Identification of Biomarkers, Pathways, Immune Properties of Mitophagy Genes, and Prediction Models for Intervertebral Disc Degeneration.

Background: Intervertebral disc degeneration (IDD) is the leading cause of low back pain (LBP). The mechanism of IDD development and progression is not fully understood. Peripheral biomarkers are increasingly vital non-radioactive methods in early detection and diagnosis for IDD. Nevertheless, less attention has been paid to the role of mitophagy genes in the progress of IDD. This study aimed to identify the mitophagy disease-causing genes in the process of IDD and mitophagy diagnostic biomarkers for IDD. Methods: Mitophagy-related differentially expressed genes (MRDEGs) related to IDD were investigated by analyzing the microarray datasets of IDD cases from GEO, PathCards and Molecular Signatures Databases. We used R software, WGCNA, PPI, mRNA-miRNA, mRNA-TF, GO, KEGG, GSEA, GSVA and Cytoscape to analyze and visualize the data. We further used ssGSEA for immunoinfiltration analysis to obtain different immune cell infiltration. LASSO model was developed to screen for genes that met the diagnostic gene model requirements. Finally, qRT-PCR, Western blotting and HE were used to verify hub genes and their expression from clinical IDD samples. Results: We identified 14 MRDEGs and 12 hub genes. GO, KEGG, GSEA and GSVA analyses demonstrated that hub genes were critical for the development of IDD. LASSO diagnostic model consisted of six hub genes, among which SQSTM1, ATG7 and OPTN were significantly different between the two IDD disease subtypes. At the same time, SQSTM1 also had a high correlation with immune characteristic subtypes. The results of qRT-PCR and Western blotting also indicated that these genes were significantly differentially expressed in nucleus pulposus cells (NPCs) of the IDD group. Conclusion: We explored an association between MRDEGs-associated signature in IDD and validated that hub genes like SQSTM1 might serve as biomarkers for diagnostic and therapeutic targets for IDD. Meanwhile, this study can provide new insights into the functional characteristics and mechanism of mitophagy in the development of IDD.

Effects of Pretreatment of the Dorsal Meningovertebral Ligaments on the Incidence of Intraoperative Cerebrospinal Fluid Leakage: A Comparative Study.

Cerebrospinal fluid leakage can lead to postoperative refractory headaches and meningitis. Dural injury is the main cause of postoperative cerebrospinal fluid leakage. Previously, we performed a comprehensive anatomic study on the dorsal meningovertebral ligaments in the lumbosacral regions and concluded that these ligaments are an anatomic factor leading to dural laceration. However, no clinical study has examined the relationship between dorsal meningovertebral ligaments and the incidence of intraoperative cerebrospinal fluid leakage. The goal of this study was to investigate the effect of prophylactic intraoperative pretreatment of the meningovertebral ligaments on the incidence of cerebrospinal fluid leakage during surgery. [Orthopedics. 2023;46(1):e66-e71.].

Tumor necrosis factor-α inhibition restores matrix formation by human adipose-derived stem cells in the late stage of chondrogenic differentiation.

BACKGROUND: Cartilage tissue engineering is a promising strategy for treating cartilage damage. Matrix formation by adipose-derived stem cells (ADSCs), which are one type of seed cell used for cartilage tissue engineering, decreases in the late stage of induced chondrogenic differentiation in vitro, which seriously limits research on ADSCs and their application. AIM: To improve the chondrogenic differentiation efficiency of ADSCs in vitro, and optimize the existing chondrogenic induction protocol. METHODS: Tumor necrosis factor-alpha (TNF-α) inhibitor was added to chondrogenic culture medium, and then Western blotting, enzyme linked immunosorbent assay, immunofluorescence and toluidine blue staining were used to detect the cartilage matrix secretion and the expression of key proteins of nuclear factor kappa-B (NF-κB) signaling pathway. RESULTS: In this study, we found that the levels of TNF-α and matrix metalloproteinase 3 were increased during the chondrogenic differentiation of ADSCs. TNF-α then bound to its receptor and activated the NF-κB pathway, leading to a decrease in cartilage matrix synthesis and secretion. Blocking TNF-α with its inhibitors etanercept (1 µg/mL) or infliximab (10 µg/mL) significantly restored matrix formation. CONCLUSION: Therefore, this study developed a combination of ADSC therapy and targeted anti-inflammatory drugs to optimize the chondrogenesis of ADSCs, and this approach could be very beneficial for translating ADSC-based approaches to treat cartilage damage.

Association between Mean Corpuscular Hemoglobin and Platelet Distribution Width Ratio and Knee Osteoarthritis Severity.

BACKGROUND: The goal was to simply and efficiently predict the indicators of disease severity in knee osteoarthritis (KOA) patients. METHODS: One hundred eighty-four patients with KOA and 126 healthy subjects were included. WOMAC (Western Ontario and McMaster Universities Osteoarthritis Index) was used as a reference index for disease severity in KOA patients, in which WOMAC < 80 was classified as mild and WOMAC ≥ 80 as moderate and severe. Blood routine parameters of the KOA and the healthy groups were analyzed by the Mann Whitney U test. Receiver operating characteristic curves were used to analyze the sensitivity and specificity of mean corpuscular hemoglobin and platelet distribution width ratio (MPR) and monocyte and hemoglobin ratio (MHR) indicators. The correlation between MPR and MHR and disease severity of KOA was determined by bivariate regression analysis. Independent predictors of disease severity in patients with KOA were assessed by multivariate regression analysis. RESULTS: MPR, MHR, and WOMAC were significantly higher in the KOA group. The ROC curve indicated that the cutoff values of MPR and MHR were 2.09 and 0.0030, respectively, with sensitivity of 86.4% and 68.5% and specificity of 99.2% and 79.4%. Bivariate regression analysis found that MPR was better correlated with disease severity than MHR. The results of multivariate regression analysis demonstrated that the MPR values of moderate and severe patients were more than 19 times that of mild patients, and the OR values were 21.695 and 19.558, respectively. CONCLUSIONS: MPR and MHR demonstrated a good correlation with disease severity in patients with KOA. MPR is a potential independent predictor of disease severity in patients with KOA.

Biomechanical effects of an oblique lumbar interbody fusion combined with posterior augmentation: a finite element analysis.

BACKGROUND: Oblique lateral interbody fusion (OLIF) is widely used to treat lumbar degenerative disc disease. This study aimed to evaluate the biomechanical stability of OLIF, OLIF including posterior pedicle screw and rod (PSR), and OLIF including cortical screw and rod (CSR) instrumentation through finite element analysis. METHODS: A complete L2-L5 finite element model of the lumbar spine was constructed. Surgical models of OLIF, such as stand-alone, OLIF combined with PSR, and OLIF combined with CSR were created in the L3-L4 surgical segments. Range of motion (ROM), end plate stress, and internal fixation peak stress were compared between different models under the same loading conditions. RESULTS: Compared to the intact model, ROM was reduced in the OLIF model under all loading conditions. The surgical models in order of increasing ROM were PSR, CSR, and stand-alone; however, the difference in ROM between BPS and CSR was less than 0.4° and was not significant under any loading conditions. The stand-alone model had the highest stress on the superior L4 vertebral body endplate under all loading conditions, whereas the end plate stress was relatively low in the BPS and CSR models. The CSR model had the highest internal fixation stress, concentrated primarily at the end of the screw. CONCLUSIONS: OLIF alone significantly reduces ROM but does not provide sufficient stability. Addition of posterior PSR or CSR internal fixation instrumentation to OLIF surgery can significantly improve biomechanical stability of the segment undergoing surgery.

Comprehensive bioinformatics analysis reveals the hub genes and pathways associated with multiple myeloma.

PURPOSE: While the prognosis of multiple myeloma (MM) has significantly improved over the last decade because of new treatment options, it remains incurable. Aetiological explanations and biological targets based on genomics may provide additional help for rational disease intervention. MATERIALS AND METHODS: Three microarray datasets associated with MM were downloaded from the Gene Expression Omnibus (GEO) database. GSE125364 and GSE39754 were used as the training set, and GSE13591 was used as the verification set. The differentially expressed genes (DEGs) were obtained from the training set, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed to annotate their functions. The hub genes were derived from the combined results of a protein-protein interaction (PPI) network and weighted gene coexpression network analysis (WGCNA). The receiver operating characteristic (ROC) curves of hub genes were plotted to evaluate their clinical diagnostic value. Biological processes and signaling pathways associated with hub genes were explained by gene set enrichment analysis (GSEA). RESULTS: A total of 1759 DEGs were identified. GO and KEGG pathway analyses suggested that the DEGs were related to the process of protein metabolism. RPN1, SEC61A1, SPCS1, SRPR, SRPRB, SSR1 and TRAM1 were proven to have clinical diagnostic value for MM. The GSEA results suggested that the hub genes were widely involved in the N-glycan biosynthesis pathway. CONCLUSION: The hub genes identified in this study can partially explain the potential molecular mechanisms of MM and serve as candidate biomarkers for disease diagnosis.

Surgical safe zones for oblique lumbar interbody fusion of L1-5: A cadaveric study.

To evaluate the operating range and morphology of the surgical safe zone for oblique lumbar interbody fusion (OLIF). Twenty embalmed full-torso cadaveric specimens were dissected. The oblique corridor and the distance between adjacent lumbar arteries were measured in a static state and with psoas major retraction. The morphology and size of the safe zone for OLIF and the location of the lumbar sympathetic trunk were also recorded. The oblique corridor of the L1-L5 segments was significantly greater in the retracted state than in the static state (p < 0.05). With psoas major retraction, the distances between adjacent lumbar arteries at L1-4 were significantly greater (p < 0.05) than those in the static state. The lumbar sympathetic trunk is just located in the safe zone and travels downward adjacent to the psoas major. The shape of the safe zone for OLIF was approximately an oblique upward parallelogram at L1/2 and L2/3, an isosceles trapezoid at L3/4, and an irregular quadrangle or triangle at L4/5. The safe zone for OLIF at L1/2, L2/3, and L3/4 was significantly larger during retraction than in the static state (p < 0.05). On the lateral side of the lumbar spine there is a natural surgical safe zone for OLIF, which can provide a sufficient operating space. The safe zone has a certain morphological pattern in L1-5 segments and psoas major retraction can significantly enlarge it.

Matrix Metalloproteinase 3: A Promoting and Destabilizing Factor in the Pathogenesis of Disease and Cell Differentiation.

Cells must alter their expression profiles and morphological characteristics but also reshape the extracellular matrix (ECM) to fulfill their functions throughout their lifespan. Matrix metalloproteinase 3 (MMP-3) is a member of the matrix metalloproteinase (MMP) family, which can degrade multiple ECM components. MMP-3 can activate multiple pro-MMPs and thus initiates the MMP-mediated degradation reactions. In this review, we summarized the function of MMP-3 and discussed its effects on biological activities. From this point of view, we emphasized the positive and negative roles of MMP-3 in the pathogenesis of disease and cell differentiation, highlighting that MMP-3 is especially closely involved in the occurrence and development of osteoarthritis. Then, we discussed some pathways that were shown to regulate MMP-3. By writing this review, we hope to provide new topics of interest for researchers and attract more researchers to investigate MMP-3.

Structure of Lumbar Intervertebral Foraminal Ligaments Based on 3-Dimensional Reconstruction Through Ultrathin Cryomilling of a Human Cadaver.

BACKGROUND: This study aimed to assess the feasibility of using the 3-dimensional (3-D) reconstruction technique based on ultrathin cryomilling to show the lumbar intervertebral foraminal ligaments in situ. METHODS: Cryomilling was performed on an embalmed human cadaver to acquire successive cross-sectional images. In each of the images, the boundaries of lumbar intervertebral foraminal ligaments and their adjacent structures were outlined, labeled, and reconstructed for 3-D modeling. The morphology, attachments, and spatial orientation of ligaments were described. RESULTS: A total of 9 ligaments in 10 lumbar intervertebral foramina (IVFs) were identified and reconstructed. These ligaments can be divided into 5 types. The IVFs were divided into 2 or 3 main portions by the first 4 types of ligaments (transforaminal ligaments, corporotransverse ligaments, "reticular" ligaments, and "Y-shaped" ligaments). The radiating ligaments (the fifth type of ligaments) attached to the surrounding structures of the IVF and were connected directly to the nerve root sleeves. Although there was no indication of neurovascular compromise in this normal specimen, these ligaments limit the space within the bony IVF such that under certain pathologic conditions (e.g., inflammation), their presence would make neurovascular compression more likely than if they were absent. CONCLUSIONS: The 3-D reconstruction technique based on ultrathin cryomilling can effectively show the lumbar intervertebral foraminal ligaments and their anatomical characteristics in situ, providing a new way to clarify the relationships between these ligaments and their adjacent structures.

Therapeutic potential of targeting HSPA5 through dual regulation of two candidate prognostic biomarkers ANXA1 and PSAT1 in osteosarcoma.

Osteosarcoma is the most common primary malignant bone tumor that mostly affects young people's health. The prognosis of patients with unresectable or recurrent osteosarcoma still remains dismal. Based on gene integration analysis from GEO and TARGET databases by R language, the differentially expressed genes of osteosarcoma patients were identified. Biological molecular function analysis indicated that these genes were importantly enriched in the process of cell adhesion molecule binding. Gene significance highly-related to clinical traits of osteosarcoma was found by weighted gene co-expression network analysis. Additionally, receiver operating characteristic curve analysis was conducted to find prognostic markers in LASSO Cox regression model. Two candidate biomarkers, ANXA1 and PSAT1, for the prognosis of osteosarcoma were detected separately on the basis of WGCNA and LASSO model. Of note, their expression profiles were interrelated with an important therapeutic target HSPA5. In vitro pharmaceutical experiments were performed to explore the biological role and prognostic benefit of candidates. Suppression of HSPA5 effectively upregulated ANXA1 and inhibited PSAT1, resulting in osteosarcoma cell proliferation arrest and apoptosis. These findings suggest that HSPA5 serves as a core molecule for osteosarcoma therapy due to its bidirectional regulation of candidate prognostic biomarkers ANXA1 and PSAT1.

Absence of VEGFR-1/Flt-1 signaling pathway in mice results in insensitivity to discogenic low back pain in an established disc injury mouse model.

Although degenerative disc disease (DDD) and related low back pain (LBP) are growing public health problems, the underlying disease mechanisms remain unclear. An increase in the vascular endothelial growth factor (VEGF) levels in DDD has been reported. This study aimed to examine the role of VEGF receptors (VEGFRs) in DDD, using a mouse model of DDD. Progressive DDD was induced by anterior stabbing of lumbar intervertebral discs in wild type (WT) and VEGFR-1 tyrosine-kinase deficient mice (vegfr-1TK-/- ). Pain assessments were performed weekly for 12 weeks. Histological and immunohistochemical assessments were made for discs, dorsal root ganglions, and spinal cord. Both vegfr-1TK-/- and WT mice presented with similar pathological changes in discs with an increased expression of inflammatory cytokines and matrix-degrading enzymes. Despite the similar pathological patterns, vegfr-1TK-/- mice showed insensitivity to pain compared with WT mice. This insensitivity to discogenic pain was related to lower levels of pain factors in the discs and peripheral sensory neurons and lower spinal glial activation in the vegfr-1TK- /- mice than in the WT mice. Exogenous stimulation of bovine disc cells with VEGF increased inflammatory and cartilage degrading enzyme. Silencing vegfr-1 by small-interfering-RNA decreased VEGF-induced expression of pain markers, while silencing vegfr-2 decreased VEGF-induced expression of inflammatory and metabolic markers without changing pain markers. This suggests the involvement of VEGFR-1 signaling specifically in pain transmission. Collectively, our results indicate that the VEGF signaling is involved in DDD. Particularly, VEGFR-1 is critical for discogenic LBP transmission independent of the degree of disc pathology.

Development and validation of a pre-scoring system for nonspecific low back pain among general population in Guangzhou: a cross-sectional study.

BACKGROUND: Nonspecific Low Back Pain (NLBP) is a common disease with a low cure rate and significant impact on the population. This study aimed to develop and validate a pre-scoring system for identifying the risk of suffering from NLBP among the general population in Guangzhou. METHODS: A total of 1439 eligible subjects were surveyed in Guangzhou by stratified random sampling and was divided randomly into the development dataset (69.6%) and validation dataset (30.4%) subsequently. Based on the development dataset, potential associated factors (average exercise times weekly, the intensity of daily work, etc.) with NLBP were tested by the sequential logistic regression, and a pre-scoring system was formulated with Sullivan's method and graded afterward. The internal validity of the system was assessed by AUC and calibration plot, and the external validation was performed in the validation dataset. RESULTS: The prevalence rates of NLBP in the development dataset and the validation dataset were 12.97 and 13.27%, respectively. Age, BMI, average exercise times weekly, gender, educational level, the intensity of daily work, place of residence, monthly income, overall evaluation of health condition and physiology health were identified as significant factors. The total risk score ranged from 0 to 38, which was split into three risk grades: low risk (0 to 18), intermediate risk (19 to 22) and high risk (23 to 38). The pre-scoring system had an adequate calibration and a good discriminating ability with bootstrap-corrected AUC equaling 0.861 in the development dataset and 0.821 in the validation dataset. CONCLUSIONS: A pre-scoring system that could help clinicians to assess the risk of NLBP in the general population was validated. Further validation of the system in a new population or prospective cohort study is suggested.

MiR-27a promotes the autophagy and apoptosis of IL-1ß treated-articular chondrocytes in osteoarthritis through PI3K/AKT/mTOR signaling.

Osteoarthritis (OA) is a common degenerative joint disorder, which involves articular cartilage degeneration as well as joint inflammatory reactions. The recent studies have identified microRNA (miRNA) as one of the epigenetic mechanisms for the regulation of gene expression. Here we aim to reveal the role of miRNA in the regulation of gene expression in articular chondrocytes and its significance in the OA pathogenesis. In the present study, miRNA profiling was performed using OA cartilage and normal healthy cartilage tissues. As compared to their levels in normal cells and tissues, miR-27a expression was found to be upregulated in OA cartilage and IL-1ß-treated articular chondrocytes. TUNEL staining, as well as flow cytometry with Annexin V-FITC/PI double labeling indicated that miR-27a inhibition reduced the apoptosis of IL-1ß-treated articular chondrocytes. Bioinformatics prediction and the dual-luciferase reporter assay indicated that miR-27a targeted the 3'-UTR of the PI3K gene to silence it. The PI3K mRNA level in OA cartilage and IL-1ß-treated articular chondrocytes was also downregulated, comparing with normal cells and tissues. Transfection of chondrocytes transfected with the miR-27a inhibitor upregulated the PI3K expression. This study demonstrated miR-27a is a regulator of the PI3K-Akt-mTOR axis in human chondrocytes and could participate in OA pathogenesis.

[Vascular endothelial growth factor/polylactide-polyethyleneglycol-polylactic acid copolymer/basic fibroblast growth factor mixed microcapsules in promoting angiogenic differentiation of rat bone marrow mesenchymal stem cells in vitro].

Objective: To observe the effect of vascular endothelial growth factor/polylactide-polyethyleneglycol-polylactic acid copolymer/basic fibroblast growth factor (VEGF/PELA/bFGF) mixed microcapsules in promoting the angiogenic differentiation of rat bone marrow mesenchymal stem cells (BMSCs) in vitro. Methods: The BMSCs were isolated by the method of whole bone marrow adherent, and sub-cultured. The passage 3 BMSCs were identified by Wright-Giemsa staining and flow cytometry, and used for subsequent experiments. VEGF/PELA/bFGF (group A), PELA/bFGF (group B), VEGF/PELA (group C), and PELA (group D) microcapsules were prepared. The biodegradable ability and cytotoxicity of PELA microcapsule were determined，and the slow-released ability of VEGF/PELA/bFGF mixed microcapsules was measured. The passage 3 BMSCs were co-cultured with the extracts of groups A, B, C, and D, separately. At 1, 3, 7, 14, and 20 days after being cultured, the morphological changes of induced BMSCs were recorded. At 21 days, the induced BMSCs were tested for DiI-labeled acetylated low density lipoprotein (Dil-ac-LDL) and FITC-labeled ulex europaeus agglutinin I (FITC-UEA-I) uptake ability. The tube-forming ability of the induced cells on Matrigel was also verified. The differences of the vascularize indexes in nodes, master junctions, master segments, and tot.master segments length in 4 groups were summarized and analyzed. Results: The isolated and cultured cells were identified as BMSCs. The degradation time of PELA was more than 20 days. There was no significant effect on cell viability under co-culture conditions. At 20 days, the cumulative release of VEGF in the mixed microcapsules exceeded 95%, and the quantity of bFGF exceeded 80%. The morphology of cells in groups A, B, and C were changed. The cells in groups A and B showed the typical change of cobble-stone morphology. The numbers of double fluorescent labeled cells observed by fluorescence microscope were the most in group A, and decreases from group B and group C, with the lowest in group D. The cells in groups A and B formed a grid-like structure on Matrigel. Quantitative analysis showed that the differences in the number of nodes, master junctions, master segments, and tot.master segments length between groups A, B and groups C, D were significant ( P<0.05). The number of nodes and the tot.master segments length of group A were more than those of group B ( P<0.05). There was no significant differences in the number of master junctions and master segments between group A and group B ( P>0.05). Conclusion: VEGF/PELA/bFGF mixed microcapsules have significantly ability to promote the angiogenic differentiation of rat BMSCs in vitro.

Controlled release of FGF-2 and BMP-2 in tissue engineered periosteum promotes bone repair in rats.

The aim of this study was to prepare chitosan-collagen (CS/COL) scaffolds that could release fibroblast growth factor-2 (FGF-2) and bone morphogenetic protein 2 (BMP-2), and to study the effect of this scaffold on bone repair. By improving the double emulsion/solvent evaporation technique, BMP-2 was encapsulated in poly(lactic acid)-poly(ethylene glycol)-poly(lactic acid) (PELA) microcapsules, to the surface of which FGF-2 was attached. The CS/COL scaffold carrying the microcapsules was prepared by freeze-drying. Periosteum derived cells (PDCs) were extracted and cultured on the scaffolds to study their proliferation and differentiation on the scaffolds. In addition, the effects of the scaffolds were investigated on rats with skull defects by micro-computed tomography and histology. We successfully prepared PELA microcapsules with external adherence to FGF-2 and encapsulated with BMP-2. The CS/COL scaffolds were porous and PDCs adhered, proliferated and underwent osteogenic differentiation on the scaffolds. The sequential release of FGF-2/BMP-2 had better osteogenic efficacy than other groups. Our results suggest that CS/COL scaffolds that bind FGF-2 and BMP-2 in combination with PDCs could be a promising new strategy for tissue engineering periosteum.

[FGF-2/PELA/BMP-2 microcapsule scaffold promotes osteogenic differentiation of rat periosteum-derived stem cells in vitro].

OBJECTIVE: To observe the effect of a microencapsule scaffold capable of sustained release of fibroblast growth factor-2 (FGF-2) and bone morphogenetic protein-2 (BMP-2) in promoting the osteogenic differentiation of rat periosteum-derived stem cells (PDSCs) in vitro. METHODS: PDSCs from 4-week-old SD rats, after identification of the surface markers using flow cytometry, were induced to differentiate into osteoblast, chondroblast, and adipocyte lineages. The differentiated cells were verified by staining with Alizarin red, toluidine blue, alcian blue, oil red O and by immunofluorescence assay. FGF-2/PELA/BMP-2, FGF-2/PELA, PELA/BMP-2 and PELA microcapsules were prepared, examined for surface morphologies using scanning electron microscopy (SEM), and tested for controlled release of FGF-2 and BMP-2 using ELISA. The third passage of PDSCs were cultured in the presence of the aqueous extracts of one of the 4 materials, and alkaline phosphatase (AKP) activity in the culture media was detected at 7 and 14 days of culture; the expression levels of osteogenesis-related genes were quantified with quantitative real-time PCR (qRT-PCR). The osteogenic differentiation ability of the PDSCs cultured with the extracts was compared. RESULTS: The PDSCs, which expressed mesenchymal stem cell surface markers, were shown to have osteogenic, chondrogenic and adipogenic differentiation potentials. The cells cultured with the extract of FGF-2/PELA/BMP-2 microcapsules showed the highest AKP activity at 7 and 14 days of culture, and their expression levels of OCN and RunX-2 mRNA were the highest among the 4 groups; RunX-2 expression reached its peak level on day 14, and OCN mRNA expression level increased progressively as the culture time extended. CONCLUSION: FGF-2/PELA/BMP-2 biomimetic controlled release microcapsules preserve the cytokine activities and are capable of promoting the osteogenic differentiation of rat PDSCs.

PELA microspheres with encapsulated arginine-chitosan/pBMP-2 nanoparticles induce pBMP-2 controlled-release, transfected osteoblastic progenitor cells, and promoted osteogenic differentiation.

Repair of the bone injury remains a challenge in clinical practices. Recent progress in tissue engineering and therapeutic gene delivery systems have led to promising new strategies for successful acceleration of bone repair process. The aim of this study was to create a controlled-release system to slowly release the arginine-chitosan/plasmid DNA nanoparticles encoding BMP-2 gene (Arg-CS/pBMP-2 NPs), efficiently transfect osteoblastic progenitor cells, secrete functional BMP-2 protein, and promote osteogenic differentiation. In this study, chitosan was conjugated with arginine to generate arginine-chitosan polymer (Arg-CS) for gene delivery. Mix the Arg-CS with pBMP-2 to condense pBMP-2 into nano-sized particles. In vitro transfection assays demonstrated that the transfection efficiency of Arg-CS/pBMP-2 nanoparticles and the expression level of BMP-2 was obviously exceed control groups. Further, PELA microspheres as the controlled-release carrier for the nanoparticles were used to encapsulate Arg-CS/pBMP-2 NPs. We demonstrated that the Arg-CS/pBMP-2 NPs could slowly release from the PELA microspheres at least for 42 d. During the co-culture with the PELA microspheres, the content of BMP-2 protein secreted by MC3T3-E1 reached the peak at 7 d. After 21d, the secretion of BMP-2 protein still maintain a higher level. The alkaline phosphatase activity, alizarin red staining, and osteogenesis-related gene expression by real-time quantitative PCR analysis all showed the PELA microspheres entrapping with Arg-CS/pBMP-2 NPs can obviously induce the osteogenic differentiation. The results indicated that the Arg-CS is a suitable gene vector which can promote the gene transfection. And the novel PELA microspheres-nanoparticle controlled-release system has potential clinical application in the future after further research.

[Analysis of clinical factors correlating with osteoporosis in patients with type 2 diabetes mellitus].

OBJECTIVE: To analyze the clinical factors that contribute to lowered bone mineral density (BMD) of the lumbar vertebrae in type 2 diabetic patients. METHODS: Forty type 2 diabetic patients with osteoporosis and 40 age- and gender-matched healthy individuals, selected using propensity score method, were examined for BMD of the L1 to L4 vertebrae and the left femur. Age, gender, course of the disease, body mass index (BMI), fasting blood glucose, serum calcium, serum phosphate, urinary calcium, and urinary phosphate were compared between the two groups. RESULTS: BMD of the L1 to L4 vertebras was significantly lower in the diabetic group than in the healthy individuals (P<0.05). In the diabetic patients, BMD showed an obvious difference between male and female patients and was negatively correlated with the course of the disease but positively with BMI, fasting blood glucose, urinary calcium and urinary phosphate; BMD was not correlated with age, serum calcium or serum phosphate in these patients. CONCLUSION: Osteoporosis in type 2 diabetic patients is closely related with gender, BMI, course of the disease and poorly controlled glucose level.

TGF-ß1, in association with the increased expression of connective tissue growth factor, induce the hypertrophy of the ligamentum flavum through the p38 MAPK pathway.

Hypertrophy of the ligamentum flavum (LF) is one of the key pathomechanisms of lumbar spinal stenosis (LSS). Transforming growth factor (TGF)-ß1 is abundantly expressed in hypertrophied degenerative LF tissues from LSS. However, the molecular mechanisms underling the association between TGF-ß1 and LF hypertrophy have not yet been fully elucidated. In this study, we investigated the important role of the mitogen-activated protein kinase (MAPK) pathway in the pathogenesis of LSS by analyzing the expression of connective tissue growth factor (CTGF) and extracellular matrix (ECM) components (collagen I and collagen III) in TGF-ß1-treated LF cells. Cell growth assay revealed that TGF-ß1, in association with CTGF, enhanced the the proliferation of LF cells, and we found that TGF-ß1 also elevated CTGF expression and subsequently enhanced the mRNA expression of collagen I and collagen III. The increased mRNA expression levels of CTGF, collagen I and collagen III were abolished by p38 inhibitors. Both immunofluorescence imaging and western blot analysis of p38 and p-p38 revealed the increased expression and phosphorylation of p38. Silencing the expression of p38 by siRNA in LF cells decreased the protein expression of p38, p-p38 and CTGF, as well as the mRNA expression of CTGF, collagen I and collagen III. Taken together, our findings indicate that TGF-ß1, in association with the increased expression of CTGF, contribute to the homeostasis of the ECM and to the hypertrophy of LF through the p38 MAPK pathway.