CD38 Drives Progress of Osteoarthritis by Affecting Cartilage Homeostasis.

OBJECTIVE: To observe expression of CD38, a key modulator of nicotinamide dinucleotide (NAD+) metabolism in mice with knee osteoarthritis, and protective effect of CD38 inhibition during the osteoarthritis (OA) development. METHOD: The destabilization of the medial meniscus (DMM) model was performed in mice to mimic the process of OA. Immunofluorescence of CD38 was performed to evaluate its response during the OA process. Limb bud-derived mesenchymal cells were isolated for micromass culture. 100 nM or 1 µM CD38 inhibitor (78c) treatment for 14 days and CD38 sgRNA infection were then used to explore the effects of chondrogenic differentiation via Alcian blue staining. The expressions of chondrogenic markers were detected using RT-PCR and Western blot. To explore the protective effect of CD38 inhibitor on cartilage degradation during OA in vivo, a CD38 inhibitor was injected into the knee joint after DMM operations. Micro-CT analysis and Safranin O-fast green staining were used to evaluate subchondral bone micro-architecture changes and cartilage degeneration. RESULTS: Compared to the control group, the CD38 expression in superficial cartilage was obviously increased in DMM group (P < 0.05). During the normal chondrogenic differentiation, the extracellular matrix formed and expression of Sox9, Col2, aggrecan increased apparently while CD38 expression decreased, which could be reversed with ablation of CD38 in limb bud-derived mesenchymal cells. Consistent with findings in vitro, CD38 blockage via CD38 inhibitor injection protected against osteosclerosis in medial subchondral bone and cartilage degeneration in DMM-induced experimental mice. Compared to the Sham group, DMM mice showed significantly increased values of BV and BV/TV in subchondral bone (P < 0.05) and Mankin score, which could be rescued by 78c treatment (P < 0.05). Also the CD38 inhibitor contributed to homeostasis of anabolism and catabolism by upregulating Sox9, Col2, aggrecan and downregulating Runx2, Col10 and Mmp13. CONCLUSION: This study primarily implicates CD38 as an important regulator of chondrogenic differentiation. Inhibition of CD38 demonstrated protection against cartilage degeneration, which suggests that CD38 could be a potential therapeutic target for OA.

[Progress on the treatment of adolescent idiopathic scoliosis].

Adolescent idiopathic scoliosis (AIS) is a common disease with the age of 10 to 18 years, the Cobb angle more than 10 ° on the coronal plane and combined with the rotation of the vertebral body without other organic lesions. The disease can lead to deformity, pain and even damage of cardiopulmonary function, which seriously affects the physical and mental health and quality of life of patients. For mild to moderate AIS patients, regular observation, braces and other conservative treatment methods can effectively delay the progress of scoliosis. For AIS patients whose conservative treatment is ineffective and reaches the surgical threshold, surgery is recommended. Currently, the prevalent surgical method is posterior vertebral body fusion represented by the pedicle screw internal fixation system, which can often achieve good clinical efficacy. In recent years, Physical Therapeutic Scoliosis Specific Exercise (PSSE) has become more and more popular because of its safety and effectiveness. At present, the specific indications for the treatment of AIS patients are gradually improving, the concept and technology of treatment are constantly updated, and the clinical efficacy is constantly improved. This article will start from two aspects of conservative treatment and surgical treatment, mainly describes the commonly used treatment methods in clinical progress and application as well as the problems faced, in order to provide a reference for the selection of clinical treatment.

A Comparative Study on the Health Status and Behavioral Lifestyle of Centenarians and Non-centenarians in Zhejiang Province, China-A Cross-Sectional Study.

Background: The growth rate of centenarians was unusually rapid in recent decades, ushering in an era of longevity. This study aims to explore the difference between centenarians and non-centenarians using quantitative research, and to scientifically guide residents to develop the correct lifestyle and health care ways. Methods: From October 2013 to August 2017. A cross-sectional survey was conducted on 271 centenarians and 570 non-centenarians by using a questionnaire to assess longevity and health issues which was developed for the needs of the study, who came from 29 counties and districts in 11 cities of Zhejiang province, China. Two hundred and fifty-five valid questionnaires were returned, with an effective response rate of 94.1%. Meanwhile, data of 526 non-centenarians from Zhejiang province was collected as a control group, with an effective response rate of 92.3%. Results: The prevalence rates of tumor, stomach and duodenal ulcer, diabetes, bronchial asthma, and chronic obstructive pulmonary disease, tuberculosis among centenarians were all lower than those among non-centenarians. The oral health of centenarians is better than that of non-centenarians. The consumption of coarse cereals, pasta, other staple foods and fruits among centenarians was higher than that of non-centenarians. The percentage of centenarians who smoke or engage in recreational activities every day was lower than that of non-centenarians. Conclusions: We should give full play to the role of preventive medicine and health management to safeguard the health of residents. Pay attention to oral health, and develop the good habit of loving teeth. The diet should be rich and varied, and increase the intake of grains and fruits. Give up smoking, limit alcohol, spirit-preserving with calming, follow the law of scientific regimen.

[Comparative study on infection rate of different adeno-associated virus for knee joint cartilage in mice].

OBJECTIVE: To explore infection rate of different adeno-associated virus (AAV) on knee joint cartilage in mice and to find a good gene editing tool for mice chondrocytes of knee joint. METHODS: Forty-five 4-week-old SPF C57BL/6 weighed(14.3±0.2) g were selected. According to different injections(6 µl) for right knee joint, mice were divided into 9 different groups, 5 mice in each group. The groups were such as following:control group (normal saline), Vigene 2 group (AAV2 from vigene biosciences, titer for 1×10¹³ vg/ml), Vigene 5 group (AAV5 from vigene biosciences, titer for 1×10¹³ vg/ml), Vigene 6 group (AAV6 from vigene biosciences, titer for 1×10¹³ vg/ml), Vigene 7 group (AAV7 from vigene biosciences, titer for 1×10¹³ vg/ml), Vigene 8 group (AAV8 from vigene biosciences, titer for 1×10¹³ vg/ml), Vigene 9 group (AAV9 from vigene biosciences, titer for 1×10¹³ vg/ml), Hanbio DJ group(AAV2-DJ from Hanbio, titer for 1×10¹² vg/ml), Hanbio 5 group (AAV5 from Hanbio, titer for 1×10¹² vg/ml). All AAVs were over-expressed green fluorescent protein(GFP). Knee joint specimens were taken and observed injury of cartilage under stereomicroscope at 30 days after injection, then 10 µm thick frozen sections were prepared. Distribution of green fluorescent protein of meniscus and cartilage of knee joint was observed under fluorescence microscope. RESULTS: Stereomicroscope observation indicated that no obvious lesion was observed in knee joint cartilage of mice after intra-articular injection of AAV. According to frozen sections of knee joints, strong green fluorescence was observed in knee joint cartilage in all AAV experimental groups. Compared with other groups, significantly stronger green fluorescence were observed both in AAV2 and AAV7 groups, whose average fluorescence density was 0.077±0.020 and 0.061±0.022. There were significant differences between two groups and other groups. CONCLUSIONS: AAV could infect chondrocyte of knee joint in vivo by injecting into knee joint cavity. Higher infection efficiency of AAV2 and AAV7 on knee joint cartilage were observed. Local injection of AAV into knee joint cavity could be used as an effective tool for gene editing of knee joint chondrocyte.

[Application status of 3D printing patient-specific instrumentation in total knee arthroplasty].

Recently, the joint replacement has gradually become a common operation of orthopedics. It's also the hot spot in current investigations. The increasing number of reports about the 3D printing technology such as patient-specific instrumentation(PSI) is applied to hip and knee replacement. It plays an important role in supporting operation especially for complex cases. However, it is still at the primary stage. This article reviews the relevant literature, analyzes and collates the research status of the application of 3D printing PSI in total knee arthroplasty, and summarizes its effectiveness in the application of knee arthroplasty from the aspects of individual preoperative planning, intraoperative operation and postoperative functional recovery, especially complex knee surgery such as obesity and knee deformity. The necessity of clinical application is discussed, and the existing problems and controversies in the current clinical application are summarized in order to provide references for the future research direction and clinical application.

[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.

[Clinical diagnosis and treatment for iliotibial band syndrome].

As an usually occurs in athletes, iliotibial band syndrome is payed more attention for people, the disease is diagnosed mainly by clinical symptoms, physical examination and MRI, but there is no uniform diagnostic criteria. The pathogenesis of iliotibial band syndrome is considered to be related to pressure and friction factors. As for the treatment, manipulation, muscle exercise, mainly drugs and physical therapy and so on both at home and abroad are recognized to use to achieve desired effect. For conservative failure, refractory iliotibial band syndrome patients, arthroscopy, or release of iliotibial band syndrome surgery are performed. While conservative local drug injection combined with muscle exercise could play a role in pain management besides, arthroscopic as operation method is more advanced, and applicable to all types of patients without absolute contraindication, so it is helpful for patients with early activity. At present, there is still a great deal of controversy about its pathogenesis, and there is no obvious limit for the specific indications of its various therapies in clinic, so it needs further specification.

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.

[Subculture, cryopreservation and recovery of osteoclasts].

OBJECTIVE: To explore the feasibility of passage, cryopreservation, and recovery of osteoclasts in order to develop new techniques facilitating osteoclast research. METHODS: Passage of osteoclasts: adult male SD rat(SPF grade, weight of 250 g) was sacrificed and the abdominal aorta was exposed for blood draw. Monocytes isolated from peripheral circulation was treated with RANKL and M-CSF for 2 weeks. After formation of osteoclasts, they were trypsinized with pipetting, centrifuged, re-suspended with α-MEM containing RANKL and M-CSF, and cultured in 6 well-plates and 35 mm culture dishes. Freezing of osteoclasts: trypsinized osteoclasts were centrifuged and resuspended with DMSO, FBS, α-MEM (1:2:7), and were stored in liquid nitrogen(-196 °C). Recovery of osteoclasts: frozen osteoclasts were taken out of liquid nitrogen tank and thawed quickly at 37 °C in water bath. After wash with PBS, the cells were resuspended with α-MEM containing RANKL and M-CSF, and were cultured in 6 well dishes and 35 mm culture dishes. Meanwhile, cells were checked with inverted phase contrast microscope and observed in the live cell station for real time imaging. TRAP staining was performed 3 days after plating. RESULTS: Trypsinization together with pipetting and shaking can detach the adherent osteoclasts, and the resuspended cells can be used for passage and storage in liquid nitrogen. The passaged cells became fully attached to the culture dishes in 2 hours, and the multinucleated feature could be clearly seen. The osteoclasts recovered from liquid nitrogen could completely spread out for 2 to 3 hours so that the multinucleated cells were clearly seen. These cells were still TRAP positive. CONCLUSIONS: Although osteoclasts strongly adhere to the bottom of culture dishes, a large majority of the osteoclasts can be detached after appropriate digestion with trypsin, pipetting and shaking. These cells can be used for passage and cryopreservation. After recovering from liquid nitrogen, these cells still preserve the viability and the feature of osteoclasts. The results provide a new and powerful tool for future study of osteoclast biology.

[Internal fixation with lag screws plus an anti-sliding plate for the treatment of Hoffa fracture of the lateral femoral condyle].

OBJECTIVE: To explore the clinical effects of internal fixation with lag screws plus an anti-sliding plate for the treatment of Hoffa fractures of the lateral femoral condyle. METHODS: From May 2006 to May 2014, 17 patients with Hoffa fractures of the lateral femoral condyle were treated with lag screws plus an anti-sliding plate. There were 13 males and 4 females, ranging in age from 27 to 59 years, with a mean of 32.5 years. All the fractures were fresh and closed fractures. According to the Letenneur's classification, 8 cases were type I, 4 cases were type II, 5 cases were type III. All the patients had no injuries of the cruciate ligament and the another part of the knee. Operative incision and fracture healing time were observed, knee joint function was evaluated by Letenneur system and HSS standard. RESULTS: The patients were followed up from 10 to 24 months with a mean of 14.6 months. All incisions achieved primary healing, and no internal fixation breakage, malunion, femoral candyle necrosis, deep vein thrombosis of lower extremity were found. Fracture healing time was from 4 to 9 months with an average of 4.7 months. According to Letenneur's functional assessment, 10 patients got an excellent results, 4 good, 3 fair. Total HSS score was 91.1 +/- 4.7 on average,15 cases obtained excellent results, 2 good. CONCLUSION: Internal fixation with lag screws and an anti-sliding plate can result in excellent effects for Hoffa fractures of the lateral femoral condyle. The key to a successful surgery is an anatomic reduction and rigid fixation of the fracture.

[Research on regulation mechanism of osteoclast differentiation].

Osteoclasts are multinucleated giant cell, which derived from mononuclear myeloid hematopoietic stem cells with the function of bone absorption. Osteoclasts plays a key role in bone metabolism, therefore the body is very strict to regulation of osteoclastogenesis. Mobilization and differentiation of osteoclast maturation is a complex and sophisticated multi-level regulatory processes. In the relevant regulatory mechanisms, OPG/RANKL/RANK system plays a pivotal role in the process of osteoclast differentiation and maturation. Recent studies revealed that immune cells and osteoclasts were closely connect with each other in the field of bone metabolism, also provide a new therapeutic target for the treatment of bone diseases. The apoptosis of osteoclasts in bone metabolism have been payed more attention,while its mechanism is still not clear, which need further research.

[Extracapsular arthroscopic excision of popliteal cysts through anterior combined with posterior approach].

OBJECTIVE: To study the clinical effect of arthroscopic excision of popliteal cysts through a combined anterior and posterior approach. METHODS: From January 2010 to December 2012,20 patients with popliteal cysts were treated with arthroscopic excision. There were 14 males and 6 females, with an average age of 49.5 years old, ranging from 45 to 65 years old. The lump has been found for 4 to 18 months,with a mean time of 12 months. Their mean sagittal diameter was 4.5 cm (ranged from 3 to 7 cm). There were 12 popliteal cysts in the left and 8 popliteal cysts in the right. The main clinical manifestation included lump at popliteal fossa, swelling and pain at knee joint and some extent of dysfunction. All diagnoses were determined according to MRI, which clearly showed the communication of cyst and articular cavity. The cyst was removed under arthroscopy, through the posterior approach and then the intra-articular lesion was treated via the anterior approach. According to Rauschning and Lindgren classification, 2 patients were grade I, 6 patients were grade II, and 12 patients were grade III. The guidelines of Rauschning and Lindgren were used to evaluate the clinical effects. RESULTS: No complications such as the injury of blood vessel and nerve, or incision infection occurred in all patients. All the patients were followed up, and the duration ranged from 8 to 24 months, with a mean time of 16 months. According to the criteria of Rauschning and Lindgren, there were 14 cases of grade 0, and 6 cases of grade I after operation, which was improved obviously compared with that pre-operation. No cyst reoccurred and the knee joint pain was relieved. CONCLUSION: Treatment of popliteal cysts with arthroscopic excision through a combined anterior and posterior approach is effective to remove the cyst sac and treat intra-articular diseases simultaneously, resulting in the decrease recurrence rate of cyst.

[Judgment of defect length of extremities artery trauma and reconstruction].

OBJECTIVE: To study the influence of actual defect length and gap width of the limbs main artery on the method selection of repairing and reconstruction. METHODS: Retrospective study was carried out for 32 patients with extremity main artery injury from 1996 to 2009, including 30 males and 2 females; 30 adults with an average age of 36 years old ranging from 18 to 51 years, 2 children of 4 and 5 years old respectively. Injured body parts involved axillary artery in 4 cases,brachial artery in 7 cases,radial artery in 2 cases, femoral artery in 4 cases, popliteal artery in 13 cases, posterior tibial artery in 2 cases. Main arterial injury defect gap width of all cases were observed and the reasons were analyzed. All cases were repaired by the method of end to end anastomosis after vessels stretch. RESULTS: The artery defect width was 3 cm to 7 cm with an average of (4.375 +/- 1.200) cm. Defect width of the upper extremity brachial artery and axillary artery group was (5.73 +/- 0.63) cm,the lower extremity femoral and popliteal artery group (3.80 +/- 0.73) cm, the posterior tibial artery group (3.25 +/- 0.35) cm, the radial artery group (3.00 +/- 0.00) cm. Defect width of upper extremity brachial artery and axillary artery group was larger than that of the other three groups (P < 0.01). End to end anastomosis was performed successfully in all cases. Blood supply recovered well. Because of the severe limb infection 2 patients had amputation in the late. All patients received follow-up. The patients without fracture were followed up to 2 weeks postoperatively, all patients with fractures were followed up to 1 year at least. Limb blood supply was good in all patients during the follow-up. CONCLUSION: Blood vessel defect gap width is different from the actual vessel defect, but is larger than the actual vessel defect. Misjudgment of the vascular defect length will lead to more vascular transplantation. The vast majority of vascular defect can be directly repaired by the method of end to end anastomosis after the vessel free and stretch.

[Balloon kyphoplasty for the treatment of osteoporotic Kummell's disease].

OBJECTIVE: To explore the clinical efficacy of the percutaneous kyphoplasty for the treatment of osteoporotic Kummell's disease. METHODS: From May 2010 to February 2012, 8 patients with osteoporotic Kummell's disease were treated with percutaneous balloon kyphoplasty. There were 2 males and 6 females, with a mean age of 73.4 years. All the patients suffered from lower back pain for 4.7 months, which affected seriously the patient's quality of life. The anterior vertebral height and Cobb angel was measured on standing lateral radiograph at pre- and post-operatively (2 days after operation) and during the final follow-up. Visual analog scale (VAS), Japanese Orthopedic Association (JOA) and Oswestry disability index (ODI) were used to evaluate pain and function. RESULTS: Incision of all patients healed normally without infection. The level of back pain decreased remarkably after surgery. The mean time of ambulation was 4.3 days (ranged, 1 to 10 days). Cement leakage occurred in 1 case with no symptom. The anterior vertebral height and Cobb angel of the fractured vertebra recovered respectively from preoperative (30.4 +/- 7.4)% and (31.3 +/- 9.9) degree to (70.3 +/- 3.3)% and (9.1 +/- 3.0) degree at the 2nd day after operation. VAS and JOA scores, ODI improved from preoperative 8.7 +/- 1.2, 12.3 +/- 1.7 and (93.3 +/- 4.6)% to 3.1 +/- 1.1, 24.9 +/- 1.6 and (32.2 +/- 5.4)% respectively at the 2nd day after operation. All the patients were followed up, and the duration ranged from 3 to 24 months, with a mean of 4.7 months. At the latest follow-up, the anterior vertebral height and Cobb angel of the fractured vertebra were (69.9 +/- 3.2)% and (10.9 +/- 2.4) degree; the VAS and JOA scores and ODI were 2.2 +/- 1.0, 26.4 +/- 1.4 and (29.2 +/- 4.5)% respectively, which had no obvious difference compared to those results at the 2nd day after operation. CONCLUSION: Balloon kyphoplasty is safe and effective treatment for osteoporotic Kummell's disease.

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

OBJECTIVE: To observe the function of wnt/ß-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. METHODS: Bone 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 ß-catenin, low density lipoprotein receptor-related protein 5 (LRP5) and T cell factor (TCF) protein, known as wnt/ß-catenin signal pathway related factors. RESULTS: EFs 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 ß-catenin, LRP5 and TCF protein which could be blocked by DKK1. CONCLUSION: EFs regulate the balance between osteogenic differentiation and adipogenic differentiation in bone marrow stromal cells of ovariectomized rats by activating wnt/ß-catenin signal pathway, which may be an important molecular mechanism of EFs on treating postmenopausal osteoporosis.

[Imaging observation of live cells originating from osteoclasts of the blood system].

OBJECTIVE: To study the application of the live cell imaging method to observe the whole process of osteoclast formation induced by monocyte macrophages in the blood system in order to clarify the origin of osteoclasts and their cytodynamics. METHODS: Blood samples (8 ml) were collected from the abdominal aorta of male SD rats weighing 280 g. Mononuclear cells were obtained by density gradient centrifugation and induced by RANKL and M-CSF. The cells were cultured and divided into four groups: inverted phase contrast microscope (IPCM) group, TRAP group, SEM group and live cell imaging (LCI) group. Images of the IPCM group were captured by a digital microscopic imaging system and recorded daily. The TRAP group was identified by enzyme activity staining after a 21-day cultivation period. The SEM group was SEM-observed after a 21-day cultivation period. The LCI group was consecutively and dynamically observed for 35 days. RESULTS: After 2-week cultivation, IPCM observations showed the formation of numerous apocytes. These cells displayed round, fusiform, fan-shaped, elliptic or irregular gibbous profiles. TRAP staining showed that most apocytes and monocytes had positive(+)reaction. SEM observations showed many bone absorption lacunae, hollows and channels, in which many osteoclasts with absorption activity were observed. Live cell imaging observations found that multinuclear osteoclasts originating from peripheral blood were generated by fusion of monocytes and apocytes and intercross fusion of monocytes and apocytes,which occurred at the adherent stage of the cells. Cytodynamic observations showed that the cell form of osteoclasts was complex and changeable. CONCLUSION: RANKL and M-CSF can induce differentiation and formation from monocytes in rat peripheral blood into multinuclear osteoclasts with bone absorption activity. The osteoclasts were formed by various cell fusion processes at the adherent stage. The adherent property of osteoclasts is important for their survival and function. Osteoclasts have phagocytosis and their morphological structure is dynamically changeable, involving not only apocytes but monocytes. The osteoclast property of multinuclear giant cells formed by cell fusion may be a special biological behavior for their adaptation of functional needs and bone absorption efficiency. This experiment has further evidenced the theory of osteoclast origination in the blood system and provided new experimental clues for clarifying the cytodynamic and cytobiological properties of osteoclasts.

[Experimental study on invigorating kidney and activating blood on preventing and curing SD rats with knee osteoarthritis].

OBJECTIVE: To explore the effects and mechanism of invigorating kidney and activating blood, invigorating kidney and expelling wind on hemorheology, IL-1ß and TNF-α of SD rats with knee osteoarthritis, then definite the evolution of muscle certified turning into heumatism and compare the effect of Chinese herbal. METHODS: One hundred and eighty SD rats with 3-month-old (each weight was 185 to 215 g) received intra-articular injection of papain solution for establishing knee OA models. All rats were randomly divided into activating blood group, preventing group, expelling wind group, invigorating kidney group, invigorating kidney and activating blood group and model group. Laboratory indexes were obtained at the 30th, 60th, 90th days after gastric perfusion, which including state of mind, activity, fur, weight, joint swelling, largely image, hemorheology, inflammation and HE pathological appearance. RESULTS: After operation, rats appeared blood stasis and swelling and difficulty crawling. There was significant difference of hemorheology in invigorating kidney and activating blood group the content of IL-1ß and TNF-α was obviously lower than model group (P < 0.05 ). While the content of IL-1ß and TNF-α on the early stage was obviously higher than late stage (P < 0.05). CONCLUSION: Knee osteoarthritis mainly show synovial inflammation at the early stage, inflammation at early stage is more severe than late; invigorating kidney and activating blood decoction can inhibit the knee cartilage injury, improve blood circulation and prevent local inflammatory reaction. Activating blood decoction and invigorating kidney and activating blood Decoction have certain curative effect in early time, but the effects of invigorating kidney and activating blood Decoction is more effective than other on the late stage.

[Experimental study on Yougui recipe in preventing osteolysis surrounding artificial prosthesis].

OBJECTIVE: To explore effects of Yougui recipe (see text) and salmon calcitonin acetate in preventing osteolysis surrounding artificial prosthesis. METHODS: Thirty-two SD male rats with weighted (250 +/- 20) g, aged 8 weeks, were randomly divided into four groups: blank group, model group, salmon calcitonin acetate group and Yougui recipe (see text) group, and 8 rats in each group. Blank group did not undergo any process, other 24 rats underwent anesthesia by chloral hydrate, their knee joints were exposed through medial patellar side,drilling from fermoral condyle nest to marrow cavity,high density of polythlene particles were injected into hole, titanium nail were put into, bone wax closed the window, then suturing step by step. After the molding, saline were used to gavaged in blank group and model group, Yougui recipe (see text) for Yougui recipe (see text) group, salmon calcitonin maximus injection for calcitonin group. After 10 weeks' mediation, rats were executed, and arterial blood and bilateral femoral organization were collected to biochemical, imaging morphology, tissue pathology and molecular biology detection. RESULTS: The key gene expression of activiting osteoclast were inhibited in Yougui recipe (see text) group and calcitonin group. The level of OPG, Ca, ALP in Yougui recipe group were higher than calcitonin group (P<0.01); the content of RANKL were lower (P<0.01). There were no significance meaning in RANK, Trap5b, P between two groups. CONCLUSION: Both of Yougui recipe (see text) and calcitonin can slow and treat surrounding osteolysis of artificial joint prosthesis, and Yougui recipe (see text) has better effect in promoting bone formation. The effect of Yougui recipe (see text) in promoting bone formation, inhibiting osteoclasts to provide a new method to treating surrounding osteolysis of artificial joint prosthesis.

Dynamic expression of DKK1 protein in the process whereby Epimedium-derived flavonoids up-regulate osteogenic and down-regulate adipogenic differentiation of bone marrow stromal cells in ovariectomized rats.

OBJECTIVE: To observe the dynamic expression of DKK1 protein in the process whereby Epimedium-derived flavonoids (EFs) regulate the balance between osteogenic and adipogenic differentiation of bone marrow stromal cells in ovariectomized rats, and to provide experimental evidence for the mechanism of EFs in the treatment of postmenopausal osteoporosis. METHODS: Bone marrow stromal cells from ovariectomized rats were separated and cultivated in osteoinductive or liquid medium for 15 days in vitro. EFs (10 µg/mL) were applied to both cultures. Alkaline phosphatase (ALP) staining, ALP activity determination, Oil Red O staining and fluorescence quantitative polymerase chain reaction were used to determine the influence of EFs on osteogenic and adipogenic differentiation of bone marrow stromal cells in ovariectomized rats. Moreover, in order to explore the exact mechanism of EFs on osteogenic and adipogenic differentiation of bone marrow stromal cells in ovariectomized rats, enzyme linked immunosorbent assay was used to determine the dynamic expression of DKK1 protein in this process. RESULTS: EFs increased activity of ALP and mRNA expression of Runx2 (early osteoblast differentiation factor) and decreased mRNA expression of PPARγ-2 (key factor of fat generation). Importantly, EFs down-regulated expression of DKK1 protein in an osteogenic induction medium and inhibited up-regulation of DKK1 protein in an adipogenic induction medium. CONCLUSION: EFs regulate the balance between osteogenic and adipogenic differentiation of bone marrow stromal cells in ovariectomized rats by down-regulating expression of DKK1 protein. This may be an important molecular mechanism of EFs in the context of treatment of postmenopausal osteoporosis.