Objective To observe the effects of mechanical stretch on cytokines release from alveolar macrophages( AMs) and the expression of macrophage inflammatory protein-2( MIP-2) induced by lipopolysaccharide( LPS) . Methods AMs were divided into the following groups: ①AMs were subjected to 20% elongation by Flexercell 4000T cell stress system for 24 hours and the supernatant was collected to detect the levels of TNF-α, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-12, IFN-γ, macrophage inflammatory protein-1α( MIP-1α) , MIP-2, monocyte chemoattractant protein-1( MCP-1) , granulocyte /macrophage colony stimulating factors( GM-CSF) , interferon inducible protein-10( IP-10) , regulated on activation in normal T-cell expressed and secreted( Rantes) and keratinocyte chemoattractant( KC) , by using LiquiChip system. ② AMs were subjected to 5% , 10% , 15% and 20% elongation for 24 hours and the supernatant was collected to detect the levels of MIP-2. ③AMs were subjected to 20% elongation and MIP-2 in supernatant was detected 1, 3,6, 12, and 24 hours later. ④ AMs were subjected to 20% elongation and/ or LPS at a concentration of 10 ng/mL, and MIP-2 in supernatant was detected 24 hours later. Unstretched AMs were used as control in all kind of test. Results ①The levels of IL-1β, IL-6,MIP-2, MCP-1, IFN-γand IP-10 secreted by stretched AMs were 8. 7, 4. 3, 38. 6, 4. 8, 14. 2 and 5. 0 times those of the control group( all P lt; 0. 001) . ② The levels of MIP-2 secreted by AMs subjected to 10% , 15% and 20% elongation were ( 480. 5 ±93. 1) pg /mL,( 806. 3 ±225. 9) pg/mL and ( 1335. 7 ±18. 5) pg/mL respectively, all significantly higher than those oft he control group [ ( 34. 6 ±11. 4) pg/mL, all P lt;0. 001] . ③ Three hours after the stimulation of stretch the level of MIP-2 began to increase gradually. And 6, 12, and 24 hours after the stimulation the levels of MIP-2 secreted by the AMs were ( 819. 4 ±147. 5) pg/mL, ( 1287. 6 ±380 ±3 ) pg/mL and ( 1455. 9 ±436. 7) pg/mLrespectively, all significantly higher than those of the control group[ ( 33. 4 ±10. 2) pg/mL, all P lt; 0. 001] . ④When the AMs were stimulated individually by LPS( 10 ng /mL) or mechanical stretch ( 20% ) , the levels of MIP-2 increased to ( 1026. 3 ±339. 5 ) pg/mL and ( 1335. 7 ±318. 5 ) pg/mL respectively( both P lt; 0. 001) . When the AMs were costimulated by LPS and mechanical stretch, the level of MIP-2 increased to ( 2275. 3 ±492. 1) pg/mL, implicating a synergistic effect between mechanical stretch and LPS ( F = 121. 983, P lt; 0. 001) . Conclusions Mechanical stretch activates AMs to produce multiple inflammatory cytokines and induce AMs to secret MIP-2 in a strength- and time-dependent manner.Mechanical stretch also has synergistic effect with LPS in inducing MIP-2 release, which might play an important role in the development of ventilator-induced lung injury.
Objective To investigate the technique of reduction by posterior approach for severe spondylolisthesis, and to discuss the method to prevent nerve stretch injury. Methods Between July 2007 and April 2011, 17 patients with severe spondylolisthesis underwent reduction, fixation, and fusion by posterior approach. There were 2 males and 15 females with a median age of 15 years (range, 8-67 years) and a median disease duration of 18 months (range, 5 months-16 years and 4 months). The level of spondylolisthesis was at L4 in 1 case and L5 in 16 cases; the spondylolisthesis was at degree III in 12 cases and degree IV in 5 cases according to Meyerding classification. There were 16 cases of developmental spondylolisthesis (high- dysplastic and low-dysplasia spondylolisthesis in 9 and 7 cases, respectively) and 1 case of traumatic spondylolisthesis; 16 cases of developmental spondylolisthesis at L5 level included 6 cases of type 4, 9 case of type 5, and 1 case of type 6 according to Spinal Deformity Study Group (SDSG) classification. All cases underwent posterior spinal decompression, Schanz screw fixation for the slipped vertebrae, the intervertebral and posterolateral fusion and reduction of the slipped vertebrae, and correction of the lumbosacral kyphosis. The reductive degree of slipped vertebrae was modulated according to the strain of exiting spinal root. The slip degree should be reduced within Meyerding degree II. The anteroposterior and lateral radiographs of whole spine were taken in a standardized standing position to observe the correction of displacement severity and lumbosacral angle. The nerve function and pain score of lower extremity were evaluated by neurological Frankel grade and visual analogue scale (VAS). Bony fusion was assessed by followed-up CT three-dimentional reconstruction. Results Exiting nerve root paralysis occurred in 1 case after operation, and released at 4 weeks after operation; no aggravation of nerve damage was observed in the other patients. The incisions primarily healed. All the patients were followed up 12-48 months (mean, 25 months). The slip percentage, the lumbosacral angle, and VAS score of lower extremity were improved from 72% ± 10%, (18.2 ± 3.5)°, and 7.0 ± 1.5 at preoperation to 12% ± 6%, ( — 7.3 ± 2.9)°, and 1.5 ± 1.3 at 12 months after operation respectively, all showing significant differences (P lt; 0.05). Osteosynthesis was seen at the bone grafting area by CT three-dimentional reconstruction at 12 months after operation. No breakage of screw and rod or reduction loss occurred. Conclusion It can obtain satisfactory clinical result to use spinal canal decompression by posterior approach, the Schanz screw fixation of the slipped vertebrae, the intervertebral and posterolateral fusion for severe spondylolisthesis. The risk of nerve stretch injury can be prevented by choosing the lowest height of intervertebral cage, modulating the reductive degree of slipped vertebrae according to the strain of exiting spinal root, and correcting lumbosacral kyphosis.
Objective To explore the effects of mechanical stretch with variant frequencies on the alignment and differentiation of the multilayer myotubes cultured in vitro, and to select the optimized cultural condition of regenerative skeletal muscle tissue with stress loading cultured in vitro. Methods C2C12 myoblasts cultured in vitro in the groove casts of Sylgard 184 were induced into the multilayer myotubes. Meanwhile the myoblasts were treated with various mechanical stretch withcells tensile instrument, at the amplitude of 10% and the frequency of 0 (group A), 0.25 (group B), 0.50 (group C), and 1.00 Hz (group D) for 1 hour, 3 times a day. The myotubes morphology was observed by inverted phase contrast microscope at 5, 7, and 10 days after continuous mechanical stretch. And the expressions of mRNA for myogenic differentiation antigen (MyoD), Myogenin, Desmin, and myosin heavy chain (MyHC) were detected by RT-PCR and real-time fluorescent quantitative PCR (QRT-PCR), respectively. Results The mechanical stretch could promote the al igned fusion and increase the number of myotubes. Indeed the multilayer myotubes arranged more closely in group B at 7 days. At the same group, as the time went on, the mRNA expressions of MyoD gradually decl ined in each group. There were significant differences in mRNA expressions of MyoD between 5 days and 7, 10 days (P lt; 0.05). The mRNA expressions of Myogenin, Desmin, and MyHC were highest at 7 days. There were significant differences between different time points (P lt; 0.05), except the mRNA expression of Desmin of group B between 7 and 10 days (P gt; 0.05). At the same time, with the increase of frequency, the highest mRNA expressions of MyoD, Myogenin, Desmin, and MyHC were in group B. There were significant differences at the same time between group B and the other groups (P lt; 0.05), except mRNA expression of Desmin at 5 days between groups B and C, and mRNA expression of MyHC at 10 days between groups A and B (P gt; 0.05). Conclusion Low frequency (0.25 Hz) and suitable time (7 days) periodic mechanical stretch is beneficial to the differentiation of the multilayer myotubes cultured in the groove casts of Sylgard 184, but as the stretch time goes on the aging of myotubes will be accelerated.
OBJECTIVE: To investigate the availability and effect of skin stretch in closing the firearm injured soft tissue defect. METHODS: Eight white pigs with firearm injured soft tissue defect were divided into 3 groups. Each group I and group II had 3 pigs which were performed skin stretch. The control group had 2 pigs without stretch. The average diameter of the defect in three groups was (7.3 +/- 0.2) cm, (9.1 +/- 0.3) cm, (7.3 +/- 0.2) cm respectively, and the site of defect was on the lateral thigh and buttock. RESULTS: Skin stretch could make a visible reduction of the wound. It was possible to close the wound by direct traction when the diameter of the buttock wound was less than 7 cm, and when the diameter of the lateral thigh wound was less than the radius of thigh. The skin stretch should not last more than 7 days and the best effect appeared in 4 to 5 days after performing the skin stretch. CONCLUSION: The skin stretch can be applied in the repair of the firearm injured soft tissue defect. It has many advantage compared with the tradtional treatment.
Bone marrow-derived mesenchymal stem cells (BMSCs) are multipotent stem cells that differentiate into a variety of cell types and widely used in tissue regeneration engineering. The purpose of this study is to investigate whether the cyclic biaxial stretching strain could promote the rat BMSCs (rBMSCs) to differentiate into cardiomyocyte-like cells in vitro. The second or third generation of rBMSCs were randomly divided into the cyclic stretching stain group, the control group and the blank group. Those rBMSCs in the cyclic stretching strain group were seeded on a silicone membrane with complete medium were exposed to biaxial stretching strain of 10% of membrane at a frequency of 1 Hz lasting for 6 h, 12 h and 24 h. Those in the control group were seeded on silicone membrane with complete medium. Those in the blank group were seeded in the 6-wells plates with complete medium. The mRNA expression of GATA4 and myocyte-specific enhancer factor 2C (MEF-2C) were detected by the real-time fluorescent quantification PCR and the protein expression of connexin 43 (Cx43) was detected by using the Western blot method. The results showed that the mRNA expression level of the GATA4 and MEF-2C, and the protein expression level of Cx43 were significantly higher in the cyclic stretching strain groups, compared with those in the relative control groups (P<0.05). It suggests that cyclic biaxial stretching strain could play a part in the induction of rBMSCs to differentiate into cardiomyocyte-like cells in vitro, but the differentiation mechanism is still unclear.
This study was aimed to design a new, accurate and easy-to-use water bath cryo-jaw, and try to solve the problems met in small animals achilles tendon mechanical testing. The muscle-tendon-bony units were fixed in the clamps. SD rats achilles tendon were randomly divided into group A and B. Group A was tested by the newly designed water bath cryo-jaw, while group B was treated by non-water bath cryo-jaw. The mechanical tests revealed that non of the samples of the newly-designed water bath cryo-jaw in group A slipped and fell off, and the achilles tendons were in a physiologically active state, but one of the group B samples slipped and fell off, and the others had the frozen phenomenon obviously. The maximum stress, fracture displacement and Young's modulus of the rats in group A were significantly different compared to those in group B (P<0.05). In conclusion, the new water bath cryo-jaw has more advantages than traditional ones. It exhibits a good simulation in vivo in the environmental conditions for testing the mechanical properties of the achilles tendon.
ObjectiveTo explore the reaction of normal skin fibroblasts from different sites of human body to cyclic stretch. MethodsThe normal skin tissues from scapular upper back and medial side of upper arm of 3 patients were cultured in vitro. Fibroblasts of experimental group were loaded by cyclic stretch with 10% amplitude for 24, 36, and 48 hours respectively. Fibroblasts of control group were cultured without cyclic stretch. The morphologic changes were observed using inverted microscope. CCK-8 method was used to detect the proliferation of the fibroblasts. The expressions of integrin β1 mRNA, p130Crk-associated substance (P130Cas) mRNA, transform growth factor β1 (TGF-β1) mRNA, and collagen type Ⅰ α1 chain (COL1A1) mRNA were detected by real-time quantitative PCR. The protein levels of collagen type Ⅰ and TGF-β1 were detected by ELISA. ResultsThe cultured cells showed a significantly increased cell proliferation ability, and apparent orientation after the applied strain. The proliferation activity, mRNA expression levels of integrin β1, P130Cas, and TGF-β1, protein levels of TGF-β1 in back skin were significantly higher than those in arm skin (P<0.05) when the fibroblasts were loaded for 36 and 48 hours, but no significant difference between back skin and arm skin at 24 hours (P>0.05). There was no significant difference in mRNA expression level of COL1A1 and protein level of collagen type Ⅰ between back skin and arm skin at 24, 36, and 48 hours (P>0.05). There was no significant difference in all above indexes between back skin and arm skin in control group (P>0.05). ConclusionFibroblasts from scapular upper back and medial side of upper arm display different reactions to cyclic stretch, which indicates that there exists site difference in the reactions of fibroblasts to cyclic stretch. It might be related with the incidence of hypertrophic scar in different sites of the body.
ObjectiveTo isolate the tendon stem cells (TSCs) from rat patellar tendon and to investigate the effect of mechanical stretching on the expression of Sox-9. MethodsTSCs were isolated from Sprague Dawley rat (12 weeks old) patellar tendon by collagenase digestion and low density culture. The cell colony morphology and number were observed by crystal violet staining;the cell morphology was observed by inverted phase contrast microscope, and the immunophenotypes of mesenchymal stem cells (MSCs) were determined by flow cytometry. The TSCs at passage 3 was given the mechanical stretching at 4%, 0.17 Hz for 4 hours and 24 hours in the experimental group, and cells without stretching was used as control. The Sox-9 gene and protein expressions were detected by real-time fluorescence quantitative PCR and Western blot. ResultsPrimary cells showed clonal growth and star shape;after subculture, cells at passage 1 showed fibroblast-like shape. The cells formed cell colonies after 7 days;the expressions were positive for CD29, CD44, and CD90 and negative for CD45. The result of real-time fluorescence quantitative PCR showed that Sox-9 gene was down-regulated at 4 hours after mechanical stretching compared with control (P<0.05), and up-regulated at 24 hours after mechanical stretching when compared with control group (P<0.05). The result of Western blot showed that Sox-9 protein expression was lower at 4 hours after stretching, but higher at 24 hours after mechanical stretching than that in control group (P<0.05). ConclusionThe rat patellar TSCs can be isolated successfully, and mechanical stretching inhibits the Sox-9 expression, but the inhibited effect might stimulate the Sox-9 expression after the mechanical stretching effect disappears.
ObjectiveTo investigate the effectiveness of distraction therapy assisted by arthroscope in the treatment of ankle traumatic osteoarthritis. MethodsBetween October 2013 and October 2014, 13 patients with ankle traumatic osteoarthritis were treated, including 8 males and 5 females with an age range of 44-63 years (mean, 55.2 years). The left ankle and the right ankle were involved in 4 and 9 cases respectively. The disease duration was 1.5-10.0 years (median, 5 years). The American Orthopaedic Foot and Ankle Society (AOFAS) ankle-hind foot scale score was 51.00±7.09; the short-form 36 health survey scale (SF-36) score was 40.82±4.62. According to Scranton grade system, 9 cases were rated as grade II and 4 cases as grade III. First, ankle hyperplasia osteophytes was removed under arthroscope, then Ilizarov apparatus was used to maintain distraction of 5-10 mm ankle space for 3 months. ResultsOne case had postoperative pin tract infection after removing the external fixation, and infection was controlled by dressing treatment; no related complications occurred in the other patients. All patients got follow-up of 12-18 months (mean, 14.7 months). Patients achieved disappearance of ankle swelling, pain relief, and were able to walk after rehabilitation. The ankle activity was obviously improved. At last follow-up, AOFAS ankel-hind foot scale score and SF-36 score were significantly increased to 85.23±6.41 and 56.29±6.20 respectively (t=20.756, P=0.025; t=11.647, P=0.018). According to AOFAS scores, the results were excellent in 4 cases, good in 8 cases, and fair in 1 case; the excellent and good rate was 92.3%. Postoperative X-ray film showed normal ankle position and alignment, osteophytes at the edges of the tibia and talus, articular surface sclerosis, normal joint space, and no joint swelling. ConclusionDistraction therapy assisted by arthroscope is an effective method for treating ankle traumatic osteoarthritis.
This study is aimed to investigate the effects of mechanical stretch on the expression of transforming growth factor-β1 (TGF-β1) and fibroblast growth factor-2 (FGF-2), and the signaling pathway in human bronchial epithelioid (16HBE) cells under mechanical stretch. Using loading device with flexible substrate (FX-4000T) to stretch 16HBE cells, we found that the stretching elongation was 15%, at frequency of 1 Hz, stretching for 0.5 h, 1 h, 1.5 h and 2 h. Choosing the higher expression of TGF-β1, FGF-2 and Ca2+ group to carry out intervention experiments, we used the cells pretreated with canonical transient receptor potential 1 (TRPC1) channel antagonist SKF96365, protein kinase C (PKC) inhibitor HA-100, and thereafter mechanical stretch to interpose. Compared with those in the blank control group, TGF-β1 and FGF-2' protein and mRNA, intracellular Ca2+ fluorescence intensity were higher, and the differences were statistically significant (P < 0.05) at the 4 time points, 0.5 h, 1 h, 1.5 h and 2 h. At 0.5 h, the increasing rate was the highest. TGF-β1 protein and mRNA, FGF-2 protein and mRNA, intracellular Ca2+ luorescence intensity in the stretch+SKF96365 and stretch+HA-100 intervented group were decreased, the differences were statistically significant than those in 0.5 h stretch group (P < 0.05) without intervention. The expression of TGF-β1, FGF-2 was up-regulated in 16HBE cells under mechanical stretch, PKC, TRPC1, and Ca2+ may participate in the signal path.