RESUMEN
INTRODUCTION: Bone decalcification is a necessary preprocessing step in histological and anatomical studies. Several solutions for decalcification with different claimed times for full decalcification are commercially available. Current literature lacks direct, quantitative measurement of calcium hydrocyapatite degradation during decalcification to compare different solutions. Therefore, the aim of this study was to test the performance of three different decalcification solutions in human bone by direct measurement of calcium hydroxyapatite using dual-X-ray-absorptiometry (DEXA) and volumetric computed tomography (CT). METHODS: Four femur slices were acquired from the proximal femur of a 76-year-old body donor. The slices were submerged in formaldehyde (control), EDTA, Osteosoft (Merck, Darmstadt, Germany) and "Rapid Bone Decalcifier" (RBD) (American MasterTech Scientific, Lodi, USA). Consecutive DEXA and CT scans were performed at 2 h, 4 h, 8 h, 11 h, 20 h, 44 h and 77 h after solutions were added. Besides the calcium hydroxyapatite concentration, the bone volume was measured each time. RESULTS: Fastest decline in volume was seen in the RBD probe. Further, RBD was the only solution, being able to fully decalcify the bone slice after 77 h. Although a steady decline in volume and hydroxyapatite concentration was seen for EDTA and Osteosoft as well, both were not able to decalcify the slices. CONCLUSION: Overall, the purely qualititve acquired literature data on bone decalcifiers was verified by our quantitative data for human, cortical-rich bones. Hydrochloric-acid based solutions seem to be preferable in order to rapidly dissolve the calcium hydroxyapatite.
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Huesos , Fémur , Absorciometría de Fotón , Anciano , Densidad Ósea , Fémur/diagnóstico por imagen , Humanos , Solventes , Tomografía Computarizada por Rayos X , Rayos XRESUMEN
Calcination and decalcification are basic procedures useful to a morphological approach of a biological, composite material like cortical bone. The study was carried out on a whole human femur conserved in liquid (from an educational collection). Cortical fracturing and SEM observation of vascular canals surface collagen texture was used to study bone deproteination at scalar temperatures (400-1,200°C) and acid bone decalcification at crescent time intervals. Heating burned and vaporized the organic matrix with shrinkage of the bone specimens as documented by the weight loss and transverse surface morphometry. SEM showed a pattern of aligned spherulites at 400°C which maintained the collagen fibrils layout (like a mineral cast), followed by a spherulites fusion progression with the temperature increments. At 1200°C a crystalline-like structure of tightly-packed trapezohendron units. XRD analysis supported the SEM morphology displaying the complete Debey rings of hydroxyapatite and spotted Debey rings of withlockite. Surface Ca and P elution was documented after 12 hr of exposition to the acid solution by dissolution of spherulites and the whole canal surface decalcified in depth after 15 days by SEM-EDAX analysis. The periodic pattern of collagen fibrils was still evident up to 15 days of decalcification together with fine granular deposits of a not-collagenic proteic material, while after 30 days no period was observed in the decalcified fibrils. Collagen mineral cast at 400°C calcination. Complete crystalline transformation at 1200°C. Up to 15 days of decalcification fibrils period maintained.
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Matriz Ósea/anatomía & histología , Hueso Cortical/ultraestructura , Fémur/anatomía & histología , Fémur/ultraestructura , Colágeno/metabolismo , Hueso Cortical/irrigación sanguínea , Hueso Cortical/fisiología , Técnica de Descalcificación/métodos , Fémur/irrigación sanguínea , Calor , Humanos , Masculino , Microscopía Electrónica de Rastreo , Minerales/metabolismoRESUMEN
OBJECTIVES: The present study aims to evaluate the preservation of the microstructure of skeletal remains collected from four different known burial sites (archaeological and contemporary). Histological analysis on undecalcified and decalcified thin sections was performed in order to assess which of the two techniques is more affected by taphonomic insults. MATERIALS AND METHODS: A histological analysis was performed on both undecalcified and decalcified thin sections of 40 long bones and the degree of diagenetic change was evaluated using transmitted and polarized light microscopy according to the Oxford Histological Index (OHI). In order to test the optical behavior of bone tissue, thin sections were observed by polarized light microscopy and the intensity of birefringence was evaluated. RESULTS: The more ancient samples are generally characterized by a low OHI (0-1) with extensive microscopic focal destruction; recent samples exhibited a better preservation of bone micromorphology. When comparing undecalcified to decalcified thin sections, the latter showed an amelioration in the conservation of microscopic structure. As regards the birefringence, it was very low in all the undecalcified thin sections, whereas decalcification process seems to improve its visibility. DISCUSSION: The preservation of the bone microscopic structure appears to be influenced not only by age, but also by the burial context. Undecalcified bones appear to be more affected by taphonomical alterations, probably because of the thickness of the thin sections; on the contrary, decalcified thin sections proved to be able to tackle this issue allowing a better reading of the bone tissue.
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Huesos/química , Huesos/patología , Calcificación Fisiológica/fisiología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Antropología Física , Birrefringencia , Entierro , Colágeno/química , Femenino , Técnicas Histológicas , Humanos , Masculino , Microscopía , Persona de Mediana Edad , Adulto JovenRESUMEN
Confocal immunofluorescence is a valuable technique for the detection of relevant molecules in the pathogenesis of arthritis in rat models; however, it requires efficient processing of tissues including bone decalcification. The decalcification process must ensure the complete removal of calcium and also a proper preservation of cellular structures and, specially, the antigenicity of the tissue to allow the immunodetection of the molecules of interest. In the present study, we evaluated the effect of four different decalcifying solutions: the Morse´s solution, 10% EDTA (pH 7.4), 7% HCl/2% EDTA and 5% Nitric acid, as well as four different treatments of the tissues (including microwave irradiation) in the processes of decalcification for large pieces of adult rat bones (hind paw, fore paw, knee and column). We assessed the time of decalcification, the easiness of slicing, the morphological preservation and finally, the antigenicity of two different bone proteins (Osteopontin (OPN) and Osteocalcin (OC)) measured by its immunofluorescence intensity under controlled confocal microscopy conditions. Our results showed that the specimen size and the presence of skin are critical factors for the rate of decalcification, and no significant benefit was found if microwave irradiation is applied to the tissue. The comprehensive statistical analysis showed that the optimal solution for the detection of OPN and OC by confocal immunofluorescence is the 5% Nitric Acid, and followed by 10% EDTA (pH 7.4), Ana Morse solution and 7% HCl/2% EDTA.