| ObjectiveIn this study, we prepared a novel nondegradable bone plate composedof nano-hydroxyapatite/polyamide66/glass fiber (n-HA/PA66/GF)composite. In vitro and in vivo experiments were performed to investigatethe biocompatibility, biomechanical properties of n-HA/PA66/GF plate andits effect of fixation.Methods1. Cell biocompatibility of n-HA/PA66/GF biocomposite. In this part,n-HA/PA66/GF disks were prepared and mesenchymal stem cells derivedfrom the marrow (BMSCs) of rat were cocultured with n-HA/PA66/GFdisks. BMSCs cultured alone and with n-HA/PA66disks were used ascontrols. We applied scanning electron microscopy (SEM) to observe theattachment and growth of BMSCs on n-HA/PA66/GF biocomposite.MTT test was performed to evaluate the influence of n-HA/PA66/GFbiocomposites on proliferation of BMSCs. Protein content and mRNA expression of alkaline phosphatase and osteocalcin measured by ELISAand RT-PCR, and Alizarin Red S staining of calcium deposits were used toexplore the influence of n-HA/PA66/GF disks on osteoblasticdifferentiation of BMSCs.2. Tissue biocompatibility of n-HA/PA66/GF biocomposite. In this part,n-HA/PA66/GF rods were fabricated and implanted into sacrospinalmuscles and tibia of New Zealand white rabbits. One, four and twelveweeks after implantation of n-HA/PA66/GF rod, tissue biocompatibilitywas evaluated by general observation, X-ray，HE staining and Van Giesonstaining of muscle and tibia samples. And histopathological examination ofmain organs was performed.3. Biomechanical properties of n-HA/PA66/GF plate. In this part,n-HA/PA66/GF plate was fabricated by extrusion method and its externalsurface and fracture surface was observed by SEM. The bending yieldstrength was determined by three-point bending test using Reger machine.Then we performed four-point bending and torsion tests using bone-plateconstructs to analysis the biomechanical properties of fractures comparingn-HA/PA66/GF plate to titanium plates.4. Effect of n-HA/PA66/GF plate on fixation of fractures. A total ofeighteen local canines with fractures of femoral shafts were randomlydivided into two groups which were fixed by n-HA/PA66/GF and titaniumplates, respectively. X-ray, CT scanning, gross examination and histological staining were performed to evaluate the influence of n-HA/PA66/GF plateon callus formation and healing of fractures. And histological staining ofmain organs was performed24and52weeks after implantation ofn-HA/PA66/GF plate.Results1. Results of SEM showed that BMSCs presented fusiform shape andadhered to the surface of the n-HA/PA66/GF disk with their pseudopodia.The number of cells increased over time with intercellular space decreasedand stratified cell layers formed. The results of MTT assay showed, the cellnumber increased with culture time and no significant difference wasdetected at each time point among n-HA/PA66/GF, n-HA/PA66compositeand the blank control during7days of culture, which demonstrated that then-HA/PA66/GF biocomposite had no negative effect on proliferation ofBMSCs. The results of ELISA and RT-PCR analysis showed the ALP andOC protein contents and mRNA ascended with culture time when BMSCswere cocultured with n-HA/PA66/GF disks in osteogenic differentiationmedia. However, no significant differences were found in comparison withthe n-HA/PA66composite and blank control group at3,7and14days.Twenty-one days after coculture with n-HA/PA66/GF biomaterial inosteogenic media, the population of the BMSCs increases manifestly andcalcium accumulations were visualized by Alizarin Red S staining. Allthese indicated n-HA/PA66/GF biomaterial had no negative influence on attachment, proliferation and osteogenic differentiation of BMSCs2. The results of muscle implantation showed the n-HA/PA66/GF rodswere wrapped in sacrospinal muscles with no obvious signs of edema,infections or necrosis of muscles at1,4, and12week. HE staining ofmuscles showed mild edema with disorganized myofiber, but no obviousmyocytolysis or necrosis at one week. Four and twelve weeks afterimplantation, HE staining showed no obvious edema and other pathologicalfindings. One, four and twelve weeks after implantation of n-HA/PA66/GFrod in tibia, we found no obvious loosening of the n-HA/PA66/GF rod, orsigns of rejection reaction and bone destruction. At one week, the gapbetween the rod and bone tissue was clear which disappeared graduallyover time. Twelve weeks after implantation the surface of n-HA/PA66/GFrod was covered by new bone tissue. X-ray also showed that no obviousloosening of the n-HA/PA66/GF rod or bone resorption occurred. At oneweek, low density area between n-HA/PA66/GF rod and bone was clearwhich had disappeared gradually over time. Van Gieson staining of bonefound no fiber grew into the space between n-HA/PA66/GF rod and boneand the morphology of bone tissue was normal. New bone trabecularformed around the n-HA/PA66/GF rod and the space disappeared over time.Besides, the histopathological examination of main organs showed notoxicity of n-HA/PA66/GF biocomposites. All these results indicated thatthe n-HA/PA66/GF biocomposite had good tissue biocompatibility. 3. SEM observation showed that n-HA/PA66/GF plate had roughexternal surface with micron size fibers. On the cross-section areas, noobvious n-HA/PA66matrix cracking was observed. When n-HA/PA66/GFplate ruptured, a large number of glass fibers breakage occurred with fewfibers pulled out at the site of fracture surface, which indicated thatadhesion between the n-HA/PA66matrix and the glass fbers was strong.The bending strength of n-HA/PA66/GF plate was184.02±5.45MPa whenthe load was located at the third hole of a plate, which was comparable tomechanical strength of human femur. In both four-point bending andtorsion tests, all the bone-plate constructs fixed n-HA/PA66/GF plate failedby breakage of the plates at the hole sites near the fracture gap, whereasconstructs fixed by titanium plate failed because of fractures of bones. Thebending (11.2±2.4Nm) and torsion (6.038±1.636Nm) strength ofbone-plate construct fixed n-HA/PA66/GF plate was equal to28.9%and49.6%of that fixed by titanium plate. And the bending (244.1±49.6N/mm)and torsion stiffness (0.159±0.039Nm/°) of bone-plate construct fixedn-HA/PA66/GF plate was reduced by34.1%and56.8%compared to thatfixed by titanium plate. All these indicated, the stiffness and failure strengthof bone-plate construct fixed n-HA/PA66/GF plate was significantly lowerthan that fixed by titanium plate, which was beneficial to decrease stressshield and facilitated interfragmentary movement. However, whether or notn-HA/PA66/GF plate can hold fractures effectively need to be studied using in vivo animal experiments.4. In animal experiments in vivo, one case with n-HA/PA66/GF plateruptured at the screw hole was found at eight week after surgery. Images ofX-ray and CT, and gross observation showed there was obvious callusformation of the fractures fixed using n-HA/PA66/GF plates. However, thehealing speed of fractures fixed by n-HA/PA66/GF plates was slower thanthose using titanium plates. When fractures were fixed by titanium plate, nocallus were formed and the fractures healed by primary union. Then-HA/PA66/GF plate did not absorb X-ray which was beneficial theobservation of healing process of fractures. Van Gieson staining showedthat obvious callus formed at the site of fractures. During the healing offractures, the number and thickness of bone trabecular increased whicharranged more regularly and compactly over time. Twelve weeks afterfixation using n-HA/PA66/GF plate, the space of bone-plate interface wasclear which had disappeared gradually with new bone formation beneaththe plate. At52week, the bonding between the bone and n-HA/PA66/GFplate was tight and some bone tissue grew into the plate. Thehistopathological examination demonstrated no toxicity of n-HA/PA66/GFbiocomposites on main organs at24and52week.ConclusionsThe nondegradable n-HA/PA66/GF plate has good biocompatibility andbiomechanical properties. It can fix fractures effectively which is beneficial to callus formation and secondary healing of fractures, which can bondwith the bone after long period of implantation. |
PDF Full Text Request