Preparation and characterization of cockle shell aragonite nanocomposite porous 3D scaffolds for bone repair

Abstract

The demands for applicable tissue-engineered scaffolds that can be used to repair load-bearing segmental bone defects (SBDs) is vital and in increasing demand. In this study, seven different combinations of 3 dimensional (3D) novel nanocomposite porous structured scaffolds were fabricated to rebuild SBDs using an extraordinary blend of cockle shells (CaCo₃) nanoparticles (CCN), gelatin, dextran and dextrin to structure an ideal bone scaffold with adequate degradation rate using the Freeze Drying Method (FDM) and labeled as 5211, 5400, 6211, 6300, 7101, 7200 and 8100. The micron sized cockle shells powder obtained (75 µm) was made into nanoparticles using mechano-chemical, top-down method of nanoparticles synthesis with the presence of the surfactant BS-12 (dodecyl dimethyl bataine). The phase purity and crystallographic structures, the chemical functionality and the thermal characterization of the scaffolds' powder were recognized using X-Ray Diffractometer (XRD), Fourier transform infrared (FTIR) spectrophotometer and Differential Scanning Calorimetry (DSC) respectively. Characterizations of the scaffolds were assessed by Scanning Electron Microscopy (SEM), Degradation Manner, Water Absorption Test, Swelling Test, Mechanical Test and Porosity Test. Top-down method produced cockle shell nanoparticles having averagely range 37.8±3-55.2±9 nm in size, which were determined using Transmission Electron Microscope (TEM). A mainly aragonite form of calcium carbonate was identified in both XRD and FTIR for all scaffolds, while the melting (Tm) and transition (Tg) temperatures were identified using DSC with the range of Tm 62.4-75.5 °C and of Tg 230.6-232.5 °C. The newly prepared scaffolds were with the following characteristics: (i) good biocompatibility and biodegradability, (ii) appropriate surface chemistry and (iii) highly porous, with interconnected pore network. Engineering analyses showed that scaffold 5211 possessed 3D interconnected homogenous porous structure with a porosity of about 49%, pore sizes ranging from 8.97 to 337 µm, mechanical strength 20.3 MPa, Young's Modulus 271±63 MPa and enzymatic degradation rate 22.7 within 14 days.

Keywords: %, Percentage; 3D porous nanocomposite scaffold; 3D, 3 Dimensional; 5211, cockle shells nanoparticles 50%, gelatin 25%, dextran 10%, and dextrin 15%; 5400, cockle shells nanoparticles 50%, gelatin 40%, dextran 5%, and dextrin 5%.; 6211, cockle shells nanoparticles 60%, gelatin 20%, dextran 10%, and dextrin 10%; 6300, cockle shells nanoparticles 60%, gelatin 30%, dextran 5%, and dextrin 5%; 7101, cockle shells nanoparticles 70%, gelatin 15%, dextran 5%, and dextrin 10%; 7200, cockle shells nanoparticles 70%, gelatin 20%, dextran 5%, and dextrin 5%; 8100, cockle shells nanoparticles 80%, gelatin 10%, dextran 5%, and dextrin 5%; ACN, Aragonite Calcium Carbonate Nanoparticles; ANOVA, One-Way Analysis of Variance; Aragonite; BS-12, dodecyl dimethyl bataine; Bone; C-H, Carbon-Hydrogen group; C-O, Carbon-Oxygen group; CCN, Calcium Carbonate Nanoparticles; Ca10PO46OH2, Chemical structure of Hydroxyapatite; CaCO3, Calcium carbonate; Characterization; Cockle shells; DSC, Differential Scanning Calorimetry; DW, Deionized Water; ECM, Extracellular Matrix; FDM, Freeze Drying Method; FTIR, Fourier Transform Infrared; HA, Hydroxyapatite; Hf, Heat of fusion; JCPDS, Joint Committee of Powder Diffraction Society; MPa, Megapascals (MPa or N/mm2) pascal (Pa) unit=one Newton per square meter; NC, Natural coral; PBS, Phosphate Buffer Solution; Pet, Density of Ethanol; R, Radius; S.E., Standard Error; SBD, Segmental Bone Defects; SEM, Scanning Electron Microscopy; T, Thickness; TEM, Transmission Electron Microscopy; Tg, Glass transition Temperature; Tm, Melting Temperature; U/mL, Unit per milliliter; W0, Dry Weight (Initial Weight); W1, Dry Weight; W2, Wet Weight; Wd, Dry Weight; Ww, Wet Weight; XRD, X-Ray Diffraction; cm, Centimeter; mL, Milliliter; min, Minutes; nm, Nanometer; °C, Degree Celsius; µm, Micrometer.

Fig. 1

Photographs show the shape and the size of cockle shells nanoparticles using TEM, (A and B), 100 nm.

Fig. 2

Photograph shows scaffolds after drying in the freeze dryer machine, which ready to be used for studying the scaffolds characterization.

Fig. 3

Photographs (A, B, C, D, E, F and G) show the superficial structure of scaffolds (5211, 5400, 6211, 6300, 7101, 7200 and 8100) using Scanning Electron Microscope. A, B, C, D and E, 200x and F and G, 100x.

Fig. 4

Photographs (A, B, C, D, E, F and G) show the internal structure of the scaffolds (5211, 5400, 6211, 6300, 7101. 7200 and 8100) using Scanning Electron Microscope. A, C, E and G, longitudinal section, B, D and F, Cross section. A, B, C, D and E 100x., F and G, 200x.

Fig. 5

Photographs (A, B, C, D, E, F and G) show the internal structure and pores diameter of the scaffolds (5211, 5400, 6211, 6300, 7101, 7200 and 8100) using Scanning Electron Microscope. A, C, D, E and G, cross section, B and F longitudinal section. 50x.

Fig. 6

Photographs (A, B, C, D, E, F, G) show the internal structure and pores of the scaffolds (5211, 5400, 6211, 6300, 7101, 7200 and 8100) using Scanning Electron Microscope. A, B, E, F and G, longitudinal section, C and D, longitudinal section. 1000x magnification showing the presences of cockle shells nanoparticles powder crystallites depositions on the internal matrix.

Fig. 7

The mean of porosity percentage of scaffolds tested through liquid displacement method. *Significant difference were observed between the scaffolds at p<0.05.

Fig. 8

The mean of medium uptake percentage and the mean of water absorption percentage (first and second 10 min) and the mean of degradation percentage using Lysozyme. *Significant difference were observed between the scaffolds at p<0.05.

Fig. 9

Photographs show the degradation manner of each scaffold using Lysozyme after 24 h.

Fig. 10

Photographs show the degradation manner of each scaffold using Lysozyme after 7 days.

Fig. 11

Photographs show the degradation manner of each scaffold using PBS after 24 h.

Fig. 12

Photographs show the degradation manner of each scaffold using PBS after 3 days.

Fig. 13

The mean of compressive strength (MPa) and young's modulus (Mpa). *Significant difference was observed between the scaffolds at p<0.05.

Fig. 14

FTIR spectra of scaffolds.

Fig. 15

X-ray diffraction analysis and the strongest three peaks.

Fig. 16

The graphs show the endothermic DSC peak (denaturation) of the scaffold materials (gelatin, dextran, dextrin and ACN powder).