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Study on Wear Reduction Mechanisms of Artificial Cartilage by

Synergistic Protein Boundary Film Formation∗

Kazuhiro NAKASHIMA∗∗, Yoshinori SAWAE∗∗ and Teruo MURAKAMI∗∗

Poly(vinyl alcohol) (PVA) hydrogel is one of the anticipated materials for artificial car-tilage. PVA hydrogel has high water content and a low elastic modulus similar to naturalcartilage, but its major disadvantage is its lower strength. PVA hydrogel experienced rapidwear under severe conditions such as mixed or boundary lubrication. Therefore, the existenceof a protective surface film with low friction becomes important to prevent surface failure.In this study, the reciprocating frictional tests for a sliding pair of PVA hydrogel and glassplate were carried out, and fluorescent observations were performed to identify the roles ofadsorbed protein film. Albumin and γ-globulin, which are contained in natural synovial fluid,were used by mixing into the lubricant. It appears that groups of albumin molecules adsorbon the smooth γ-globulin adsorbed layer at content of 2.1 wt% of proteins with an appropriateratio. But in the case of a lubricant which has excessive protein at 2.8 wt%, albumin and γ-globulin adsorbed separately. Considering the wear reduction at 2.1 wt% content of protein,albumin and γ-globulin constituted synergistic adsorbed film for wear reduction. It is indi-cated that albumin constructs a low shear layer and γ-globulin forms a layer protecting PVAhydrogel from wear. It is considered that wear and friction of PVA hydrogel were reduced dueto slip of the boundary of adsorbed albumin and γ-globulin layer. Content of protein and ratioof albumin to γ-globulin (AG ratio) are important to constitute the appropriate protein film.

Key Words: Biomechanics, Biotribology, Lubrication, Wear, Artificial Cartilage,Poly(vinyl alcohol) Hydrogel, Wear Reduction, Protein Film, ProteinAdsorption

1. Introduction

For many years joint prostheses were employed torevive daily motional activities of patients with severearthritis and to release their pain. In not few cases,however, patients required revisional treatments due toproblems derived from the deterioration of implants. Itis said that most problems of artificial joints occur dueto tribological occasions. The common rubbing materi-als that compose joint prostheses are ultra high molec-ular weight polyethylene (UHMWPE) and corrosion-resistant alloys or bioceramics. The tribological prob-lems of joint prostheses appear to be caused by wear ofUHMWPE(1) – (3). Macrophages phagocytize the wear par-ticles of UHMWPE, but their unusual reactions attack the

∗ Received 30th May, 2005 (No. 05-4050)∗∗ Department of Intelligent Machinery and Systems, Fac-

ulty of Engineering, Kyushu University, 744 Motooka,Nishi-ku, Fukuoka 819–0385, Japan.E-mail: nakaji@mech.kyushu-u.ac.jp

natural living tissues and lead to osteolysis or the loos-ening(4). Therefore the wear of UHMWPE must be re-duced for the implants to function for a longer period.Much research has been carried out to protect UHMWPEfrom wear(5). However, Dowson et al.(6) suggested that thecushion-form bearings for joint implants would be able tochange the mode of lubrication fundamentally, not to en-hance the wear resistance of UHMWPE. By using the softmaterials like articular cartilage as rubbing surfaces, thesoft EHL (elastohydrodynamic lubrication) effect is ex-pected and sufficient fluid film will be formed so that themode of lubrication will become fluid film lubrication.

Poly(vinyl alcohol) (PVA) hydrogel is one of the an-ticipated materials for artificial cartilage. The final goal ofthis study is to apply PVA hydrogel to the rubbing mate-rial of joint prosthesis. PVA hydrogel has high water con-tent and a low elastic modulus similar to natural cartilage.Under normal walking conditions in a knee joint simula-tor, PVA hydrogel showed low friction(7), (8). Thus, PVAhydrogel is expected to be usable as a frictional material

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under steady state. However, in daily use, artificial jointsundergo not only steady state but also severe conditionssuch as extended standing or running. Therefore, the ex-tent of the wear of PVA hydrogel under severe conditionsmust be investigated before being employed in artificialcartilage as clinical use.

The evaluation of the wear of PVA hydrogel in the se-vere conditions such as the mixed or thin film lubricationis important to use as artificial cartilage. The wear prop-erty of PVA hydrogel was evaluated in a previous study(9).PVA hydrogel experienced rapid wear under severe condi-tions such as mixed or boundary lubrication. However,application of some lubricants with appropriate contentof proteins at 2.1 wt%, i.e., 2/1 or 1/2 ratio of albuminand γ-globulin (AG ratio) which are contained in natu-ral synovial fluid, reduced the wear of PVA hydrogel(9).Proteins adsorbed on PVA hydrogel as a boundary filmcontributed to reduce friction and protect the rubbing sur-face from wear. Although some lubricants with proteins at2.1 wt% showed low wear, other lubricants with proteinsshowed severe wear. Especially total content of proteins at2.8 wt% showed destructive severe wear. It is consideredthat the formation of protein film is different in each condi-tion. Therefore, protein films on glass plate after recipro-cating test were observed using atomic force microscopy(AFM)(9). Glass plates were used as a hydrophilic substi-tute for PVA hydrogel because of the difficulty of observ-ing PVA hydrogel with AFM. The observation was carriedout after washing in which water was dropped on the glassplate fixed at an inclination of 45 degrees. The AFM im-ages showed that disparities of surface film constituentsexisted among different types of proteins. Although al-bumin adsorbed as cohesion film, γ-globulin covered theglass plate entirely. Furthermore, the adsorption amountof albumin was smaller than that of γ-globulin. It is sup-posed that albumin is easily washed off by water, resultingin its weaker adsorption strength compared to that of γ-globulin. Adsorbed layer of γ-globulin will protect PVAhydrogel from wear and that of albumin will play a role inmaintaining low shear. The specific structures of proteinboundary film at 2.1 wt% responsible for reduced wearand friction are unidentified.

Considering the above-mentioned facts, it is neces-sary to discriminate the types of proteins and to distinguishtheir roles in friction and wear. AFM images can discrim-inate height and surface profile, but they can distinguishneither the type of proteins nor the adsorption state of pro-teins. Therefore, reciprocating tests and fluorescent obser-vation were performed to identify the roles of the proteins.

2. Experimental Method

2. 1 PVA hydrogelPVA hydrogel was prepared by freezing-thawing

cyclic processing(7), (10). Poly(vinyl alcohol) (polymeriza-

tion degree 2 000, Kishida Chemical Co., Ltd.) was dis-solved in boiling water at 20 wt% and then the PVA solu-tion was cast into a sheet 2 mm in thickness. The mold wasfrozen at −20 degree centigrade for 10 hours and thawedat 3 degree centigrade for 20 hours. The freezing-thawingcycle was repeated for 5 times. The elastic modulus ofPVA hydrogel was 1.2 MPa and equivalent water contentwas 79 wt%, similar to natural cartilage.

2. 2 LubricantsThe applied lubricants are shown in Table 1 corre-

sponding to Fig. 1. Bovine serum albumin (Wako PureChemical Industries, Ltd.) and human serum γ-globulin(Wako Pure Chemical Industries, Ltd.) were mixed inhyaluronic acid (molecular weight 9.2×105, supplied fromSeikagaku Co., Ltd.) solution of 0.5 wt% as lubricants.The content of proteins was selected from the correspond-ing values in the previous wear tests as shown in Fig. 1(9),where higher value in wear grade indicates higher wear.Albumin and γ-globulin were dyed by rhodamine B isoth-iocyanate (RITC) (Sigma chemical Co.) and fluorescein(FITC) (Sigma chemical Co.) respectively before beingmixed into the lubricant.

2. 3 Reciprocating frictional tests and observationof fluorescent images

The reciprocating frictional tests were carried out in

Table 1 Protein content in lubricants

Fig. 1 Selection of lubricants A to F corresponding to previouswear tests for PVA hydrogel against itself

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Fig. 2 Schematic of reciprocating tester

Fig. 3 Fixed glass plate after reciprocating test and distilledwater dropped on center of glass plate

a reciprocating tester as shown in Fig. 2. The sliding sur-faces are composed of PVA hydrogel and a glass plate.PVA hydrogel is the stationary upper specimen of ellipti-cal geometry with diameters of 25 mm and 40 mm, and theglass plate is the reciprocating lower one. The glass plateswere used as hydrophilic substitutes for PVA hydrogel be-cause of the difficulty of observing PVA hydrogel. Thefrictional test conditions were chosen from the previousstudy(9) and the frictional conditions realized the thin filmor the mixed lubrication to evaluate the properties of pro-tein film adsorbed on rubbing surfaces in severe conditionas follows;load: 2.94 N, sliding speed: 20 mm/s, sliding distance:3 km.

After the reciprocating test, the glass plate was fixedon the holder with an inclination of 45 degrees, as shownin Fig. 3. Next, 50 mL of water was dropped on the cen-ter of the glass plate to remove the superfluous lubricant,hyaluronate and proteins in the lubricant from remainingfilm on the glass plate. Then the lower specimen was driedout in vacuum oven for 1 hour. Water not only washes off

Fig. 4 Example of profile of glass plate experiencedreciprocating test with lubricant A

Fig. 5 Fluorescent images with lubricant A (left to right:surface, middle and deep layers)

Fig. 6 Fluorescent images with lubricant B (left to right:surface, middle and deep layers)

superfluous constituents on a glass plate but also changesthe thickness of the remaining film as shown in Fig. 4.Therefore, the internal structure of protein film could beobserved along the long axis of lower specimens. Fluores-cent images in each condition were taken at three pointsin thickness, i.e. surface, middle and deep layers as shownin Fig. 4. The fluorescent images were acquired by a flu-orescent microscope (OLYMPUS IX71, OLYMPUS Co.,Ltd.).

3. Results

The fluorescent images tested with lubricants A andB are shown in Figs. 5 and 6. The images in lubricant Athat contains only albumin showed adsorptive structure ascohesive form. But the adsorbed film was worn off par-tially in the middle layer. In the deep layer the glass platesurface appeared as a result of the removal of adsorbed al-bumin molecules. In the case of lubricant B, the glass platewas covered with γ-globulin in each layer. Especially the

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Fig. 7 Fluorescent images with lubricant C (top to bottom:surface, middle and deep layers, left to right: albumin,γ-globulin and superposition images)

middle and deep layers showed smooth and uniform struc-ture. Therefore it was confirmed that the strength of ad-sorption of γ-globulin is stronger than that of albumin.

Figure 7 shows images in lubricant C which con-tains mixed proteins at 2/1 of AG ratio at 2.1 wt%. Itappears that groups of albumin molecules adsorb on thesmooth γ-globulin adsorbed layer. This condition resultedin lower wear of PVA hydrogel(9). As shown in Fig. 8, sim-ilar structures composed of albumin and γ-globulin layerswere observed with lubricant D at 1/2 of AG ratio. Thecohesive form of albumin was observed in the middle anddeep layers and larger than a case of lubricant C. In theseconditions that showed low wear, albumin and γ-globulincooperated to make low wear by forming layered struc-ture.

Figure 9 shows fluorescent images experienced withlubricant E. There were some disparities in lubricant Ecompared with lubricants C and D. γ-globulin adsorbedpartially in the surface layer, but uniformly in the middlelayer. Protein films were scarcely detected as adsorbedfilms in the deep layer.

Test with lubricant F at 1/1 of AG ratio at 2.8 wt%especially resulted in singular protein film as shown inFig. 10. Albumin and γ-globulin absorbed separately inboth surface and middle layers. And γ-globulin adsorbedthickly in deep layer. This lubricants was showed signifi-cant wear.

4. Discussions

Murakami et al.(11) suggested that the adaptive mul-timode lubrication mechanism facilitates both low fric-

Fig. 8 Fluorescent images with lubricant D (top to bottom:surface, middle and deep layers, left to right: albumin,γ-globulin and superposition images)

Fig. 9 Fluorescent images with lubricant E (top to bottom:surface, middle and deep layers, left to right: albumin,γ-globulin and superposition images)

tion and low wear in natural synovial joints. In natu-ral joints, various supplemental lubrication mechanismssuch as weeping, boundary and gel-film lubrication modesseem to protect articular cartilage even under severe thinfilm conditions with local contacts. Therefore, artificialcartilage materials that have not only a low elastic mod-ulus, but also high water content are suitable as rubbing

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materials for artificial joints to mimic the excellent lubri-cation mechanism of natural joints. And it is indicated thatadsorbed film of proteins is effective in reducing frictionand wear in natural joints.

Gu et al.(12) investigated the mechanical propertiesof PVA hydrogel prepared by the process of repeatedfreezing-thawing. They concluded that PVA hydrogel is apromising material for artificial cartilage. Oka et al.(13), (14)

investigated the biocompatibility of PVA hydrogel by im-

Fig. 10 Fluorescent images with lubricant F (top to bottom:surface, middle and deep layers, left to right: albumin,γ-globulin and superposition images)

(a) Lubricant A (b) Lubricant B

(c) Lubricant C (d) Lubricant F

Fig. 11 AFM images in previous study(9) (left to right: middle layer and deep layer)

planting PVA hydrogel into the knees of Japanese whiterabbits. They reported that there was no inflammatory re-action even in implantation for 52 weeks, whereas slight tomild inflammation was evident in UHMWPE groups(14).As they used PVA hydrogel that has low content of water,it is supposed that weeping lubrication will be hard to ex-hibit. It is considered that PVA hydrogel with high contentof water in our study has advantages in lubrication. How-ever, because PVA hydrogel with high water content has aweak point as lower strength, a protective surface film withlow friction becomes important to prevent surface failure.

In this study, the glass plates were used as hydrophilicsubstitutes for PVA hydrogel. For clinical use, the pairof PVA hydrogel and the clinical bionic ceramics such asalmina or zirconia ceramics as the rubbing surface of jointprosthesis is probable.

Experimental results in this study indicated that pro-tein films have two roles. The first role is to maintain lowshear layer of albumin and the second is protection fromwear by adsorbed film of γ-globulin. If lubricant has onetype of protein alone or excessive content of protein, wearreduction is little expected due to adverse adhesive effectof protein between rubbing surfaces. The conformation ofeach protein will be different in adsorption on rubbing sur-face. The difference of adsorptive strength is considered tobe caused by conformational changes of proteins. Ishiharaet al.(15) and Tanaka et al.(16), (17) suggested that free waterfraction on the polymer surface makes protein reversibleadsorption. Spencer et al.(18), (19) indicated the efficacy ofalbumin boundary film for friction. They pointed out theimportance of surface hydrophilicity and albumin of na-tive conformation on the lubricating ability. Albumin will

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(a) (b) (c)

Fig. 12 Adsorbed protein model observed in this study (a) layered protein film experiencedin lubricant C and D that has an effect of reducing wear, (b) protein film that reducedwear little, (c) increasing wear due to addition of excessive proteins to lubricant

contact the surface reversibly in such as cohesion form,and γ-globulin will adsorb with significant conformationalchange. Therefore, albumin constructs a low shear layerand γ-globulin creates a protective layer from wear.

Figure 11 shows AFM images in the previousstudy(9). In the deep layer, the adsorbed albuminmolecules exist separately on the glass surface in lubri-cant A, but the uniform γ-globulin film covered the en-tire surface in lubricant B. Lubricant C of minimum wearshows mixture film structure in Fig. 11 (c). Consideringthe fluorescent images in Fig. 7, the cohesive structures inFig. 11 (c) were discovered as cohesion of albumin. It isidentified that forming layer structure of adsorbed proteinfilm is necessary in order to reduce wear of PVA hydrogel.Lubricants with excessive proteins construct a mass of onetype of protein as shown in Figs. 10 and 11 (d), which re-sulted in high wear. Therefore, there is suitable contentor mixed ratio of protein as shown in Fig. 1 to constituteappropriate protein film. It is supposed that synergisticlayer structure was constituted in lubricant C and D. At thesame total content of protein, changes of AG ratio showeddifferent wear and adsorbed film. 2/1 or 1/2 of AG ratioshowed minimal wear but 1/1 resulted in an increase ofwear compared with 2/1 or 1/2. The experimental resultsindicated that protein ratio is important to form an idealadsorbed film. It is considered that protein films consistof plural layers and the adsorptive strength will be differ-ent on rubbing surfaces and on protein layer. If proteinadsorbed film is rubbed, at the boundary of adsorbed albu-min and γ-globulin layer, the slip is likely to occur.

To protect the rubbing surface from wear, it is neces-sary to form synergistic adsorbed film structure of albuminand γ-globulin. Figure 12 shows a model of protein filmbased on experimental results. The boundary film withwear reduction in lubricants C and D forms an optimumlayered structure composed of albumin and γ-globulin(Fig. 12 (a)) as a result of repeated rubbing process in so-lution containing an appropriate content of both proteins.

But boundary film with wear increase constructs sepa-rately adsorbed protein structure as shown in Fig. 12 (c).Figure 12 (b) corresponds to lubricant E that had wear re-duction little.

Protein adsorption depends on hydrophilicity and freewater fraction on surface(15) – (17). It is essential for longendurance of compliant artificial cartilage to form accom-modating boundary film of proteins. The influence of freewater and hydrophilicity of PVA hydrogel on adsorptionwill be evaluated in the next study.

The protein content resulted in the least wear of PVAhydrogel is rich compared with natural synovial fluid.Therefore it is required to adjust the content of proteinsby treatments such as injections. But it is reported that thecontent of proteins in synovial fluid is enhanced after thejoint surgery(20). The appropriate formations of adsorbedprotein film on rubbing surfaces is expected by a sophis-ticated self-organization mechanism assisted by rubbingaction in natural synovial fluid.

5. Conclusions

The frictional test of PVA hydrogel as artificial carti-lage were conducted using hyaluronate solutions contain-ing different protein constituents and the protein bound-ary film was observed by fluorescent microscopy. Pro-teins of albumin and γ-globulin have different roles. It isindicated that albumin constructs low shear layer and γ-globulin layer protect PVA hydrogel from wear. It is im-portant for actualization of low wear to form appropriatelayer structure of albumin and γ-globulin.

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