Intermittent Hydrostatic Pressure Research Articles

Intermittent Hydrostatic Pressure Promotes Cartilage Repair in an Inflammatory Environment through Hippo-YAP Signaling In Vitro and In Vivo.

The study of chondrogenic progenitor cells (CPCs) as seed cells has become a new focus of cartilage regeneration. The inflammatory environment of osteoarthritis (OA) inhibits the repair ability of CPCs. But the OA patients' CPCs showed an excellent regeneration ability with intermittent hydrostatic pressure (IHP). However, the mechanism is unclear. We compared the expression of the Hippo signaling effect factor YAP between OA and normal cartilages. Then, the relationship between the Kellgren-Lawrence (K-L) score of OA and the rate of YAP-positive cells was analyzed. The changes of CPCs after IHP and IL-1β applications were observed. The OA model was established by cutting the anterior cruciate ligament of rats. The knee joint of the OA rats was distracted by hinged external fixator to create suitable IHP, named as the IHP group. The IHP group plus intra-articular injection of Verteporfin (VP) was named as the IHP+VP group, and the untreated rat group was named as the CON group. Four and 8 weeks after the operation, the reparative effect was evaluated by MASSON staining and immunohistochemical staining. Lower levels of YAP1 and higher expressions of p-YAP1 were found in the OA group as compared to the normal group. IHP inhibited the Hippo signaling in an inflammatory environment and promoted the proliferation of CPCs. The cartilage deterioration in the CON group progressed more significantly than that in the IHP+VP group. The best reparative effect was observed in the IHP group with increased expression of YAP1 and decreased p-YAP1. These results hint that mechanical stress can activate CPCs and promote cartilage repair in an inflammatory environment through inhibiting Hippo signaling.

Effect of intermittent hydrostatic pressure on aging human chondrocyte cells.

In this study, a hydrostatic pressure chamber (HPC) is designed and developed to host chondrocyte cell culture. External stimuli such as forces, pressure, vibration etc. are found to be significant factors on upregulating the relevant proteins for constructing the extracellular matrix (ECM) during culturing. The aim of this paper is to design a system which provides external stimuli during chondrocyte cell culture as well as to discover the relevant gene which can generate the repair and regeneration of aging cells. The system consists of a controllable HPC that provides intermittent hydrostatic pressure (HP) on the cultured cells. The chamber is capable of applying intermittent HP in the range: 0 to1 MPa, at a frequency of 0.5-1Hz. An investigation was undertaken to determine the improvement of human chondrocyte cells viz. of 3 sub-jects whose ages are 60 and above. The effect of HP on the aging cells is observed through the extracted ribonucleic acid (RNA) after the cell is treated with HP for two hours, each day, over four days. The experiments were conducted to observe the effect of HP on the level of collagen type I, collagen type II, and aggrecan. Results show that HP did little to help in upregulating the aggrecan and collagen type II in aged-chondrocyte cells. Further, it was found that the application of HP depended on the number of days applied. The results presented the possibility of ap-plying HP in regeneration of damaged cartilage in elderly.

Eliminating senescent chondrogenic progenitor cells enhances chondrogenesis under intermittent hydrostatic pressure for the treatment of OA

BackgroundOsteoarthritis (OA) is a major cause of limb dysfunction, and distraction arthroplasty which generates intermittent hydrostatic pressure (IHP) is an effective approach for OA treatment. However, the result was not always satisfactory and the reasons remained unresolved. Because aging is recognized as an important risk factor for OA and chondrogenic progenitor cells (CPCs) could acquire senescent phenotype, we made a hypothesis that CPCs senescence could have harmful effect on chondrogenesis and the outcome of distraction arthroplasty could be improved by eliminating senescent CPCs pharmacologically.MethodsThe role of senescent CPCs on distraction arthroplasty was first determined by comparing the cartilage samples from the failure and non-failure patients. Next, the biological behaviors of senescent CPCs were observed in the in vitro cell culture and IHP model. Finally, the beneficial effect of senescent CPCs clearance by senolytic dasatinib and quercetin (DQ) on cartilage regeneration was observed in the in vitro and in vivo IHP model.ResultsLarger quantities of senescent CPCs along with increased IL-1 β secretion were demonstrated in the failure patients of distraction arthroplasty. Senescent CPCs revealed impaired proliferation and chondrogenic capability and also had increased IL-1 β synthesis, typical of senescence-associated secretory phenotype (SASP). CPCs senescence and SASP formation were mutually dependent in vitro. Greater amounts of senescent CPCs were negatively correlated with IHP-induced chondrogenesis. In contrast, chondrogenesis could be significantly improved by DQ pretreatment which selectively induced senescent CPCs into apoptosis in the in vitro and in vivo IHP model. Mechanistically, senescent CPCs elimination could decrease SASP formation and therefore promote the proliferation and chondrogenic regeneration capacity of the surrounding survived CPCs under IHP stimulation.ConclusionsEliminating senescent CPCs by senolytics could decrease SASP formation and improve the result of joint distraction arthroplasty effectively. Our study provided a novel CPCs senescence-based therapeutic target for improving the outcome of OA treatment.

A Platform for Studying of the Three-Dimensional Migration of Hematopoietic Stem/Progenitor Cells.

Hematopoietic stem/progenitor cells (HSPCs) have the property to return to the bone marrow, which is believed to be critical in situations such as HSPC transplantation. This property plays an important role in the stemness, viability, and proliferation of HSPCs, also. However, most in vitro models so far have not sufficiently simulated the complicate environment. Here, we proposed a three-dimensional experimental platform for the quantitative study of the migration of HSPCs. After encapsulating osteoblasts (OBs) in alginate beads, we quantified the migration of HSPCs into the beads due to the physical environment using digital image processing. Intermittent hydrostatic pressure (IHP) was used to mimic the mechanical environment of human bone marrow without using any biochemical factors. The expression of stromal cell-derived factor 1 (SDF-1) under IHP was measured. The results showed that the presence of OBs in the hydrogel scaffold initiate the movement of HSPCs. Furthermore, the IHP promotes the migration of HSPCs, even without the addition of any biochemical factors, and the results were confirmed by measuring SDF-1 levels. We believe this suggested three-dimensional experimental platform consisting of a simulated in vivo physical environment and encapsulated OBs should contribute to in vitro migration studies used to investigate the effects of other external factors.

Mechanical stress promotes cartilage repair in inflammatory environment

To investigate the effect and mechanism of mechanical stress on cartilage repair in inflammatory environment. The chondrogenic progenitor cells (CPCs) were isolated from the knee joint cartilage of patients with osteoarthritis (OA) undergoing total knee arthroplasty. The CPCs were cultured and expanded in a 3-D scaffold constructed with alginate. Intermittent hydrostatic pressure (IHP) was applied in a inflammatory environment induced by IL-1β, and Western blot was used to detect the expression of MAPK signaling pathway proteins. Cell proliferation was detected by CCK-8 method, and the expression of related genes like matrix metallo-proteinases 13 (MMP-13) and a disintegrins and metalloproteinase with thrombospondin motif 5 (ADAMTS-5) was detected by real-time RT-PCR. The anterior cruciate ligament of the rats was cut to construct the knee joint OA model, and the appropriate mechanical stress was constructed with external fixation to distract the knee joint in order to observe the repair of the cartilage and to explore its mechanism. Adding 0.01 ng/ml IL-1β in cell culture inhibited the proliferation of CPCs. After IHP application, the expression of MAPK pathway protein was decreased, the mRNA expression of MMP-13 and ADAMTS-5 was reduced. The inhibition of IL-1β on CPCs was counteracted by IHP. Four weeks after the anterior cruciate ligament resected, the articular cartilage degeneration was observed in rats. The Mankin score in the OA treatment (joint distraction) group was lower, and the cartilage repair was better than that of the control group (P<0.01). Animal experiments found that the suitable mechanical stress reduced the expression of P-p38, MMP-13 and COLL-X, inhibited cartilage cells apoptosis and promoted the repair of OA cartilage. Mechanical stress can promote the proliferation of CPCs, reduce the expression of matrix degrading enzymes, and promote the repair of OA cartilage by inhibiting MAPK signaling pathway.

The combined effects of hierarchical scaffolds and mechanical stimuli on ex vivo expansion of haematopoietic stem/progenitor cells

We describe the ex vivo expansion of haematopoietic stem/progenitor cells (HSPCs) with consideration of their eventual in-vivo niche. We firstly fabricated hierarchically structured scaffolds (lattices derived via three-dimensional plotting combined with electrospun submicron fibers coated with vitronectin to increase cell affinity). We also applied intermittent hydrostatic pressure (IHP) to mimic the physical environment of the in vivo niche. In the absence of mechanical stimuli, the cell phenotype (CD34+, CD34+CD38–) remained excellent in the vitronectin-treated group. Two IHP regimens were tested; optimally, cells were pressurized (20 kPa) for 2 min and then rested for 13 min. On day 7 of culture, the total cell number had increased 21.2-fold and that of CD34+ cells 10.94-fold. CD34+ and CD34+CD38– cells constituted 44.50 and 44.07% of total cells, respectively. Colony-forming counts and the long-term culture-initiating cell assay showed that clonogenic potential was greatly improved under our experimental conditions. Scaffolds with hierarchical structures were valuable in this context. Furthermore, ex vivo expansion of HSPCs was improved by physical stimulation.

Combinational effects of mechanical forces and substrate surface characteristics on esophageal epithelial differentiation.

Even the efficacy of substrate and mechanical stimuli in addition to biochemical cues have been recognized in many studies of stem cell differentiation, few studies have been reported on the differentiation into esophageal epithelial cells. Therefore, the aim of this study was set to propose a method of differentiating stem cells into esophageal epithelial cells according to biochemical reagent concentration, substrate properties, and mechanical forces. After the concentration of all-trans retinoic acid was determined as 5 μM by a baseline experiment, the degree of differentiation was compared in three different kinds of substrates: cover glass, polyurethane (PU) membrane, and electrospun PU sheet (ePU). Then, on the substrate showing the more positive results, that is, ePU, two types of mechanical forces, intermittent hydrostatic pressure (IHP), and shear stress (SS), were applied individually at different magnitudes for the latter 7 days of an overall incubation period of 14 days. Following various biological assays, the lower IHP (50 mmHg) resulted in greater positive effects than the others. Even with cessation of the mechanical force, the relevant markers were remarkably increased. Although the range of factors regulating differentiation was limited, this study nonetheless demonstrated the combinational effects of mechanical force along with substrate type for the first time in related studies. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 552-560, 2019.

Hydrostatic pressure in combination with topographical cues affects the fate of bone marrow-derived human mesenchymal stem cells for bone tissue regeneration.

Topographical and mechanical cues are vital for cell fate, tissue development in vivo, and to mimic the native cell growth environment in vitro. To date, the combinatory effect of mechanical and topographical cues as not been thoroughly investigated. This study investigates the effect of PCL nanofiber alignment and hydrostatic pressure on stem cell differentiation for bone tissue regeneration. Bone marrow‐derived human mesenchymal stem cells were seeded onto standard tissue culture plastic and electrospun random and aligned nanofibers. These substrates were either cultured statically or subjected to intermittent hydrostatic pressure at 270 kPa, 1 Hz for 60 min daily over 21 days in osteogenic medium. Data revealed higher cell metabolic activities for all mechanically stimulated cell culture formats compared with non‐stimulated controls; and random fibers compared with aligned fibers. Fiber orientation influenced cell morphology and patterns of calcium deposition. Significant up‐regulation of Collagen‐I, ALP, and Runx‐2 were observed for random and aligned fibers following mechanical stimulation; highest levels of osteogenic markers were expressed when hydrostatic pressure was applied to random fibers. These results indicate that fiber alignment and hydrostatic pressure direct stem cell fate and are important stimulus for tissue regeneration. © 2017 The Authors Journal of Biomedical Materials Research Part A Published by Wiley Periodicals, Inc. J Biomed Mater Res Part A: A: 629–640, 2018.

Intermittent hydrostatic pressure maintains and enhances the chondrogenic differentiation of cartilage progenitor cells cultivated in alginate beads.

The objective of this study was to explore the effects of intermittent hydrostatic pressure (IHP) on the chondrogenic differentiation of cartilage progenitor cells (CPCs) cultivated in alginate beads. CPCs were isolated from the knee joint cartilage of rabbits, and infrapatellar fat pad-derived stem cells (FPSCs) and chondrocytes (CCs) were included as the control cell types. Cells embedded in alginate beads were treated with IHP at 5Mpa and 0.5Hz for 4h/day for 1, 2, or 4weeks. The cells' migratory and proliferative capacities were evaluated using the scratch and Live/Dead assays, respectively. Hematoxylin and eosin staining, safranin O staining, and immunohistochemical staining were performed to determine the effects of IHP on the synthesis of extracellular matrix (ECM) proteins. Real-time polymerase chain reaction analysis was performed to measure the expression of genes related to chondrogenesis. The scratch and Live/Dead assays revealed that IHP significantly promoted the migration and proliferation of FPSCs and CPCs to different extents. The staining experiments showed greater production of cartilage ECM components (glycosaminoglycans and collagen II) by cells exposed to IHP, and the gene expression analysis demonstrated that IHP stimulated the expression of chondrocyte-related genes. Importantly, these effects of IHP were more prominent in CPCs than in FPSCs and CCs. Considering all of our experimental results combined, we conclude that CPCs demonstrated a stronger chondrogenic differentiation capacity than the FPSCs and CCs under stimulation with IHP. Thus, the use of CPCs, combined with mechanical stimulation, may represent a valuable strategy for cartilage tissue engineering.

Cartilage regeneration and intermittent hydrostatic pressure: A role for MSCs

Cartilage regeneration and intermittent hydrostatic pressure: A role for MSCs

Involvement of Smad pathway in proteoglycan 4 expression induced by hydrostatic pressure in temporomandibular synovial fibroblasts

To examine the expression of proteoglycan 4 (PRG-4) induced by hydrostatic pressure in rat temporomandibular synovial fibroblasts and investigate the possible mechanism. The cultured rat temporomandibular synovial fibroblasts were subjected to 100 kPa magnitude intermittent hydrostatic pressure (IHP) at frequency of 4 h/day, and the static group served as control. The expressions of Smad pathway proteins and p38MAPK pathway proteins were analyzed by Western blot and immunofluorescence staining. Then the cells were incubated with SB431542, the inhibitor of transforming growth factor (TGF)-β receptor. Western blot and reverse transcription PCR were used to detect the PRG-4 expression after 72 h. The expression of phosphorylated Smad-2 and phosphorylated Smad-3 were increased after 1 h of IHP, reaching a maximum after 2 h and 4 h of IHP, respectively.However, the protein content of phosphorylated p38 did not vary significantly. In addition, IHP induced nuclear translocation of Smad-2/-3, and the immunofluorescence staining signal intensity markedly increased (24.11 ± 4.70)(P < 0.05). The levels of PRG-4 mRNA were significantly increased by IHP (1.48 ± 0.08)(P < 0.05). Treatment of cells with SB431542 could decrease the expression of PRG-4 mRNA significantly after IHP (0.47 ± 0.05)(P < 0.05). In addition, SB431542 inhibited the expression of PRG-4 protein induced by IHP. Smad signal acts as an essential signal pathway to regulate PRG-4 expression induced by IHP.

Effects of various patterns of intermittent hydrostatic pressure on the osteogenic differentiation of mesenchymal stem cells

This study investigated the effects of intemittent hydrostatic pressure (IHP) patterns on the responses of mesenchymal stem cells (MSCs) such as osteogenic differentiation, proliferation and senescence. For these experi- mental groups were set based on IHP patterns: (1) C_BM: control group with no stimulation in basal media; (2) C_OM: control group with no stimulation in osteogenic media (OM); (3) S_2H/5M: longer pressurizing (2hours) and shorter resting (5minutes) time in OM; (4) S_1H/1H: equal pressurizing and resting time (1hour) in OM; (5) S_2M/15M: shorter pressurizing (2minutes) and longer resting (15minutes) time in OM. The magnitude of IHP was 0.15 MPa. IHP was applied to corresponding groups for 4 hours a day from 3 days starting at 48 hours after seeding. Examination of DNA contents, ALP activity and its staining, quantitative real-time PCR, and β-galactosidase stain- ing were performed. ALP amount normalized by corresponding DNA content in S_2H/5M, C_BM was significantly lower than that of the other group at day 5, which was more observable even at day 7 while S_2H/5M significantly had lower ALP count than other groups at day 5 and day 7. Other groups (S_1H/1H and S_2M/15M) showed sig- nificantly higher ALP amounts indicating the positive effect on osteogenic differentiation. Other markers indicating the degrees of differentiation showed comparable results. Based on β-galactosidase staining, it appeared that mechanical stimuli did not affect cell senescence significantly. From this study, we concluded that engagement of IHP has a potential of controlling osteogenic differentiation depending on its pattern: it can promote or suppress dif- ferentiation.

Mechanical stimulation and the presence of neighboring cells greatly affect migration of human mesenchymal stem cells

There are few studies regarding the effects of mechanical stimulation on cell migration although biochemical factors have been widely studied. We have investigated the effects of intermittent hydrostatic pressure (IHP) on mesenchymal stem cell migration with or without neighboring endothelial cells (EC). IHP promoted MSCs migration and the neighboring ECs helped with this. However, when IHP was applied to MSCs cultured with ECs, the opposite effect was observed. The concentration of stromal-derived factor-1 culture in medium was measured to explain the obtained results. SDF-1 concentration increased as IHP increased when MSCs were cultured alone. However, it decreased as IHP increased when MSCs and ECs were co-cultured. These results indicate that the mechanical environment should be considered when studying the migration of a cell type along with its biochemical environment.

Synergistic Effects of Conditioned Media and Hydrostatic Pressure on the Differentiation of Mesenchymal Stem Cells

The objective of this study was to investigate the effect of mechanical stimuli in combination with conditioned media (CM) on the osteogenic and chondrogenic differentiation of mesenchymal stem cells (MSCs). Two “opposing” groups were defined for the study. The first, designated the bone group, was formed by seeding MSCs onto porous poly(l-lactide-co-e-caprolactone)/hydroxyapatite scaffolds. The second group, designated the cartilage group, was formed by encapsulating MSCs in 2% agarose. The samples were either subjected to hydrostatic pressure (HP), administered CM from the opposing group at a concentration of 50%, or given a combination of the two stimuli. The sulfated glycosaminoglycan (sGAG) content and SOX9, aggrecan, and collagen type II expressions suggest that both osteoblast CM and intermittent HP individually enhanced the chondrogenic differentiation of MSCs. In addition, there is evidence of a synergistic interaction between CM and HP on chondrogenic differentiation. The alkaline phosphatase (ALP) activity and osteocalcin and bone sialoprotein expression suggest that the combination of chondrocyte CM and HP significantly enhanced the osteogenic differentiation of MSCs. However, the individual stimuli did not have a significant effect on osteogenic differentiation. Enzyme-linked immunosorbent assay (ELISA) results revealed that both the chondrocyte CM and osteoblast CM contained considerable amounts of TGF-β1 and BMP-2.

Intermittent Hydrostatic Pressure Enhances Growth Factor‐Induced Chondroinduction of Human Adipose‐Derived Mesenchymal Stem Cells

Hydrostatic pressure (HP) plays an essential role in regulating function of chondrocytes and chondrogenic differentiation. The objective of this study was to examine effects of intermittent HP on chondrogenic differentiation of human adipose-derived mesenchymal stem cells (hASCs) in the presence or absence of chemical chondrogenic medium. Cells were isolated from abdominal fat tissue and confirmed for expression of ASC surface proteins and differentiation potential. Passage 3 pellets were treated with chemical (growth factor), mechanical (HP of 5 MPa and 0.5 Hz with duration of 4 h/day for 7 consecutive days), and combined chemical-mechanical stimuli. Using real-time polymerase chain reaction, the expression of Sox9, collagen II, and aggrecan as three major chondrogenic markers were quantified among three experimental groups and compared to those of stem cells and human cartilage tissue. In comparison to the chemical and mechanical groups, the chemical-mechanical group showed the highest expression for all three chondrogenic genes close to that of cartilage tissue. Results show the beneficial role of intermittent HP on chondrogenic differentiation of hASCs, and that this loading regime in combination with chondrogenic medium can be used in cartilage tissue engineering.

Effects of intermittent hydrostatic pressure magnitude on the chondrogenesis of MSCs without biochemical agents under 3D co-culture

Without using biochemical agents, in this study, we sought to investigate the potential of controlling the differentiation of mesenchymal stem cells (MSCs) into a specific cell type through the use of 3D co-culturing and mechanical stimuli. MSCs and primary cultured chondrocytes were separately encapsulated into alginate beads, and the two types of beads were separated by a membrane. For the investigation a computer-controllable bioreactor was designed and used to engage intermittent hydrostatic pressure (IHP). Five different magnitudes (0.20, 0.10, 0.05, 0.02 MPa and no stimulation) of IHP were applied. The stimulation pattern was the same for all groups: 2 h/day for 7 days starting at 24 h after seeding; 2 and 15 min cycles of stimulating and resting, respectively. Biochemical (DNA and GAG contents), histological (Alcian blue), and RT-PCR (Col II, SOX9, AGC) analyses were performed on days 1, 5, 10, and 20. The results from these analyses showed that stimulation with higher magnitudes of IHP (≥0.10 MPa) were more effective on the proliferation and differentiation of co-cultured MSCs. Together, these data demonstrate the potential of using mechanical stimulation and co-culturing for the proliferation and differentiation of MSCs, even without biochemical agents.

Combination of intermittent hydrostatic pressure linking TGF-β1, TNF-α on modulation of proteoglycan 4 metabolism in rat temporomandibular synovial fibroblasts

This study was designed to evaluate the combined effects of intermittent hydrostatic pressure (IHP) and TGF-β1 or TNF-α on proteoglycan4 (PRG4) expression in rat temporomandibular synovial fibroblasts (SFs). Rat SFs were isolated and expanded in monolayer cultures and subjected to IHP in the presence of TGF-β1 or TNF-α. Quantitative real-time RT-PCR was applied to analyze the PRG4 expression levels. Enzyme-linked immunosorbent assay was also used for the quantification of PRG4 accumulation in the culture medium while immunofluorescence staining was used to detect intracellular PRG4 protein expression. The combination of IHP and TGF-β1 induced greater PRG4 expression than either stimulus alone. In contrast, TNF-α inhibited PRG4 expression, and this was partially alleviated by IHP. Our study demonstrates a beneficial role of IHP, which can be used successfully in combination with TGF-β1 to enhance PRG4 production, and can partially counteract TNF-α-induced PRG4 inhibition in isolated rat SFs.

Effects of intermittent hydrostatic pressure and BMP‐2 on osteoarthritic human chondrocyte metabolism in vitro

This study examined effects of intermittent hydrostatic pressure (IHP) and a chondrogenic growth factor, bone morphogenetic protein-2 (BMP-2), on anabolic, catabolic, and other metabolic markers in human osteoarthritic (OA) chondrocytes in vitro. Articular chondrocytes, isolated from femoral OA cartilage and maintained in high-density monolayer culture, were examined for effects of BMP-2 and IHP on gene expression of matrix-associated proteins (aggrecan, type II collagen, and SOX9) and catabolic matrix metalloproteinases (MMP-2 and MMP-3) and culture medium levels of the metabolic markers MMP-2, nitric oxide (NO), and glycosaminoglycan (GAG). The results were analyzed using a mixed linear regression model to investigate the effects of load and growth factor concentration. IHP and BMP-2 modulated OA chondrocyte metabolism in accordance with growth factor concentration independently, without evidence of synergism or antagonism. Each type of stimulus acted independently on anabolic matrix gene expression. Type II collagen and SOX9 gene expression were stimulated by both IHP and BMP-2 whereas aggrecan was increased only by BMP-2. IHP exhibited a trend to decrease MMP-2 gene expression as a catabolic marker whereas BMP-2 did not. NO production was increased by addition of BMP-2 and IHP exhibited a trend for increased levels. GAG production was increased by BMP-2. This study confirmed the hypothesis that human OA chondrocytes respond to a specific type of mechanical load, IHP, through enhanced articular cartilage macromolecule gene expression and that IHP, in combination with a chondrogenic growth factor BMP-2, additively enhanced matrix gene expression without interactive effects.

Physiological loading of joints prevents cartilage degradation through CITED2

Both overuse and disuse of joints up-regulate matrix metalloproteinases (MMPs) in articular cartilage and cause tissue degradation; however, moderate (physiological) loading maintains cartilage integrity. Here, we test whether CBP/p300-interacting transactivator with ED-rich tail 2 (CITED2), a mechanosensitive transcriptional coregulator, mediates this chondroprotective effect of moderate mechanical loading. In vivo, hind-limb immobilization of Sprague-Dawley rats up-regulates MMP-1 and causes rapid, histologically detectable articular cartilage degradation. One hour of daily passive joint motion prevents these changes and up-regulates articular cartilage CITED2. In vitro, moderate (2.5 MPa, 1 Hz) intermittent hydrostatic pressure (IHP) treatment suppresses basal MMP-1 expression and up-regulates CITED2 in human chondrocytes, whereas high IHP (10 MPa) down-regulates CITED2 and increases MMP-1. Competitive binding and transcription assays demonstrate that CITED2 suppresses MMP-1 expression by competing with MMP transactivator, Ets-1 for its coactivator p300. Furthermore, CITED2 up-regulation in vitro requires the p38δ isoform, which is specifically phosphorylated by moderate IHP. Together, these studies identify a novel regulatory pathway involving CITED2 and p38δ, which may be critical for the maintenance of articular cartilage integrity under normal physical activity levels.

Development and Validation of a System for the Growth of Cells and Tissues Under Intermittent Hydrostatic Pressure

Various cell populations have been shown to respond to hydrostatic pressure; however, many of the culture systems suffer from shortcomings in design or methodology. Of particular interest to us is the potential role of pressure and other environmental factors in modulating stem cell behavior in intervertebral disk repair. A system was developed for the growth of cells and tissues under intermittent hydrostatic pressure. The system was validated with NIH 3T3 fibroblasts for sterilizability and cytotoxicity. Further experiments were conducted with canine mesenchymal stem cells under various levels of pressure, oxygen, glucose, and conditioned medium. The culture system showed no cytotoxicity and was able to demonstrate that the proliferation and metabolism of mesenchymal stem cells are sensitive to medium glucose and oxygen concentration and hydrostatic pressure. The cells exposed to hydrostatic pressure differed in their morphology from nonexposed cells. The system is capable of supporting long-term cell culture and examining the role of mechanical and environmental stimulation in vivo.