Tensile strain versus LSJL genes

LSJL causes dynamic tensile strain on the growth plates.  How much does dynamic tensile strain explain the effects of LSJL?

Cyclic tensile strain facilitates the ossification of ligamentum flavum through β-catenin signaling pathway: in vitro analysis.

"Histological, immunohistochemical, and real-time reverse transcription-polymerase chain reaction analyses of the expression of cell signaling and transcriptional factors in human ossification of ligamentum flavum (OLF).

To test the hypothesis that β-catenin plays a role in the ossification of OLF cells in response to cyclic tensile strain.

Several studies have investigated the roles of biomechanical and metabolic factors in the development and progression of OLF, based on the importance of genetic and biological factors. The process of ossification includes enchondral ossification.

Using real-time reverse transcription-polymerase chain reaction, we analyzed the mRNA expression levels of signaling factors known to be involved in the ossification process (β-catenin, Runx2, Sox9, and osteopontin) in cultured OLF cells subjected to cyclic tensile strain. Cyclic tensile strain was produced by Flexercell FX-3000 (Flexercell International, Hillsborough, NC), applied for 0, 6, 12, or 24 hours. The localization of these factors was examined in decalcified paraffin OLF sections by immunohistochemistry. Controlled samples were harvested from nonossified ligamentum flavum of patients who underwent thoracic posterior surgical procedures.

Under resting conditions (no tensile strain), the mRNA levels of β-catenin, Runx2, Sox9, and osteopontin in cultured OLF cells were significantly higher than in the control non-OLF cells. Application of cyclic tensile strain to OLF cells resulted in significant increases in mRNA expression levels of β-catenin, Runx2, Sox9, and osteopontin at 24 hours. Hypertrophic chondrocytes present around the calcification front were immunopositive for Runx2 and osteopontin. Immunoreactivity of β-catenin and Sox9 was strongly present in premature chondrocytes in the fibrocartilage area.

Our results indicated that cyclic tensile strain applied to OLF cells activated their ossification through a process mediated by the β-catenin signaling pathway."

"From a histological point of view, the progression of OLF correlates with enchondral ossification. Structurally, pathological specimens exhibit an ossification front, including a calcified cartilage layer, calcification front, and fibrocartilage layer, between the bone formation and the ligamentous fiber area."

Sox9 increased to 8 fold at 6 to 24 hour time points.

"non-OLF cells showed no immunoreactivity for [beta]-catenin, Runx2, Sox9, and osteopontin without strain but became positive for Sox9 after tensile strain application"<-Thus tensile strain may be able to induce ectopic chondrogenesis.

Effects of tensile strain and fluid flow on osteoarthritic human chondrocyte metabolism in vitro.

"Primary high-density monolayer chondrocytes cultures were exposed to varying magnitudes of tensile strain and fluid-flow using a four-point bending system. Metabolic changes were quantified by real-time PCR measurement of aggrecan, IL-6, SOX-9, and type II collagen gene expression, and by determination of nitric oxide levels in the culture medium. A linear regression model was used to investigate the roles of strain, fluid flow, and their interaction on metabolic activity. Aggrecan, type II collagen, and SOX9 mRNA expression were negatively correlated to increases in applied strain and fluid flow. An effect of the strain on the induction of nitric oxide release and IL-6 gene expression varied by level of fluid flow (and visa versa). This interaction between strain and fluid flow was negative for nitric oxide and positive for IL-6. These results confirm that articular chondrocyte metabolism is responsive to tensile strain and fluid flow under in vitro loading conditions. Although the articular chondrocytes reacted to the mechanically applied stress, it was notable that there was a differential effect of tensile strain and fluid flow on anabolic and catabolic markers."

" In porcine articular cartilage, cyclic tensile strain upregulates the catabolic mediators, nitric oxide (NO), prostaglandin E2, and MMP-1. Aggrecan and type II collagen were also upregulated at 3 h, but this effect was muted at all later time points (up to 24 h). One confounding variable of previous tensile strain loading systems is the unintended effect of fluid motion of the culture medium on the cells."

"The three anabolic markers (aggrecan, type II collagen, SOX9) were independently responsive to strain and fluid flow. The two catabolic markers (NO and IL-6) were responsive to the interaction of strain and fluid flow. NO production was also affected independently by strain and fluid flow."

The minimum microstrain here is 850 which is much higher than that generated in LSJL which was 20.

"For mechanical loading, each chondrocyte-seeded substrate was placed in a loading chamber containing 15 mL of serum-free medium. Homogenous cyclic tensile strain was applied to the chondrocyte-seeded substrate by using a custom-made four-point bending device, which was driven by computer-controlled linear actuator assembly with an interface controller to regulate vertical displacement and displacement rate"

Enhancement of nitric oxide and proteoglycan synthesis due to cyclic tensile strain loaded on chondrocytes attached to fibronectin.

"Cyclic tensile strain was applied to bovine articular chondrocytes. PG and NO synthesis were determined by [35S] sulfate incorporation and chemiluminescence analysis, respectively. To determine the expression of inducible NO synthase (iNOS), quantitative RT-PCR was used.

Enhanced PG and NO synthesis were evident when cyclic tensile strain was applied to chondrocytes seeded on fibronectin-coated plates. When NO production was inhibited, PG synthesis was further enhanced.

Cyclic tensile strain loaded on the chondrocytes enhanced NO synthesis and this enhanced NO inhibited PG synthesis."

"a low frequency and magnitude of cyclic tensile strain applied to chondrocytes increased proteoglycan (PG) synthesis, a high frequency and magnitude of strain decreased its synthesis"

"In articular cartilage, NO production is increased in chondrocytes exposed to fluid shear stress. Static and intermittent compression loaded on articular cartilage explants enhanced NO production"

a5b1 is the major fibronectin receptor in chondrocytes and the major mechanoreceptor.

"there is a linear relationship between the vacuum level (kPa) and maximal percent elongation of cells. In this study, the frequency (10 cycles per minute, i. e. three seconds of strain followed by three seconds of relaxation) and magnitude (10 kPa, 7%) of strain"

NO production was low until 12 hours.

According to Mechanical Stress inhibits chondrogenesis through ERK-1/2 phosphorylation in micromass culture, mechanical stress can inhibit chondrogenesis on embryonic like stem cells.


Mechanical Stretch Induces ERK1/2 Phosphorylation in Micromass Culture

"Our previous study demonstrated that tension inhibited chondrocyte differentiation through integrin mediated cell-extracellular matrix adhesion in micromass culture derived from embryonic rat limb bud cells. In the present study, we investigated the phosphorylation of extracellular signal-regulated kinase (ERK)-1/2 in micromass cultures under a hypothesis that stretch induces ERK-1/2 phosphorylation in short term immediately after force loading. 

Embryonic limb bud cells were isolated from embryonic day 12 Sprague-Dawley rats. Dissociated cells by 0.25mg/ml collagenase and 0.25mg/ml trypsin EDTA were assembled to micromass culture on a Flexcer cell plate. After two drops of 50μl of 4.66x106 cells/ml suspension were plated around the center of wells of the dish and maintained for 5 hours in a CO2 incubator at 37°C, 2ml of 10% fetal bovine serum supplemented DMEM was added. After 4 days, micromass cultures were stretched for 0.5, 1.0, 1.5, 3.0, 6.0 and 12.0 hours prior to the isolation of protein samples. Western blot analysis for phosphorylated and non-phosphorylated ERK-1/2 was performed on samples from non-stretched control and stretched cultures. 

The expression level of ERK-1/2 was maintained to be constant level throughout the experimental period in rat limb bud derived micromass cultures. Phosphorylation of ERK-1/2 increased and peaked at 1.0 hour after stretch loaded, while basal level of the phosphorylarion of ERK-1/2 was maintained in the control non-stretched culture. 

Signaling through ERK-1/2 was activated by stretch-based shearing stress in micromass cultures and was suggested to be involved in the inhibition of chondrogenesis by shearing stress generated by stretching."

Molecular mechanisms of the response to mechanical stimulation during chondrocyte differentiation

"During embryonic development, mesenchymal stem cells congregate along with fibronectin and tenascin in the location where future long bone will be formed in the immature tissue-specific ECM. Once cellular condensation begins using these ECM molecules as a scaffold, the cells in the aggregation begin to express cell–cell adhesion molecules such as N-cadherin and N-CAM. After cellular condensation, mesenchymal cells start to differentiate into chondrocytes by firstly expressing the transcription factors Sox-5, -6, and -9, which regulate the gene expression of the phenotypic genes, Col2a1 and aggrecan. By producing these cartilage-specific ECM macromolecules, cell shape changes from ovoid to round, and polarity is obtained by arranging the positions of the nucleus and a lipid storage area in the cytosol, and the intercellular space is enlarged through the progression of chondrocyte differentiation. Further hypertrophic differentiation continues as endochondral bone formation progresses during growth. Chondrocytes terminally differentiate into hypertrophic chondrocytes expressing the Col10a1 gene and induce calcification of the cartilaginous matrix, which is later replaced by bone."

" a recent study revealed the structural changes that occur in integrin-related molecules after mechanical stimulation lead to the activation of downstream signaling mediated by the MAPK pathway and/or small GTPases such as Cdc42, Ras, and Rho"

"Mechanical stretching directly activates ERK-1/2, but not JNK or p38 MAPK. The phosphorylation of ERK-1/2 even increased under the inhibition of protein production, which can be interpreted as showing that the signal was transferred to the nucleus after the cell had sensed it."<-LSJL increases p38 MAPK phosphorylation which could be why ERK1/2 was not chondroinhibitory in LSJL.

Intermittent Cyclic Mechanical Tension-Induced Calcification and downregulation of ankh gene expression of end plate chondrocytes.

"Intermittent Cyclic Mechanical Tension (ICMT) was applied to end plate chondrocytes by using an FX-4000T Flexercell Tension Plus unit (Flexcell International Corporation, Hillsborough, NC). 

Rat end plate chondrocytes were cultured and ICMT (strain at 0.5 Hz sinusoidal curve at 10% elongation) was applied for 25 days, 4 hours a day and continued to culture for 5 days. End plate chondrocytes were incubated for 12 hours in the presence or absence of 10 ng/mL of transforming growth factor-β1 (TGF-β1) (prepared from a stock solution at 10 μg/mL in 2 mM citric acid containing 2 mg/mL bovine serum albumin) in MEM/F-12 containing a final concentration of 1% FCS. End plate chondrocytes calcification was stained by alizarin red S (AR-S). End plate chondrocytes viability was examined by LIVE/DEAD viability/cytotoxicity kit.

LIVE/DEAD assay verified that the nonloading (NC) group and the ICMT group end plate chondrocytes remained adherent, with no change in viability after the application of ICMT. Alizarin red staining showed that ICMT induced the calcification of end plate chondrocytes. Real-time reverse transcription-polymerase chain reaction showed that mRNA expression of endogenous TGF-β1 decreased and mRNA expression of type I, type X, osteocalcin and osteopontin increased after ICMT. The ankh gene expression of both mRNA and protein levels decreased in the ICMT stimulation. The ankh gene expression of both mRNA and protein levels increased in TGF-β1 stimulation. Compared with NC group, the alkaline phosphatase activities significantly increased in ICMT group.

Our results directly showed that ICMT induced the calcification and downregulation of ankh gene expression of end plate chondrocytes, which may be caused by the endogenous TGF-β1."

"One of the main pathways for nutrients to reach the avascular nucleus pulpous is by diffusion from the blood supply of the vertebral body through the end plate cartilage. The end plate cartilage is a layer of hyaline cartilage lying between the vertebral body and the intervertebral disc. End plate calcification could impede the passage of nutrients from the blood to the intervertebral disc and lead to the alterations in mechanical material properties of disc and make the end plate fail to maintain the nucleus pulposus and could also accelerate degenerative process of intervertebral disc."

"Many genes associated with cartilage calcification have been confirmed, such as COL9A2, COL9A3{up in LSJL}, AGCI, CLIP, TNAP, ANK, and transforming growth factor-[beta]1 (TGF-[beta]1)"

"A multipass transmembrane protein, ANK, seemed to function as an inorganic pyrophosphate (PPi) transporter or regulator of PPi transport, and PPi is a potent inhibitor of basic calcium phosphate (BCP) crystals formation. The ANK protein is a transporter able to export iPPi from the cells and is known to be upregulated in osteoarthritis."


Mechanical stretch enhances COL2A1 expression on chromatin by inducing SOX9 nuclear translocalization in inner meniscus cells.

"We investigated mechanical stretch-regulated gene expression in human meniscus cells. Human inner and outer meniscus cells were prepared from the inner and outer halves of the lateral meniscus. The gene expressions of Sry-type HMG box (SOX) 9 and α1(II) collagen (COL2A1) were assessed by real-time PCR analyses after cyclic tensile strain (CTS) treatment (0.5 Hz, 5% stretch). The localization and phosphorylation of SOX9 were evaluated by immunohistochemical and Western blot (WB) analyses. Chromatin immunoprecipitation (IP) analysis was performed to assess the stretch-related protein-DNA complex formation between SOX9 and the COL2A1 enhancer on chromatin. Type II collagen deposition and SOX9 production were detected only in inner menisci. CTS treatments increased expression of the COL2A1 and SOX9 genes in inner meniscus cells, but not in outer meniscus cells. In addition, CTS treatments stimulated nuclear translocalization and phosphorylation of SOX9 in inner meniscus cells. Chromatin IP analyses revealed that CTS increased the association between SOX9 and its DNA-binding site, included in the COL2A1 enhancer, on chromatin. Our results indicate that inner and outer meniscus cells have different properties in mechanical stretch-induced COL2A1 expression. In inner meniscus cells, mechanical stretch may have an essential role in the epigenetic regulation of COL2A1 expression."

"the total amounts of endogenous SOX9 were similar between non-stretched and 4-h-stretched cells"

"5% uniaxial CTS (0.5 Hz, 4 h) induced the highest expression of COL2A1 and SOX9 genes in human inner meniscus cells"

Cyclic tensile strain and cyclic hydrostatic pressure differentially regulate expression of hypertrophic markers in primary chondrocytes.

"Chondrocyte-seeded alginate constructs were exposed to one of the two loading modes for a period of 3 h per day for 3 days. Gene expression was analyzed using real-time RT-PCR. Cyclic tension upregulated the expression of Cbfa1, MMP-13, CTGF, type X collagen and VEGF and downregulated the expression of TIMP-1. Cyclic tension also upregulated the expression of type 2 collagen, COMP and lubricin, but did not change the expression of SOX9 and aggrecan. Cyclic hydrostatic pressure downregulated the expression of MMP-13 and type I collagen and upregulated expression of TIMP-1 compared to the unloaded controls. Hydrostatic pressure may slow chondrocyte differentiation and have a chondroprotective, anti-angiogenic influence on cartilage tissue. Our results suggest that cyclic tension activates the Cbfa1/MMP-13 pathway and increases the expression of terminal differentiation hypertrophic markers."

"Sinusoidal strains of 9% peak-to-peak were applied to the alginate specimens"  HP was 5Mpa.  Both were loaded at 0.5Hz, 3 hrs per day for 3 days.


Effect of uniaxial stretching on rat bone mesenchymal stem cell: orientation and expressions of collagen types I and III and tenascin-C.

"Rat BMSCs were harvested from femoral and tibial bone marrow by density gradient centrifugation. Cells from passages 1-6 were characterized by flow cytometry using monoclonal antibodies. The recovered cells were stably positive for the markers CD90 and CD44 and negative for CD34 and CD45. A cyclic 10% uniaxial stretching at 1Hz was applied on rat BMSC for different time-courses. The length, width, and orientation of the cells were subsequently determined. Expression of collagen types I{up} and III{up} and tenascin-C mRNAs was measured by real-time RT-PCR, and the synthesis of these receptors was determined by radioimmunoassay. Results showed that uniaxial stretching lengthened and rearranged the cells. Compared with control groups, expression of collagen types I and III mRNAs was up-regulated after 12-h of stretching, while significant increase in synthesis of the two collagen protein types was not observed until after 24-h stretching. The expression of tenascin-C mRNA was significantly increased after a 24-h stretching. These data suggest that cyclic stretching promotes the synthesis of collagen types I and III and tenascin-C by the rat BMSC."

[Strain-induced tenogenic differentiation of bone marrow mesenchymal stem cells].

Both Scx and Tnmd were upregulated by LSJL.

"BMSCs were isolated by adherent culture from the bone marrow of 1-week-old SD rats. Inducing method of multiple differentiation and flow cytometry were applied to identify the cells. The stress-strain curve of SIS was measured with Instron machine. Purified BMSCs (2nd passage, 2.5 x 10(5) cells/cm2) were seeded on SIS (3 cm x 1 cm at size) and cultured for 2 days and then continued for another 5 days under strain stimulation (stretching frequency was 0.02 Hz, action time was 15 minutes/hour and 12 hours/day, strain amplitude was 5%) as experimental group, while the BMSCs-SIS composites were sustained static culture as control group. TCs were isolated from tail of 1-week-old SD rats. TCs-SIS composites were cultured under non-strained as positive control group. Scanning electron microscope (SEM) was used to examine the morphological changes of BMSCs after strain stimulation. The contents of Scleraxis and Tenomodulin in supernatant were tested by ELISA kit. Results The BMSCs could be induced to differentiate into osteoblasts and lipocytes, and showed the results of CD34-, CD45-, and CD90+, which were accorded with the biological characteristics of BMSCs. The failure test of SIS showed that the average elastic strain was 39.5%. SEM observation showed that the strain-stimulated BMSCs had the TCs-like morphological characteristics. The contents of Scleraxis and Tenomodulin in supernatant of experimental group, control group, and positive control group were (3.56 +/- 0.91) micromol/L and (4.27 +/- 1.10) micromol/L, (0.23 +/- 0.14) micromol/L and (0.16 +/- 0.10) micromol/L, and (14.73 +/- 2.30) micromol/L and (10.65 +/- 1.51) micromol/L, respectively. There were significant differences among 3 groups (P < 0.05)."

This validates that stretching plays a large role in LSJL response based on the effects of stretching on tendon related gene expression.

Bending loading produces tensile strain on the bone.

Mechanical stimulation alters tissue differentiation and molecular expression during bone healing.

"This study investigated the use of mechanical stimulation to promote cartilage rather than bone formation within an osteotomy. Retired breeder Sprague-Dawley rats (n = 85) underwent production of a mid-diaphyseal, transverse femoral osteotomy followed by external fixation. Beginning on postoperative day 10 and continuing for 1, 2, or 4 weeks, a cyclic bending motion (+35 degrees/-25 degrees at 1 Hz) was applied in the sagittal plane for 15 min/day for 5 consecutive days/week. Control animals experienced continuous rigid fixation. Histological and molecular analyses indicated that stimulation substantially altered normal bone healing. Stimulated specimens exhibited an increase in cartilage volume over time, while control specimens demonstrated bony bridging. Stimulation induced upregulation of cartilage-related genes (COL2A1 and COL10A1){both up in LSJL} and downregulation of bone morphogenetic proteins (BMPs) -4, -6 and -7. However, BMP-3 was upregulated with stimulation."

"a cyclic, axial, compressive displacement applied to a diaphyseal fracture or osteotomy enhances healing via formation of a larger cartilaginous callus and earlier bony bridging."

"in distraction osteogenesis, a series of tensile, step displacements is applied to the osteotomy gap to promote ossification and achieve bone lengthening."

"In fracture healing, less stable fixation induces expression of genes associated with chondrogenesis, cartilage extracellular matrix components, cell division, and inflammation."

"BMP-3 has been shown to antagonize the osteo-inductive effects of BMP-2."

Tensile strain may inhibit osteogenic genes like Runx2:

Continuous cyclic mechanical tension inhibited Runx2 expression in mesenchymal stem cells through RhoA-ERK1/2 pathway.

"CCMT[Continuous cyclic mechanical tension} [inhibits] the expression of osteogenic genes such as key transcription factor Runx2. RhoA regulates cell differentiation in response to mechanical stimuli. MAPK signaling acts as a downstream effector of RhoA. In MSCs, CCMT regulates the osteogenic master gene Runx2 through RhoA-ERK1/2 pathway. There is a decrease in RhoA activity after CCMT stimulation. Pre-treatment of CCMT-loaded MSCs with LPA, a RhoA activator, restores ALP activity and significantly rescues Runx2 expression, while pre-treatment with C3 toxin, a RhoA inhibitor, further decreases the activity of ALP and down-regulates the expression of Runx2. The inhibition of Runx2 expression after CCMT stimulation is mediated by RhoA-ERK1/2 pathway."

"Different from intermittent tensile loading, CCMT applies continuous stimulation throughout the study period, which could partially simulate mechanical overuse in vitro."  Intermittent tensile loading has been found to be pro-osteogenic.

CCMT also inhibited Osteopontin and Type I Collagen.  CCMT decreased ERK1/2 phosphorylation.

Tensile strain increases expression of CCN2 and COL2A1 by activating TGF-β-Smad2/3 pathway in chondrocytic cells.

"Physiologic mechanical stress stimulates expression of chondrogenic genes, such as multifunctional growth factor CYR61/CTGF/NOV (CCN) 2 and α1(II) collagen (COL2A1), and maintains cartilage homeostasis. Cyclic tensile strain (CTS) induces nuclear translocation of transforming growth factor (TGF)-β receptor-regulated Smad2/3 and the master chondrogenic transcription factor Sry-type HMG box (SOX) 9. CTS may induce TGF-β1 release and stimulate Smad-dependent chondrogenic gene expression in human chondrocytic SW1353 cells. Uni-axial CTS (0.5Hz, 5% strain) stimulated gene expression of CCN2 and COL2A1 in SW1353 cells, and induced TGF-β1 secretion. CCN2 synthesis and nuclear translocalization of Smad2/3 and SOX9 were stimulated by CTS. In addition, CTS increased the complex formation between phosphorylated Smad2/3 and SOX9. The CCN2 promoter activity was cooperatively enhanced by CTS and Smad3 in luciferase reporter assay. Chromatin immunoprecipitation revealed that CTS increased Smad2/3 interaction with the CCN2 promoter and the COL2A1 enhancer. CTS epigenetically stimulates CCN2 transcription via TGF-β1 release associated with Smad2/3 activation and enhances COL2A1 expression through the complex formation between SOX9 and Smad2/3."

The epigenetic stimulation of CCN2 may affect how applicable this could be to mesenchymal stem cells.

"immoderate cyclic tensile strain (CTS, 0.5 Hz, 10% strain) induces expression of catabolic factors, such as MMP-13 and ADAMTS-4/5/9, and inhibits expression of cartilage-specific ECM molecule α1(II) collagen (COL2A1) in human chondrocytic SW1353 cells. In addition, excessive CTS (10% strain) stimulates expression of MMP-3/13 and ADAMTS-5 in human articular chondrocytes. On the other hand, appropriate stretching force (5-6% strain) increases expression of anabolic factors, such as COL2A1 and multifunctional growth factor CYR61/CTGF/NOV (CCN) 2, in human fibrochondrocytes derived from the inner meniscus and chondrocytic HCS-2/8 cells"

"CTS (0.5 Hz, 5% strain) induces the nuclear translocation of phosphorylated Smad2/3 and enhances Smad3-dependent CCN2 expression in inner meniscus cells"<-maybe this translocation can occur in MSCs.

Interesting that no CCN2 was detected above threshold in LSJL.

TGFB1 levels continued to increase with duration of tensile strain up to 120 minutes.