Research Progress in the Treatment of New Bone Formation of Ankylosing Spondylitis

Ankylosing spondylitis(AS)has a very high disability rate.How to effectively inhibit the formation of new bones has become a difficult point in clinical treatment.In recent years,research has shown that different treatment plans can have an impact on inhibiting new bone formation.In this paper,the different effects of new bone formation in the treatment of AS with traditional Chinese and Western medicine are systematically listed.

TGF-β and BMP signaling in osteoblast,skeletal development,and bone formation,homeostasis and disease

INTRODUCTIONThe transforming growth factor-β （TGF-β） superfamily com- prises TGF-βs, Activin, bone morphogenetic proteins （BMPs） and other related proteins. TGF-β superfamily members act through a heteromeric receptor complex,, comprised of type I and type II receptors at the cell surface that transduce intracellular signals via Smad complex or mitogen-activated protein kinase （MAPK） cascade.

TGF-β/BMP signaling and other molecular events: regulation of osteoblastogenesis and bone formation

Transforming growth factor-beta（TGF-β）/bone morphogenetic protein（BMP） plays a fundamental role in the regulation of bone organogenesis through the activation of receptor serine/threonine kinases. Perturbations of TGF-β/BMP activity are almost invariably linked to a wide variety of clinical outcomes, i.e., skeletal, extra skeletal anomalies, autoimmune, cancer, and cardiovascular diseases. Phosphorylation of TGF-β（I/II） or BMP receptors activates intracellular downstream Smads, the transducer of TGF-β/BMP signals. This signaling is modulated by various factors and pathways, including transcription factor Runx2. The signaling network in skeletal development and bone formation is overwhelmingly complex and highly time and space specific.Additive, positive, negative, or synergistic effects are observed when TGF-β/BMP interacts with the pathways of MAPK, Wnt, Hedgehog（Hh）, Notch, Akt/m TOR, and mi RNA to regulate the effects of BMP-induced signaling in bone dynamics. Accumulating evidence indicates that Runx2 is the key integrator, whereas Hh is a possible modulator, mi RNAs are regulators, and b-catenin is a mediator/regulator within the extensive intracellular network. This review focuses on the activation of BMP signaling and interaction with other regulatory components and pathways highlighting the molecular mechanisms regarding TGF-β/BMP function and regulation that could allow understanding the complexity of bone tissue dynamics.

LRP6 in mesenchymal stem cells is required for bone formation during bone growth and bone remodeling

Lipoprotein receptor-related protein 6 （LRP6） plays a critical role in skeletal development and homeostasis in adults. However, the role of LRP6 in mesenchymal stem cells （MSCs）, skeletal stem cells that give rise to osteoblastic lineage, is unknown. In this study, we generated mice lacking LRP6 expression specifically in nestin＋ MSCs by crossing nestin-Cre mice with LRP6 flox mice and investigated the functional changes of bone marrow MSCs and skeletal alterations. Mice with LRP6 deletion in nestin＋ cells demonstrated reductions in body weight and body length at I and 3 months of age. Bone architecture measured by microCT （uCT） showed a significant reduction in bone mass in both trabecular and cortical bone of homozygous and heterozygous LRP6 mutant mice. A dramatic reduction in the numbers of osteoblasts but much less significant reduction in the numbers of osteoclasts was observed in the mutant mice. Osterix＋ osteoprogenitors and osteocalcin＋ osteoblasts significantly reduced at the secondary spongiosa area, but only moderately decreased at the primary spongiosa area in mutant mice. Bone marrow MSCs from the mutant mice showed decreased colony forming, cell viability and cell proliferation. Thus, LRP6 in bone marrow MSCs is essential for their survival and proliferation, and therefore, is a key positive regulator for bone formation during skeletal growth and remodeling.

RANKL-induced M1 macrophages are involved in bone formation

The activation of M1 macrophages can be achieved by stimulating them with lipopolysaccharide （LPS） and interferon-γ （IFN-γ）. However, M1 can be found under physiological conditions without any pathological stimuli. This study aimed to understand the involvement of RANKL-induced M1 macrophages in bone formation compared with pathologically induced macrophages. Fischer rats were used to investigate macrophage distribution in normal and injured femoral condyles in vivo. Bone marrow-derived macrophages （BMDMs） were activated with LPS＋IFN-γ and RANKL to achieve M1 activation in vitro. Gene expression related to inflammation, osteoclastogenesis, angiogenesis, and migration was determined by reverse transcription-quantitative polymerase chain reaction （RT-qPCR） and fluorescence-activated cell sorting （FACS）. Tissue macrophages showed distinct expression patterns at different bone regions. RANKL was found in close proximity to inducible nitric oxide synthase-positive （iNOS＋） cells in vivo, suggesting an association between RANKL expression and iNOS＋ cells, especially in trabecular bone. RANKL-induced macrophages showed a different cytokine secretion profile compared with pathologically induced macrophages. Both osteoclasts and M1 macrophages peaked on day 7 during bone healing. RANKL could trigger Ml-like macrophages with properties that were different from those of LPS＋IFN-γ-induced macrophages. These RANKL-activated M1 macrophages were actively involved in bone formation.

Bone Formation Process of β-TCP Ceramics with Tetracycline Tracing

To study the new bone formation in the bone defect area after implantation, the tetracycline tracing method was used. The results show that new bone formed in 1 month, and the formation rate of new bone was very high (8.164μm/day),considerably faster than that of control groups (3.219μm/day).The new bone grew up quickly and β-TCP particles were surrounded by double fluorescence bands which became more obvious. The new bone formation rate was maximal at 2 months, and then gradually reduced. The rate was steady at 4 months, and then reduced to resembling as the normal physiologic metabolism of bone, which indicated the implanted materials were completely replaced by bone. Calcium phosphate materials had the ability of osteoconduction.

New simulation model for bone formation markers in osteoporosis patients treated with once-weekly teriparatide

Daily 20-mg and once-weekly 56.5-mg teriparatide（parathyroid hormone 1–34） treatment regimens increase bone mineral density（BMD） and prevent fractures, but changes in bone turnover markers differ between the two regimens. The aim of the present study was to explain changes in bone turnover markers using once-weekly teriparatide with a simulation model. Temporary increases in bone formation markers and subsequent decreases were observed during once-weekly teriparatide treatment for 72 weeks. These observations support the hypothesis that repeated weekly teriparatide administration stimulates bone remodeling, replacing old bone with new bone and leading to a reduction in the active remodeling surface. A simulation model was developed based on the iterative remodeling cycle that occurs on residual old bone. An increase in bone formation and a subsequent decrease were observed in the preliminary simulation. For each fitted time point, the predicted value was compared to the absolute values of the bone formation and resorption markers and lumbar BMD. The simulation model strongly matched actual changes in bone turnover markers and BMD. This simulation model indicates increased bone formation marker levels in the early stage and a subsequent decrease. It is therefore concluded that remodeling-based bone formation persisted during the entire treatment period with once-weekly teriparatide.

Intracystic negative pressure may promote bone formation around jaw cysts

The growth and enlargement of jaw cysts are associated with raised intracystic pressure and bone resorption surrounding the cysts.The major bone-resorbing cells are the osteoclasts.They are acting under the influence of local bone-resorbing factors: prostaglandins,proteinases and cytokines.It was found that positive pressure enhanced the expression of IL-1αmRNA and protein in epithelial cells of odontogenic keratocyst,and increased the secretion of matrix metalloproteinase and PGE2 in a co-culture of odontogenic keratocyst fibroblasts and epithelial cells.However,the signal intensities for IL-1α mRNA and protein in the epithelium were significantly decreased after marsupialization which relived intracystic pressure.Experimental study indicated that intermittent negative pressure could promote osteogenesis in human bone marrow-derived stroma cells(BMSCs) in vitro.We propose a hypothesis that bone formation around the cyst of the jaws would be stimulated by intracystic negative pressure.

In Vitro and In Vivo Effects of Puerarin on Promotion of Osteoblast Bone Formation

Objective： To assess the effect of puerarin, a natural fiavonoid found in Chinese Pueraria Lobata （Wild.） Ohwi, on promotion of new bone formation. Methods： Osteoblasts isolated from calvarial of newborn rats were cultured in vitro in the presence of puerarin at various concentrations. The viability of osteoblasts and alkaline phosphotase activity and mineral node formation were determined. In addition, osteoblasts seeded in the β -tricaclium phosphate scalfolds as bone substitute were implanted in rat dorsal muscles. Half of the recipient rats received intramuscular injection of pueradn at 10 mg/（kg.d） for 7 days. Osteogenesis was analyzed by examining the histology after 4 weeks of implantation. Results： The viability of osteoblasts treated with puerarin at either 40 or 80 umol/L was significantly higher than that of the control （P〈0.05 and P〈0.01, respectively）. Alkaline phosphatase and mineral modules were significantly increased in osteoblasts cultured with puerarin at 40 or 80 mol/L when compared with that of the untreated cells. The pueradn-treated rats had a higher rate of bone formation in the osteoblast implants than the control rats （6.35% vs. 1.32%, respectively, P〈0.05）. Conclusion： Puerarin was able to affect osteoblast proliferation and differentiation, and promote the new bone formation in osteoblast implants.

Role of Epithelium Sodium Channel in Bone Formation

Objective：To review the recent developments in the mechanisms of epithelium sodium channels （ENaCs） induced bone formation and regulation.Data Sources：Studies written in English or Chinese were searched using Medline,PubMed and the index of Chinese-language literature with time restriction from 2005 to 2014.Keywords included ENaC,bone,bone formation,osteonecrosis,estrogen,and osteoporosis.Data from published articles about the structure of ENaC,mechanism of ENaC in bone formation in recent domestic and foreign literature were selected.Study Selection：Abstract and full text of all studies were required to obtain.Studies those were not accessible and those did not focus on the keywords were excluded.Results：ENaCs are tripolymer ion channels which are assembled from homologous α,β,and γ subunits.Crystal structure of ENaCs suggests that ENaC has a central ion-channel located in the central symmetry axis of the three subunits.ENaCs are protease sensitive channels whose iron-channel activity is regulated by the proteolytic reaction.Channel opening probability of ENaCs is regulated by proteinases,mechanical force,and shear stress.Several molecules are involved in regulation of ENaCs in bone formation,including nitride oxide synthases,voltage-sensitive calcium channels,and cyclooxygenase-2.Conclusion：The pathway of ENaC involved in shear stress has an effect on stimulating osteoblasts even bone formation by estrogen interference.

The effect of carbon nanotubes on osteogenic functions of adipose-derived mesenchymal stem cells in vitro and bone formation in vivo compared with that of nano-hydroxyapatite and the possible mechanism

It has been well recognized that the development and use of artificial materials with high osteogenic ability is one of the most promising means to replace bone grafting that has exhibited various negative effects.The biomimetic features and unique physiochemical properties of nanomaterials play important roles in stimulating cellular functions and guiding tissue regeneration.But efficacy degree of some nanomaterials to promote specific tissue formation is still not clear.We hereby comparatively studied the osteogenic ability of our treated multiwalled carbon nanotubes(MCNTs)and the main inorganic mineral component of natural bone,nano-hydroxyapatite(nHA)in the same system,and tried to tell the related mechanism.In vitro culture of human adiposederived mesenchymal stem cells(HASCs)on the MCNTs and nHA demonstrated that although there was no significant difference in the cell adhesion amount between on the MCNTs and nHA,the cell attachment strength and proliferation on the MCNTs were better.Most importantly,the MCNTs could induce osteogenic differentiation of the HASCs better than the nHA,the possible mechanism of which was found to be that the MCNTs could activate Notch involved signaling pathways by concentrating more proteins,including specific bone-inducing ones.Moreover,the MCNTs could induce ectopic bone formation in vivo while the nHA could not,which might be because MCNTs could stimulate inducible cells in tissues to form inductive bone better than nHA by concentrating more proteins including specific bone-inducing ones secreted from M2 macrophages.Therefore,MCNTs might be more effective materials for accelerating bone formation even than nHA.

Heterotopic bone formation in the musculus latissimus dorsi of sheep usingβ-tricalcium phosphate scaffolds:evaluation of different seeding techniques

Osseous reconstruction of large bone defects remains a challenge in oral and maxillofacial surgery.In addition to autogenous bone grafts,which despite potential donor-site mobility still represent the gold standard in reconstructive surgery,many studies have investigated less invasive alternatives such as in vitro cultivation techniques.This study compared different types of seeding techniques on pureβ-tricalcium phosphate scaffolds in terms of bone formation and ceramic resorption in vivo.Cylindrical scaffolds loaded with autologous cancellous bone,venous blood,bone marrow aspirate concentrate or extracorporeal in vitro cultivated bone marrow stromal cells were cultured in sheep on a perforator vessel of the musculus latissimus dorsi over a 6-month period.Histological and histomorphometric analyses revealed that scaffolds loaded with cancellous bone were superior at promoting heterotopic bone formation and ceramic degradation,with autogenous bone and bone marrow aspirate concentrate inducing in vivo formation of vital bone tissue.These results confirm that autologous bone constitutes the preferred source of osteoinductive and osteogenic material that can reliably induce heterotopic bone formation in vivo.

Low-intensity pulsed ultrasound prompts tissue-engineered bone formation after implantation surgery

Background A practical problem impeding clinical translation is the limited bone formation seen in artificial bone grafts.Low-pressure/vacuum seeding and dynamic culturing in bioreactors have led to a greater penetration into the scaffolds,enhanced production of bone marrow cells,and improved tissue-engineered bone formation.The goal of this study was to promote more extensive bone formation in the composites of porous ceramics and bone marrow stromal cells （BMSCs）.Methods BMSCs/β-tricalcium phosphate （β-TCP） composites were subcultured for 2 weeks and then subcutaneously implanted into syngeneic rats that were split into a low-intensity pulsed ultrasound （LIPUS） treatment group and a control group.These implants were harvested at 5,10,25,and 50 days after implantation.The samples were then biomechanically tested and analyzed for alkaline phosphate （ALP） activity and osteocalcin （OCN） content and were also observed by light microscopy.Results The levels of ALP activity and OCN content in the composites were significantly higher in the LIPUS group than in the control group.Histomorphometric analysis revealed a greater degree of soft tissue repair,increased blood flow,better angiogenesis,and more extensive bone formation in the LIPUS groups than in the controls.No significant difference in the compressive strength was found between the two groups.Conclusion LIPUS treatment appears to enhance bone formation and angiogenesis in the BMSCs/β3-TCP composites.

JMJD3 promotes chondrocyte proliferation and hypertrophy during endochondral bone formation in mice

JMJD3(KDM6B)is an H3K27me3 demethylase and counteracts polycomb-mediated transcription repression.However,the function of JMJD3 in vivo is not well understood.Here we show that JMJD3 is highly expressed in cells of the chondrocyte lineage,especially in prehypertrophic and hypertrophic chondrocytes,during endochondral ossification.Homozygous deletion of Jmjd3 results in severely decreased proliferation and delayed hypertrophy of chondrocytes,and thereby marked retardation of endochondral ossification in mice.Genetically,JMJD3 associates with RUNX2 to promote proliferation and hypertrophy of chondrocytes.Biochemically,JMJD3 associates with and enhances RUNX2 activity by derepression of Runx2 and Ihh transcription throughits H3K27me3 demethylase activity.These results demonstrate that JMJD3 is a key epigenetic regulator in the process of cartilage maturation during endochondral bone formation.

Exercise promotes osteogenic differentiation by activating the long non-coding RNA H19/microRNA-149 axis

BACKGROUND Regular physical activity during childhood and adolescence is beneficial to bone development,as evidenced by the ability to increase bone density and peak bone mass by promoting bone formation.AIM To investigate the effects of exercise on bone formation in growing mice and to investigate the underlying mechanisms.METHODS 20 growing mice were randomly divided into two groups:Con group(control group,n=10)and Ex group(treadmill exercise group,n=10).Hematoxylin-eosin staining,immunohistochemistry,and micro-CT scanning were used to assess the bone formation-related indexes of the mouse femur.Bioinformatics analysis was used to find potential miRNAs targets of long non-coding RNA H19(lncRNA H19).RT-qPCR and Western Blot were used to confirm potential miRNA target genes of lncRNA H19 and the role of lncRNA H19 in promoting osteogenic differentiation.RESULTS Compared with the Con group,the expression of bone morphogenetic protein 2 was also significantly increased.The micro-CT results showed that 8 wk moderate-intensity treadmill exercise significantly increased bone mineral density,bone volume fraction,and the number of trabeculae,and decreased trabecular segregation in the femur of mice.Inhibition of lncRNA H19 significantly upregulated the expression of miR-149 and suppressed the expression of markers of osteogenic differentiation.In addition,knockdown of lncRNA H19 significantly downregulated the expression of autophagy markers,which is consistent with the results of autophagy-related protein changes detected in mouse femurs by immunofluorescence.CONCLUSION Appropriate treadmill exercise can effectively stimulate bone formation and promote the increase of bone density and bone volume in growing mice,thus enhancing the peak bone mass of mice.The lncRNA H19/miR-149 axis plays an important regulatory role in osteogenic differentiation.

Effects of phosvitin phosphopeptide-Ca complex prepared by efficient enzymatic hydrolysis on calcium absorption and bone deposition of mice

Phosvitin(PV)was treated with high-temperature,mild pressure(HTMP),and enzyme combination,and then phosvitin phosphopeptides-calcium(PPP-Ca)complexes were prepared.The low-calcium specific pathogen free-Kunming(SPF-KM)mice were used to determine the effect of PPP-Ca complexes on intestinal calcium absorption and their utilization for bone formation.The serum calcium content was the highest with the HTMP-Enz-PPP-Ca treatment(2.19 mmol/L),and it significantly down-regulated the abnormal elevation of serum alkaline phosphatase(AKP)caused by calcium deficiency.The low-calcium control group had the lowest calcium deposited to the femur(80.41 mg/g)and the lowest femur bone mineral density(BMD)(0.17 g/cm^(3)),while HTMP-Enz-PPP-Ca significantly improved bone calcium content(94.33 mg/g)and BMD(0.29 g/cm^(3)).The micro-computed tomography(MCT)images showed that the femur with the normal control,PV-Ca,and HTMP-Enz-PPP-Ca treatments had a more compact,complete,and thicker trabecular network than the low-calcium and CaCl_(2)treatments.These results indicated that the organic calcium(HTMP-Enz-PPP-Ca)promoted calcium absorption and bone deposition,and the effect of HTMP-Enz-PPP-Ca was better than the inorganic CaCl_(2).

Molecular mechanisms of intermuscular bone development in fish:a review

Intermuscular bones(IBs)are slender linear bones embedded in muscle,which ossify from tendons through a process of intramembranous ossification,and only exist in basal teleosts.IBs are essential for fish swimming,but they present a choking risk during human consumption,especially in children,which can lead to commercial risks that have a negative impact on the aquaculture of these fish.In this review,we discuss the morphogenesis and functions of IBs,including their underlying molecular mechanisms,as well as the advantages and disadvantages of different methods for IB studies and techniques for breeding and generating IB-free fish lines.This review reveals that the many key genes involved in tendon development,osteoblast differentiation,and bone formation,e.g.,scxa,msxC,sost,twist,bmps,and osterix,also play roles in IB development.Thus,this paper provides useful information for the breeding of new fish strains without IBs via genome editing and artificial selection.

Induced Bone by Calcium Ohosphate (Ca-P) Ceramics after 2-years Implantation

The changes of induced bone with four kinds of Ca-P ceramics after 2 years implantation in the dorsal muscles of rabbits were investigated. After 2 years implantation, mature bone （with a lamellar structure after the remodeling process） with bone marrow was observed in HA1100 （HA, micro＋macro porous）, HA900 （HA, micro＋macro porous） and BCP （HA/TCP=7：3, micro＋macro porous）, and no bone formation was observed in HA1200 （HA, macro porous） ceramics. The induced bone neither disappeared nor grew uncontrollably, and was seen inside the pores of the implants or on the outer surface under the fibrous layer after 2 years. No bone formation was observed in the soft tissues distant from the implants. These results indicate that heterotopic bone formation induced by Ca-P materials did not give rise to uncontrolled growth over time, and the induced bone is limited to the inside or around the implants. Moreover, ectopic bone formation on the outer surface of the implants was detected in rabbits after 2 years.

The Transformation of Calcium Phosphate Bioceramics in Vivo

To study the transformation process of calcium phosphate bioceramic in vivo,biodegradable porous β-tricalcium phosphate ceramics (β-TCP) were used in this experiment. The materials (5×8mm) were implanted in the tibia of rabbits. The β-TCP ceramics with bone tissue were retrieved and treated for histology, and then observed by using a scanning electron microscope (SEM) and an electron probe X-ray microanalyzer (EMPA) every month. The results show that β-TCP ceramics bond to bone directly,new bones are forming and maturing with materials continuous degrading,and the materials are nearly replaced by the formed bone finally.Parts of the materials were degraded,absorpted and recrystallized,the others dispersped on the cancellous bone and the Haversian lamella with an irregular arrangement incorporating in bone formation directly by remodeling structure.

In Vivo Estimation of Osteogenesis by Bone Marrow Cells in a Bi-Phasic Scaffold and in Each of Its Components

A bi-phasic scaffold consisting of a columnar formaldehyde-acetalized polyvinyl alcohol (PVF) sponge and a cylindrical porous hydroxyapatite (HA) with a hollow center was devised. Rat bone marrow cells (rBMCs) were seeded into the sponge placed in the hollow center of the cylindrical porous HA. The bi-phasic scaffold, a cylindrical porous HA and a PVF sponge separated from a bi-phasic scaffold after rBMC seeding, and a PVF sponge without rBMCs as a negative control, were implanted for 6 weeks into rat dorsal subcutaneous tissue. In each construct, bone formation was examined histologically and osteocalcin was measured immunochemically. Bone formation was observed in the bi-phasic scaffold and also in the cylindrical porous HA isolated from the bi-phasic scaffold. A significant difference in the quantity of osteocalcin was observed between the bi-phasic scaffold and the isolated cylindrical porous HA. No bone formation was found in the isolated PVF sponge. The bi-phasic scaffold as an outer layer of the scaffold seemed to inhibit the outflow of rBMCs from the PVF sponge. This type of bi-phasic scaffold may have two specific characteristics: Attachment of cells both in PVF sponge and cylindrical porous HA.