Hydroxyapatite-alginate biocomposite promotes bone mineralization in different length scales <italic>in vivo

doi:10.1007/s11706-009-0029-9

Front Mater Sci Chin

2009, Vol. 3

Issue (2) : 145-153 https://doi.org/10.1007/s11706-009-0029-9

RESEARCH ARTICLE

Hydroxyapatite-alginate biocomposite promotes bone mineralization in different length scales in vivo

F. L. DE PAULA¹, I. C. BARRETO¹, M. H. ROCHA-LE?O², R. BOROJEVIC¹, A. M. ROSSI³, F. P. ROSA⁴, M. FARINA¹(

)

1. Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil; 2. Escola de Química, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil; 3. Centro Brasileiro de Pesquisas Físicas, Rio de Janeiro, RJ, Brazil; 4. Instituto de Ciências da Saúde, Universidade Federal da Bahia, Salvador, Brazil

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Abstract

Tissue engineering is a multidisciplinary research area that aims to develop new techniques and/or biomaterials for medical applications. The objective of the present study was to evaluate the osteogenic potential of a composite of hydroxyapatite and alginate in bone defects with critical sizes, surgically made in the calvaria region of rats. The rats (48 adult males), Rattus norvegicus Wistar, were divided into two groups: control (without composite implantation) and experimental (with composite implantation) and analyzed by optical microscopy at the biological time points 15, 45, 90 and 120 d, and transmission electron microscopy 120 d after the implantation of the biomaterial. It was observed that the biomaterial presented a high degree of fragmentation since the first experimental points studied, and that the fragments were surrounded by new bone after the duration of the project. These areas were studied by analytical transmission electron microscopy using an energy dispersive X-ray spectrometer. Three regions could be distinguished: (1) the biomaterial rich in hydroxyapatite; (2) a thin contiguous region containing phosphorus but without calcium; (3) a region of initial ossification containing mineralizing collagen fibrils with a calcium/phosphorus ratio smaller than the particles of the composite. The intermediate region (without calcium or containing very low amounts of calcium), which just surrounded the composite had not been described in the literature yet, and is probably associated specifically to the biocomposite used. The high performance of the biomaterial observed may be related to the fact that alginate molecules form highly anionic complexes and are capable of adsorbing important factors recognized by integrins from osteoblasts. Regions of fibrotic tissue were also observed mainly in the initial experimental points analyzed. However, it did not significantly influence the final result. In conclusion, the biomaterial presents a great potential for application as bone grafts in the clinical area.

Keywords bone engineering bone healing hydroxyapatite alginate biocomposite analytical microscopy

Corresponding Author(s): FARINA M.,Email:mfarina@anato.ufrj.br

Issue Date: 05 June 2009

Cite this article:

F. L. DE PAULA,I. C. BARRETO,M. H. ROCHA-LE?O, et al. Hydroxyapatite-alginate biocomposite promotes bone mineralization in different length scales in vivo[J]. Front Mater Sci Chin, 2009, 3(2): 145-153.

URL:

https://academic.hep.com.cn/foms/EN/10.1007/s11706-009-0029-9
https://academic.hep.com.cn/foms/EN/Y2009/V3/I2/145

Fig.1 Control group 15 d: Region near the pre-existing bone tissue (right in the figure), where it is possible to observe the formation of a mineralized tissue (central region). Left region stained in blue corresponds to the non mineralized defect area. Toluidine blue – basic fucsin staining. Image obtained, from the same region observed in the previous figure, by using polarization light microscopy. Note that the region corresponding to the new bone formed (diffuse birefringence at the central region of the figure) does not present organization of the collagen fibrils similar to the original bone tissue (parallel bright lines).

Fig.2 Experimental group 15 d: Image of a polished section cut transversely to the calvaria region, near the border of the defect (bottom left), where it is observed a fragmentation of the biomaterial (top right). Some particles extruded from the defect region (top left). The sample was post-fixed with osmium tetroxide.

Fig.3 Control group 45 d: Transverse semithin section near the border of the defect. Formation of new bone (bottom left) in direct contact with the remaining tissue is observed. Toluidine blue – basic fucsin stain. Image obtained using polarization light microscopy of the same region shown in the previous figure. We observed the formation of the new bone tissue (irregular pattern of brightness) in direct contact with the remaining bone tissue (brighter area with fibers oriented in parallel). From these figures we can infer that the thickness of the tissue covering the central region of the defect diminished significantly relatively to the original calvaria bone tissue. Fibrous tissue covered the contours of the new formed bone.

Fig.4 Experimental group 45 d: Image of a polished section of epoxi-embedded sample near the border of the defect, where it is observed the formation of mineralized tissue surrounding particles and in the spaces between particles of the biomaterial. Polarization light microscopy image from the same region as in the previous figure. In the central area of the figure several fragments of the original biomaterial are seen with surrounding new bone tissue formed (brighter contour with a circular appearance around each fragment of the material). This shows the osteogenic potential of the biomaterial, particularly when it is fragmented. Richardson stain.

Fig.5 Control group 90 d: Formation of new bone tissue (top right in the figure) near the border of the defect (bottom left and central region). Toluidine blue – basic fucsin stain. Polarization light microscopy image of the same region observed in the previous figure. We observed new bone formation (top right) in contact with the remaining bone tissue (bottom left and central region) brighter areas in the figure.

Fig.6 Experimental group 90 d: Image of a polished section of epoxi-embedded material where it is observed new bone formed (bottom central) as well as blood vessels (regions in red) surrounding the highly fragmented biomaterial. Post-fixed with osmium tetroxide.

Fig.7 Control group 120 d: Formation of new bone (left half of the figure) near the border of the defect. Previously formed calvaria bone at top right. Nonmineralized tissue and cells are stained in blue. Toluidin blue – basic fucsin stain.

Fig.8 Experimental group 120 d: Image of a polished section of rat calvaria (top view) where it is observed that some of the spheres disaggregated and were surrounded by new bone formed in different length scales. Polarization light microscopy image of the same region as in the previous figure. This image confirms the hypothesis posed in the previous sentence. The brighter regions correspond to the new bone formed. Note that both intact and disaggregated spheres in the central and periphery areas are surrounded by new bone.

Fig.9 Transmission electron microscopy and analysis of experimental group after 120 d: Region near the implanted biomaterial where mineralization (dark regions) is occurring on the collagen fibrils and in the gap regions (arrows). Three regions of distinct nature related to the biomaterial, found in regions where there was mineralization of new bone: the biomaterial itself (dark regions at the upper part of the figure); cement layer (LC); mineralized matrix rich in type I collagen (COL and arrows).

Fig.10 EDS spectra of the regions described in the previous figure: Hydroxyapatite nanoparticles from the biomaterial. Region just surrounding the nanoparticle-containing biomaterial. Region corresponding to the forming new bone tissue. Note that the region that surrounds the biomaterial does not contain calcium, and that the region of forming new bone tissue presents a Ca/P ratio smaller than the one for hydroxyapatite. The samples were stained with uranil acetate and lead citrate.

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