The potential role of mechanosensitive ion channels in substrate stiffness-regulated Ca2+ response in chondrocytes.

PURPOSE: The stiffness of the pericellular matrix (PCM) decreases in the most common degenerative joint disease, osteoarthritis (OA). This study was undertaken to explore the potential functional role of transient receptor potential vanilloid 4 (TRPV4), Piezo1, and Piezo2 in transducing different PCM stiffness in chondrocytes. METHODS AND RESULTS: Polydimethylsiloxane (PDMS) substrates with different stiffness (designated 197 kPa, 78 kPa, 54 kPa, or 2 kPa, respectively) were first prepared to simulate the decrease in stiffness of the PCM that chondrocytes encounter in osteoarthritic cartilage. Next, the TRPV4-, Piezo1-, or Piezo2-knockdown primary chondrocytes (designated TRPV4-KD, Piezo1-KD, or Piezo2-KD cells) were seeded onto these different PDMS substrates. Then, using a Ca2+-imaging system, substrate stiffness-regulated intracellular Ca2+ influx ([Ca2+]i) in chondrocytes was examined to investigate the role of TRPV4, Piezo1, and Piezo2 in Ca2+ signaling in response to different stiffness. Results showed that the characteristics of intracellular [Ca2+]i in chondrocytes regulated by PDMS substrate exhibited stiffness-dependent differences. Additionally, stiffness-evoked [Ca2+]i changes were suppressed in TRPV4-KD, Piezo1-KD, or Piezo2-KD cells compared with control siRNA-treated cells, implying that any channel is fundamental for Ca2+ signaling induced by substrate stiffness. Furthermore, TRPV4-mediated Ca2+ signaling played a central role in the response of chondrocytes to 197 kPa and 78 kPa substrate, while Piezo1/2-mediated Ca2+ signaling played a central role in the response of chondrocytes to 54 kPa and 2 kPa substrate. CONCLUSIONS: Collectively, these findings indicate that chondrocytes might perceive and distinguish the different PCM stiffness by using different mechanosensitive ion channels.

Substrate stiffness-dependent regulatory volume decrease and calcium signaling in chondrocytes.

The pericellular matrix stiffness is strongly associated with its biochemical and structural changes during the aging and osteoarthritis progress of articular cartilage. However, how substrate stiffness modulates the chondrocyte regulatory volume decrease (RVD) and calcium signaling in chondrocytes remains unknown. This study aims to investigate the effects of substrate stiffness on the chondrocyte RVD and calcium signaling by recapitulating the physiologically relevant substrate stiffness. Our results showed that substrate stiffness induces completely different dynamical deformations between the cell swelling and recovering progresses. Chondrocytes swell faster on the soft substrate but recovers slower than the stiff substrate during the RVD response induced by the hypo-osmotic challenge. We found that stiff substrate enhances the cytosolic Ca oscillation of chondrocytes in the iso-osmotic medium. Furthermore, chondrocytes exhibit a distinctive cytosolic Ca oscillation during the RVD response. Soft substrate significantly improves the Ca oscillation in the cell swelling process whereas stiff substrate enhances the cytosolic Ca oscillation in the cell recovering process. Our work also suggests that the TRPV4 channel is involved in the chondrocyte sensing substrate stiffness by mediating Ca signaling in a stiffness-dependent manner. This helps to understand a previously unidentified relationship between substrate stiffness and RVD response under the hypo-osmotic challenge. A better understanding of substrate stiffness regulating chondrocyte volume and calcium signaling will aid the development of novel cell-instructive biomaterial to restore cellular functions.

Combined effects of interleukin-1ß and cyclic stretching on metalloproteinase expression in corneal fibroblasts in vitro.

BACKGROUND: Corneal tensile strain increases if the cornea becomes thin or if intraocular pressure increases. However, the effects of mechanical stress on extracellular matrix (ECM) remodelling in the corneal repair process and the corneal anomalies are unknown. METHODS: In this study, the combined effects of interleukin-1ß (IL-1ß) on matrix metalloproteinases (MMPs) in corneal fibroblasts under cyclic stretching were investigated in vitro. Cultured rabbit corneal fibroblasts were subjected to 5, 10 or 15 % cyclic equibiaxial stretching at 0.1 Hz for 36 h in the presence of IL-1ß. Conditioned medium was harvested for the analysis of MMP2 and MMP9 protein production using the gelatin zymography and western blot techniques. RESULTS AND CONCLUSIONS: Cyclic equibiaxial stretching changed the cell morphology by increasing the contractility of F-actin fibres. IL-1ß alone induced the expression of MMP9 and increased the production of MMP2, and 5 % stretching alone decreased the production of MMP2, which indicates that a low stretching magnitude can reduce ECM degradation. In the presence of IL-1ß, 5 and 10 % stretching increased the production of MMP2, whereas 15 % stretching increased the production of MMP9. These results indicate that MMP expression is enhanced by cyclic mechanical stimulation in the presence of IL-1ß, which is expected to contribute to corneal ECM degradation, leading to the development of post-refractive surgery keratectasia.

Development of alginate-collagen interpenetrating network for osteoarthritic cartilage by in situ softening.

This study presents an alginate-collagen interpenetrating network (IPN) matrix of incorporating collagen fibrils into an alginate hydrogel by physical mixing and controlled gelation. The resulting matrix closely mimics the physiological and pathological stiffness range of the chondrocyte pericellular matrix (PCM). Chondrocytes were cultured within three-dimensional (3D) alginate-collagen IPN matrices with varying stiffness, namely Firm, Medium, and Soft. Alginate lyase was introduced to study the effects of the changes in stiffness of the Firm on chondrocyte response by in situ softening. The developed alginate-collagen IPN matrix displayed good cell-biocompatibility. Compared with stiffer tissue culture plastic (TCP), chondrocytes grown within Firm displayed a stabilized differentiated phenotype characterized by higher expression levels of aggrecan, collagen II, and SOX-9. Moreover, the developed alginate-collagen IPN matrix exhibited a gradually increased percentage of propidium iodide (PI)-positive dead cells with decreasing stiffness. Softer matrices directed cells towards higher proliferation rates and spherical morphologies while stimulating chondrocyte cluster formation. Furthermore, reducing Firm stiffness by in situ softening decreased aggrecan expression, contributing to matrix degradation similar to that seen in osteoarthritis (OA). Hence, the 3D alginate-collagen IPN constructs hold significant potential for in vitro replicating PCM stiffness changes observed in OA cartilage.

Resolving the formidable barrier of oxygen transferring rate (OTR) in ultrahigh-titer bioconversion/biocatalysis by a sealed-oxygen supply biotechnology (SOS).

BACKGROUND: The critical issue in the competitiveness between bioengineering and chemical engineering is the products titer and the volume productivity. The most direct and effective approach usually employs high-density biocatalyst, while the weakened mass transfer and evoked foam problem accompany ultrahigh-density biocatalyst loading and substrate/product titer. In high-density obligate aerobic bioconversion, oxygen as electron acceptor is a speed-limiting step in bioprocesses, but sufficient oxygen supply will lead to the foaming which results in a significant reduction in oxygen utilization and the use of additional defoamers. In this study, we designed a novel sealed-oxygen supply (SOS) biotechnology to resolve the formidable barrier of oxygen transferring rate (OTR), for bio-based fuels and chemical production process. RESULTS: Based on systemic analysis of whole-cell catalysis in Gluconobacter oxydans, a novel sealed-oxygen supply technology was smartly designed and experimentally performed for biocatalytic oxidation of alcohols, sugars and so on. By a simple operation skill of automatic online supply of oxygen in a sealed stirring tank bioreactor of SOS, OTR barrier and foaming problem was resolved with great ease. We finally obtained ultrahigh-titer products of xylonic acid (XA), 3-hydroxypropionic acid (3-HPA), and erythrulose at 588.4 g/L, 69.4 g/L, and 364.7 g/L, respectively. Moreover, the volume productivity of three chemical products was improved by 150-250% compared with normal biotechnology. This SOS technology provides a promising approach to promote bioengineering competitiveness and advantages over chemical engineering. CONCLUSION: SOS technology was demonstrated as an economic and universally applicable approach to bio-based fuels and chemicals production by whole-cell catalysis. The novel technology greatly promotes the competitiveness of bioengineering for chemical engineering, and provides a promising platform for the green and environmental use of biofuels.

Upregulated SET Promotes Cell Survival Through Activating Akt/NF-κB Signal in Colorectal Carcinoma.

PURPOSE: SET has been proven to be an oncogene, which promotes the initiation and progression in several kinds of malignant carcinomas. However, the expression and its functional roles in colorectal carcinoma (CRC) remained unknown. MATERIALS AND METHODS: CRC tissues samples, CRC cell lines and xenograft mouse tumors were used in this study. The mRNA and protein expressions were detected by quantitative real-time polymerase chain reaction (qRT-PCR), immunohistochemistry (IHC), and Western blot (WB), respectively. siRNAs were used to silence the gene expression. Cell viability, cell proliferation, colony formation, and apoptosis were measured by MTS assay, EdU incorporation assay, plated colony formation assay, and flow cytometry, respectively. Western blot was applied to evaluate the levels of Akt, p-Akt, c-Myc and cyclin D1. Xenograft mouse model was performed to observe the role of SET in vivo. RESULTS: Our results revealed that SET was up-regulated in CRC, and the expression of SET was increased with the development of CRC. SET knockdown in vitro attenuated cell proliferation activity, and increased cell apoptosis in CRC cells. Moreover, the knockdown of SET reduces tumorigenic potential in nude mice. For the mechanism, knockdown of SET promoted the dephosphorylation of Akt, followed by suppressing the translocation of NF-κB to nucleus. In addition, SET knockdown-mediated dephosphorylation of Akt downregulated the expression of c-Myc and Cyclin D1, which inhibited the cell survival in CRC. CONCLUSION: Our results indicated that SET promoted cell survival via activating Akt/NF-κB signaling pathway in CRC, which strongly suggested that SET might be a potential therapeutic target in the colorectal carcinoma treatment.

A techno-practical method for overcoming the biotoxicity and volatility obstacles of butanol and butyric acid during whole-cell catalysis by Gluconobacter oxydans.

BACKGROUND: Butyric acid is a platform chemical material, the production of which has been greatly stimulated by the diverse range of downstream applications in many industries. In particular, higher quality butyric acid used in food and medicine, is more dependent on microbiological production methods. Hence, the bio-oxidation of butanol to butyric acid has been identified as a promising method with good potential economic and environmental benefits. However, both butanol and butyric acid are usually intensively toxic to most microorganisms as well as the bio-oxidation pathway. To develop a green, efficient and competitive microbiological method is the primary work to overcome the bottleneck of butyric acid industry. RESULT: A combined bioprocess was designed with alternative whole-cell catalysis for butyric acid bio-conversion from butanol by Gluconobacter oxydans in a sealed-oxygen supply bioreactor (SOS). In the operation system, the escape of volatile substrates and toxic chemicals to cells can be avoided by the use of a sealed bioreactor, combined with the rejuvenation of cells by supplying energy co-factors. Finally, during a one-batch whole-cell catalysis, the utilization rate of substrate increased from 56.6 to 96.0% by the simple skill. Additionally, the techno-practical bioprocess can realize the purpose of cell-recycling technology through the rejuvenation effect of co-factor. Finally, we obtained 135.3 g/L butyric acid and 216.7 g/L sorbose during a 60-h whole-cell catalysis. This techno-practical technology provides a promising approach to promote the industrial production of butyric acid with more competitiveness. CONCLUSION: The techno-practical biotechnology has powerfully promoted the process of butyric acid production by microorganisms, especially makes up for the lack of aerobic fermentation in the industry, and surmounts the shortcomings of traditional anaerobic fermentation. At the same time, this technically practical system provides a promising approach for the promotion of the industrial production of butyric acid in a more competitive manner.

Cost-practical of glycolic acid bioproduction by immobilized whole-cell catalysis accompanied with compressed oxygen supplied to enhance mass transfer.

Bioprocess for Glycolic acid (GA) production from ethylene glycol by whole-cell catalysis of Gluconobacter oxydans is restrained by various biological impediments and high production costs. In this study, these limitations were subsided through the implementation of immobilized whole-cell bio-catalysis combined with increased oxygen supply. Results indicated that this strategy noticeably enhanced mass transfer efficiency, and prolonged cell life that significantly reduced the cost of biomass. Ultimately, with immobilized whole-cell catalysis in air-open and oxygen-open bioreactor, 41.3 and 66.9 g/L of GA was obtained within 48 h, with an increment of 62.0%. Additionally, in oxygen-compressed bioreactor, 63.3 g/L of GA was accumulated with the yield of 97.2%. Subsequently, 605.7 g of GA was produced after 10 rounds of recovery experiments. Although there was a slight decrease in GA production compared with pure-oxygen supply, production cost was reduced with limited oxygen supply. This strategy commendably demonstrated cost-practical bioprocess for GA production.

Combined effect of TNF-α and cyclic stretching on gene and protein expression associated with mineral metabolism in cementoblasts.

OBJECTIVE: This work aims to investigate how the combination of TNF-α and cyclic stretching affects the expression of gene and protein associated with mineral metabolism in cementoblasts in vitro. DESIGN: Cementoblasts were cyclically stretched using the Flexcell tension system 4000 in the presence of 10 ng/ml TNF-α. Subsequently, the gene and protein expression CAP, Col I, BSP, OPG, RANKL and RUNX2 were detected using RT- PCR and ELISA/Western immunoblotting methods, respectively. RESULTS: Cyclic stretching alone enhanced CAP, Col I, OPG, RANKL and RUNX2 expression in an amplitude manner, while decreased BSP expression. Expression of all these proteins was attenuated in the presence of TNF-α whether the cells were exposed to cyclic stretching or not. The ratio of RANKL/OPG was increased at any stimulation. CONCLUSION: This results suggest that TNF-α affected the regulation of gene and protein expression induced by mechanical stimulation in cementoblasts. This may suppress anabolism and promote catabolism of cementum. It suggests that inflammatory cytokine may impair the cementum remodeling under mechanical stimulation.

Induction of MMP­1 and ­3 by cyclical mechanical stretch is mediated by IL­6 in cultured fibroblasts of keratoconus.

In order to understand the effect of mechanical stretch on corneal extracellular matrix remodeling, human keratoconus fibroblasts (HKCFBs) were subjected to cyclic stretch in vitro and the expression of matrix metalloproteinases (MMPs), tissue inhibitor of metalloproteinases (TIMPs), and inflammatory cytokines were evaluated. HKCFBs were seeded into a flexible membrane base and subjected to a cyclic stretch regimen of 10% equibiaxial stretch at a stretching frequency of 1 Hz for 6 h using a Flexcell tension unit. An antibody directed against interleukin­6 (IL­6 Ab) was used to investigate the roles of IL­6 on mechanical stretch mediated regulation of MMP in HKCFBs. Culture supernatants were assayed using an enzyme­linked immunosorbent assay for MMP­1 and ­3, TIMP­1 and ­2, and IL­6. Total RNA from the cells was extracted, and quantitative polymerase chain reaction was used to determine mRNA for MMP­1 and ­3, TIMP­1 and ­2, and IL­6. In stretched cells, levels of MMP­1 and ­3 demonstrated an increase compared with unstretched cells, but levels of TIMP­1, and ­2 revealed a decrease. Mechanical stretch significantly increased the mRNA expression and protein synthesis of IL­6 compared with unstretched cells. IL­6 induced MMP­1 and ­3 expression, whereas no significant effects were observed in levels of TIMP­1 and ­2 compared with the untreated control groups. Additionally, the IL­6 Ab markedly inhibited the stretch­induced increase in MMP­1 and ­3 in culture supernatants in a dose­dependent manner. No significant differences in TIMP­1 and ­2 protein levels were detected between stretched cells treated with IL­6 Ab and stretched cells without IL­6 Ab treatment. These results indicate that cyclical mechanical stretch augments IL­6 production and MMP expression, and reduces levels of TIMP in HKCFBs. Thus, it is suggested that IL­6 mediates the stretch­induced MMP expression.

Induction of matrix metalloproteinase-1 by tumor necrosis factor-α is mediated by interleukin-6 in cultured fibroblasts of keratoconus.

Inflammatory molecules and matrix metalloproteinase (MMPs) have been found over-expressed in the tear film of patients with keratoconus. However, the mechanistic link between inflammatory molecules and MMPs in the pathogenesis of keratoconus remains still elusive. Therefore, we investigated the effect of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) on MMP-1 expression and used IL-6 antibody (IL-6 Ab) to examine the role of IL-6 on TNF-α mediated regulation of MMP-1 in fibroblasts of normal cornea and keratoconus. Real-time polymerase chain reaction, Enzyme-linked immunosorbent assay, and Western blot data demonstrated that MMP-1 and IL-6 were expressed in fibroblasts of normal cornea and keratoconus. Levels of MMP-1 and IL-6 were significantly higher in keratoconus than normal cornea. TNF-α treatment led to a significant increase in IL-6 levels. IL-6 treatment induced MMP-1 synthesis in normal cornea and keratoconus. TNF-α increased MMP-1 expression in a dose- and time-dependent manner and this response was completely inhibited by the IL-6 Ab. In conclusion, these results indicate that fibroblasts of keratoconus shows increased levels of IL-6 and MMP-1 gene and protein expression and IL-6 mediates the TNF-α-induced MMP-1 expression.