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made at 24 and 72 h; the implants were then processed for SEM. At 24 h, only sur-

faces scaled with a stainless-steel curette showed a significant reduction in num-

ber of attached cells relative to untreated control surfaces. It was found that, at 72

h, both stainless steel and Ti-6Al-4V alloy curette instrument surfaces showed sig-

nificantly fewer attached cells than untreated control surfaces, with the greatest

reduction in cell attachment observed on stainless-steel curette instrument sur-

faces [8-82]. Ryhänen et al. [8-83] evaluated the new bone formation, modeling,

and cell–material interface responses induced by the NiTi SME alloy after

periosteal implantation. The regional acceleratory phenomenon (RAP) model was

employed, in which a periosteal contact stimulus provokes an adaptive modeling

response. The test implant was placed in contact with the intact femur periosteum,

but it was not fixed inside the bone. It was found that (i) a typical peri-implant

bone wall modulation was seen due to the normal RAP, (ii) the maximum new

woven bone formation started earlier (2 weeks) in the Ti-6Al-4V group than the

NiTi group, but also decreased earlier, and at 8 weeks the NiTi and stainless steel

groups had greater cortical bone width, and (iii) at 12 and 26 weeks no statistical

significances were seen in the histomorphometric values. On the basis of these

findings, it was concluded that NiTi had no negative effect on total new bone for-

mation or normal RAP after periosteal implantation during a 26-week follow-up

[8-83]. The bone formation around CpTi implants with varied surface properties

was tested by Larsson et al. [8-84]. Machined and electropolished CpTi samples

with and without anodically formed surface oxides were prepared and inserted

into the cortical bone of rabbits (1, 3, and 6 weeks). It was reported that (i) the

smooth, electropolished implants, irrespective of anodic oxidation, were sur-

rounded by less bone than the machined implants after 1 week, (ii) after 6 weeks,

the bone volume as well as the bone–implant contact, was lower for the merely

electropolished implants than for other three groups; however (iii) the result with

the smooth (electropolished) implants indicates that a reduction of surface rough-

ness, in the initial phase, decreases the rate of bone formation in rabbit cortical

bone [8-84]. Larsson et al. [8-85], in another experimental study, analyzed the

bone formation around systematically modified CpTi implants which were

machined, electropolished, and anodically oxidized, and were inserted in the cor-

tical bone of rabbits (7 and 12 weeks). It was found that (i) light microscopy

revealed that all implants were in contact with bone, and a large proportion of bone

within the threads, and (ii) the smooth, electropolished implants were surrounded

by less bone than the machined implants with similar oxide thickness (4–5 nm)

and the anodically oxidized implants with 21–180 nm after 7 weeks. It was further

reported that a high degree of bone contact and bone formation could be achieved

with CpTi implants which are modified with respect to oxide thickness and sur-

face topography; however, it appears that a reduction of surface roughness may

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influence the rate of bone formation in rabbit cortical bone [8-85]. Since interac-

tions among cell/matrix/substrate, which are associated with cell signaling, occur

in the nanoscale dimension, Oliveira and Nanci [8-86] evaluated the influence of

nanotexturing of Ti-based surfaces (CpTi and Ti-6Al-4V, treated by H

and

30% H

) on the expression of matrix proteins by cultures osteogenic cells at

initial time points. It was reported that (i) after 6 h, nanotextured surfaces exhib-

ited up to a nine-fold increase in the proportion of cells with peripheral osteopon-

tin labeling, (ii) at day 3, the proportion of osteopontin and bone sialoprotein

labeled cells was higher, and the intensity of immunoreactivity dramatically

increased, and (iii) no significant differences were observed in the expression pat-

tern and the proportion of cells immunoreactive for fironectin [8-86].

Biological performance of dental and orthopedic implants depends on factors

such as composition, design, surface topography, sterilization, and surgical tech-

nique. Common dental implants are made of different grades of CpTi. Ahmad et al.

[8-87] conducted in vitro tests to determine if human osteoblast-like cells, Saos-2,

would respond differently when plates on disks of CpTi grades 1 and 4, with glass

disks serving as controls. It was reported that (i) rhodamine phalloidin fluorescence

microscopy showed variations in the actin-based cytoskeleton between grades 1 and

4 CpTi in cell spreading, shape, and the organization of stress fibers, (ii) immuno-

fluorescent staining showed differential expression of vinculin, a focal adhesion

protein, on the substrates, (iii) at 24 h, the percent of collagen synthesized was sig-

nificantly more on grade 1 than on grade 4 and on glass, and (iv) the calcium con-

tent was significantly higher on grade 1 than on grade 4 and on glass at 24 h and at

4 weeks. It was, thus, concluded that commonly used CpTi induced differential

morphologic and phenotypic changes in human osteoblast-like cells depending on

the grade of the material [8-87]. The spreading and orientation of human gingival

fibroblasts in relation to the rim of smooth-surfaced and porous-coated Ti disks was

in vitro investigated. It was found that (i) the cells migrated from the multilayer onto

the smooth-surfaced disks forming bridges between them, and orientated along par-

allel circumferential grooves in the rim of the disks, and (ii) cellular bridges were

also formed between the porous-coated disks and the multilayer, but because the

cells that migrated onto and between the sphere of the porous-coated showed no

preferred orientation, the bridges retained their orientation at right angles to surface

of the rim. This suggests that the geometrical configuration of the surface of the

implants could influence whether a capsule or an orientated fibrous attachment is

developed in relation to implants in vivo [8-88].

Proliferation and adhesion of mouse osteoblastic cells and primary osteoblastic

cells were carried out on Ti-6Al-4V samples with varied surface roughness [8-89].

Mechanically or manually polished surfaces were prepared to produce, respectively,

non-orientated or orientated polishing grooves. Sand-blasted surfaces were

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prepared using 500 m or 3 mm alumina particles. It was found that (i) surface

roughness parameters showed a negative correlation in comparison to proliferation

and adhesion parameters, and (ii) X-ray microprobe chemical microanalysis

showed complete disturbance of the surface element composition of the Ti-6Al-4V

following sand-blasting treatment, on which an AlO

-enriched layer was observed.

This may lead to the suspicion that the concomitant effect of surface roughness

amplitude and AlO

surface concentration has an effect on osteoblastic cell prolif-

eration and adhesion. It was demonstrated that usual surface preparation for

increasing the bone integration of Ti-6Al-4V surgical implants (i.e., sand-blasting)

may extensively transform their chemical surface composition. Therefore, the

expected bone integration of implants may be extensively impaired by surface treat-

ments [8-89]. The possible contamination by used blasting media on Ti surfaces

will be discussed in Chapter 11. Mustafa et al. [8-90] analyzed variations in the

oxide films on CpTi surfaces blasted with TiO

particles of various sizes after cul-

tures with cells derived from human mandibular bone. Turned Ti surfaces, and

sand-blasted with 63–90, 106–180, and 180–300 m TiO

particles were cultured

with osteoblast-like cells. The surfaces were characterized before and after the cell

culture with electrochemical impedance spectroscopy (EIS). It was found that (i)

EIS revealed, with respect to the turned surfaces, the effective surface area was

about 5, 6, and 4 times larger on the surfaces blasted with 63–90, 106–180, and

180–300 m particles, respectively, (ii) after 28 days of the cell culture, the corro-

sion resistance on all sample types was unaffected; the impedance characteristics

suggest a considerable effect of ion incorporation and precipitation during cultur-

ing, (iii) XPS revealed that before the cell culture, a typical surface layer consisted

of TiO

, and (iv) after the culture, the surface oxide film contained both P and Ca,

along with large amounts of oxidized carbon (carbonate) and nitrogen [8-90]. Lee

et al. [8-91] examined the cell attachment and proliferation of neonatal rat calvar-

ial osteoblasts on Ti-6Al-4V as affected by the surface modifications (as polished,

heated in vacuum at 970°C for 8 h to have 300 m thick brazed, at 970

C for 8 h to

have 600 m brazed). During the brazing, a foil of Ti-15Cu-15Ni with melting

point of 934

C was placed top of the Ti-6Al-4V. All samples were passivated by

boiling them in distilled water for 24 h, followed by autoclaving at 121°C for 15

min. The modifications could alter simultaneously the surface chemistries of the

alloy, as well as the surface characteristics of oxides on the metal. It was found that

a slight change of the surface characteristics of oxides as a result of the modifica-

tions neither alters the in vitro capacity of Ca and P ion adsorption nor affects the

metal ion dissolution behavior of the alloy, suggesting that any influence on the

cytocompatibilities of the materials should only be correlated to the effect of sur-

face chemistries of the alloy and the associated metal ion dissolution behavior of

the alloy. It was therefore concluded that the cell response of neonatal rat calvarial

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osteoblasts on the Ti-6Al-4V alloy should not be affected by the variation of sur-

face chemistries of the alloy in a range studied [8-91]. To investigate the roles of

compositions and properties of Ti surface oxides in cellular behavior of osteoblasts,

the surface oxides of Ti were modified in compositions and topography by anodic

oxidation in two kinds of electrolytes, 0.1 M H

and a mixture of 0.03 M cal-

cium glycerophosphate and 0.15 M calcium acetate. It was found that (i) phospho-

rous (P: ~ 10 at%) or both Ca (1–6 at%) and P (3–6 at%) were incorporated into

the anodized surfaces in the form of phosphate and calcium phosphate, and (ii) con-

tact angles of all the anodized oxides were in the range of 60–90

, indicating rela-

tively high hydrophobicity. It was also mentioned that cell culture experiments

demonstrated absence of cytotoxicity and an increase of osteoblast adhesion and

proliferation by the anodic oxides [8-92].

Bony ingrowth to control (non-treated) and heat-treated (autoclaved at 280

for 3 h) stainless-steel and Ti-6Al-4V implants into the medullary canal of the

femur in rats was studied by Hazan et al. [8-93]. It was found that (i) at all time

intervals up to 3 days post-implantation, the shear strengths of heat-treated Ti-6Al-

4V and stainless-steel implants were significantly higher (1.6- and 3.4-fold) than

the controls, and (ii) the heat treatment of metal implants prior to their insertion

alters their chemical surface properties and augments bony ingrowth to them

[8-93]. Characteristics of the porous surfaces may be important in improving the

bone ingrowth into the porous coatings. Quicker and more mature interstitial bone

formation was obtained using a porous rather than a solid structure, due to differ-

ences of penetration by some growth factors and bone marrow cells [8-94–8-96].

Li et al. [8-97] examined the effect of the surface macrostructure of a dimpled

CpTi implant on bone ingrowth in vivo by means of histological examination and

a push-out test. Dimples had diameters of 100, 140, and 160 m, and distance

between dimple centers of about 400 m. Cylindrical implants were inserted in

rabbits for 1.5, 3, and 13 months. The femur with the implant of each animal was

then examined in a push-out test. It was found that the dimpled CpTi surface

results in an increased retention of the implant in bone due to interlocking between

vital bone and the dimples [8-97].

NiTi SME alloy makes it possible to prepare functional implants that apply a

continuous benign force to the bone. Kujala et al. [8-98] investigated if bone mod-

eling can be controlled with a functional intramedullary NiTi nail. Preshaped NiTi

nail with a curvature radius of 25–37 mm were implanted in the cooled martensite

phase form in the medullary cavity of the right femur in 8 rats, where they restored

their austenite form, causing a bending force. After 12 weeks, the operated femurs

were compared with their non-operated contralateral counterpairs. It was found

that (i) quantitative densitometry showed a significant increase in the average

cross-sectional cortical area and cortical thickness, which were most obvious in

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the mid-diaphyseal area, (ii) cortical bone mineral density increased in the proxi-

mal part of the bone and decreased in the distal part, and (iii) polarized light

microscopy of the histological samples revealed that the new bone induced by the

functional intramedullary nail was mainly woven bone. It was therefore concluded

that bone modeling can be controlled with a functional intramedullary nail made

of NiTi SME alloy [8-98]. The biocompatibility of NiTi as a potential implant

material was investigated through in vivo studies on beagles. A high-purity alloy

was fabricated into prototype bone plates and implanted into the femurs of bea-

gles. Commercial Co-Cr alloy bone plates served as reference controls. The bone

plates were removed from the animals and examined after exposures of 3, 6, 12,

and 17 months. It was reported that (i) there was no evidence of localized or gen-

eralized corrosion on the surfaces of the bone plates and screws, (ii) gross clini-

cal, radiological, and morphological observations of the tissue at the implantation

sites during the autopsies uncovered no signs of adverse tissue reactions resulting

from the implants, and (iii) a histological analysis was performed on samples of

muscle and bone adjacent to the implantation sites and of tissues removed from

such organs as the liver, spleen, brain, and kidneys. It was further found that there

was no metallic contamination in the organs due to the implants; however, there

does appear to be some chromium contamination from the Co-Cr alloy implants

in the adjacent bone, and concluding that NiTi alloy is sufficiently compatible with

dog tissue to warrant further investigation of its potential as a biomaterial [8-99].

Nishiguchi et al. [8-100] evaluated the bone-bonding ability of three differently

treated samples of CpTi: (1) as a smooth surface control, (2) treated in 5 M NaOH

solution for 24 h at 60

C, and (3) plus heated at 600

C for 1 h. The plates were

inserted transcortically into the proximal metaphyses of bilateral rabbit tibiae. The

tensile failure loads between implants and bones were measured at two intervals

using a detaching test. It was reported that (i) the tensile failure loads of the alkali-

and alkali-heat-treated group were 27 and 40 MPa, at 8 and 16 weeks, and signif-

icantly higher than those of the other Ti groups, (ii) histological examination

revealed that alkali- and heat-treated Ti was in direct contact with bone, but the

other Ti groups had a thin intervening fibrous tissue, and (iii) the alkali-treated Ti

without heat treatment had no bone-bonding ability due to the unstable reactive

surface layer of alkali-treated Ti. It was therefore concluded that both alkali and

heat treatment are essential for preparing bioactive Ti and this bioactive Ti is

thought to be useful for orthopedic implants with cementless fixation [8-100].

There is an increasing attention given to the influence of surface condition, and

in particular on methods to control and improve titanium implant surfaces; such

techniques should include laser surface treatment [8-101], strain hardening [8-

102] and chemical passivation methods [8-103]. From histological examinations,

it has been found that implant loosening is generally associated with the formation

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of fibrous tissue at the bone–implant interface. To improve implant biocompati-

bility and osseointegration, Ti-6Al-4V alloy was treated by passivation by immer-

sion in 30% HNO

for 1 h, or passivation in boiling distilled water for 10 h

[8-104], resulting in an increase in the oxide layer thickness of Ti-6Al-4V alloy as

orthopedic implant material. The kinetics of gene expression over 120 h was fol-

lowed for 58 genes to quantify the effect of he developed surface treatment.

Twenty eight genes were further selected to compare the effects of surface treat-

ments on osteoblasts. On the basis of the genes studies, a general pathway for the

cell reaction was proposed according to the surface treatment used: (1) metal ion

release changes the time course of gene expression in the focal adhesion kinase

pathway; (2) once the accumulation of metal ions released from the Ti surface

exceeds a threshold value, cell growth is diminished and apoptosis may be acti-

vated; (3) protein tyrosine kinase upregulation is also induced by metal ion

release; (4) expression of the Bc1-2 family and Bax may suggest that metal ions

induce apoptosis [8-104].

The cleanliness of Ti dental implant surfaces is considered to be important

for achieving osseointegration, and it has been hypothesized that the presence of

inorganic contaminants could lead to a lack of clinical success. As Anselme et al.

[8-89] pointed out that aluminum ions are suspected of impairing bone formation

by a possible competitive action to calcium. Piattelli et al. [8-105] investigated the

effects of residual Al

particles on the implant surface on the integration of Ti

dental implants as compared to decontaminated implants in a rabbit experimental

model. Threaded screw-shaped machined grade 3 CpTi dental implants were used.

The implants were sand-blasted with 100–120 m Al

particles at 1.5 atm pres-

sure for 1 min, then 24 implants (control implants) underwent the ASTM 86-68

decontamination process in an ultrasonic bath. The other 24 implants (test

implants) were washed in saline solution for 15 min. Both test and control

implants were air-dried and sterilized at 120

C for 30 min. After sterilization, the

implants were inserted into the tibiae of New Zealand white mature rabbits. All

animals were euthanized after 4 weeks. A total of 48 implants were retrieved. It

was found that (i) percentage of the implant surface covered by inorganic particles

was significantly higher than that of control implants, (ii) no statistically signifi-

cant differences were found in the bone-implant contact percentage of test and

control implants, and (iii) no differences were found in the number of multinucle-

ated cells and osteoblasts in contact with the implant surface. Hence, it was con-

cluded that histological results do not provide evidence to support the hypothesis

that the residual aluminum oxide particles on the implant surface could affect the

osseointegration of Ti dental implants [8-105].

Primary stability and an optimized load transfer are assumed to account for an

undistributed osseointegration process of implants. Immediate-loading type newly

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designed Ti dental implants inserted in the mandible of minipigs, were used for the

characterization of the interface area between the implant surface and the sur-

rounding bone tissue during the early healing phase. Histological and SEM studies

were done. Two different load regimes were applied to test the load-related tissue

reaction. It was shown that (i) a direct bone apposition on the implant surfaces, as

well as the attachment of cells and matrix protein in the early loading phase, and

(ii) a striking finding of the ultrastructural immunocytochemical investigations was

the synthesis and deposition of bone proteins (osteonection, fibronectin,

fibronectin receptor) by osteoblasts from day one of bone/biomaterials interaction.

It was therefore suggested that loadings of specially designed implants can be per-

formed immediately after insertion without disturbing the biological osseointegra-

tion process [8-106]. Apatite formation on implants is important in achieving a

direct bonding to bone tissue. It was shown that a Ti surface which was chemically

treated with a H

solution containing tantalum chloride had the ability to form a

HA layer in simulated body fluid (SBF) that had an inorganic ion composition sim-

ilar to human blood plasma [8-107]. A CpTi cylinder treated with a H

solution

with tantalum chloride was inserted into a hole in a rabbit’s tibia for 16 weeks.

It was found that (i) 8 weeks after surgery, the shearing force of the treated Ti

implanted in the 4.2 mm hole was significantly higher than that of the non-treated

Ti, although the surface roughness was not changed after the treatments, and (ii) the

shearing force was higher for the implanted sample in the 4.0 mm hole than that in

the 4.2 mm hole [8-107].

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