Biological Evaluation Of A Novel Injectable Alveolar Bone Substitiute For Controlled Delivery Of Tetracycline

PART ONE In Vitro Cytotoxicity of a Novel Injectable and BiodegradableAlveolar Bone SubstitiuteObjective Currently, the unsaturated polyphosphoester (UPPE) polymer is beinginvestigated as an injectable and biodegradable system for alveolar bone repair in thetreatment of periodontal diseases. The incorporation of beta-tricalcium phosphate(β-TCP) particles into the UPPE polymer was previously shown to significantlyincrease the material's mechanical properties. Moreover, in vitro experimentsdemonstrated that the UPPE/β-TCP composite was capable of release of tetracyclinefor over 2 weeks (w). This study aimed to investigate the in vitro cytotoxicity of eachindividual component, the resulting cross-linked network, and the eventualdegradation products of the UPPE/β-TCP composite. Methods Each individualcomponent of the UPPE/β-TCP composite was immersed in culture media andincubated, respectively. The culture media without extracts severed as the control.Using cell line L929 as model cells, the individual cytotoxicity of each constituentwas evaluated by an AlamarBlue viability assay. The effects of the leachable productsfrom the cross-linked UPPE polymer and UPPE/β-TCP composite, the in vitrodegradation products obtained from the complete breakdown of the cross-linkedcomposite under accelerated conditions on the cytotoxicity were also investigated.Single factor analysis of variance and Tukey's HSD multiple comparison tests wereused to determine statistical significance of results. Resultsβ-TCP demonstratedcell viability comparable to the control (P＞0.05). The other components of the UPPE/β-TCP composite exhibited significantly higher cytotoxicity at the highestconcentration (P＜0.05), and dilutions of these extracts produced an improvement incell viability. Once cross-linked, however, the cytotoxicity of the leachable productsfrom the cross-linked UPPE polymer and UPPE/β-TCP composite was comparable tothat of the control (P＞0.05) because most of the individual constituents may beincorporated into the cross-linked network. The degradation products of thecross-linked composite displayed a dose-dependent cytotoxic response with only thetwo highest concentration solutions demonstrating cell viability significantly lowerthan the control (P＜0.05). Conclusion The results suggest that although there areconcerns regarding the biocompatibility of the novel UPPE/β-TCP composite, it holdsgreat promise for use as an injectable and biodegradable alveolar bone substitute inthe treatment of periodontal diseases.PART TWO Investigation of MC3T3-E1 Cell Behavior on the Surface of aBiodegradable Alveolar Bone Substitiute Containing TetracyclineObjecitve The early osseointegration of bone substitutes is an important factorfor their clinical success. The challenge in the engineering of bone substitute'ssurfaces is to attract, above all, osteoblasts that produce a bone extracellular matrix,which will ensure a high bone-implant contact. Cell adhesion is one of the initialstages for subsequent proliferation and differentiation of osteoblastic cells producingbone tissue. The aim of this present study is to investigate the behavior of osteoblasticMC3T3-E1 cells on four different bone grafts surfaces in vitro:β-TCP, UPPE polymer,UPPE/β-TCP composite and UPPE/β-TCP containing 1% tetracycline(UPPE/β-TCP+1% TTC) composite. Methods MC3T3-E1 cells were culturedeither on theβ-TCP, UPPE polymer, UPPE/β-TCP composite and UPPE/β-TCP+1%TTC composite discs or in the absence of material (plastic) for 4, 8 and 15 days (d).The cell viability of each group was measured as optical density by an AlamarBlueviability assay, and normalized to cell culture medial (the control) in order to demonstrate differences in cell viability on the different substrates. After 1 or 14 dincubations of the cells on the four materials or the control, the cellswere double-stained with Acridine orange/Ethidium bromide (AO/EB) for 5 minutes(min) and viewed by epifluorescence microscopy to simultaneously examine of bothlive and dead cells on the materials. The morphology of osteoblastic cells MC3T3-E1after culturing for 2 d on the different substrates was examined using a scanningelectron microscope (SEM). The differentiation function of osteoblastic cellsMC3T3-E1 was assessed by measuring alkaline phosphatase (ALP) activity. Singlefactor analysis of variance and Tukey's HSD multiple comparison tests were used todetermine statistical significance of results. Results For all substrates, the resultsshow an increase in cell viability indicating cell proliferation with culture time. After4 d of culture, the cell viability was slightly inferior for all groups compared with thecontrol, but there was no statistical differences between groups (P＞0.05). The dataappeared similar for the UPPE/β-TCP composite after 8 and 15 d of culture, andslightly superior for theβ-TCP or UPPE/β-TCP+1% TTC composite after 15 dcompared with the culture. Nevertheless, the statistical analysis did not showsignificant differences between groups (P＞0.05). The cell viability remainedslightly lower in UPPE compared with the control. Visual examination revealed thatthe density of live cells adherent to each material was similar after 2 and 15 d ofculture. Live cells, stained green, appeared to have adhered and attained a normalpolygonal morphology on all materials. Dead cells (stained red) were very few on allthree materials. SEM micrographs show similar cell attachment on the four materials.ALP activity increased with culture time whatever the substrate was and appeared tobe at its maximum after 21 d. Conculsion These data show that a UPPE/β-TCPcontaining 1% TTC composite is biocompatible and supports the adhesion, spreading,proliferation and viability of osteoblast cells. PART THREE In Vivo Bone and Soft Tissue Response to an Injectable,Biodegradable Alveolar Bone Substitiute Containing TetracyclineObjective Earlier investigations have characterized some of the physical andbiological properties of UPPE/β-TCP composite. It is worth noting that sometimes thebone substitute is not only in contact with bone, but also with the surrounding softtissues. To ensure the safety and effectiveness of this technique, this study wasdesigned to assess in vivo soft and bone tissue response to to the UPPE/β-TCPcomposite and UPPE/β-TCP+1% TTC composite after implantation using a rabbitmodel. Methods Twenty-five male New Zealand White rabbits were used asexperimental animals. For the insertion of the subcutaneous implants, fourssubcutaneous pockets at the dorsum of the rabbits were created. The implants (β-TCP UPPE polymer, UPPE/β-TCP composite and UPPE/β-TCP+1% TTC) wereinserted in these pockets. UPPE/β-TCP composite and UPPE/β-TCP+1% TTC wereinjected in circular defects as created in the femoral condyles of rabbits and were leftin place for 2, 4, 8, 12 and 24 w. The specimens were evaluated morphologically(histology, and histomorphometry). Single factor analysis of variance and Tukey'sHSD multiple comparison tests were used to determine statistical significance ofresults. Results The histologic evaluation in vivo after 2 w showed a mild infiltrateof inflammatory cells for most of the groups. UPPE/β-TCP+1% TTC compositeexhibited no signal of inflammatory response. After 4 w. the tissue showed nearnormal pattern for all the groups. The soft tissue capsule had a non-uniformdistribution in thickness, which increased most significantly between 4 w and 12 wafter implantation. After 8 weeks, the capsule thickness did not change much. None ofpolymorphic cells, osteoblast cells or bone cells adjacent to the implant were observed.The surface of theβ-TCP, UPPE/β-TCP composite and UPPE/β-TCP+1% TTCcomposite themselves remained substantially intact without noticeable cracking,chipping or dissolution. However, at 8 w, UPPE polymer showed extensive surfaceerosion and superficial fragmentation that was surrounded by a few inflammatory cells. Gross examination of retrieved implant/bone composite samples indicated thatthe UPPE/β-TCP composite and UPPE/β-TCP+1% TTC composite did not evokeinflammatory response, necrosis or fibrous encapsulation in surrounding bony tissues.Histological examination revealed excellent composite/host bone bonding. At 4 w, theresorption induced voids between terminals of bone defects and implants were largelyfilled with new bone. Composite resorption, new blood vessels, osteocytes, osteonsand osteoblast-like cells lining up with active new bone were observed at remodelingsites. At 12 w, a new bone network was developed within femoral defect, whilecomposite became islands incorporated in the new bone. At 24 w, bone ingrowth andremodeling activities became so extensive that the interface between residual cementand new bone became less identifiable. In general, the resorption ratio valuesincreased with implantation time. Conclusion TC addition optimizedbiocompatibility of the UPPE/β-TCP composite, contributing to anti-inflammatoryresponse during the early phases of the wound healing process. These results showedthat UPPE/β-TCP composite holds promise for use as a syntheticbiodegradable scaffolds for tissue engineering.PART FOUR The Influence of Novel Injectable Alvelor Bone Substitutes onRegeneration of Periodontonal Defects with Experimental Periodontitis in DogsObjective Earlier studies showed that an injectable UPPE/β-TCP composite wasbiocompatible and promoted histocompatible healing of bone tissue in rabbits. Thesemisolid consistency of UPPE/β-TCP composite containing TTC shows sustainedand almost constant in vitro drug release in phosphate buffer, pH 7.4 at 37℃, for upto 14 days. The purposes of this present study were (1) to assess the in vivo releaseof drug in the periodontal pockets from the UPPE/β-TCP composite containing 1%,5% or 10% (wt/wt) TTC and (2) to evaluate influence of the formulation onregeneration of periodontal defects with experimental periodontitis in dogs.Methods Experimental periodontitis was induced by placing stainless-steel mesh on the mesial side of maxillary canines for 6 w in fifteen adult, healthy dogs.Subsequently, intrabony defects were resized so as to be standard, and UPPE/β-TCPcomposite containing 1%, 5% and 10% TTC were directly injected in theexperimental bone defects. Non-grafted defects on the contralateral side served ascontrols. The retention time of the antibiotic product was determined indirectly bymeasuring the concentration of tetracycline in gingival crevicular fluid (GCF) usinghigh-performance liquid chromatography (HPLC) at 3, 7, and 14 d after the placementof the UPPE/β-TCP + TTC composites. Sixteen weeks after surgery, the animals weresacrificed and histologic specimens were prepared. Periodontal tissue healing wasevaluated histologically and histometrically. Results The TTC concentrations inthe GCF well above the minimum inhibitory concentration (MIC) of most periodontalpathogens were seen at 3, 7, and 14 d following the injection of theUPPE/β-TCP+TTC composite formulations. Healing of periodontal tissues, in termsof bone and cementum formation, was consistently observed in theUPPE/β-TCP+TTC composite-applied sites. UPPE/β-TCP+TTC composites werepartly replaced by new bone. New cementum and periodontal ligament-like tissuewere observed between UPPE/β-TCP+TTC composites and the root surface. Newbone (P＜0. 05), new cementum (P＜0.05), and new connective tissue attachment andadhesion (P＜0.05) were significantly enhanced in the experimental sites.Conclusion The UPPE/β-TCP+TTC composite formulations can be retained in theperiodontal pocket for a prolonged period, releasing a sufficient amount oftetracycline to eliminate pathogenic bacteria. The composites containing TTCprovides stable wound healing and enhanced periodontal regeneration in periodontaldefects in dogs with experimental periodontitis. The local application of injectablebioerodible UPPE/β-TCP+TTC composites appears to be promising in the context ofperiodontal treatment.