8/3/2019 Direct Bone Formation - Piattelli Et Al (1996)

1/4

ELSEVIER

Biomateriols 17 (1996) 1015-10180 1996 Elsevier Science Limited

Printed in Great Britain. All rights reserved014%9612/96/$15.00

Direct bone format ion on sand-blast edtitanium implants: an experimentalstudyA. Piattelli*, A. Scam.no*, M. Piattelli* and L. Calabrese+Dental School, Universit y of Chiefi, Italy ;tDenta/ School, University of Cagliari, ltalySurface modifications of an implant have been demonstrated to be important in influencing the tissuereactions around the implant. Recently, osteoblasts have been shown to be capable of laying down amineralized matrix in direct contact with the titanium surface. The aim of the present study was toanalyse the early bone responses to titanium implants with an aluminium dioxide sand-blastedsurface. Microscopical analysis showed that in the first week it was possible to observe the presenceof mineralized bone in direct contact with the metal surface, while in other portions of the interface,osteoblasts were seen at the implant surface. These results were confirmed in the 2 and 4 wkobservations. Our results could help to explain the increased removal torque forces reported in theliterature concerning sand-blasted implants.Keywords: Bone tissue, cell attachment, surface morphology, titanium implantsReceived 3 April 1995; accepted 30 June 1995

It is said that bone formation on metal implants derivesfrom the bone bed surrounding the implant with adirection from it towards the implant surface1X2, i.e. animplantopetal kind of bone growth. On the contrary,hydroxyapatite determines the formation of bonedirectly on the ceramic surface. Different interfacesbetween mineralized tissues and dental implants havebeen proposed in various experimental and clinicalconditions. Some authors have found that between thesetwo structures it is possible to find an amorphous,unmineralized layer of different thickness3, while otherinvestigators report evidence of direct contact ofmineralized bone matrix with the titanium layer4*.Recently, Davies et c11.~-~and Lowenberg et ~1.~demonstrated that differentiating osteoblasts werecapable of laying down a mineralized collagen-freematrix in direct contact with the metal oxide surface oftitanium. A major consideration to be done certainlyrelates to the surface topography of the implants, in thatcell behaviour around implants is modified by it5,6.

microscopy analysis of bone growth on sand-blastedtitanium implants in the first 4 weeks after implantplacement.

MATERIALS AND METHODSThreaded smooth titanium screw-shaped implants withan outer diameter of 3.7 mm, a core diameter of 3.2 mmand a total length of 8 mm were manufactured with highprecision instruments (AT, Padova, Italy), and wereinserted in rabbit tibiae. Six New Zealand white maturemale rabbits were used for this study. Each rabbit tibiareceived one implant. The implants had been sand-blasted with aluminium dioxide of 150 pm diameter.

Recently papers have been published about thetissue responses to titanium implants with differentsurface morphologies10-13. In previous works fromour laboratory we have found different types ofinterfaces between mineralized tissues and titanium,with some areas in direct contact at the light andlaser scanning microscopy level, while in other areasa thin unmineralized layer was interposed indicatingprobably different states of dynamic activity at theinterface.

The implants were made by commercially pureGrade 2 titanium (Fe, 0.20%; 0, 0.20%; H, 0.015%, C,0.10% and N, 0.03%). Prior to insertion, implants wereultrasonicated with freon (5 min), ethanol (5 min),distilled water (5 min) and finally sterilized in anautoclave (135C, 20 min).

The aim of our study was to carry out light

The rabbits were anaesthetized with intramuscularinjections of fluanizone (0.7 mg kg- body weight) anddiazepam (1.5 mgkg- body weight); local anaesthesiawas given using 1 ml of 2% lidocainladrenalinsolution. A skin incision with a periosteal flap wasused to expose the tibia1 metaphysis. Preparation ofthe bone sites was done with burs under generoussaline irrigation. The implant insertion was performedby hand. The periosteum and fascia were sutured withcatgut and the skin with silk. No postoperativecomplications or death occurred.Correspondence to Professor Adrian0 Piattelli. postoperatively the animals received intramuscular

1015 Biomaterials 1996, Vol. 17 No. 10

8/3/2019 Direct Bone Formation - Piattelli Et Al (1996)

2/4

1016 Direct bone formation on titanium implants: A. Piattelli et al.injections of penicillin (2000000 units per 5 ml;0.1ml kg- body weight). Two of the animals werekilled with an overdose of intravenous pentobarbitalafter 1 wk, two after 2 wk and two after 1 month. Atotal of 12 implants were retrieved.Implants and surrounding tissues were washed insaline solution and immediately fixed in 4%paraformaldehyde and 0.1% glutaraldehyde in 0.15 Mcacodylate buffer at 4C and pH 7.4, to be processed forhistology. The specimens were processed to obtain thinground sections with the Precise 1 Automated System(Precise, Pescara, Italy). The specimens were dehydratedin an ascending series of alcohol rinses and embedded ina glycohnethacrylate resin (Technovit 7200 VLC, Kulzer,Wehrheim, Germany). After polymerization, thespecimens were sectioned along their longitudinal axiswith a high precision diamond disc at about 150 pm andground down to about 30 pm with a specially designedgrinding machine. A total of three slides were obtainedfor each implant. The slides were stained with acidfuchsin and toluidine .blue. These were observed innormal transmitted light under a Leitz Laborluxmicroscope (Leitz, Wetzlar, Germany).

RESULTSAt 1 weekIt was possible to observe the presence of a smallquantity of bone that appeared to be growing directlyon the metal surface (Figure 1). Osteoblasts appearedto be in close and direct contact with the titaniumsurface (Figure I). An osteoblastic rim was present onthe external side of this bone (Figure z), that waspositive for acid fuchsin. Islands of woven immaturebone, surrounded by osteoblasts, were present verynear to the implant surface (Figure 3). At lowmagnification it was possible to observe osteoblaststhat seemed to migrate from the endosteal surface ofbone towards the implant surface (Figure 4).At 2 weeksIn one of the spires it was possible to see the presenceof mineralized bone with wide osteocytic lacunae,

Figure 1 It is possible to observe osteoblasts (arrows) thathave bone deposited directly on the surface of the implant;in other portions it is possible to see osteoblasts(arrowheads) that are in direct contact with the titanium.(Acid fuchsin-toluidine blue, original magnification x1200.)

Figure 2 It is possible to observe the implant surface (i),bone (b) and an osteoblastic rim (arrows) on the externalsurface. (Acid fuchsin-toluidine blue, original magnificationx 1200.)

Figure 3 At low magnification it is possible to observe thepresence, near the implant surface, of small bone islands.(Acid fuchsin-toluidine blue, original magnification x200.)

Figure 4 At low magnification it is possible to seeosteoblasts (arrows) that appear to migrate, from theendosteal surface of bone (b) towards the implant surface.(Acid fuchsin-toluidine blue, original magnification x200.)

deposited directly on the metal surface, and lined onthe external side by a rim of osteoblasts activelysecreting bone matrix (Figure 5). In other regions thebone appeared to be more mature and the osteocyticlacunae appeared to be narrower (Figure 6). At highermagnification it was possible to observe mineralizedbone lined on the external surface by osteoblasts, andother portions where the osteoblasts, in direct contactBiomaterials 1996, Vol. 17 No. 10

8/3/2019 Direct Bone Formation - Piattelli Et Al (1996)

3/4

Direct bone formation on titanium implants: A. Piatfelli et al. 1017

Figure 5 Mineralized bone, with wide osteocytic lacunae,is in direct contact with the implant surface, and is lined byan osteoblastic rim. (Acid fuchsin-toluidine blue, originalmagnification x200.)

lacunae, compact with the presence of o&eons (Figure 8).At higher magnification it was possible to observe, inother portions of the implant perimeter, the presence ofimmature bone; osteoblasts, actively secretingumnineralized matrix, were present on the external partof this bone (Figure 9). In other areas a more maturebone, with osteocytic lacunae, was present (Figure 10).DISCUSSIONMany studies have demonstrated that bone can bedeposited in close approximation to implants.Histological examination usually provides the bestevidence of the type of tissue present at the interface,even if the use of light microscopy in thick groundsections probably cannot visualize details of the bone-implant interface14. In previous studies from ourlaboratory it has been possible to observe the presenceof a thin unmineralized layer interposed, in manyareas, between mineralized bone and implant. Thislayer had staining properties similar to the osteocytelamina limitans and bone osteoid.

Figure 6 In other areas of the interface it is possible toobserve the presence of more mature bone. (Acid fuchsin-toluidine blue, original magnification x200.)

Figure 6 At low magnification it is possible to observe,after 4 wk, the presence of mature osteonic bone that linesthe implant perimeter. (Acid fuchsin-toluidine blue, originalmagnification x200.)

Figure 7 At higher magnification it is possible to observebone (b), lined by osteoblasts (arrows) and osteoblasts inclose contact with the implant surface (arrowheads). (Acidfuchsin-toluidine blue, original magnification x1200.)

with the titanium surface, appeared to be in activeproduction of bone matrix (Figure 7).At 4 weeksIt was possible to observe in some areas a rim ofosteoblasts that appeared to be in a resting phase. Thebone appeared to be mature with narrow osteocytic

Figure 9 At higher magnification, it is possible to observeimmature bone (b) in some portions of the interface, linedby osteoblasts (arrows) that appear to be secreting bonematrix. (Acid fuchsin-toluidine blue, original magnificationx 1200.)

Biomaterials 1996. Vol. 17 No. 10

8/3/2019 Direct Bone Formation - Piattelli Et Al (1996)

4/4

1018 Direct bone formation on titanium implants: A. Piattelli et al.

Figure 10 In other portions of the implant perimeter it ispossible, on the other hand, to observe more mature bone(b) with osteocytic lacunae. (Acid fuchsin-toluidine blue,original magnification x 1200.)

Recently, some investigato&, in a series of in vitrostudies, have demonstrated deposition of an extracel-lular matrix comprising afibrillar calcium phosphateglobular accretions on a variety of substrata, such aspolystyrene or titanium. This matrix becamemineralized with time and was produced by cellsidentified as differentiating osteoblasts. Theseosteoblasts possessed numerous cell processes towhich were associated small globules of material ofabout 1 pm diameter. These globules appearedattached to the underlying substratum.Previous studies have shown that by altering thesurface characteristics of an implant it is possible toselect certain populations of cells: macrophages havebeen shown to prefer rough surfaces to smooth ones,while fibroblasts tended to accumulate on smoothportions of the culture dishes5. Furthermore it hasbeen demonstrated that different types of cells canorient in the grooves of different micromachinedsurfacesg. In an in vitro study osteoblast-like cellswere shown to attach more to rough titaniumsurfaces produced by sand-blasting than to smootherones15.In an in vitro study Bowers et a1.l showed that thehighest percentage of cell attachment was obtained onthe rough surfaces produced by sand-blasting;according to these authors sand-blasted surfaces couldprovide a unique environment for attachment not onlyof cells but also of the extracellular matrix. Gotfredsenet al. demonstrated significantly higher removaltorque forces for Ti02-blasted titanium surfacescompared with machine-produced implants. Theseresults could be related either to greater bone-implantcontact or to interlocking with the ingrowing bone.Ericsson et ~1.~ found that at 4 month after implantplacement Ti02-blasted implants showed a significantincrease in the percentage of bone-implant contact.The results of the present study could also help toexplain the phenomenon of increased removal torqueforces because early direct bone growth on the implantsurface could improve the implant anchorage in bone.Further studies are certainly needed in order to seewhether a variation of the implant surface can lead to

better results regarding the bone anchorage of titaniumimplants, and subsequent implant longevity.

ACKNOWLEDGEMENTSThis work was supported in part by the NationalResearch Council (CNR) and by the Ministry ofUniversity, Research, Science and Technology(MURST).

REFERENCES1

23

4

5

6

Weinlaender M. Bone growth around dental implants.Dent Chin N Am 1991; 35: 585-601.Zablotsky MH. Hydroxyapatite coatings in implantdentistry. Implant Dent 1992; 1: 53-257.Sennerby L, Ericson LE, Thomsen P, Lekholm U,Astrand P. Structure of the bone-titanium interface inretrieved clinical oral implants. CJin Oral Imp1 Res1991; 2: 103-111.Listgarten MA, Buser D, Steinemann SG, Donath K,Lang NP, Weber HP. Light and transmission electronmicroscopy of the intact interfaces between non-submerged titanium-coated epoxy resins implants andbone or gingiva. JDent Res 1992; 71: 64-371.Brunette DM. The effects of implant surface topographyon the behaviour of cells. Int J Oral Maxillofac Implant1988; 3: 231-246.Brunette DM, Ratkay J, Chehroudi B. Behaviour ofosteoblasts on micromachined surfaces. In: Davies JE,ed. The Bone-Biomat erial Inte$ace. Toronto: TorontoUniversity Press, 1991: 170-180.Davies JE, Ottensmeyer P, Shen X, Hashimoto M, PeelSAF. Early extracellular matrix synthesis by bone cells.In: Davies JE, ed. The Bone-Biomat erial Interface.Toronto: Toronto University Press, 1991: 214-228.Davies JE, Chernecky R, Lowenberg B, Shiga A. Deposi-tlon and resorption of calcified matrix in vitro by ratmarrow cells. Cell Mater 1991; : -15.Lowenberg B, Chernecky R, Shiga A, Davies JE. Minera-lized matrix production by osteoblasts on solid titaniumin vitro. CeJJ Mater 1991; 1: 77-187.Bowers KT, Keller JC, Randolph BA, Wick DG, MichaelsCM. Optimization of surfaces micromorphology forenhanced osteoblast responses in vitro. Int J OralMaxi llofa c Im plant s 1992; 7: 302-310.Cook SD, Baffes GC, Palafox AJ, Wolfe MW, Burgess A.Torsional stability of HA-coated and grit-blastedtitanium dental implants. J Oral ImplantoJ 1992; 18:354-358.Gotfredsen K, Nimb L, Hjorting-Hansen E, Jensen JS,Holmen A. Histomorphometric and removal torqueanalysis for TiO,-blasted titanium implants. Clin OralImp] Res 1992; 3: 77-84.Ericsson I, Johansson CB, Bystedt H, Norton MR. Ahistomorphometric evaluation of bone-to-implantcontact on machine-prepared and roughened titaniumdental implants. A pilot study in the dog. Clin OralImp1 Res 1994; 5: 202-206.Weinlaender M, Kenney EB, Lekovic V, Beumer J, MoyPK, Lewis S. Histomorphometry of bone appositionaround three types of endosseous dental implants. Int JOral Maxillofac Implants 1992; 7: 491-496.Michaels CM, Keller JC, Stanford CM, Solorush M.In vitro cell attachment of osteoblast-like cells totitanium. J Dent Res 1989; 68: 276.

Biomaterials 1996, ol. 17 No. 10