The let-7f-5p-Nme4 pathway mediates tumor necrosis factor α-induced impairment in osteogenesis of bone marrow-derived mesenchymal stem cells.

Although tumor necrosis factor α (TNF-α)-mediated inflammation significantly impacts osteoporosis, the mechanisms underlying the osteogenic differentiation defects of bone marrow-derived mesenchymal stem cells (BM-MSCs) caused by TNF-α remain poorly understood. We found that TNF-α stimulation of murine BM-MSCs significantly upregulated the expression levels of several microRNAs (miRNAs), including let-7f-5p, but this increase was significantly reversed by treatment with the kinase inhibitor BAY 11-7082. To study gain- or loss of function, we transfected cells with an miRNA inhibitor or miRNA mimic. We then demonstrated that let-7f-5p impaired osteogenic differentiation of BM-MSCs in the absence and presence of TNF-α, as evidenced by alkaline phosphatase and alizarin red staining as well as quantitative assays of the mRNA levels of bone formation marker genes in differentiated BM-MSCs. Moreover, let-7f-5p targets the 3' untranslated region of Nucleoside diphosphate kinase 4 (Nme4) mRNA and negatively regulates Nme4 expression in mouse BM-MSCs. Ectopic expression of Nme4 completely reversed the inhibitory effects of the let-7f-5p mimic on osteogenic differentiation of mouse BM-MSCs. Furthermore, inhibition of let-7f-5p or overexpression of Nme4 in BM-MSCs restored in-vivo bone formation in an ovariectomized animal model. Collectively, our work indicates that let-7f-5p is involved in TNF-α-mediated reduction of BM-MSC osteogenesis via targeting Nme4.

Transforaminal endoscopic decompression for thoracic spinal stenosis under local anesthesia.

BACKGROUND: Thoracic spinal stenosis is a common vertebral degenerative disease, and treatment remains challenging. In recent years, transforaminal endoscopic decompression has been widely used for treating lumbar degenerative diseases. However, the efficacy of this procedure for thoracic spinal stenosis has yet to be established. Herein, we report a case of thoracic spinal stenosis treated with transforaminal endoscopic decompression under local anesthesia. CASE REPORT: An 88-year-old man presented with a 1-month history of progressive paralysis and dysesthesia in the bilateral lower extremities. A diagnosis of thoracic spinal stenosis was made, based on physical examination. A two-step percutaneous transforaminal endoscopic thoracic decompression was performed for spinal canal decompression. Over a follow-up of 1 year, a favorable outcome was noted. CONCLUSION: Transforaminal endoscopic decompression is a safe and an effective surgical approach for the treatment of thoracic spinal stenosis. For patients with thoracic spinal stenosis, accurate diagnosis and elaborate surgical planning should be highlighted, and the surgical outcome can be favorable.

Neuroprotective effects of ginsenoside Rg1 on oxygen-glucose deprivation reperfusion in PC12 cells.

The present study was aimed to investigate the protective effects of ginsenoside Rg1 (GRg1), an important component of ginseng, in oxygen-glucose deprivation/reperfusion (OGDR) and to elucidate the related mechanisms. PC12 cells were used as the model of OGDR. GRg1 administration was started 12 h before OGD and lasted for 12 h. After OGD, the cells were incubated in drug-free and full culture medium under normoxic condition for 24 h. Cell viability was then measured using MTT assay. Cell morphology was studied under a microscope. The expressions of survivin, caspase-3 and Terminal dUTP nick-end labeling (TUNEL) were measured by immunocytology. Results showed that pretreatment with GRg1 significantly increased the viability and survivin expression, and decreased the expressions of caspase-3 and TUNEL in a dose-dependent manner. In addition, it dramatically increased the number of cells and improved the cellular morphology. These results demonstrate the effect of GRg1 in preventing OGDR-induced PC12 cell apoptosis and partly reveal the mechanisms of the protective effect. It is suggested that GRg1 has potential beneficial effects in ischemic diseases.

[Therapeutic effect and mechanism of piracetam for the treatment of spinal cord injury in rats through MAPK pathway].

OBJECTIVE: To explore mechanism of piracetam for the treatment of spinal cord injury in rats through mitogen-activated protein kinase (MAPK) pathway. METHODS: Fifty-four healthy 6-week-old SD female rats with body weight of 80 to 100 g were divided into sham operation group, spinal cord injury group and piracetam group by random number table method, with 18 rats in each group. Spinal cord injury model was established in spinal cord injury group and piracetam group using percussion apparatus, while sham operation group did not damage spinal cord. Piracetam group was injected with piracetam injection through tail vein according to 5 ml·kg-1 standard, once a day for 3 days;the other two groups were injected with normal saline at the same dose, the same frequency and the same duration. The rats were sacrificed at 1, 3, and 7 days after surgery, and changes of Basso, Beattie and Bresnahan (BBB) locomotor rating scale was observed and compared. Enzyme-linked immunosorbent assay (ELISA) was used to detect spinal cord inflammatory factors, such as interleukin-6 (IL-6), interleukin-10 (IL-10), interleukin-1ß (interleukin-1ß), necrosis factor-α (IL-1ß) and tumor necrosis factor-α (TNF-α);HE staining was used to observe morphological changes of rats with spinal cord injury, and immunohistochemistry was used to observe expression level of aquaporin 4 (AQP4). The activation of MAPK signaling pathway in spinal cord of rats after spinal cord injury was observed by western blotting (WB). RESULTS: BBB scores of sham operation group on 1, 3 and 7 day were 21 points. In spinal cord injury group, the scores were (1±1), (4±1) and (7±2);piracetam group was (1±1), (5±1), (9±2), respectively;the difference between spinal cord injury group and sham operation group was statistically significant (P<0.05). HE staining showed that no abnormality was found in sham operation group. In spinal cord injury group, bleeding and degeneration of spinal cord tissue appeared at 1 day after operation; flaky necrotic areas were appeared in spinal cord at 3 days after surgery, and spinal cord tissue began to slowly repair at 7 days after surgery. In piracetam group, the bleeding area was less than that of spinal cord injury group at 1 day after surgery;at 3 days after operation, the necrotic area was reduced and the range of nuclear disappearance was reduced; and the spinal cord began to recover slowly at 7 days after surgery. AQP4 staining of spinal cord of rats in sham operation group was weak at 1, 3 and 7 days after modeling, AQP4 staining was deepened and area increased in spinal cord injury group, AQP4 staining of piracetam group was lighter than that of spinal cord injury group, and the positive cells were slightly increased and the staining was slightly darker than that of sham operation group. At 1, 3 and 7 days, the level of IL-6, IL-10, IL-1ß and TNF-α in spinal cord injury group were higher than those in sham operation group and piracetam group(P<0.05). Compared with spinal cord injury group, the area of spinal cord bleeding and necrosis were decreased by HE staining in piracetam group, and AQP4 staining was decreased by immunohistochemistry. WB results showed that P-ERK, P-JNK and P-P38 levels in spinal cord injury group at 3 days were higher than those in sham operation group and piracetam group(P<0.05). CONCLUSION: Piracetam not only showed significant effect in promoting motor function recovery after spinal cord injury, but also showed positive therapeutic potential in reducing lesion area, regulating AQP4 expression to reduce edema, and reducing inflammatory response by regulating MAPK signaling pathway.

[Development and in vivo biomechanics of goat mobile artificial lumbar spine complex].

OBJECTIVE: Mobile artificial lumbar complex (MALC) which suitable for reconstruction after subtotal lumbar resection in goats was developed,and to test stability of the complex and postoperative lumbar segmental motor function. METHODS: Eighteen male boer goats aged from 1 to 2 years old (weighted from 35 to 45 kg) were selected and divided into control group,fusion group and non-fusion group,with 6 goats in each group. According to preoperative CT scans and MRI examinations of lumbar,the goat MALC was designed and performed by 3D printed for non-fusion group. Operation was performed on three groups respectively,and only vertebral body and disc were exposed in control group. In fusion group,L4 part of vertebral body and the upper and lower complete disc tissues were removed,and the lumbar spine bone plate fixation was performed with titanium mesh bone grafting. In non-fusion group,vertebral body and disc were removed in the same way,and MALC was implanted. AP and lateral X-rays of lumbar vertebrae in goat were taken at 6 months after surgery,in order to understand whether the plant was dislocated,displaced and fractured. Biomechanical tests were performed on the specimens by mechanical instrument to measure range of motion (ROM) of L2,3,L3,4,L4,5 intervertebral space and the overall ROM of L2-5 lumbar vertebrae. RESULTS: MALC of lumbar vertebra was designed by 3D printing,and its component artificial vertebrae and upper and lower artificial end plates were manufactured. The semi-spherical structure was fabricated by precision lathe using high-crosslinked polyethylene material,and the prosthesis was assembled. Postoperative AP and lateral X-rays of lumbar vertebra at 6 months showed the implant position of implant and MALC were good without displacement and dislocation. In vitro biomechanical test of lumbar vertebrae specimens:(1) There were no statistical significance in ROM of lumbar intervertebral space flexion and extension,lateral flexion and rotation on L3,4 and L4,5,between non-fusion group and control group (P>0.05),while ROM of fusion group was significantly reduced compared with the other two groups (P<0.05). There were no significant difference in ROM of L2,3 intervertebral flexion and extension,lateral flexion and rotation between non-fusion group and control group (P>0.05),while fusion group was significantly increased compared with the other two groups (P<0.001). (2) There was no significant difference in overall lumbar ROM of L2-5 (P> 0.05). CONCLUSION: The individual MALC could restore intervertebral height of lumbar vertebra while maintaining the stability of lumbar vertebra and re-establishing motor function of lumbar space.

A novel primary culture method for rat choroidal epithelial cells.

OBJECTIVE: To establish a method for the culture of primary choroidal epithelial cells. METHODS: This descriptive experimental study was carried out in Xi`an Jiaotong University, Xi`an, China from September 2009 to August 2012. Choroidal epithelial cells were isolated from the choroid plexus tissues of the lateral ventricles from neonatal rats (n=36). The tissues were dissociated into small cell aggregates by a mechanical method, and cultured on plastic culture dishes containing Dulbecco`s modified Eagle`s medium with 10% fetal bovine serum and 10 ng/ml epidermal growth factor at 37 degrees C in an incubator with 5% humidified carbon dioxide. The cultured cells were examined by phase contrast microscope, electron microscopy, and immunocytochemistry. RESULTS: The cells showed typical morphologic characteristics of epithelial phenotypes with a cobblestone appearance in monolayer 7-9 days post-seeding. The electron microscopy spotted typical choroidal epithelial cells with microvilli on the cytomembrane, organelles in the cytoplasm, and tight junctions welding 2 adjacent cells. They were positive against anti-transthyretin immunostaining. CONCLUSION: This culture technique, which does not require complex equipment and operation skills, might be a simple and efficient method for obtaining choroidal epithelial cells in sufficient number and purity from mixed primary cultures of rat tissue.

Conspicuity of breast lesions at different b values on diffusion-weighted imaging.

BACKGROUND: Diffusion-weighted (DW) imaging has shown potential to differentiate between malignant and benign breast lesions. However, different b values have been used with varied sensitivity and specificity. This study aims to prospectively evaluate the influence of b value on the detection and assessment of breast lesions. METHODS: Institutional review board approval and informed patient consent were obtained. Between February 2010 and September 2010, sixty women suspected of having breast cancer by clinical examination and mammography underwent bilateral breast MRI and DW imaging (with maximum b values of 600, 800, and 1000 s/mm(2)). Conspicuity grades of lesions at different b values on DW images were performed. Signal intensity and apparent diffusion coefficient (ADC) values were recorded and compared among different b values by the signal-to-noise ratio (SNR), contrast-to-noise ratio (CNR) and receiver operating characteristic (ROC) curve. RESULTS: Fifty-seven lesions from 52 recruited patients including 39/57 (68%) malignant and 18/57 (32%) benign were confirmed with pathology. DCE MRI accurately detected 53 lesions with the sensitivity of 93.0% and specificity of 66.7%, and DW imaging accurately detected 51 lesions with the sensitivity of 89.5% and specificity of 100%. There were no significant differences in conspicuity grades compared among the three b values (P = 0.072), although the SNR and CNR of breast lesions decreased significantly with higher b values. Mean ADCs of malignant lesions (b = 600 s/mm(2), 1.07 ± 0.26 × 10-3 mm(2)/s; b = 800 s/mm(2), 0.96 ± 0.22 × 10-3 mm(2)/s; b = 1000 s/mm(2), 0.92 ± 0.26 × 10-3 mm(2)/s) were significantly lower than those of benign lesions (b = 600 s/mm(2), 1.55 ± 0.40 × 10-3 mm(2)/s; b = 800 s/mm(2), 1.43 ± 0.38 × 10-3 mm(2)/s; b = 1000 s/mm(2), 1.49 ± 0.38 × 10-3 mm(2)/s) with all P values <0.001, but there were no significant differences among the three b values (P = 0.303 and 0.840 for malignant and benign lesions, respectively). According to the area under the ROC curves, which were derived from ADC and differentiate malignant from benign lesions, no significant differences were found among the three b values (P = 0.743). CONCLUSIONS: DW imaging is a potential adjunct to conventional MRI in the differentiation between malignant and benign breast lesions. Varying the maximum b value from 600 to 1000 s/mm(2) does not influence the conspicuity of breast lesions on DW imaging at 1.5 T.

[Research progress of Notch signaling pathway in spinal cord injury].

Spinal cord injury is a severe central nervous system disease, which will cause a series of complex pathophysiological changes and activate a variety of signaling pathways including Notch signaling. Studies have evidenced that activation of the Notch signaling pathway is not conducive to nerve repair and symptom improvement after spinal cord injury. Its mechanisms include inhibiting neuronal differentiation and axon regeneration, promoting reactive astrocyte proliferation, promoting M1 macrophage polarization and the release of proinflammatory factors, and inhibiting angiogenesis. Therefore, it has become a promising therapeutic strategy to inhibit Notch signal as a target in the treatment of spinal cord injury. In recent years, some researchers have used drugs, cell transplantation or genetic modification to regulate Notch signaling, which can promote the recovery of nerve function after spinal cord injury, thereby providing new treatment strategies for the treatment of spinal cord injury. This article will summarize the mechanism of Notch signaling pathway in spinal cord injury, and at the same time review the research progress in the treatment of spinal cord injury by modulating Notch signaling pathway in recent years, so as to provide new research ideas for further exploring new strategies for spinal cord injury.

Lithium promotes recovery after spinal cord injury.

Lithium is associated with oxidative stress and apoptosis, but the mechanism by which lithium protects against spinal cord injury remains poorly understood. In this study, we found that intraperitoneal administration of lithium chloride (LiCl) in a rat model of spinal cord injury alleviated pathological spinal cord injury and inhibited expression of tumor necrosis factor α, interleukin-6, and interleukin 1 ß. Lithium inhibited pyroptosis and reduced inflammation by inhibiting Caspase-1 expression, reducing the oxidative stress response, and inhibiting activation of the Nod-like receptor protein 3 inflammasome. We also investigated the neuroprotective effects of lithium intervention on oxygen/glucose-deprived PC12 cells. We found that lithium reduced inflammation, oxidative damage, apoptosis, and necrosis and up-regulated nuclear factor E2-related factor 2 (Nrf2) and heme oxygenase-1 in PC12 cells. All-trans retinoic acid, an Nrf2 inhibitor, reversed the effects of lithium. These results suggest that lithium exerts anti-inflammatory, anti-oxidant, and anti-pyroptotic effects through the Nrf2/heme oxygenase-1 pathway to promote recovery after spinal cord injury. This study was approved by the Animal Ethics Committee of Xi'an Jiaotong University (approval No. 2018-2053) on October 23, 2018.

[miRNA-181a-5p inhibits proliferation and migration of osteosarcoma cell line HOS by targeting HOXB4].

OBJECTIVE: To study the effects and mechanisms of miR-181a-5p on the proliferation, cycle and migration of HOS osteosarcoma cells. METHODS: Real-time quantitative PCR was used to detect the expression of miR-181a-5p and HOXB4 in osteoblast hFOB1.19 cell line and osteosarcoma cell lines (HOS, U2OS, MG63). miR-181a-5p mimics and miR-181a-5p inhibitors were respectively transfected into HOS cells by Lipofectamine 2000, and miR NC group was set as control group. CCK-8 method was used to detect the change in cell proliferation. Flow cytometry was used to detect the changes in cell cycles. Wound healing experiments and Transwell migration experiments were used to detect the changes in cell migration ability. The target gene of miR-181a-5p was predicted by Targetscan website and validated by Dual-luciferase reporter gene system and Western blot. RESULTS: Compared with osteoblast hFOB1.19, miR-181a-5p was low expressed in osteosarcoma cells HOS, U2OS, and MG63(P<0.05), while HOXB4 was high expressed in osteosarcoma cells HOS, U2OS, and MG63(P<0.05). Compared with the miR NC group, over expression of miR-181a-5p inhibited the proliferation and migration of osteosarcoma HOS cells, and the number of cells in S phase decreased(P<0.05). However, knockdown miR-181a-5p promoted the proliferation and migration of osteosarcoma HOS cells, the cells in S phase increased(P<0.05). Bioinformatics prediction and Dual-luciferase reporter gene system validate HOXB4 as a downstream target gene of miR-181a-5p(P<0.05). Western blot showed that miR-181a-5p over expression or knockdown significantly down-regulated or up-regulated HOXB4 expressions in the HOS cells respectively(P<0.05). CONCLUSION: miR-181a-5p is down expressed in osteosarcoma cells, and over-expression miR-181a-5p inhibits the proliferation, cell cycle and migration ability of osteosarcoma cells by targeting HOXB4.

Change of neural stem cells in the choroid plexuses of developing rat.

Neural stem cells reside in various brain regions. However, neural stem cells in the choroid plexuses are poorly understood. This study was conducted to corroborate the hypotheses that there are neural stem cells in the choroid plexuses, and the change of neural stem cells is age dependent. We examined neural stem cells from rats at postnatal 1, 3, 7 days, 2, 4, 6, and 8 weeks to investigate the distribution and change of the cells in the choroid plexuses. We found nestin-positive cells in the choroid plexuses and these nestin-expressing cells were located principally at the boundary between the choroid plexus epithelium and the underlying connective tissue stroma. Some choroid plexuses of the postnatal 1-, 3-, 7-day, and 2-week rats were stained with line-like markers of nestin. We also observed nestin-positive cells in 4-week rats, but no such cells were detected in the 6- and 8-week rats. These findings indicate that neural stem cells exist in the rat choroid plexuses, and the change of neural stem cells is age-dependent.

[Expression of Semaphorin 3A after spinal cord injury].

OBJECTIVE: To investigate expression of Semaphorin 3A in rats after spinal cord injury and explore possible mechanism of inhibiting of axonal regeneration after SCI. METHODS: Forty healthy female SD rats, 8 weeks old, weighing (210.00±9.88) g, were randomly divided into control group(20 rats in group A) and model group(20 rats in group B). In control group, removal of T10 lamina and partial removal of T9 and T11 lamina were performed, and no further operation was performed on spinal cord (pseudo operation). In model group, the total T10 and partial T9, T11 partial lamina were incised and the spinal cord transection was performed to create animal models of spinal cord injury. The rats were perfused and spinal cord tissue obtained at 3, 7, 14, 28 and 42 days after surgery (4 rats in each group at each time point), respectively, and then HE staining was performed. Meanwhile, the expression of Semaphoring 3A was detected in accordance with the protocol of SP kit. RESULTS: After a simple spinal cord transection injury, hemorrhagic necrosis, localized edema, neurodegeneration, necrosis, and cyst formation occurred in the injured area, and glial scar formation occurred in glial cells. Semaphorin 3A expression levels in control group was low in the gray matter area. There was no expression of Semaphorin 3A in the injured area of spinal cord injury in model group 3 days after operation. On the 14th day, the expression of Semaphorin 3A in the injured area of spinal cord injury increased significantly and was at a high level. On the 28th day, the expression of Semaphorin 3A was moderate. On the 42th day, the positive expression of Semaphorin 3A returned to normal level. CONCLUSION: The increased expression of Semaphorin 3A after spinal cord injury may be one of the mechanisms that inhibit axonal regeneration.

[Advances in olfactory ensheathing cells for the treatment of spinal cord injury].

Spinal cord injury is a highly disabled neurological disease, and there is still a lack of effective treatments. Studies have proved that olfactory ensheathing cells are one of the ideal seed cells for promoting nerve regeneration after spinal cord injury. Olfactory ensheathing cells can promote axonal germination and elongation through secretion, interaction with astrocytes, regulation of inflammatory reaction, migration characteristics, myelination, anti-oxidation, lipid regulation and other channels. Thus olfactory ensheathing cells play the role of neuroprotection and nerve repair. In recent years, some studies have used bioengineering, tissue engineering, reprogramming and other technologies to enhance the efficacy of olfactoryensheathing cells from different aspects, thereby providing new therapeutic strategies for optimizing the cell therapy of spinal cord injury. This article will summarize the mechanism of olfactory ensheathing cells in repairing spinal cord injury, and review the progress of optimizing strategy of olfactory ensheathing cells in treating spinal cord injury recently, so as to provide new research ideas for the further developing the repair potential of olfactory ensheathing cells and optimize the cell therapy effect of spinal cord injury.

Olfactory ensheathing cell transplantation alters the expression of chondroitin sulfate proteoglycans and promotes axonal regeneration after spinal cord injury.

Cell transplantation is a potential treatment for spinal cord injury. Olfactory ensheathing cells (OECs) play an active role in the repair of spinal cord injury as a result of the dual characteristics of astrocytes and Schwann cells. However, the specific mechanisms of repair remain poorly understood. In the present study, a rat model of spinal cord injury was established by transection of T10. OECs were injected into the site, 1 mm from the spinal cord stump. To a certain extent, OEC transplantation restored locomotor function in the hindlimbs of rats with spinal cord injury, but had no effect on the formation or volume of glial scars. In addition, OEC transplantation reduced the immunopositivity of chondroitin sulfate proteoglycans (neural/glial antigen 2 and neurocan) and glial fibrillary acidic protein at the injury site, and increased the immunopositivity of growth-associated protein 43 and neurofilament. These findings suggest that OEC transplantation can regulate the expression of chondroitin sulfate proteoglycans in the spinal cord, inhibit scar formation caused by the excessive proliferation of glial cells, and increase the numbers of regenerated nerve fibers, thus promoting axonal regeneration after spinal cord injury. The study was approved by the Animal Ethics Committee of the Medical College of Xi'an Jiaotong University, China (approval No. 2018-2048) on September 9, 2018.

[A case-control study of cervical spine Key-hole technique and anterior cervical Zero-P system in the treatment of cervical spondylotic radiculopathy].

OBJECTIVE: To explore the short-term clinical efficacy of single-stage cervical spondylotic radiculopathy (CSR) between the minimally invasive Key-hole technique and anterior cervical Zero profile intervertebral fusion system (Zero-P). METHODS: A retrospective analysis was performed on 45 patients who underwent surgical treatment for CSR from January 2017 to January 2020, including 21 in Key hole group (12 males and 9 females), followed up for 10-22(13.2±2.3) months;24 cases in Zero-P group (14 males and 10 females), and the follow up period was 10 to 23(12.7±1.9) months. Perioperative conditions (incision length, intraoperative blood loss, operation time, length of hospital stay, and complications) were compared between two groups, and X-rays of cervical spine before and after surgery and at the final follow-up were taken to analyzed curvature of the cervical spine, visual analogue scale(VAS) of pain before and after surgery, Oswestry Disability Index(ODI) and Japanese Orthopaedic Association (JOA) score of cervical spine were recorded to evaluate clinical efficacy. RESULTS: In Key-hole group and Zero-P group, the surgical incision length, intraoperative blood loss, operation time, final follow-up Cobb angle and immediate postoperative VAS score respectively were (1.2±0.2) cm, (5.3±0.3) cm;(35.3±9.7) ml, (120.2±13.5) ml;(56.4±11.3) min, (90.6±12.6) min;(3.2±3.9)°, (7.3±3.8)°;(2.8±1.2)points, (3.8±1.1) points;the Zero-P group was larger than the Key hole group, with statistical significance(P<0.05) . There were no statistically significant difference in length of hospital stay, ODI and JOA scores between two groups (P>0.05). After the follow-up, 1 case of neurostimulation symptoms in Key-hole group was relieved by conservative treatment, 2 cases improved after reoperation due to recurrence of cervical disc herniation;2 cases of neurostimulation symptoms in Zero-P group, 2 cases of throat discomfort, and 1 case dural tears were all relieved by conservative treatment. CONCLUSION: The cervical spine Key-hole technology is similar to the anterior cervical Zero-P system in the treatment of CSR. The Key-hole technique has certain advantages in incision length, intraoperative blood loss, and operation time. It is a safe, effective and can be widely used cervical spine surgery method.

Lentivirus-mediated knockdown of aggrecanase-1 and -2 promotes chondrocyte-engineered cartilage formation in vitro.

Chondrocyte-based tissue engineering has emerged as a promising approach for repair of injured cartilage tissues that have a poor self-healing capacity. However, this technique faces a major limitation: dedifferentiation of chondrocytes occurs following several passages in culture. Aggrecan, a major component of cartilage extracellular matrix, plays an essential role in chondrocyte differentiation. The aim of this study is to determine whether inhibition of chondrocyte aggrecanases, key degradative enzymes for aggrecan in cartilage, could benefit chondrocyte differentiation and the preservation of chondrocyte phenotype within a long-term period. Lentivirus-mediated RNA interference (RNAi) was employed to target both aggrecanase-1 and -2 in primary rat chondrocytes, and the transduced cells were seeded into chitosan-gelatin three-dimensional scaffolds. Histological, morphological, and biochemical analyses were performed at 1-8 weeks post-implantation to study chondrocyte survival, differentiation, and function. We found that lentivirus-mediated RNAi notably decreased the abundance of aggrecanase transcripts in chondrocytes but did not affect cell viability. Most importantly, compared to the control constructs seeded with untransduced chondrocytes, the aggrecanase inhibition increased chondrocyte proliferation and reinforced the production of glycosaminoglycans and total collagen, indicative of chondrocyte differentiation. The mRNA expression of chondrocyte marker genes (collagen II and aggrecan) was enhanced by aggrecanase silencing relative to the control. Together our data demonstrate that inhibition of endogenous aggrecanases facilitates chondrocyte differentiation and chondrocyte-engineered cartilage formation in vitro. The combination of lentiviral delivery system and genetic manipulation techniques provides a useful tool for modulation of chondrocyte phenotype in cartilage engineering.

Cartilage tissue engineering with demineralized bone matrix gelatin and fibrin glue hybrid scaffold: an in vitro study.

To develop a cartilage-like tissue with hybrid scaffolds of demineralized bone matrix gelatin (BMG) and fibrin, rabbit chondrocytes were cultured on hybrid fibrin/BMG scaffolds in vitro. BMG scaffolds were carefully soaked in a chondrocyte-fibrin suspension, which was polymerized by submerging the constructs into thrombin-calcium chloride solution. Engineered cartilage-like tissue grown on the scaffolds was characterized by histology, immunolocalization, scanning electron microscopy, biochemical assays, and analysis of gene expression at different time points of the in vitro culture. The presence of proteoglycan in the fibrin/BMG hybrid constructs was confirmed by positive toluidine blue and alcian blue staining. Collagen type II exhibited intense immunopositivity at the pericellular matrices. Chondrogenic properties were further demonstrated by the expression of gene-encoded cartilage-specific markers, collagen type II, and aggrecan core protein. The glycosaminoglycan production and hydroxyproline content of tissue grown on the fibrin/BMG hybrid scaffolds were higher than that of the BMG group. In conclusion, the fibrin/BMG hybrid scaffolds may serve as a potential cell delivery vehicle and a structural basis for cartilage tissue engineering.

Increased expression of collagens, transforming growth factor-beta1, and -beta3 in gluteal muscle contracture.

BACKGROUND: Gluteal muscle contracture (GMC) is a multi-factor human chronic fibrotic disease of the gluteal muscle. Fibrotic tissue is characterized by excessive accumulation of collagen in the muscle's extracellular matrix. Transforming growth factor (TGF)-beta1 and -beta2 are thought to play an important role in fibrogenesis, while TGF-beta3 is believed to have an anti-fibrotic function. We hypothesize that the expression of collagen and TGF-betas would be up-regulated in GMC patients. METHODS: The expression of collagen type I, type III and TGF-betas were studied in 23 fibrotic samples and 23 normal/control samples in GMC patients using immunohistochemistry, reverse transcription and polymerase chain reaction (RT-PCR) and western bolt analysis. RESULTS: Compared to the unaffected adjacent muscle, increased expression of TGF-beta1 and -beta3 was associated with deposition of collagen type I and type III in the fibrotic muscle of the GMC patients at the mRNA level. Strong up-regulation of these proteins in fibrotic muscle was confirmed by immunohistochemical staining and western blot analysis. TGF-beta2 was not up-regulated in relation to GMC. CONCLUSION: This study confirmed our hypothesis that collagen types I, III, TGF-beta1 and TGF-beta3 were up-regulated in biopsy specimens obtained from patients with GMC. Complex interaction of TGF-beta1 with profibrotic function and TGF-beta3 with antifibrotic function may increase synthesis of collagens and thereby significantly contribute to the process of gluteal muscle scarring in patients with GMC.

Butylphthalide has an Anti-Inflammatory Role in Spinal Cord Injury by Promoting Macrophage/Microglia M2 Polarization via p38 Phosphorylation.

STUDY DESIGN: An experimental animal study of treatment of spinal cord injury (SCI). OBJECTIVE: This report aims to evaluate the in vivo effects of butylphthalide NBP on SCI biology and to explore its potential mechanism. SUMMARY OF BACKGROUND DATA: SCI causes great damage to humans. The inflammatory and reconstructive processes after SCI is regulated by activation of astroglial and microglial cells. Activated microglia/macrophages can be divided into M2 (anti-inflammatory) and M1 (pro-inflammatory) phenotypes. Butylphthalide (3-n-butylphthalide or NBP) treatment can significantly alleviate ischemic brain damage, and further study has confirmed that central neuroprotective effects can be realized by converting M1 polarized microglia/macrophages to the M2 phenotype. Thus far, it remains unknown whether NBP can modulate the transition of macrophages/microglia between the M1 and M2 phenotypes. METHODS: We randomly divided male mice into three groups (sham group, SCI group, SCI+ NBP group). Molecular and histological tests were performed to detect the macrophage/microglia polarization as well as the potential mechanism of NBP in vivo and in vitro. RESULT: It was found that NBP treatment significantly attenuated the motor dysfunction and neuronal apoptosis induced by SCI. Treatment with NBP could also reduce pro-inflammatory cytokine release after SCI and could facilitate macrophage/microglia M2 polarization and inhibit M1 polarization after SCI. To verify the findings in animal experiments, we examined the effect of NBP on BV2 cell polarization, the results showed that NBP treatment could enhance M2 polarization and inhibit M1 polarization, and that M2 polarization occurred in a p38-dependent manner. CONCLUSION: NBP plays an important role in the anti-inflammatory response in SCI via the facilitation of macrophage/microglia M2 polarization as well as the inhibition of macrophage/microglia M1 polarization. The M2 polarization of macrophages/microglia occurs via activation of p38 pathway. LEVEL OF EVIDENCE: 3.

Artificial atlanto-odontoid joint replacement through a transoral approach.

Resection of the odontoid process and anterior arch of the atlas results in atlantoaxial instability, which if left uncorrected may lead to severe neurological complications. Currently, such atlantoaxial instability is corrected by anterior and/or posterior C1-C2 fusion. However, this results in considerable loss of rotation function of the atlantoaxial complex. From the viewpoint of retaining the rotation function and providing stability, we designed an artificial atlanto-odontoid joint based on anatomical measurements of 50 pairs of dry atlantoaxial specimens by digital calipers and 10 fresh cadaveric specimens by microsurgical techniques. The metal-on-metal titanium alloy joint has an arc-shaped atlas component, and a hollow cylindrical bushing into which fits a rotation axle of an inverted v-shaped axis component and is implanted through a transoral approach. After the joint was implanted onto specimens with anterior decompression, biomechanical tests were performed to compare the stability parameters in the intact state, after decompression, after artificial joint replacement, and after fatigue test. Compared to the intact state, artificial joint replacement resulted in a significant decrease in the range of motion (ROM) and neutral zone (NZ) during flexion, extension, and lateral bending (P < 0.001); however, with regard to axial rotation, there was no significant difference in ROM (P = 0.405), a significant increase in NZ (P = 0.008), and a significant decrease in stiffness (P = 0.003). Compared to the decompressed state, artificial joint replacement resulted in a significantly decreased ROM (P B 0.021) and NZ (P B 0.002) and a significantly increased stiffness (P \ 0.001) in all directions. Following artificial joint replacement, there was no significant difference in ROM (P C 0.719), NZ (P C 0.580), and stiffness (P C 0.602) in all directions before and after the fatigue test. The artificial joint showed no signs of wear and tear after the fatigue test. This artificial atlanto-odontoid joint may be useful in cases of odontoid resection due to malunion or nonunion of odontoid fracture, atraumatic odontoid fracture, irreducible atlas dislocation, posterior atlantoaxial subluxation, or congenital skull base abnormalities.