Intermittent Hydrostatic Pressure Research Articles

Induction of chondrogenic phenotype in synovium-derived progenitor cells by intermittent hydrostatic pressure

The aim of this study was to investigate the effect of intermittent hydrostatic pressure (IHP) on chondrogenic differentiation of synovium-derived progenitor cells (SPCs). SPCs, bone marrow-derived progenitor cells and skin fibroblasts from rabbits were subjected to IHP ranging from 1.0 to 5.0 MPa. The mRNA expression of proteoglycan core protein (PG), collagen type II and SOX-9 was examined using real-time reverse transcriptase-polymerase chain reaction (RT-PCR). The production of SOX-9 protein and glycosaminoglycan (GAG) by SPCs was analyzed by Western blot and the dimethylmethylene blue assay. In addition, mitogen-activated protein (MAP) kinase inhibitors for c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and the p38 pathway were used to identify the signal transduction pathways. Real-time RT-PCR showed that mRNA expression of PG, collagen type II and SOX-9 was significantly enhanced only in SPCs receiving 5.0 MPa of IHP. The production of SOX-9 protein and GAG by SPCs was also increased by exposure to 5.0 MPa of IHP. These up-regulated expressions were suppressed by pretreatment with an inhibitor of JNK, but not with inhibitors of ERK or p38. Our results demonstrated that the exposure of SPCs to 5.0 MPa of IHP could facilitate induction of the chondrogenic phenotype by the MAP kinase/JNK pathway. This finding suggests the potential for IHP utilization in regenerative treatments for cartilage injuries or osteoarthritis.

Coordinate Regulation of IL-1β and MMP-13 in Rat Tendons Following Subrupture Fatigue Damage

Mechanical overloading is a major causative factor of tendinopathy; however, its underlying mechanisms are unclear. We hypothesized mechanical overloading would damage tendons and alter genes associated with tendinopathy in a load-dependent manner. To test this hypothesis, we fatigue loaded rat patellar tendons in vivo and measured expression of the matrix-degrading enzyme MMP-13 and the inflammatory cytokine IL-1beta. We also examined these responses in cultured tenocytes exposed to intermittent hydrostatic pressure in vitro. Additionally, we hypothesized load-induced changes in tenocyte MMP-13 expression would be dependent on expression of IL-1beta. In vivo fatigue loading at 1.7% strain caused overt microstructural damage and upregulated expression of MMP-13 and IL-1beta, while 0.6% strain produced only minor changes in matrix microstructure and downregulated expression of both MMP-13 and IL-1beta. Loading of cultured tenocytes at 2.5 and 7.5 MPa produced comparable changes in expression to those of in vivo tendon loading. Blocking IL-1beta expression with siRNA suppressed load-induced both MMP-13 mRNA expression and activity. The data suggest fatigue loading alters expression of MMP-13 and IL-1beta in tendons in vivo and tenocytes in vitro in a load-dependent manner. The data also suggest MMP-13 is regulated by both IL-1beta-dependent and IL-1beta-independent pathways.

Hydrostatic Pressure Enhances Chondrogenic Differentiation of Human Bone Marrow Stromal Cells in Osteochondrogenic Medium

This study demonstrated the chondrogenic effect of hydrostatic pressure on human bone marrow stromal cells (MSCs) cultured in a mixed medium containing osteogenic and chondrogenic factors. MSCs seeded in type I collagen sponges were exposed to 1 MPa of intermittent hydrostatic pressure at a frequency of 1 Hz for 4 h per day for 10 days, or remained in identical culture conditions but without exposure to pressure. Afterwards, we compared the proteoglycan content of loaded and control cell/scaffold constructs with Alcian blue staining. We also used real-time PCR to evaluate the change in mRNA expression of selected genes associated with chondrogenic and osteogenic differentiation (aggrecan, type I collagen, type II collagen, Runx2 (Cbfa-1), Sox9, and TGF-beta1). With the hydrostatic pressure loading regime, proteoglycan staining increased markedly. Correspondingly, the mRNA expression of chondrogenic genes such as aggrecan, type II collagen, and Sox9 increased significantly. We also saw a significant increase in the mRNA expression of type I collagen, but no change in the expression of Runx2 or TGF-beta1 mRNA. This study demonstrated that hydrostatic pressure enhanced differentiation of MSCs in the presence of multipotent differentiation factors in vitro, and suggests the critical role that this loading regime may play during cartilage development and regeneration in vivo.

Effects of intermittent hydrostatic pressure on cell adhesive forces and other related parameters under various resting periods

The purpose of this study is to investigate the effects of intermittent hydrostatic pressure with various resting periods on the cell adhesive forces and other parameters related to spreading in early stage of cell adhesion. For this, bovine pulmonary arterial endothelium (CPAE, cell line), porcine articular chondrocytes, and human endothelial cells (HECs) were used. The cells were divided into six different experimental groups. Control group was cultured without stimulation, while the constant pressure was applied to group 1 for 2 h. Groups 2-5 were intermittently pressurized for 2 min at a time over a 2-h period with 5, 10, 15, and 20-min resting periods, respectively. Each group was then split into two subgroups, depending whether it experienced extra 60 min stabilization period after stimulation. The average adhesive force and the number and area of focal contacts were significantly higher in the group 4 subgroup, which received an extra 60 min of culture than in the other groups. Similarly, other parameters in this subgroup were significantly different from those in the other groups. The focal contact area and adhesive force were closely related (r = 0.990). We concluded that the mechanical stimuli affect cell adhesion and that the length of the resting period influences the adhesive forces generated at the early stages of adhesion.

Effects of cyclic hydrostatic pressure on the brain biogenic amines concentrations in the flounder, Platichthys flesus

The present study evaluated the effects of cyclic variations of hydrostatic pressure (HP) on neurotransmitters in the whole brain of flounder. The concentrations of the biogenic amines L-3,4-dihydroxyphenylalanine (L-DOPA), dopamine (DA), norepinephrine (NE), epinephrine (E), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), 3-methoxytyramine (3-MT), 5-hydroxytryptamine (5-HT) and 5-hydroxyindolacetic acid (5-HIAA) were measured. Fish were subjected to HP cyclic variations which mimic naturally occurring conditions for a period of 14 days. DA, NE and 5-HT concentrations were significantly smaller by 21, 24 and 36%, respectively, compared to control fish. The concentrations of monoamine metabolites HVA, 3-MT and 5-HIAA were also smaller than those in control fish. These results suggest that central monoaminergic systems were influenced during long exposure to cyclic HP. The decreases of central neurotransmitters content might be involved in the physiological and behavioral responses to intermittent HP in fish.

Redifferentiation of chondrocytes and cartilage formation under intermittent hydrostatic pressure

Since articular cartilage is subjected to varying loads in vivo and undergoes cyclic hydrostatic pressure during periods of loading, it is hypothesized that mimicking these in vivo conditions can enhance synthesis of important matrix components during cultivation in vitro. Thus, the influence of intermittent loading during redifferentiation of chondrocytes in alginate beads, and during cartilage formation was investigated. A statistically significant increased synthesis of glycosaminoglycan and collagen type II during redifferentiation of chondrocytes embedded in alginate beads, as well as an increase in glycosaminoglycan content of tissue-engineered cartilage, was found compared to control without load. Immunohistological staining indicated qualitatively a high expression of collagen type II for both cases.

Effects of Hydrostatic Pressure and Transforming Growth Factor-β3 on Adult Human Mesenchymal Stem Cell Chondrogenesis In Vitro

This study examined the effects of intermittent hydrostatic pressure (IHP) and transforming growth factor-beta 3 on chondrogenesis of adult human mesenchymal stem cells (hMSCs) in vitro. Chondrogenic gene expression was determined by quantifying mRNA signal levels for SOX9, a transcription factor critical for cartilage development and the cartilage matrix proteins, aggrecan and type II collagen. Extracellular matrix production was determined by weight and histology. IHP was applied to hMSCs in pellet culture at a level of 10 MPa and a frequency of 1 Hz for 4 h per day for periods of 3, 7, and 14 days. hMSCs responded to addition of TGF-beta 3 (10 ng/mL) with a greater than 10-fold increase (p < 0.01) in mRNA levels for each, SOX9, type II collagen, and aggrecan during a 14-day culture period. Applying IHP in the presence of TGF-beta 3 further increased the mRNA levels for these proteins by 1.9-, 3.3-, and 1.6-fold, respectively, by day 14. Chondrogenic mRNA levels were increased with just exposure to IHP. Extracellular matrix deposition of type II collagen and aggrecan increased in the pellets as a function of treatment conditions and time of culture. This study demonstrated adjunctive effects of IHP on TGF-beta 3-induced chondrogenesis and suggests that mechanical loading can facilitate articular cartilage tissue engineering.

Dose- and Time-Dependent Effects of Cyclic Hydrostatic Pressure on Transforming Growth Factor-β3-Induced Chondrogenesis by Adult Human Mesenchymal Stem Cellsin Vitro

This study examined effects of varying magnitudes of intermittent hydrostatic pressure (IHP) applied for different times on chondrogenesis of adult human mesenchymal stem cells (hMSCs) in vitro. hMSCs were exposed to 0.1, 1, and 10 MPa of IHP at a frequency of 1 Hz for 4 h/day for 3, 7, and 14 days in the presence of transforming growth factor (TGF-beta3). Chondrogenesis was characterized by gene expression, macromolecule production, and extracellular matrix deposition. Exposure of hMSCs to 0.1 MPa of IHP increased SOX9 and aggrecan mRNA expression by 2.2- and 5.6-fold, respectively, whereas type II collagen mRNA expression responded maximally at 10 MPa. Production of sulfated glycosaminoglycan responded to IHP of 1 MPa and 10 MPa, whereas collagen levels increased only at 10 MPa. Morphologically, matrix condensation occurred with increased IHP, concomitant with collagen expression. This study demonstrated that different levels of IHP differentially modulate hMSC chondrogenesis in the presence of TGF-beta3. The data suggest that tissue engineering of articular cartilage through application or recruitment of hMSCs can be facilitated by mechanical stimulation.

The Effects of Intermittent Hydrostatic Pressure on Self-Assembled Articular Cartilage Constructs

The Effects of Intermittent Hydrostatic Pressure on Self-Assembled Articular Cartilage Constructs

Effects of Hydrostatic Pressure on TMJ Disc Cells

The effects of mechanical stimuli on TMJ disc cells have yet to be investigated. This study examined for the first time the effect of constant and intermittent hydrostatic pressure (HP) on TMJ disc cells. Guided by studies on articular chondrocytes, the chosen amplitude was 10 MPa, the frequency was 1 Hz for intermittent HP, and the duration was 4 h. A one-time application of the HP stimulus was applied in 2-D and 3-D for gene expression studies. A duty cycle of 2 days on, 1 day off for 1 week of HP stimulus was used for biochemical content studies. In monolayer, the intermittent HP regimen increased collagen II expression, while constant HP increased collagen I expression when compared to the non-loaded control. However, the overall expression of collagen I was much higher than collagen II in both constant and intermittent HP. The expression results correlated well with gross morphology, histology, and biochemical content. At Week 1, the intermittent HP group had a lower content of collagen, 7.5 +/- 0.2 microg/construct, than the non-loaded control group, 18.2 +/- 4.0 microg/construct. The constant HP group showed the highest amount of collagen, 24.5 +/- 1.6 microg/construct. These data show that constant HP at 10 MPa for 4 h produces more collagen I than do the non-loaded control or intermittent HP at 10 MPa and 1 Hz.

The Effects of Intermittent Hydrostatic Pressure on Self-Assembled Articular Cartilage Constructs

To date, static culture for the tissue engineering of articular cartilage has shown to be inadequate in conferring functionality to constructs. Various forms of mechanical stimuli accompany articular cartilage development in vivo, and one of these is hydrostatic pressure. This study used histology, biochemistry, and biomechanics to examine the effects of intermittent hydrostatic pressure, applied at 10 MPa and 1 Hz for 4 h per day for 5 days per week for up to 8 weeks on self-assembled chondrocyte constructs. The self-assembling process is a novel approach that allows engineering of articular cartilage constructs without the use of exogenous scaffolds. The self-assembled constructs were found to be capable of enduring this loading regimen. Significant increases in collagen production were only observed in pressurized samples. Intermittent hydrostatic pressure prevented a significant decrease in total GAG, which was significant in controls. Aside from the beneficial effects intermittent hydrostatic pressure may have on ECM synthesis, its effects on mechanical properties may require longer culture periods to manifest. This study demonstrates the successful use of the self-assembling process to produce articular cartilage constructs. It also shows for the first time that long-term culture of tissue-engineered articular cartilage construct benefits from intermittent hydrostatic pressure.

Electrospun PLGA nanofiber scaffolds for articular cartilage reconstruction: mechanical stability, degradation and cellular responses under mechanical stimulation in vitro

We investigated the potential of a nanofiber-based poly(DL-lactide-co-glycolide) (PLGA) scaffold to be used for cartilage reconstruction. The mechanical properties of the nanofiber scaffold, degradation of the scaffold and cellular responses to the scaffold under mechanical stimulation were studied. Three different types of scaffold (lactic acid/glycolic acid content ratio = 75 : 25, 50 : 50, or a blend of 75 : 25 and 50 : 50) were tested. The tensile modulus, ultimate tensile stress and corresponding strain of the scaffolds were similar to those of skin and were slightly lower than those of human cartilage. This suggested that the nanofiber scaffold was sufficiently mechanically stable to withstand implantation and to support regenerated cartilage. The 50 : 50 PLGA scaffold was degraded faster than 75 : 25 PLGA, probably due to the higher hydrophilic glycolic acid content in the former. The nanofiber scaffold was degraded faster than a block-type scaffold that had a similar molecular weight. Therefore, degradation of the scaffold depended on the lactic acid/glycolic acid content ratio and might be controlled by mixing ratio of blend PLGA. Cellular responses were evaluated by examining toxicity, cell proliferation and extracellular matrix (ECM) formation using freshly isolated chondrocytes from porcine articular cartilage. The scaffolds were non-toxic, and cell proliferation and ECM formation in nanofiber scaffolds were superior to those in membrane-type scaffolds. Intermittent hydrostatic pressure applied to cell-seeded nanofiber scaffolds increased chondrocyte proliferation and ECM formation. In conclusion, our nanofiber-based PLGA scaffold has the potential to be used for cartilage reconstruction.

A three-dimensional collagen matrix as a suitable culture system for the comparison of cyclic strain and hydrostatic pressure effects on intervertebral disc cells

To study intervertebral disc cell mechanobiology, the authors developed experimental systems that allow the application of cyclic strain and intermittent hydrostatic pressure (IHP) on isolated disc cells under equal three-dimensional (3D) culture conditions. The purpose of the study was to characterize disc cell proliferation, viability, morphology, and gene expression in 3D collagen matrices. The effects of cyclic strain (1, 2, 4, and 8% strain; 1 Hz) and IHP (0.25 MPa, 0.1 Hz) on gene expression (real-time polymerase chain reaction) of anabolic and catabolic matrix proteins were investigated and compared with those derived from mechanically unstimulated controls. Intervertebral disc cells proliferated in the collagen gels (mean viability 91.6%) and expressed messenger RNA for collagen I, collagen II, aggrecan, matrix metalloproteinase (MMP)-2, and MMP-3. Morphologically, both spindle-shaped cells with longer processes and rounded cells were detected in the collagen scaffolds. Cyclic strain increased collagen II and aggrecan expression and decreased MMP-3 expression of anulus fibrosus cells. No significant difference between the four strain magnitudes was found. Intermittent hydrostatic pressure tended to increase collagen I and aggrecan expression of nucleus cells and significantly decreased MMP-2 and -3 expression of nucleus cells, whereas aggrecan expression of anulus cells tended to decrease. Based on these results, the collagen matrix appeared to be a suitable substrate to apply both cyclic strain and IHP to intervertebral disc cells under 3D culture conditions. Individual variations may be influenced by the extent of degeneration of the disc specimens from which the cells were isolated. This experimental setup may be suitable for studying the influence of degeneration on the disc cell response to mechanical stimuli.

Cyclic hydrostatic compression stimulates chondroinduction of C3H/10T1/2 cells

While the potential for intermittent hydrostatic pressure to promote cartilaginous matrix synthesis is well established, its potential to influence chondroinduction remains poorly understood. This study examined the effects of relatively short- and long-duration cyclic hydrostatic compression on the chondroinduction of C3H/10T1/2 murine embryonic fibroblasts by recombinant human bone morphogenetic protein-2 (rhBMP-2). Cells were seeded at high density into round bottom wells of a 96-well plate and supplemented with 25 ng/ml rhBMP-2. Experimental cultures were subjected to either 1,800 cycles/day or 7,200 cycles/day of 1 Hz sinusoidal hydrostatic compression to 5 MPa (applied 10 min on/10 min off) for 3 days. Non-pressurized control and experimental cultures were maintained in static culture for an additional 5 days. Cultures were then analyzed for alcian blue staining intensity, DNA and sulfated glycosaminoglycan (sGAG) content, and for the rate of collagen synthesis. Whereas cultures subjected to 1,800 pressure cycles exhibited no significant differences (statistical or qualitative) compared to controls, those subjected to 7,200 cycles stained more intensely with alcian blue, contained nearly twice as much sGAG, and displayed twice the rate of collagen synthesis as non-pressurized controls. This study demonstrates the potential for cyclic hydrostatic compression to stimulate chondrogenic differentiation of the C3H/10T1/2 cell line in a duration-dependent manner.

P217 Intermittent hydrostatic pressure induces the expression of chondrocyte-specific genens in rabbit synovium-derived progenitor cells

P217 Intermittent hydrostatic pressure induces the expression of chondrocyte-specific genens in rabbit synovium-derived progenitor cells

129 間欠的静水圧負荷およびその周波数変化が軟骨細胞の遺伝子発現に与える効果(OS1-10 : 細胞の物理刺激応答(3),マイクロ・ナノバイオメカニクス)

129 間欠的静水圧負荷およびその周波数変化が軟骨細胞の遺伝子発現に与える効果(OS1-10 : 細胞の物理刺激応答(3),マイクロ・ナノバイオメカニクス)

Hydrodynamic Stimulation and Long Term Cultivation of Nucleus Pulposus Cells: A New Bioreactor System to Induce Extracellular Matrix Synthesis by Nucleus Pulposus Cells Dependent on Intermittent Hydrostatic Pressure

A novel bioreactor system was constructed to induce extracellular matrix (ECM) synthesis by intervertebral disc (ID) cells due to intermittent hydrostatic pressure. The developed system is completely sterilizable and reusable. It is viable for cultivation, immobilization, and stimulation of various other cell types and tissues especially for cartilage. The custom made lid allows long-run cultivation through semi-continuous operation. Manual interferences and therefore the risk of contamination are reduced. Sampling, medium changing and addition of supplements are easily performed from the connected conditioning vessel, which could be placed in an incubator. For the present investigations nucleus pulposus cells from pigs were taken and immobilized in agarose to obtain three-dimensional cell matrix constructs which were subjected to intermittent hydrostatic pressure. Afterwards the construct was biochemically examined. The proven constituents of ECM were found to be released in dependence of the magnitude and profile of the applied pressure.

Intermittent hydrostatic pressure inhibits matrix metalloproteinase and pro-inflammatory mediator release from human osteoarthritic chondrocytes in vitro

Objective This study tested the hypothesis that intermittent hydrostatic pressure applied to human osteoarthritic chondrocytes modulates matrix metalloproteinase and pro-inflammatory mediator release in vitro. Design Human osteoarthritic articular chondrocytes were isolated and cultured as primary high-density monolayers. For testing, chondrocyte cultures were transferred to serum-free medium and maintained without loading or with exposure to intermittent hydrostatic pressure (IHP) at 10 MPa at a frequency of 1 Hz for periods of 6, 12 and 24 h. Levels of matrix metalloproteinase-2, -9 (MMP-2, -9), tissue inhibitor of metalloproteinase-1 (TIMP-1), and the pro-inflammatory mediators, interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1), released into the culture medium were assessed by ELISA. Matrix metalloproteinase activity was confirmed by zymographic analysis. Results In the absence of IHP, levels of MMP-2, TIMP-1, IL-6, and MCP-1 in the chondrocyte culture medium increased in a time-dependent manner. Application of IHP decreased MMP-2 levels at all time periods tested, relative to unloaded control cultures maintained for the same time periods. Although 84/82 kDa bands were faintly detectable by zymography, MMP-9 levels were not quantifiable in medium from loaded or unloaded cultures by ELISA. TIMP-1 levels were not altered in response to IHP at any time period tested. IL-6 and MCP-1 levels decreased in cultures exposed to IHP at 12 and 24 h, relative to unloaded control cultures maintained for the same time periods. Conclusion IHP decreased release of MMP-2, IL-6 and MCP-1 by osteoarthritic chondrocytes in vitro suggesting that pressure influences cartilage stability by modulating chondrocyte expression of these degradative and pro-inflammatory proteins in vivo.

Modelling cartilage mechanobiology.

The growth, maintenance and ossification of cartilage are fundamental to skeletal development and are regulated throughout life by the mechanical cues that are imposed by physical activities. Finite element computer analyses have been used to study the role of local tissue mechanics on endochondral ossification patterns, skeletal morphology and articular cartilage thickness distributions. Using single-phase continuum material representations of cartilage, the results have indicated that local intermittent hydrostatic pressure promotes cartilage maintenance. Cyclic tensile strains (or shear), however, promote cartilage growth and ossification. Because single-phase material models cannot capture fluid exudation in articular cartilage, poroelastic (or biphasic) solid/fluid models are often implemented to study joint mechanics. In the middle and deep layers of articular cartilage where poroelastic analyses predict little fluid exudation, the cartilage phenotype is maintained by cyclic fluid pressure (consistent with the single-phase theory). In superficial articular layers the chondrocytes are exposed to tangential tensile strain in addition to the high fluid pressure. Furthermore, there is fluid exudation and matrix consolidation, leading to cell 'flattening'. As a result, the superficial layer assumes an altered, more fibrous phenotype. These computer model predictions of cartilage mechanobiology are consistent with results of in vitro cell and tissue and molecular biology experiments.

Mechanical factors influence the expression of endostatin––an inhibitor of angiogenesis––in tendons

Avascular zones of tendons are predisposed for degenerative changes and spontaneous rupture. Therefore, we analyzed the expression of the endogenous angiogenesis inhibiting factor endostatin in human fetal and adult tendons by immunohistochemical and biochemical methods. Moreover, to elucidate factors involved in the regulation of vascularity, we exposed primary cultures of rat tendon cells to intermittent hydrostatic pressure (0.2 MPa, 0.1 Hz for 24 h), and measured the endostatin content by ELISA and the effect of the conditioned medium to the proliferation of human umbilical vein endothelial cells (HUVEC). In fetal tendons high endostatin levels could be quantified by ELISA whereas low levels were found in adult tissue. In fetal tendons endostatin could also be immunostained in endothelial cells but mainly in fibroblasts. In adult Achilles tendons endostatin immunostaining was restricted to endothelial cells. In the tibialis posterior tendon––as an example for “wrap around”––endostatin immunostaining remained positive in the fibrocartilage adjacent to the medial malleolus. Fibrochondrocytes of the type II collagen positive fibrocartilage were intensively stained with the endostatin antibody. Factor VIII immunostaining showed that this region was largely avascular. Monolayer cultures of tendon cells released measurable amounts of endostatin into their culture supernatants. Application of intermittent hydrostatic pressure increased endostatin expression significantly. The conditioned media of tendon fibroblasts cultivated under intermittent hydrostatic pressure inhibited the proliferation of HUVEC in a dose dependent way. The spatial expression of endostatin in adult gliding tendons suggests that mechanical factors are involved in the regulation of this anti-angiogenic factor. In accordance, tendon cells exposed to intermittent hydrostatic pressure inhibit endothelial cell proliferation via humoral factors and produce endostatin. These findings support the view that the development and maintenance of avascular zones in tendons might be caused by a mechanically induced upregulation of anti-angiogenic factors.