki-67, and Caspase-3. We measured matrix metalloproteinase (MMP) levels in the conditioned media and in blood plasma from patients with adenocarcinoma of the lung. Organized tumor nodules with intact vascular space formed in the ex vivo 3D lung model but not in 2D culture. Proliferation and apoptosis were greater in the ex vivo 3D lung model compared to the 2D culture. After 15 days, there were significantly more cells in the 2D culture than the 3D model. MMP-1, MMP-9, and MMP-10 production were significantly greater in the ex vivo 3D lung model. There was no production of MMP-9 in the 2D culture. The patient samples contained MMP-1, MMP-2, MMP-9, and MMP-10. The human lung cancer cells grown on ex vivo 3D model form perfusable nodules that grow over time. It also produced MMPs that were not produced in 2D culture but seen in human lung cancer patients. The ex vivo 3D lung model may more closely mimic the biology of human lung cancer development than the 2D culture.     

人胚髁状突软骨细胞的改良培养及生物学特性的研究

目的:探讨行之有效的人胚髁状突软骨细胞体外分离培养方法及研究其生物学特性.方法:用0.25%胰蛋白酶和0.2%Ⅱ型胶原酶分阶段联合消化法分离髁状突软骨细胞,将分离的软骨细胞与未消化完的小片软骨共同置入预涂多聚赖氨酸的培养瓶中培养,通过倒置显微镜,免疫组织化学染色、电镜观察等方法与传统培养方法对比,检测软骨细胞的分离后存活率、贴壁生长速度及传代7次内髁状突软骨细胞表型改变相伴随的形态学及生物学特征.结果:本方法培养的人髁突软骨细胞存活率可达98%以上,细胞贴壁能力强,与传统培养方法形成细胞单层的速度相比具有统计学意义(p<0.05).7代以内培养的髁状突软骨细胞免疫组织化学染色阳性,透射电镜可见细胞内有丰富的粗面内质网及线粒体.而传统培养方法所获得的人髁状突软骨细胞存活率为90%左右,培养3代以后免疫组织化学染色显示其软骨细胞表型逐渐减弱,培养至第7代其免疫组织化学染色为阴性.结论:本方法培养的髁状突软骨细胞存活率高,且能维持软骨细胞的特有表型,至少能保持软骨细胞7代稳定,是一种简便、有效的培养方法.     

Synthesis and biologic activity of 3 beta-thiovitamin D3

We synthesized 3 beta-thiovitamin D3 from 7-dehydrocholesterol and tested its biological activity and protein binding properties. The thiovitamin was found to be a weak vitamin D agonist at high doses in vivo. It was poorly bound by both vitamin D-binding protein as well as by the intestinal cytosol receptor for 1,25-dihydroxyvitamin D. It did not increase the synthesis of calcium binding protein in the chick embryonic duodenum and did not block the activity of 1,25-dihydroxyvitamin D3 in this system. We conclude that 3 beta-thiovitamin D3 is a weak vitamin D agonist in vivo with no agonist activity or antagonist activity to 1,25-dihydroxyvitamin D3 in the chick embryonic duodenum.     

三维支架材料中软骨细胞生长过程的数值模拟

组织工程是临床上用于修复以及重建受损软骨的一项有广泛应用前景的方法。但是在支架材料内部物质传递仅仅依赖于扩散,这是支架材料中细胞生长的主要限制因素。利用氧扩散-反应基本原理建立了软骨细胞生长过程的数学模型,同时考虑了由于细胞生长引起的扩散系数下降以及空间抑制因素对细胞生长的影响。模拟结果与实验数据吻合良好,表明该模型对材料内部的细胞生长的分析具有较高的可靠性,可用于组织工程生物反应器以及三维多孔支架材料的优化设计。