Peijian Tong

Chondrocyte BMP2 signaling plays an essential role in bone fracture healing.

The specific role of endogenous Bmp2 gene in chondrocytes and in osteoblasts in fracture healing was investigated by generation and analysis of chondrocyte- and osteoblast-specific Bmp2 conditional knockout (cKO) mice. The unilateral open transverse tibial fractures were created in these Bmp2 cKO mice. Bone fracture callus samples were collected and analyzed by X-ray, micro-CT, histology analyses, biomechanical testing and gene expression assays. The results demonstrated that the lack of Bmp2 expression in chondrocytes leads to a prolonged cartilage callus formation and a delayed osteogenesis initiation and progression into mineralization phase with lower biomechanical properties. In contrast, when the Bmp2 gene was deleted in osteoblasts, the mice showed no significant difference in the fracture healing process compared to control mice. These findings suggest that endogenous BMP2 expression in chondrocytes may play an essential role in cartilage callus maturation at an early stage of fracture healing. Our studies may provide important information for clinical application of BMP2.

Anti-DKK1 antibody promotes bone fracture healing through activation of β-catenin signaling

In this study we investigated if Wnt/β-catenin signaling in mesenchymal progenitor cells plays a role in bone fracture repair and if DKK1-Ab promotes fracture healing through activation of β-catenin signaling. Unilateral open transverse tibial fractures were created in CD1 mice and in β-catenin(Prx1ER) conditional knockout (KO) and Cre-negative control mice (C57BL/6 background). Bone fracture callus tissues were collected and analyzed by radiography, micro-CT (μCT), histology, biomechanical testing and gene expression analysis. The results demonstrated that treatment with DKK1-Ab promoted bone callus formation and increased mechanical strength during the fracture healing process in CD1 mice. DKK1-Ab enhanced fracture repair by activation of endochondral ossification. The normal rate of bone repair was delayed when the β-catenin gene was conditionally deleted in mesenchymal progenitor cells during the early stages of fracture healing. DKK1-Ab appeared to act through β-catenin signaling to enhance bone repair since the beneficial effect of DKK1-Ab was abrogated in β-catenin(Prx1ER) conditional KO mice. Further understanding of the signaling mechanism of DKK1-Ab in bone formation and bone regeneration may facilitate the clinical translation of this anabolic agent into therapeutic intervention.

The efficacy of targeted intraarterial delivery of concentrated autologous bone marrow containing mononuclear cells in the treatment of osteonecrosis of the femoral head: a five year follow-up study.

OBJECTIVEnTo investigate the efficacy and safety of targeted delivery of autologous bone marrow mononuclear cells (BMMCs), which are highly enriched with mesenchymal stem cells (BMMSCs), via medial circumflex femoral artery in the treatment of osteonecrosis of the femoral head (ONFH).nnnMETHODSn62 patients (78 hips) with ONFH were recruited in this study. All of these patients were treated with BMMCs perfusion via medial circumflex femoral artery. The concentrated BMMCs (30-60ml) were gained from autologous bone marrow (100-200ml) harvested from anterior iliac crest and then were intra-arterially perfused into the femoral head. Ficat stage was used to classify the radiological stage of ONFH. Harris hip score was used to evaluate the clinical symptoms of osteonecrosis. Ficat stage and Harris hip scores were assessed at onset of treatment at 6, 12, 24, 36, 48 and 60months after the initial treatment. Total hip arthroplasty (THA) was also assessed as an endpoint at each follow-up.nnnRESULTSnA follow-up on the patient was done at the end of five years, and 92.31% (72 of 78) of hips achieved a satisfactory clinical result while only 6 hips (7.69%) progressed to clinical failure and required THA. Radiological progression was noted in 34 of 78 hips (43.59%); the overall rate of collapse was 38.24% (26 of 68 hips) in stage-I and stage-II hip combinations and 12.5% (2 of 16) in stage-I hips and 46.15% (24 of 52) in stage-II hips. The mean time of conversion to THA was 3years (1 to 5years) and the average time to collapse were 3.5years (1-5years). The mean Harris hip score increased from 59 points at baseline to 75 points at 12months, 82 points at 24months, 81 points at 36months, 79 points at 48months and 74 points at 60months. Five years after the treatment, 3 of 10 hips (30%) in stage-III had deteriorated to clinical failure whereas only 3 of 68 hips (4.41%) in stage-I and II combination had progressed to clinical failure (p<0.05). Kaplan-Meier survival analysis showed a significant difference in the time to failure between the pre-collapse hips (Ficat stage-I and II) and the post-collapse hips (Ficat stage-III) at five years follow-up (Log-rank test; p<0.01). No complication was found in any patients.nnnCONCLUSIONSnAutologous BMMSC perfusion via the medial circumflex femoral artery can relieve symptoms, improve hip function and delay the progression of ONFH. The clinical outcome is better when it is applied prior to the collapse. This work demonstrates that autologous BMMSC perfusion via the medial circumflex femoral artery is a safe, effective and minimally invasive treatment strategy for early-stage ONFH.

Development and assessment of a complete-detoxication strategy for Fuzi (lateral root of Aconitum carmichaeli) and its application in rheumatoid arthritis therapy

ETHNOPHARMACOLOGICAL RELEVANCEnFuzi (lateral root of Aconitum carmichaeli) is a popular traditional Chinese medicine well known for its both therapeutic and high-toxic activities. Its toxic alkaloid ingredients, mainly aconitine, mesaconitine, and hypaconitine, are responsible for the high toxicity. However, to date, no detoxication strategy is available to completely eliminate Fuzis toxicity, and, whether Fuzis efficacy could be kept after detoxication, remain unknown and debatable.nnnMATERIALS AND METHODSnThe purpose of this study was to establish and validate a complete-detoxication strategy for Fuzi via acute toxicity test, to clarify the detoxication mechanism by HPLC and titrimetric analyses, and to evaluate the therapeutic effect of detoxicated Fuzi on adjuvant arthritis (AA). Three processed Fuzi (Bai-fu-pian) with 30-min, 60-min, and 120-min decoctions, respectively, named dBfp-30, dBfp-60, and dBfp-120, were prepared for this study. For the acute toxicity test, their oral doses to male and female Kunming mice were up to 70-190g/kg body weight, and their toxicological profiles were evaluated by median lethal dose (LD50), maximal tolerance dose (MTD), minimal lethal dose (MLD), no-observed-adverse-effect-level (NOAEL), and time-concentration-mortality (TCM) modeling methods using a 14-day schedule with up to five doses. The HPLC analysis was performed to determine the detoxication-induced changes in composition and amount of aconitine, mesaconitine and hypaconitine in Fuzi, whilst the titrimetric method was adopted to estimate the amount changes of Fuzis total alkaloids. AA model was established by incomplete Freunds adjuvant injection in Wistar rats, and the animals physiological (body weight, food intake, etc.), clinical (hind paw volume), and immunological (IL-1 and TNF-α) parameters were assessed as markers of inflammation and arthritis.nnnRESULTSnWith increasing decoction time, the acute toxicity of detoxicated Fuzi became decreased in the following order: dBfp-30 (LD50 of 145.1g/kg; MTD of 70g/kg; MLD of 100g/kg; NOAEL of 70g/kg) >dBfp-60 (too large LD50; MTD of 160g/kg; MLD of 190g/kg; NOAEL of 100g/kg) >dBfp-120 (no LD50; unlimited MTD; unlimited MLD; NOAEL of 130g/kg). dBfp-30 and dBfp-60 displayed the toxicity at a dose-dependent manner with maximum mortalities reaching 100% and 50% respectively, whereas no mortality or signs of intoxication was induced by dBfp-120. The chemical analyses revealed a dramatic reduction of the toxic alkaloids as well as total alkaloids in Fuzi after the detoxication, from which no level of aconitine and only minimum residual of mesaconitine (0.56±0.02μg/g) and hypaconitine (8.73±0.13μg/g) were detected in dBfp-120. However, no significant difference of total alkaloid amount was found among dBfp-30, dBfp-60, and dBfp-120 (P>0.05), suggesting an equivalent conversion from toxic alkaloids to its non-toxic derivants in dBfp-120. Further, also no significant differences were seen among dBfp-30, dBfp-60, and dBfp-120 for the therapeutic effects on physiological, clinical, and immunological parameters in AA rat, indicating that dBfp-120 is of non-toxicity and efficacy.nnnCONCLUSIONSnA complete-detoxication strategy has been developed successfully for ensuring the safe and effective use of Fuzi. The detoxication mechanism associated with elimination of toxic alkaloids has kept Fuzis efficacy, indicating a non-interdependent relationship between its efficacy and toxicity. This is the first report on such an optimal detoxication strategy and on the application of detoxicated Fuzi in AA. It may provide in depth understanding to the toxicological and pharmacological profiles of Fuzi and further benefit the herbal drug development with safety and efficacy for disease especially RA therapy.

Osteoarthritis Pathogenesis: A Review of Molecular Mechanisms

Osteoarthritis (OA), the most prevalent chronic joint disease, increases in prevalence with age, and affects majority of individuals over the age of 65 and is a leading musculoskeletal cause of impaired mobility in the elderly. Because the precise molecular mechanisms which are involved in the degradation of cartilage matrix and development of OA are poorly understood and there are currently no effective interventions to decelerate the progression of OA or retard the irreversible degradation of cartilage except for total joint replacement surgery. In this paper, the important molecular mechanisms related to OA pathogenesis will be summarized and new insights into potential molecular targets for the prevention and treatment of OA will be provided.

The effects of Liuwei Dihuang on canonical Wnt/β-catenin signaling pathway in osteoporosis.

ETHNOPHARMACOLOGICAL RELEVANCEnThe Liuwei Dihuang (LWDH), a wellknown classic traditional Chinese medicine formula, consists of six herbs including Rehmannia glutinosa Libosch. (family: Scrophulariaceae), Cornus officinalis Sieb. (family: Cornaceae), Dioscorea opposite Thunb. (family: Dioscoreaceae), Alisma orientale (G. Samuelsson) Juz (family: Alismataceae), Poria cocos (Schw.) Wolf (family: Polyporaceae) and Paeonia suffruticosa Andrews (family: Paeoniaceae). It has been used clinically in the treatment of many types of diseases with signs of deficiency of Yin in the kidneys for more than 1000 years in China. The purpose of this study was to observe the effects of LWDH on canonical Wnt/β-catenin signaling pathway in osteoporosis.nnnMATERIALS AND METHODSnOsteoporosis model was induced by ovariectomy (OVX) in 8-week-old female Sprague-Dawley (SD) rats. After 12 weeks of treatment with LWDH by intragastric administration, the rats were put to death in batch. The changes of alkaline phosphatase (ALP), osteocalcin (BGP) and estradiol (E2) in serum were determined, bone mineral density (BMD) and histomorphology of right femur were observed, biomechanics of lumbar vertebra were measured, and the expression of Lrp-5, β-catenin, Runx2, Osx involving the canonical Wnt/β-catenin signaling pathway were detected by RT-PCR. In addition, osteoblasts isolated from neonatal rat calvariae were used in this study to investigate the effects of LWDH on the canonical Wnt/β-catenin signaling pathway. Cell proliferation and differentiation were observed by the MTT test, ALP activity and calcified nodules. The expression of Lrp-5, β-catenin, Runx2, Osx mRNA of cells were also detected. All the data were analyzed by SPSS 13.0.nnnRESULTSnTwelve weeks of treatment with LWDH could significantly decrease the level of ALP and BGP in serum, increase the BMD of femurs, and improve the biomechanical capabililty of vertebral body in maximum loading and elastic modulus. Concerning histomorphology, we found ordered arrangement of trabeculae, slightly thinning of trabeculae and none obvious slight fractures in femurs after twelve weeks of treatment with LWDH. In osteoblast, serum containing LWDH elicited significantly increase in cell viability (at day 6), alkaline phosphatase activity (at days 2, 4 and 6) and amount of calcified nodules. The expression of Lrp-5, β-catenin, Runx2 and Osx involved in the canonical Wnt/β-catenin signaling pathway were significantly up-regulated in the presence of LWDH both in vivo and in vitro experiment.nnnCONCLUSIONSnOur results suggest that Liuwei Dihuang could alleviate osteoporosis induced by ovariectomy, in part, through up-regulation of canonical Wnt/β-catenin signaling pathway of osteoblast.

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 (pu2009=u20090.031). Kaplan‐Meier survival analysis showed a significant difference in the survival time between the two groups (log‐rank test; pu2009=u20090.025). Compared to the control group, combination treatment significantly improved the HHS at 36 months (pu2009=u20090.003). 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 (pu2009=u20090.005). 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.

Epimedium-derived flavonoids modulate the balance between osteogenic differentiation and adipogenic differentiation in bone marrow stromal cells of ovariectomized rats via Wnt/β-catenin signal pathway activation

ObjectiveTo observe the function of wnt/β-catenin signal pathway on the process that epimedium-derived flavonoids (EFs) regulate the balance between osteogenic differentiation and adipogenic differentiation in bone marrow stromal cells of ovariectomized rats, and to provide an experimental evidence for the mechanism of EFs on treating postmenopausal osteoporosis.MethodsBone marrow stromal cells from ovariectomized rats were separated and cultivated in the condition of osteoinductive medium or liquid medium for 15 days. Low- (1 μg/mL), medium- (10 μg/mL) and high- (100 μg/mL) dose EFs were administrated correspondingly. Alkaline phosphatase (ALP) staining, ALP activity determination, oil red O staining and realtime polymerese chain reaction (RT-PCR) were used to determine the effect of EFs on osteogenic differentiation and adipogenic differentiation in bone marrow stromal cells of ovariectomized rats. Moreover, in order to explore the mechanism of EFs on osteogenic differentiation and adipogenic differentiation in bone marrow stromal cells of ovariectomized rats, Dickkopf-related protein 1 (DKK1) was used in the medium group. Enzymelinked immunosorbent assay (ELISA) and RT-PCR were used to determine mRNA levels of β-catenin, low density lipoprotein receptor-related protein 5 (LRP5) and T cell factor (TCF) protein, known as wnt/β-catenin signal pathway related factors.ResultsEFs increased mRNA expression levels of ALP and early osteoblast differentiation factors, such as runt-related transcription factor 2 (Runx2), osteocalcin and collagen I, and decreased mRNA expression levels of fat generation factors, such as peroxisome proliferator activated receptor gamma 2 (PPARγ-2) and CCAAT enhancer-binding protein-α (C/EBPα) in a dose-dependent manner. While osteoblast differentiation factors were down-regulated, fat generation factors were up-regulated when DKK1 was applied. Also EFs up-regulated mRNA expression levels of β-catenin, LRP5 and TCF protein which could be blocked by DKK1.ConclusionEFs regulate the balance between osteogenic differentiation and adipogenic differentiation in bone marrow stromal cells of ovariectomized rats by activating wnt/β-catenin signal pathway, which may be an important molecular mechanism of EFs on treating postmenopausal osteoporosis.

Leptin promotes apoptosis and inhibits autophagy of chondrocytes through upregulating lysyl oxidase-like 3 during osteoarthritis pathogenesis

OBJECTIVEnLeptin has been found highly expressed in human osteoarthritis. We aimed to explore the possible effects and mechanisms of leptin on the apoptosis and autophagy of chondrocytes during osteoarthritis pathogenesis.nnnMETHODSnGene expression profile from osteoarthritis affected and preserved cartilage were downloaded from NCBIs Gene Expression Omnibus database (GSE57218). Lysyl oxidase-like 3 (LOXL3) mRNA expression in cartilage tissues and leptin concentration in joint synovial fluid (SF) was measured in samples from 45 osteoarthritis patients and 25 healthy donors by real-time PCR and radioimmunoassay, respectively. Rat osteoarthritis model was induced by anterior cruciate ligament transection (ACLT). The expression of apoptosis regulators and autophagy markers were detected by Western blot. Cell survival and cell apoptosis were identified by CCK-8 and flow cytometry, respectively.nnnRESULTSnRe-analysis on GSE57218 indicated that LOXL3 mRNA was upregulated in osteoarthritis affected cartilage. LOXL3 mRNA was upregulated in osteoarthritis patients, which was positively correlated with SF leptin concentration. Similar results were obtained in rat osteoarthritis model. Moreover, ACLT surgery led to a significant increase in the protein levels of cleaved caspase 3, and a notable decrease in the protein levels of Bcl-2, LC3 II/LC3 I and Beclin1. Silencing of LOXL3 in ACLT and leptin treated primary chondrocytes significantly inhibited cell apoptosis, and promoted cell proliferation and autophagy. Moreover, overexpression of LOXL3 remarkably inhibited autophagy of chondrocytes via activating mTORC1.nnnCONCLUSIONSnLOXL3, a downstream of leptin, stimulated the apoptosis, but inhibited the autophagy of chondrocytes. LOXL3 is a potential therapy target for osteoarthritis.

Chondroprotective activity of a detoxicated traditional Chinese medicine (Fuzi) of Aconitum carmichaeli Debx against severe-stage osteoarthritis model induced by mono-iodoacetate.

ETHNOPHARMACOLOGICAL RELEVANCEnFuzi is an effective but toxic traditional Chinese medicine (TCM) derived from Aconitum carmichaeli. In our previous study, detoxicated Fuzi (d-Fuzi) has been originally developed with no toxicity but significant efficacy. However, whether d-Fuzi can be used for therapy of osteoarthritis (OA), remain unknown.nnnMATERIALS AND METHODSnSevere OA model was established by intra-articular mono-iodoacetate (MIA) injection (1.25mg) into rats and orally treated with 2g/ml d-Fuzi at a dosage of 7 ml/kg body weight for 28 days. In vivo, the articular radiographic and histopathologic analyses were performed to qualitatively assess the chondroprotective effect of d-Fuzi, followed by quantitative measurements of bone density and Mankin scores. In vitro, such effect on chondrocyte viability after MIA attack was evaluated. Hybrid quadrupole time-of-flight mass spectrometry (QTOF-MS) was performed for chemical analysis of d-Fuzi.nnnRESULTSnd-Fuzi was demonstrated to possess chondroprotective activity on MIA-induced OA model by in vivo preventing the articular degeneration and the reducing of bone density and Mankin score, as well as by in vitro promoting the chondrocyte proliferation and inhibiting the MIA-induced chondrocyte damage. A total of 23 compounds were identified in d-Fuzi, most of which were deduced as the non-toxic derivatives of aconite alkaloids.nnnCONCLUSIONSnThis is the first report regarding chondroprotective effect and chemical profile of d-Fuzi, originally revealing its great anti-OA potential and thereby providing a promising TCM candidate for OA therapy.