Chemical induced demineralization study in cortical bone

Abstract

In this work we present a study of demineralization in bovine cortical bone. We selected 9 fresh cortical bone samples from 2 diaphyseal femurs for analysis. Samples were demineralized for 24 h, 48 h, 72 h and 96 h using two concentrations of EDTA with different pH: EDTA 0.1 M (pH 10, alkaline) and EDTA 0.5 M (pH 7.4, neutral). We have employed µ-X-ray fluorescence (µ-XRF) and X-ray diffraction (XRD) to assess the degree of demineralization. EDTA solutions were analyzed for Calcium (Ca) and Phosphorous (P) extractions by Atomic Absorption Spectrophotometry (AAS) and Ion Chromatography (IC), respectively. Results from AAS and IC showed that EDTA 0.5 M (pH 7.4) removed two times more Ca and 3 times more P than EDTA 0.1 M (pH 10) in the first 24 hours. µ-XRF results presented that EDTA has a high capacity to bind Calcium and Phosphorus. On the other hand, despite the differences in concentration and pH, EDTA did not bind Zn and Sr. Results from XRD showed that EDTA with high concentration had a greater impact to the samples' crystallinity causing a severe damage.

Factors influencing DNA extraction from human skeletal remains: bone characteristic and total demineralization process

“…This process is based on the use of EDTA to chelate some mineral composition in specimens (Balayan et al 2015). The concentration and pH of EDTA were previously reported to affect the demineralization process of the teeth or on the bovine cortical bone (Sales et al 2018). Hasap et al (2020) found that total demineralization combined with DNA purification by commercial kit improved DNA yield and STR profiles (Hasap et al 2020).…”

“…To determine the calcium level chelated from the bone in demineralization buffer after the completed demineralization process, the calcium concentration was quantified by atomic absorption spectrophotometry (Sales et al 2018). The amount of calcium in the EDTA solution was calculated comparing the calcium standard curve and expressed as mg/dL.…”

“…This substance supports the hard structure of the bone, resulting in limited DNA extraction (Loreille et al 2007). Hence, several researchers have discovered the appropriate techniques to eliminate calcium phosphate from the bone structure (Pérez et al 1989;Serper and Çalt 2002;Sousa and Silva 2005;Sales et al 2018).…”

“…Ethylene-diaminetetra-acetic acid (EDTA), ethyleneglycol-ether-diaminetetra-acetic acid (EGTA), and 1,2 cyclohexane-diaminetetra-acetic acid (CDTA) have also been recommended for total demineralization (Sousa and Silva 2005). Although many studies have attempted to find appropriate methods for sample preparation prior to DNA extraction, there was still no clear procedure for sample preparation from skeletal remains (Pérez et al 1989;Serper and Çalt 2002;Sousa and Silva 2005;Sales et al 2018). A highly efficient DNA extraction method has been developed such as total demineralization plus silica membrane columns method or modified Hi-Flow® silica method, and PrepFiler® BTA kit.…”

“…Only a few studies have been carried out on all influencing factors on human skeletal remains. Previous studies related to the demineralization process of EDTA have been conducted on human teeth (Pérez et al 1989;Serper and Çalt 2002;Sousa and Silva 2005) or bovine bones (Sales et al 2018); however, the application in old human skeletons has been less performed. Thus, this study was conducted to study the factors influencing total demineralization process in DNA extraction from human skeletal remains and developed effective methods with less time-consuming, reasonable cost, and compatible for laboratory to obtain qualified DNA for DNA profiling.…”