Characterization and evaluation of mechanical properties of injectable porous spherical nano-hydroxyapatite/chitosan particles for craniofacial bone regeneration

Abstract

Event Abstract Back to Event Characterization and evaluation of mechanical properties of injectable porous spherical nano-hydroxyapatite/chitosan particles for craniofacial bone regeneration Suren Uswatta1 and Ambalangodage C. Jayasuriya2 1 University of Toledo, Bioengineering, United States 2 University of Toledo, Department of Orthopaedic Surgery, United States Introduction: Craniofacial bone surgery and regeneration is a major challenge in regenerative medicine. As a solution injectable microscale particles have been fabricated and characterized since they can be implanted with a minimally invasive surgery[1]. Although craniofacial bone is not a load bearing bone, injectable particles should possess stability towards certain physiologic conditions and retain its mechanical strength at abruptly occurring external loading applications. In this study we have fabricated porous particles using chitosan biopolymer and nano-hydroxyapatite (nHA). We hypothesized that incorporating nHA into chitosan could emulate the mineralized cortical bone structure, thus improve the mechanical strength of the particles. Materials and Methods: Medium molecular weight chitosan, hydroxyapatite nanopowder (nHA, <200nm particle size), sodium tripolyphosphate (TPP, technical grade, 85%) and acetic acid (A.C.S reagent, 99.7%) 2% (w/v) chitosan solution was prepared by dissolving chitosan and 1% (v/v) acetic acid. Then nHA has been added to 2% (w/v) chitosan solution to make 0.5%, 1% and 2% (w/v) nHA/chitosan composite mixtures. These mixtures were dripped into 27.18mM TPP/Deionized water solution using 30 gauge needle and stirred at 600RPM. After 30 minutes TPP cross-linked nHA/chitosan beads filtered out from TPP solution and lyophilized at -52oC temperature and 0.02 mbar pressure for 24 hours. Portion of each batch were treated further by soaking followed by drying and named as Lyophilized Soaked and Dried (LSD) particles. Finally particles were characterized for their morphology, chemical structure, physical structure, cell cytotoxicity and attachment. Finally particles were tested for its ultimate compressive strength (UCS) at dry and wet states using ADMET’s MTESTQuattro universal testing machine using 0.05mm/s cross head movement. Statistical data were analyzed using IBM SPSS Statistics version 21. Figure 1- Scanning electron microscopy images of 2% nHA/Chitosan particle surfaces Results and Discussion: Fabricated lyophilized and LSD particles had a mean diameter of 1.33 and 0.93 mm respectively which is promising for injectability of the particles via needles. SEM images showed porous surface morphology and interconnected pore structures inside the particles. Lyophilized and LSD particles had surface pores with diameters less than 10 and 2 µm respectively (Figure 1.A and 1.B). This highly porous surface morphology improves the osteoconductivity of scaffolds by facilitating osteoblast cell adhesion and proliferation. One-way ANOVA results showed that, there is significant increase in UCS of 1% and 2% nHA compared to 0% nHA lyophilized particles at (p<0.001) significance level (Figure 2.A). Further increase in nHA percentage affects the dripping process due to high clogging factor in the needle and higher viscosity of the solution. Moreover, 2% nHA LSD particles had increased their mean UCS by 120% compared to 2% nHA lyophilized particles at (p<0.005) significance level. In a drawback, all particles have lost their mechanical properties by 95% at (p<0.001) significance level on the 2nd day when fully immersed in phosphate buffered saline (PBS). However, particles preserved its initial shape and showed minimal degradation at the end of 100th day in PBS solution. Additionally live and dead cell assay showed zero cytotoxicity and good osteoblast cell attachment to both lyophilized and LSD particles at the end of 6th day of in vitro studies (Figure 2.B and 2.C). Figure 2- Ultimate compressive strengths of nHA/Chitosan lyophilized particles at different nHA compositions are shown in 2.A. Live and Dead cell assay results are shown in 2.B and 2.C. Live cells proliferation can be seen on 2.B FITC image. Presence of dead cells in the same region is captured using TRITC filter as shown in 2.C Conclusion: Experiments results shows that incorporating nHA can improve the mechanical properties of lyophilized chitosan particles. This result is consistent with a study that HA can improve the mechanical properties of electrospun fibers[2]. No cell cytotoxicity and higher cell attachment shows improved osteoconductivity of the particles. However further modifications on particles is required to improve to wet state mechanical properties. National Institute of Health - Grant account number R01DE023356