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.

Exploring the mechanism of Buyang Huanwu decoction in the treatment of lumbar disc herniation based on network pharmacology and molecular docking.

Buyang Huanwu decoction (BYHWD), as one of the traditional Chinese medicine formulas, is widely used in the clinical treatment of lumbar disc herniation (LDH) with curative effect. It has the characteristics of multi-component, multi-target, and mutual synergy, but the mechanism of action is often unclear. It needs some research to explore the molecular mechanism of BYHWD in the treatment of LDH based on network pharmacology and molecular docking. Screen the active compounds of BYHWD and predict drug-related gene/protein targets, which could determine the specific target of BYHWD in the treatment of LDH. Construct the "Drugs-Compounds-Targets" network and search for the core targets. Use Gene Ontology functional enrichment analysis, Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis, and molecular docking verification to explore the possible molecular mechanism. Eighty-two effective compounds and 666 targets of BYHWD, 187 targets for LDH treatment, and 20 core candidate targets were excavated. A total of 3414 entries were identified by Gene Ontology enrichment analysis, 173 related signal pathways were identified by Kyoto Encyclopedia of Genes and Genomes enrichment analysis, and 5 core compounds were identified by molecular docking, which had a good affinity with core genes STAT3, JUN, AKT1, MAPK1, RELA, and PIK3CA. BYHWD may play the role of analgesic and improving function by synergistic anti-inflammatory and analgesic compounds, regulating cell metabolic differentiation, regulating immunity, and anticoagulation. BYHWD in the treatment of LDH may play a role in analgesia and improve function through multiple signaling pathways, including PI3K-Akt, mitogen-activated protein kinase, tumor necrosis factor, and interleukin-17. The PI3K-Akt signaling may be one of the key mechanisms.

Editorial Commentary: The Indication and Technique of Iliotibial Band Release for External Snapping Hip During Hip Arthroscopy in Patients With Femoroacetabular Impingement Syndrome.

Patients with femoroacetabular impingement syndrome (FAIS) often have extra-articular disorders, such as external snapping hip (ESH). We recommend that obvious ESH be addressed by endoscopic transversal iliotibial band (ITB) release during hip arthroscopy for FAIS because the residual serious snapping caused by ESH negatively affects the outcome of hip arthroscopy. However, for mild ESH without indications for severe trochanteric bursitis on magnetic resonance imaging, we still propose that physical therapy, extracorporeal shock wave therapy, or local injection be performed for pain relief. Surgical interventions for ESH including the Z-plasty technique and the modified Z-plasty technique for lengthening the ITB, as well as endoscopic cruciate or transversal incision in the ITB for release, have been reported with good results. Every technique has advantages and disadvantages, and we believe that surgeons should perform ITB release for ESH at the time of hip arthroscopy for FAIS based on their personal experience and inclination. In any case, excessive release of the ITB should be avoided. Finally, we wish to propose that more attention should be paid to the peri-greater trochanter (GT) space, an anatomic space between the ITB and the GT, which is similar to the subacromial space in the shoulder joint. Greater trochanteric pain syndrome (GTPS), related to the peri-GT space, is a spectrum of disorders, including trochanteric bursitis, abductor tendon pathology, and ESH. Precise diagnosis and proper procedures for concurrent GTPS during surgery may improve the outcome of arthroscopy in patients with both FAIS and GTPS.

The Evaluation of Sagittal Pelvic-Femoral Kinematics in Patients with Cam-Type Femoracetabular Impingement.

OBJECTIVE: To investigate the sagittal hip-pelvic kinematics in symptomatic cam-type femoroacetabular impingement (FAI) patients in the process of sitting down and compare their difference between patients with sitting pain complaint and those without. METHODS: Twenty-nine symptomatic cam-type FAI patients were recruited from our clinic between May 2018 and October 2018. Patients were categorized into two groups depending on whether they complain of pain in prolonged sitting or not. The pelvic-femoral measurements were assessed with a set of lateral pelvic radiography in sitting and standing respectively. Pelvic incidence (PI), sacral slope (SS), and proximal femoral shaft angle (PFSA) were measured on lateral pelvic radiography, and then pelvic tilting, apparent hip flexion, true hip flexion, and the pelvic-femoral ratio were calculated to investigate the kinematic change from standing to sitting position. Demographic measurements, hip morphology measurements, functional measurements, visual analog scale (VAS), and pelvic-femoral measurements were compared between the two groups. RESULTS: Thirteen cases without sitting pain complaint and 16 cases with sitting pain complaint were stratified to Group N and Group P respectively. No was significant difference in age, body mass index (BMI), and gender between the two groups. Hip morphology measurements (α angle and lateral center-edge angle) and functional measurements (iHOT-12) showed no significant difference between the two groups. However, the mean VAS of pain while sitting was 0.5 ± 0.4 and 1.6 ± 0.6 in Group N and Group P respectively (P = 0.005). Patients with sitting pain complaint have increased pelvic PI compared to those without (50.1° ± 6.5° and 44.2° ± 7.6°, P = 0. 042). The changes in SS (pelvic tilting) from standing to sitting in Group N was significantly larger than that in Group P (21.8° ± 7.0° and 15.1° ± 6.5°, P = 0.012). Although no significant difference in apparent hip flexion and true hip flexion was found. Patients without sitting pain complaint demonstrated a higher pelvic-femoral ratio (22.8% ± 7.9% and 16.1% ± 7.5%, P = 0.010) compared to those with sitting pain complaint. CONCLUSION: Sagittal pelvic-femoral kinematics could have an influence on the symptomology of cam-type FAI. The small PI and insufficient sagittal pelvic tilting in the process of sitting down could be related to the complaint of sitting pain in patients with symptomatic cam-type FAI.

Arthroscopic Treatment for Femoroacetabular Impingement Syndrome with External Snapping Hip: A Comparison Study of Matched Case Series.

OBJECTIVE: To determine the effectiveness of hip arthroscopy combined with endoscopic iliotibial band (ITB) release in patients with both femoroacetabular impingement (FAI) syndrome and external snapping hip (ESH). METHODS: Retrospectively review the preoperative and minimum of 2-year follow-up data of patients with both FAI syndrome and ESH who underwent endoscopic ITB release during hip arthroscopy (FAI + ESH group) from January 2014 to December 2018. The same number of age- and gender-matched FAI syndrome patients without ESH undergoing hip arthroscopy were enrolled in the control group (FAI group). Patient-reported outcomes (PROs) including international Hip Outcome Tool (iHOT-33), modified Harris Hip Score (mHHS), visual analog scale for pain (VAS-pain), and abductive force of affected hip at 3 month and 2 years postoperatively were comparatively analyzed. The VAS-satisfaction score of two groups at 2 years postoperatively were also analyzed. RESULTS: The prevalence of ESH in FAI syndrome patients undergoing hip arthroscopy in our institution was 5.5% (39 of 715 hips), including nine males (10 hips) and 29 females (29 hips). The mean age at the time of surgery was 32.1 ± 6.9 years (range, 22-48 years). According to inclusion and exclusion criteria, 23 patients were enrolled in FAI + ITB group. Twenty-three age- and sex-matched FAI syndrome patients were enrolled in FAI group. At 24 months postoperatively, no patient still suffered ESH symptoms and painful palpation at lateral region in FAI + ITB group. The iHOT-33, mHHS, and VAS-pain score of patients in FAI + ESH group were significantly severer than patients in FAI group preoperatively (41.6 ± 7.5 vs 48.8 ± 7.2, 54.8 ± 7.2 vs 59.2 ± 6.9, 5.5 ± 0.9 vs 4.7 ± 1.0; P < 0.05), while there was no significant difference in these scores between the patients in FAI + ESH group and FAI group at 3-month and 24-month follow-up (73.6 ± 8.5 vs 76.1 ± 6.9, 85.3 ± 7.8 vs 84.2 ± 6.6, 0.8 ± 0.9 vs 0.6 ± 0.9; P > 0.05). At 3 months after surgery, the abductive force of operated hip was significantly smaller than that in FAI group (82.4 ± 12.4 N vs 91.9 ± 16.1 N, P < 0.05), whereas there was no significant difference at 24 months after surgery (101.6 ± 14.9 N vs 106.5 ± 13.7 N, P > 0.05). The VAS-satisfaction scores of patients in the two groups were at a similarly high level (90.5 ± 6.8 vs 88.8 ± 7.3, P > 0.05). There was no complication and no arthroscopic revision in either group until 2-year follow-up. CONCLUSION: Although abductive force recovery of the hip was delayed, hip arthroscopy combined with endoscopic ITB release addressed hip snapping in patients with both FAI syndrome and ESH, and could get similar functional improvement, pain relief, recovery speed, as well as patient satisfaction compared with the pure hip arthroscopy in FAI syndrome patients without ESH.

Inflammation, angiogenesis and sensory nerve sprouting in the synovium of bony ankylosed and not bony ankylosed knees with end-stage haemophilic arthropathy.

INTRODUCTION: Clinical practice showed that patients with haemophilia (PwH) with bony ankylosed end-stage haemophilic arthropathy knees reported milder pain than those with not bony ankylosed knees. AIM: To compare the differences in pain sensation and the histopathological differences in synovial samples of affected knee joints between PwH with bony ankylosed end-stage haemophilic arthropathy knees and those with not bony ankylosed knees. METHODS: From January 2011 to December 2019, the synovial samples of knee joints were collected during total knee arthroplasty (TKA) surgery for end-stage haemophilic arthropathy. The visual analogue scale (VAS, 0-10) pain score was reviewed from the chart data of the patients. The thickness of the inner layer of the synovium in haematoxylin and eosin (H&E) staining sections was measured. The expression levels of Ki67, IL-1ß, TNF-α, CD31, VEGF, NGF and PGP9.5 in the synovium were detected by immunohistochemistry (IHC) method. RESULTS: Fifty-two end-stage haemophilic arthropathy knee synovial samples from 36 male PwH (34 type A and 2 type B) were collected. Fifteen knees had bony ankylosed (BA-group), and 37 were not bony ankylosed (Not-BA-group). The mean age of patients at TKA surgery of BA-group and Not-BA-group was 32 years (15) and 32 years (10), respectively (p = 0.824). Before TKA surgery, the mean VAS pain scores of patients in the Not-BA-group were significantly higher than those in the BA-group (p < 0.001). The mean thickness of the inner layer of the synovium, the mean rate of Ki67+ cells, the mean density of CD31+ vascular endothelial cells and the expression levels of IL-1ß, TNF-α, VEGF and NGF in samples in the Not-BA-group was significantly higher than those in samples in the BA-group (p < 0.001, p = 0.02, p = 0.001, p = 0.117, p < 0.001, p = 0.003 and p = 0.008), respectively. The mean density of PGP9.5+ sensory neural fibres in the Not-BA-group was slightly higher than in the BA-group (p = 0.131). Linear regression analysis showed a significant positive correlation between the VAS pain score and indicators including the synovial thickness, the rate of Ki67+ cells, the expression level of IL-1ß, TNF-α, VEGF, NGF and the densities of CD31+ vascular endothelial cells and PGP9.5+ nerve fibres (p < 0.05). CONCLUSIONS: Worsened hypertrophic synovitis, angiogenesis and sensory nerve sprouting in the synovium may play a critical role in causing worse pain sensation in PwH with not bony ankylosed haemophilic arthropathy knees than in those with bony ankylosed knees.

Endoscopic Iliotibial Band Release During Hip Arthroscopy for Femoroacetabular Impingement Syndrome and External Snapping Hip Had Better Patient-Reported Outcomes: A Retrospective Comparative Study.

PURPOSE: To compare patient-reported outcomes (PROs) in patients with femoroacetabular impingement (FAI) syndrome and external snapping hip (ESH) treated with hip arthroscopy with or without endoscopic iliotibial band (ITB) release. METHODS: Retrospective review case series with both FAI syndrome and ESH who underwent surgical treatment under same indications. According to the primary operation that was determined by patients themselves, the patients undergoing ITB release during hip arthroscopy for FAI syndrome were enrolled in the ITB-R group, and patients undergoing hip arthroscopy without ITB release were enrolled in non-ITB-R group. Patients with dysplasia, severe osteoarthritis, revision, and bilateral surgery were excluded. PROs including international Hip Outcome Tool (iHOT-33), modified Harris Hip Score (mHHS), visual analog scale for pain (VAS-pain) and VAS-satisfaction, and the rates of achieving minimal clinically important difference, patient acceptable symptomatic state (PASS), and substantial clinical benefit for the PROs at 2 years operatively were comparative analyzed. RESULTS: The prevalence of ESH in patients with FAI syndrome who underwent hip arthroscopy in our institution was 4.9% (30 of 612 hips). The mean age at the time of surgery was 33.1 ± 6.9 years (range 22-48 years). After exclusion, 16 patients (16 hips) were enrolled into ITB-R group and 11 patients (11 hips) enrolled into non-ITB-R group. PROs including iHOT-33, mHHS, VAS-pain, and VAS-satisfaction in patients in ITB-R group were better than that in non-ITB-R group at 2 years postoperatively (P = .013, .016, .002, and .005, respectively). The rates of achieving PASS for mHHS, PASS for VAS-pain, and substantial clinical benefit for iHOT-33 of patients in ITB-R group were significantly better than that in non-ITB-R group (P = .009, .006, and .027, respectively). CONCLUSIONS: Patients with both FAI syndrome and ESH undergoing ITB release during hip arthroscopy had better PROs than those undergoing hip arthroscopy without ITB release. LEVEL OF EVIDENCE: Level III, retrospective comparative study.

Pathological mechanism of joint destruction in haemophilic arthropathy.

Haemophilic arthropathy (HA), caused by intra-articular haemorrhage, is one of the most common complications in patients with haemophilia. Factor replacement therapy provides missing coagulation factors to prevent children with haemophilia from joint bleeding and decreases their risk for HA. However, haemophilia patients in developing countries are still suffering from HA due to insufficient replacement therapy. Symptoms such as pain and activity limitations caused by HA seriously affect the functional abilities and quality of life of patients with HA, causing a high disability rate in the haemophilia cohort. The pathological mechanism of HA is complicated because the whole pathological mainly involves hypertrophic synovitis, osteopenia, cartilage and bone destruction, and these pathological changes occur in parallel and interact with each other. Inflammation plays an important role in the whole complex pathological process, and iron, cytokines, growth factors and other factors are involved. This review summarizes the pathological mechanism of HA to provide background for clinical and basic research.

Evaluation of Long-Time Decoction-Detoxicated Hei-Shun-Pian (Processed Aconitum carmichaeli Debeaux Lateral Root With Peel) for Its Acute Toxicity and Therapeutic Effect on Mono-Iodoacetate Induced Osteoarthritis.

BACKGROUND: As a degenerative joint disease with severe cartilage destruction and pain, osteoarthritis (OA) has no satisfactory therapy to date. In traditional Chinese medicine (TCM), Aconitum carmichaeli Debeaux derived Hei-shun-pian (Hsp) has been developed for joint pain treatment. However, it causes adverse events in OA patients. Long-time decoction has been traditionally applied to reduce the aconite toxicity of Hsp and other aconite herbs, but its detoxifying effect is uncertain. METHODS: Hsp was extracted with dilute decoction times (30, 60, and 120 min) and evaluated by toxicological, chemical, pharmacological assays. Acute toxicity assay and chemical analysis were employed to determine the toxicity and chemoprofile of Hsp extracts, respectively. Since the detoxified Hsp (dHsp) was defined, its therapeutic effect was evaluated by using an OA rat model induced by monosodium iodoacetate. dHsp at 14 g/kg was orally administered for 28 days, and the pain assessments (mechanical withdrawal threshold and thermal withdrawal latency) and histopathological analyses (HE and safranin-O staining) were performed. Real-time PCR (qPCR) was applied to determine the molecular actions of dHsp on cartilage tissue and on chondrocytes. MTT assay was conducted to evaluate the effect of dHsp on the cell viability of chondrocytes. The cellular and molecular assays were also conducted to analyze the functions of chemical components in dHsp. RESULTS: The chemoprofile result showed that the contents of toxic alkaloids (aconitine, mesaconitine, and hypaconitine) were decreased but that of non-toxic alkaloids (benzoylaconitine, benzoylmesaconitine, and benzoylhypaconitine) were increased with increasing decoction time. Acute toxicity assay showed that only Hsp extract with 120 min decoction was non-toxic within the therapeutic dose range. Thus, it was defined as dHsp for further experiment. In OA experiment, dHsp significantly attenuated joint pain and prevented articular degeneration from MIA attack. qPCR data showed that dHsp restored the abnormal expressions of Col10, Mmp2, Sox5, Adamts4/5/9, and up-regulated Col2 expression in rat cartilage. In vitro, dHsp-containing serum significantly proliferated rat chondrocytes and regulated the gene expressions of Col2, Mmp1, Adamts9, and Aggrecan in a similar way as the in vivo data. Moreover, aconitine, mesaconitine, and hypaconitine exerted cytotoxic effects on chondrocytes, while benzoylaconitine and benzoylhypaconitine except benzoylmesaconitine exhibited similar molecular actions to dHsp, indicating contributions of benzoylaconitine and benzoylhypaconitine to dHsp. CONCLUSIONS: This study defined dHsp and demonstrated dHsp as a potential analgesic and disease modifying agent against OA with molecular actions on the suppression of chondrocyte hypertrophy and extracellular matrix degradation, providing a promising TCM candidate for OA therapy.

Acute Synovitis after Trauma Precedes and is Associated with Osteoarthritis Onset and Progression.

Osteoarthritis (OA) is a whole-joint disease characterized by cartilage destruction, subchondral bone sclerosis, osteophyte formation, and synovitis. However, it remains unclear which part of the joint undergoes initial pathological changes that drives OA onset and progression. In the present study, we investigated the longitudinal alterations of the entire knee joint using a surgically-induced OA mouse model. Histology analysis showed that synovitis occurred as early as 1 week after destabilization of the medial meniscus (DMM), which preceded the events of cartilage degradation, subchondral sclerosis and osteophyte formation. Importantly, key pro-inflammatory cytokines such as IL-1ß, IL-6, TNFα, and Ccl2, major matrix degrading enzymes including Adamts4, Mmp3 and Mmp13, as well as nerve growth factor (NGF), all increased significantly in both synovium and articular cartilage. It is notable that the inductions of these factors in synovium are far more extensive than those in articular cartilage. Results from behavioral tests demonstrated that sensitization of knee joint pain developed after 8 weeks, later than histological and molecular changes. In addition, the nanoindentation modulus of the medial tibiae decreased 4 weeks after DMM surgery, simultaneous with histological OA signs, which is also later than appearance of synovitis. Collectively, our data suggested that synovitis precedes and is associated with OA, and thus synovium may be an important target to intervene in OA treatment.

Silent Mating Type Information Regulation 2 Homolog (SIRT1) Influences Osteogenic Proliferation and Differentiation of MC3T3-E1 Cells via Regulation of miR-132-3p.

BACKGROUND The essence of osteoporosis is mainly the imbalance of bone formation and absorption. Previous studies indicated that SIRT1 is closely related to bone metabolism and bone mass as a regulator of bone mass. The literature reports that microRNAs are significant regulators of osteoblast proliferation and differentiation. MATERIAL AND METHODS In this study, SIRT1 protein and mRNA levels were examined by Western blot and RT-PCR. Osteogenic proliferation was examined by CCK8 assay and osteogenic markers, including ALP, OCN, and RUNX2, were examined by ELISA. The target of miR-132-3p was identified by luciferase reporter assay. RESULTS LPS downregulated the SIRT1 protein level and ß-glycerophosphate upregulated the SIRT1 protein level. The results demonstrated that SIRT1 overexpression promoted the proliferation and differentiation in MC3T3-E1 cells, and SIRT1 interference had the opposite effect. Luciferase reporter assay revealed that miR-132-3p inhibited the reporter gene activity of SIRT1. LPS upregulated the mRNA level of miR-132-3p, and ß-glycerophosphate downregulated the mRNA level of miR-132-3p. CONCLUSIONS miR-132-3p is a pivotal regulator in osteogenic proliferation and differentiation by targeting SIRT1.

Deletion of Runx2 in condylar chondrocytes disrupts TMJ tissue homeostasis.

Runt-related transcription factor-2 (Runx2) is essential for chondrocyte maturation during cartilage development and embryonic mandibular condylar development. The process that chondrocytes, especially a subgroup of hypertrophic chondrocytes (HC), could transform into bone cells in mandibular condyle growth makes chondrocytes crucially important for normal endochondral bone formation. To determine whether Runx2 regulates postnatal condylar cartilage growth and tissue homeostasis, we deleted Runx2 in chondrocytes in postnatal mice and assessed the consequences on temporomandibular joint (TMJ) cartilage growth and remodeling. The cell lineage tracing data provide information demonstrating the role of chondrocytes in subchondral bone remodeling. The histologic and immunohistochemical data showed that Runx2 deficiency caused condylar tissue disorganization, including loss of HC and reduced hypertrophic zone, reduced proliferative chondrocytes, and decreased cartilage matrix production. Expression of Col10a1, Mmp13, Col2a1, Aggrecan, and Ihh was significantly reduced in Runx2 knockout mice. The findings of this study demonstrate that Runx2 is required for chondrocyte proliferation and hypertrophy in TMJ cartilage and postnatal TMJ cartilage growth and homeostasis, and that Runx2 may play an important role in regulation of chondrocyte-derived subchondral bone remodeling.

Activation of ß-catenin signaling in aggrecan-expressing cells in temporomandibular joint causes osteoarthritis-like defects.

ß-Catenin plays a critical role in cartilage formation and development. To further understand the role of ß-catenin in osteoarthritis (OA) development in temporomandibular joint (TMJ), we have generated ß-catenin conditional activation mice (ß-cat(ex3) Agc1CreER ) by breeding Agc1-CreER mice with ß-cateninflox(ex3)/+ mice. Results of histologic analysis showed the progressive TMJ defects in 3- and 6-month-old ß-cat(ex3) Agc1CreER mice (tamoxifen induction was performed at 2 weeks of age), including decreased chondrocyte numbers in the superficial layer associated with less Alcian blue staining, increased numbers of hypertrophic chondrocytes in deep layers, and rough articular surface. Compared to the TMJ phenotype of ß-cat(ex3) Col2CreER mice, ß-cat(ex3) Agc1CreER mice showed much severe morphological defects in the superficial layer of TMJ. This may reflect that Agc1-CreER mice could efficiently target cells in the superficial layer of TMJ. Results of immunostaining showed significantly increased expression of MMP13, Col-X, Adamts4, and Adamts5 in TMJ of ß-cat(ex3) Agc1CreER mice. Results of proliferating cell nuclear antigen (PCNA), Ki67, and terminal deoxinucleotidyl transferase-mediated dUTP-fluorescein nick end labeling (TUNEL) staining further demonstrated that cell proliferation was decreased and cell apoptosis was increased in condylar cartilage of ß-cat(ex3) Agc1CreER mice. Our findings indicate that abnormal upregulation of ß-catenin in TMJ leads to defects assembling to OA-like phenotype, further demonstrating that ß-catenin plays a critical role in TMJ pathogenesis.

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.

Administration of erythropoietin prevents bone loss in osteonecrosis of the femoral head in mice.

Long­term administration of glucocorticoid hormones is considered one of predominant pathological factors inducing osteonecrosis of the femoral head (ONFH) development and progression, in which reduction of blood supply leads to a progressive bone loss and impairment of bone structure in the majority of cases. In a non­hematopoietic system, erythropoietin (EPO) can stimulate angiogenesis and bone regeneration. However, the specific mechanism underlying the role of EPO in ONFH remains to be elucidated. Therefore, the purpose of this study was to determine the effect of EPO on the prevention of bone loss in ONFH. Male C57BL/6J mice 3 months old were divided into two groups: EPO group and control groups. ONFH was established by the administration prednisolone (PDS, 100 mg/kg) with co­treatment of lipopolysaccharide (LPS, 1 mg/kg). ONFH mice received recombinant mouse EPO (500 U/kg/day) or saline intramuscularly. The mice were sacrificed at 2, 4, 6 and 8 weeks following the initiation of treatment. Alterations in the general architecture and histomorphology of the right femoral head were determined by hematoxylin and eosin staining and micro computed tomography (micro­CT). The expression of runt­related transcription factor 2 (Runx2), osteocalcin, vascular endothelial growth factor (VEGF) and platelet endothelial cell adhesion molecule (CD31) in the femoral head was tested by immunohistochemistry. Terminal deoxynucleotidyl­transferase­mediated dUTP nick end labeling (TUNEL) assay was performed to detect apoptosis in femoral heads. Micro­CT data revealed that EPO significantly improved bone volume/total volume and bone mineral density following 6 and 8 weeks of treatment. Histological analysis further demonstrated that EPO treatment improved the arrangement of trabeculae, thinning of trabeculae and other fractures in femoral heads, especially following 6 and 8 weeks of treatment. Immunohistochemical analysis suggested that EPO treatment up­regulated the expressions of Runx2, osteocalcin, VEGF and CD31 at 4 and 8 weeks. The TUNEL apoptosis assay suggested that EPO intervention reduced apoptosis in avascular ONFH. Therefore, EPO prevents bone loss in ONFH in mice through enhancing Runx2­mediated osteogenesis, VEGF­mediated angiogenesis and inhibition of cell apoptosis.

Osthole Promotes Bone Fracture Healing through Activation of BMP Signaling in Chondrocytes.

Osthole is a bioactive coumarin derivative and has been reported to be able to enhance bone formation and improve fracture healing. However, the molecular mechanism of Osthole in bone fracture healing has not been fully defined. In this study we determined if Osthole enhances bone fracture healing through activation of BMP2 signaling in mice. We performed unilateral open transverse tibial fracture procedure in 10-week-old C57BL/6 mice which were treated with or without Osthole. Our previous studies demonstrated that chondrocyte BMP signaling is required for bone fracture healing, in this study we also performed tibial fracture procedure in Cre-negative and Col2-Cre;Bmp2flox/flox conditional knockout (KO) mice (Bmp2Col2Cre ) to determine if Osthole enhances fracture healing in a BMP2-dependent manner. Fracture callus tissues were collected and analyzed by X-ray, micro-CT (µCT), histology, histomorphometry, immunohistochemistry (IHC), biomechanical testing and quantitative gene expression analysis. In addition, mouse chondrogenic ATDC5 cells were cultured with or without Osthole and the expression levels of chondrogenic marker genes were examined. The results demonstrated that Osthole promotes bone fracture healing in wild-type (WT) or Cre- control mice. In contrast, Osthole failed to promote bone fracture healing in Bmp2Col2Cre conditional KO mice. In the mice receiving Osthole treatment, expression of cartilage marker genes was significantly increased. We conclude that Osthole could promote bone strength and enhance fracture healing by activation of BMP2 signaling. Osthole may be used as an alternative approach in the orthopaedic clinic for the treatment of fracture healing.

Deletion of Runx2 in Articular Chondrocytes Decelerates the Progression of DMM-Induced Osteoarthritis in Adult Mice.

Runx2 may play an important role in development of osteoarthritis (OA). However, the specific role of Runx2 in articular chondrocyte function and in OA development in adult mice has not been fully defined. In this study, we performed the destabilization of the medial meniscus (DMM) surgery at 12-week-old mice to induce OA in adult Runx2 Agc1CreER mice, in which Runx2 was specifically deleted in Aggrecan-expressing chondrocytes by administering tamoxifen at 8-weeks of age. Knee joint samples were collected 8- and 12-weeks post-surgery and analyzed through histology, histomorphometry and micro-computed tomography (µCT). Our results showed that severe OA-like defects were observed after DMM surgery in Cre-negative control mice, including articular cartilage degradation and subchondral sclerosis, while the defects were significantly ameliorated in Runx2 Agc1CreER KO mice. Immunohistochemical (IHC) results showed significantly reduced expression of MMP13 in Runx2 Agc1CreER KO mice compared to that in Cre-negative control mice. Results of quantitative reverse-transcription PCR (qRT-PCR) demonstrated that expression of the genes encoding for matrix degradation enzymes was significantly decreased in Runx2 Agc1CreER KO mice. Thus, our findings suggest that inhibition of Runx2 in chondrocytes could at least partially rescue DMM-induced OA-like defects in adult mice.

Runx2 and microRNA regulation in bone and cartilage diseases.

The homeostasis of skeletal tissues requires tight regulation of a variety of signaling pathways, and the onset and progression of skeletal diseases are often caused by signaling abnormalities. MicroRNAs (miRNAs) are short noncoding RNA molecules that have emerged as a new dimension of gene regulation. MiRNAs have been shown to play an important role in the regulation of the differentiation of embryonic and hematopoietic stem cells. However, the role of specific miRNAs and their target genes has not been fully defined in the regulation of mesenchymal stem cells. Runx2 is a key transcription factor controlling MSC differentiation and bone and cartilage function. This article reviews work on Runx2 and miRNA regulation in bone and cartilage diseases.

The Fate and Distribution of Autologous Bone Marrow Mesenchymal Stem Cells with Intra-Arterial Infusion in Osteonecrosis of the Femoral Head in Dogs.

This study aimed to investigate if autologous bone marrow mesenchymal stem cells (MSCs) could treat osteonecrosis of the femoral head (ONFH) and what the fate and distribution of the cells are in dogs. Twelve Beagle dogs were randomly divided into two groups: MSCs group and SHAM operated group. After three weeks, dogs in MSCs group and SHAM operated group were intra-arterially injected with autologous MSCs and 0.9% normal saline, respectively. Eight weeks after treatment, the necrotic volume of the femoral heads was significantly reduced in MSCs group. Moreover, the trabecular bone volume was increased and the empty lacunae rate was decreased in MSCs group. In addition, the BrdU-positive MSCs were unevenly distributed in femoral heads and various vital organs. But no obvious abnormalities were observed. Furthermore, most of BrdU-positive MSCs in necrotic region expressed osteocalcin in MSCs group and a few expressed peroxisome proliferator-activated receptor-γ (PPAR-γ). Taken together, these data indicated that intra-arterially infused MSCs could migrate into the necrotic field of femoral heads and differentiate into osteoblasts, thus improving the necrosis of femoral heads. It suggests that intra-arterial infusion of autologous MSCs might be a feasible and relatively safe method for the treatment of femoral head necrosis.

Combination treatment of biomechanical support and targeted intra-arterial infusion of peripheral blood stem cells mobilized by granulocyte-colony stimulating factor for the osteonecrosis of the femoral head: a randomized controlled clinical trial.

The objective of this study was to determine the benefits of combination treatment with mechanical support and targeted intra-arterial infusion of peripheral blood stem cells (PBSCs) mobilized by granulocyte-colony stimulating factor (G-CSF) via the medial circumflex femoral artery on the progression of osteonecrosis of the femoral head (ONFH). Fifty-five patients (89 hips) with early and intermediate stage ONFH were recruited and randomly assigned to combination treatment or mechanical support treatment (control group). All hips received mechanical support treatment (porous tantalum rod implantation). Then, hips in the combination treatment group were performed targeted intra-arterial infusion of PBSCs. At each follow-up, Harris hip score (HHS) and Association Research Circulation Osseous (ARCO) classification were used to evaluate the symptoms and progression of osteonecrosis. Total hip arthroplasty (THA) was assessed as an endpoint at each follow-up. At 36 months, 9 of the 41 hips (21.95%) in the control group progressed to clinical failure and underwent THA whereas only 3 of the 48 hips (6.25%) in the combination treatment group required THA (p = 0.031). Kaplan-Meier survival analysis showed a significant difference in the survival time between the two groups (log-rank test; p = 0.025). Compared to the control group, combination treatment significantly improved the HHS at 36 months (p = 0.003). At the final follow-up examination, radiological progression was noted in 13 of 41 hips (31.71%) for the control group, but in only 4 of 48 hips (8.33%) for the combination treatment group (p = 0.005). The overall collapse rates were 15.15% (5/33 hips) and 8.11% (3/37 hips) in the control and combination treatment groups, respectively. Targeted intra-arterial infusion of PBSCs is capable of enhancing the efficacy of biomechanical support in the treatment of ONFH. This clinical trial confirmed that the combination treatment might be a safe and feasible choice for the treatment of early or intermediate stages of ONFH.