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.
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 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 study the effect of mechanical stretch on the microenvironment of BEAS-2B on macrophage polarization and the role of polarized macrophages in the epithelial-mesenchymal transition (EMT) of BEAS-2B. Methods Using enzyme linked immunosorbent assay to detect the changes in the levels of cytokines such as interferon-γ, granulocyte-macrophage colony stimulating factor, tumor necrosis factor-α, interleukin (IL)-4, IL-6, IL-10 in the supernatant of lung epithelial cells cultured statically and mechanically stretched. The M0 macrophages (derived from THP-1) were stimulated by stretch/static conditioned medium of BEAS-2B. The surface markers of M1 (CD197) /M2 (CD206) macrophages were detected by flow cytometer. Stretch/static conditioned medium were used to stimulate the co-culture system of macrophages and BEAS-2B in the presence or absence of platelet-derived growth factor receptor inhibitor (PDGFRi), then the protein expression level of EMT makers was examined by Western blot. Results Exposure of BEAS-2B to mechanical stretch resulted in significantly higher production of the pro-M1/M2 polarized factor. The EMT of the co-culture system of M0 and BEAS-2B could be induced by stretch conditioned medium, epithelial marker cytokeratin (CK)-8 and E-cadherin were decreased, while mesenchymal marker α-smooth muscle actin, N-cadherin and vimentin were increased in stretch conditioned medium group. The expression of platelet-derived growth factor (PDGF) was significantly higher in stretch conditioned medium group. The PDGFRi can block the EMT in stretch conditioned medium group. Conclusions The lung epithelial cell supernatant induced by mechanical stretch can promote the polarization of macrophages to M1 and M2. Polarized macrophages promote EMT in human lung epithelial cells via PDGF, and blocking PDGF might attenuate the VILI-associated lung fibrosis.
Objective To investigate the application of modified adjustable skin stretching and secure wound-closure system in repairing of skin and soft tissue defect. Methods Between March 2016 and April 2017, 21 cases of skin and soft tissue defects were repaired with the modified adjustable skin stretching and secure wound-closure system (the size of regulating pressure and the times of adjustment were determined according to the color, temperature, capillary response, and swelling degree of the skin edge). There were 11 males and 10 females, with an average age of 49.2 years (range, 21-67 years). Among them, 1 case was the residual wound after amputation of leg; 18 cases were the wounds after traumatic injury operation, including 4 cases in the lower leg, 3 cases in the knee joint, 7 cases in the upper limb, and 4 cases in the foot; and 2 cases were diabetic feet. The skin defect area ranged from 4.0 cm×2.5 cm to 21.0 cm×10.0 cm. Results Skin defect wounds closed directly in one stage in 4 cases; 12 cases were closed after continuously stretching for 5-14 days (mean, 10 days); 5 cases were reduced to less than one-half area, and the wound healed after the second skin grafting or flap repairing. All the 21 patients were followed up 3-12 months (mean, 5.2 months). The wound was linear healing with small scar, and no invasive margin, poor blood flow, necrosis, and poor sensory function happened. Conclusion The modified adjustable skin stretching and secure wound-closure system can reduce the skin and soft tissue defects or close the wound directly, and even replace the skin graft and skin flap repairing. It was a good method for the treatment of skin and soft tissue defect.
Objective To explore the components of passive movement resistance in the wrist flexor in subjects after stroke, and investigate the correlations between these components and clinical scales such as Modified Ashworth Scale (MAS) and Fugl-Meyer Assessment (FMA). Methods From March to August 2017, a cross-sectional study was performed in 15 stroke survivors in the Department of Rehabilitation Medicine, the First Affiliated Hospital, Sun Yat-sen University. MAS and FMA were assessed by an experienced physical therapist. Components of passive movement resistance in the flexors of wrist and finger were recorded by NeuroFlexor (Aggro MedTech AB, Solna, Sweden), then the average resisting force in one second ensued the passive stretch at 5°/s was took as peak resisting force (PRF). The PRF between paretic side and non-paretic side was compared. Spearman’s rank correlation was used to test the relation between the components and clinical scales. Results The PRF of the paretic side during the slow passive stretch (5°/s) was significantly higher than that of the non-paretic side [(10.49±1.65) vs. (8.98±1.11) N, P<0.05]. Correlations between MAS and the components/PRF were insignificant (P>0.05). FMA had a significant correlation with neural component of the paretic side (rs=–0.645, P=0.009). Conclusions The higher PRF of slow passive stretch in the paretic side may be attributed to the higher muscle stiffness. Neural component of the paretic wrist is correlated with FMA. These findings could be applied in clinical evaluation of functional performance of the wrist muscle of stroke survivors.
Objective To observe the effectiveness of disposable skin stretch closure in the treatment of wounds with skin and soft tissue defects that were difficult to close. Methods The clinical data of 13 patients with skin and soft tissue defects that were difficult to close treated with disposable skin stretch closure and met the selection criteria between July 2021 and February 2022 were retrospectively reviewed. There were 9 males and 4 females, the age ranged from 15 to 71 years with a mean of 39.8 years. The causes of injury included falling injury in 5 patients, traffic accident injury in 5 patients, and falling from height injury in 3 patients. The causes of skin soft tissue defects included open fractures in 4 patients, wound infection in 4 patients, osteomyelitis in 3 patients, degloving injury in 1 patient, and necrosis of skin graft in 1 patient. The injury was located at calf in 8 patients, calcaneus in 3 patients, pelvis in 1 patient, and plantar in 1 patient. The skin and soft tissue defects ranged from 5.0 cm×2.0 cm to 10.5 cm×6.5 cm. Wound conditions (wound closure and wound healing) and the presence or absence of complications were recorded. Results All 13 patients were followed up 32-225 days with a median of 164 days. The wound closure time ranged from 5 to 14 days, with a mean of 8.8 days. The wound closure speed ranged from 0.7 to 13.7 cm2/day, with a mean of 3.6 cm2/day. All wounds healed at grade A, and no complication such as skin edge injury, wound necrosis, infection, dehiscence, and edema occurred. No patient complained of pain or discomfort, and no obvious scarring was found during follow-up. The wound healing time ranged from 17 to 28 days, with a mean of 21.7 days. One of them was transferred to other department due to lung cancer condition changes after using disposable skin stretch closure, and the wound had directly healed without suturing at 17 days after operation. Conclusion The effectiveness of disposable skin stretch closure in the treatment of wounds with skin and soft tissue defects that were difficult to close was exact, with short wound closure time, few complications, and easy operation.
Objective To investigate the effects of different mechanical stretch conditions on the differentiation of rat tendon stem cells (TSCs), to find the best uniaxial cyclic stretching for TSCs tenogenic differentiation, osteogenic differentiation, and adipogenic differentiation. Methods TSCs were isolated from the Achilles tendons of 8-week-old male Sprague Dawley rats by enzymatic digestion method and cultured. The TSCs at passage 3 were randomly divided into 5 groups: group A (stretch strength of 4% and frequency of 1 Hz), group B (stretch strength of 4% and frequency of 2 Hz), group C (stretch strength of 8% and frequency of 1 Hz), group D (stretch strength of 8% and frequency of 2 Hz), and group E (static culture). At 12, 24, and 48 hours after mechanical stretch, the mRNA expressions of the tenogenic differentiation related genes [Scleraxis (SCX) and Tenascin C (TNC)], the osteogenic differentiation related genes [runt related transcription factor 2 (RUNX2) and distal-less homeobox 5 (DLX5)], and the adipogenic differentiation related genes [CCAAT-enhancer-binding protein-α (CEBPα) and lipoprteinlipase (LPL)] were detected by real-time fluorescent quantitative PCR and the protein expressions of TNC, CEBPα, and RUNX2 were detected by Western blot. Results The mRNA expressions of SCX and TNC in group B were significantly higher than those in groups A, C, D, and E at 24 hours after mechanical stretch (P<0.05). The mRNA expressions of CEBPα and LPL in group D were significantly higher than those in groups A, B, C, and E at 48 hours after mechanical stretch (P<0.05). The mRNA expressions of RUNX2 and DLX5 in group C were significantly higher than those in groups A, B, D, and E at 24 hours after mechanical stretch (P<0.05). Western blot detection showed that higher protein expression of TNC in group B than group E at each time point after mechanical stretch (P<0.05), and the protein expression of CEBPα was significantly inhibited when compared with group E at 24 hours after mechanical stretch (P<0.05). At 24 hours after mechanical stretch, the protein expression of RUNX2 in group C was significantly higher than that in group E (P<0.05); and the protein expression of TNC was significantly lower than that in group E at 24 and 48 hours after mechanical stretch (P<0.05). At 48 hours after mechanical stretch, the protein expression of CEBPα was significantly increased and the protein expression of TNC was significantly decreased in group D when compared with group E (P<0.05), but no significant difference was found in the protein expression of RUNX2 between groups D and E (P>0.05). Conclusion The mechanical strain could promote differentiation of TSCs, and different parameter of stretch will lead to different differentiation. The best stretch condition for tenogenic differentiation is 4% strength and 2 Hz frequency for 24 hours; the best stretch condition for osteogenic differentiation is 8% strength and 1 Hz frequency for 24 hours; and the best stretch condition for adipogenic differentiation is 8% strength and 2 Hz frequency for 48 hours.
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.