Preparation And Evaluation Of Poly(L-lactic Acid) Internal Fixation Composites Containing Heterogeneity Bone Particles

ObjectiveIn order to avoid the disadvantages of traditional metallic internalfixation, such as second operation imposed on fracture patient for takingout the internal fixation, stress sheltering and metallical toxicity et al,many attempts have been made to obtain a novel material for fixation inthe last 20 years. Poly(lactic acid) (PLA) and their copolymers haveattracted wide attention for their biodegradability in the human body.However, the mechanical strength, toughness, and elastic modulus ofthese polymers were lower than those of natural cortical bones. A cavityis left when polymers are absorbed. As a degradable product, the acidproduced by degradation can erode the new bone. Thus, for preparing adesired material that presents high mechanical performance to matchnatural bones, a bioactive and biocompatible composite was fabricatedbased on poly (l-lactic acid) (PLLA) and bovine bone particles (averageparticle size: 1.39μm) by a solvent evaporation technique. At the sametime, systemic studies including phase structure, microstructure,mechanical properties, degradation properties and biocompatibility assayswere carried out to this composite rods, which were used as internalfixation materials of bone fracture.Methods 1. Bovine Bone Particles (BBP) were prepared by milling the degreased anddeproteined bovine bone ,and then characterized. The BBP andPLLA(MW=200000) solution were mixed by ultrasound then themixture was dried. The mixture was further heating-molded in 185℃and under the pressure of 110Mpa to produce the composite rods. Themechanical properties of the rods, such as bending strength and elasticmodules, shear strength, torsion strength were measured. The rods withdifferent proportion of bone particles were statically degraded insimulated body fluid for 12 weeks to investigate the effect of BBP andacid as degradation production. The rods with the best mechanicproperties were selected to investigate the degradation in solution ofsimulated body fluid for2w, 4w, 6w, 8w, 10w, 12Wo Concentrationof H⁺, Ca²⁺ in these fluids were analyzed every two weeks. Thecubage, quality, bending strength, molecular weight of the degraded rodswere also analyzed. The surface and cross section of the rods wereinvestigated by SEM.2. The cytotoxicity of the BBP/PLLA mixture was analyzed by MTrcytotoxicity assay. The shape changes of the cells were analyzed underthe vertical microscope. The adherences of the cells to the materials wereinvestigated under the SEM. The L929 fibroblasts were cultured inleaching liquor of BBP/PLLA. On day 18 and 35, the tetracycline, VonKossa and alkaline phosphatase staining were performed, and the amounts of alkaline phosphatease and osteocalcin were measured. Afterbone particles were imbedded in the musculature and subcutaneous of rabbitsfor 12w, the CD3,CD4,CD8,CD38 lymphopoiesis wereinvestigated by immunohistochemical. BBP/PLLA was imbedded insubcutaneous stratum and femur condyloid of rabbit, the histology wasdetected by hematoxylin and eosin (HE) staining while osteoinductivitywas evaluated by staining of Von Kossa,alizarin red,tetracyclinefluorescence. Biological safty tests of BBP/PLLA internal fixation, suchas acute toxicity, sub—acute toxicity, pyrogen,hemolysis, hypersusceptibility, antibacterial, and hereditytoxicity wereperformed according to the biomedical salty assessment standards (GB/T16886).3. Twelve medial femoral condyle osteotomies of rabbits were fixedwith 12 composite screws made of BBP/PLLA. The fracturehealing and treatment results were analyzed by radiograph and roughview of the samples.Results:1 The average granularity of BBP is 1.39um, which is mainlymade up of lower crystallinity HA, microelement and a few proteins.With the percentage of bone powder increasing, the elasticity modules ofthe composites increase, and their bending strength, shear strength, andtorsion strength increased at first and then decreased. SEM shows BBP are distributed homogeneously in the composite. Thirty percent of bonepowder in the rods is considered as the best percentage because thebending strength is 102Mpa and the elastic module is 4.5Gpa. The PHvalue of the BBP/PLLA degradation solution was 7.10-7.15, which waslower than the PH value of the degradation solution with only PLLA.The BBP/PLLA with 30% bone powder degrades slowly in thesimulated body fluid during the first 3 months. The bending strength ofthe composites implanted in the SBF was maintained at more than 70Mpaafter 3 months . The molecular weight dropped to 38ï¼…of the initialvalues at 8 week and to approximately 30ï¼…at 12 week. Differentquality percentage BBP/PLLA rods all losed the strength after 4months in the SBF acidified by degradation product. The rods' surfacesand cross sections showed degradation. With the increasing quality ofBBP, the strength left didn't show difference even if the drop ofmolecular weight decelerated.2. The MTr assay showed that the BBP/PLLA composites arecompatible with the tested cells without cytotoxicity. Calcificationnodules were discovered in the fibroblast which were cultured in theleaching liquor. The calcification nodes were showed as strong florescentnodes under tetracycline fluorescence staining and as black nodes underVon Kossa staining. Alkaline phosphatase staining showed that therewere positive or strong positive particles in the cells. The amount of alkaline phosphatase and osteocalcin went up to the maximum in 15days and then went down. These results are significantly different fromthe control group (P＜0.05). Samples taken from the rabbits' subcutaneousat 12 weeks were used to measure the amount of CD³⁺, CD4⁺, CD8⁺ Tcells and CD38⁺ B cells by immunohistochemical staining. The resultsindicated that there was no difference between the BBP group and thehydroxyapatite control group. The initial cellular immune responsedecreased after 12 weeks. The HE staining results indicated that theBBP/PLLA material has good tissue biocompatibility. The amount ofmonocytes and macrophages decreased and the inflammatory responsedisappeared after 8 weeks. Ossifications (Calcifications) of thematerial-tissue interfaces were detected by tetracycline fluorescence, VonKossa, alizarin red, and tri-fluorescence staining. No toxicity of theBBP/PLLA, such as acute toxicity, sub-acute toxicity, pyrogen,hemolysis, hypersusceptibility, antibacterial, heredity toxicity to theanimal were detected by the toxicology studies.3. The BBP/PLLA screws successfully fixed 11 rabbits'intercondyloid fractures and the fractures healed after 8 weeks.ConclusionsThe BBP/PLLA material is a novel and excellent material forinternal fixation. The elastic modulus was enhanced because of BBP. Theinitial BBP/PLLA strength and its strength after 3 months' degradation were strong enough to maintain the stability of the cancellous bonefracture fixation. It has excellent cellular and tissue biocompatibilitieswithout any acute and chronic toxicity. The extraction solution of theBBP/PLLA can induce the fibroblast to express ossification activity. Thein vitro results agreed well with the in vivo data. New bone forming onthe material-tissue interface was detected by in vivo histology. The screws made by BBP/PLLA are suitable for secure fixation of jointfractures.X ray results showed that the density of the BBP/PLLA with 30ï¼…bone power range between the density of the cortical bone and cancellousbone. Although further long-term degradation studies are needed, thecomposites have promising mechanical strength and no adverse tissuereaction for use as fracture fixation devices during the experimentalperiods.