Minimally invasive transforaminal lumbar interbody fusion by a novel two-medium compatible bichannel endoscopy system, technique note and preliminary clinical results.

PURPOSE: Our team designed a novel two-medium compatible bichannel endoscopy system for spinal surgery, V-shape bichannel endoscopy (VBE) system. Hereby, this study will introduce minimally invasive transforaminal lumbar interbody fusion (TLIF) with VBE system and report its preliminary clinical results. METHODS: Fifty-two participants, who accepted VBE-assisted TLIF surgery (VBE-TLIF) in our hospital were included in this study. The duration of operation, off-bed time, and days of hospitalization were recorded. Besides, the patient's preoperative and postoperative pain were evaluated via visual analog scale (VAS), the functional status was evaluated via Oswestry dysfunction index (ODI) and modified MacNab criteria. Patients were asked to follow-up in the outpatient department at the 3rd, 6th, 12th, and 24th month after surgery. X-ray or CT was examined to evaluate the internal fixation position and interbody fusion result. RESULTS: All patients received unilateral decompression with an average operation duration of 178.49 ± 27.49 min. After the surgery, their VAS score of leg pain and back pain reduced significantly. At the last follow-up, the VAS score of leg pain and back pain was 0.80 ± 0.69 and 0.86 ± 0.75 separately. The difference shows statistically significant with p < 0.05. At the last follow-up, the ODI was 15.20 ± 5.75. According to modified MacNab criteria, 39 patients rated their function as excellent, and 10 patients were good. The overall satisfaction rate reached 94%. CONCLUSION: The VBE system reported in the current study can complete TLIF surgery safely and effectively.

TNF-α promotes osteoclastogenesis through JNK signaling-dependent induction of Semaphorin3D expression in estrogen-deficiency induced osteoporosis.

Tumor necrosis factor α (TNF-α)-induced osteoclast formation have been demonstrated to play an important role in the pathogenesis of estrogen deficiency-mediated bone loss, but the exact mechanisms by which TNF-α enhanced osteoclast differentiation were not fully elucidated. The class III semaphorins members were critical to regulate bone homeostasis. Here, we identified a novel mechanism whereby TNF-α increasing Semaphorin3D expression contributes to estrogen deficiency-induced osteoporosis. In this study, we found that Semaphorin3D expression was upregulated by TNF-α during the process of RANKL-induced osteoclast differentiation. Inhibition of Semaphorin3D in pre-osteoclasts could attenuate the stimulatory effects of TNF-α on osteoclast proliferation and differentiation. Mechanistically, blocking of the Jun N-terminal kinase (JNK) signaling markedly rescued TNF-α-induced Semaphorin3D expression, suggesting that JNK signaling was involved in the regulation of Semaphorin3D expression by TNF-α. In addition, silencing of Semaphorin3D in vivo could alleviate estrogen deficiency-induced osteoporosis. Our results revealed a novel function for Semaphorin3D and suggested that increased Semaphorin3D may contribute to enhanced bone loss by increased TNF-α in estrogen deficiency-induced osteoporosis. Thus, Semaphorin3D may provide a potential therapeutic target for the treatment of estrogen-deficiency induced osteoporosis.

Estrogen inhibits RANKL-induced osteoclastic differentiation by increasing the expression of TRPV5 channel.

The inhibitor effect of estrogen on osteoclasts differentiation is very important in the etiology of estrogen protecting the adult skeleton against bone loss. However, the precise molecular events underlying the effect of estrogen on osteoclasts differentiation are not known. Recent studies implicated an important role of transient receptor potential vanilloid 5 (TRPV5) in osteoclast differentiation and bone resorption. Furthermore, some studies have confirmed that estrogen is involved in the regulation of calcium ion (Ca(2+)) influx in many cells via TRPV5 channel. Therefore, we hypothesize that TRPV5 channel may be implicated in the process of estrogen-inhibited osteoclastogenesis and bone resorption. Western blot, quantitative real-time PCR, tartrate-resistant acid phosphatase (TRAP) staining, and pit formation assay were employed to investigate the role of TRPV5 in estrogen decreasing osteoclast differentiation and bone resorption. We found that the expression of TRPV5 is significantly down-regulated during estrogen deficiency-induced osteoclastogenesis. Furthermore, TRAP staining and pit formation assay showed that the depletion of TRPV5 significantly blocks the inhibitor effects of estrogen on osteoclasts differentiation and bone resorption activity. Further studies confirmed that estrogen regulates the expression of TRPV5 channel via estrogen receptor. Based on these results above, we can draw conclusion that TRPV5 may contribute to the process of estrogen-inhibited osteoclastogenesis and bone resorption activity.

Knockout of TRPV6 causes osteopenia in mice by increasing osteoclastic differentiation and activity.

BACKGROUND: Calcium ion (Ca(2+)) signals are required for osteoclast differentiation. Previous study showed that transient receptor potential vanilloid 5 (TRPV5) is an essential Ca(2+) transporter in osteoclastogenesis and bone resorption. TRPV5 and TRPV6 represent two highly homologous members within the transient receptor potential (TRP) superfamily. However, the role of TRPV6 in bone metabolism is still controversial and little is known about the involvement of TRPV6 in receptor activator of nuclear factor κ-B ligand (RANKL)-induced osteoclastogenesis. METHODS: In our study, gene knockout mice, RNA interference, western blot, quantitative real-time PCR, tartrate-resistant acid phosphatase (TRAP) staining, pit formation assay, histomorphometry and measurement of serum parameters were employed to investigate the role of TRPV6 in bone homeostasis, osteoclastogenesis and bone resorption. RESULTS: We found that TRPV6 depletion results in noticeable destruction of bone microarchitecture in TRPV6 knockout mice (TRPV6(-/-)), suggesting that TRPV6 is a critical regulator in bone homeostasis. Inactivation of Trpv6 had no effect on osteoblastic bone formation. However, quantification of the TRAP staining showed a significantly increased osteoclast number and surface area in the metaphyseal area of femurs bone sections derived from TRPV6(-/-) mice. In agreement with our observations from TRPV6(-/-) mice, TRPV6 depletion in vitro significantly increased osteoclasts differentiation and bone resorption activity. CONCLUSION: Based on these results above, we can draw conclusions that TRPV6 plays an essential role in bone metabolism and is a critical regulator in osteoclasts differentiation and bone resorption.

Glucocorticoid induced osteoblast apoptosis by increasing E4BP4 expression via up-regulation of Bim.

It is well known that glucocorticoid (GC)-induced bone loss is caused primarily by hypofunction and apoptosis of osteoblasts. However, the precise molecular events underlying the effect of GC on osteoblast apoptosis are not fully understood. Recent studies implicated an important role of E4BP4 in the regulation of osteoblast apoptosis and differentiation. Furthermore, E4BP4 is a GC-regulated gene required for GC-induced apoptosis in many cells. Therefore, we hypothesize that E4BP4 may be implicated in the process of GC-induced osteoblast apoptosis. Western blot, reverse-transcription-PCR, flow cytometry, and Hoechst 33258 staining were employed to investigate the role of E4BP4 in dexamethasone (DEX)-induced osteoblast apoptosis. We found that the expression of E4BP4 is significantly up-regulated in osteoblasts exposed to DEX. Furthermore, the depletion of E4BP4 significantly decreased DEX-induced osteoblast apoptosis. In addition, E4BP4 plays a crucial role in GC-evoked apoptosis of osteoblasts by enabling induction of Bim. On the basis of these results above, we can draw the conclusion that E4BP4 may contribute to the process of DEX-induced osteoblast apoptosis.

MicroRNA-224-5p nanoparticles balance homeostasis via inhibiting cartilage degeneration and synovial inflammation for synergistic alleviation of osteoarthritis.

MicroRNAs play a crucial role in regulating cartilage extracellular matrix (ECM) metabolism and are being explored as potential therapeutic targets for osteoarthritis (OA). The present study indicated that microRNA-224-5p (miR-224-5p) could balance the homeostasis of OA via regulating cartilage degradation and synovium inflammatory simultaneously. Multifunctional polyamidoamine dendrimer with amino acids used as efficient vector to deliver miR-224-5p. The vector could condense miR-224-5p into transfected nanoparticles, which showed higher cellular uptake and transfection efficiency compared to lipofectamine 3000, and also protected miR-224-5p from RNase degradation. After treatment with the nanoparticles, the chondrocytes showed an increase in autophagy rate and ECM anabolic components, as evidenced by the upregulation of autophagy-related proteins and OA-related anabolic mediators. This led to a corresponding inhibition of cell apoptosis and ECM catabolic proteases, ultimately resulting in the alleviation of ECM degradation. In addition, miR-224-5p also inhibited human umbilical vein endothelial cells angiogenesis and fibroblast-like synoviocytes inflammatory hyperplasia. Integrating the above synergistic effects of miR-224-5p in regulating homeostasis, intra-articular injection of nanoparticles performed outstanding therapeutic effect by reducing articular space width narrowing, osteophyte formation, subchondral bone sclerosis and inhibiting synovial hypertrophy and proliferation in the established mouse OA model. The present study provides a new therapy target and an efficient intra-articular delivery method for improving OA therapy. STATEMENT OF SIGNIFICANCE: Osteoarthritis (OA) is the most prevalent joint disease worldwide. Gene therapy, which involves delivering microRNAs, has the potential to treat OA. In this study, we demonstrated that miR-224-5p can simultaneously regulate cartilage degradation and synovium inflammation, thereby restoring homeostasis in OA gene therapy. Moreover, compared to traditional transfection reagents such as lipofectamine 3000, G5-AHP showed better efficacy in both microRNA transfection and protection against degradation due to its specific surface structure. In summary, G5-AHP/miR-224-5p was developed to meet the clinical needs of OA patients and the high requirement of gene transfection efficiency, providing a promising paradigm for the future application and development of gene therapy.

TNF-α suppresses osteogenic differentiation of MSCs by accelerating P2Y2 receptor in estrogen-deficiency induced osteoporosis.

Tumor Necrosis Factor-α (TNF-α)-inhibited osteogenic differentiation of mesenchymal stem cells (MSCs) contributes to impaired bone formation, which plays a central role in the pathogenesis of postmenopausal osteoporosis. However, the exact mechanisms of TNF-α-inhibited osteoblast differentiation have not been fully elucidated. Multiple P2 purinoceptor subtypes are expressed in several species of osteoblasts and are confirmed to regulate bone metabolism. The purpose of this study is to investigate whether P2 purinoceptors are involved in TNF-α-inhibited osteoblast differentiation. This study shows TNF-α increased P2Y2 receptor expression in the differentiation of MSCs into osteoblasts in a noticeable manner. Overexpressing or silencing of the P2Y2 receptor either impaired or promoted osteogenic differentiation of MSCs significantly. Silencing of the P2Y2 receptor attenuated the inhibitory effects of TNF-α on osteoblastic differentiation of MSCs. In addition, silencing of the P2Y2 receptor evidently alleviated TNF-α-inhibited MSC proliferation. P2Y2 receptor expression was mechanistically upregulated by TNF-α mainly through extracellular regulated protein kinase (ERK) and c-Jun N-terminal kinase (JNK) signaling pathways. Overall, our results revealed a novel function of the P2Y2 receptor and suggested suppressing the P2Y2 receptor may be an effective strategy to promote bone formation in estrogen deficiency-induced osteoporosis.

Differential Characterization of Two Kinds of Stem Cells Isolated from Rabbit Nucleus Pulposus and Annulus Fibrosus.

Objective. Nucleus pulposus (NP) and annulus fibrosus (AF) are two main components of intervertebral disc (IVD). We aimed to figure out whether NP and AF also contain stem cells and whether these stem cells share common properties with chondrocytes and/or fibroblasts in their phenotypes or whether they are completely different types of cells with different characteristics. Design. The disk cells were isolated from AF and NP tissues of the same lumbar spine of the rabbits. The properties of these disk cells were characterized by their morphology, population doubling time (PDT), stem cell marker expression, and multidifferentiation potential using tissue culture techniques, immunocytochemistry, and RT-PCR. Results. Both disk cells formed colonies in culture and expressed stem cell markers, nucleostemin, Oct-4, SSEA-4, and Stro-1, at early passages. However, after 5 passages, AFSCs became elongated and NPSCs appeared senescent. Conclusion. This study indicated that IVD contains stem cells and the characteristics of AFSCs and NPSCs are intrinsically different. The findings of this study may provide basic scientific data for understanding the properties of IVD cells and the mechanisms of lower back pain.

Tumor necrosis factor alpha suppresses osteogenic differentiation of MSCs by inhibiting semaphorin 3B via Wnt/ß-catenin signaling in estrogen-deficiency induced osteoporosis.

The proinflammatory cytokines, especially tumor necrosis factor alpha (TNF-α), have been shown to inhibit osteogenic differentiation of mesenchymal stem cells (MSCs) and bone formation in estrogen-deficiency-induced osteoporosis, but the mechanisms of TNF-α impaired bone formation remain poorly understood. Semaphorins have been shown to regulate cell growth, cell migration, and cell differentiation in a variety of tissues, including bone tissue. Here, we identified a novel mechanism whereby TNF-α, suppressing Semaphorin3B expression contributes to estrogen-deficiency-induced osteoporosis. In this study, we found that TNF-α could decrease Semaphorin3B expression in osteogenic differentiation of MSCs. Overexpression of Semaphorin3B in MSCs attenuated the inhibitory effects of TNF-α on MSCs proliferation and osteoblastic differentiation. Mechanistically, activation of the Wnt/ß-catenin signaling markedly rescued TNF-α-inhibited Semaphorin3B expression, suggesting that Wnt/ß-catenin signaling was involved in the regulation of Semaphorin3B expression by TNF-α. Taken together, our results revealed a novel function for Semaphorin3B and suggested that suppressed Semaphorin3B may contribute to impaired bone formation by elevated TNF-α in estrogen-deficiency-induced osteoporosis. This study may indicate a therapeutic target gene of Semaphorin3B for osteoporosis.

Effects of RNA interference-mediated knockdown of livin and survivin using monomethoxypolyethylene glycol-chitosan nanoparticles in MG-63 osteosarcoma cells.

MG-63 human osteosarcoma cells were transfected with short hairpin RNA (shRNA) against livin and survivin using monomethoxypolyethylene glycol­chitosan (mPEG­CS) nanoparticles (NPs) as carriers, with the aim of evaluating the effect on cell proliferation and apoptosis. mPEG­CS NPs sized ~100 nm were prepared by ionic crosslinking. mPEG­CS­livin shRNA, mPEG­CS­survivin shRNA and mPEG­CS­(livin shRNA + survivin shRNA) NPs were constructed by electrostatic adsorption at NP suspension/gene solution ratios of 3:1 to transfect MG­63 cells. The expression levels of livin and survivin mRNA and protein were measured by reverse transcription­polymerase chain reaction and western blotting, respectively. The inhibitory effects of downregulated livin and survivin expression on cell proliferation were measured using an MTT assay. The apoptosis­inducing effects of livin and surivin knockdown were investigated using a Hoechst staining kit. All shRNA groups resulted in reduced expression of livin and survivin mRNA and protein in MG­63 cells. The MTT assay and Hoechst staining indicated that simultaneous knockdown of livin and survivin genes inhibited the proliferation of MG­63 cells and promoted their apoptosis, to a greater extent than knocking down either gene individually. The simultaneous interference mediated by mPEG­CS NPs significantly reduced livin and survivin expression in MG­63 cells, suppressed proliferation and facilitated apoptosis, to a greater extent than knockdown of either livin or survivin alone were. Thus the results indicate a synergistic effect of livin and survivin.

Comparison of plate-cage construct and stand-alone anchored spacer in the surgical treatment of three-level cervical spondylotic myelopathy: a preliminary clinical study.

BACKGROUND CONTEXT: Although stand-alone cages were advocated to be superior to plate-cage construct (PCC) because of comparable clinical outcomes and fewer plate-related complications, cage dislocation and subsidence were frequently mentioned in multilevel fusion. There are some concerns about whether these issues can be effectively prevented in multilevel anterior cervical discectomy and fusion (ACDF) by stand-alone anchored spacer (SAAS). PURPOSE: The aim was to compare clinical outcomes, radiologic parameters, and complications of PCC and SAAS in the treatment of three-level cervical spondylotic myelopathy (CSM). STUDY DESIGN/SETTING: This was a retrospective comparative study. PATIENT SAMPLE: A total of 38 consecutive patients with three-level CSM (ACDF with PCC, 20 patients; ACDF with SAAS, 18 patients) were reviewed. OUTCOME MEASURES: Clinical outcomes were assessed using Japanese Orthopaedic Association and Neck Disability Index. The radiologic evaluations included cervical alignment (CA), segmental angle (SA), postoperative curvature loss (PCL), and incidence of subsidence. METHODS: All the aforementioned parameters were compared before and after surgery between two groups. Besides, the aforementioned results were also compared between the two groups. The complications were also recorded. RESULTS: The mean follow-up period was 30.3 months. No significant differences were observed in clinical outcomes between the two groups (p>.05). Additionally, no significant differences existed in fusion rate between the two groups. There were significant differences in PCL of SA and CA and correction of SA between the two groups (p<.05). Besides, the incidence of subsidence (9 of 54 levels, 16.7%) was recorded in the SAAS group, and the potential of SAAS to reduce the incidence of postoperative dysphagia was not proven. No other complications were observed in this study. CONCLUSIONS: In the surgical treatment of three-level CSM, PCC is superior to SAAS in correction and maintenance of SA and avoiding cage subsidence, although the technique of ACDF with SAAS yielded encouraging clinical outcomes and high fusion rate.