Controlled Release of TGF-ß3 for Effective Local Endogenous Repair in IDD Using Rat Model.

Introduction: Intervertebral disc (IVD) degeneration (IDD) is one of the most widespread musculoskeletal diseases worldwide and remains an intractable clinical challenge. Currently, regenerative strategies based on biomaterials and biological factors to facilitate IVD repair have been widely explored. However, the harsh microenvironment, such as increased ROS and acidity, of the degenerative region impedes the efficiency of IVD repair. Here, an intelligent biodegradable nanoplatform using hollow manganese dioxide (H-MnO2) was developed to modulate the degenerative microenvironment and release transforming growth factor beta-3 (TGF-ß3), which may achieve good long-term therapeutic effects on needle puncture-induced IDD. Methods: Surface morphology and elemental analysis of the MnO2 nanoparticles (NPs) were performed by transmission electron microscopy and an energy-dispersive X-ray spectroscopy detector system, respectively. The biological effects of MnO2 loaded with TGF-ß3 (TGF-ß3/MnO2) on nucleus pulposus cells (NPCs) were assessed via cytoskeleton staining, EdU staining, qPCR and immunofluorescence. The efficacy of TGF-ß3/MnO2 on needle puncture-induced IDD was further examined using MRI and histopathological and immunohistochemical staining. Results: The MnO2 NPs had a spherical morphology and hollow structure that dissociated in the setting of a low pH and H2O2 to release loaded TGF-ß3 molecules. In the oxidative stress environment, TGF-ß3/MnO2 was superior to TGF-ß3 and MnO2 NPs in the suppression of H2O2-induced matrix degradation, ROS, and apoptosis in NPCs. When injected into the IVDs of a rat IDD model, TGF-ß3/MnO2 was able to prevent the degeneration and promote self-regeneration. Conclusion: Use of an MnO2 nanoplatform for biological factors release to regulate the IDD microenvironment and promote endogenous repair may be an effective approach for treating IDD.

[Expression of B-cell lymphoma-2 protein multisite phosphorylation in autophagy after spinal cord injury in rats].

OBJECTIVE: To investigate the changes of autophagy after spinal cord injury (SCI) in rats and its relationship with multisite phosphorylation of B-cell lymphoma-2 (Bcl-2) protein. METHODS: Forty male Sprague-Dawley rats aged 8 weeks were used to prepare SCI models by modified Allen method, and the SCI model were prepared successfully in 36 rats. The 36 SCI models were randomly divided into SCI group, autophagy inhibitor group, and autophagy promoter group, with 12 rats in each group. Another 12 rats were selected as sham operation group with only laminectomy and no spinal cord injury. At the end of modeling, the autophagy inhibitor group and the autophagy promoter group were intrathecally injected with 20 µL of 600 nmol/L 3-methyladenine and 25 nmol/L rapamycin, respectively, once a day for 4 weeks. The sham operation group and the SCI group were injected with only 20 µL of normal saline at the same time point. The motor function of rat in each group was evaluated by the Basso-Beattie-Bresnahan (BBB) score at 1 day and 1, 2, 4 weeks after modeling. The rats in each group were sacrificed at 24 hours after the last injection and the spinal cord tissues were taken. ELISA assay was used to detect the levels of inflammatory factors in spinal cord tissues, including myeloperoxidase (MPO), tumor necrosis factor α (TNF-α), and interleukin 1ß (IL-1ß); the morphological changes of spinal cord were observed by HE staining; the autophagy of mitochondria in spinal cord tissues was observed by transmission electron microscopy; the expressions of Beclin1 and microtubule-associated protein light chain 3 (LC3) were detected by immunofluorescence staining; neuronal apoptosis in spinal cord tissues were observed by TUNEL staining; LC3/TUNEL positive cells were calculated by immunofluorescence double staining; the expressions of Bcl-2 associated X protein (Bax), Bcl-2, p-Bcl-2 (Ser87), and p-Bcl-2 (Ser70) were detected by Western blot. RESULTS: Compared with sham operation group, BBB score of SCI group decreased at each time point, while the levels of MPO, TNF-α, and IL-1ß increased; peripheral space of nerve cells enlarged, cells swelled, vacuoles appeared, and autophagic bodies appeared in mitochondria; the positive rates of Beclin1 and LC3 proteins, and apoptotic rate of neurons significantly increased; the LC3/TUNEL positive cells significantly increased; the expressions of Bax, p-Bcl-2 (Ser87), and p-Bcl-2 (Ser70) proteins increased, while the expression of Bcl-2 protein decreased; all showing significant differences ( P<0.05). Compared with SCI group, BBB score in autophagy inhibitor group decreased at each time point, while the levels of MPO, TNF-α, and IL-1ß increased; a few autophagic vesicles appeared in mitochondria; the positive rates of Beclin1 and LC3 proteins decreased and the apoptotic rate of neurons increased significantly; the LC3 positive cells decreased and the TUNEL positive cells increased; the expressions of Bax, p-Bcl-2 (Ser87), and p-Bcl-2 (Ser70) proteins increased, while the expression of Bcl-2 protein decreased. The results of autophagy promoter group were opposite to those of autophagy inhibitor group; all showing significant differences between groups ( P<0.05). CONCLUSION: Induction of autophagy after SCI in rats can reduce neuronal apoptosis and protect spinal cord function, which may be related to the inhibition of Bcl-2 protein multisite phosphorylation.

[The role and mechanism of S100 calcium binding protein B in osteoarthritis cartilage damage repair].

Objective: To investigate the role and mechanism of S100 calcium binding protein B (S100B) in osteoarthritis (OA) cartilage damage repair. Methods: Twenty New Zealand rabbits were randomly divided into control group and model group, with 10 rabbits in each group. Rabbits in the model group were injured by the right knee joint immobilization method to make the artilage injury model, while the control group did not deal with any injury. After 4 weeks, the levels of interleukin-1ß (IL-1ß) and tumor necrosis factor α (TNF-α) in synovial fluid were detected by ELISA method; the mRNA and protein expressions of S100B, fibroblast growth factor 2 (FGF-2), and FGF receptor 1 (FGFR1) in cartilage tissue were examined by real-time fluorescence quantitative PCR (qRT-PCR) and Western blot assay. Human synovial fibroblasts (SF) were isolated and cultured in vitro. The effects of S100B overexpression and knockdown on the levels of IL-1ß and TNF-α (ELISA method) and the expressions of FGF-2 and FGFR1 gene (qRT-PCR) and protein (Western blot) were observed. Moreover, the effects of FGFR1 knockdown in above S100 overexpression system on the levels of IL-1ß and TNF-α (ELISA method) and the expressions of FGF-2 and FGFR1 gene (qRT-PCR) and protein (Western blot) were observed. Results: ELISA detection showed that the expressions of IL-1ß and TNF-α in the synovial fluid of the model group were significantly higher than those of the control group ( P<0.05); qRT-PCR and Western blot detection showed that the mRNA and protein expressions of S100B, FGF-2, and FGFR1 in cartilage tissue were significantly higher than those of the control group ( P<0.05). Overexpression and knockdown S100 could respectively significantly increase and decrease lipopolysaccharides (LPS) induced IL-1ß and TNF-α levels elevation and the mRNA and protein expressions of FGF-2 and FGFR1 ( P<0.05); whereas FGFR1 knockdown could significantly decrease LPS induced IL-1ß and TNF-α levels elevation and the mRNA and protein expressions of FGF-2 and FGFR1 ( P<0.05). Conclusion: S100B protein can regulate the inflammatory response of SF and may affect the repair of cartilage damage in OA, and the mechanism may be related to the activation of FGF-2/FGFR1 signaling pathway.

S100B regulates inflammatory response during osteoarthritis via fibroblast growth factor receptor 1 signaling.

The present study aimed to investigate the role of S100B in the inflammation process during osteoarthritis (OA). OA cartilage samples were collected for S100B expression analysis. S100B expression levels were significantly increased in patients with OA compared with the Controls (1.28±0.66 vs. 0.42±0.31; P=0.01) and were determined to be correlated with TNF­α (r=0.42; P=0.04) and IL­1ß (r=0.73; P=0.001) expression levels. Orthopedic casting tape was used to immobilize the right knee at 180˚ extension of adult female New Zealand white rabbits for 4 weeks to establish an OA model. Cartilage specimens from the medial femoral condyle of these rabbits were used for histological confirmation and immunohistochemical analyses, whereas synovial fluid was used in ELISA assays for tumor necrosis factor (TNF)­α and interleukin (IL)­1ß expression levels. Human synovial fibroblasts from the knee synovial tissues of normal patients with traumatic injury were transfected with S100B overexpression and knockdown plasmids and subjected to lipopolysaccharide (LPS) stimulation; subsequently, TNF­α and IL­1ß expression levels in conditioned medium were determined by ELISA; S100B overexpression and knockdown significantly increased and decreased the TNF­α and IL­1ß expression levels, respectively. Increased TNF­α (573.3±15.4 vs. 102.6±8.7 pg) and IL­1ß (378.6±7.2 vs. 170.1±5.8 pg) expression levels were detected in OA model rabbits compared with the Control rabbits. Additionally, S100B, fibroblast growth factor (FGF)­1 and FGF receptor (FGFR)­1 mRNA and protein expression levels were increased in OA model rabbits compared with the Control group. FGFR1 knockdown significantly decreased TNF­α and IL­1ß expression levels in LPS­stimulated S100B­overexpressing human synovial fibroblasts. S100B is involved in FGFR1 signaling­mediated inflammatory response during OA, which may be considered as a potential therapeutic target.

Application value of expansive pedicle screw in the lumbar short-segment fixation and fusion for osteoporosis patients.

Clinical value of expansive pedicle screw in lumbar short-segment fixation and fusion for patients with osteoporosis was investigated. A total of 80 patients with lumbar compression fracture but without obvious nerve compression were selected and divided into the observation group (n=40) and the control group (n=40) using a random number table. The observation group used the expansive pedicle screw, and the control group received conventional pedicle screw fixation and bone graft fusion. In the observation group, the operation and hospitalization time after operation were shorter and the intraoperative bleeding amount was less than that in control group (p<0.05). At 1 week, 1, 3 and 6 months after operation, the observation group had better straight leg raising test (SLRT) scores, higher lower limb sensory scores but lower visual analogue scale (VAS) scores than control group (p<0.05). Besides, the proportions of postoperative infection, dural mater tear, nerve root injury and spinal cord injury during operation in the observation group were lower than those in the control group (p<0.05), and the bone graft fusion rates at 3 and 6 months after operation were obviously superior to those in control group (p<0.05). Moreover, after operation, the spinal stenosis rate in the observation group was lower than that in control group (p<0.05), the vertebral height ratio was larger than that in control group (p<0.05), and the Cobb's angle was smaller than that in the control group (p<0.05). In addition, there was a negative correlation between bone mineral density (BMD) and hospitalization time after operation in the observation group (p<0.05). In conclusion, the internal fixation with expansive pedicle screw for osteoporosis patients with lumbar compression fracture is characterized by short operation time, less intraoperative bleeding, few complications, quick recovery of postoperative neurological function and satisfactory surgical effect. However, reasonable intervention in osteoporosis is also necessary.

Three-dimensional morphological analysis of acromioclavicular joint in patients with and without subacromial erosion after hook plate fixation.

Objective To investigate the role of acromioclavicular joint morphology in the presence of subacromial erosion after hook plate fixation. Methods We retrospectively analyzed the clinical data of 36 patients (17 men, 19 women; mean age, 48.7 years; range, 21-76 years) treated with hook plate fixation for distal clavicular fractures (n = 20) or acromioclavicular joint dislocation (n = 16) from August 2011 to March 2013. The patients were divided into two groups: the subacromial erosion group (18 patients) and the normal group (18 patients). Differences in multiple anatomical parameters between the two groups were measured and compared. Results The distal clavicle-acromion angle was significantly larger in the subacromial erosion group (mean, 51.37° ± 5.59°) than in the normal group (mean, 44.20° ± 3.83°), as was the distal clavicle-coronal angle (mean, 25.44° ± 2.51° vs. 21.67° ± 4.06°, respectively). The thickness of the acromion was significantly different between men and women (9.72 ± 1.13 vs. 8.16 ± 1.89 mm, respectively). Conclusion The results of this study indicate that the distal clavicle-acromion angle and distal clavicle-coronal angle are closely correlated with the occurrence of subacromial erosion after hook plate fixation.

Oxidized low density lipoprotein facilitates tumor necrosis factor­α mediated chondrocyte death via autophagy pathway.

We aimed to investigate the role of oxidized low density lipoprotein (ox­LDL) in tumor necrosis factor­α (TNF­α) mediated chondrocyte death and explore the mechanisms. Ten osteoarthritis (OA) and normal control cartilage tissue and synovial fluid (SF) samples were collected, and the expression of lectin­like ox­LDL receptor­1 (LOX­1) and ox­LDL level was examined by real time quantitative PCR and enzyme­linked immunosorbent assay (ELISA). An in vitro chondrocyte model was established by the introduction of TNF­α and ox­LDL, cell death was analyzed by trypan blue assay and the mechanisms were explored based on the apoptosis related pathway and autophagy pathway. Significantly increased ox­LDL level (70.30±17.83 vs. 37.22±19.97, P<0.05) in SF sample and LOX­1 expression level (0.51±0.10 vs. 0.32±0.04, P<0.05) in cartilage tissue was found in OA patients compared to those corresponding samples from control subjects. Ox­LDL could facilitate TNF­α mediated chondrocyte death and this effect could be blocked by LOX­1 antibody neutralization. Autophagy inhibition by 3­MA and Atg­5 siRNA could reverse the cell death effect mediated by TNF­α and ox­LDL co­treatment on chondrocytes. Oxidized low density lipoprotein facilitates tumor necrosis factor­α mediated chondrocyte death via its interaction with LOX­1, and autophagy is involved in the mechanisms.

[Impact of joint capsule repair and external rotators suture on prognosis in primary total hip arthroplasty by posterolateral approach].

OBJECTIVE: To investigate the impact of joint capsule repair and external rotators suture on the prognosis in primary total hip arthroplasty (THA) by posterolateral approach. METHODS: Between January 2006 and June 2009, 159 patients with femoral neck fracture underwent primary THA by posterolateral approach, and were divided into 4 groups according to different treatments: joint capsule repair and external rotators suture were given in group A (n=38), only joint capsule repair in group B (n=39), only external rotators suture in group C (n=41), and no joint capsule repair or external rotators suture in group D (n=41). There was no significant difference in gender, age, cause of injure, disease duration, type of fracture, combined medical disease, or prosthesis selection among 4 groups (P > 0.05). The bleeding volume, drainage, postoperative hip dislocation rate, hip Harris score, and the hip range of motion (ROM) in internal rotation and external rotation were compared. RESULTS: There was no significant difference in operative time, bleeding volume, or drainage among 4 groups (P > 0.05). Postoperative hip dislocation occurred in 0, 0, 4 (9.8%), and 4 (9.8%) cases of groups A, B, C, and D, respectively, showing significant difference in incidence of postoperative hip dislocation among 4 groups (chi2=7.910, P=0.048). The hip Harris scores were significantly improved after operation when compared with preoperative scores in 4 groups (P < 0.05). Significant differences were found in hip Harris score at 6 weeks and 6 months after operation among 4 groups (P < 0.05); group D was significantly lower than groups A, B, and C, and groups B and C were significantly lower than group A (P < 0.05). There was no significant difference in the hip ROM in internal rotation among 4 groups at 6 weeks and 6, 12 months after operation (P > 0.05); but the hip ROM in external rotation were significantly bigger in groups A and C than in groups B and D at 6 weeks and 6 months after operation (P < 0.05). CONCLUSION: Joint capsule repair and external rotators suture in primary THA by posterolateral approach do not increase the bleeding volume and drainage, but can reduce the early postoperative hip dislocation risk, increase the Harris score, and recover the external rotation function of involved hip. So joint capsule and external rotators should be repaired in THA by posterolateral approach.