Fabrication Of Cu-MBG/Porous Hydroxyapatite Composite Scaffolds For Orbital Implants

Part Ⅰ Hydroxyapatite powder and porous hydroxyapatite scaffolds preparationPurposeCoralline hydroxyapatite (CHA) derived from the naturally occurring corals by the hydrothermal conversion of the calcium carbonate skeleton of coral to hydroxyapatite, are increasingly used as orbital implants to partially replace the volume of the eye ball after enucleation. However, concerns about the high cost, the potential detrimental effects on marine environments and the shortcomings of CHA led us to design a new implant material.MethodsThe hydroxyapatite powder and porous hydroxyapatite are prepared using Ca(NO3)2·4H2O and (NH4)2HPO4as the starting material, by chemical precipitation method. The structure of hydroxyapatite (HA) powder is characterized by means of XRD. To investigate the biocompatibility of HA, dense HA ceramic was prepared and used to culture the human umbilical vein endothelial cells (HUVEC). Then, the MTT method was used to calculate the relative growth rate (RGR) and the results were evaluated according to the National Standard GB/T1688615-1997for the biocompatibility evaluation. The morphological characteristics of the attached HUVECs were observed by SEM. Porous scaffolds of HA were fabricated by a paraffin microsphere approach. Then, its porosity was characterized by scanning electron microscopy (SEM) and Archimedes method.ResultsHydroxyapatite was successfully prepared by aqueous precipitation methods. XRD patterns indicated that the HA powder was pure and with high crystallinity. The particle size distribution was concentrated, ranging from400-900nm, the average particle size of the HA powder was577.4nm. It is met the requirement of preparation of porous scaffolds. MTT results showed the biocompatibility belong to grade1, according to the national standard, implying a fine biocompatibility. Porous scaffolds of HA were fabricated by a paraffin microsphere approach. SEM analysis showed that the HA scaffolds had a highly porous structure with a large-pore size of200-500um. The results of Archimedes method imply a good porosity about75%.ConclusionsProportion of Ca/P was controlled to be1.67was the key of preparation of pure HA powder. Hydroxyapatite powders with high crystallinity and small particle size was prepared. The HA dense ceramic piece has good biocompatibility. HA porous scaffolds fabricated by a paraffin microsphere approach have a good porosity. This porous hydroxyapatite could be used as orbital implant. Part II. The preparation and study of copper-containing mesoporous bioactive glassPurposeConventional treatments to prevent orbital implant-related infections include systemic antibiotic administration, and soaking the orbital implant in antibiotic solution prior to implantation. However, the soaking practice is ineffective in perfusing the drug to the center of implant. The purpose of this study was to prepare mesoporous bioactive glass (MBG), which could work as a promising bioactive drug delivery system, and study its morphology. Furthermore, different doses of copper-doped MBG were prepared and the effects of copper doped were studied, which will provide an experimental basis for the design and development of a new orbital implant.MethodsUsing non-ionic block copolymers (P123) as structure-directing agents through an evaporation-induced self-assembly (EISA) process to prepare mesoporous bioactive glass (MBG). By tuning the chemical compositions, different doses of copper-containing MBGs were prepared (xCu-MBG, x=0,2,5). The composition and morphology were analyzed by using FE-SEM and energy dispersive X-ray spectroscopy (EDX). Then, the mesoporous structure was characterized by high resolution transmission electron microscopy (HRTEM) and N2adsorption-desorption analysis. Leaching liquor of the xCu-MBG was used to culture the rat bone mesenchymal stem cells (rBMSCs) to evaluate the biocompatility of the xCu-MBG. The cell viability was measured by the MTT test.ResultsHighly ordered copper-containing mesoporous bioactive glasses (xCu-MBG, x=0,2,5) were successfully prepared through sol-gel and EISA method. The EDX showed that the doses of copper were met the amount of raw material. The HRTEM showed that all of the three kind of MBG had uniform mesoporous structure. The N2adsorption-desorption analysis demonstrated that all the isotherms were identified as type IV according to IUPAC classification with H2-type hysteresis loops. The BET specific ranged from385to413m2/g, pore size was about3-6nm. The doping of Cu into MBG slightly decreases the BET specific surface area and mesopore volume. The MTT test results implied a fine biocompatibility of the xCu-MBG.ConclusionsIn this study, highly ordered copper-containing mesoporous bioactive glasses (xCu-MBG, x=0,2,5) were successfully prepared. With the increase of the copper-doped amount, mesoporous pore size and specific surface area was decreased. By biomaterials biocompatibility evaluation criteria, the present study confirms obtained xCu-MBG has good biocompatibility. Thanks to its extensive mesoporous structure, it could be design as a drug delivery carrier, which could endow a controlled drug delivery mode in orbital implants. Part III Fabrication of Cu-MBG/porous hydroxyapatite composite scaffolds for orbital implant and the study on its propertiesPurpose:A porous implant is usually inserted after enucleation to partially replace the volume of the eye socket which facilitates the retention of an ocular prosthesis. However, Anophthalmic orbit restoration with artificial implants is usually accompanied with the risks of bacterial penetration and implant exposure. Here, we want to develop a facile evaporation-inducing self-assembly (EISA)-high sintered approach to modify the porous hydroxyapatite (pHA) scaffolds with CuO-containing mesoporous bioactive glass (Cu-MBG). Further investigation was performed to study the drug loading compatibility, the releasing properties and the antibacterial activity of the Cu-MBG/pHA composite scaffold, which will establish a substantial foundation for the manufacture of new orbital implant with multifunctional properties and will provide a new research approach in designing controlled releasing materials, scaffolds for clinical application.MethodsMesoporous bioactive glass (MBG) sol-gel was prepared as described in part II. pHA scaffolds coated with MBG on pore walls (MBG/pHA) were designed and fabricated by combining an evaporation-induced self-assembly (EISA) and high sintering process. Vacuum treatment was applied to remove the excess sol in the porous structure, reducing the impact on the porosity. Ofloxacin was chosen as a model drug to investigate the drug delivery property of the xCu-MBG/pHA scaffolds (x=0,2,5). The loading amount was determined by UV analysis (at wavelength293nm) through calculating the difference of ofloxacin-PBS concentration before and after loading. According to the method of "Chinese Pharmacopoeias" in accessing the controlled releasing property, samples were fetched at different time point and measured by UV-Spectrophotometer, then accumulated releasing rates were calculated. At last, Oxford cup method was used to research the antibacterial activity of xCu-MBG/pHA.ResultsThe Cu-MBG/porous HA composite scaffolds was designed and fabricated through evaporation-induced self-assembly (EISA) and high sintering process. The porosity of scaffolds decreased slightly after the xCu-MBG modified. These new composite scaffolds had a significant drug loading capacity. The OCu-MBG/pHA showed the maximum ofloxacin-loading capacity (-60%), while the2Cu-MBG/pHA and5Cu-MBG/pHA showed a small reduction. However, to compare with that of pure pHA (-14.4%), the drug-loading capacities for2Cu-MBG/pHA (52.2%) and5Cu-MBG/pHA (35.6%) were still significantly higher (P<0.05). All kinds of the xCu-MBG/pHA scaffolds maintained a sustained release of ofloxacin over168h. Localized the antibiotics delivery is very important for the antibacterial ability of the implant. The result of oxford cup method showed that the OCu-MBG/HA had no antibacterial ability, while the5Cu-MBG/HA significantly inhibited the bacterial viability (Staphylococcus aureus and Escherichia coli).ConclusionThis study has demonstrated that it is possible to develop xCu-MBG/pHA composite scaffolds by EISA-high sintered approach. This new composite scaffolds have significant drug loading capacity and could maintain a sustained release of ofloxacin. With increasing CuO content, the Cu-MBG/pHA scaffolds significantly inhibited the bacterial viability. This study provided a substantial experimental foundation for the further research on manufacture of a new orbital implant with multifunctional properties, which potentially reduce implant-related side effects.