The use of Raman spectroscopy in the analysis of UHMWPE uni-condylar bearing systems after run on a force and displacement control knee simulators

Wear 297 (2013) 781–790

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The use of Raman spectroscopy in the analysis of UHMWPE uni-condylar bearing systems after run on a force and displacement control knee simulators Saverio Affatato a,n, Enrico Modena b, Simone Carmignato c, Paola Taddei b a

Laboratorio di Tecnologia Medica, Istituto Ortopedico Rizzoli, Via di Barbiano, 1/10, Bologna, 40136, Italy Dipartimento di Biochimica ‘‘G. Moruzzi’’, Sezione di Chimica e Propedeutica Biochimica, Universita di Bologna, Bologna, Italy c DTG, Padova University, Padova, Italy b

a r t i c l e i n f o

abstract

Article history: Received 28 October 2011 Received in revised form 17 August 2012 Accepted 15 October 2012 Available online 29 October 2012

The complications associated with prosthetic failure and revision surgery still constitute the main clinical problem. The objective of wear evaluation is to determine the wear rate and its dependence on the test conditions. To obtain realistic results, a wear test can be performed to reproduce in vivo working conditions and compare the wear characteristics of various total knee prostheses designs. Two knee wear simulators with different input control mechanisms, displacement and force controlled, were used to assess the wear behavior of ultra-high molecular weight polyethylene uni-condylar knee prostheses. The differences in wear behavior were assessed using a state-of-art coordinate measuring machine; Raman spectroscopy was used to evaluate the possible crystallinity changes on the uni-condylar menisci induced by mechanical stress. These results were compared with available uni-condylar retrievals. Scratches were visible along the anterior-posterior direction emphasizing that the motion was constant during the movements under the displacement control simulation. On the contrary, different kinematics schemes were observed under the force controlled simulation. The structural Raman markers showed a good correlation with the coordinate measuring machine data, and in particular with the depth of the concavity formed upon in vitro testing or in vivo service. With regards to the in vitro tested components, the specimens tested under force controlled simulator, which underwent a higher volumetric loss, showed a higher increase of the amorphous content. At a molecular level, the wear mechanism did not appear significantly different for the three sets of specimens, with the exception of the amorphous content which, upon wear, increased in the in vitro tested components, while decreased in the retrievals. & 2012 Elsevier B.V. All rights reserved.

Keywords: UKP DC simulator FC simulator Knee retrievals Raman spectroscopy Coordinate measuring machine

1. Introduction The unicompartmental knee arthroplasty (UKA) represents an alternative approach to the total knee osteotomy for patients with a localized tibio-femoral non-inflammatory disease (localized osteoarthritis). UKA is a minimal invasive surgical operation in which only one compartment (medial or lateral) is replaced, resulting in a wider preservation of healthy tissue and bone, a reduction of surgical time, a better range of motion and an improved patient’s life quality [1–5]. It has been accepted that in vitro wear tests are necessary to know the performance of the biomaterials used in the orthopedic field; however, while procedures for testing total knee replacements

n

Corresponding author. Tel.: þ39 051 6366864; fax: þ39 051 6366863. E-mail address: [email protected] (S. Affatato).

0043-1648/$ - see front matter & 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.wear.2012.10.002

(TKR) are consolidated (ISO 14243) [6,7], there is no indication for pre-clinical assessment of unicompartmental knee prosthesis (UKP). Some in vitro protocols have been developed to test UKP using a total knee wear simulator to test contemporary such bi-unicondylar prostheses [8,9]. Another ‘complication’ in testing uni- or total knee prostheses is the knee simulator used. Currently, two philosophies are adopted in knee simulation: in some simulators the force (FC) is controlled, in others the displacement (DC). In both, level walking is the sole activity of daily living that is represented for testing. Four degrees of freedom of knee joint motion are actively controlled while the remaining two are free to move passively. The difference between the two simulator concepts lies within the control mode of anterior-posterior movement (AP) and internal–external rotation (IE). Using the FC strategy, the power control employs the force generated at the interface between the tibial and femoral components, because there is no continuous linear relationship between the control output (displacement and/or velocity) and the controlled

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parameter (force). The motion of the tibia, relative to the femur, resulting from the force on the tibia is complex and sensitive to small variations in force. Conversely, with DC strategy, the AP displacement and IE rotation are close-loop controlled by displacement feedback and closely follow the displacement command profile. Contact kinematics has been identified as the dominant factor affecting ultra-high molecular weight polyethylene (UHMWPE) wear in TKR [8,9], but often, contradictory results have been reported in the literature as far as UHMWPE wear is concerned when FC or DC simulator is used [10,11]. In the present study, the wear behavior of UHMWPE menisci tested under DC and FC conditions was assessed to find a correlation between microstructural changes and macroscopic wear/deformation. The former were investigated by microRaman spectroscopy, the latter by using a coordinate measuring machine (CMM). In particular, CMM allowed to assess the volumetric loss of the specimens, while micro-Raman spectroscopy was used to investigate on a micro-scale the possible changes in crystallinity induced by mechanical stress. Micro-Raman spectroscopy is used to investigate on a micro-scale the possible changes in crystallinity induced by mechanical stress. Actually, it is well known that wear is preceded and accompanied by microstructural changes in the polymer. Edidin et al. [12] have suggested that the mechanical loading conditions at the articulating surface may induce the development of a plasticity-induced damage layer (characterized by a high level of crystalline lamellae orientation), precursor to wear and wear debris. The Raman spectrum of PE has been largely investigated and various authors have tried to correlate the spectral features with the crystallinity of the polymer to obtain information on chemical and structural degradation in the polyethylene structure [13–15]. The results of these analyses were compared with the results obtained from available UKP retrievals. In summary, our study was aimed:  at assessing, using a metrological CMM-based procedure, the damage patterns of commercially available, non-congruous UKP design tested using two different simulators design (DC and FC simulation).  at evaluating, using the Raman spectroscopy, the possible crystallinity changes on the UHMWPE menisci induced by mechanical stress. Therefore, the correlation between structural changes at molecular level and macroscopic wear represents the novelty of this study.

symmetric commercial UHMWPE tibial inserts, size maxi (EFDIOS, Citieffe srl, Italy) were tested. These menisci (hereinafter called DC–PE) were EtO sterilized and tested on a DC ‘‘three-plus-one’’ knee joint simulator (Shore Western Mfg., Monrovia, CA, USA) in conjunction with six lateral and six medial CoCr alloy femoral size large components (Citieffe srl, Italy). Alignments and load components (axial load, anterior/ posterior translation, intra/extra-rotation, flexion/extension) replicated a simplified gait cycle according to ISO 14243-3. In particular, load was applied vertically (perpendicular to the tibial tray), oscillating between 168 and 2600 N following a physiological profile. The applied kinematics was in displacement control for the flexion/extension angle oscillating between 01 (neutral) and 581 (flexion) synchronously with the load; for the anterior/ posterior translation oscillating between 0.0 mm (neutral) and 5.2 mm (posterior); and for the intra/extra-rotation oscillating between 21.91 (extra-rotation) and 5.71 (intra-rotation). The implants were changed periodically between the three running stations in order to reduce eventual different load conditions between the stations. Major details are available in literature [17]. During the second phase, six medial and six lateral commercial symmetric commercial UHMWPE tibial inserts, size maxi (EFDIOS, Citieffe srl, Italy) were tested. These menisci (hereinafter called FC–PE) were EtO sterilized and tested on a force-controlled knee joint wear simulator (Model KC Instron/Stanmore) in conjunction with six lateral and six medial CoCr alloy femoral size large components (Citieffe srl, Italy). This kind of simulator replicates a simplified gait cycle according to ISO 14243-1; it allows the sixdegrees-of-freedom articulation of the prosthesis according to the geometric constraints of the individual articular surface design. It utilizes pneumatic force actuators to simulate knee kinematics. A holding frame is mounted on a fixed flexion axis with a specimen chamber mounted below. The holding frame hosts the femoral components, and the chamber is mounted on the end of an actuator and contains tibial components. The holding frame rotates on the tibia at a flexion angle up to 581. An axial force is applied in a sinusoidal form with a maximum of approximately 2600 N. The A/P forces span from 2170 to 110 N, with I/E torque controlled from 21 to 6 Nm. The machine was run at 1 Hz. Major details are available in literature [18]. After testing, all the specimens were washed for 10 min by immersion in an ultrasonic bath filled with a special detergent (Clean 70, Elma GmbH, Germany). After which they were rinsed with distilled water for another 15 min, dried with a jet of filtered inert gas (nitrogen), and further dried in a vacuum drying system for 30 min, prior to Raman and CMM analyses. 2.1. Retrieval knee unicompartimental prostheses

2. Materials and methods In this study, two wear tests were performed; two groups of unicompartmental knee prostheses having the same design, shape, size, and the same metallic holder fixation were evaluated for two millions cycles using two different knee simulators. All the polyethylene inserts were UHMWPE (ASTM F-648, ISO 5834-1-2), GUR1050 derived from ram extrusion. The simulations were conducted in a physiological environment maintained at 3772 1C with 25% (v/v) sterile bovine calf serum lubrication and 0.2% sodium azide to eliminate bacterial growth. Following a standardized protocol [16], all the UHMWPE menisci inserts were pre-soaked in distilled water for four weeks prior to the wear tests in order to achieve a steady level of fluid sorption as recommended by the international standard (ISO 14243/2). The tests were performed in two different phases: in particular, during the first phase, six medial and six lateral commercial

Due to the unavailability of EFDIOS retrievals, some available UKP retrievals similar to those in vitro tested in shape and design were taken into account. Retrievals consisted of UHMWPE menisci (hereinafter called EX–PE) inserts, and obtained from six patients who underwent revision at the Rizzoli Orthopaedic Institute, Bologna, Italy, from 2000 to 2010. The retrievals belonged to two female and four male patients with a median age of 71 years (range: from 55 to 82) at the time of the revision surgery; they were implanted in the medial compartment during knee arthroplasty and later explanted 1.3 to 4 years after implantation (mean 2.8 years). Reasons for revision included pain and polyethylene wear. 2.2. Wear damage and CMM assessment Volumetric wear of the menisci was calculated using a coordinate measuring machine (CMM, ZEISS Prismo VAST 7, Germany).

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The CMM is equipped with a scanning probe head, capable of measuring a high point density with high accuracy. The maximum scanning probing error of the CMM was tested to be below 1 mm, according to ISO 10360-4:2000. The CMM was in a temperature and humidity controlled room; the measured specimens were left in the room before running the CMM measurements for a sufficient time, so that they stabilized at 20 70.1 1C. All surfaces were measured with respect to a reference CAD datum aligned on the unloaded areas of the specimens. The comparison between measured points and reference datum was performed using a dedicated elaboration algorithm implemented in a three dimensional-data modeling and evaluation software (PolyWorks, InnovMetric Software Inc., Canada). The evaluation software was used in order to obtain: (i) the deviation maps associated to the worn surfaces with respect to the datum, and, (ii) consequently calculate the volume of material worn away. 2.3. Micro-Raman crystallinity measurements Micro-Raman spectra were obtained using a Jasco NRS-2000C instrument with a microscope of 100  magnification. All the spectra were recorded in back-scattering conditions with 5 cm  1 spectral resolution using the 514 nm line (Innova Coherent 90) with a power of ca. 20 mW. The detector was a 160 K cooled digital CCD from Princeton Inc. A confocal pinhole with an aperture diameter of 200 mm was placed in the optical circuit to obtain signals from a limited in-depth region. All the Raman measurements were made in a fully non-destructive way, without any sample manipulation. Under these experimental conditions, the laser spot size was about 1 mm; in a previous paper [19] the field depth for our confocal setup using the 50 mm-pinhole has been calculated as about 5 mm. Therefore, it can be concluded, according to Zerbi et al. [36] that no deep inside of UHMWPE significantly contributes to the Raman signal. Therefore, the data obtained from the Raman spectra can be considered really corresponding to the surface of the samples. Two components of each group (i.e., the most representative after CMM measurements) were analysed only in their upper surface, in the most worn area. The unworn border of a tested component was measured as reference for FC–PE and DC–PE groups. With regards to the EX–PE, a new unworn Link prosthesis was analyzed as control for the corresponding retrieval (EX–PE2); with regards to the EX–PE1 retrieval, its unworn border was analyzed as reference, due to the unavailability of a new unworn component. On each area of each component 15 spectra at least were recorded. UHMWPE can be described, at a first approximation, as a composite of three different phases: a crystalline phase, a meltlike amorphous one (with a prevailing gauche conformation of the chains), and an anisotropic disordered phase (with a prevailing trans conformation of the chains). The crystalline phase contains chains folded into highly oriented lamellae, with the crystals being primarily orthorhombic in structure (although monoclinic structures have been claimed to coexist in certain conditions, including uniaxial deformation [15]). The fraction of orthorhombic (ao), amorphous (aa) and intermediate anisotropic disordered (ab) phases were calculated from the relative intensities of selected Raman bands, according to Strobl and Hagedorn [20]: A1416 ao ¼ 0:46  A1295 þ 1305

ð1Þ

A1080 0:79  A1295 þ 1305

ð2Þ

aa ¼

ab ¼ 1ðao þ aa Þ

ð3Þ

783

where A1416 and A1080 are the areas of the Raman bands at 1416 and 1080 cm  1, respectively; A1295 þ 1305 is the area of the internal standard (i.e., independent of chain conformation) band group. The A1080 band area was determined after a curve fitting analysis of the 1040–1105 cm  1 range by means of a commercial software (Opus 5.0 from Bruker Optik GmbH, Germany). Curve fitting was performed on the original spectra after baseline correction, using the Levenberg–Marquardt algorithm. The Raman components were described as linear combinations of Gaussian and Lorentzian functions. The full-width at half maximum (FWHM) of the bands at 1130 and 1060 cm  1 was calculated to evaluate residual strain in polyethylene, according to previous studies [14,21]. The A1130/ A1060 band area ratio has been used to evaluate the occurrence of orientation upon wear testing, according to a method proposed by Pigeon et al. [22]; if the molecules are oriented in a preferred direction, the 1130 cm  1 band has been reported to become stronger with respect to the 1060 cm  1 band. The A1060 band area has been determined by curve fitting, as above described. To evaluate the occurrence of an orthorhombic to monoclinic phase transformation, the I1416/(I1440 þI1460) and I1416/I1295 intensity ratios were calculated according to previous studies [23–25]; I1416, I1440, I1460 and I1295 were calculated as peak heights.

3. Results In spite of the differences between the overall damage patterns in both UKP simulations and the retrievals (Fig. 1), there were notable similarities for the observed main damage. In particular, for DC–PE it is possible to observe (Fig. 1c) that kinematics motion follows a constant scheme. Scratches were visible along the AP direction and emphasize that the motion was constant during the movements. On the contrary, for the FC–PE it is possible to observe (Fig. 1d) that kinematics motion follows different schemes. In particular, since the load is applied in a constant direction, probably the AP translation was not coincident with changes in IE rotation producing different scratches on the UHMWPE menisci components. The retrieved specimens showed a greater variety of damage modes in the focal scars, especially compared to the DC–PE. 3.1. CMM measurements The volumetric mass loss measured using the CMM showed a wide range of values: from 68 mm3 for the DC–PE, to 110 and 150 mm3 for the EX and FC–PE, respectively. Fig. 2 shows the CMM pictures of two components of each analyzed group. A dispersed damage scar pattern dominated by abrasive modes was observed in the FC–PE and EX–PE. Fig. 3 (volume vs. depth) shows the trend of the volumetric loss as a function of the depth of the concavity formed upon in vitro testing or in vivo service, as obtained from CMM measurements. The DC–PE and FC–PE appear well distinct each other; the EX–PE appears constituted by two sub-groups that differ for the value of the concavity depth. Two of them appear ‘‘outliers’’, while the rest of the components fall within a distribution characterized by a linear association between wear volume and penetration depth. 3.2. Micro-Raman crystallinity measurements The Raman spectrum of UHMWPE, being sensitive to the polymer crystalline state [13–15,26] allows to detect variations in phase composition due to mechanically induced wear. Two components of each set were analyzed; they were chosen as the most representative at CMM measurements (Fig. 3). As an example, Fig. 4 shows the Raman spectra recorded on the unworn

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Fig. 1. Visual examination of one component of each analysed group. A new UKP is shown for comparison.

control and worn areas of an FC–PE component (FC–PE1). As can be easily seen, the bands prevalently due to crystalline PE at 1416, 1130 cm  1 (and to a lower extent the component at 1060 cm  1) are stronger in the worn area; the bands prevalently due to amorphous PE at about 1460, 1440, 1305 and 1080 cm  1 showed an analogous behavior, being significantly higher in the same area. From a qualitative point of view, it is possible to affirm that mechanical stress has altered PE crystallinity of the analysed components. From a quantitative point of view, the fractions of orthorhombic (ao), amorphous (aa) and intermediate anisotropic disordered (ab) phases have been determined for all the tested components and their values are reported in Fig. 5 together with the other calculated spectroscopic markers. The values of the FWHM of the bands at 1130 and 1060 cm  1 have not been reported since they did not undergo any significant changes upon wear testing. The data reported in Fig. 5 shows that upon wear testing of both DC and FC groups, the fraction of orthorhombic (ao) content significantly increased, while the intermediate anisotropic disordered (ab) phase decreased; the amorphous content (aa) increased to different extents in all the components with the exception of DC–PE1. The increase of both the I1416/(I1440 þI1460) and I1416/I1295 ratios reflected the increase in the orthorhombic content; in particular, the increase of the I1416/(I1440 þI1460) ratio showed that the orthorhombic content increased at a meanly higher rate than the amorphous content. As reported above, the 1130 cm  1 band increased more strongly than the 1060 cm  1 component upon wear testing (see Fig. 4); from a quantitative point of view, this behavior was reflected in the increase of the A1130/A1060 ratio (Fig. 5). Although the changes induced by wear testing on this spectral marker did not appear significant, its general increase suggested the occurrence of orientation upon wear. The most significant difference between the FC–PE and DC–PE concerned the amorphous content (aa), which increased more significantly in the FC–PE. Fig. 6 reports the values of the selected spectroscopic markers obtained from the spectra recorded on the control and worn areas

of the retrievals. The two retrievals showed a similar behavior upon in vivo wear, although EX-2 underwent more significant changes than EX–PE1 in all the spectroscopic markers, in agreement with its higher CMM volumetric loss (85 mm3 vs. 69 mm3). On the other hand, EX–PE1 underwent a significant increase in the FWHM of both the 1060 and 1130 cm  1 bands (Fig. 7). On average, if compared with the DC–PE and FC–PE components, the retrievals showed a different trend for the amorphous content upon wear: the latter displayed a general decrease in aa (Fig. 6), the former an average increase (Fig. 5). Fig. 8 reports the trend of the main Raman spectroscopic markers recorded on the analysed specimens as a function of the depth of the concavity formed upon wear, as calculated from CMM measurements. As can be easily seen, all the spectroscopic markers (with the exception of aa) showed a fairly good correlation.

4. Discussion Pre-clinical testing is important for the accurate assessment of wear, in order to improve UKP functionality. In this study we asked whether commercially available non-congruous UKP design tested on two different simulators design (DC and FC simulation) would generate a similar wear pattern. Volumetric mass loss was assessed using a metrological CMM-based procedure while Raman spectroscopy was used to measure the possible crystallinity changes on the UHMWPE menisci induced by mechanical stress. As a novelty of the present study, the coupling of these two techniques allowed to correlate macroscopic wear/deformation to structural changes at a molecular level. Even if UKP has been implanted since the 1990s and represents a valid alternative procedure to TKR, this surgical technique is utilized much less frequently than TKR and, luckily, has provided few retrievals. Therefore, this study is definitely limited by the scarce availability of UKP retrievals.

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Fig. 2. CMM characterisation of two components of each analysed group (i.e., the same analysed by micro-Raman spectroscopy).

Both the DC and FC simulators completed the planned millions cycles, as reported previously [17,18]; the DC- and FC-tested UKP specimens and a subset of EX–PE bearings showed different damage patterns. In the DC-Simulation, kinematics motion schemes were uniformly consistent with combined sliding and rolling over the entire gait cycle from terminal stance through swing-phase. It is likely that such kinematics differences contributed to the vastly different wear rates reported for the same materials and designs. Using DC strategy, Affatato et al. [17] reported medial and lateral wear rates of 0.6 mg/Mc for UKP simulation of the same monocompartmental design evaluated in the current study. On the contrary, using FC strategy and the same UKP design, Spinelli, et al. [18] reported average medial and lateral wear rates of 10.27 71.83 mg/Mc and 4.4970.53 mg/Mc, respectively.

In our knowledge no report are available to compare displacement or force control simulation. Other authors compared fixed vs. mobile unicondylar knee specimens reporting different wear behavior. In particular, Kretzer et al. [27] have compared the in vitro wear behaviour of fixed and mobile unicondylar bearing designs and have reported significantly higher wear rates for the latter. Laurent et al. [28] compared medial vs. lateral compartments founding substantial differences: the medial compartment worn more than the lateral one. The volumetric mass loss measured using the CMM machine showed a wide range of values: from 68 mm3 for the DC–PE, to 110 and 150 mm3 for the EX–PE and FC–PE, respectively (Fig. 2). A dispersed damage pattern dominated by abrasive modes was observed on the FC–PE and on retrieved UKP (consistent with in vivo contact with abrasive bone and/or cement). CMM findings were in agreement with the gravimetric data: actually, the FC–PE

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Fig. 3. Trend of the volumetric loss as a function of the depth of the concavity, as obtained from CMM measurements. The samples analysed by micro-Raman spectroscopy (DC-1, DC-2, FC-1, FC-2, EX-1, EX-2) are indicated. It appears that there are two ‘‘outliers’’, while the rest of the components fall within a distribution characterized by a linear association between wear volume and penetration depth.

Fig. 4. Average Raman spectra recorded on the on the unworn control (black) and worn (red) areas of an FC component (FC-1) (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

underwent a higher average volumetric loss than the DC–PE. The data reported in Fig. 3 show that the DC–PE and FC–PE appear well distinct each other; the EX–PE appears constituted by two subgroups that differ for the value of the concavity depth. In the current study, the damage pattern observed on all simulated UHMWPE UKP menisci evidenced surface deformation; such deformation was also observed on all explanted UKP specimens (Fig. 1). CMM measurements (Fig. 2) confirmed this trend with larger linear penetration and a wider worn area for the FC- and EX–PE. In the DC–PE, the wear tracks are situated in the centre of the tibial insert with a marked area that shows one wear direction; in the FC- and EX–PE, the wear pattern takes a wider zone on the tibial insert and even shows a medio/lateral wear direction. Micro-Raman spectroscopy allowed to gain insights into this subject at a molecular level. At a first rough analysis, Raman measurements showed that the calculated spectroscopic markers, with the exception of the amorphous content (ao), had similar trends in the three analyzed groups (Figs. 5 and 6). This result did

not appear surprising since the components were all analyzed in the deepest area of the concavity formed upon in vitro testing or in vivo service. The orthorhombic content (ao) increased upon either in vitro testing or in vivo service (Figs. 5 and 6); the increase in crystallinity upon wear did not appear unexpected and has been already reported in previous Raman studies [25,29]. This result can be explained according to other studies. Jasty et al. [30] have found that PE surfaces undergo large-strain plastic deformation during in vivo function. Edidin et al. [31] have suggested that the mechanical loading conditions at the articulating surface of PE components may be conducive to the development of a plasticityinduced damage layer, which has been found to be the precursor to wear and wear debris. In UHMWPE components tested under uniaxial and multiaxial loading conditions, the crystalline lamellae reorganize and a region of lamellar orientation near the articulating surface (termed plasticity-induced damage layer) has been observed. The increase in the order of the polymeric chains was confirmed in all the analysed components by the increase of the I1416/(I1440 þI1460) and I1416/I1295 intensity ratios, as well as the general increase of the A1130/A1060 band area ratio (Figs. 5 and 6), which confirmed the occurrence of orientation upon wear. The bands at 1130 and 1060 cm  1 have different vibrational symmetries; if the molecules are oriented in a preferred direction, the 1130 cm  1 band has been reported to become stronger with respect to the 1060 cm  1 band [22], so that the A1130/A1060 band area ratio can be considered a marker of molecular orientation. The bands at 1130 and 1060 cm  1 were analysed also with regards to their FWHM. Actually, both bands (due to C–C symmetric and antisymmetric stretching modes, respectively) have been reported to split under strain into two components, which originates a broadening of the Raman bands. This has been interpreted as being due to a bimodal stress distribution of the C–C bonds in the material [32]. Both tensile [21] and compressive [14] plastic strain have been directly measured in medical grades of UHMWPE by using a direct proportionality relationship between uniaxial strain and the increase in the FWHM of the bands at 1060 and 1130 cm  1. No significant band wavenumber shifts were observed upon wear; the only sample which showed a significant increase in the FWHM of the above mentioned bands was EX–PE1 (Fig. 7). With regards to the I1416/(I1440 þI1460) and I1416/I1295 intensity ratios, they increased in the three sets of samples by effect of mechanical stress (Figs. 5 and 6). In contrast, some previous studies on UHMWPE acetabular cups [15,25] have shown that these ratios decreased upon wear; the latter trend has been explained with the occurrence of a phase transformation of orthorhombic into monoclinic. As above stated, the trend obtained in the present study reflected the increase in the orthorhombic content; this results suggests that a different wear mechanism at molecular scale would occur in UKPs. The intermediate anisotropic phase content (ab) decreased upon either in vitro testing or in vivo service (Figs. 5 and 6). This result did not appear surprising in the light of nature of this phase as well as the above proposed wear mechanism. Several authors to explain that the sum of ao and aa is generally different from 1 have hypothesized the presence of the intermediate anisotropic phase. This ‘‘third phase’’ has been defined as a short-range crystalline structure (i.e., between the amorphous and crystalline phases) [20]. Evidently, according to the mechanism proposed by Edidin et al., the mechanical loading conditions at the articulating surface of the components causes a rearrangement of the lamellae also in the intermediate third phase, resulting in the decrease of its content. The amorphous content (aa) showed a different behavior for the in vitro tested and explanted UKPs. In fact, the DC–PE

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Fig. 5. Values of the main Raman spectroscopic markers (average7 standard deviation) calculated from the spectra recorded on the unworn control (CTRL) and worn areas of the analysed FC-1, FC-2, DC-1 and DC-2 components.

and FC–PE components showed an average increase of aa upon wear (which was particularly evident for the FC–PE1 and DC–PE2 components, Fig. 5), while an opposite result was obtained for the EX–PE1 and EX–PE2 retrievals (Fig. 6). This appeared the most significant difference between the retrievals and the samples tested in vitro. Probably, according to a previous study [25,33], it can be ascribed to the different sterilization method of the two sets of samples: the in vitro tested components were sterilized with EtO, while the retrievals were gamma-irradiated. According to Kyomoto et al. [21], the different trend observed for the in vitro tested and in vivo worn components could be ascribed to differences in the oxidation degree of the components. Actually, these authors have reported that the amorphous content is influenced by oxidation; they have affirmed that the evaluation of the oxidation index by indirectly using the amount of amorphous phase can be a viable choice to non-destructively characterize the degree of oxidation from Raman assessments rather than destructive IR measurements. The latter explanation is not in disagreement with the former, since it is well known that EtO and gamma-irradiated UHMWPE components have a significantly

different oxidation degree [34]. It is interesting to note that the Raman findings here reported about retrievals are in agreement with the results obtained by Medel et al. [35] on retrieved UHMWPE total knee prostheses sterilized by gamma-irradiation. These authors have ascribed the increase in the orthorhombic content as well as the decrease in the amorphous and third phase fractions to the substantial chain scission induced by the high extent of oxidation. This process allows secondary crystallization to start probably both at the crystal interfaces and the amorphous regions. For a deeper analysis, the possible correlation between CMM and micro-Raman data was investigated and the obtained results are reported in Fig. 8. As can be easily seen, all the spectroscopic markers (with the exception of aa) showed a fairly good correlation with the depth of the concavity as obtained by CMM measurements, i.e., with the depth of the area were Raman spectra were taken. The orthorhombic content (ao) as well as the I1416/(I1440 þI1460), I1416/I1295 and A1130/A1060 ratios linearly increased with the depth of the concavity: the EX-2 retrieval, which was characterized by the highest concavity depth, showed

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Fig. 6. Values of the main Raman spectroscopic markers (average 7standard deviation) calculated from the spectra recorded on the control (CTRL) and worn areas of the EX-1 and EX-2 explants.

Fig. 7. Average Raman spectra recorded on the unworn control (black) and worn (red) areas of the EX-1 retrieval (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

the highest values of the ao, I1416/(I1440 þI1460), I1416/I1295 and A1130/A1060 values. Interestingly, the slope of the plots corresponding to ao and A1130/A1060 was nearly the same (about 0.20), suggesting that the increase in the orthorhombic content proceeds at the same rate as molecular orientation. The slope of the plot corresponding to the I1416/(I1440 þI1460) ratio was higher than that for the I1416/I1295 ratio (0.13 vs. 0.08) confirming that the former marker was more sensitive than the latter to structural rearrangements [15,25,33]. The increase of the orthorhombic content (ao) was accompanied by a decrease of the anisotropic intermediate phase (ab), which occurred at nearly the same rate (the slope of the plot was 0.15 vs. 0.20, Fig. 8). With regards to the amorphous content (aa), its correlation with the concavity depth did not appear good as much (Fig. 8); this result reflected the different behavior observed for the in vitro tested and explanted UKPs. In fact, the DC–PE and FC–PE components showed an average increase of aa upon wear (Fig. 5), while an opposite result was obtained for the EX–PE1 and EX–PE2 retrievals (Fig. 6).

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Fig. 8. Trend of the main Raman spectroscopic markers recorded on the analysed specimens as a function of the depth of the concavity formed upon wear, as calculated from CMM measurements.

5. Conclusions The approach described in the current study combined retrieval and experimental methods. Unicompartmental knee prostheses necessitate pre-clinical assessments capable of varying the physiological factors affecting prosthesis performance. As a novelty of the present study, the coupled use of CMM and Raman spectroscopy allowed to disclose a tight correlation between structural changes at a molecular scale and macroscopic wear. Actually, the structural Raman markers showed a good correlation with the CMM data, and in particular with the depth of the concavity formed upon in vitro testing or in vivo service. The retrieval that showed the highest concavity depth (i.e., EX–PE2) underwent the highest changes at molecular level. With regards to the in vitro tested components, the FC–PE, which underwent a higher volumetric loss, showed a higher increase of the amorphous content. At a molecular level, the wear mechanism did not appear significantly different for the three sets of specimens, with the exception of the amorphous content which, upon wear, increased in the in vitro tested components, while decreased in the retrievals, probably due to the differences in the sterilization method. In contrast, the orthorhombic and third phase contents had the same trend in the three sets of samples. The results reported in the present study show that the multivariable approaches (CMM and micro-Raman techniques) proved suitable for the comparative characterization of the wear behavior of in vitro tested components and retrievals.

Acknowledgments The authors acknowledge Mara Zavalloni and Michele Spinelli for their contribution in the initial planning phase and during the experiments. Thanks are also due to Citieffe srl, Bologna Italy that promoted this work. The authors wish to thank again John Desjardins and Melinda Harman for their availability in the use of

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