MD Matthieu Ollivier

PhD, Se ´bastien Parratte

PhD Ludovic Lino

MD Xavier Flecher

MD Jean-Noe ¨l Argenson
email: jean-noel.argenson@ap-hm.fr

No Benefit of Computer-assisted TKA: 10-year Results of a Prospective Randomized Study

Background Previously published studies reported benefits of computer-assisted surgery (CAS) in terms of ra-diographic implant position in TKA, but whether these improvements result in clinically important survival differ-ences or functional differences that a patient might perceive at a minimum 10-year followup remains largely unknown.

Questions/purposes

We performed a prospective randomized trial and asked whether CAS (1) improved survival free from aseptic loosening; and (2) demonstrated any clear dif-ference in patientreported outcomes at latest followup using validated outcome measures at minimum 10-year followup. Methods Between January 2004 and December 2005, 80 patients sched-uled for TKA were randomly assigned either to the CAS group or to the conventional technique group by the Hospital Informatics De-partment. The patient inclusion cri-teria were age 20 to 80 years old, weight < 100 kg, and consent to re-ceive a primary knee arthroplasty performed through a medial parapatellar approach by the senior au-thor. The exclusion criteria were a history of prior knee surgery, TKA performed for a posttraumatic indication, or revision knee surgery. The first 80 patients meeting these criteria were included in the study. There were 21 women and 19 men and in each group; mean age was 66 years (range, 58-77 years), and mean body mass index was 27 6 4 kg/m

. An initial published study using this patient group investigated only dif-ferences regarding implant position-ing in the coronal and sagittal planes. This is a secondary analysis of patients from the earlier study pro-tocol at a minimum of 10year fol-lowup with different endpoints.

Kaplan-Meier survivorship was compared between groups, and functional patient-reported outcome measures (PROMs) were evaluated using the SF-12, Knee Injury and Osteoarthritis Outcome Score (KOOS), Forgotten Joint Score, and the new Knee Society Score. Those PROMs were not available at the time of the original randomized controlled trial and we therefore do not have baseline preoperative values demonstrating that our two groups were comparable. However, our groups were created using strict randomiza-tion and were similar in terms of de-mographic parameters and knee deformities. Our secondary analysis was not powered for survival analysis Each author certifies that neither he, nor any member of his immediate family, has funding or commercial associations (consultancies, stock ownership, equity interest, patent/licensing arrangements, etc) that might pose a conflict of interest in connection with the submitted article. Clinical Orthopaedics and Related Research® neither advocates nor endorses the use of any treatment, drug, or device. Readers are encouraged to always seek additional information, including FDA-approval status, of any drug or device prior to clinical use. Each author certifies that his institution approved for the human protocol for this investigation and that all investigations were conducted in conformity with ethical principles of research. This work was performed at the Institute for Locomotion, Aix-Marseille University, Marseille, France.

C

but had 80% power to detect a dif-ference > 6 points on the SF-12 components and > 6 points out of 100 on the KOOS subscores (pub-lished minimal clinically important difference: 8 points) at the p < 0.05 level.

Results

With the numbers available, there was no difference between the CAS group and the conventional TKA group in terms of survivorship free from aseptic loosening 10 years after TKA (97%, 95% confidence interval [CI], 95%-99% versus 97%, 95% CI, 95%-99%; p = 0.98). Investigation of the latest followup PROM scores showed no difference between SF-12 scores (respectively, for CAS and control patients, physical SF-12: 72 6 12 versus 73 6 13 mean difference 0, 95% CI -3 to 3 , p = 0.9; mental SF-12: 75 6 8 versus 73 6 10, mean difference 2, 95% CI 024, p = 0.3) as well as for all KOOS subscores (all p > 0.1). Forgotten Joint Scores were similar in both groups with 83 6 4 for CAS and 82 6 5 for control patients (mean difference 1, 95% CI 022, p = 0.2). Finally, the new Knee Society Scores were not statistically different between groups with a mean objective score of 82 6 13 for CAS patients versus 79 6 12 for control patients (mean difference 2, 95% CI 025, p = 0.5) and a mean subjective score of 83 6 11 versus 85 6 12, respectively (mean difference 2, 95% CI 025, p = 0.5). [START_REF] Pitto | Accuracy of a computer-assisted navigation system for total knee replacement[END_REF]. However, the role that coronal align-ment of TKA plays in these areas remains somewhat controversial [START_REF] Parratte | Effect of postoperative me-chanical axis alignment on the fifteenyear survival of modern, cemented total knee replacements[END_REF]. Although several studies have indicated that coronal plane alignment beyond the range of 0° 6 3° is a risk factor for impaired results, much of the literature on alignment and survivorship is historical and surprisingly weak in terms of lower limb alignment definitions [START_REF] Abdel | Coronal alignment in total knee replacement: historical review, contemporary analysis, and future di-rection[END_REF]. In 2008, Parratte et al. [START_REF] Parratte | Effect of postoperative me-chanical axis alignment on the fifteenyear survival of modern, cemented total knee replacements[END_REF] reported that TKAs that are mechanically aligned (0° 6 3°) versus those that are outside that range have identical survivorship 15 years after surgery. This research opened the debate on alternatives to the mechanical axis as a target for contem-porary TKAs [START_REF] Howell | Does a kinematically aligned total knee arthroplasty restore function without failure regardless of alignment category?[END_REF].

Previously published studies dem-onstrated benefits of CAS in terms of radiographic implant position, partic-ularly with regard to outliers [10, 12], but whether these improvements result in clinically important differences that patients might perceive at a minimum 10-year followup remains largely de-bated [5,[START_REF] Lu ¨ Tzner | No dif-ference between computerassisted and conventional total knee arthroplasty: fiveyear results of a prospective randomised study[END_REF]. Although some series demonstrated clinical benefits of TKA performed using CAS [3-5], most of them are shortto midterm studies with followup < 60 months [4, 5]. Series with longer followup failed to demon-strate clinical benefits or improved implant survivorship when comparing CAS and conventionally instrumented TKA [11,[START_REF] Kim | Computer-navigated versus conventional total knee arthroplasty: a prospective randomized trial[END_REF].

We earlier designed a randomized controlled trial to compare implant positioning using CAS versus a conventional surgical technique [START_REF] Lino | Computer-assisted total knee arthroplasty with and without pre-operative imaging[END_REF]. Us-ing this same study group, we performed a secondary analysis 10 years after that study and asked whether CAS (1) improved implant survival free from aseptic loosening; and (2) demonstrated any clear difference in patient-reported outcomes at latest followup using validated out-come measures at minimum 10-year followup.

Patients and Methods

Between January 2004 and December 2005, we performed 373 elective TKAs for primary or secondary ar-thritis. Of those, 86 (23%) were per-formed using the CAS technique [START_REF] Kim | Computer-navigated versus conventional total knee arthroplasty: a prospective randomized trial[END_REF]. Between April 2004 and April 2005, a randomized study was designed to assess implant positioning using CAS versus conventional instrumentation TKA (defined as control patients). The patient inclusion criteria were age 20 to 80 years old, weight < 100 kg, and consented for primary knee arthro-plasty performed through a medial parapatellar approach by the senior author (J-NA). The exclusion criteria were a history of prior knee surgery, TKA performed for a posttraumatic indication, or revision knee surgery (Fig. 1). All patients provided in-formed consent to participate in the study.

The first 80 patients meeting the inclusion criteria of this study were randomly assigned to the CAS or control group by the Hospital

In-formatics Department with use of a systematic sampling method. Patients were randomized after strat-ification on age and gender to try to ensure comparability of control and CAS patients (Fig. 1). The study protocol (including the use of navi-gation and postoperative evaluation) and consent forms were approved by the local ethical committee, and this study was subsequently pub-lished [START_REF] Lino | Computer-assisted total knee arthroplasty with and without pre-operative imaging[END_REF].

Ten years after initiation of this study, we performed a secondary analysis of this group. Of those who were treated with CAS, three patients (7.5%) had died, whereas 37 patients (37 knees) were available for followup at a minimum of 10 years (median, 11 years; range, 11-13 years); no patients were lost to followup. Of those treated with conventional instrumentation, four patients died (10%) leaving 36 patients (36 knees) for clinical and ra-diologic evaluation at a minimum of 10 years (median, 11 years; range, 11-13 years). There were no differences in demographic data between the two study groups with the numbers available. There ) in the control group (p = 0.44). The etiologies were primary osteoarthritis in 32 knees and eight with secondary arthritis in the CAS group and primary osteoarthritis in 29 knees and 11 with secondary arthritis in the control group.

One surgeon with subspecialty training and interest in TKA (J-NA) performed all the surgeries. Traditional mechanical guides were used in the control group. Before the beginning of the study, the senior author had performed > 2000 TKAs, which included > 50 computer-assisted procedures.

NexGen® LPS Flex (Zimmer, Warsaw, IN, USA) posteriorstabilized femoral components and NexGen all-polyethylene patella components were cemented in all patients. All TKAs were done without a tourniquet using a standard paramedian incision and a medial parapatellar approach. The tibial resection was performed with an extramedullary guide and the distal femoral resection with an intra-medullary guide. Femoral rotation was set according to a previously described technique [START_REF] Springer | Measured resection versus gap balancing for total knee arthroplasty[END_REF]. In the CAS group, an imageless femoral and tibial implant positioning, computerbased naviga-tion system was used following a pre-viously described protocol [START_REF] Lino | Computer-assisted total knee arthroplasty with and without pre-operative imaging[END_REF]. Two additional skin incisions were made to position the rigid bodies required for navigation in the CAS group.

Once the standard approach was done and the knee exposed, the tibial and femoral rigid bodies were implanted through separate incisions. After calibration of the instruments, the first step of the CAS procedure was the acquisition of predetermined landmarks and surfaces on the distal femur and the proximal tibia using a bone morphing technique. The mechanical axis of the limb and of the femur and tibia was then determined with the registration of the center of the femoral head (circum-duction of the entire limb), the center of the trochlear notch, the center of the tibial spines, and palpation of the me-dial and lateral malleoli to determine the center of the ankle. After this registration process, the tibial cut was then made using the specific navigated tibial jig following the indications of frontal alignment and slope given by the software. The femoral cuts were then done following the same principles. Both cuts were then checked using the dedicated navigated probe. Size of the femoral implant was given by the computer, but standard instruments were used to determine femoral rota-tion. At the time of the trial, ROM, frontal alignment, and varus/valgus laxity were assessed using the software. Using this information, comple-ligament release was performed when needed [START_REF] Springer | Measured resection versus gap balancing for total knee arthroplasty[END_REF]. Varus and valgus stresses were performed to assess the stability of the knee at the time of the trials. The goal of the surgeon was to obtain a neutral postoperative mechanical axis as defined by the center of the hip, center of the knee, and center of the ankle. Femoral and tibial rotations were set according to a previously de-scribed technique mainly based on a measured resection technique. The same combination of classic bony landmarks was used in both groups. CAS was only used to determine the frontal and sagittal alignment of the implants as a result of the limited ac-curacy of CAS to determine femoral and tibial rotation. The same postoperative rehabilitation protocol was used for all patients.

As part of our earlier study on alignment with these same groups, we observed a difference in implant positioning referenced by the hip-kneeangle (evaluated on long-axis radiographs (CAS: 179° 6 2° versus control: 177° 6 4°, mean difference 2° 6 3°, 95% confidence interval [CI] -2° to 5°, p = 0.031) and tibial implant position in the coronal plane (CAS: 89° 6 2° versus control: 87° 6 3°, mean difference 2° 6 4°, 95% CI -1° to 6°, p = 0.044; Table 1).

Survivorship was compared in the two groups using the Kaplan-Meier method [START_REF] Kaplan | Nonparametric es-timation from incomplete observations[END_REF] defining the endpoint as revision of one or more of the compo-nents resulting from aseptic loosening or mechanical failure (fracture excep-ted) defined as substantial migration of one or more component(s), substantial wear, or osteolysis at last followup, potentially calling for a revision [8]. Septic revision or revision resulting from periprosthetic fracture was not included in our survivorship analysis.

We also elected to compare the subjective functional outcome and quality of life of the two groups, be-cause they were judged to be equiva-lent based on the earlier randomization process. However, because the original randomization had been performed for an alignment study, preoperative patient-reported outcome measures (PROMs) had not been obtained. Fur-thermore, the PROMs in question were not common, available, and/or validated in French at the time of the Values are mean 6 SD; HKA = hip-knee-ankle angle; CAS = computer-assisted surgery; Gamma = femoral sagittal mechanical angle; Beta = tibial frontal mechanical angle.

[26], KOOS [START_REF] Roos | Knee Injury and Osteoarthritis Outcome Score (KOOS)-development of a selfadministered out-come measure[END_REF], FJS [START_REF] Thienpont | Joint awareness in different types of knee arthroplasty evaluated with the Forgotten Joint score[END_REF], and the new KSS [7]).

Statistical Analysis

A comparison of the functional out-comes at last followup between the two groups was performed using either a parametric or nonparametric test (depending on parameter distribution). Our initial protocol was designed to detect differences in terms of lower limb alignment between two groups of patients undergoing CAS or conven-tional TKA. The present research protocol aimed to evaluate clinical and radiologic outcomes of those two groups 10 years after surgery.

A post hoc analysis confirmed that regarding our primary outcome with our avail-able sample size and postoperative score SD, this study had 80% power to detect a difference > 6 points out of 100 on the KOOS subscores (published minimally clinically important differ-ence [MCID]: 8 points) and > 6 points on the SF-12 components (published MCID: 5 points) at the p < 0.05 level [4, 6]. Our study was not adequately powered for survival analysis.

Analysis was performed using SPSS software (Version 12; SPSS Inc, Chicago, IL, USA). All calculations assumed two-tailed tests.

Results

With the numbers available, there was no difference between the CAS group and the conventional TKA group in terms of survivorship free from aseptic loosening 10 years after TKA (97%, 95% CI 95%-99% versus 97%, 95% CI 95%-99%; p = 0.98; Fig. 2).

When investigating latest followup scores (improvement of those scores was not assessed because they were not available preoperatively), we found no difference between groups regarding SF-12 score (respectively, for CAS and control patients, physical SF-12: 72 6 12 versus 73 6 13 mean difference 0, 95% CI -3 to 3, p = 0.9; mental SF-12: 75 6 8 versus 73 6 10, mean differ-ence 2, 95% CI 024, p = 0.3) as well as for all KOOS subscores (all p > 0.1; Fig. 3). FJS scores were similar in both groups with 83 6 4 for CAS and 82 6 5 for control patients (mean difference 1, 95% CI 022, p = 0.2) at last fol-lowup. Finally, the new KSS scores were not statistically different between groups with a mean objective score of 82 6 13 for CAS patients versus 79 6 12 for control patients (mean differ-ence 2, 95% CI 025, p = 0.5) and a mean subjective score of 83 6 11 versus 85 6 12, respectively (mean difference 2, 95% CI 025, p = 0.5; Table 2).

Discussion

The premise of CAS in TKA was to improve implant positioning and re-store a neutral mechanical axis with the goal of improving function and im-plant survivorship [START_REF] Pitto | Accuracy of a computer-assisted navigation system for total knee replacement[END_REF]. The benefits of CAS to improve implant positioning around a defined target goal and to decrease outliers have been demon-strated, but whether improved posi-tioning will impact these other goals remains uncertain [10,12]. The results of our earlier randomized controlled trial focusing on postoperative implant positioning in these two groups of patients randomly assigned to CAS or conventional TKA showed improved lower limb alignment and tibial im-plant position in the coronal plane for the CAS group [START_REF] Lino | Computer-assisted total knee arthroplasty with and without pre-operative imaging[END_REF]. To evaluate any potential benefits of this improved alignment, in this secondary analysis, we compared clinical outcomes and implant survivorship between the same groups with a minimum followup of 10 years [4,[START_REF] Lino | Computer-assisted total knee arthroplasty with and without pre-operative imaging[END_REF]. Specifically, we asked whether CAS would improve 10-year survivorship and certain PROMs as compared with conventional in-strumentation. Those PROMs were not included in our initial study protocol and thus the functional scores we assessed in the present study were only available at the latest followup period. The results of our study suggest that Fig. 2 Kaplan-Meier analysis shows survivorship using revision for mechanical failure as the endpoint. as the target goal did not confer any substantial advantage in survivorship, function, or quality of life 10 years after TKA. Several limitations can be outlined in our study. First, the study is un-derpowered to detect all but very large differences in survivorship. When the study protocol was developed, the groups were powered for radiographic analysis of alignment; functional and survivorship data were not available to calculate sample size because all PROMs used in this secondary analy-sis were either not available or not translated into French in the preoperative/postoperative period. Al-though our groups were initially comparable in terms of demographics and preoperative knee deformities, we can only suspect that they were also comparable in terms of pain and func-tion at baseline. However, with the numbers available and a power of 80%, we could demonstrate a clinically important difference for the SF-12 and the KOOS (minimal clinically important Controversy surrounds the ideal alignment goal in TKA. Historically "restoring" mechanically neutral alignment through bone cuts aligned to the limb's mechanical axis has been suggested to offer patients the best chance for long-term prosthetic knee function and survival [START_REF] Abdel | Coronal alignment in total knee replacement: historical review, contemporary analysis, and future di-rection[END_REF][START_REF] Allen | Neutral mechanical alignment: is it necessary[END_REF]. More recently, ques-tions have been raised as to whether these targets should shift to achieve more patient-specific alignment goals, restoring the individual's premorbid alignment and thus restoring knee kinematics [START_REF] Howell | Does a kinematically aligned total knee arthroplasty restore function without failure regardless of alignment category?[END_REF][START_REF] Parratte | Effect of postoperative me-chanical axis alignment on the fifteenyear survival of modern, cemented total knee replacements[END_REF]. We might have chosen the wrong target in our series; however, to date no scientific evidence of a better "systematic option" has been demonstrated [START_REF] Young | The Chi-tranjan S. Ranawat Award: No difference in 2-year functional outcomes using kinematic versus mechanical alignment in TKA: a randomized controlled clinical trial[END_REF].

CAS used for TKA placement with restoration of a neutral mechanical axis

With the numbers available, we found no survivorship advantage in patients treated with CAS TKA 10 years after surgery. Although our results are consistent with the results from others authors [3,9,11,[START_REF] Ishida | Mid-term out-comes of computer-assisted total knee arthroplasty[END_REF][START_REF] Kim | Computer-navigated versus conventional total knee arthroplasty: a prospective randomized trial[END_REF][START_REF] Lu ¨ Tzner | Functional outcome after computer-assisted versus conventional total knee arthroplasty: a randomized controlled study[END_REF][START_REF] Marques | No differences in clinical out-comes between fixedand mobile-bearing computerassisted total knee arthroplasties and no correlations be-tween navigation data and clinical scores[END_REF] (Table 3), our series was not adequately powered for survival anal-ysis. Because we found the same im-plant survivorship between the groups (if aseptic loosening was defined as the endpoint), a post hoc analysis evalu-ated that 1500 patients would be needed in each group to be able to de-tect a 2% survivorship difference 10 years after surgery. Those numbers demonstrate the difficulty in planning a controlled trial powered to distin-guish such survivorship improvement. Parratte et al. also showed that the restoration of a neutral mechanical axis is perhaps not the best predictor of survivorship [START_REF] Parratte | Effect of postoperative me-chanical axis alignment on the fifteenyear survival of modern, cemented total knee replacements[END_REF]. More specific tar-gets of alignment may be required and several new concepts have emerged recently such as kinematic alignment or constitutional alignment [START_REF] Abdel | Coronal alignment in total knee replacement: historical review, contemporary analysis, and future di-rection[END_REF][START_REF] Allen | Neutral mechanical alignment: is it necessary[END_REF]. These techniques are too recent to have long-term survivorship; and if some clinical improvements have been shown at midterm followup [START_REF] Howell | Does a kinematically aligned total knee arthroplasty restore function without failure regardless of alignment category?[END_REF], a re-cent randomized controlled study failed to demonstrate any clinical benefit of the kinematic alignment concept 2 years after TKA [START_REF] Young | The Chi-tranjan S. Ranawat Award: No difference in 2-year functional outcomes using kinematic versus mechanical alignment in TKA: a randomized controlled clinical trial[END_REF].

The results of the clinical evaluation showed no benefit for navigated patients. This is in agreement with other publications with at least 5 years of followup [9, 11, [START_REF] Kim | Computer-navigated versus conventional total knee arthroplasty: a prospective randomized trial[END_REF][START_REF] Lu ¨ Tzner | No dif-ference between computerassisted and conventional total knee arthroplasty: fiveyear results of a prospective randomised study[END_REF][START_REF] Lu ¨ Tzner | Functional outcome after computer-assisted versus conventional total knee arthroplasty: a randomized controlled study[END_REF]. Harvie et al. [9] found no differences in patient satisfaction 5 years postoperatively and Kim et al. [START_REF] Kim | Computer-navigated versus conventional total knee arthroplasty: a prospective randomized trial[END_REF] did not find any differences in KSS or Hospital for Special Surgery score evaluation at 10 years. Similar KSS scores were repor-ted by Lu ¨ tzner et al. [START_REF] Lu ¨ Tzner | No dif-ference between computerassisted and conventional total knee arthroplasty: fiveyear results of a prospective randomised study[END_REF][START_REF] Lu ¨ Tzner | Functional outcome after computer-assisted versus conventional total knee arthroplasty: a randomized controlled study[END_REF] and Herna ´ndez-Vaquero et al. [11] at, re-spectively, 5 years and 7 years of fol-lowup. We did not find any long-term report demonstrating a clinically important difference between CAS and conventional TKA. However, Ishida et al. [START_REF] Ishida | Mid-term out-comes of computer-assisted total knee arthroplasty[END_REF]

Fig. 1

 1 Fig. 1 This is a flow diagram illustrating patients' enrollment, allocation, followup, and analysis.

2 (

 2 were 21 women and 19 men. The median age of the patients was 66 years (range, 40-80 years) in the CAS group and 64 years (range, 46-78 years) in the control conventional technique group (p = 0.31). The mean body mass index was 27 6 4 kg/m

  original randomization. Nevertheless, PROMs of the following scores (SF-12, Knee Injury and Osteoarthritis Outcome Score [KOOS], Forgotten Joint Score [FJS], new Knee Society Score [KSS]) were assessed at an in-dividual clinical examination to offer a general basis of comparison be-tween the groups, accepting the ob-vious limitation of not having preoperative and serial scores for comparison. Subjective functional outcome and quality of life were assessed during individual clinical evaluation, per-formed by two authors not involved in the surgical interventions (MO, SP), using validated questionnaires (SF-12 mentary releases were performed and a thicker liner was tried when needed. At the time of inclusion, the instru-mented ligament tensor was not avail-able. A pure measured resection technique was used in both groups. The flexion and extension gaps were tested after the cuts and

Fig. 3

 3 Fig. 3 These results show a comparison of the KOOS score between CAS and control patients. *All p > 0.1. ADL = activities of daily living; QOL = quality of life.

Table 1 .

 1 appropriate Summary of postoperative implant position analysis

	Groups	HKA	Gamma	Beta	Tibial
					slope
	CAS	179 6 2	6 6 2	89 6 2	4 6 3
	Control	177 6 4	6 6 3	87 6 3	3 6 3
	p value	0.031	0.6	0.044	0.2

Table 2 .

 2 Clinical score evaluation at last followup

	Clinical outcomes	CA	Control	Mean diff erence absolute value (SD)	p
		S			value
	New KSS subjective score	83 6 11	85 6 12	2 (3)	0.5
	New KSS objective score	82 6 13	79 6 12	2 (3)	0.5
	KOOS symptoms	94 6 8	95 6 8	1 (2)	0.4
	KOOS pain	92 6 7.8	94 6 4.7	2 (1)	0.1
	KOOS ADL	90 6 5	90 6 3	1 (1)	0.5
	KOOS sport	66 6 12	68 6 11	2 (3)	0.4
	SF-12 physical	72 6 12	73 6 13	0 (3)	0.9
	SF-12 mental	75 6 8	73 6 10	2 (2)	0.4
	FJS	83 6 4	82 6 5	1 (1)	0.2

Values are mean 6 SD; CAS = computer-assisted surgery; KSS = Knee Society Score; KOOS = Knee injury and Osteoarthritis Outcome Score; ADL = activities of daily living; FJS = Forgotten Joint Score.

Table 3 .

 3 Level I and II studies reporting clinical and radiologic outcomes for CAS versus conventional TKA

	Survivorship or
	Main clinical


Christophe Jacquet

MD Roger Erivan

Jean-Noël Argenson

PhD Sebastien Parratte

PhD Matthieu Ollivier
email: ollivier.matthieu@yahoo.fr

MD Jean-Noe ¨ L Argenson

on Human Menisci Ultrastructure An Ex Vivo Comparative Study With Fresh Tissue as a Gold Standard

Keywords: meniscus allograft, irradiation, cryopreservation, freezing, histologic, collagen

published or not. The documents may come   L'archive ouverte pluridisciplinaire

to restore knee joint mechanics and potentially slow the onset of osteoarthritis. [START_REF] Lewis | Multiple freeze-thaw cycled meniscal allograft tissue: a biomechanical, biochemical, and histologic analysis[END_REF] Verdonk et al [START_REF] Verdonk | Meniscal allograft transplantation: long-term clinical results with radiological and magnetic resonance imaging correlations[END_REF] described midterm results showing a significant improvement in patients' pain associated with 5-year allograft survival .85%. [START_REF] Verdonk | Transplantation of viable meniscal allograft: survivorship analysis and clinical outcome of one hundred cases[END_REF][START_REF] Verdonk | Meniscal allograft transplantation: long-term clinical results with radiological and magnetic resonance imaging correlations[END_REF][START_REF] Wirth | Longterm results of meniscal allograft transplantation[END_REF] As those interesting results imply a meniscal graft equivalent to the native fibrocartilage, graft preservation methods play a vital role in the clinical and biological success of meniscal allograft techniques. Three main menisci preservation methods were advocated: freezing, freezing with gamma irradiation, and cryopreservation. [START_REF] Vangsness | Allograft transplantation in the knee: tissue regulation, procurement, processing, and sterilization[END_REF] Simple freezing brings the material to 280°C, but deep modification of the collagen network and global architecture was reported with this preservation. [START_REF] Gelber | Freezing causes changes in the meniscus collagen net: a new ultrastructural meniscus disarray scale[END_REF][START_REF] Giannini | Effects of freezing on the biomechanical and structural properties of human posterior tibial tendons[END_REF] Freezing associated with irradiation allows a deep sterilization of the graft but also compromises allograft microarchitectural and biomechanical properties. [START_REF] Fideler | Gamma irradiation: effects on biomechanical properties of human bone-patellar tendon-bone allografts[END_REF][START_REF] Vangsness | Allograft transplantation in the knee: tissue regulation, procurement, processing, and sterilization[END_REF] Cryopreservation techniques require cryoprotectants (dimethylsulfoxide) and bring the tissue to 2145°C. Gelber et al [START_REF] Gelber | Cryopreservation does not alter the ultrastructure of the meniscus[END_REF] demonstrated that cryopreservation was superior to freezing to maintain the meniscus ultrastructure during the storage process. To date, studies investigating human meniscus preservation had 2 main limitations: first, they did not compare preservation processes with the same meniscus sample; second, conserved tissues are not compared with fresh meniscus samples. As meniscus ultrastructure depends on various demographic and pathologic confounding factors, comparing potential detrimental effects of various conservation processes should be performed on ''identical'' tissue samples. [START_REF] Choi | Collagenous ultrastructure of the discoid meniscus: a transmission electron microscopy study[END_REF][START_REF] Delrio | Structural organization of the menisci of the human knee: scanning electron microscopy study [in Italian][END_REF][START_REF] Ghadially | Ultrastructure of normal and torn menisci of the human knee joint[END_REF][START_REF] Katsuragawa | Changes of human menisci in osteoarthritic knee joints[END_REF] Therefore, we aimed to analyze and compare 3 preservation processes with a ''fresh tissue'' control group using samples harvested from the same human meniscus. We hypothesized that all preservation methods would result in structural and architectural properties similar to those of fresh meniscus, defined as the gold standard. We aimed to estimate the effects of these methods on meniscus ultrastructure by using electronic microscopy to compare collagen fiber diameters in longitudinal and transverse sections and by calculating a validated architectural score per sample. [START_REF] Gelber | Freezing causes changes in the meniscus collagen net: a new ultrastructural meniscus disarray scale[END_REF][START_REF] Gelber | Cryopreservation does not alter the ultrastructure of the meniscus[END_REF]

METHODS

After local review board approval, 5 human lateral menisci were collected from patients who had total knee arthroplasty between September and October 2017. All patients signed an informed consent form before their inclusion in our study. Inclusion criteria were as follows: patient aged \70 years undergoing total knee arthroplasty because of isolated internal femorotibial arthritis or femoropatellar and internal femorotibial joint degeneration (but with an external femorotibial compartment graded Kellgren and Lawrence \2) [START_REF] Kohn | Classifications in brief: Kellgren-Lawrence classification of osteoarthritis[END_REF] and no surgery, trauma, or developmental disease of the operated knee. Table 1 summarizes patients' characteristics.

Sample Creation

The anterior and posterior horns were sectioned to retain only the median horn of the meniscus. Four cubes (9 mm 3 ) were cut inside of the white, or avascular, area of each specimen and divided into 4 groups: ''fresh'' control, freezing, cryopreserved, and freezing 1 irradiation. Histologic fixation of the control specimens was performed directly in the operating theater under a hood, which consisted of the following steps based on a previously validated protocol: to 100% ethanol before inclusion in Spurr. [START_REF] Wallis | A routine method for embedding animal tissues in Spurr resin for electron microscopy[END_REF]

Resin fixation for transmission electron microscopy

Control specimens were analyzed immediately after those steps in a delay \6 hours. The other 3 fragments were placed in physiologic saline in a cryokit and kept at 8°C until their transport to our local tissue bank (Etablissement Franxcais du Sang) (\6 hours). The 3 samples were then prepared with the following steps: (1) graft reception in clean room (controlled atmosphere zone), (2) decontamination of the graft with an antibiotic solution (rifampicin 1 thiamphenicol), (3) rinsing with 0.1M cacodylate buffer for 5 minutes, and (4) bacteriologic sampling. Then, the 3 conservation methods were applied (1 sample for each). For the cryopreservation group, cryoprotective solution (10% of DMSO 1 SCOT 30) was added, and the bag was vacuumed to extract the residual air and the temperature progressively decreased (staring at 24°C and then decreasing at 22°C per minute to 240°C and then 25°C per minute to 2140°C). Samples were stored in a nitrogen tank in vapor phase at 2145°C. For the freezing group, a simple freezing process was used by progressively decreasing the temperature (staring at 24°C and then decreasing at 22°C per minute to 240°C and then 25°C per minute to 280°C). For the freezing 1 irradiation group, a simple freezing process with a progressive decrease in temperature was performed (starting at 24°C and then decreasing at 22°C per minute to 240°C and then 25°C per minute to 280°C). The grafts were then transported in a dry ice-controlled container (stored at 280°C) to be irradiated by gamma ray by IONI-SOS. The doses received ranged between 23.9 and 26.5 kGy (2.4-2.6 Mrad). After this treatment, the samples were again stored at 280°C until analysis.

After 1 month, the samples were transported to the electronic microscopy laboratory to be analyzed. The fixing steps (steps 1-7) were the same as those described for the control group. For all samples, ultrafine 60-nm sections were made with an Ultracut ultramicrotome (Reichert-Jung); contrast of the sections was made with uranyl acetate and lead citrate. Ultrastructure pictures were obtained with a transmission electron microscope (JEM 1400; JEOL) at 80 kV with a Megaview III camera and iTEM Five software (SIS Imaging). For each sample, we took 10 pictures with a magnification of 60003 and 40,0003. The longitudinal (Figure 1) and transverse diameters of the collagen fibers were measured at 70 points on a picture taken at 40,0003 magnification. The collagen meniscal architecture scoring system was calculated for each sample on 5 pictures with 60003 magnification (Table 2). [START_REF] Gelber | Cryopreservation does not alter the ultrastructure of the meniscus[END_REF]

Statistics

Before the initiation of the study, a sample analysis estimated that 5 samples for each group was necessary to be powered to distinguish D 20 6 10 nm regarding collagen transverse or longitudinal diameters. [START_REF] Fabbriciani | Meniscal allografts: cryopreservation vs deep-frozen technique. An experimental study in goats[END_REF][START_REF] Fideler | Gamma irradiation: effects on biomechanical properties of human bone-patellar tendon-bone allografts[END_REF][START_REF] Gelber | Cryopreservation does not alter the ultrastructure of the meniscus[END_REF] Patient characteristics were expressed with the appropriate descriptive statistics for the type of variables. Descriptive statistics included mean with SD or median with interquartile range as appropriate for continuous variables. The intraclass correlation coefficient with 95% CIs was calculated to assess intra-and interobserver reproducibility for the transverse and longitudinal diameter values. Student t tests were used to compare the distribution of continuous parameters between groups (or the Mann-Whitney test when the data were not normally distributed or when the homoscedasticity assumption was rejected). All reported P values were 2-sided, with a significance threshold of .05. Statistical analyses were performed with SPSS/JMP software (v 13; Microsoft).

RESULTS

Transverse Diameter

No significant difference was found between the control and cryopreserved groups regarding mean transverse There was a significant difference between the control and frozen groups (95.39 6 15.87 nm vs 70.20 6 13.94 nm, P \ .001) and between the control and frozen 1 irradiated groups (95.39 6 15.87 nm vs 63.1 6 15.57 nm, P \ .001) (Table 3). The mean difference between the frozen and frozen 1 irradiated groups was also significant: D 7.1 nm (95% CI, 4.6-9.5 nm; P \ .023).

Longitudinal Diameter

We found no significant difference in mean longitudinal diameter between the control and cryopreserved groups (96.3 6 14 nm vs 94.6 6 16.4 nm, P = .1). There was a significant difference in mean longitudinal diameter between the control and frozen groups (96.3 6 14 nm vs 71.3 6 10.6 nm, P \ .001) as well as the frozen 1 irradiated groups (96.3 6 14 nm vs 60.9 6 14.8 nm, P \ .001) (Table 4).

The mean difference between the frozen and frozen 1 irradiated groups was also significant: D 10.6 nm (95% CI, 8.2-12.5 nm; P \ .011).

Collagen Meniscal Scoring System

The mean values of the collagen meniscal architecture scoring system are summarized in Table 5. No difference was found between the control and cryopreserved groups (4.5 6 1.3 vs 4.3 6 1.6 points, P = .9). There were significant differences between the control and frozen groups (4.5 6 1.3 vs 2.3 6 1.4 points, P = .02) and between the control and frozen 1 irradiated groups (4.5 6 1.3 vs 1.4 6 0.9, P = .02).

DISCUSSION

The main finding of the current study was that cryopreservation preserves meniscus histologic ultrastructure, unlike simple freezing or freezing 1 irradiation. We rejected our hypothesis that all preservation methods will result in structural and architectural properties similar to those of fresh meniscus. Cryopreservation does not entail a significant modification in terms of collagen fiber diameter or architectural organization as compared with fresh tissue. We did find, however, significant differences regarding those 2 measurements when we compared freezing and freezing 1 irradiation processes with fresh tissue or cryopreserved samples. Irradiation was the more detrimental process in terms of tissue preservation, as all of our measurements were inferior to those taken for fresh tissue, cryopreservation, and freezing samples.

Our study is limited in that our patients are older than usual donors 2 (mean age: 63.8 years in our study vs 53.5 in the register). Because of this, menisci evaluated during our analyses might have been altered by aging and degeneration processes. We tried to avoid limitations related to this methodological bias by studying nonarthritic joints (lateral compartment) from subjects having only medial femorotibial degeneration. Moreover, our direct comparative design allowed us to think that potential degenerative changes would have similarly affected our evaluation of the 3 preservation methods, since the compared samples were created from the same meniscus. Our relatively short storage period before the analysis could also be considered a limitation. We supposed that most of the ultrastructure alteration related to the preservation method occurred during the decreasing-of-temperature steps, including direct chemical effects, ice formation, and dehydration, as described by Pegg. [START_REF] Pegg | Principles of cryopreservation[END_REF] Our evaluation method involved manual measurement of collagen fiber diameters; as such, 1 senior technician trained in transmission electron microscopy carried out the entire collection of data and was blinded regarding the preservation process of the specimens. The measurements were always made at the same magnification (40,0003) and at 70 random points of the picture, with consideration to only the most circular fibers for the transverse diameter and the most linear for the longitudinal diameter. Our protocol did not aim to investigate cell viability. It was impossible for us to perform both ultrastructure analysis and flow cytometric cell counts on the same sample. Gelber et al [START_REF] Gelber | Cryopreservation does not alter the ultrastructure of the meniscus[END_REF] were the first to demonstrate that cell survival was possible in cryopreserved samples, but in a study of 15 meniscal goat allografts, Fabbriciani et al [START_REF] Fabbriciani | Meniscal allografts: cryopreservation vs deep-frozen technique. An experimental study in goats[END_REF] showed that even if cryopreservation made it possible to maintain partial cell viability in the tissue, the morphologic and biochemical characteristics of the graft were not improved. Finally, our sample size might seem low, but the numbers of specimens included was decided before the initiation of the study to compare groups upon collagen thickness evaluation. Despite these limitations, our study is the first to directly compare the influence of preservation processes on 4 samples prepared from the same meniscus, permitting us to assert that the differences observed are mostly related to preservation methods and their specificities more than demographic confounding factors.

We used the collagen meniscal scoring system by Gelber et al [START_REF] Gelber | Freezing causes changes in the meniscus collagen net: a new ultrastructural meniscus disarray scale[END_REF][START_REF] Gelber | Cryopreservation does not alter the ultrastructure of the meniscus[END_REF] to compare our results with the existing literature. Gelber et al [START_REF] Gelber | Freezing causes changes in the meniscus collagen net: a new ultrastructural meniscus disarray scale[END_REF][START_REF] Gelber | Cryopreservation does not alter the ultrastructure of the meniscus[END_REF] advocated the superiority of cryopreservation on meniscus ultrastructure as compared with freezing, with 2 studies corroborating our results, even if they did not use a control group. In another study, Vangsness et al [START_REF] Vangsness | Allograft transplantation in the knee: tissue regulation, procurement, processing, and sterilization[END_REF] demonstrated that the use of gamma irradiation caused clear alteration of musculoskeletal tissues mechanical properties.

Bone sample load-to-failure behavior was significantly lower when exposed to .3 Mrad of irradiation. [START_REF] Vangsness | Allograft transplantation in the knee: tissue regulation, procurement, processing, and sterilization[END_REF] Fideler et al 5 determined that .2 Mrad of irradiation of bonepatellar tendon-bone allografts adversely affected 4 of the 7 structural properties that were analyzed. Thus, they found that all structural parameters were deeply affected at irradiation levels of 3.0 and 4.0 Mrad. This conclusion is supported by our data: we found the highest rate of collagen disorganization for samples exposed to 2.5 Mrad (25 kGy: freezing 1 irradiation group). Advantages in terms of sterilization allowed by gamma irradiation should be balanced with those deep architectural and potentially mechanical consequences.

With regard to the meniscus, Lewis et al [START_REF] Lewis | Multiple freeze-thaw cycled meniscal allograft tissue: a biomechanical, biochemical, and histologic analysis[END_REF] studied 7 human menisci and demonstrated that samples that underwent a single freeze-thaw cycle had a significantly higher Young modulus than did those undergoing multiple freeze-thaw cycles (Young modulus: 1.2 3 10 7 for 1 cycle vs 8.5 3 10 6 for multiple cycles).

Our histologic results must be confirmed by mechanical trials to better understand potential consequences of ultrastructure alteration on meniscal graft mechanical properties. In the same manner, only in vivo studies will be able to assess the real clinical relevance of our ex vivo conclusion.

CONCLUSION

Cryopreservation is the only method that preserves fresh meniscus architectural specificities. Freezing and freezing 1 irradiation methods modify histologic properties of meniscal allograft. Irradiation deeply alters diameters and organization of collagen fibers, and this method should be used with caution to preserve and sterilize menisci tissue.

Figure 1 .

 1 Figure 1. Example of longitudinal diameter measurement (cryopreserved sample diameter estimated on a picture at 40,0003 magnification).

TABLE 1

 1

			Patients' Demographics a
	Patient	Age, y	Sex	Weight, kg	Size, cm	BMI, kg.m -2
	1	61	M	76	181	22.6
	2	67	M	82	184	24.2
	3	62	F	67	172	22.1
	4	60	F	60	162	22.9
	5	69	M	77	178	24.3
	a BMI, body mass index.

TABLE 2 Collagen

 2 Meniscal Architecture Scoring System

		0 Point	1 Point	2 Points
	Disrupt/periodicity	Mild	Moderate	Severe
	Intrafibrillary edema	No	Yes	-
	Packing	High density Intermediate Low density
	Banding	Yes	No	-
	Fibril size variability	Low	High	-

TABLE 3 Collagen

 3 Fiber Transverse Diameter Comparison Among Groups

	Group Comparison	Mean Difference, nm	95% CI, nm	P Value
	Control	Cryopreserved	4.22	1.36-7.08	.1
	Control	Irradiated	32.27	29.55-34.99	\.001
	Control	Frozen	25.191	22.61-27.77	\.001
	Cryopreserved	Frozen	29.413	26.5-32.2	\.001
	Cryopreserved	Irradiated	36.4	33.34-39.65	\.001
	Frozen	Irradiated	7.08	4.64-9.52	.023
			TABLE 4
		Collagen Fiber Longitudinal Diameter Comparison Among Groups
	Group Comparison	Mean Difference, nm	95% CI, nm	P Value
	Control	Cryopreserved	1.738	0.55-4.03	.1
	Control	Irradiated	35.39	32.79-37.98	\.001
	Control	Frozen	25.02	22.86-27.19	\.001
	Cryopreserved	Frozen	23.29	20.87-25.70	\.001
	Cryopreserved	Irradiated	33.65	30.99-36..31	\.001
	Frozen	Irradiated	10.36	8.24-12.49	.011

diameter (95.39 6 15.87 nm vs 99.62 6 19.23 nm, P = .1).

TABLE 5

 5

	Collagen Meniscal Architecture Scoring Comparison
	Between Preserved and Control Samples
	Group	Score, Mean 6 SD	P Value
	Control vs	4.5 6 1.3	-
	Cryopreservation	4.3 6 1.6	.9
	Frozen	2.3 6 1.4	.02
	Irradiation	1.4 6 0.9	.02

ACKNOWLEDGMENT

The authors thank Alexandre Altie for his support during electron microscopy analysis.


M Escudier

Matthieu Ollivier
email: ollivier.mt@gmail.com

S Donnez

P Parratte

Jean-Noël Lafforgue

Argenson

J.-C Escudier

M Donnez

S Parratte

P Lafforgue

J.-N Argenson

Superimposition of Maximal Stress and Necrosis Areas at the Top of the Femoral Head in Hip Aseptic Osteonecrosis

Keywords: osteonecrosis of the femoral head, necrosis area, maximal stress area, 3D finite element model

Superimposition of maximal stress and necrosis areas at the top of the femoral head in hip aseptic osteonecrosis

Introduction

Recent reports described possible mechanical factors in the development and aggravation of osteonecrosis of the femoral head (OFH), but these have yet to be confirmed on dedicated mechanical study. We therefore developed a 3D finite element model based on in-vivo data from patients with incipient OFH, with a view to determining whether the necrosis area was superimposed on the maximal stress area on the femoral head.

Hypothesis

The location of the necrosis area is determined by stress on the femoral head.

Material and method

All patients from the rheumatology department with early stage OFH in our center were investigated. Analysis of CT scans showed stress distribution on the head by 3D finite elements models, enabling determination of necrosis volume within the maximal stress area and of the percentage intersection of necrosis within the stress area (%I n/s: necrosis volume in stress area divided by total stress area volume and multiplied by 100) and of stress within the necrosis area (%I s/n: stress volume in necrosis area divided by total necrosis area volume and multiplied by 100). Results

Nineteen of the 161 patients assessed retrospectively for the period between 2006 and 2015 had incipient unilateral OFH, 10 of whom (4 right, 6 left) had CT scans of sufficient quality for inclusion. Mean age was 52 years (range, 37-81 years). Mean maximal stress was 1.63MPa, mean maximal exported stress volume was 2,236.9 mm³ and mean necrosis volume 6,291.1 mm³. Mean %I n/s was 83% and mean %I s/n 35%, with no significant differences according to gender, age, side or stress volume. There was a strong inverse correlation between necrosis volume and %I s/n (R 2 = -0.92) and a strong direct correlation between exported stress volume and %I s/n (R 2 =0.55). %I s/n was greater in small necrosis (<7,000mm 3 ). Conclusion OFH seems to develop within the maximal stress area on the femoral head. The present results need confirmation by larger-scale studies. We consider it essential to take account of these mechanical parameters to reduce failure rates in conservative treatment of OFH.

Level of evidence: IV. Case series

Introduction

Osteonecrosis of the femoral head (OFH) consists in impaired femoral head perfusion due to intravascular coagulation. Part of the subchondral bone supporting the joint surface becomes necrotic, causing collapse of the head and secondary osteoarthritis [START_REF] Guerado | The physiopathology of avascular necrosis of the femoral head: an update[END_REF][START_REF] Fraitzl | Reduced head-neck offset in nontraumatic osteonecrosis of the femoral head[END_REF]. Many etiologies and risk factors have been identified, including chronic alcoholism, medication (including longcourse corticosteroids, with dose-dependent risk), and metabolic diseases such as gout, hyperlipidemia and thrombophilia [START_REF] Fraitzl | Reduced head-neck offset in nontraumatic osteonecrosis of the femoral head[END_REF]. In 30% of cases, however, no etiology can be established, and the osteonecrosis is diagnosed as "idiopathic" [START_REF] Dudkiewicz | Total hip arthroplasty after avascular necrosis of the femoral head: does etiology affect the results?[END_REF][START_REF] Assouline-Dayan | Pathogenesis and natural history of osteonecrosis[END_REF].

The pathophysiology of osteonecrosis has been well described and is of vascular origin [START_REF] Guerado | The physiopathology of avascular necrosis of the femoral head: an update[END_REF]. However, recent studies have shown a critical role of hip-joint loading, with anatomic variations in the development of OFH [START_REF] Fraitzl | Reduced head-neck offset in nontraumatic osteonecrosis of the femoral head[END_REF][START_REF] Ollivier | Anatomical Findings in Patients Undergoing Total Hip Arthroplasty for Idiopathic Femoral Head Osteonecrosis[END_REF][START_REF] Kim | Effects of non-weight-bearing on the immature femoral head following ischemic osteonecrosis: an experimental investigation in immature pigs[END_REF][START_REF] Nishimura | The role of mechanical stress on the femoral head in the occurrence of femoral head lesions in spontaneously hypertensive rats[END_REF][START_REF] Mankin | The reaction of articular cartilage to injury and osteoarthritis (first of two parts)[END_REF][START_REF] Mankin | The reaction of articular cartilage to injury and osteoarthritis (second of two parts)[END_REF]. Stress location in the femoral head may thus account for necrosis location, in the summit [START_REF] Yang | Mechanics of femoral head osteonecrosis using three-dimensional finite element method[END_REF][START_REF] Kaushik | Osteonecrosis of the femoral head: An update in year 2012[END_REF]. However, this remains to be confirmed on mechanical study, and we therefore developed a 3D finite element model based on in-vivo data in patients with incipient OFH, to determine whether the necrosis area is superimposed on the area of maximal stress. The study hypothesis was that the location of the necrosis area is determined by mechanical factors: i.e., stress exerted by the weight of the pelvis over the femur.

Material and method

Patients

With institutional review board approval, a prospective data-base was used to analyze patients assessed for incipient OFH in our rheumatology department between 2006 and 2015. Patients were followed up on 3D imaging of the hip or pelvis to confirm diagnosis of osteonecrosis and assess extension.

Inclusion criteria comprised: early-stage OFH (stage 1 or 2 on Ficat's classification [START_REF] Ficat | Idiopathic bone necrosis of the femoral head. Early diagnosis and treatment[END_REF]: i.e., without head deformity), with hip or pelvis CT of sufficient quality for segmentation and meshing.

Methods

3D finite element models of the hip were constructed in 3 stages, using 3 different software applications. a) Hip or pelvis CT scans were first imported to the Mimics 18™ segmentation package (Materialise, Leuven, Belgium). The software's semi-automatic tools created 3D models of the patient's pelvic and proximal femoral bone, and then created cartilage, unseen on CT, as an intermediate object by addition and subtraction operations on the pelvis and femur. The three resulting objects (pelvis, cartilage and femur) were first smoothed in Mimics to obtain an optimally homogeneous and anatomically faithful 3D structure (Figure 1). In parallel, the necrotic area of the head was located as the least dense area, and a corresponding 3D object was then created using the same software tools (Figure 2). b) Pelvis, cartilage and femur were then exported to the 3-matic™ meshing application (Materialise, Leuven, Belgium). Each object was surface-meshed by triangles. Target size was 2.5 mm for the pelvis, 2 mm for the femur and 1.5 mm for the cartilage (Figure 3). Linear tetrahedral volume meshing was then applied.

c) The three meshed objects were then imported to Abaqus 14™ (Simulia, RI) to construct the finite element model. For modeling, each object was assumed to be homogeneously isotropic, with linear elastic behavior (Table 1). Various interactions between the objects were added. Pelvis and cartilage were taken to be mutually adherent (tie interaction). Between cartilage and femur, a friction interaction was modeled: normal behavior was linear, with 100 MPa/mm penetration coefficient, and tangential behavior was frictionless. Pelvis and distal femoral surface were embedded; the femur was allowed only translation movements along the X and Y axes and rotation around Z. Uniform vertical 600N pressure was applied on the superior side of the pelvis, to simulate bipedal loading (Figure 4). These successive manipulations enabled stress distribution on each femoral head to be obtained. The volume showing stress greater than 50% of the mean value for the femoral head was then exported to the initial 3D model. The intersection between necrosis volume and maximal stress volume was thus obtained and assessed as percentage necrosis/stress intersection (%I n/s) and as percentage stress/necrosis interaction (%I s/n) (Figure 5). %I n/s = necrosis volume within the maximal stress area / total necrosis volume x 100 %I s/n = stress volume within the necrosis area / total stress volume x 100

Statistical analysis

Quantitative variables were expressed as mean±standard deviation, and qualitative variables as percentage; in view of the sample size, the non-parametric Wilcoxon test was used for quantitative variables and the Fisher test for qualitative variables. The significance threshold was set at p = 0.05. To limit disease progression related bias (volume effect), 2 subgroups were distinguished: small OFH (volume < 7,000 mm 3 ) and large OFH (volume > 7,000mm 3 ).

Intra-and inter-observer reproducibility was assessed by intra-class correlation coefficients for %I s/n and %I n/s by 2 different blinded observers delineating the necrosis area on 2 analyses at 7 days' interval, stress areas being determined automatically on the 2 analyses (Table 2).

Results

Only 19 of the 161 patients showed incipient (stage 1 or 2) OFH, and only 10 of these had imaging of sufficient quality for inclusion: 5 males and 5 females, with a mean age of 52 years (range, 37-81 years). OFH was unilateral in all cases: 4 right (40%) and 6 left (60%). Three patients had chronic alcohol abuse (30%), and 5 were taking long-course corticosteroids (50%); 1 had HIV infection (10%), and 1 had idiopathic OFH (10%). There were no significant differences in %I s/n on univariate analysis according to gender (p=0.7), side (p=0.8), or etiology (p=0.7). Likewise, there were no significant differences in %I n/s according to gender (p=0.8), side (p=0.7) or etiology (p=0.8) (Table 4). I.e., necrosis and stress area superimposition seemed independent of gender, side and etiology.

There was a strong inverse correlation between necrosis volume and %I s/n (R 2 = -0.92) and a strong direct correlation between exported stress volume and %I s/n (R 2 = 0.55): the greater the necrosis volume the smaller the %I s/n, and the greater the stress volume the greater the %I s/n. There were no correlations between %I n/s and stress or necrosis volume.

Five of the 10 OFHs were small (< 7,000 mm 3 ) and 5 large (> 7,000 mm 3 ). Table 3 shows subgroup analysis; there were no significant differences according to maximal stress (p=0.682), exported stress volume (p=0.345), or necrosis/stress area intersection or %I n/s (p=0.087). %I s/n was significantly greater in small OFH (n 20n10%; p=0.006) (Table 5). Stress volume within necrosis volume seemed greater in small OFH.

Discussion

To our knowledge, this was the first study to investigate a possible relation between necrosis and stress area locations on 3D finite element analysis. Results seemed to show that the necrosis area is superimposed on the maximal stress area, with percentage necrosis/stress intersection exceeding 80% and percentage stress/necrosis intersection of 35%, especially in small OFH. This finding was independent of biometric factors (gender, etiology, side) and of the factors controlled for in the model (maximal stress, exported volume). An influence of the mechanical environment on the development and/or aggravation of OFH was supported by several previous experimental studies showing that femoral head loading influenced the development of OFH [START_REF] Kim | Effects of non-weight-bearing on the immature femoral head following ischemic osteonecrosis: an experimental investigation in immature pigs[END_REF][START_REF] Nishimura | The role of mechanical stress on the femoral head in the occurrence of femoral head lesions in spontaneously hypertensive rats[END_REF][START_REF] Mihara | Standing is a causative factor in osteonecrosis of the femoral head in growing rats[END_REF][START_REF] Hungerford | The importance of increased intraosseous pressure in the development of osteonecrosis of the femoral head: implications for treatment[END_REF]. Kim et al. [START_REF] Kim | Effects of non-weight-bearing on the immature femoral head following ischemic osteonecrosis: an experimental investigation in immature pigs[END_REF] reported a series of animal models of OFH and concluded that, in pigs with induced OFH, non-weight-bearing slowed the natural progression of osteonecrosis. In a recent study, Ollivier et al. [START_REF] Ollivier | Anatomical Findings in Patients Undergoing Total Hip Arthroplasty for Idiopathic Femoral Head Osteonecrosis[END_REF] likewise found that OFH patients presented anatomic abnormalities liable to modify stress on the summit of the femoral head.

The present study has several limitations. Firstly, the design was single-center and retrospective, limiting extrapolation. Moreover, it was a preliminary study; only 10 scans were included in 3D analysis, which highlights two points: the difficulty of large-scale inclusion in this rare pathology, and the infraclinical progression and diagnostic nomadism of some patients who consult only after long disease progression and serious deterioration of bone tissue. The 3D finite element model is also debatable: it was not always possible to differentiate between cartilage of the head and of the labrum due to the pathology and to the poor resolution of CT without contrast enhancement. This led us to create an intermediate object to represent the cartilage; this compromise, however, has been validated in the recent literature on a similar subject [START_REF] Sanchez Egea | Impact of hip anatomical variations on the cartilage stress: a finite element analysis towards the biomechanical exploration of the factors that may explain primary hip arthritis in morphologically normal subjects[END_REF]. We did not differentiate cortical and trabecular bone on Young's modulus or Poisson coefficient, assuming the bone to be smooth and homogeneous throughout, as it is difficult to reconstruct and model femoral trabecular bone, which differs between head, neck and trochanteric region, and as the properties of necrotic trabecular bone have not been described. The present method was used in other 3D models of the hip [START_REF] Anderson | Effects of idealized joint geometry on finite element predictions of cartilage contact stresses in the hip[END_REF][START_REF] Chen | A Three-Dimensional Finite Element Model for Biomechanical Analysis of the Hip[END_REF]. The present mechanical model was, however, robust, including interactions between the objects (tie interaction between pelvis and cartilage, and linear friction between cartilage and femur) [START_REF] Sanchez Egea | Impact of hip anatomical variations on the cartilage stress: a finite element analysis towards the biomechanical exploration of the factors that may explain primary hip arthritis in morphologically normal subjects[END_REF]18], and the permitted movements [START_REF] Chen | A Three-Dimensional Finite Element Model for Biomechanical Analysis of the Hip[END_REF][18][START_REF] Yoshida | Three-dimensional dynamic hip contact area and pressure distribution during activities of daily living[END_REF][START_REF] Anderson | Validation of Finite Element Predictions of Cartilage Contact Pressure in the Human Hip Joint[END_REF] and loads [START_REF] Anderson | Validation of Finite Element Predictions of Cartilage Contact Pressure in the Human Hip Joint[END_REF][START_REF] Grecu | Numerical simulations of the 3D virtual model of the human hip joint, using finite element method[END_REF][START_REF] Recnik | Higher peak contact hip stress predetermines the side of hip involved in idiopathic osteoarthritis[END_REF][START_REF] Bergmann | Hip contact forces and gait patterns from routine activities[END_REF] validated in the literature. Moreover, it comprised 10 3D analyses, ensuring greater clinical relevance than models extrapolated from a single hip as in all other finite element modeling studies. Reconstructions were based on pathological femurs under normal therapeutic conditions rather than experimental conditions, further enhancing clinical relevance. And lastly, all patients had very early necrosis, without femoral deformity, thus providing "cleaner" models without the alterations in stress distribution associated with advanced necrosis.

The present mean maximal stress value on finite element analysis (1.63 MPa) was low in comparison to certain reported values, which range between 3 and 6 MPa [START_REF] Sanchez Egea | Impact of hip anatomical variations on the cartilage stress: a finite element analysis towards the biomechanical exploration of the factors that may explain primary hip arthritis in morphologically normal subjects[END_REF][START_REF] Yoshida | Three-dimensional dynamic hip contact area and pressure distribution during activities of daily living[END_REF][START_REF] Recnik | Higher peak contact hip stress predetermines the side of hip involved in idiopathic osteoarthritis[END_REF][START_REF] Genda | Normal hip joint contact pressure distribution in single-leg standing--effect of gender and anatomic parameters[END_REF][START_REF] Steinberg | Determining lesion size in osteonecrosis of the femoral head[END_REF]; this was due to greater pressure being applied directly on the femur in these studies (Table 6): these previous studies aimed to model forces passing through the hip during daily life activity, representing loading equivalent to 4 times the body-weight [START_REF] Recnik | Higher peak contact hip stress predetermines the side of hip involved in idiopathic osteoarthritis[END_REF]. We chose to apply a 600N load, modeling standard bipedal weight-bearing, exerted vertically downward on the superior side of the pelvis, which seemed more physiological than force applied directly on the femur, as we sought to study stress transmitted from the pelvis to the femoral head; moreover, increasing the load would doubtless have increased the stress area, enhancing %I values but introducing a measurement bias similar to that we described in patients with large necrosis volume. Comparison between the present results and the literature is difficult, as previous studies focused rather on the optimal means of quantifying necrosis volume [START_REF] Bergmann | Hip contact forces and gait patterns from routine activities[END_REF], the influence of necrosis volume on fracture risk [START_REF] Genda | Normal hip joint contact pressure distribution in single-leg standing--effect of gender and anatomic parameters[END_REF], the impact of anatomic variants on OFH [START_REF] Fraitzl | Reduced head-neck offset in nontraumatic osteonecrosis of the femoral head[END_REF][START_REF] Ollivier | Anatomical Findings in Patients Undergoing Total Hip Arthroplasty for Idiopathic Femoral Head Osteonecrosis[END_REF], or the effects of different treatments [START_REF] Bergmann | Hip contact forces and gait patterns from routine activities[END_REF]. Anderson et al. [START_REF] Anderson | Validation of Finite Element Predictions of Cartilage Contact Pressure in the Human Hip Joint[END_REF] reported only contact areas ranging between 304.2 and 366.1 mm 2 in their finite element analysis. To our knowledge, the maximal stress volume on the femoral head has never been calculated or studied elsewhere, and nor has superimposition between necrosis and stress areas. This may explain the better results found with aggressive treatment of OFH such as femoral varization or rotation osteotomy [START_REF] Genda | Normal hip joint contact pressure distribution in single-leg standing--effect of gender and anatomic parameters[END_REF][START_REF] Steinberg | Determining lesion size in osteonecrosis of the femoral head[END_REF][START_REF] Volokh | Prediction of femoral head collapse in osteonecrosis[END_REF][START_REF] Baba | Long-term results of rotational acetabular osteotomy for osteonecrosis with collapse of the femoral head in young patients[END_REF][START_REF] Yamamoto | Results of the Sugioka transtrochanteric rotational osteotomy for osteonecrosis: Frequency and role of a defect of the quadratus femoris muscle in osteonecrosis progression[END_REF][START_REF] Sugioka | Transtrochanteric posterior rotational osteotomy for osteonecrosis[END_REF] than with conservative treatments such as decompression [START_REF] Schneider | The value of core decompression in treatment of femur head necrosis[END_REF][START_REF] Bellot | Core decompression of the femoral head for avascular necrosis[END_REF] or stem-cell injection [START_REF] Rosset | Cell therapy for bone repair[END_REF] that do not modify stress distribution on the head. This theory, however, would not be applicable to certain OFH etiologies such as sickle-cell disease, where the necrosis area is usually posterior [START_REF] Mukisi-Mukaza | Risk factors and impact of orthopaedic monitoring on the outcome of avascular necrosis of the femoral head in adults with sickle cell disease: 215 patients case study with control group[END_REF].

Conclusion

The results of the present finite element model of OFH support the mechanical theory of the pathophysiology of osteonecrosis of the hip. Hip anatomy and biomechanics seem to play a nonnegligible role in the genesis of OFH: in the present study, necrosis developed in the area of the femoral head in which stress was greatest, although no direct causal relation can be affirmed. These findings need confirmation in larger-scale multicenter studies using a finite element model even closer to clinical reality. It seems to us to be primordial to take these mechanical parameters into account to limit failure of conservative treatment of OFH.

Figure 1 :

 1 Figure 1: Creation of objects (pelvis, cartilage and femur) smoothed under Mimics™ (Materialise, Leuven, Belgium) to obtain a 3D structure optimally homogeneous and faithful to patient anatomy.

Figure 2 :

 2 Figure 2: Location and definition of necrosis area.

Figure 3 :

 3 Figure 3: Surface-meshing by triangles. Element target size was 2.5 mm for the pelvis, 2 mm for the femur and 1.5 mm for the cartilage.

Figure 4 :

 4 Figure 4: Application of uniform vertical downward 600N pressure on the superior side of the pelvis, to simulate bipedal loading.

Figure 5 :

 5 Figure 5: Visualization of the intersection between necrosis and maximal stress volumes.

Table 3 shows

 3 %I results in the overall series. Mean maximal stress was 1.628 nnnnnnn MPa. Mean necrosis/maximal stress intersection volume was 1,837.2 nn409.1 mm 3 , mean %I n/s 82.1 nn14%, and mean %I s/n 35.3 nn13.1%.

Table 1 :

 1 Material properties used in finite element model

		Type of material	Young's modulus (MPa) Poisson coefficient
	Pelvis	Bone	10,000	0.3
	Femur	Bone	10,000	0.3
	Cartilage	Cartilage	10	0.4

Table 2 :

 2 Intra-and inter-observer reproducibility was assessed by intra-class correlation coefficients (ICC) with 95% CI for %I s/n and %I n/s by 2 different blinded observers delineating the necrosis area on 2 analyses at 7 days' interval.

	Intra-observer reproducibility	ICC	95% CI
	Observer 1	0.95	0.89-0.99
	Observer 2	0.91	0.85-0.97
	Inter-observer reproducibility	0.89	0.84-0.94

Table 3 :

 3 Analysis of superimposition in the overall series. (percentage necrosis/stress intersection) = necrosis volume within maximal stress area / total necrosis volume x 100. %I s/n (percentage stress/necrosis intersection) = stress volume within necrosis area / total stress volume x 100.

	Patient	Ficat stage [12]	Max. stress (MPa)	Exported stress volume (mm 3 )	Necrosis volume (mm 3 )	Necrosis/stress (mm 3 ) intersection	%I s/n	%I n/s
	1	1	1.48	1,652.4	3,038	1,510,1	50	91
	2	2	0.93	3,064.2	7,213.5	2,524.3	35	82
	3	2	1.38	2,645.7	3,423.7	1,823.3	53	69
	4	1	1.18	2,580	2,961.7	1,501.9	51	58
	5	1	3.09	2,019	13,947.5	1,792.5	13	89
	6	2	1.12	2,350	7,849.8	2,274.5	29	97
	7	2	1.56	2,356.5	8,635.3	2,036.9	24	86
	8	2	1.89	2,103.1	7,789	2,082.7	27	99
	9	2	2.13	1,858.4	5,196	1,696.7	33	91
	10	1	1.52	1,739.5	2,856.1	1,129.1	40	65
	Mean		1.628	2,236.8	6291.1	1,837.2	35	83
	Standard deviation	0.624	446.474	3,535.961	409.097	13	14
	%I n/s

Table 4 :

 4 Influence of confounding demographic and etiologic factors on endpoints. %I n/s (percentage necrosis/stress intersection) = necrosis volume within maximal stress area / total necrosis volume x 100. %I s/n (percentage stress/necrosis intersection) = stress volume within necrosis area / total stress volume x 100.

Table 5 :

 5 Superimposition rate (mean±SD) by subgroup.