TGFß attenuates cartilage extracellular matrix degradation via enhancing FBXO6-mediated MMP14 ubiquitination.

OBJECTIVES: FBXO6, a component of the ubiquitin E3 ligases, has been shown to bind high mannose N-linked glycoproteins and act as ubiquitin ligase subunits. Most proteins in the secretory pathway, such as matrix metalloproteinases, are modified with N-glycans and play important roles in the development of osteoarthritis (OA). However, whether FBXO6 exerts regulatory effects on the pathogenesis of OA remains undefined. METHODS: The expression of FBXO6 was examined in the cartilage of human and multiple mouse OA models. The role of FBXO6 in cartilage degeneration was analysed with global FBXO6-/- mice, transgenic Col2a1-CreERT2;FBXO6f/f mice. The FBXO6 interacting partner MMP14 and its regulatory transcriptional factor SMAD2/3 were identified and validated in different pathological models as well as SMAD2-/- mice. RESULTS: The expression of FBXO6 decreased in the cartilage from human OA samples, anterior cruciate ligament transaction (ACLT) -induced OA samples, spontaneous OA STR/ort samples and aged mice samples. Global knockout or conditional knockout of FBXO6 in cartilage promoted experimental OA process. The molecular mechanism study revealed that FBXO6 decreased MMP14 by ubiquitination and degradation, leading to inhibited proteolytic activation of MMP13. Interestingly, FBXO6 expression is regulated by transforming growth factor ß (TGFß)-SMAD2/3 signalling pathway. Therefore, the overexpression of FBXO6 protected mice from post-injury OA development. CONCLUSIONS: TGFß-SMAD2/3 signalling pathway suppressed MMP13 activation by upregulating of FBXO6 transcription and consequently promoting MMP14 proteasomal degradation. Inducement of FBXO6 expression in OA cartilage might provide a promising OA therapeutic strategy.

Circular RNA circTADA2A promotes osteosarcoma progression and metastasis by sponging miR-203a-3p and regulating CREB3 expression.

BACKGROUND: As a subclass of noncoding RNAs, circular RNAs (circRNAs) have been demonstrated to play a critical role in regulating gene expression in eukaryotes. Recent studies have revealed the pivotal functions of circRNAs in cancer progression. However, little is known about the role of circTADA2A, also named hsa_circ_0043278, in osteosarcoma (OS). METHODS: CircTADA2A was selected from a previously reported circRNA microarray comparing OS cell lines and normal bone cells. QRT-PCR was used to detect the expression of circTADA2A in OS tissue and cell lines. Luciferase reporter, RNA immunoprecipitation (RIP), RNA pull-down and fluorescence in situ hybridization (FISH) assays were performed to confirm the binding of circTADA2A with miR-203a-3p. OS cells were stably transfected with lentiviruses, and Transwell migration, Matrigel invasion, colony formation, proliferation, apoptosis, Western blotting, and in vivo tumorigenesis and metastasis assays were employed to evaluate the roles of circTADA2A, miR-203a-3p and CREB3. RESULTS: Our findings demonstrated that circTADA2A was highly expressed in both OS tissue and cell lines, and circTADA2A inhibition attenuated the migration, invasion and proliferation of OS cells in vitro as well as tumorigenesis and metastasis in vivo. A mechanistic study revealed that circTADA2A could readily sponge miR-203a-3p to upregulate the expression of CREB3, which was identified as a driver gene in OS. Furthermore, miR-203a-3p inhibition or CREB3 overexpression could reverse the circTADA2A silencing-induced impairment of malignant tumor behavior. CONCLUSIONS: CircTADA2A functions as a tumor promoter in OS to increase malignant tumor behavior through the miR-203a-3p/CREB3 axis, which could be a novel target for OS therapy.

CircSERPINE2 protects against osteoarthritis by targeting miR-1271 and ETS-related gene.

OBJECTIVES: Circular RNAs (circRNA) expression aberration has been identified in various human diseases. In this study, we investigated whether circRNAs could act as competing endogenous RNAs to regulate the pathological process of osteoarthritis (OA). METHODS: CircRNA deep sequencing was performed to the expression of circRNAs between OA and control cartilage tissues. The regulatory and functional role of CircSERPINE2 upregulation was examined in OA and was validated in vitro and in vivo, downstream target of CircSERPINE2 was explored. RNA pull down, a luciferase reporter assay, biotin-coupled microRNA capture and fluorescence in situ hybridisation were used to evaluate the interaction between CircSERPINE2 and miR-1271-5 p, as well as the target mRNA, E26 transformation-specific-related gene (ERG). The role and mechanism of CircSERPINE2 in OA was also explored in rabbit models. RESULTS: The decreased expression of CircSERPINE2 in the OA cartilage tissues was directly associated with excessive apoptosis and imbalance between anabolic and catabolic factors of extracellular matrix (ECM). Mechanistically, CircSERPINE2 acted as a sponge of miR-1271-5 p and functioned in human chondrocytes (HCs) through targeting miR-1271-5 p and ERG. Intra-articular injection of adeno-associated virus-CircSERPINE2-wt alleviated OA in the rabbit model. CONCLUSIONS: Our results reveal an important role for a novel circRNA-CircSERPINE2 in OA progression. CircSERPINE2 overexpression could alleviate HCs apoptosis and promote anabolism of ECM through miR-1271-ERG pathway. It provides a potentially effective therapeutic strategy for OA progression.

The PPAR-γ antagonist T007 inhibits RANKL-induced osteoclastogenesis and counteracts OVX-induced bone loss in mice.

BACKGROUND: Osteoclasts are key determinant cellular components implicated in the development and progression of disorders driven by bone damage. Herein, we studied the upshot of T007, an antagonist of peroxisome proliferator-activated receptor-gamma (PPARγ), on osteoclastogenesis using cell and animal models. RESULTS: The in vitro assays revealed that T007 hindered the osteoclastogenesis caused by the treatment with the receptor activator of nuclear factor-κB ligand (RANKL) through inhibiting the levels of PPARγ in cells. The PPARγ siRNA partially reproduced the inhibitory action of T007. The opposite findings were produced after PPARγ overexpression. Furthermore, T007 prevented from bone loss in a mouse model of osteoporosis induced by ovariectomy (OVX). These findings implied that T007 is a potential efficient drug for the prophylaxis and cure of osteoclast-related disorders. CONCLUSIONS: Taken together, our findings demonstrated that T007 impedes osteoclastogenesis and will be useful for the therapy of bone related diseases, essentially osteoporosis.

Lycorine Suppresses Endplate-Chondrocyte Degeneration and Prevents Intervertebral Disc Degeneration by Inhibiting NF-κB Signalling Pathway.

BACKGROUND/AIMS: Cartilaginous endplate (CEP) degeneration is an important cause for intervertebral disc (IVD) degeneration that leads to low-back pain. The identification of compounds that may prevent CEP degeneration is of interest for the prevention of IVD degeneration. METHODS: Catabolic protease expression in the CEP of disc degeneration patients was first assessed. The toxicity, function and underlying mechanism of lycorine (LY) on CEP-derived chondrocytes degeneration were assessed in vitro by flow cytometry analysis and western blotting. The concentration and function of LY in rat-tail disc-degeneration models were also assessed by HPLC (High Performance Liquid Chromatography) quantification and histological analysis. RESULTS: In CEP cells, Interleukin (IL)-1ß upregulated the expression of matrix metalloproteinase (MMP)-3, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4 and ADAMTS-5 that is critical for the degradation of cartilage extracellular matrix. Interestingly, LY suppressed the expression of these enzymes via the inhibition of nuclear factor-κB (NFκB) signalling and thus prevented IL-1ß-induced endplate cell degeneration in vitro. More importantly, LY also reduced the expression of MMP-3, MMP-13, ADAMTS-4 and ADAMTS-5 in CEP and exerted a protective effect on both CEP and nucleus pulposus (NP) degeneration. In addition to its inhibitory effect on matrix-degrading protease expression, LY treatment also reduced positive regulators of proinflammatory cytokines, such as MIF, which can be secreted by CEP cells and subsequently target NP cells. CONCLUSION: LY could serve as a potential drug for treating IVD disease.

TGF-ß synergizes with ML264 to block IL-1ß-induced matrix degradation mediated by Krüppel-like factor 5 in the nucleus pulposus.

Intervertebral disc degeneration causes low back pain.Interleukin-1ß (IL-1ß) is a well-known inflammatory mediator that is involved in disc degeneration but its molecular mechanisms on catabolic and anabolic events in nucleus pulposus (NP) cells remain unclear. Krüppel-like factor 5 (KLF5) is associated with inflammation and was previously shown to cause cartilage degradation. In this study, we revealed that KLF5 is involved in IL-1ß activated NF-kB cascade by enhancing both p65 phosphorylation and p65 acetylation. Moreover, the catabolic effect of KLF5 can be abolished by transforming growth factor-ß (TGF-ß) via promoting the proteasomal degradation of KLF5. Therefore, a KLF5 inhibitor ML264 was further proved to synergize with TGF-ß to attenuate IL-1ß-induced intervertebral disc degeneration. These results indicate the critical role of KLF5 in regulating intervertebral disc metabolism and suggest KLF5 inhibitor such as ML264 as potential compound for treatment of degenerative disc disease.

Combined use of leptin and mechanical stress has osteogenic effects on ossification of the posterior longitudinal ligament.

PURPOSE: To evaluate the effects of leptin/leptin receptor (LepR) combined with mechanical stress on the development of ossification of the posterior longitudinal ligament (OPLL), which is a disease characterized by ectopic bone formation of the posterior longitudinal ligament (PLL) and can lead to radiculopathy and myelopathy. METHODS: Six human samples of the PLL were analyzed for the expression of leptin and LepR by RT-PCR and western blotting. PLL cells were stimulated with leptin and mechanical stress delivered via a Flexcell tension system, and osteogenic differentiation was evaluated by RT-PCR and western blotting analysis of osteogenic marker expression as well as by alkaline phosphatase (ALP) staining and alizarin red S staining. Activation of mitogen-activated protein kinase (MAPK), Janus kinase (JAK) 2-signal transducer, activator of transcription (STAT) 3 and phosphatidylinositol 3-kinase (PI3K)-Akt was evaluated by western blotting. RESULTS: Samples from the OPLL group had higher LepR mRNA and protein levels and lower leptin levels than those from healthy controls. Exposure to leptin and Flexcell increased the number of ALP-positive cells and calcium nodules in a dose-dependent manner; this effect was accompanied by upregulation of the osteogenic markers osteocalcin, runt-related transcription factor 2 (RUNX2) and osteopontin. Extracellular signal-regulated kinase, P38 MAPK, JAK2, STAT3, PI3K and Akt signaling, was also activated by the combined effects of leptin and mechanical stress. CONCLUSIONS: Leptin and LepR are differentially expressed in OPLL tissues, and the combined use of leptin/LepR and mechanical stress promotes osteogenic differentiation of PLL cells via MAPK, JAK2-STAT3 and PI3K/Akt signaling. These slides can be retrieved under Electronic Supplementary Material.

Effects of oral tauroursodeoxycholic acid and/or intestinal probiotics on serum biochemical indexes and bile composition in patients with cholecystolithiasis.

Background: In recent years, gallstones have become a major condition affecting people's health. Cholecystectomy remains an effective treatment method, but it has large risk factors. It is well known that the hepatoenteric axis plays a key role in gallstone formation, and it is gradually becoming a research focus. Cholesterol homeostasis can be regulated by the liver and intestinal tract in our bodies, and intestinal flora can regulate the digestion and absorption of cholesterol. These two factors are closely related to the formation of gallstones. Aim: To investigate the effects of tauroursodeoxycholic acid (TUDCA) and/or intestinal probiotics on serum biochemical indexes and bile composition in patients with cholecystolithiasis. Methods: For this study, 96 patients with cholecystolithiasis were recruited at our hospital. The patients were randomly divided into four groups according to a random number table: group Ⅰ (TUDCA, 24 cases), group Ⅱ (intestinal probiotics, 24 cases), group Ⅲ (TUDCA and intestinal probiotics, 24 cases) and group Ⅳ (control group, 24 cases). All patients underwent laparoscopic gallbladder-preserving lithotomy or laparoscopic cholecystectomy. Bile samples were identified and extracted during the operation. Results: The results revealed that the levels of serum total bile acid (TBA), serum total cholesterol (TCHOL) and serum triglyceride in groups I, II and III before and after the intervention were statistically significant (p < 0.05). There were significant differences in serum low-density lipoprotein cholesterol (LDL-C) between groups I and II before and after the intervention (p < 0.05), but the serum LDL-C level in group Ⅲ before and after the intervention was similar (p > 0.05). Regarding bile, TBA levels demonstrated no significant difference between groups I and III (p > 0.05), and the differences between the other two groups were statistically significant (p < 0.05). No significant difference was identified in phospholipid and TCHOL levels between groups I and Ⅲ (p > 0.05), and the differences between the other two groups were statistically significant (p < 0.05). There were significant differences in the levels of free Ca2+, pH value and glycoprotein in bile among the four groups (p < 0.05). The levels of cholic acid, chenodeoxycholic acid and deoxycholic acid in bile were significantly different among the four groups (p < 0.05). The level of lithocholic acid (LCA) in groups Ⅱ and Ⅲ was similar, as was the level of LCA in groups I and ⅠV, but the difference in level between the other two groups was statistically significant (p < 0.05). Conclusion: The combination of TUDCA and intestinal probiotics did not enhance the effect of either treatment. The use of intestinal probiotics alone can maximise the reverse development of bile composition in patients with cholecystolithiasis compared with TUDCA alone and a combination of TUDCA and intestinal probiotics, thereby reducing gallstone formation.

USP1 inhibition suppresses the progression of osteosarcoma via destabilizing TAZ.

Mutations and altered expression of deubiquitinating enzymes (DUBs) profoundly influence tumor progression. Ubiquitin-specific protease 1 (USP1) is a well-characterized human DUB reportedly overexpressed in and associated with maintaining the mesenchymal stem cell status of osteosarcoma (OS); however, the potential mechanisms of USP1 in OS remain poorly understood. In this study, we identified that USP1 directly interacts with Transcriptional Co-Activator With PDZ-Binding Motif (TAZ) in OS cell lines, and with mechanistic analysis indicating that the anti-OS effects of USP1 inhibition could be partially attributed to TAZ instability, with its reduced nuclear accumulation responsible for a subsequent decrease in the expression of downstream genes associated with the Hippo signaling pathway. Moreover, pharmacological inhibition USP1 by ML323 presented the similar effects on Hippo signaling pathway and suppressed OS growth and metastasis both in vitro and in vivo. Taken together, our results revealed a novel molecular mechanism underlying the function of USP1 in OS and a potential role of ML323 as a therapeutic strategy for the clinical treatment of OS.

CircRNF111 Protects Against Insulin Resistance and Lipid Deposition via Regulating miR-143-3p/IGF2R Axis in Metabolic Syndrome.

Abnormal expression of circRNAs (circular RNAs), a subclass of non-coding RNAs, has been documented in numerous human diseases. Herein, we explored whether circRNAs act as ceRNAs (competing endogenous RNAs) to modulate the pathological process-insulin resistance, as well as dyslipidemia of MetS (Metabolic Syndrome). The profile of circRNAs in serume of MetS and control samples was characterized by circRNA deep sequencing. We identified circRNF111 as a key downregulated circRNA involved in MetS. The decreased expression of circRNF111 in the serum samples of MetS was directly linked to excessive insulin resistance and dyslipidemia. Loss-of-function experiments showed that circRNF111 knockdown inhibited the glucose uptake and the Akt signaling pathway, meanwhile increased the deposition of triglycerides in adipogenic differentiated hADSCs (human adipose-derived stem cells). Mechanistically, circRNF111 sponged miR-143-3p and functioned via targeting miR-143-3p along with its downstream target gene IGF2R. The role along with the mechanism of circRNF111 sponging miR-143-3p in MetS was also explored in obese mice triggered by high-fat die. Therefore, our data suggest a protective role of the novel circRNA-circRNF111 in MetS progression. CircRNF111 inhibition enhances insulin resistance and lipid deposition in MetS through regulating miR-143-3p-IGF2R cascade. This provides a promising therapeutic approach for MetS.

Oxidative Stress-Induced Hypermethylation of KLF5 Promoter Mediated by DNMT3B Impairs Osteogenesis by Diminishing the Interaction with ß-Catenin.

Aims: Emerging evidence suggests that the pathogenesis of osteoporosis, characterized by impaired osteogenesis, is shifting from estrogen centric to oxidative stress. Our previous studies have shown that the zinc-finger transcription factor krüppel-like factor 5 (KLF5) plays a key role in the degeneration of nucleus pulposus and cartilage. However, its role in osteoporosis remains unknown. We aimed to investigate the effect and mechanism of KLF5 on osteogenesis under oxidative stress. Results: First, KLF5 was required for osteogenesis and stimulated osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). KLF5 was hypermethylated and downregulated in ovariectomy-induced osteoporosis mice and in BMSCs treated with H2O2. Interestingly, DNA methyltransferases 3B (DNMT3B) upregulation mediated the hypermethylation of KLF5 induced by oxidative stress, thereby impairing osteogenic differentiation. The inhibition of KLF5 hypermethylation using DNMT3B siRNA or 5-AZA-2-deoxycytidine (5-AZA) protected osteogenic differentiation of BMSCs from oxidative stress. Regarding the downstream mechanism, KLF5 induced ß-catenin expression. More importantly, KLF5 promoted the nuclear translocation of ß-catenin, which was mediated by the armadillo repeat region of ß-catenin. Consistently, oxidative stress-induced KLF5 hypermethylation inhibited osteogenic differentiation by reducing the expression and nuclear translocation of ß-catenin. Innovation: We describe the novel effect and mechanism of KLF5 on osteogenesis under oxidative stress, which is linked to osteoporosis for the first time. Conclusion: Our results suggested that oxidative stress-induced hypermethylation of KLF5 mediated by DNMT3B impairs osteogenesis by diminishing the interaction with ß-catenin, which is likely to contribute to osteoporosis. Targeting the hypermethylation of KLF5 might be a new strategy for the treatment of osteoporosis. Antioxid. Redox Signal. 35, 1-20.

SREBP-2 aggravates breast cancer associated osteolysis by promoting osteoclastogenesis and breast cancer metastasis.

Bone is one of the most common sites of breast cancer metastasis and a major cause of high mortality in these patients. Thus, further understanding the molecular mechanisms regulating breast cancer-induced osteolysis is critical for the development of more effective treatments. In this study, we demonstrated that important roles sterol regulatory element-binding protein 2 (SREBP-2) play in osteoclast formation a function, and in breast cancer metastasis. SREBP-2 expression was found to be induced during the early stages of osteoclast formation under the control of the RANKL/cAMP-response element binding protein (CREB) signaling cascade. SREBP-2 is subsequently translocated into the nucleus where it participates with other transcriptional factors to induce the expression of NFATc1 required for mature osteoclast formation. Additionally, SREBP-2 was also found to be highly expressed in breast cancer tissues and correlated with a poor prognosis. SREBP-2 was similarly under the transcriptional control of CREB and its induction regulates the expression of matrix metalloproteinases (MMPs), key degradative enzymes involved in bone metastases by breast cancer cells. Accordingly, targeting of SREBP-2 with Fatostatin which specifically inhibits SCAP (SREBP cleavage-activating protein) and prevents SREBP activation, attenuated breast cancer-induced osteolysis in vivo. Collectively, our results suggest that SREBP-2 plays a critical role in regulating osteoclastogenesis and contributes to breast cancer-induced osteolysis. Thus, SREBP-2 inhibition is a potential therapeutic approach for breast cancer patients with osteolytic bone lesions.

Prospective Study of Long-term Effect between Multifidus Muscle Bundle and Conventional Open Approach in One-level Posterior Lumbar Interbody Fusion.

OBJECTIVE: To compare postoperative imaging results, clinical outcomes and complications between the multifidus muscle bundle (MMB) approach and the conventional open (CO) approach in one-level posterior lumbar interbody fusion (PLIF). METHODS: Based on the inclusion and exclusion criteria, 201 of 351 patients in our hospital were enrolled in this prospective study and underwent MMB-PLIF or CO-PLIF randomly: 111 patients in the MMB-PLIF group and 90 patients in the CO-PLIF group. A total of 100 patients failed to be followed up in the following 7-9 years. Therefore, in this study, 52 patients of the MMB group and 49 patients of the CO group were included. We evaluated the differences in terms of multifidus atrophy rate, intervertebral disc height and segmental lordosis restoration of the operation segment, lumbar lordosis restoration, fusion rate, visual analogue scale (VAS) for back and leg pain, Oswestry disability index (ODI), complication rates, and patient satisfaction rates between the two groups. Correlation between multifidus muscle degeneration and the incidence of complications was investigated, and we compared the multifidus muscle degeneration rate between patients with or without intractable back pain or adjacent segment degeneration. RESULTS: There were no significant differences in age, sex, body mass index (BMI), diagnosis, segments distribution, and mean follow-up time between the MMB-PLIF group and the CO-PLIF group. In addition, no differences regarding sex, age, or BMI were found between the lost follow-up group and the successful follow-up group. In regard to imaging and clinical evaluation, at the final follow-up, there were significant differences in multifidus atrophy rates (27.0% ± 6.8% vs 38.7% ± 10.9%), lumbar lordosis restoration (4.6° ± 2.5° vs 3.0° ± 1.9°), postoperative VAS for back pain (1.1 ± 0.9 vs 1.8 ± 1.2), ODI (7.7 ± 5.0 vs 12.4 ± 6.7), and patient satisfaction rates (86.5% vs 61.2%) between MMB-PLIF and CO-PLIF groups. However, there were no significant differences in segmental lordosis, intervertebral height restoration, postoperative VAS for leg pain or fusion rate between the two groups. In regards to complications, there were significant differences in the incidence of adjacent segment degeneration (3.8% vs 14.3%), intractable back pain (3.8% vs 22.4%), and residual neurological symptoms (5.8% vs 20.4%) between the two groups (P < 0.05) at the final follow-up. In addition, patients with adjacent segment degeneration and intractable back pain were observed with more significant multifidus muscle atrophy than those without these two complications (31.9% ± 1.1% vs 39.6% ± 2.1% and 30.9% ± 1.1% vs 42.8% ± 2.1%). CONCLUSION: Compared with CO-PLIF, MMB-PLIF had advantages in relation to protection of the multifidus muscle, better maintenance of lumbar lordosis, reduced lower back pain and ODI score, fewer complications, and a higher patient satisfaction rate. Protection of the multifidus muscle in lumbar surgery is an important aspect of minimally invasive surgery.

Expression of Matrix Metalloproteinases, Tissue Inhibitors of Metalloproteinases, and Interleukins in Vertebral Cartilage Endplate.

OBJECTIVE: Turnover of cartilage endplate extracellular matrix (ECM) may play an important role in disc degeneration and low back pain (LBP). However, the expression pattern of pro-inflammatory factors, matrix metalloproteinases (MMP), and tissue inhibitors of metalloproteinases (TIMP) in the cartilage endplates (CEP) of intervertebral discs (IVD) is not understood. We aimed to examine the transcriptional levels of MMP, TIMP, and interleukins (IL), and the correlations between them. METHODS: Thirty degenerated cartilage endplate samples from patients with LBP who underwent lumbar fusion surgery were included in the degenerated group. Ten patients without LBP history who underwent lumbar surgery because of vertebral burst fractures were included in the control group. The degenerative severity of the samples was evaluated by MRI, and hematoxylin-eosin and safranin O-fast green (SO-FG) staining. Real-time polymerase chain reaction (RT-PCR) was used to detect the mRNA levels of MMP-1, MMP-3, MMP-9, MMP-13, TIMP-1, TIMP-2, TIMP-3, IL-1α, IL-1ß, and IL-6. The correlations between the levels of these genes were tested using Spearman's rho test. RESULTS: Hematoxylin-eosin and SO-FG staining confirmed a decrease in cell number and proteoglycans in the degenerated cartilage endplate. MRI showed significant signal changes in degenerated cartilage endplates. Patients in the degenerated group showed a higher rate of endplate Modic changes when compared with the control group. MMP-3, MMP-9, TIMP-3, IL-1α, and IL-1ß were elevated with statistical significance, while MMP-1, MMP-13, TIMP-1, TIMP-2, and IL-6 were changed without statistical significance or remained unchanged. Expression of MMP-3 was positively correlated with IL-1α (Spearman coefficient, 0.486; P < 0.05); expression of TIMP-3 was positively correlated with MMP-9, IL-1α, and IL-1ß (Spearman coefficient, 0.577, 0.407, and 0.571, respectively; P < 0.05). CONCLUSION: MMP-3, MMP-9, TIMP-3, IL-1α, and IL-1ß may play a role in the process of cartilage endplate degeneration. MMP-3 may be regulated by IL-1α, and TIMP-3 might be associated with MMP-9 and regulated by IL-1α and IL-1ß.

Preparation of decellularized biphasic hierarchical myotendinous junction extracellular matrix for muscle regeneration.

Muscle injury and defect affect people's quality of life, and effective treatment is lacking. Herein, we generated a scaffold to obtain decellularized porcine Achilles tendon myotendinous junction (D-MTJ) extracellular matrix (ECM) with well-preserved native biphasic hierarchical structure, biological composition, and excellent mechanical properties for muscle regeneration. The combined use of potassium chloride, potassium iodide, Triton-X 100, and sodium-dodecyl sulfate (SDS) can completely remove the main immunogenicity, while maintaining the major biological components and microstructure. The specific biomechanics of D-MTJ is comparable to the native muscle-tendon physiological conditions. Additionally, the D-MTJ ECM scaffold induced minimal immunological reaction (histology analysis) through rat subcutaneous implantation. Moreover, in vitro, muscle satellite cells adhered, proliferated, and infiltrated into the D-MTJ scaffold, and myofiber-like cell differentiation was observed as shown by increased expression of myogenesis-related genes during culture. In vivo, newly formed myofibers were observed in a muscle defect model with D-MTJ orthotopic transplantation, while the control group presented mostly with fibrous tissue deposition. Additionally, the number of Myod and MyHC-positive cells in the ECM scaffold group was higher at day 30. We preliminary explored the mechanisms underlying D-MTJ-mediated muscle regeneration, which may be attributed to its specific biphasic hierarchical structure, bio-components, and attractiveness for myogenesis cells. In conclusion, our findings suggest the D-MTJ ECM scaffold prepared in this study is a promising choice for muscle regeneration. STATEMENT OF SIGNIFICANCE: This study is the first to use decellularization technology obtaining the specifically decellularized myotendinous junction (D-MTJ) with well-preserved biphasic hierarchical structure and constituents, excellent mechanical properties and good biocompatibility. The D-MTJ was further proved to be efficient for muscle regeneration in vitro and in vivo, and the underlying mechanisms may be attributed to its specifically structure and constituents, improved myogenesis and good preservation of repair-related factors. Our study may provide basis for the decellularization of other biphasic hierarchical tissues and a platform for further studies on muscle fiber and tendon integrations in vitro.

Preparation and Evaluation of Tibia- and Calvarium-Derived Decellularized Periosteum Scaffolds.

The periosteum plays a key role in bone regeneration and an artificial bionic material is urgently required. The periostea on the tibia and skull differ with respect to the types of cells, microstructure, and components, leading to different biological functions and biomechanical properties. We aimed to prepare decellularized periosteum scaffolds derived from different origins and evaluate their angiogenic and osteogenic activities. Histological assessment of α-smooth muscle actin, bone morphogenetic protein-2, and alkaline phosphatase in tibial and calvarial periosteum tissues provided preliminary information on their differing angiogenic and osteogenic properties. We developed decellularization protocols to completely remove the periosteum cellular components and for good maintenance of the hierarchical multilayer structures and components of the extracellular matrix (ECM) with no cytotoxicity. Moreover, using a chicken egg chorioallantoic membrane assay and a nude mouse implantation model, we found that tibia-derived periosteum ECM had superior osteogenic activity and calvarium-derived ECM had good angiogenic activity. The preliminary mechanisms of differing activities were then evaluated by osteogenesis- and angiogenesis-related gene expression in human umbilical vein endothelial cell- and MC-3T3 cell-seeded ECM scaffolds. Thus, this study provides periosteum biomaterials that are derived from specific tissues and have different functional properties and structures, for use in bone regeneration.

Quantitative MRI and X-ray analysis of disc degeneration and paraspinal muscle changes in degenerative spondylolisthesis.

BACKGROUND AND OBJECTIVE: The intervertebral disc degeneration changes and paraspinal muscles changes are believed to be risk factors for lumbar degenerative spondylolisthesis (LDS). But there is limited quantitative information about this progression. This study is to reveal their changes the in the progression of LDS. METHODS: Data were gathered from 149 middle-aged degenerative spondylolisthesis patients and same amount of age- and sex-matched control group with both lumber spine MRI and X-ray. Narrowed disc space were measured in percent as anterior inferior disc height (DHIA)/anterior superior disc height (DHSA), inferior disc height (DHI)/superior disc height (DHS), and posterior inferior disc height (DHIP)/posterior superior disc height (DHSP). Signal intensity ratio of multifidus muscle (RM) and erector spinae (RES) to psoas muscle, muscle atrophy ratio of lean CSA (LCSA) to gross CSA (GCSA) of paraspinal muscles were calculated. RESULTS: In the case group the most common slipped vertebra was L4 (75.84%). Disc height (DHIA/DHSA, DHI/DHS) and multifidus muscle atrophy ratio (M-LCSA/M-GCSA) tended to be lower than those in the control group, whereas the disc degeneration degree and T2 signal intensity ratio (RM,RES) of the paraspinal muscles and erector spinae muscle atrophy ratio were higher than control group. The difference between the two groups was statistically significant (P< 0.05). Using multivariate logistic regression analysis, it was confirmed that ES-LCSA/ES-GCSA, especially RM are independent predisposing factors to lumbar spondylolisthesis (OR > 1, P< 0.05) while DHIA/DHSA, M-LCSA/M-GCSA are independent protective factors (OR < 1, P< 0.05). CONCLUSIONS: Decreased anterior disc height and multifidus muscle atrophy are found in the LDS patients and thy could be the cause of LDS. The presence of erector spinae hypertrophy could be a compensatory mechanism to compensate for the instability.