The Study On Self-etch Adhesive As A Carrier For ACP Nanoprecursors To Deliver Biomimetic Remineralization

Part1 The preparation of amorphous calcium phosphate(ACP)particles and their biomimetic remineralization ability of type I collagen fibrilsObjectives:This study was to synthesize PAsp(polyaspartic acid)-stabilized and Si-containing ACP particles(PAsp-Si-ACP)and evaluate their remineralization ability of reconstituted type I collagen fibrils and demineralized dentin.The biocompatibility of PAsp-Si-ACP particles was also evaluated.Materials and Methods:PAsp-stabilized and Si-containing ACP particles(PAsp-Si-ACP)and PAsp-stabilied ACP particles(PAsp-ACP)were prepared by a solution precipitation method.PAsp-Si-ACP particles were synthesized by mixing the equal volumes(10 ml)of 10 mM CaC12/480 ?g/ml PAsp and 6 mM Na2HPO4/12 mM Na2SiO3·9H2O solutions and stirring for 10 min.PAsp-ACP paticles were synthesized by mixing the equal volumes(10 ml)of 10 mM CaCl2/480 ?g/ml PAsp and 6 mM Na2HPO4 solutions and stirring for 10 min.The precipitates(ACP nano-particles)were collected by centrifugation(15000 rpm)for 5 min and were dried at 30? in a vacuum drying oven overnight.The characterizations of PAsp-Si-ACP and PAsp-ACP paricles were analyzed by transmission electron microscopy(TEM),scanning electron microscopy(SEM),Fourier transform infrared spectroscopy(FTIR),X-ray diffraction(XRD)and X-ray photoelectron spectroscopy(XPS).A single-layer reconstituted collgen model was used to test the biomimetic remineralization ability of PAsp-Si-ACP and PAsp-ACP particles.Type I collagen fibrils derived from rat tail were reconstituted on 400-mesh Ni TEM grids.Eight milligram of PAsp-ACP was suspended in 240 ?l of 1M HEPES buffer solution(pH=7.4)as a control group.Eight milligram of PAsp-Si-ACP was suspended in 240 ?l of 1M HEPES buffer solution(pH=7.4)as an experimental group.The mixture(ACP/HEPES)was placed in a petri dish to form a small droplet on which four grids covered with reconstituted collagen were floated upside down.Three droplets per group were prepared.The grids were incubated in a 100%humidity chamber at 37 ? for 1,4,and 7 day.Four grids per group were retrieved and analyzed by TEM with selected area electron diffraction(SAED).Six human dentin disks were prepared as approximate 3 mm×3 mm×1mm.The dentin disks were demineralized by 37%phosphoric acid for 30 s and rinsed with deionized water to produce a mineral-free collagen matrix.Randomly-selected two dentin disks were used for TEM examination after deminineralization.The other four dentin disks were used to test the remineralization ability by PAsp-Si-ACP partilces.Thirty-three point three milligram of PAsp-Si-ACP was suspended in 1ml of 1M HEPES buffer solution(pH=7.4).The demineralized dentin disks were respectively put into the suspension(PAsp-Si-ACP/HEPES)and incubated in a 100%humidity chamber at 37? for 1 month.The dentin disks were retrieved and also examined by TEM with SAED.The cytotoxicity of PAsp-Si-ACP particles was assessed by CCK-8 assay.Results:SEM and TEM images indicated that ACP particles(PAsp-Si-ACP and PAsp-ACP)were globular with a size of approximately 20-80 nm.Both the FTIR and XRD spectrums indicated that the ACP particles were amorphous.The XPS spectrum of PAsp-Si-ACP particles indicated that silicon,oxygen,calcium,phosphate,carbon and nitrogen were present in PAsp-Si-ACP particles.The XPS spectrum of PAsp-ACP particles indicated oxygen,calcium,phosphate,carbon and nitrogen were present in PAsp-ACP particles.The grids in the experimental group(PAsp-Si-ACP)showed that most of the collagen fibrils were heavily remineralized after 1-day incubation,exhibiting intra-and extra-fibrillar mineralization.The grids in the control group(PAsp-ACP)showed that the collagen fibrils were not remineralized after 1-day and 4-day incubation and only a meagre amount remineralization was found even for 7 days.The SAED patterns showed that the intrafibrillar minerals within collagen were aligned along the longitudinal axis of the collagen fibril.TEM images showed that the demineralized dentin prepared by the use of 37%phosphoric acid for 30 s produced a 10 to 15 ?m thick layer of completely demineralized collagen.After 1-month incubation,the demineralized dentins were remineralized.The remineralization layer was approximately 3-5 ?m.CCK-8 assay indicated that PAsp-Si-ACP particles had no adverse effect on cell viability.Conclusions:PAsp-Si-ACP particles showed a biomimetic remineralization ability of type I collagen fibrils and Si could promote the remineralization.PAsp-Si-ACP particles had good biocompatibility,indicating potential for use in biomedical applications.Part 2 Self-etch adhesive as a carrier for ACP nanoprecursors to deliver biomimetic remineralization of single-layer reconstituted type?collagenObjectives:This study evaluated self-etch adhesive as a carrier for ACP nanoprecursors(solid PAsp-Si-ACP particles)to deliver biomimetic remineralization of single-layer reconstituted type ? collagen fibrils.Materials and Methods:PAsp-Si-ACP particles and a single-layer reconstituted collgen model were prepared as in Part 1.The biomimetic remineralization of single-layer reconstituted type?collagen fibrils was analyzed by using two one-bottle self-etch dental adhesives(Clearfil S3 Bond(S3),Kurraray-Noritake;Adper Easy One(AEO),3M ESPE)as a carrier loaded(or not,in the case of the control)with 5wt%,15wt%,25wt%of solid PAsp-Si-ACP particles.Self-etch adhesives loaded with or without PAsp-Si-ACP particles were each dropped in approximately 13 mm diameter rings with 0.1 mm height,before they were light-cured(Radii plus,1500 mW/cm2,SDI,Australia)for 80 s(each quadrant for 20 s)to obtain four films each group,namely,remineralization film(adhesive film containing PAsp-Si-ACP particles)and pure adhesive film(blank control).Subsquently,a remineralization film or an adhesive film was placed at the bottom of a 12-well plate.Then,240 ?l of artificial saliva was dropped onto the surface of a film.A total of 128 collagen-coated grids(n= 16),with 4 grids each well,were floated upside-down on top of the artificial saliva.After the grids in each group were incubated in a 100%humidity chamber at 37? for 1,4,7,and 14 days,4 grids per group were retrieved and analyzed by TEM with SAED.The capacity for Ca and P ions release from the remineralization films which exhibited the remineralization ability was evaluated by inductively coupled plasma-atomic emission spectrometry(ICP-AES)in sodium chloride solution(133mmol/L,pH 7.4)for 28 days.Results:The collagen fibrils in the control groups and in experimental groups containing 5wt%and 15wt%of PAsp-Si-ACP were not remineralized after 14-day incubation.In experimental groups containing 25wt%of PAsp-Si-ACP,a few collagen fibrils were mineralized but in a non-crystalline or amorphous state after 4-day incubation in artificial saliva.After further incubation for 7 days,the collagen extra-and intra-fibrils were partially mineralized with a distinct borderline between the mineralized part and the un-mineralized.Most of the collagen fibrils were heavily mineralized after 14-day incubation.SAED of the mineralized collagen fibrils produced an arc-shaped diffraction pattern of the(002)growth face,indicating that the intrafibrillar minerals within collagen were aligned along the longitudinal axis of the collagen fibril.The amounts of Ca and P ions released from 25wt%PAsp-Si-ACP remineralization films increased successively during the period of 28 days.Conclusions:Self-etch adhesives loaded with 25wt%of PAsp-Si-ACP particles could remineralize self-assembly type I collagen fibrils and successively release Ca and P ions for 28 days.Therefore,self-etch adhesives could be used as a novel carrier for ACP nanoprecursors to continuously deliver biomimetic remineralization.Part 3 Self-etch adhesives as a carrier for ACP nanoprecursors to deliver biomimetic remineralization of demineralized dentinObjectives:This study evaluated self-etch adhesive as a carrier for ACP nanoprecursors(solid PAsp-Si-ACP particles)to deliver biomimetic remineralization of demineralized dentin.Materials and Methods:PAsp-Si-ACP particles and the demineralized dentin disks were prepared as described in Part 1.Forty-eigtht demineralized dentin disks were randomly divided into four groups as following(n= 12):Group 1:Clearfil S3 Bond(S3,control)Group 2:Adper Easy One(AEO,control)Group 3:The adhesive(S3)was sufficiently mixed with 25wt%of PAsp-Si-ACP particles before use(S3+25wt%PAsp-Si-ACP).Group 4:The adhesive(AEO)was sufficiently mixed with 25wt%of PAsp-Si-ACP particles before use(AEO+25wt%PAsp-Si-ACP).Briefly,for groups 1 and 2,the adhesives were applied to the surface of the demineralized dentin disks strictly according to manufacturer' instructions.For groups 3 and 4,the adhesives containing 25wt%of PAsp-Si-ACP particles were applied to the surface of the demineralized dentin disks,agitated for 20 s,air-dried for 5 s and light-cured for 10 s.Subsequently,a thin layer of flowable composite resin(3M ESPE,Germany)was placed on the pretreated surface of all the demineralized dentin disks.All the samples were each incubated in 15 ml of artificial saliva(changed weekly)in a 100%humidity chamber at 37? for 1 day,1 month,and 3 months,and subsequently four samples from each group were retrieved for TEM analysis with SAED.Results:In the control groups 1&2,the demineralized dentins were not remineralized after they were incubated in the artificial saliva for 1 day,1 month,and even 3 months.In the experimental group(Group 3:S3+25wt%PAsp-Si-ACP),TEM images show that a thin remineralization layer of approximately 200-300 nm thickness at the bottom of the demineralized dentin collagen was detected after the dentin samples were incubated in artificial saliva for 1 day.SAED patterns indicate that calcium phosphate mineral is weakly crystalline.In the experimental group(Group 4:AEO+25wt%PAsp-Si-ACP),remineralization was not apparent after incubation in artificial saliva for 1 day.However,remineralization layers of approximately 1.5 ?m and 2?m thickness in group 3 and group 4 could be discerned after incubation in artificial saliva for 1 month and 3 months.SAED patterns showed that the mineral phase was apatite.Conclusions:Self-etch adhesives containing 25wt%PAsp-Si-ACP particles could result in the remineralization of demineralized dentin.Therefore,self-etch adhesives could be used as a novel carrier for ACP nanoprecursors to continuously deliver biomimetic remineralization of demineralized dentin.That might be used to manage initial caries and dentin hypersensivtity.