Detection Of Alkaline Phosphatase Research Articles

The effect of strontium ranelate on titanium particle-induced periprosthetic osteolysis regulated by WNT/β-catenin signaling in vivo and in vitro.

Aseptic loosening following periprosthetic osteolysis is the primary complication that limits the lifetime of total joint arthroplasty (TJA). The wear particles trigger a chronic inflammation response in the periprosthetic tissue and turn over the bone balance to bone resorption. The present study aimed to investigate the possible effect and mechanism of strontium ranelate (SR), a clinically safe drug for osteoporosis, on particle-induced periprosthetic osteolysis. Thirty-six female C57BL/6j mice underwent tibial Ti-nail implantation to establish an animal model of aseptic loosening. After 12 weeks, micro-CT results showed that strontium ranelate could inhibit periprosthetic bone resorption. In vitro, Ti particles were used to stimulate RAW264.7 cell line to collect conditioned medium, and co-culture MC3T3-E1 cell line with conditioned medium to establish a cell model of aseptic loosening. The results of alkaline phosphatase (ALP) detection, immunofluorescence, and flow cytometry demonstrated that strontium ranelate could regulate the expression of OPG/RANKL, promote differentiation and mineralization, and inhibit apoptosis in osteoblasts. Moreover, we revealed that SR’s exerted its therapeutic effect by down-regulating sclerostin, thereby activating the Wnt/β-catenin signal pathway. Therefore, this research suggests that strontium ranelate could be a potential drug for the prevention and treatment of particle-induced aseptic loosening post-TJA.

Sensitive Fluorescence Assay for the Detection of Alkaline Phosphatase Based on a Cu2+-Thiamine System.

The authors describe a novel, facile, and sensitive fluorometric strategy based on a Cu2+-thiamine (Cu2+-TH) system for the detection of alkaline phosphatase (ALP) activity and inhibition. The principle of the method is as follows. Under a basic conditions, TH, which does not exhibit a fluorescence signal, is oxidized into fluorescent thiochrome (TC) by Cu2+. Ascorbic acid 2-phosphate (AAP), which is the enzyme substrate, is hydrolyzed to produce ascorbic acid (AA) by ALP. The newly formed AA then reduces Cu2+ to Cu+, which prevents the oxidation of TH by Cu2+; as a result, the fluorescent signal becomes weaker. On the contrary, in the absence of ALP, AAP cannot reduce Cu2+; additions of Cu2+ and TH result in a dramatic increase of the fluorescent signal. The sensing strategy displays brilliant sensitivity with a detection limit of 0.08 U/L, and the detection is linear in the concentration range of 0.1 to 100 U/L. This approach was successfully applied to ALP activity in human serum samples, indicating that it is reliable and may be applied to the clinical diagnosis of ALP-related diseases.

Deferasirox (ExJade): An FDA-Approved AIEgen Platform with Unique Photophysical Properties.

Deferasirox, ExJade, is an FDA-approved iron chelator used for the treatment of iron overload. In this work, we report several fluorescent deferasirox derivatives that display unique photophysical properties, i.e., aggregation-induced emission (AIE), excited state intramolecular proton transfer, charge transfer, and through-bond and through-space conjugation characteristics in aqueous media. Functionalization of the phenol units on the deferasirox scaffold afforded the fluorescent responsive pro-chelator ExPhos, which enabled the detection of the disease-based biomarker alkaline phosphatase (ALP). The diagnostic potential of these deferasirox derivatives was supported by bacterial biofilm studies.

Visual detection of alkaline phosphatase based on ascorbic acid-triggered gel-sol transition of alginate hydrogel

Stimuli-responsive hydrogel has been emerged as a popular tool for chemical sensing due to its unique mechanical properties. In this work, we fabricated an ascorbic acid (AA)-responsive alginate hydrogel for the visual detection of alkaline phosphatase (ALP). This alginate hydrogel (RhB@Alg/Fe3+) was crosslinked with Fe3+, and rhodamine B (RhB) was encapsulated into the hydrogel as an indicating reagent to assistant visual detection. Because of the weak affinity of Fe2+ to alginate, the presence of reductive AA can trigger the dissolution of RhB@Alg/Fe3+ to give an observable red color in the sol solution. On this basis, by using ascorbic acid 2-phosphate as a substrate of ALP, which can be hydrolyzed by ALP to produce AA, the gel-sol transition process of RhB@Alg/Fe3+ was further modulated by ALP. This finding leads to a simple visual method for ALP detection with a low detection limit of 0.37 mU/mL and an excellent selectivity over other proteins. Compared with conventional colorimetric assays, this visual sensor shows the distinct advantages of simple fabrication, cost-effectiveness and easy to implement. We believe that this study can provide a new insight into the fabrication of responsive alginate hydrogel for promising applications in chemical sensing and biomedical fields.

A novel copper ion sensing fluorescent probe for fast detection of pyrophosphate and alkaline phosphatase

A Cu2+ sensing fluorescent probe is synthesized via a Mannich reaction and is applied in the fluorescence detection of pyrophosphate and alkaline phosphatase.

Digital counting of single semiconducting polymer nanoparticles for the detection of alkaline phosphatase.

Alkaline phosphatase (ALP) as a necessary hydrolase in phosphate metabolism is closely related to various diseases. Ultrasensitive detection of ALP with a convenient and sensitive method is of fundamental importance. In this work, a fluorescence resonance energy transfer (FRET)-based single-particle enumeration (SPE) method is proposed for the quantitative analysis of ALP. This strategy is based on the effective fluorescence suppression by a polydopamine (PDA) shell on the surface of semiconducting polymer nanoparticles (SPNs). PDA with broadband absorption in the UV-vis region can serve as an excellent quencher for SPNs. However, ascorbic acid (AA), the product of the hydrolysis of 2-phosphate-l-ascorbic acid trisodium salt (AAP) in the presence of ALP, can effectively inhibit the self-polymerization of dopamine (DA) to form a PDA layer. Therefore, ALP can be accurately quantified by counting the concentration-related fluorescent particles in the fluorescence image. A linear range from 0.031 to 12.4 μU mL-1 and a limit-of-detection (LOD) of 0.01 μU mL-1 for ALP determination are achieved. The spiked recoveries for ALP determination in a human serum sample are between 90% and 108% with RSD less than 3.1%. In summary, this convenient and sensitive approach proposed here provides promising prospects for ALP detection in a complex biological matrix.

Preadministration of yerba mate (Ilex paraguariensis) helps functional activity and morphology maintenance of MC3T3-E1 osteoblastic cells after in vitro exposition to hydrogen peroxide.

Natural substances with antioxidant effects may benefit prevention and treatment of people with or prone to bone diseases after menopause, such as osteoporosis. This study aimed to evaluate the in vitro effect of preadministration of yerba mate extract (YM) in the metabolism of MC3T3-E1 osteoblasts exposed to hydrogen peroxide (H2O2). The cells (MC3T3-E1) were cultured in 24-well plates with the concentration of 1μg/mL yerba mate extract dissolved in culture medium throughout the culture period. Four hours before each experiment, 400μmol/L H2O2 was added per well to simulate oxidative stress. There were evaluated cell adhesion and proliferation, in situ detection of alkaline phosphatase (ALP), mineralized nodules, and immunolocalization of osteocalcin (OCN), bone sialoprotein (BSP) and alkaline phosphatase (ALP) proteins. The results showed that YM preadministration to H2O2 exposition significatively increased cell adhesion after 3days as well as proliferation and in situ ALP detection after 10 and 7days respectively, when compared to H2O2 group. Besides, staining of OCN and BSP proteins was less intense and scattered in poor spread cells with cytoskeletal changes in H2O2 group when compared to control and YM H2O2 group. ALP staining was restrained to intracellular regions and similar in all experimental groups. Our results suggest that preadministration of yerba mate extract may prevent deleterious effects in the morphology and functional activity of osteoblasts exposed to H2O2, which could enable the maintenance of extracellular matrix in the presence of oxidative stress.

A redox modulated fluorescence nanoplatform for the detection of alkaline phosphatase activity with fluorescent polydopamine nanoparticles.

Herein, we simply synthesized intrinsic fluorescent polydopamine nanoparticles (PDA NPs) in sodium hydroxide solution (NaOH, pH 11), and constructed a new fluorescence nanoplatform for the detection of alkaline phosphatase (ALP) using PDA NPs as an effective signal reporter. The nanoplatform was constructed by the combination of enzymatic hydrolysis of ALP to the substrate l-ascorbic acid-2-phosphate (AA2P) and the chemical redox reaction between l-ascorbic acid (AA) and mercury ion (Hg2+). The fluorescence of PDA NPs could be effectively quenched by Hg2+ through the coordination effect between Hg2+ and the functional groups on the surface of PDA NPs. However, the quenching effect was greatly inhibited by the addition of AA into the solution. Based on this point, the activity of ALP could be monitored by hydrolysis of the substrate AA2P to AA and the fluorescence output of PDA NPs. The nanoplatform exhibited high sensitivity and desirable selectivity for ALP detection. With a wide linear range of 0 to 18 U L-1, a detection limit of 0.4 U L-1 was achieved using the developed nanosensor. The proposed method could not only be used to screen the inhibitor of ALP but also be used to detect ALP activity in human serum samples successfully. Moreover, the strategy can easily be expanded to determining other kinds of enzymes participating in AA-generation reactions.

Fe-N-C single-atom nanozymes with peroxidase-like activity for the detection of alkaline phosphatase.

Single-atom nanozymes have drawn wide attention in bio-sensing for their remarkable merits such as low cost, high stability, and maximum atom utilization. Herein, a colorimetric strategy based on Fe-N-C single-atom nanozymes (Fe/NC-SAs) was established for the detection of alkaline phosphatase (ALP) activity. The Fe/NC-SAs prepared by pyrolysis have excellent peroxidase-like activity and can oxidize 3,3',5,5'-tetramethylbenzidine (TMB) to a blue color product in the presence of hydrogen peroxide (H2O2). When ascorbic acid (AA) is added to the system, the blue color fades, and the absorbance has a linear relationship with the concentration of AA. Alkaline phosphatase (ALP) can catalyze the hydrolysis of ascorbic acid 2-phosphate (AAP) to produce AA. Thus, a strategy based on Fe/NC-SAs for the detection of ALP activity was established, which provided a linear range of 0.1-1.5 U L-1 and a limit of detection as low as 0.05 U L-1. Besides, Fe/NC-SAs showed high stability under harsh conditions. Moreover, an Fe/NC-SA-based assay was successfully validated using human serum samples for ALP determination with satisfactory results, and has broad prospects in the field of biosensing.

Ratiometric fluorescence determination of alkaline phosphatase activity based on dual emission of bovine serum albumin-stabilized gold nanoclusters and the inner filter effect.

A novel and convenient method for the ratiometric fluorescence detection of alkaline phosphatase (ALP) activity was proposed based on dual emission of bovine serum albumin-templated gold nanoclusters (BSA-AuNCs) and the mechanism of the inner filter effect between BSA-AuNCs and p-nitrophenol (PNP). First, ALP catalyzed the hydrolysis of the substrate p-nitrophenyl phosphate (PNPP) to produce PNP. PNP effectively quenched the emission peak of BSA-AuNCs at 410 nm because of the overlap in absorbance feature of PNP and the fluorescence spectrum of BSA-AuNCs, and the peak at 650 nm was almost unaffected. Thus, a sensitive ratiometric method for detection of ALP activity was developed using the fluorescence intensity of BSA-AuNCs at 650 nm as a reference signal. ALP activity versus the ratio of fluorescence intensities at 410 and 650 nm showed good linearity between 0.2 and 5 mU mL-1 (R2 = 0.9931) and high sensitivity with a detection limit of 0.03 mU mL-1 (S/N = 3). The developed sensing method was successfully applied to investigate ALP inhibitors and detect ALP in serum samples.

OSTEOPLASTIC PROPERTIES OF MULTIPOTENT MESENCHYMAL STROMAL CELLS OF ADIPOSE TISSUE

The aim: Determining the ability of samples based on MMSC – AT differentiating in the osteogenic direction. Materials and methods: The study was conducting at Bukovinian State Medical University, Chernivtsi, Ukraine. Adipose tissue samples were obtaining from the neck of 60 experimental animals (white Wistar rats). Multipotent mesenchymal stromal cells of adipose tissue were obtained by grinding adipose tissue of rats in 0.1% collagenase 1A . Alkaline phosphatase activity was assessing by using the Alkaline Phosphatase Detection Kit (Sigma, USA) according to the manufacturer’s protocol. Osteopontin gene expression was determining by immunocytochemical method. To determine the mRNA used the PCR method, which is associated with reverse transcription (RT-PCR) in the area of quantification of gene expression to the marker BGP. Results: On the 21st day of observations, the expression of mRNA encoding the BGP gene decreased in samples № 1 and № 3 to 35,800 ± 420.0 copies and to 35,000 ± 400.0 copies, p1<0.01, p>0.05. Also was observing growth of copies of the BGP gene in samples № 2 and № 4 in 2.1, р<0.01 and 2.2 times, р-р2<0.05, relative to the data in sample № 1. Conclusions: Comparative study of osteoplastic properties samples MMSC-AT showed that a larger number of cells differentiate into the osteoblasts in samples containing MMSC-AT + PRP (№ 2) and MMSC-AT + PRP + «Kolapan» (№ 4). This has been proven higher alkaline phosphatase activity, higher levels osteopontin expression, and higher levels BGP gene expression.

Ultrasensitive Electrochemiluminescence Biosensor Based on Closed Bipolar Electrode for Alkaline Phosphatase Detection in Single Liver Cancer Cell.

An ultrasensitive electrochemiluminescence (ECL) biosensor was proposed based on a closed bipolar electrode (BPE) for the detection of alkaline phosphatase (ALP). For most of the BPE-ECL biosensors, an effective signal amplification strategy was the key to enhance the sensitivity of the system. Herein, the signal amplification strategy of the enzyme catalysis was utilized in the BPE-ECL system. Au nanoparticles (NPs) were electrodeposited on the cathode surface of the ITO electrode to improve the stability and sensitivity of the signal. Compared with the previous BPE-ECL biosensors, the sensitivity was increased by at least 3 orders of magnitude. The biosensor showed high sensitivity and specificity of ALP detection with a detection limit of as low as 3.7 aM. Besides, it was further applied to the detection of ALP in different types of cells and successfully realized ALP detection in single Hep G2 cell, which had a huge application prospect in single biomolecule detection or single cell analysis.

A novel aggregation-induced dual emission probe for in situ light-up detection of endogenous alkaline phosphatase

Abnormal level of alkaline phosphatase (ALP) activity has been linked to many diseases in human. The development of fluorescent molecular probes that can report the expression and activity of ALP in various biological systems will be extremely valuable. However, the in vivo monitoring for ALP in living cells and more complex biological systems remains a great challenge. The excited-state intramolecular proton transfer (ESIPT) probe with proportional fluorescence has low background noise, while the aggregation induced emission (AIE) probe has the advantages of signal amplification and good light stability. Herein, an “AIE + ESIPT” fluorescent probe 2-(benzo[d]thiazol-2-yl)-4-(1,4,5-triphenyl-1H-imidazole-2-yl)phenyl dihydrogen phosphate (THP) was constructed for the highly selective and sensitive detection of ALP. By introducing a phosphate ester at the hydroxyl position of the solid fluorophore 2-(benzo[d]thiazol-2-yl)-4-(1,4,5-triphenyl-1H-imidazole-2-yl)phenol, ESIPT was hindered and the probe present a faint blue fluorescence in DMSO solution. While ALP was introduced, causing the phosphate in THP hydrolyzed, and the ESIPT process was restored to yield a yellow fluorescence at 550 nm, thereby achieving proportionality detection. THP exhibited high selectivity and sensitively to ALP with low limit of detection (1.21228 U/L), and the reaction completed within 20 min. In addition, with its outstanding advantages of low biological toxicity and enzyme conversion characteristics, THP has been successfully applied to ALP imaging in living cells (Hela cells, A549 cells and Hek293 cells), and can provide in situ information on the reaction site. Therefore, THP has the potential for detecting ALP activity in biomedical application.

In Vitro Induction of Pluripotency from Equine Fibroblasts in 20% or 5% Oxygen.

The cellular reprogramming into pluripotency is influenced by external and internal cellular factors, such as in vitro culture conditions (e.g., environmental oxygen concentration), and the aging process. Herein, we aimed to generate and maintain equine iPSCs (eiPSCs) derived from fibroblasts of a horse older than 20 years and to evaluate the effect of different levels of oxygen tension (atmospheric 20% O2, 5% O2, or 20% to 5% O2) on these cells. Fibroblasts were reprogrammed, and putative eiPSCs were positive for positive alkaline phosphatase detection; they were positive for pluripotency-related genes OCT4, REX1, and NANOG; immunofluorescence-positive staining was presented for OCT4 and NANOG (all groups), SOX2 (groups 5% O2 and 20% to 5% O2), and TRA-1-60, TRA-1-81, and SSEA-1 (only in 20% O2); they formed embryoid bodies; and there is spontaneous differentiation in mesoderm, endoderm, and ectoderm embryonic germ layers. In addition to the differences in immunofluorescence analysis results, the eiPSC colonies generated at 20% O2 presented a more compact morphology with a well-defined border than cells cultured in 5% O2 and 20% to 5% O2. Significant differences were also observed in the expression of genes related to glucose metabolism, mitochondrial fission, and hypoxia (GAPDH, GLUT3, MFN1, HIF1α, and HIF2α), after reprogramming. Our results show that the derivation of eiPSCs was not impaired by aging. Additionally, this study is the first to compare high and low oxygen cultures of eiPSCs, showing the generation of pluripotent cells with different profiles. Under the tested conditions, the lower oxygen tension did not favor the pluripotency of eiPSCs. This study shows that the impact of oxygen atmosphere has to be considered when culturing eiPSCs, as this condition influences the pluripotency characteristics.

Alkaline phosphatase-responsive Zn2+ double-triggered nucleotide capped gold nanoclusters/ alginate hydrogel with recyclable nanozyme capability

A novel hydrogel loaded with adenosine 5′-monophosphate capped Au nanoclusters (AuAMP NCs) is fabricated in a gentle route by double-triggering of Zn2+. This built-in fluorescent hydrogel material not only has good optical properties of Au NCs, but also possesses excellent mechanical structure of hydrogel materials. Free phosphate ions may trigger the devastation of the “egg-box” structure of the as-prepared ZnSA-AuAMP hydrogel, thus releasing the immobilized fluorescent AuAMP NCs, with a release efficiency up to 93.62% within 3 h. On this basis, a fast, sensitive fluorescent detection method for alkaline phosphatase (ALP) is achieved, with a linear detection of ALP in the range of 0.47–10.03 U/L and a limit of detection of 0.09 U/L. This allows the accurate detection of ALP in diluted human serum samples. Last but not least, the ZnSA-AuAMP hydrogel also exhibits peroxidase-like activity with good recyclability, because it is facile to be separated and extracted from catalytic reaction buffer. This work suggests that hydrogels may act as an inexpensive container for controllable regulation of nanozyme activity.

Osteoporosis Affects Functional Activity and Gene Expression of Osteoblastic Cells Derived from Rat Alveolar Bone.

Recent studies suggest that osteoporosis, in addition to the damage caused in long bones, may cause deterioration in the jaws, especially in alveolar bone sites, with effects in the progress of periodontal disease as well as in bone healing. The aim of this study was to evaluate the effect of osteoporosis in the metabolism of rat alveolar bone osteoblasts. There were used 10 female rats divided in two experimental groups (Sham and OVX), which were ovariectomized and after 8 weeks euthanized to collect mandibular bone samples in order to isolate osteoblastic cells. The cells were cultured in 24-well plates to perform the in vitro experiments. After 7, 10 and 14 days, there were evaluated cell proliferation by MTT assay, in situ detection of alkaline phosphatase (ALP) as well as mineralized nodules and expression of genes associated to bone remodeling. Results showed that at 7, 10 and 14 days cell proliferation was lower for OVX group. In situ detection of ALP was higher at 7 days and lower at 10 and 14 days in OVX group. At 17 and 21 days, OVX group had a significative decrease of mineralization nodules. There was a downregulation in the expression of Alp, Bglap and Runx2 genes and an upregulation of Opg in OVX group, whereas Opn and Rankl modulation was similar between the evaluated groups. Our results suggest that osteoporosis has a deleterious effect on alveolar bone cells from ovariectomized rats, which might affect the treatment of diseases associated to the jaw bones.

Magnetically controlled 2D nano-DNA fluorescent biosensor for selective and sensitive detection of alkaline phosphatase activity

In this work, a novel magnetically controlled two-dimensional nano-DNA fluorescence sensor for sensitive and selective detection of alkaline phosphatase (ALP) activity has been developed by combining the λ exonuclease (λ exo) cleavage reaction and a δ-FeOOH nanosheet-based platform. A 5ʹ-phosphoryl-modified double-stranded DNA probe (5ʹ-P-dsDNA) was designed as a substrate for ALP, and a 5ʹ-dye-labeled single-stranded DNA/δ-FeOOH system was designed as a detection unit. In the absence of ALP, 5ʹ-P-dsDNA was degraded by λ exo to yield mononucleotides. Thus, the fluorescence of the detection unit was quenched. However, in the presence of ALP, the 5ʹ-phosphate functional group of 5ʹ-P-dsDNA was efficiently removed to form 5ʹ-hydroxyl-dsDNA, which could no longer be degraded by λ exo. Then, the dye-labeled single-stranded DNA hybridized with the 5ʹ-hydroxyl-dsDNA to form a triplex DNA (tsDNA), resulting in increased fluorescence due to the weak binding of tsDNA with the δ-FeOOH nanosheet. More importantly, a high signal-to-background ratio was obtained through magnetic separation. The detection limit for ALP was 0.02 mU/mL (3σ/S), which is much lower than that previously reported for fluorescence methods. This work revealed the application of δ-FeOOH nanosheets in the design of magnetically controlled biosensors for biomolecular detection.

In Situ Controllable Generation of Copper Nanoclusters Confined in a Poly-l-Cysteine Porous Film with Enhanced Electrochemiluminescence for Alkaline Phosphatase Detection.

Copper nanoclusters (Cu NCs) as emerging luminescent metal NCs are gaining increasing attention owing to the comparatively low cost and high abundance of the Cu element in nature. However, it remains challenging to manipulate the optical properties of Cu NCs. Unlike most dispersed Cu NCs, whose luminescence efficiency was restricted by nonexcited relaxation, the Cu NCs confined in a porous poly-l-cysteine (poly-l-Cys) film were generated controllably with enhanced electrochemiluminescence (ECL) by in situ electrochemical reduction. Specifically, poly-l-Cys provided a porous structure to regulate the generation of Cu NCs within its holes, which not only increased the restriction on the intramolecular vibration and rotation of the ligands but also expedited the electron transfer near the electrode surface, reflecting in an enhancement of the ECL signal and efficiency. As an application of the confined Cu NCs, an ECL biosensor with high performance was constructed skillfully for highly sensitive detection of alkaline phosphatase (ALP), which adopted Cu NCs as the ECL luminophore and poly-l-Cys as a coreaction accelerator in a novel ECL ternary system (Cu NCs/S2O82-/poly-l-Cys). Furthermore, an ingenious target amplification based on the combination of a DNA walker and click chemistry was developed to convert ALP to DNA strands efficiently, achieving great improvement in the recognition efficiency. As a result, the biosensor had a low detection limit (9.5 × 10-7 U·L-1) and a wide linear range (10-8-10-2 U·L-1) for ALP detection, which showed great promise for the detection of non-nucleic acid targets and the diagnosis of diseases.

Recent advances in fluorescence imaging of alkaline phosphatase

Phosphatase plays a vital important role in many biological functions due to the dephosphorylation serves varied roles in cellular regulation and signaling. Among the family of phosphatase, alkaline phosphatase (ALP) could act as crucial prognostic indicators for many diseases such as bone diseases and cancer. However, the detection of ALP is mainly limited to in vitro colorimetric method in clinic. Therefore, huge efforts have been paid on the fluorescence imaging that provides a reliable method to detect the real-time and in vivo changes of the level of ALP. In this review, we summarize recent advances in fluorescence imaging of phosphatase, mainly focused on ALP. The imaging probes of phosphatase are mainly classified according to their luminescence mechanisms. In the end, we assessed the challenges and future prospects of phosphatase probes.

236. Ectopic bone formation by submicron structured calcium phosphates: role of the innate immune system

BACKGROUND CONTEXT Ectopic implantation of calcium phosphates (CaP) with an advanced submicron surface topography can lead to de novo bone formation. Since implantation initiates a wound healing response, the innate immune system may play a role in this process. PURPOSE Tis study is to investigate the role of immune cells (ie, macrophages) and osteoclasts in material-driven bone formation. STUDY DESIGN/SETTING TCP-B with micron-scaled surface (1-2mm, 1cc/implant) and BCP with submicron-scaled surface (MagnetOs, 1-2mm, 1cc/implant) were implanted in the dorsal muscles of nine beagles for 3 days, 1, 3, 6 and 12 weeks. Histology, immune-histochemistry and bioassays were performed to detect alkaline phosphatase (ALP), tartrate-resistant acid phosphatase (TRAP), Cathepsin K (CTSK), Tumor necrosis factor-alpha (TNF-alpha), Interleukin-10 (IL-10) and bone formation. PATIENT SAMPLE N/A OUTCOME MEASURES Bone formation was identified by histology and ALP expression. M1/M2 macrophages were characterized by IL-10 level (M2) and TNF-alpha level (M1) and the presence of osteoclasts was determined by TRAP level, TRAP staining and CTSK staining. Histomorphometry was also performed to quantify bone formation. One-way ANOVA with Tukey pos-hoc test was applied to distinguish differences between groups (p METHODS At harvesting, samples (n=4 per condition) were cut into halves for bioassays, histology and immune-histochemistry (IHC). Bioassays (Elisa for IL-10 and TNF-alpha; pNPP method for ALP and TRAP) were performed with lysates and the data were normalized to the mass of the samples. Histology (HE, Masson-Golder, ALP and TRAP) and IHC stain (CTSK) were applied on undecalcified sections. HE-stained sections were used to quantify bone (area percentage of bone in available space). RESULTS Bone was absent in TCP-B implants at all time-point, while bone was formed in BCP at week 6 (3/4, CONCLUSIONS Higher IL-10 level in BCP than TCP-B up to week 3, indicates that BCP favors pro-healing M2 macrophage polarization. The higher TNF-alpha level in BCP than TCP-B up to week 1, showed that initially more pro-inflammatory M1 macrophages are present in BCP compared to TCP-B. TRAP/CTSK-positive multinucleated cells were present at week 3, before bone formation (at week 6), which suggests that osteoclastogenesis occurs prior to osteogenesis. These data imply that calcium phosphates with a submicron topography facilitate macrophage recruitment/proliferation, enhance pro-healing M2 macrophage polarization and osteoclast-like cell formation, ultimately leading to ectopic bone formation. FDA DEVICE/DRUG STATUS MagnetOs Granules (Not approved for this indication)