Leukocyte-poor platelet-rich plasma and leukocyte-rich platelet-rich plasma promote myoblast proliferation through the upregulation of cyclin A, cdk1, and cdk2.

Muscle injuries are common among athletes and often treated with platelet-rich plasma (PRP). However, whether the leukocyte concentration affects the efficacy of PRP in treating muscle injuries remains unclear. This study investigated the effects of leukocyte-poor platelet-rich plasma (LP-PRP) and leukocyte-rich platelet-rich plasma (LR-PRP) on myoblast proliferation and the molecular mechanisms underlying these effects. Myoblasts were treated with 0.5% LP-PRP, 0.5% LR-PRP, 1% LP-PRP, or 1% LR-PRP for 24 h. The gene expression of the LP-PRP- and LR-PRP-treated myoblasts was determined using RNA sequencing analysis. Cell proliferation was evaluated using an bromodeoxyuridine (BrdU) assay, and cell cycle progression was assessed through flow cytometry. The expression of cyclin A, cyclin-dependent kinase 1 (cdk1), and cdk2 was examined using Western blotting. The expression of myoblast determination protein 1 (MyoD1) was examined through Western blotting and immunofluorescence staining. The LP-PRP and LR-PRP both promoted the proliferation of myoblasts and increased differential gene expression of myoblasts. Moreover, the LP-PRP and LR-PRP substantially upregulated the expression of cyclin A, cdk1, and cdk2. MyoD1 expression was induced in the LP-PRP and LR-PRP-treated myoblasts. Our results corroborate the finding that LP-PRP and LR-PRP have similar positive effects on myoblast proliferation and MyoD1 expression.

Lidocaine Inhibited Tendon Cell Proliferation and Extracellular Matrix Production by Down Regulation of Cyclin A, CDK2, Type I and Type III Collagen Expression.

Lidocaine injection is a common treatment for tendon injuries. However, the evidence suggests that lidocaine is toxic to tendon cells. This study investigated the effects of lidocaine on cultured tendon cells, focusing on the molecular mechanisms underlying cell proliferation and extracellular matrix (ECM) production. Tendon cells cultured from rat Achilles tendons were treated with 0.5, 1.0, or 1.5 mg/mL lidocaine for 24 h. Cell proliferation was evaluated by Cell Counting Kit 8 (CCK-8) assay and bromodeoxyuridine (BrdU) assay. Cell apoptosis was assessed by Annexin V and propidium iodide (PI) stain. Cell cycle progression and cell mitosis were assessed through flow cytometry and immunofluorescence staining, respectively. The expression of cyclin E, cyclin A, cyclin-dependent kinase 2 (CDK2), p21, p27, p53, matrix metalloproteinases-2 (MMP-2), matrix metalloproteinases-9 (MMP-9), type I collagen, and type III collagen were examined through Western blotting, and the enzymatic activity of MMP-9 was determined through gelatin zymography. Lidocaine reduced cell proliferation and reduced G1/S transition and cell mitosis. Lidocaine did not have a significant negative effect on cell apoptosis. Lidocaine significantly inhibited cyclin A and CDK2 expression but promoted p21, p27, and p53 expression. Furthermore, the expression of MMP-2 and MMP-9 increased, whereas that of type I and type III collagen decreased. Lidocaine also increased the enzymatic activity of MMP-9. Our findings support the premise that lidocaine inhibits tendon cell proliferation by changing the expression of cell-cycle-related proteins and reduces ECM production by altering levels of MMPs and collagens.

Simvastatin Downregulates Cofilin and Stathmin to Inhibit Skeletal Muscle Cells Migration.

Statins are the most effective therapeutic agents for reducing cholesterol synthesis. Given their widespread use, many adverse effects from statins have been reported; of these, musculoskeletal complications occurred in 15% of patients after receiving statins for 6 months, and simvastatin was the most commonly administered statin among these cases. This study investigated the negative effects of simvastatin on skeletal muscle cells. We performed RNA sequencing analysis to determine gene expression in simvastatin-treated cells. Cell proliferation and migration were examined through cell cycle analysis and the transwell filter migration assay, respectively. Cytoskeleton rearrangement was examined through F-actin and tubulin staining. Western blot analysis was performed to determine the expression of cell cycle-regulated and cytoskeleton-related proteins. Transfection of small interfering RNAs (siRNAs) was performed to validate the role of cofilin and stathmin in the simvastatin-mediated inhibition of cell migration. The results revealed that simvastatin inhibited the proliferation and migration of skeletal muscle cells and affected the rearrangement of F-actin and tubulin. Simvastatin reduced the expression of cofilin and stathmin. The knockdown of both cofilin and stathmin by specific siRNA synergistically impaired cell migration. In conclusion, our results indicated that simvastatin inhibited skeletal muscle cell migration by reducing the expressions of cofilin and stathmin.

Ibuprofen inhibited migration of skeletal muscle cells in association with downregulation of p130cas and CrkII expressions.

BACKGROUND: Nonsteroidal anti-inflammatory drugs (NSAIDs) are commonly used to treat sports-related muscle injuries. However, NSAIDs were recently shown to impede the muscle healing process after acute injury. Migration of skeletal muscle cells is a crucial step during the muscle healing process. The present study was performed to investigate the effect and molecular mechanisms of action of ibuprofen, a commonly used NSAID, on the migration of skeletal muscle cells. METHODS: Skeletal muscle cells isolated from the gastrocnemius muscle of Sprague-Dawley rats were treated with ibuprofen. MTT assay (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) was used to evaluate cell viability, and cell apoptosis was evaluated by TUNEL assay, after ibuprofen treatment. Skeletal muscle cell migration and spreading were evaluated using the transwell filter migration assay and F-actin staining, respectively. The protein expression of p130cas and CrkII, which are cell migration facilitating genes, was determined by western blot analysis. The overexpression of p130cas of muscle cells was achieved by p130cas vector transfection. RESULTS: The results demonstrated that ibuprofen did not have a significant negative effect on cell viability and apoptosis. Ibuprofen inhibited the migration and spreading of skeletal muscle cells in a dose-dependent manner. Ibuprofen also dose-dependently decreased the protein expression of p130cas and CrkII. Furthermore, overexpression of p130cas resulted in the promotion of cell migration and spreading and counteracted ibuprofen-mediated inhibition. CONCLUSION: This study suggested that ibuprofen exerts a potentially adverse effect on the migration of skeletal muscle cells by downregulating protein expression of p130cas and CrkII. These results indicate a possible mechanism underlying the possible negative effect of NSAIDs on muscle regeneration.

Platelet-Rich Plasma Releasate Promotes Regeneration and Decreases Inflammation and Apoptosis of Injured Skeletal Muscle.

BACKGROUND: Platelet-rich plasma (PRP) contains various cytokines and growth factors that may be beneficial to the healing process of injured muscle. Based on the authors' previous study, PRP releasate can promote proliferation and migration of skeletal muscle cells in vitro, so animal studies are performed to support the use of PRP to treat muscle injury in vivo. PURPOSE: To investigate the effect of PRP releasate on regeneration of injured muscle, as well as its effect on inflammatory reaction and cell apoptosis, in the early stages of the muscle-healing process. STUDY DESIGN: Controlled laboratory study. METHODS: The gastrocnemius muscles of Sprague-Dawley rats were injured by partial transverse incision and then treated with PRP releasate. Hematoxylin and eosin stain was used to evaluate the healing process of injured muscle at 2, 5, and 10 days after injury. TUNEL assay was used to evaluate the cell apoptosis of injured muscle after PRP releasate treatment. Immunohistochemistry was used to stain the CD68-positive cells during the healing process. Muscle contractile properties, including fast-twitch and tetanic strength, were evaluated by electric stimulation. RESULTS: The results revealed that PRP releasate treatment could enhance the muscle-healing process and decrease CD68-positive cells and apoptotic cells. Furthermore, the tetanic strength was significantly higher in injured muscle treated with PRP releasate. CONCLUSION: In conclusion, PRP releasate could enhance the healing process of injured muscle and decrease inflammatory cell infiltration as well as cell apoptosis. CLINICAL RELEVANCE: PRP promotes skeletal muscle healing in association with decreasing inflammation and apoptosis of injured skeletal muscle. These findings provide in vivo evidence to support the use of PRP to treat muscle injury.

Rhythmic Neck Muscle Spasms and Upper Limb Muscle Myoclonic Jerks as an Unusual Initial Presentation of Posttraumatic Cervicothoracic Syringomyelia: A Case Report.

Posttraumatic syringomyelia with an initial presentation of involuntary movement is rare. We describe a 25-year-old patient who sustained complete traumatic spinal cord injury at the thoracic level and presented with rhythmic neck muscle spasms and upper limb muscle myoclonic jerks 1 month after trauma. Magnetic resonance imaging revealed syrinx formation between C3 and T1. Lumbar-peritoneal shunt and decompression were performed. The symptoms completely disappeared after surgery. This report highlights that rhythmic neck muscle spasms and upper limb muscle myoclonic jerks can be the initial and only manifestations of syringomyelia. LEVEL OF EVIDENCE: V.

Comparative Effectiveness of Nonoperative Treatments for Chronic Calcific Tendinitis of the Shoulder: A Systematic Review and Network Meta-Analysis of Randomized Controlled Trials.

OBJECTIVE: To investigate the effectiveness of various nonoperative treatments for chronic calcific tendinitis of the shoulder, a systematic review and network meta-analysis of randomized trials was performed to evaluate changes in pain reduction, functional improvements in patients with calcific tendinitis, and the ratio of complete resolution of calcific deposition. DATA SOURCES: Studies were comprehensively searched, without language restrictions, on PubMed, Embase, Cochrane Controlled Trials Register, the Cochrane, and other databases. The reference lists of articles and reviews were cross-checked for possible studies. STUDY SELECTION: Randomized controlled trials from before August 2016 were included. Study selection was conducted by 2 reviewers independently. DATA EXTRACTION: The quality of studies was assessed and data extracted by 2 independent reviewers. Disagreements were settled by consulting a third reviewer to reach a consensus. DATA SYNTHESIS: Fourteen studies with 1105 participants were included in the network meta-analysis that used a random-effect model to investigate the mean difference of pooled effect sizes of the visual analog scale, Constant-Murley score, and the ratio of complete resolution of calcific deposition on native radiographs. CONCLUSIONS: The present network meta-analysis demonstrates that ultrasound-guided needling (UGN), radial extracorporeal shockwave therapy (RSW), and high-energy focused extracorporeal shockwave therapy (H-FSW) alleviate pain and achieve complete resolution of calcium deposition. Compared with low-energy focused extracorporeal shockwave therapy, transcutaneous electrical nerve stimulation, and ultrasound therapy, H-FSW is the best therapy for providing functional recovery. Physicians should consider UGN, RSW, and H-FSW as alternative effective therapies for chronic calcific tendinitis of the shoulder when initial conservative treatment fails.

Low-level laser irradiation stimulates tenocyte migration with up-regulation of dynamin II expression.

Low-level laser therapy (LLLT) is commonly used to treat sports-related tendinopathy or tendon injury. Tendon healing requires tenocyte migration to the repair site, followed by proliferation and synthesis of the extracellular matrix. This study was designed to determine the effect of laser on tenocyte migration. Furthermore, the correlation between this effect and expression of dynamin 2, a positive regulator of cell motility, was also investigated. Tenocytes intrinsic to rat Achilles tendon were treated with low-level laser (660 nm with energy density at 1.0, 1.5, and 2.0 J/cm(2)). Tenocyte migration was evaluated by an in vitro wound healing model and by transwell filter migration assay. The messenger RNA (mRNA) and protein expressions of dynamin 2 were determined by reverse transcription/real-time polymerase chain reaction (real-time PCR) and Western blot analysis respectively. Immunofluorescence staining was used to evaluate the dynamin 2 expression in tenocytes. Tenocytes with or without laser irradiation was treated with dynasore, a dynamin competitor and then underwent transwell filter migration assay. In vitro wound model revealed that more tenocytes with laser irradiation migrated across the wound border to the cell-free zone. Transwell filter migration assay confirmed that tenocyte migration was enhanced dose-dependently by laser. Real-time PCR and Western-blot analysis demonstrated that mRNA and protein expressions of dynamin 2 were up-regulated by laser irradiation dose-dependently. Confocal microscopy showed that laser enhanced the expression of dynamin 2 in cytoplasm of tenocytes. The stimulation effect of laser on tenocytes migration was suppressed by dynasore. In conclusion, low-level laser irradiation stimulates tenocyte migration in a process that is mediated by up-regulation of dynamin 2, which can be suppressed by dynasore.

Determination of the augmentation effects of hyaluronic acid on different heel structures in amputated lower limbs of diabetic patients using ultrasound elastography.

This study measured tissue properties of different anatomies of heels in amputated lower limbs of diabetic patients before and after hyaluronic acid (HA) or normal saline (NS) injections. Seven amputated lower limbs from six diabetic patients constituted the experimental group and one amputated lower limb from a diabetic patient served as the control. The limbs were placed in a fixation platform. A 5-12 MHz linear-array ultrasound transducer controlled by a stepping motor was used to load and unload tested heels. The loading-unloading velocity was 6 mm/s and the maximum loading stress was 178 kPa. Loading-unloading tests were performed before and after 1 mL HA injections into heels in the experimental group. The control limb underwent the same test before and after 1 mL NS injection. The unloaded thickness and Young's modulus of the macrochambers, microchambers and heel pads were determined before and after the interventions. The unloaded thickness of the macrochambers and the heel pad increased significantly (p = 0.012) after HA injection. The Young's modulus of the macrochambers decreased nonsignificantly after HA injections. Similar thickness and tissue stiffness changes were observed in the control limb. The baseline heel-pad energy dissipation ratio (EDR(hp)) was 81.3 ± 1.3% and decreased significantly (p = 0.012) to 73.1 ± 1.7% after HA injections. The EDR(hp) in the control increased after NS injection. Histologic examinations revealed localized HA accumulation in the macrochambers with an extension into the adjacent fibrous septa. Injection of HA can increase tissue thickness and enhance heel-pad tissue resilience.

Effects of negative pressures on epithelial tight junctions and migration in wound healing.

Negative-pressure wound therapy has recently gained popularity in chronic wound care. This study attempted to explore effects of different negative pressures on epithelial migration in the wound-healing process. The electric cell-substrate impedance sensing (ECIS) technique was used to create a 5 x 10(-4) cm(2) wound in the Madin-Darby canine kidney (MDCK) and human keratinocyte (HaCaT) cells. The wounded cells were cultured in a negative pressure incubator at ambient pressure (AP) and negative pressures of 75 mmHg (NP(75)), 125 mmHg (NP(125)), and 175 mmHg (NP(175)). The effective time (ET), complete wound healing time (T(max)), healing rate (R(heal)), cell diameter, and wound area over time at different pressures were evaluated. Traditional wound-healing assays were prepared for fluorescent staining of cells viability, cell junction proteins, including ZO-1 and E-cadherin, and actins. Amount of cell junction proteins at AP and NP(125) was also quantified. In MDCK cells, the ET (1.25 +/- 0.27 h), T(max) (1.76 +/- 0.32 h), and R(heal) (2.94 +/- 0.62 x 10(-4) cm(2)/h) at NP(125) were significantly (P < 0.01) different from those at three other pressure conditions. In HaCaT cells, the T(max) (7.34 +/- 0.29 h) and R(heal) (6.82 +/- 0.26 x 10(-5) cm(2)/h) at NP(125) were significantly (P < 0.01) different from those at NP(75). Prominent cell migration features were identified in cells at the specific negative pressure. Cell migration activities at different pressures can be documented with the real-time wound-healing measurement system. Negative pressure of 125 mmHg can help disassemble the cell junction to enhance epithelial migration and subsequently result in quick wound closure.

Sonographic features of soft tissue tumors in the hand and forearm.

BACKGROUND: High-resolution sonography is well suited for screening soft tissue masses because of its safety, low cost, and real-time, dynamic imaging. The purpose of our study was to elaborate the preoperative sonographic features of soft tissue tumors of the hand and forearm and the corresponding histologic results. METHODS: Thirty-one soft tissue tumors of the hand and forearm were evaluated by ultrasound preoperatively. The mobility, consistency, echogenicity, margin, and color Doppler signal of each tumor were assessed. Dynamic study was also performed. The pathologic diagnosis was obtained after subsequent surgery. RESULTS: The pathologic diagnoses of these soft tissue lesions were lipoma (n = 6), ganglion cyst (n = 6), neurilemmoma (n = 3), neurofibroma (n = 3), giant cell tumor (n = 10), tenosynovitis (n = 2), and malignant lymphoma (n = 1). An adjacent tendon or communication duct extending to the joint space could be found in most giant cell tumors and ganglion cysts; a traceable nerve could be found in most nerve sheath tumors. All benign tumors appeared well-defined. The only malignant tumor appeared ill-defined without a color Doppler signal. CONCLUSION: Sonography enables a reliable diagnosis of the cystic or solid nature of soft-tissue lesions, accurate estimation of the volume, and precise three-dimensional localization of the abnormality. Examiners should perform a dynamic examination and trace the adjacent structure to obtain more diagnostic clues.

Ultrasonographic examination of the normal and injured posterior cruciate ligament.

PURPOSE: The purpose of the study was to determine the echogenicity and thickness of both the normal and injured posterior cruciate ligament (PCL). METHODS: Eight patients with anterior cruciate ligament injury received ultrasonographic evaluation during arthroscopic examination. With the aid of the comet-tail artifact produced by the metal hook during arthroscopic examination, the normal PCL was located on sonograms. Thereafter, 11 patients with PCL injury were examined. In all subjects, the PCL thickness was measured at 2.0 cm proximal from posterior end of the distal PCL inserting onto the tibia. RESULTS: The normal PCL was located just posterior to the posterior tibial intercondylar area. It was hypo-echoic and was thickened proximally and tapered distally. The mean thickness of the injured PCL was 0.71 +/- 0.12 cm, which was significantly (p < 0.05) greater than that of the normal ligament (0.52 +/- 0.08 cm). Different appearances could be observed, including ligamental rupture and avulsion fracture of the tibial insertion of the PCL. CONCLUSIONS: The normal PCL appears on longitudinal sonograms as a hypoechoic fan-shape structure. Sonographic examination can identify different types of PCL lesions.

Ultrasound guidance in caudal epidural needle placement.

BACKGROUND: This study was conducted to investigate the feasibility of using ultrasound as an image tool to locate the sacral hiatus accurately for caudal epidural injections. METHODS: Between August 2002 and July 2003, 70 patients (39 male and 31 female patients) with low back pain and sciatica were studied. Soft tissue ultrasonography was performed to locate the sacral hiatus. A 21-gauge caudal epidural needle was inserted and guided by ultrasound to the sacral hiatus and into the caudal epidural space. Proper needle placement was confirmed by fluoroscopy. RESULTS: In all the recruited patients, the sacral hiatus was located accurately by ultrasound, and the caudal epidural needle was guided successfully to the sacral hiatus and into the caudal epidural space. There was 100% accuracy in caudal epidural needle placement into the caudal epidural space under ultrasound guidance as confirmed by contrast dye fluoroscopy. CONCLUSIONS: Ultrasound is radiation free, is easy to use, and can provide real-time images in guiding the caudal epidural needle into the caudal epidural space. Ultrasound may therefore be used as an adjuvant tool in caudal needle placement.

Ibuprofen inhibition of tendon cell proliferation and upregulation of the cyclin kinase inhibitor p21CIP1.

Sports-related tendon injuries are commonly treated with nonsteroidal antiinflammatory drugs. This study was designed to determine the in vitro effect of ibuprofen on the proliferation of tendon cells intrinsic to rat Achilles tendon. Furthermore, the existence of a correlation between this effect and the expression of the cyclin kinase inhibitor p21(CIP1) and retinoblastoma (Rb) protein was also examined. Using cultured tendon cells, cell viability was evaluated by MTT assay. To determine whether apoptosis was related to the effect of ibuprofen, terminal deoxynucleotidyl transferase nick-end labeling (TUNEL) assay was used. The mitotic index (MI) was calculated from the number of cells in the mitotic phase as stained and identified by propidium iodide. The mRNA expression of p21(CIP1) was determined by reverse transcription-polymerase chain reaction (RT-PCR). Protein expressions of p21(CIP1) and Rb protein were determined by Western blot analysis. A dose-dependent decrease in the cellularity of tendon cells by ibuprofen was demonstrated by MTT assay (p<0.001). However, TUNEL assay revealed no evidence of apoptosis. Ibuprofen dose-dependently reduced the MI (p<0.001). Upregulation of p21(CIP1) both at the levels of mRNA expression and protein was revealed from RT-PCR and Western blot analyses. The inhibition of Rb protein phosphorylation was also noted in ibuprofen-treated cells. In conclusion, ibuprofen inhibits tendon cell proliferation in a process that is probably mediated by the upregulation of p21(CIP1) and reduced phosphorylation of Rb protein.