Biocompatibility Studies on a Collagen-Hydroxyapatite Biomaterial

Abstract

The current treatment of osteomyelitis includes systemic antibiotic therapy and a debridement procedure of the formed biofilm and necrotic tissue. Moreover, cements and three-dimensional scaffolds are used both for the delivery of therapeutic agents and as fillers for bone defects. The aim of our research was to test, on cellular cultures, the biocompatibility of a previously synthesized microporous biocomposite containing hydroxyapatite and a collagen matrix including a therapeutic agent (ciprofloxacin and gentamicin). The scaffold was obtained by direct mineralization namely co-precipitation of hydroxyapatite on a collagen matrix.

Keywords: Biocomposite; biocompatibility; collagen-hydroxyapatite.

Figure 1

SEM images for the scaffolds tested: A. Col-HA, B. Col-HA-Gentamicin, C. Col-HA-Ciprofloxacin

Figure 1

SEM images for the scaffolds tested: A. Col-HA, B. Col-HA-Gentamicin, C. Col-HA-Ciprofloxacin

Figure 1

SEM images for the scaffolds tested: A. Col-HA, B. Col-HA-Gentamicin, C. Col-HA-Ciprofloxacin

Figure 2

Cytotoxicity evaluation of Col-HA scaffolds both with and without antibiotics (ciprofloxacin and gentamicin) using the MTS test. A. Sample images after the MTS reaction and supernatant removal. B.Graphic representation of the absorbance signal obtained following the MTS reaction

Figure 3

Mesenchymal stem cells viability after being cultured together with the Col-HA scaffold. Calcein (green) and ethidium homodimer-1 (red) were used to stain the viable cells and the dead cells, respectively inside the cellular culture. Hoechst was used to stain the nuclei. The upper image represents an overlap of images taken on 3 fluorescence channels (calcein/FITC+EthD-1/TxRed+Hoechst/DAPI). Below the first image are two decomposed images: left-calcein / FITC and right-EthD-1/TxRed+Hoechst/DAPI). Scale=100μm

Figure 4

Viability test performed on mesenchymal stem cells stained with calcein and co-cultivated with the scaffolds. After three days the samples were evaluated using the fluorescence microscope. Cell nuclei were identified by Hoechst labelling on the DAPI channel (left)

Figure 5

Cell adhesion analysis after staining MSC with AlexaFluor488 phalloidin which labels actin cytoskeleton (green); cell nuclei were stained with Hoechst (blue). The images were obtained using the TissueFAXSiPlus system which automatically scans the entire surface of the samples and then renders a virtual reconstruction of the sample (left); images on the right side represent selected detailed visual fields

Figure 6

SEM images of the MSC morphology 14 days after cell culture in osteoinduction medium compared to expansion medium

Figure 7

Analysis of the alkaline phosphatase enzymatic activity after 14 days of cultivating MSC in osteoinduction medium (OIM) compared to expansion medium (DMEM) by incubating samples with NBT-BCIP substrate (the result of the colorimetric reaction is highlighted in purple)