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In Situ Nanotechnology-Derived Sensors for Ensuring Implant Success
Published in Šeila Selimovic, Nanopatterning and Nanoscale Devices for Biological Applications, 2017
Sirinrath Sirivisoot, Thomas J. Webster
To study osteoblast differentiation between non-Ca- and Ca-depositing cells, osteo-blasts were seeded at a density of 40,000 cells/cm2 in DMEM supplemented with 10% FBS, 1% P/S, 50 nM β-glycerophosphate (Sigma), and 50 μg/mL ascorbic acid (Sigma) under standard cell culture conditions (a humidified 5% CO2 and 95% air environment at 37°C) for 7, 14, and 21 days. The cell media were changed every other day. At the end of each time point, an alkaline/acid phosphatase assay kit (Upstate) was used to determine the concentration of alkaline phosphatase in cell lysates. The cell lysates were prepared by first rinsing all samples three times with Tris-buffered saline (TBS; 42 mM Tris-HCl, 8 mM Tris base, and 0.15 M NaCl; a pH of 7.4; Sigma-Aldrich) and then subjecting the cells to three freeze–thaw cycles using distilled water. A Ca quantification kit (Sigma) was used to determine the amount of Ca deposited by the osteoblasts seeded on each sample. An acidic super-natant solution for a Ca deposition assay was prepared by incubating all the samples with 0.6 N HCl (Sigma) for 24 h. The light absorbance was measured by a spectrophotometer (SpectraMAX 340PC384; Molecular Devices) at 650 nm for alkaline phosphatase activity and 570 nm for Ca deposition. Long-term cytocompatible testing was conducted in triplicate and was repeated three different times. The numerical data were analyzed using standard Student’s t-test.
Anatomy, physiology and disease
Published in C M Langton, C F Njeh, The Physical Measurement of Bone, 2016
Matrix vesicles formed from bone cells were once thought to be the controlling determinants of crystallization during modelling and remodelling [57]. These extracellular vesicles, which are pinched away from osteoblasts, contain enzymes such as alkaline phosphatase which are essential for proper mineralization, and other proteins such as bone sialoprotein, which appears very early in the course of osteoblast mineralization in vitro. Indeed, deficiencies in alkaline phosphatase expression result in syndromes of osteomalacia, or ‘soft bone’, characterized by large amounts of osteoid that is not mineralized. However, it is now apparent, notwithstanding the phenotype of deficient alkaline phosphatase, that matrix vesicles are a function of mineralization only during the earliest phases of bone development, when so-called woven bone is produced. During later stages of postnatal development, the bone tissue containing matrix vesicles is resorbed and new tissue is formed which does not contain these vesicles, but which is appropriately mineralized. Hence, only during a specific developmental time period, or in the case of fracture during the production of woven bone, are the matrix vesicles important in mineralization [58, 59].
Polyphosphazenes as Biomaterials
Published in Severian Dumitriu, Valentin Popa, Polymeric Biomaterials, 2020
Meng Deng, Cato T. Laurencin, Harry R. Allcock, Sangamesh G. Kumbar
Since the first report on the biodegradable blends of poly[(amino acid ester)phosphazenes] and polyesters, a series of polyphosphazene blends have been developed and characterized for biocompatibility as prospective biomaterials. Most amino acid ester–based polyphosphazene blends were found to be biocompatible. Nair et al. blended PNEA with PLAGA (85:15) at two weight ratios of 1:3 and 1:1. Significantly higher PRO adhesion and proliferation were observed for both the blends compared to the parent polymers (Nair et al. 2005). Meanwhile, Qiu et al. have characterized a series of degradable blends comprising PNEG and polyesters or polyanhydrides for tissue biocompatibility using a rat subcutaneous implantation model (Qiu 2002a). The tissue biocompatibility of PNEG-PLAGA was found to be better than that of PNEG-PSTP. Moreover, the tissue biocompatibility of PNEG-PSTP blends was improved by increasing the weight percentage of PNEG in blends. These findings suggested the blends of PNEG-PLAGA or -PSTP may be used as drug delivery matrices or for other potential biomedical applications. Deng et al. (2008) developed high-strength materials for tissue engineering applications from the blends of PNEAPhPh and PLAGA at three weight ratios of 1:3, 1:1, and 3:1. In an in vitro osteocompatibility study using PRO culture, these blends not only supported the PRO proliferation to the same extent as PLAGA but also showed enhanced differentiation characterized by increased levels of alkaline phosphatase activity and mineralized matrix synthesis compared to parent polymers.
Sludge: next paradigm for enzyme extraction and energy generation
Published in Preparative Biochemistry and Biotechnology, 2019
Santosh Kumar Karn, Awanish Kumar
Alkaline phosphatase has widespread use in research and industry, specifically in protein labeling and dephosphorylation of nucleic acid. Alkaline phosphatase is a useful tool in the molecular biology lab since DNA possesses a phosphate group at 5′ end. Eliminating this phosphate prevents the ligation and circularization of DNA molecules, easy for radiolabelling, etc. Other important applications of alkaline phosphatase enzyme for immune assays are blotting, sequencing, enzyme-linked immunoabsorbent assays (ELISA), and nonisotopic probing. Alkaline phosphatase is apparently ubiquitous in nature. These properties and their tendency of structure and regulation in multiple forms, often make the isolation of alkaline phosphatases complicated. Studies on pure alkaline phosphatase focused mainly on the clinical significance[17,18] from animal sources. Among possible role of alkaline phosphatases might be providing inorganic phosphate for metabolic excretory and some secretory purpose in plants. Alkaline phosphatases probably have the role in the reproductive system of the mammal; possible human prostatic alkaline phosphatase may be in the dephosphorylation of esters to liberate fructose.[19]
The effect of multi-walled carbon nanotubes/hydroxyapatite nanocomposites on biocompatibility
Published in Advanced Composite Materials, 2018
Jung-Eun Park, Yong-Seok Jang, Il-Song Park, Jae-Gyu Jeon, Tae-Sung Bae, Min-Ho Lee
The Alkaline phosphatase (ALP) activity was evaluated using a TRACP & ALP assay kit (TaKaRa, Japan). MC3T3-E1 cells were cultured for 7 and 10 d at 1.5 × 104 cells ml−1 on a powder-coated 24-well plate. After incubation, the medium was removed and the plate was washed with saline. Then, 1 ml of p-nitrophenyl phosphate (pNPP) solution was added, followed by the ALP buffer, and incubated at 30 °C for 1 h. The resulting solution (200 μl) was transferred to a 96-well plate, and the absorbance at 405 nm was measured.
Synthesis, spectroscopic characterization, X-ray crystal structure, antimicrobial, DNA-binding, alkaline phosphatase and insulin-mimetic studies of oxidovanadium(IV) complexes of azomethine precursors
Published in Journal of Coordination Chemistry, 2020
Khurram Shahzad Munawar, Saqib Ali, Muhammad Nawaz Tahir, Nasir Khalid, Qamar Abbas, Irfan Zia Qureshi, Shabbir Hussain, Muhammad Ashfaq
Human alkaline phosphatase (ALP) can be classified into at least four tissue-specific forms or isozyme mainly according to the specificity of the tissue to be expressed, termed as placental alkaline phosphatase (PLALP or Regan isozyme), intestinal alkaline phosphatase (IALP), liver/bone/kidney alkaline phosphatase (L/B/K ALP) and germ cell ALP (GCALP or NAGAO isozyme).