Aberrant methylation and expression of TNXB promote chondrocyte apoptosis and extracullar matrix degradation in hemophilic arthropathy via AKT signaling.

Recurrent joint bleeding in hemophilia patients frequently causes hemophilic arthropathy (HA). Drastic degradation of cartilage is a major characteristic of HA, but its pathological mechanisms has not yet been clarified. In HA cartilages, we found server matrix degradation and increased expression of DNA methyltransferase proteins. We thus performed genome-wide DNA methylation analysis on human HA (N=5) and osteoarthritis (OA) (N=5) articular cartilages, and identified 1228 differentially methylated regions (DMRs) associated with HA. Functional enrichment analyses revealed the association between DMR genes (DMGs) and extracellular matrix (ECM) organization. Among these DMGs, Tenascin XB (TNXB) expression was down-regulated in human and mouse HA cartilages. The loss of Tnxb in F8-/- mouse cartilage provided a disease-promoting role in HA by augmenting cartilage degeneration and subchondral bone loss. Tnxb knockdown also promoted chondrocyte apoptosis and inhibited phosphorylation of AKT. Importantly, AKT agonist showed chondroprotective effects following Tnxb knockdown. Together, our findings indicate that exposure of cartilage to blood leads to alterations in DNA methylation, which is functionally related to ECM homeostasis, and further demonstrate a critical role of TNXB in HA cartilage degeneration by activating AKT signaling. These mechanistic insights allow development of potentially new strategies for HA cartilage protection.

Liquiritin reduces chondrocyte apoptosis through P53/PUMA signaling pathway to alleviate osteoarthritis.

AIMS: The main pathological features of osteoarthritis (OA) include the degeneration of articular cartilage and a decrease in matrix synthesis. Chondrocytes, which contribute to matrix synthesis, play a crucial role in the development of OA. Liquiritin, an effective ingredient extracted from Glycyrrhiza uralensis Fisch., has been used for over 1000 years to treat OA. This study aims to investigate the impact of liquiritin on OA and its underlying mechanism. MATERIALS AND METHODS: Gait and hot plate tests assessed mouse behavior, while Micro-CT and ABH/OG staining observed joint morphological changes. The TUNEL kit detected chondrocyte apoptosis. Western blot and immunofluorescence techniques determined the expression levels of cartilage metabolism markers COL2 and MMP13, as well as apoptosis markers caspase3, bcl2, P53, and PUMA. KEGG analysis and molecular docking technology were used to verify the relationship between liquiritin and P53. KEY FINDINGS: Liquiritin alleviated pain sensitivity and improved gait impairment in OA mice. Additionally, we found that liquiritin could increase COL2 levels and decrease MMP13 levels both in vivo and in vitro. Importantly, liquiritin reduced chondrocyte apoptosis induced by OA, through decreased expression of caspase3 expression and increased expression of bcl2 expression. Molecular docking revealed a strong binding affinity between liquiritin and P53. Both in vivo and in vitro studies demonstrated that liquiritin suppressed the expression of P53 and PUMA in cartilage. SIGNIFICANCE: This indicated that liquiritin may alleviate OA progression by inhibiting the P53/PUMA signaling pathway, suggesting that liquiritin is a potential strategy for the treatment of OA.

ß-catenin inhibition disrupts the homeostasis of osteogenic/adipogenic differentiation leading to the development of glucocorticoid-induced osteonecrosis of the femoral head.

Glucocorticoid-induced osteonecrosis of the femoral head (GONFH) is a common refractory joint disease characterized by bone damage and the collapse of femoral head structure. However, the exact pathological mechanisms of GONFH remain unknown. Here, we observed abnormal osteogenesis and adipogenesis associated with decreased ß-catenin in the necrotic femoral head of GONFH patients. In vivo and in vitro studies further revealed that glucocorticoid exposure disrupted osteogenic/adipogenic differentiation of bone marrow mesenchymal cells (BMSCs) by inhibiting ß-catenin signaling in glucocorticoid-induced GONFH rats. Col2+ lineage largely contributes to BMSCs and was found an osteogenic commitment in the femoral head through 9 mo of lineage trace. Specific deletion of ß-catenin gene (Ctnnb1) in Col2+ cells shifted their commitment from osteoblasts to adipocytes, leading to a full spectrum of disease phenotype of GONFH in adult mice. Overall, we uncover that ß-catenin inhibition disrupting the homeostasis of osteogenic/adipogenic differentiation contributes to the development of GONFH and identify an ideal genetic-modified mouse model of GONFH.

Protective effects of Shensuitongzhi formula on intervertebral disc degeneration via downregulation of NF-κB signaling pathway and inflammatory response.

Low back pain (LBP) is a common orthopedic disease over the world. Lumbar intervertebral disc degeneration (IDD) is regarded as an important cause of LBP. Shensuitongzhi formula (SSTZF) is a drug used in clinical treatment for orthopedic diseases. It has been found that SSTZF can have a good treatment for IDD. But the exact mechanism has not been clarified. The results showed that SSTZF protects against LSI-induced degeneration of cartilage endplates and intervertebral discs. Meanwhile, SSTZF treatment dramatically reduces the expression of inflammatory factor as well as the expression of catabolism protein and upregulates the expression of anabolism protein in LSI-induced mice. In addition, SSTZF delayed the progression of LSI-induced IDD via downregulation the level of NF-κB signaling key gene RELA and phosphorylation of key protein P65 in endplate chondrocytes. Our study has illustrated the treatment as well as the latent mechanism of SSTZF in IDD.

Ambient PM2.5 Exposure and Bone Homeostasis: Analysis of UK Biobank Data and Experimental Studies in Mice and in Vitro.

BACKGROUND: Previous evidence has identified exposure to fine ambient particulate matter (PM2.5) as a leading risk factor for adverse health outcomes. However, to date, only a few studies have examined the potential association between long-term exposure to PM2.5 and bone homeostasis. OBJECTIVE: We sought to examine the relationship between long-term PM2.5 exposure and bone health and explore its potential mechanism. METHODS: This research included both observational and experimental studies. First, based on human data from UK Biobank, linear regression was used to explore the associations between long-term exposure to PM2.5 (i.e., annual average PM2.5 concentration for 2010) and bone mineral density [BMD; i.e., heel BMD (n=37,440) and femur neck and lumbar spine BMD (n=29,766)], which were measured during 2014-2020. For the experimental animal study, C57BL/6 male mice were assigned to ambient PM2.5 or filtered air for 6 months via a whole-body exposure system. Micro-computed tomography analyses were applied to measure BMD and bone microstructures. Biomarkers for bone turnover and inflammation were examined with histological staining, immunohistochemistry staining, and enzyme-linked immunosorbent assay. We also performed tartrate-resistant acid phosphatase (TRAP) staining and bone resorption assay to determine the effect of PM2.5 exposure on osteoclast activity in vitro. In addition, the potential downstream regulators were assessed by real-time polymerase chain reaction and western blot. RESULTS: We observed that long-term exposure to PM2.5 was significantly associated with lower BMD at different anatomical sites, according to the analysis of UK Biobank data. In experimental study, mice exposed long-term to PM2.5 exhibited excessive osteoclastogenesis, dysregulated osteogenesis, higher tumor necrosis factor-alpha (TNF-α) expression, and shorter femur length than control mice, but they demonstrated no significant differences in femur structure or BMD. In vitro, cells stimulated with conditional medium of PM2.5-stimulated macrophages had aberrant osteoclastogenesis and differences in the protein/mRNA expression of members of the TNF-α/Traf6/c-Fos pathway, which could be partially rescued by TNF-α inhibition. DISCUSSION: Our prospective observational evidence suggested that long-term exposure to PM2.5 is associated with lower BMD and further experimental results demonstrated exposure to PM2.5 could disrupt bone homeostasis, which may be mediated by inflammation-induced osteoclastogenesis. https://doi.org/10.1289/EHP11646.

Glucocorticoid-induced activation of NOX/ROS/NF-κB signaling in MSCs contributes to the development of GONFH.

BACKGROUND: This study aimed to investigate the pathogenic factors of glucocorticoids (GCs)-induced osteonecrosis of the femoral head (GONFH) and its underlying pathogenesis in vivo and in vitro. METHODS: Radiographical (µCT) scanning, histopathological, immunohistochemical, reactive oxygen species (ROS) and tunel staining were conducted on GONFH patients and rats. ROS, tunel, flow cytometry, alkaline phosphatase, Oil red O staining, reverse transcription­quantitative PCR and western blotting were applied to elucidate the exact pathogenesis mechanism. RESULTS: Clinical and animal studies demonstrated increased levels of ROS, aggravated oxidative stress (OS) microenvironment, augmented apoptosis and imbalance in osteogenic/lipogenic in the GONFH group compared to the control group. The fate of mesenchymal stem cells (MSCs) directed by GCs is a crucial factor in determining GONFH. In vitro studies further revealed that GCs promote excessive ROS production through the expression of NOX family proteins, leading to a deterioration of the OS microenvironment in MSCs, ultimately resulting in apoptosis and imbalance in osteogenic/lipogenic differentiation. Furthermore, our results confirmed that the NOX inhibitor-diphenyleneiodonium chloride and the NF-κB inhibitor-BAY 11-7082 ameliorated apoptosis and osteogenic/lipogenic differentiation imbalance of MSCs induced by an excess of GCs. CONCLUSION: We demonstrated for the first time that the aggravation of the OS microenvironment in MSCs caused by high doses of GCs leading to apoptosis and differentiation imbalance is a crucial factor in the pathogenesis of GONFH, mediated through activating the NOX/ROS/NF-κB signaling pathway.

Protein phosphatase PPM1A inhibition attenuates osteoarthritis via regulating TGF-ß/Smad2 signaling in chondrocytes.

TGF-ß signaling is crucial for modulating osteoarthritis (OA), and protein phosphatase magnesium-dependent 1A (PPM1A) has been reported as a phosphatase of SMAD2 and regulates TGF-ß signaling, while the role of PPM1A in cartilage homeostasis and OA development remains largely unexplored. In this study, we found increased PPM1A expression in OA chondrocytes and confirmed the interaction between PPM1A and phospho-SMAD2 (p-SMAD2). Importantly, our data show that PPM1A KO substantially protected mice treated with destabilization of medial meniscus (DMM) surgery against cartilage degeneration and subchondral sclerosis. Additionally, PPM1A ablation reduced the cartilage catabolism and cell apoptosis after the DMM operation. Moreover, p-SMAD2 expression in chondrocytes from KO mice was higher than that in WT controls with DMM induction. However, intraarticular injection with SD-208, repressing TGF-ß/SMAD2 signaling, dramatically abolished protective phenotypes in PPM1A-KO mice. Finally, a specific pharmacologic PPM1A inhibitor, Sanguinarine chloride (SC) or BC-21, was able to ameliorate OA severity in C57BL/6J mice. In summary, our study identified PPM1A as a pivotal regulator of cartilage homeostasis and demonstrated that PPM1A inhibition attenuates OA progression via regulating TGF-ß/SMAD2 signaling in chondrocytes and provided PPM1A as a potential target for OA treatment.

Glycyrrhizic acid alters the hyperoxidative stress-induced differentiation commitment of MSCs by activating the Wnt/ß-catenin pathway to prevent SONFH.

This study aimed to examine the in vivo and in vitro therapeutic effects of glycyrrhizic acid (GA) on steroid-induced osteonecrosis of the femoral head (SONFH), which is caused by the overuse of glucocorticoids (GCs). Clinically, we identified elevated oxidative stress (OS) levels and an imbalance in osteolipogenic homeostasis in SONFH patients compared to femoral neck fracture (FNF) patients. In vivo, we established experimental SONFH in rats via lipopolysaccharides (LPSs) combined with methylprednisolone (MPS). We showed that GA and Wnt agonist-S8320 alleviated SONFH, as evidenced by the reduced microstructural and histopathological alterations in the subchondral bone of the femoral head and the decreased levels of OS in rat models. In vitro, GA reduced dexamethasone (Dex)-induced excessive NOX4 and OS levels by activating the Wnt/ß-catenin pathway, thereby promoting the osteogenic differentiation of mesenchymal stem cells (MSCs) and inhibiting lipogenic differentiation. In addition, GA regulated the expression levels of the key transcription factors downstream of this pathway, Runx2 and PPARγ, thus maintaining osteolipogenic homeostasis. In summary, we demonstrated for the first time that GA modulates the osteolipogenic differentiation commitment of MSCs induced by excessive OS through activating the Wnt/ß-catenin pathway, thereby ameliorating SONFH.

Mid-term outcomes of microfragmented adipose tissue plus arthroscopic surgery for knee osteoarthritis: A randomized, active-control, multicenter clinical trial.

BACKGROUND: Osteoarthritis (OA) is the most prevalent form of degenerative whole-joint disease. Before the final option of knee replacement, arthroscopic surgery was the most widely used joint-preserving surgical treatment. Emerging regenerative therapies, such as those involving platelet-rich plasma, mesenchymal stem cells, and microfragmented adipose tissue (MFAT), have been pushed to the forefront of treatment to prevent the progression of OA. Currently, MFAT has been successfully applied to treat different types of orthopedic diseases. AIM: To assess the efficacy and safety of MFAT with arthroscopic surgery in patients with knee OA (KOA). METHODS: A randomized, multicenter study was conducted between June 2017 and November 2022 in 10 hospitals in Zhejiang, China. Overall, 302 patients diagnosed with KOA (Kellgren-Lawrence grades 2-3) were randomized to the MFAT group (n = 151, were administered MFAT following arthroscopic surgery), or the control group (n = 151, were administered hyaluronic acid following arthroscopic surgery). The study outcomes were changes in the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) score, the visual analog scale (VAS) score, the Lequesne index score, the Whole-Organ Magnetic Resonance Imaging Score (WORMS), and safety over a 24-mo period from baseline. RESULTS: The changes in the WOMAC score (including the three subscale scores), VAS pain score, and Lequesne index score at the 24-mo mark were significantly different in the MFAT and control groups, as well as when comparing values at the posttreatment visit and those at baseline (P < 0.001). The MFAT group consistently demonstrated significant decreases in the WOMAC pain scores and VAS scores at all follow-ups compared to the control group (P < 0.05). Furthermore, the WOMAC stiffness score, WOMAC function score, and Lequesne index score differed significantly between the groups at 12 and 24 mo (P < 0.05). However, no significant between-group differences were observed in the WORMS at 24 mo (P = 0.367). No serious adverse events occurred in both groups. CONCLUSION: The MFAT injection combined with arthroscopic surgery treatment group showed better mid-term clinical outcomes compared to the control group, suggesting its efficacy as a therapeutic approach for patients with KOA.

Elucidation of the Underlying Mechanism of Gujian Oral Liquid Acting on Osteoarthritis through Network Pharmacology, Molecular Docking, and Experiment.

Gujian oral liquid (GJ), a traditional herbal formula in China, has been widely used to treat patients with osteoarthritis (OA). Nevertheless, the active component and potential mechanism of GJ are not fully elucidated. Thus, we investigate the effect of GJ and explore its underlying mechanism on OA through network pharmacology and experimental validation. First, a total of 175 bioactive compounds were identified, and 134 overlapping targets were acquired after comparing the targets of the GJ with those of OA. 8 hub targets, including IL6 and AKT1, were obtained in PPI network analysis. Then, we built up GJ-target-OA network and protein-protein interaction (PPI) network, followed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. The results underlined inflammatory tumor necrosis factor (TNF) as a promising signaling pathway of GJ for OA treatment. Moreover, molecular docking also verified the top two active compounds had direct bindings with the top three target genes. Finally, we verified the effect of GJ on OA in vivo and in vitro. In vivo experiments validated that GJ not only significantly attenuated OA phenotypes including articular cartilage degeneration and subchondral bone sclerosis but also reduced the expressions of tumor necrosis factor-α (TNF-α) and p-p65 in articular chondrocytes. Besides, GJ serum also had a protective effect on chondrocytes against inflammation caused by TNF-α in vitro. Hence, our study predicted and verified that GJ could exert anti-inflammation and anticatabolism effects partially via regulating TNF-α/NF-kappa B (NF-κB) signaling.

TGF-ß/Smad2 signalling regulates enchondral bone formation of Gli1+ periosteal cells during fracture healing.

OBJECTIVES: Most bone fracture heals through enchondral bone formation that relies on the involvement of periosteal progenitor cells. However, the identity of periosteal progenitor cells and the regulatory mechanism of their proliferation and differentiation remain unclear. The aim of this study was to investigate whether Gli1-CreERT2 can identify a population of murine periosteal progenitor cells and the role of TGF-ß signalling in periosteal progenitor cells on fracture healing. MATERIALS AND METHODS: Double heterozygous Gli1-CreERT2 ;Rosa26-tdTomatoflox/wt mice were sacrificed at different time points for tracing the fate of Gli1+ cells in both intact and fracture bone. Gli1-CreERT2 -mediated Tgfbr2 knockout (Gli1-CreERT2 ;Tgfbr2flox/flox ) mice were subjected to fracture surgery. At 4, 7, 10, 14 and 21 days post-surgery, tibia samples were harvested for tissue analyses including µCT, histology, real-time PCR and immunofluorescence staining. RESULTS: Through cell lineage-tracing experiments, we have revealed that Gli1-CreER T2 can be used to identify a subpopulation of periosteal progenitor cells in vivo that persistently reside in periosteum and contribute to osteochondral elements during fracture repair. During the healing process, TGF-ß signalling is continually activated in the reparative Gli1+ periosteal cells. Conditional knockout of Tgfbr2 in these cells leads to a delayed and impaired enchondral bone formation, at least partially due to the reduced proliferation and chondrogenic and osteogenic differentiation of Gli1+ periosteal cells. CONCLUSIONS: TGF-ß signalling plays an essential role on fracture repair via regulating enchondral bone formation process of Gli1+ periosteal cells.

Radix Rehmanniae Praeparata promotes bone fracture healing through activation of TGF-ß signaling in mesenchymal progenitors.

BACKGROUND: Radix Rehmanniae Praeparata (RR), the steamed roots of Rehmannia glutinosa, is a traditional Chinese medicine with the function of kidney-nourishing, and it has been safety used for centuries to treat bone-related disorders. The aim of this study is to investigate the positive effect and underlying mechanism of RR enhancing bone fracture healing in mouse model. METHODS: Ten-week-old C57BL/6J mice were subjected to a unilateral open transverse tibial fracture and provided a daily treatment of RR. Bone samples were harvested for tissue analyses including x-ray, µCT, histology, histomorphometry, biomechanical testing, immunohistochemical (IHC) and quantitative gene expression analysis. To determine the role of TGF-ß in accelerating fracture healing effect of RR, aforementioned experiments were performed on Gli1-CreER; Tgfbr2 flox/flox (Tgfbr2Gli1ER) conditional knockout mice. RESULTS: RR promoted bone fracture healing and strengthened bone intensity in wild-type and Cre- mice with the activation of TGF-ß/Smad2 signaling, on the contrary, RR failed to accelerating fracture healing in Tgfbr2Gli1ER mice. CONCLUSION: RR promotes bone fracture healing by intensify the contribution of Gli1+ cells on bone and cartilage formation mainly in TGF-ß-dependent manner. RR is an alternative option for clinical treatment of fracture.

Bushenhuoxue formula promotes osteogenic differentiation of growth plate chondrocytes through ß-catenin-dependent manner during osteoporosis.

BACKGROUND: Bushenhuoxue formula (BSHXF) has shown excellent clinical effects on the treatment of osteoporosis in China. The aim of this study is to determine the anti-osteoporosis effects and precise molecular mechanisms of BSHXF on mouse models. METHODS: Ten-week-old female C57BL/6 J mice were subjected to ovariectomy and provided a daily treatment of BSHXF. At 8 weeks post-surgery, the femurs were harvested for tissue analyses including µCT, histology, qRT-PCR and immunohistochemical (IHC) staining of ß-catenin, ALP and FABP4. To investigate the role of ß-catenin in the anti-osteoporosis effects of BSHXF, relative experiments mentioned above were performed in ß-catenin conditional knockout mice. RESULTS: Ovariectomized (OVX) mice presented severe bone loss and excessive fat accumulation in the chondro-osseous junction underneath the growth plate, with decreased expression of ALP and increased expression of FABP4. BSHXF significantly recovered the OVX-induced abnormal osteogenesis and adipogenesis with the activation of ß-catenin in growth plate chondrocytes. Further, we generated growth plate chondrocyte-specific ß-catenin knockout (ß-cateninGli1ER) mice that exhibited bone loss and fat accumulation in the chondro-osseous junction, similar to the OVX mice. However, BSHXF failed to rescue the osteoporosis-like phenotype in ß-cateninGli1ER mice, indicating the anti-osteoporosis effects of BSHXF act mainly through ß-catenin signaling. No significant restoration of ALP and FABP4 was observed in ß-cateninGli1ER mice after the treatment of BSHXF. CONCLUSIONS: BSHXF attenuates osteoporosis by promoting osteogenic differentiation of growth plate chondrocytes mainly in ß-catenin-dependent manner. BSHXF is considered as a new candidate for the treatment of osteoporosis.

Peripheral Blood Stem Cell Therapy Does Not Improve Outcomes of Femoral Head Osteonecrosis With Cap-Shaped Separated Cartilage Defect.

A combination treatment with porous tantalum rod implantation and intra-arterial infusion of peripheral blood stem cells (PBSCs) provides a promise for treating early and intermediate stages of osteonecrosis of the femoral head (ONFH). However, its clinical indications and application restrictions remain unclear. This study aims to determine the clinical, histological, and radiological outcomes of a combination treatment using mechanical support and a targeted intra-arterial infusion of PBSCs for painful ONFH with a cap-shaped separation (CSS) cartilage defect. Compared with the standard pain management (control group), this combination treatment did not improve the Harris Hip Score (HHS) at 36 months. Micro-CT and histologic analyses showed severe focal destruction in all CSS-ONFH femoral heads in both the combination and control groups. Femoral heads showed a higher percentage of bone lesions in the combination treatment group than in the control group. There was no significant difference in osteoclast number in the subchondral bone areas between the two groups. A high level of expression of inflammatory cytokines, including tumor necrosis factor-α and interleukin-1ß, was detected in blood vessels around the subchondral bone in both groups. The RANKL/OPG (receptor activator of the nuclear factor-kB ligand/osteoprotegerin) ratio was also similar between the control and combination treatment groups. Our results indicate that this combination treatment is not an effective method for the treatment of patients with painful CSS-ONFH. Moreover, this combination treatment did not inhibit inflammatory osteoclastogenesis in patients with more advanced disease. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:269-276, 2020.

Genome-wide microRNA screening reveals miR-582-5p as a mesenchymal stem cell-specific microRNA in subchondral bone of the human knee joint.

Emerging evidence suggests that microRNAs (miRNAs) may be pathologically involved in osteoarthritis (OA). Subchondral bone (SCB) sclerosis is accounted for the knee osteoarthritis (KOA) development and progression. In this study, we aimed to screen the miRNA biomarkers of KOA and investigated whether these miRNAs regulate the differentiation potential of mesenchymal stem cells (MSCs) and thus contributing to SCB. We identified 48 miRNAs in the blood samples in KOA patients (n = 5) through microarray expression profiling detection. After validation with larger sample number, we confirmed hsa-miR-582-5p and hsa-miR-424-5p were associated with the pathology of SCB sclerosis. Target genes prediction and pathway analysis were implemented with online databases, indicating these two candidate miRNAs were closely related to the pathways of pluripotency of stem cells and pathology of OA. Surprisingly, mmu-miR-582-5p (homology of hsa-miR-582-5p) was downregulated in osteogenic differentiation and upregulated in adipogenic differentiation of mesenchymal progenitor C3H10T1/2 cells, whereas mmu-mir-322-5p (homology of hsa-miR-424-5p) showed no change through the in vitro study. Supplementing mmu-miR-582-5p mimics blocked osteogenic and induced adipogenic differentiation of C3H10T1/2 cells, whereas silencing of the endogenous mmu-miR-582-5p enhanced osteogenic and repressed adipogenic differentiation. Further mechanism studies showed that mmu-miR-582-5p was directly targeted to Runx2. Mutation of putative mmu-miR-582-5p binding sites in Runx2 3' untranslated region (3'UTR) could abolish the response of the 3'UTR-luciferase construct to mmu-miR-582-5p supplementation. Generally speaking, our data suggest that miR-582-5p is an important biomarker of KOA and is able to regulate osteogenic and adipogenic differentiation of MSCs via targeting Runx2. The study also suggests that miR-582-5p may play a crucial role in SCB sclerosis of human KOA.

Treatment with Vancomycin Loaded Calcium Sulphate and Autogenous Bone in an Improved Rabbit Model of Bone Infection.

Bone infection results from bacterial invasion, which is extremely difficult to treat in clinical, orthopedic, and traumatic surgery. The bone infection may result in sustained inflammation, osteomyelitis, and eventual bone non-union. Establishment of a feasible, reproducible animal model is important to bone infection research and antibiotic treatment. As an in vivo model, the rabbit model is widely used in bone infection research. However, previous studies on rabbit bone infection models showed that the infection status was inconsistent, as the amount of bacteria was variable. This study presents an improved surgical method for inducing bone infection on a rabbit, by blocking the bacteria in the bone marrow. Then, multi-level evaluations can be carried out to verify the modelling method. In general, debriding necrotic tissue and implantation of vancomycin-loaded calcium sulphate (VCS) are predominant in antibiotic treatment. Although calcium sulphate in VCS benefits osteocyte crawling and new bone growth, massive bone defects occur after debriding. Autogenous bone (AB) is an appealing strategy to overcome bone defects for the treatment of massive bone defects after debriding necrotic bone. In this study, we used the tail bone as an autogenous bone implanted in the bone defect. Bone repair was measured using micro-computed-tomography (micro-CT) and histological analysis after animal sacrifice. As a result, in the VCS group, bone non-union was consistently obtained. In contrast, the bone defect areas in the VCS-AB group were decreased significantly. The present modeling method described a reproducible, feasible, stable method to prepare a bone infection model. The VCS-AB treatment resulted in lower bone non-union rates after antibiotic treatment. The improved bone infection model and the combination treatment of VCS and autogenous bone could be helpful in studying the underlying mechanisms in bone infection and bone regeneration pertinent to traumatology orthopedic applications.

[Research status of correlation between type IX collagen gene and musculoskeletal diseases].

As an important component of articular cartilage, type IX collagen plays an important role in regulating homeostasis of bone and cartilage. The mutation or deletion of gene could disequilibrate homeostasis leads to deformity of corresponding bone and joint, and finally causes multiple epiphyseal dysplasia. Moreover, anatomic variation also leads to biomechanics change of corresponding functional unit, combine with micro-environment change caused by change of genes, osteoarthritis and disc degeneration were occurred under the function of stress over and over again. In addition, lack of type IX collagen could effect repair of articular cartilage, intervertebral disc tissue injury. However, musculoskeletal diseases related with type IX collagen has so much not limited this, the reports about it is less for lack of evidence, and need further work to study. Clear relationship of type IX collagen and its disease could provide an effective diagnostic method, and develop a new pathway for follow-up treatment.

Bushenhuoxue formula attenuates cartilage degeneration in an osteoarthritic mouse model through TGF-ß/MMP13 signaling.

BACKGROUND: Articular cartilage degeneration plays a key role in the pathogenesis of osteoarthritis (OA). Bushenhuoxue formula (BSHXF) has been widely used in the treatment of OA in clinics. However, the molecular mechanisms responsible for the chondroprotective effect of BSHXF remain to be elucidated. The purpose of this study was to explore the effects of BSHXF on OA mice model. METHODS: In this study, we investigated the effects of BSHXF on destabilization of the medial meniscus (DMM)-induced chondrocyte degradation in OA mice model. At 12 weeks post-surgery, the joints were harvested for tissue analyses, including histology, histomorphometry, TUNEL, OARSI scoring, micro-CT and immunohistochemistry for COL2, TGFBR2, pSMAD2 and MMP13. Additionally, we also evaluated the effects of BSHXF on Mmp13 mRNA and protein expression in chondrogenic ATDC5 cells through real-time PCR and Western blot respectively. Moreover, we investigated the chondroprotective effect of BSHXF on mice with Tgfbr2 conditional knockout (Tgfbr2 Col2ER mice) in chondrocyte, including the relative experiments mentioned above. We transfected Tgfbr2 siRNA in ATDC5 to further evaluate the changes of Mmp13 mRNA and protein expression followed by BSHXF treatment. RESULTS: Amelioration of cartilage degradation and chondrocyte apoptosis were observed in DMM-induced mice, with increases in cartilage area and thickness, proteoglycan matrix, COL2 content and decreases in OARSI score at 12 weeks post surgery. Moreover, the elevated TGFBR2 and pSMAD2, and reduced MMP13 positive cells were also revealed in DMM-induced mice treated with BSHXF. Besides, decreased Mmp13 mRNA and protein expression were observed inchondrogenic ATDC5 cells culture in serum containing BSHXF. As expected, Tgfbr2 Col2ER mice exhibited significant OA-like phenotype. Interestingly, obvious improvement in articular cartilage structure was still observed in Tgfbr2 Col2ER mice after BSHXF treatment via up-regulated pSMAD2 and down-regulated MMP13 expressional levels in articular cartilage. CONCLUSIONS: BSHXF could inhibit cartilage degradation through TGF-ß/MMP13 signaling, and be considered a good option for the treatment of OA.

Accumulated Spinal Axial Biomechanical Loading Induces Degeneration in Intervertebral Disc of Mice Lumbar Spine.

OBJECTIVE: To investigate the effect of accumulated spinal axial biomechanical loading on mice lumbar disc and the feasibility of applying this method to establish a mice intervertebral disc degeneration model using a custom-made hot plate cage. In previous studies, we observed that the motion pattern of mice was greatly similar to that of humans when they were standing and jumping on their lower limbs. There is little data to demonstrate whether or not accumulated spinal axial biomechanical loading could induce intervertebral disc degeneration in vivo. METHODS: Twenty-four 0-week-old mice were randomly divided into model 1-month and 3-month groups, and control 1-month and 3-month groups (n = 6 per group). The model groups was transferred into the custom-made hot plate cage three times per day for modeling. The control group was kept in a regular cage. The intervertebral disc samples of the L3 -L5 were harvested for histologic, molecular, and immunohistochemical studies after modeling for 1 and 3 months. RESULTS: Accumulated spinal axial biomechanical loading affects the histologic, molecular, and immunohistochemical changes of mice L3- L5 intervertebral discs. Decreased height of disc and endplate, fissures of annulus fibrosus, and ossification of cartilage endplate were found in morphological studies. Immunohistochemical studies of the protein level showed a similar expression of type II collagen at 1 month, but a slightly decreased expression at 3 months, and an increased expression level of type X collagen and matrix metalloproteinase 13 (MMP13). Molecular studies showed that ColIIa1 and aggrecan mRNA expression levels were slightly increased at 1 month (P > 0.05), but then decreased slightly (P > 0.05). ColXa1, ADAMTS-5, and MMP-13 expression levels werer increased both at 1 and 3 months (P < 0.05). In addition, increased expression of Runx2 was observed. CONCLUSION: Accumulated spinal axial loading provided by a custom-made hot plate accelerated mice lumbar disc and especially endplate degeneration. However, this method requires further development to establish a lumbar disc degeneration model.

Transforming growth factor-beta1 promotes articular cartilage repair through canonical Smad and Hippo pathways in bone mesenchymal stem cells.

AIMS: Transforming growth factor-ß1 (TGF-ß1) is a chondrogenic factor and has been reported to be able to enhance chondrocyte differentiation from bone marrow mesenchymal stem cells (BMSCs). Here we investigate the molecular mechanism through which TGF-ß1 chronically promotes the repair of cartilage defect and inhibit chondrocyte hypertrophy. MAIN METHODS: Animal models of full thickness cartilage defects were divided into three groups: model group, BMSCs group (treated with BMSCs/calcium alginate gel) and BMSCs+TGF-ß1 group (treated with Lentivirus-TGF-ß1-EGFP transduced BMSCs/calcium alginate gel). 4 and 8weeks after treatment, macroscopic observation, histopathological study and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) were done to analyze phenotypes of the animals. BMSCs were transduced with Lentivirus-TGF-ß1-EGFP in vitro and Western blot analysis was performed. KEY FINDINGS: We found that TGF-ß1-expressiing BMSCs improved the repair of the cartilage defect. The impaired cartilage contained higher amount of GAG and type II collagen and was integrated to the surrounding normal cartilage and higher content of GAG and type II collagen. The major events include increased expression of type II collagen following Smad2/3 phosphorylation, and inhibition of cartilage hypertrophy by increasing Yes-associated protein-1 (YAP-1) and inhibiting Runx2 and Col10 after the completion of chondrogenic differentiation. SIGNIFICANCE: We conclude that TGF-ß1 is beneficial to chondrogenic differentiation of BMSCs via canonical Smad pathway to promote early-repairing of cartilage defect. Furthermore, TGF-ß1 inhibits chondrocyte hypertrophy by decreasing hypertrophy marker gene expression via Hippo signaling. Long-term rational use of TGF-ß1 may be an alternative approach in clinic for cartilage repair and regeneration.