OA-4 inhibits osteoclast formation and bone resorption via suppressing RANKL induced P38 signaling pathway.

Osteoclasts are one of the key therapeutic targets for a variety of orthopedic diseases such as osteoporosis and osteoarthritis. In this study, we synthesized a novel compound N-(3-(cyclohexylcarbamoyl) phenyl)-1H-indole-2- carboxamide (termed as OA-4) and investigated the effects of OA-4 on the differentiation and function of osteoclasts. OA-4 markedly diminished osteoclast differentiation and osteoclast specific gene expression in a dose-dependent manner. In addition, OA-4 dose-dependently suppressed osteoclastic bone resorption. Furthermore, we found OA-4 attenuated RANKL-induced p38 phosphorylation without affecting JNK or NF-κB signaling pathways. Collectively, we synthesized a novel compound OA-4 which can inhibit osteoclast formation and functions via the suppression of p38 signaling pathway.

Andrographolide suppresses RANKL-induced osteoclastogenesis in vitro and prevents inflammatory bone loss in vivo.

BACKGROUND AND PURPOSE: Osteoclasts play a pivotal role in diseases such as osteoporosis, rheumatoid arthritis and tumour bone metastasis. Thus, searching for natural compounds that may suppress osteoclast formation and/or function is promising for the treatment of osteoclast-related diseases. Here, we examined changes in osteoclastogenesis and LPS-induced osteolysis in response to andrographolide (AP), a diterpenoid lactone isolated from the traditional Chinese and Indian medicinal plant Andrographis paniculata. EXPERIMENTAL APPROACH: Effects of AP on osteoclast differentiation and bone resorption were measured in vitro. Western blots and RT-PCR techniques were used to examine the underlying molecular mechanisms. The bone protective activity of AP in vivo was assessed in a mouse model of osteolysis. KEY RESULTS: AP concentration-dependently suppressed RANKL-mediated osteoclast differentiation and bone resorption in vitro and reduced the expression of osteoclast-specific markers, including tartrate-resistant acid phosphatase, calcitonin receptors and cathepsin K. Further molecular analysis revealed that AP impaired RANKL-induced NF-κB signalling by inhibiting the phosphorylation of TGF-ß-activated kinase 1, suppressing the phosphorylation and degradation of IκBα, and subsequently preventing the nuclear translocation of the NF-κB p65 subunit. AP also inhibited the ERK/MAPK signalling pathway without affecting p38 or JNK signalling. CONCLUSIONS AND IMPLICATIONS: AP suppressed RANKL-induced osteoclastogenesis through attenuating NF-κB and ERK/MAPK signalling pathways in vitro, thus preventing bone loss in vivo. These data indicated that AP is a promising natural compound for the treatment of osteoclast-related bone diseases.

OA10 is a novel p38alpha mitogen-activated protein kinase inhibitor that suppresses osteoclast differentiation and bone resorption.

In search of anti-bone resorbing agents for the potential treatment of osteoporosis, we synthesized a novel compound Tert-butyl 4-(3-[1H-indole-2-carboxamido]benzoyl)piperazine-1-carboxylate (OA10) and found that OA10 is capable of inhibiting RANKL-mediated osteoclast formation and osteoclastic bone resorption in a dose-dependent manner. This biological effect is further supported by the fact that OA10 suppressed osteoclastic-specific gene expression, including tartrate-resistant acid phosphatase, cathepsin K receptor, and calcitonin receptor. Further molecular mechanism investigation revealed OA10 inhibited p38 phosphorylation, suppressed c-fos and NFATc1 expression without affecting NF-κB or JNK signaling pathways. Taken together, this study suggested that OA10 can inhibit osteoclastogenesis by suppressing p38-c-Fos-NFATc1 cascade. OA10 may be developed as a therapeutic drug for osteoclast-related osteolytic diseases.

V-ATPases in osteoclasts: structure, function and potential inhibitors of bone resorption.

The vacuolar-type H(+)-ATPase (V-ATPase) proton pump is a macromolecular complex composed of at least 14 subunits organized into two functional domains, V(1) and V(0). The complex is located on the ruffled border plasma membrane of bone-resorbing osteoclasts, mediating extracellular acidification for bone demineralization during bone resorption. Genetic studies from mice to man implicate a critical role for V-ATPase subunits in osteoclast-related diseases including osteopetrosis and osteoporosis. Thus, the V-ATPase complex is a potential molecular target for the development of novel anti-resorptive agents useful for the treatment of osteolytic diseases. Here, we review the current structure and function of V-ATPase subunits, emphasizing their exquisite roles in osteoclastic function. In addition, we compare several distinct classes of V-ATPase inhibitors with specific inhibitory effects on osteoclasts. Understanding the structure-function relationship of the osteoclast V-ATPase may lead to the development of osteoclast-specific V-ATPase inhibitors that may serve as alternative therapies for the treatment of osteolytic diseases.

Biologic effect and immunoisolating behavior of BMP-2 gene-transfected bone marrow-derived mesenchymal stem cells in APA microcapsules.

We investigated the encapsulation of BMP-2 gene-modified mesenchymal stem cells (MSCs) in alginate-poly-L-lysine (APA) microcapsules for the persistent delivery of bone morphogenic protein-2 (BMP-2) to induce bone formation. An electrostatic droplet generator was employed to produce APA microcapsules containing encapsulated beta-gal or BMP-2 gene-transfected bone marrow-derived MSCs. We found that X-gal staining was still positive 28 days after encapsulation. Encapsulated BMP-2 gene-transfected cells were capable of constitutive delivery of BMP-2 proteins for at least 30 days. The encapsulated BMP-2 gene-transfected MSCs or the encapsulated non-gene transfer MSCs (control group) were cocultured with the undifferentiated MSCs. The gene products from the encapsulated BMP-2 cells could induce the undifferentiated MSCs to become osteoblasts that had higher alkaline phosphatase (ALP) activity than those in the control group (p<0.05). The APA microcapsules could inhibit the permeation of fluorescein isothiocyanate-conjuncted immunoglobulin G. Mixed lymphocyte reaction also indicates that the APA microcapsules could prevent the encapsulated BMP-2 gene-transfected MSCs from initiating the cellular immune response. These results demonstrated that the nonautologous BMP-2 gene-transfected stem cells are of potential utility for enhancement of bone repair and bone regeneration in vivo.

Treatment of osteonecrosis of the femoral head with hBMP-2-gene-modified tissue-engineered bone in goats.

The efficacy of beta-tricalcium phosphate (beta-TCP) loaded with bone morphogenetic protein-2 (BMP-2)-gene-modified bone-marrow mesenchymal stem cells (BMSCs) was evaluated for the repair of experimentally-induced osteonecrosis of the femoral head in goats. Bilateral early-stage osteonecrosis was induced in adult goats three weeks after ligation of the lateral and medial circumflex arteries and delivery of liquid nitrogen into the femoral head. After core decompression, porous beta-TCP loaded with BMP-2 gene- or beta-galactosidase (gal)-gene-transduced BMSCs was implanted into the left and right femoral heads, respectively. At 16 weeks after implantation, there was collapse of the femoral head in the untreated group but not in the BMP-2 or beta-gal groups. The femoral heads in the BMP-2 group had a normal density and surface, while those in the beta-gal group presented with a low density and an irregular surface. Histologically, new bone and fibrous tissue were formed in the macropores of the beta-TCP. Sixteen weeks after implantation, lamellar bone had formed in the BMP-2 group, but there were some empty cavities and residual fibrous tissue in the beta-gal group. The new bone volume in the BMP-2 group was significantly higher than that in the beta-gal group. The maximum compressive strength and Young's modulus of the repaired tissue in the BMP-2 group were similar to those of normal bone and significantly higher than those in the beta-gal group. Our findings indicate that porous beta-TCP loaded with BMP-2-gene-transduced BMSCs are capable of repairing early-stage, experimentally-induced osteonecrosis of the femoral head and of restoring its mechanical function.

BMP2 gene therapy on the repair of bone defects of aged rats.

Age-related decline in the number of mesenchymal stem cells (MSCs) and their reduced capability to differentiate osteogenically, along with diminished availability of growth factors, may be major factors accounting for reduced bone formation in the aging mammalian body. In the first part of the study, we compared the number of MSCs in bone marrow (BM) and the content of bone morphogenetic protein 2 (BMP2) in cortical bone tissue in juvenile, adult, and aged (1, 9, and 24 months, respectively) male rats. To assay the influence of aging on osteogenic differentiation ability, MSCs from the three age groups were transduced with the BMP2 gene. Following gene transduction, the production of BMP2 in culture media, expression of osteogenic proteins (e.g., alkaline phosphatase, type Ialpha1 collagen, osteopontin, and bone sialoprotein), as well as ectopic bone formation in athymic mice were compared. Results showed that the number of MSCs in BM as well as the content of BMP2 in cortical bone tissue decreased with age, but no significant differences between the three age groups were found with regard to production of BMP2 or capability of BMP2 gene-modified MSCs to differentiate osteogenically. The second part of the study applied BMP2 gene-modified autologous MSCs/beta-tricalcium phosphate for repair of bone defects in aged rats with positive results. Our data indicate that the osteogenic potential of MSCs of aged rats can be restored following BMP2 gene transduction and that this technique may be a useful approach in the future planning of gene therapy for age-related osteoporotic fractures.

Repairing of goat tibial bone defects with BMP-2 gene-modified tissue-engineered bone.

Bone defects larger than a critical size are major challenges in orthopedic medicine. We combined tissue-engineered bone and gene therapy to provide osteoprogenitor cells, osteoinductive factors, and osteo-conductive carrier for ideal bone regeneration in critical-sized bone defects. Goat diaphyseal bone defects were repaired with tissue and genetically engineered bone implants, composed of biphasic calcined bone (BCB) and autologous bone marrow derived mesenchymal stem cells (BMSC) transduced with human bone morphogenetic protein-2 (hBMP-2). Twenty six goats with tibial bone defects were divided into groups receiving implants by using a combination of BCB and BMSCs with or without the hBMP-2 gene. In eight goats that were treated with BCB that contained hBMP-2 transduced BMSC, five had complete healing and three showed partial healing. Goats in other experimental groups had only slight or no healing. Furthermore, the area and biochemical strength of the callus in the bone defects were significantly better in animals treated with genetically engineered implants. We concluded that the combination of genetic and tissue engineering provides an innovative way for treating critical-sized bone defects.

Novel method for correction of x-ray fluoroscopic image.

X-ray fluoroscopic images have been widely used in orthopedic surgery. Unfortunately, the inherent distortion deteriorates the quality of fluoroscopic image. To avoid the discontinuities of local correction techniques and achieve good accuracy in present global correction method, a novel approach for distortion correction is proposed which allows good image quality in relatively acceptable time by combining both global and local methods, and a new local interpolation method is also proposed. Computer simulation and experimental test on fluoroscopic image have been carried out.

[The effect of stress-relaxation plate fixation on the remodeling of the cortex under plate].

OBJECTIVE: To explore the influence of stress-relaxation plate(SRP) fixation on the remodeling of cortex under plate. METHODS: Twenty-eight New Zealand rabbits were used in this study, the bilateral tibia were osteotomized in the middle and fixed with SRP (experimental group) and rigid plate (control group) respectively. The scanning electron microscopy was used to observe the bone remodeling process from 2 to 48 weeks after operation. RESULTS: There was cortex osteoporosis beneath plate in different degree in both experimental and control groups before 8 weeks, it showed as the disorganization of collagen fiber structure and formation of resorption cavities. In comparison, the osteoporosis degree in experimental group showed milder than that of the control group. After 12 weeks, the resorption cavities became smaller, and the structure of collagen fibers became regular with the alignment parallel to the long axis of cortex. In contrast to the experimental group, the bone osteoporosis under plate of control group exacerbated continuously. CONCLUSION: Without removal of the bone plate, SRP fixation not only reduce the degree of plated bone osteoporosis, but also make the osteoporosic bone return to normal.

[The gene expression of some cytokines and collagen proteins in rat bone tissue is related to estradiol (E2) and age].

30 female SD rats (3 months old) are equally divided into three groups: ovariectomy (OVX) rats, sham-operated (SHO) rats and 17 beta estradiol (E2) treated OVX rats. For each group, mRNA was isolated from long bone at one month and three months after surgery, respectively. mRNA was reverse transcribed into single strand cDNA and then used as a probe hybridizing to the DNA fragments of col I alpha(1), col I alpha(2), col III, col V, fibronectin, IL-1, IL-6, TGF-beta, LIF, TNF-alpha, TNF-beta by reverse northern and dot blot hybrization. The housekeeping gene, gapdh, was used as an internal control. The results show that in bone of rat, the stable expression of col I alpha (1), col I alpha(2) and col III are related to age not ovariectomy, while supplement with E2 can inhibit the expression of col III and col I alpha(2) completely. The expression of col V, IL-1, IL-6 can be inhibited by estrogen and recovered by removal of estrogen by OVX, then addition of E2 decreased it to the normal level. The expression of TGF-beta is also inhibited by estrogen. It increased during one month after overiectomy, and partially decreased in E2 complemented rat. Three months after surgery, the level of increasing and decreasing is less evident as two months ago. It seems that in young SD rat, the expression of TGF-beta is related to both estrogen and age.

Application of a NiTi staple in the metatarsal osteotomy.

Thirty-six metatarsal osteotomies using internal fixation of a shape memory metal compression staple for hallux valgus were performed in 21 women with a mean age of 42.3 years (30-66 years). The time for return to light work averaged 19.0 days, and to full work and normal walk 41.2 days after the operation. Twenty patients (35 feet) had complete relief from pain. Only in one foot, the pain was transferred under the second metatarsal head. The appearance of the feet was apparently improved and the width of the forefeet was decreased by a mean of 0.90 cm after the operation. Radiographic analysis of feet showed that all osteotomies united, and the average angle of hallux valgus was improved from 31.2 degrees to 16.3 degrees and the mean intermetatarsal angle from 12.0 degrees to 6.4 degrees. No external fixation of plaster splintage was needed and the distal fragment during healing of the osteotomy was stable. The time of bone healing was shortened and patients were allowed to bear weight at an early time.

Effect of internal fixation plates on microcirculation in under-plate cortical bones. Microangiography and scanning electronmicroscopy.

To elucidate the effect of the internal fixation plates on the local bone blood supply, we used microangiography and scanning electron microscopy to observe the morphological changes of microcirculation in the cortical bones obtained from intact rabbit tibiae on which plates of two different stiffness had been fixed for comparison. The results indicated that both rigid stainless steel plate and less rigid methyl methacrylate plate could induce the bone microcirculation under the plate to undergo a process from early depression to late reactive recruitment. The features of the microcirculation recruitment such as vascular number, arrangement and dilatation varied with plates of different stiffness and were more obvious in the cortex fixed by rigid stainless steel plate.

Bone remodelling after internal fixation with different stiffness plates: ultrastructural investigation.

Forty adult New Zealand rabbits were used in this experiment. Four rabbits served as control and the rest 36 being divided into 3 groups, each of which was fixed respectively with methylmethacrylate, titanium alloy and stainless steel plate on the left intact tibiae. The cortical bone beneath the plate was harvested at 2, 4, 8 and 12 weeks after operation and observed by means of using transmission electron microscope. The major ultrastructural changes after internal fixation with different stiffness plates were alteration of osteocyte life cycle, perilacunar matrix and disorder of osteoblast and osteoblast coupling. The greater the stiffness of the plate, the greater the ultrastructural change.

[An experimental study and preliminary clinical report of shape-memory sawtooth-arm embracing internal fixator].

The sawtooth-arm internal embracing fixator is made of Nickel-Titanium shape memory alloy and consists of 3 components: body, arms and sawteeth. In cross section a 2/3 circumference is constructed by body and arms of the fixator. The free ends of the arms which exceed the semi-circle are bent more medially so that it can match the requirement of fixation of long tubular bone whose cross section is not a regular circle. The embracing fixator has two types: the cylinder type is for use in the middle part and the cone type is either for the proximal or distal 1/3 of the shaft of long tubular bone. The animal experimental studies and in vitro mechanical tests demonstrated that the embracing fixator possesses good anti-bending and anti-torsion effects, and its resisting compression effect was much lower than that of bone plate. These characteristics are beneficial for enhancing fracture healing, reducing post fixation osteoporosis and providing a new, simple and effective method for the treatment of long tubular bone fracture. The fixator has already been used in the treatment of fractures of femur, humerus, radius and ulna with good results.

[Comparative study of three different hip function evaluation systems].

Harris's, Charnley's and Beijing's numerical rating systems were used to evaluate the functional results of 42 cases of bipolar type artificial femoral head replacement. The results of assessment of the three methods were compared statistically and proved that the results are comparable. Before the overall evaluation, the patients should be divided into three groups (according to Charnley's method) on the basis of their original capacity of locomotion. The score results of the three groups should not be mixed up. The calculation method of total score of Beijing system may need certain modification. The advantages and disadvantages of the three systems, the significance of capacity assessment of daily activity and radiological evaluation are discussed on the basis of the results of analysis.

Repair of regional osteoporosis after removal of rigid fixing plate. An experimental investigation.

Twenty-four New Zealand white rabbits were plated on their intact left tibiae with stainless steel plates and 4 animals served as controls. The plates were removed 2 months after implantation in 20 plated animals. Of them, 4 were sacrificed immediately after plate removal and the other 16 were killed in successive groups with 4 in each group 1,2,3 and 4 months after plate removal respectively. The remaining 4 plated animals were killed at 6 months after implantation. Bone samples under the plates were harvested and prepared for polarized light microscopy to investigate the repair of the regional osteoporosis induced by rigid implant. The results indicated that the regional osteoporosis could recover gradually after plate removal. The repair process manifested itself mainly as the repair of resorption cavities and the remodeling of collagen fibers. The orientation of the collagen fibers remained disorganized when the resorption cavities had been repaired. It is suggested that delayed restoration of bone structure might be one of the potential causes of refracture after removal of the rigid implant.

[Clinical analysis of femoral neck fracture in 188 adult cases].

There were 188 cases of femoral neck fracture of adult below the age of fifty-five years old admitted to our hospital from 1974 to 1989, including 152 cases of fresh fracture, 36 old fracture. The rate of femoral head necrosis after fracture was 11.8 percent in the group of patients under fifty years of age, and 9.8 percent in patients between fifty-one and fifty five years old. The operation adopted in our series was mainly the percutaneous screw-thread pinning for internal fixation. Bone graft transplantation with the pedicle of quadratus femoris muscle was simultaneously performed in some cases, 26.7 percent in patients between the age of fifteen and twenty, along with the increase of age the patient number for transplantation decreased, only 7.8 percent in patients between the age of fifty-one and fifty-five. The author suggests that in treatment of femoral neck fracture of adult generally should not consider the use of artificial prosthesis replacement. We should decide according to the patients general and local status as the basis for the choice of a reasonable internal fixation, and consider whether an operation should be performed to further improve blood circulation of the femoral head.

Treatment of intra-articular fractures with shape memory compression staples.

The management of intra-articular fractures remains difficult. Shape memory compression staples fulfil nearly all the special treatment requirements of intra-articular fractures. Early bone union and 93.5 per cent satisfactory function were achieved in a series of 121 cases. Stable fixation, 'early' movement and continuous compressive force produced by the staple are the main factors contributing to good results.