Titanium SurfacesProblems Bone anchorage with machined surfaces was not ideal in bone sites exhibiting thin cortical layers combined with poor quality trabecular bone, such as the posterior maxilla. •Some of the initial titanium plasma spray surfaces were excessively rough and giant cells and macrophages were seen phagocytizing portions of the surface (particle disease). •It was very difficult to remove the contaminants from the original sand blasted surfaces. •The original plasma spray HA-CaP surfaces were not predictable
Hydroxyl Apatite - CaP CoatingsCoatings of hydroxyl apatite, because oftheir chemical similarity to bone, werethought to be advantageous.The HA – CaP surface is moreosteoinductive than titanium and thisleads to more rapid bone depositionfollowing implant placement.However the original so-called HAsurfaced implants were mostlycomposed of tri-calcium phosphate,
Mechanism of Action: HA-CaP Coatings v During healing Calcium and Phosphate ions are released from the HA-CaP coating in the peri-implant region v This leads to the precipitation of a biological apatite with various proteins which serves as a substrate for osteoblastic cells producing bone v The biological apatite substrate promotes cell adhesion, cell differentiation and the synthesis of mineralized collagen
HA-CaP CoatingsHA surfaces were very osteo-conductive. v At six weeks following placement the bone appositional index is close to 70% for HA coated implants compared to 30-50% for original titanium surfaces (machined and TPS) (Weinlander et al, 1993).However: v When the so-called plasma spray HA - Cap surface becomes exposed to the oral cavity it becomes contaminated with oral bacteria
HA-CaP Coatings Clinical Problemsl Colonization by microorganisms leads to circum-implant infections.l Cracks and fissures in some cases led to the entire loss of the HA coating.l These factors predisposed to a higher rate of implant failure Note the loss of bone than seen with titanium around these HA coated implants 4 implants (Wheeler, 1996). years after insertion.
HA-CaP Coatingsv These data have led most clinicians to switch to the titanium surfaces.v However, new methods of applying the HA-CaP coatings with nano-sized crystals are evolving which result in direct bonding of bone to the surface of the implant eliminating the cement line. In summary, HA coated systems have provoked great interest and enthusiasm because of there osteo- conductivity. The initial problems associated with plasma spray application are in the process of being overcome and this type of surface may be the surface of the future.
Ideal Implant Surface Propertiesv Promote adsorption of proteinsv Promote adhesion and differentiation of bone producing cellsv Tissue integration
Implant Surface Sciencev The biological events leading to osseointegration are influenced by: v Surface chemistry v Surface topography of the implant v Hydrophilicity of the surface (wetability)v Research efforts in the last twenty years have attempted to idealize these properties on dental implants in order to: v Decrease healing time v Enable use in compromised bone sites v Improve the quality of the bone implant interface
The Jane and Jerry Weintraub Center for Reconstructive Biotechnology UCLA School of Dentistry Ichiro Nishimura DDS, PhD, Director Takahiro Ogawa DDS, PhD Neil Garrett PhD Anna Jewitt PhD Kumar Shah DDS Eleni Roumanas DDS Ting Ling Chang DDS Evelyn Chung DDS James Kelley DDS, MS John Beumer DDS, MS
Uncovering the secrets ofl Discover insights into the molecular basis of osseointegrationl Develop the next generation of implant surfacesl Objectives l Earlierloading l Immediate loading l Predictability in compromised sites*Ichiro Nishimura DDS, PhD and Takahiro Ogawa DDS, PhD
Titanium Implants - Surface Modifications 2nd Generation 1st break through The micro-rough surface implantsHow are the new surfaces different from the old surfaces?Why do we get better bone anchorage with these newsurfaces?Are these surfaces more bioreactive? Do they accelerate theprocess of osseointegration?If so, what has been the impact on implant biomechanics, earlyand immediate loading and treatment planning?
Current Implant Surfaces Micro-rough surface textures – Why are they a significant improvement? Initial stabilization is improved Maximize the volume of interlocking mineralized bone with the implant surface Double acid MachinedTorque Removal etched surface v Reverse torque test v Data recorded in N/cm v Double acid etched vs Machined v 2 month data (1997) v 1, 2, 3 months (2001) Klokkevold et al, 1997, 2001
Initial anchorage -Torque removal studiesv 10 New Zealand White Rabbitsv 2 Custom-designed implants (distal femur) v Machined v Double acid etched (Osseotite) Klokkevold et al, 1997, 2001)
Initial anchorage -Torque removal studies Reverse torque rotation to removal (failure) Klokkevold et al,1997; 2001)
Initial anchorage -Torque removal studies Why the difference? vBetter mechanical anchorage? vBone contact area? vQuality of the bone at the interface? Klokkevold et al, 1997
Titanium Implants - Surface Modification Surface roughness and the bone contact areaAnimal studies have shown that the bone contactarea achieved is 50% greater with micro -roughsurfaces as compared to machined surfaces(Buser et al, 1991, Weinlander, 1993, Hamada, 1995,Nishimura and Ogawa, 2000, 2003).
50 µm HistomorphometryAcid etched vs Machine surface Near zone Far zone Machined Acid etched (%) 80 * 60 40 * 20 (Ogawa and 0 W2 W4 Nishimura, 2000, Bone-implant 2003), contact ratio
Bone contact area Microrough surfaces (Weinlander et al, 2004)Electrolytically Titanium Double acid Sandblast acid Modified plasma spray etched etched
Implant anchorage dataShear Strength obtained with a push out test StudiesAcid etched Machined surface surface Experimental Rat implant
Implant anchorage data obtained with a push out test This test measures the strength of implant tissue interface rather thanPush-in value (N) the strength of the surrounding bone. 60 Machined * Etched 40 * 20 * 0 0 2 4 8 *p<0.05 Healing period (week)
Current Implant SurfacesMicro-rough surfaces and osseointegration What biologic phenomenon are affected by the changes in surface micro topography?
Current Implant Surfaces Why are these surfaces more bioreactive? vRate of plasma protein absorption vFibrin clot retention vCell Adhesion vCell differentiation vGene expression
Rate of plasma protein absorptionv Rate of plasma protein absorption v Enhanced by hydrophilic surfaces v Enhanced by micro-rough surfaces v Reduced as the surface agesv Promotes cell adhesionv Promotes cell differentiation
Fibrin Clot Retention Micro-rough surfacesvDavies (1998) showed that micro-roughsurfaces captured and retained the fibrinclot initially deposited on the implantsurface more effectively than machinedsurfacesv As a result the initial crticial events(plasma protein adsorption, clot formation,angiogenesis, mesenchymal stem cellmigration etc.) associated withosseointegration were facilitated.
Differentiation of mesenchymal stem cells (MSC) into functioning osteoblasts Functioning osteoblastvStem cells migrate to the implant surface and into the osteotomy site via the fibrin networkvThe micro-rough facilitates this process
Micro – rough Implant SurfacesOgawa and Nishimura set out to determine whether ornot the double acid etched surface result in: Earlier osseointegration? Better bone anchorage? Determine what factors are responsible for the different boneproﬁles seen on different surface textures of titanium implants.They were particularly interested in the gene expression of thedifferentiating osteoblasts Osseotite- Double Acid Etched
Gene expression data obtained with a T-cell model developed by DaviesT-cell implant Machined Acid-etched v They implanted T-cell implants into the femurs of rats and retrieved the specimens at various time intervals. vThey hypothesized that gene expression is controlled at local levels by the surface texture of the implant.
Why was the double acid etched surfacesuperior to the machined surface?Why was the bone different?Nishimura and Ogawa suggestedseveral reasons including: Bone repair and generation may not be the primary prerequisite for osseointegration Might it be an implant dependent mechanism? Hypothesis: A set of genes that are NOT involved in bone repair initiate and/or regulate the process of osseointegration Ogawa and Nishimura, 2000, 2002, and 2003
Purpose of the studyIdentify the genes that are expressed around implantsbut not in non-implant wound healing of bone. Non-implant defect Turned implant Etched implant Screening of candidate osseointegration-specific genes Differential display polymerase chain reaction (DD-PCR)
Testing the candidate DD-PCR products From 1853 DD-PCR products, 19 implant-specific (- + +) 2 acid-etched-specific (- - +) 42 different clones3 Osseointegration-specific genes (TO1, TO2, TO3)
mRNA expression of TO1 Day 3 Week 1 Week 2 Week 4 Non-implant defect Turned implant Etched implant Untreated control TO1GAPDH 0.2 Etched implant Turned implant 0.1 Non-implant defect Day 3 Week 1 Week 2 Week 4
mRNA expression of TO2 Day 3 Week 1 Week 2 Week 4 Non-implant defect Turned implant Etched implant Untreated control TO2GAPDH 0.6 0.4 Turned implant Etched implant 0.2 Non-implant defect Day 3 Week 1 Week 2 Week 4
mRNA expression of TO3 Day 3 Week 1 Week 2 Week 4 Non-implant defect Turned implant Etched implant Untreated control TO3GAPDH 1.2 Etched implant 0.8 Turned implant 0.4 Non-implant defect Day 3 Week 1 Week 2 Week 4
TO genes showedOsseointegration-specific expressionUpregulation in early stages of implantationAccelerated expression for the double acid etched surface
TO3 happens to be P4H Enhanced gene expression of prolyl 4-hydroxylase (P4H) Collagen synthesis considerably higher
Why was the accelerated expression of P4H on micro- rough surfaces significant?Collagen density and orientation, as well as thedegree of mineralization are contributingfactors relative to the microhardness and elasticmodulus of bone
Bone Implant Interface Double Acid Etched SurfacesA different combination of collagenous andnoncollagenous proteins make up the bone depositedon the dual acid etched surface as compared to amachined surface.Resorption and remodeling of bone deposited on acidetched surfaces appeared to be different than boneon machined surfaces.
Nano Indentation Test 3 times stiffer bone on dual acid etched 2000nm indentation depth P=0.0252Elastic modulus P=0.0339 (GPa) 0.2 0.1 0 Bone on Bone on Bone on Polystyrene Machined Ti DAE
Nano indentation test 2 times harder bone on dual acid etched 200mN maximum load P=.0005 P=0.0153 P=0.0130Nanohardness (GPa)0.8 0.6 0.4 0.2 0 Bone on Bone on Bone on Polystyrene Machined Ti DAE
Nanoindentation: in vivo boneBone around machined surfaces is as hard as the trabecularbone, while the bone around the DAE surfaces is as hard asthe cortical bone. Ogawa et al, 2005
Day 28 Day 14 7 Day 21 0 3Distinct pattern of osteogenesis on DAE Osteoblast Non-collagenous matrix Mineral deposition Collagen matrix
Current Implant SurfacesMicro-rough surface textures – Why are they a significant improvementShape of the cell affects its gene expressionand the micro-environment affects cell behavior. Improved adsorption of plasma proteins
Micro-rough surfaces – Why are they superior?vImproved clot retention (Davies, 1998)vInitial absorption of plasma proteins isenhanced (fibronectin, vitronectin etc) (Kohavi(2010)vMSC differentiate much faster on micro-rough surfaces as compared to smoothsurfaces (Ogawa et al, 2003)vMicro-rough surfaces changes geneexpression of the differentiating osteoblasts(Ogawa and Nishimura (2000,2003 and 2004, 2006)vBone deposited on micro-rough surfaces isharder and stiffer than bone deposited onmachined surfaces (Butz,2006; Takeuchi et al,2005)
Impact of Strengthened Peri-implant Bone Trabecular bone Cortical boneCortical bone: l Very dense l Less subject to resorption or remodeling
Enhancement of current titanium surfacesSLA active (implant packaged in saline) (Strauman) Maintains the wetability of the surface Wetable surfaces significantly enhances initial adsorption of plasma proteins This, in turn facilitates migration, adhesion and differentiation of mesenchymal stem cells
Other means of increasing wetability of the implant surfacev Incorporate calcium, magnesium or fluoride ions into the titanium oxide surface v Thisis referred to as “electro-wetting” and allows the plasma proteins to flow freely onto the implant surface and into the irregularities of the micro-roughened surface immediately upon insertion of the implant
Enhancement of titanium surfacesFluoride treated surfaces (Astra) vImproves the wetability of the surface vCbfa expression is high for the grit blasted fluoride prepared surface (Isa et al, 2006) Cbfa is a transcription protein that promotes cell differentiation of osteoprogenitor cells) Accelerates the events leading to deposition of bone on the implant surface
Biological aging andphotofunctionalization of TiO2 UV-treatment Tak Ogawa DDS, PhD Weintraub LA, UCLA
Biological aging and photofunctionalization of TiO2v Present day implants are packaged in plasticv They are then sterilized with gamma radiationv This process releases hydrocarbons which contaminate the implant surface Bioreactivity of the implant surface is impaired The surface charge is changed from positive to negative The surface becomes less wetable Adsorption of plasma proteins is inhibited
Impact of UV-treatment on biomechanical strength *p<0.0001 Original Light-treated **p<0.05Push-in value N=9(N) 50 50 * 40 40 * 30 30 20 ** 20 ** 10 10 0 0 Day 14 Day 28 Day 14 Day 28 Machined surface Acid-etched surface
Week 4 peri-implant bone morphogenesis Bone contact area *p<0.0001Light-treated acid-etched surface Original Light N=4 100 * 80 * 60 40 20 0 Day 14 Day 28 Bone–implant contact
Control UV-Effects (6 h)v UV Enhanced protein absorptionvUV Enhanced migration of MSC to Ti surfacev UV Promotes more rapid differentiation of MSC into functioning osteoblastsv UV Enhanced adhesion, spreading behavior and cytoskeleton arrangement of osteoblasts Light treated
Proposed mechanism for improved osteoblast affinity on UV treated TiO2 Cx Hy UV-pretreated TiO2 TiO2 R C O O O Ti4+ Ti4+ O (h+) O hv Ti4+ Ti4+ OTitanium Oxide is photocatalytic. Following UV exposurefree radicals are formed which absorb the hydrocarbons onthe implant surface.
Benefits of UV Lightv Improved wetabilityv Changes the surface charge from negative to positivev These factors dramatically improve initial absorption of plasma proteins during the initial stages of healing (10-20 minutes immediately following implant placement) Aita et al, 2009; Atta et al, 2009
Benefits of UV Light Possible future clinical applicationsTreatment of the failing implant v Decontaminating implant surfaces in vivo with UV light Courtesy G Perri
3rd Generation of Implant Surfaces Genetically engineered implant surfaces Adding recombinant peptides to the surface (BMP’s etc) Nano-enhanced surface topography Coatings to enhance surface chemistry HA-CaP crystal deposition Pico to nanometer thin TiO2 coating
Application of TO genes: Non-viral Plasmid DNA Gene Transfer TO2
Impact of TO2 gene delivery (1 week) P=.0055Push-in value (N) 16 14 DAE 12 Machined 10 8 6 4 2 0 Untreated Matrix control TO2
Genetically Engineered Implant SurfacesPotential advantages of gene delivery Doesn’t degrade Low doses Associated with the ! normal cell cascadeDisadvantages Regulatory issues Time to market Cost/Benefit ratio
Surfaces enhanced with recombinant peptidesRecombinant peptide delivery (BMP-2) has not proven to be effecticve. Why? Production issues During sterilization much of BMP may be deactivated They require high doses which may be toxic They are not associated with the normal ! cellular cascade Retention and release difficult to control High cost Schliephake et al, 2005
Nano-enhancement of the implant surfacev Increased surface areav Enhanced wetability and adsorption of plasma proteinsv More favorable surface chemistry with HA- CaP coatings and TiO2 pico-nanometer coatings
Nanotechnology Current Deﬁni6on 1. Size: 1 to 100 nanometer range 2. Novel proper6es due to its small size 3. Incorporated into large material componentsl Research and technology development at the atomic, molecular or macromolecular levels, in the length scale of approximately 1 - 100 nanometer range, to provide a fundamental understanding of phenomena and materials at the nanoscale and to create and use structures, devices and systems that have novel properties and functions because of their small and/or intermediate size. The novel and differentiating properties and functions are developed at a critical length scale of matter typically under 100 nm.l Nanotechnology research and development includes manipulation under control of the nanoscale structures and their integration into larger material components, systems and architectures. Within these larger scale assemblies, the control and construction of their structures and components remains at the nanometer scale. In some particular cases, the critical length scale for novel properties and phenomena may be under 1 nm (e.g., manipulation of atoms at ~0.1 nm) or be larger than 100 nm (e.g., nanoparticle reinforced polymers have the unique feature at ~ 200-300 nm as a function of the local bridges or bonds between the nano particles and the polymer).l DEFINITION of NANOTECHNOLOGY by NSET, 2002: Subcommittee on Nanoscale Science, Engineering and Technology for the U.S. National Science and Technology Council under President G.W. Bush
Effect of Nano-Structure: Cell response Overwhelming numbers of studies report significant effect of nano-structure on cellular behaviorsHuman corneal epithelial cells with Fibroblast growth was 70nm groove (A) or ﬂat surface (B) prohibited on nano-‐structured surface
Effect of Nano-Structure: Cell response On nanometer particle size ceramics such as alumina, titania and hydroxyapatite, osteoblast adhesion increased while fibroblast adhesion decreased. Osteoblast Fibroblast Webster et al, Biomaterials, 1999 Richert et al, Orthoped Res Soc abstract, 2006
Effect of Nano-Structure: Controlled protein adsorption v Protein adsorption to nano-structured surfaces requires less energy than to flat surface v Nano-structure orients the direction of adsorbed proteinSabirianov et al, Enhanced iniJal protein adsorpJon on engineered nanostructured cubic zirconia
Effect of Nano-Structure: Controlled protein adsorption v Protein adsorption increased significantly on ~30nm structured TiOx surface. v Surface nano- structure determines the protein adsorptionScopelliJ et al, The eﬀect of surface nanometre-‐scale morphology on protein adsorpJon, PlosOne, 2010
Effect of Nano-Structure:Long-term stability of osseointegration v Recent theoretical models indicates increased mechanical interlocking of bone with nano-structured surfaces. Loberg et al, Open Biomater J, 2010 Hansson et al, Open Biomater J, 2010
Conclusionv In the advent of Nanotechnology development, dental implant surface modifications now employ a variety of new processing methods at nano-scales.v While micro-structured implants entered in the discount, generic manufacturing, nano-structured implant surfaces present the new generation of dental implants.v Advantages of nano-structured implants include selective cellular behaviors and controlled protein adsorption leading to a “tailored” biological regulation.v Once osseointegration is established, bone-implant mechanical interlocking may be better maintained on the nano-structured implant, potentially contributing to the long-term stability.
Nano-coating of HA-CaP Crystalsapplied to the surface of the implant
Changes Nano-Surface Topography and Chemistry Before After
HA-CaP CoatingsMechanism of Action v During healing Calcium and Phosphate ions are released from the HA-CaP coating in the peri-implant region v This leads to the precipitation of a biological apatite with various proteins incorporated which serves as a substrate for osteoblastic cells producing bone v The biological apatite substrate promotes cell adhesion, cell differentiation and the synthesis of mineralized collagen v The CaP coatings promote direct bone bonding as compared to noncoated surfaces
Shear Strength (MPa) DAE Ti-nanoHA Chemical Bonding? Machined Ti DAE Ti DAE Ti-nanoHA Shear strength at 2 wk S=F/A [N/mm2]
Synergistic effect of DAE topography and HA – CaP nano-crystal deposition Nishimura and Butz et al, 2004When nano-HA coating was added to conventional smooth andDAE implants, bone anchorage was increased over 100%. In factDAE Ti-nanoHA implant showed the accelerated bone-implantintegration at the level that has never been reported.
Bone-implant integration Machined DAE+HA-nano-topography boneWeak Link – Cement Line Smooth implant was almost naked because surrounding bonedid not stay on implant. DAE plus nano-HA was covered by the surrounding boneindicating that bonding was so strong the push-in forcefractured the bone.
Bone-implant integration Machined DAE+HA-nano-topography boneThe bond between the bone andimplant surface was greater thanbetween the new bone and old bone. No cement line?
HA – CaP Enhanced Surfaces HA-Ca P crystal depositionAdditional advantages for potential futureapplications v Gene delivery v Growth factors, osteogenic protein, antibiotic delivery etc.
Titanium Implants - Surface ModificationsNano-surface modification of titanium surface Further enhancement of the surface topography by physical vapor deposition (Ogawa et al, 2007; Sugita et al, 2011) l Increased surface area for bone deposition l Surface topography created similar to mineralized bone matrix l Enhanced osteoblast adhesion, proliferation and differentiation l Promotion of osteoblast function l Greater strength of osseointegration
Pico-super-thin surface modification of Tiv Ogawa and associates have shown that a pico- meter thin TiO2 coating improves the bioreactivity of microrough implant surfaces by modulating its surface chemistry while preserving the existing surface morphology Sugita et al, 2011
Pico-super-thin surface modification of TiO2 Method Slow-rate sputter coating of liquidified nanoscale TiO2 particles Optimal exposure time – 15 min Liquidiﬁed TiO2- Control Ti coated Ti Sugita et al, 2011
No surface thin as 300 pm change before and after As topography The coating is as thin as 300 pmControl Ti Liquid TiO2 - 15 minThe micro-rough topgraphy is unchanged by the coating Sugita et al, 2011
Enhanced bone cell attachment (6 h) P<0.05 WST-1/cell 0.2 0.1 0 Control Ti Liquid TiO2 coated N=3 Sugita et al, 2011
Expedited and enhanced Enhanced Cell spreading and cellular settlement and spread cytoskeleton arrangement of osteoblasts Control Ti Liquid TiO2 coated Overlay Overlay min 15 50µm 50µmUncoated surface 15 minute TiO2 coated surface Sugita et al, 2011
Rate of Osteoblastic Differentiation Enhanced bone cell function (Day 5) P<0.05ALP activity 0.15 0.1 0.05 0 Untreated Liquid TiO2 coated N=3 Sugita et al, 2011
Bone-related gene expression (Days 7 and 14) Sugita et al, 2011
Mineral deposition (Day 14) Sugita et al, 2011
Control Liquid TiO2 coatedMineralized nodule area at day 14 (arizarin red) Control Liquid TiO2 coated Sugita et al, 2011
Significance:vFor the first time, pico-super-thincoating of TiO2 was applied, whichdid not to alter the existing micro -topography of Ti.vSurface chemistry of TiO2 is animportant factor determining thebioreactivity of the implant surface.vThis technology may have openeda new avenue of surfaceenhancement for endosseousimplants. Sugita et al, 2011
Clinical Impact of advances in implant surface science?The biologic events leading toosseointegration have been acceleratedBetter bone anchorage
Implants in Compromised Sites Can we use shorter implants?Posterior maxillaPosterior mandible over theinferior alveolar nerve in partiallyedentulous patientsCraniofacial application Theoretically perhaps. However we need well designed clinical outcome studies to determine predictability
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