Knee Surgery, Sports Traumatology, Arthroscopy https://doi.org/10.1007/s00167-022-06935-2 KNEE The morphology of the femoral footprint of the anterior cruciate ligament changes with aging from a large semicircular shape to a small flat ribbon‑like shape Rodolfo Morales‑Avalos1,2,3 · Simone Perelli1,2 · Félix Vilchez‑Cavazos3 · Tadeo Castillo‑Escobedo3 · Víctor M. Peña‑Martínez3 · Rodrigo Elizondo‑Omaña4 · Santos Guzmán‑López4 · José Ramón Padilla‑Medina3 · Juan Carlos Monllau1,2 Received: 9 October 2021 / Accepted: 4 March 2022 © The Author(s) 2022 Abstract Purpose Compare the differences in the morphology of the ACL femoral footprint between the cadavers of the young and elderly in consideration of the degenerative physiological process that occurs with aging. Methods The femoral footprint of the ACL was dissected in 81 knees of known gender and age (45 male/36 female). They were divided into four groups by age and gender, establishing 50 years as the cut-off point to divide patients by age. Three observers analyzed the femoral footprint dissections, and the shapes were described and classified. The area and morpho- metric characteristics of the femoral insertion of the ACL were determined and these were compared between genders and age groups. Results The femoral footprint of the ACL from the cadavers of males younger than 50 years of age presented a semicircular morphology in 90% of the cases. In males aged more than 50 years, a ribbon-like morphology was found in 96% of the cases. In women less than 50 years old, the semicircular morphology was observed in 93.7% of the cases. In women aged over 50 years old, the ribbon-like morphology was found in 95% of the cases. A significant difference was observed between the prevalence rates of the morphologies, area size and measurements of the younger and older groups (p < 0.001 for both genders). Conclusions The femoral insertion of the ACL presents variations in its morphology, area and morphometric characteristics over time. It goes from a large semicircular shape that almost contacts the posterior articular cartilage to a smaller, flattened ribbon-like shape that moves away from the edge of the articular cartilage. It is bounded anteriorly by the lateral intercondy- lar ridge. These findings should be considered to avoid employing reconstruction techniques in which femoral tunnels with oval or rectangular shapes are used in patients under 50 years of age because they do not correspond to the morphology of the femoral insertion of the ACL in this age group. Keywords Anterior cruciate ligament · Aging · Area · Attachment · Femoral · Footprint · Insertion site · Morphology · Shape · Variations 3 * Rodolfo Morales‑Avalos Knee Unit, Department of Orthopedic Surgery rodolfot59@hotmail.com and Traumatology, School of Medicine and University Hospital “Dr. José Eleuterio González”, Universidad 1 Knee and Arthroscopy Unit (ICATKNEE‑EQUILAE), Autonoma de Nuevo León (U.A.N.L), Monterrey, Department of Orthopedic Surgery, Institut Catalá de Nuevo León, México Traumatologia i Medicina de l’Esport (I.C.A.T.M.E.), 4 Department of Human Anatomy, School of Medicine Hospital Universitari Dexeus, Universitat Autonoma de , Universidad Autonoma de Nuevo León (U.A.N.L.), Barcelona (U.A.B.), Barcelona, Catalunya, Spain Monterrey, Nuevo León, México 2 Department of Surgery and Morphologic Science, Orthopaedic Surgery Service, Hospital del Mar, Universitat Autònoma de Barcelona, Barcelona, Spain 13 Vol.:(0123456789) Knee Surgery, Sports Traumatology, Arthroscopy Introduction Materials and methods Anatomic anterior cruciate ligament (ACL) reconstruction This study was approved by the Institutional Review Board attempts to recreate the native dimensions of the ACL, colla- and Ethics Committee of the School of Medicine and Uni- gen orientation and insertion sites to recreate the function of versity Hospital “Dr. Jose Eleuterio Gonzalez” of the Uni- the native ligament [32, 42]. It has already been shown that versidad Autonoma de Nuevo Leon with registration number anatomic ACL reconstruction, in which the graft is placed OR16-00007. The study was carried out in accordance with within the native ACL insertions, might result in better knee the World Medical Association Declaration of Helsinki of stability when compared to nonanatomic techniques [6, 24]. 1964 and its revision in 2013 [45]. An anatomical, descrip- Furthermore, the correct placement of the femoral tunnel is tive, observational and comparative study was performed known as one of the most important factors influencing knee with 88 non-paired, formalin-fixed human cadaver knees kinematics and clinical results in anatomic ACL reconstruc- from the Mexican population is included. tion [23, 34]. The cadavers were collected from three distinct depart- ACL reconstruction was conventionally performed with ments of human anatomy between 2016 and 2020. All a single-round femoral tunnel. However, some anatomical specimens were over 18 years of age at the time of death. studies have revealed that the ACL femoral footprint is rela- The gender, weight, and height of each cadaver were docu- tively elliptical in shape, not round [19]. Therefore, the use mented. The cause of death and the most important clinical of bi-fascicular techniques or the realization of rectangu- information for each corpse was known and varied between lar or oval tunnels have been proposed to try to imitate the the cadavers of the young and elderly. Head trauma was native anatomy of the femoral insertion of the ACL in a predominant in the young group and myocardial infarction more precise way. Still, some studies have described mor- and metabolic disorders in the elderly group. Unequivocally phologies of the femoral insertion as being crescent-shaped, so, none of the causes of death or medical history of the circular/round [12], semi-circular [10] or ovalized [10, 11]. included cadavers influenced the execution and results of Moreover, differences in the size of the femoral insertion this study. have been described. A few studies have reported a long The sample size was determined based on the availability femoral insertion that extends backward to the articular car- of cadavers of individuals under 50 years of age. Data from tilage margin [3, 7, 12, 18, 29], whereas other studies have a previous study of ACL morphology confirmed that the described a relatively narrow insertion area [11, 26, 40]. sample size was sufficient to detect differences among ages Due to this confusion, a large number of anatomical stud- [27]. Furthermore, a post-hoc power analysis was performed ies have been carried out that have tried to precisely define to assess the adequacy of the sample size. Additionally, the the morphological characteristics of the femoral insertion goal of the study was for it to be done with the largest sam- of the ACL [7, 10–12, 16, 19, 34, 39, 40]. However, most of ple reported in the literature. The sample was divided into those studies were carried out with small samples and with four groups according to age and gender: males and females elderly cadavers, which could lead to errors in the deter- younger and older than 50 years. This cut-off age was used mination of the true morphological characteristics of the based on a previous study that showed statistically signifi- ACL. In addition, on other occasions, they do not make a cant differences in the tibial footprint morphology of the clear distinction between the analysis of the direct insertion ACL [27]. (mid-substance insertion) and the indirect insertion (fan-like All the knees were analyzed in phases to make sure extension) of the ACL. they fulfilled the inclusion criteria. Before being included A recent study determined that the tibial insertion of the in the study, every specimen underwent an anteroposterior ACL presents significant morphological variations with age radiograph to exclude knees that presented an intraarticu- [27]. Therefore, it is thought that similar changes may occur lar fracture sequela, the presence of orthopedic implants or at the femoral insertion. The aim of the study was to com- congenital bone anomalies. However, they were not used pare the differences in the morphology of the ACL femoral for the radiographic classification of osteoarthritis. In the footprint between the cadavers of the young and elderly due inspection, those that presented scars from previous surgical to a degenerative physiological process that occurs over interventions were excluded. Those knees that, at the time time. The hypothesis is that there are significant variations of the dissection, presented an ACL or macroscopic rupture in the shape of the femoral insertion of the ACL between the of the posterior cruciate ligament (PCL), advanced osteoar- cadavers of younger people and older people. The results throsis (Outerbridge Grade IV) or macroscopic degenerative of this study will help to determine the normal shape and changes in the synovial membrane that impaired adequate position that the femoral tunnel should have in a more indi- observation or dissection of structures of interest were vidualized way. excluded. Furthermore, the presence of osteophytes in the 13 Knee Surgery, Sports Traumatology, Arthroscopy medial region of the lateral femoral condyle, which could tension pattern in the range-of-motion. The AM bundle interfere with the measurements of distances between the fibers become taut during flexion, while PL bundle fib- ACL attachment and the articular cartilage of the condyle, ers become taut by extension. It is important to mention was considered criteria for exclusion. A knee surgeon (SP) that it was not possible to show a division into fascicles and an anatomist (SGL) independently reviewed the speci- in all cases. Thus, it was only done on specimens where mens for inclusion. it was possible. It is worth noting that it was particularly difficult in the elderly specimens. The synovial envelope of the ACL bundles was carefully excised with a surgical blade and then sutures with different colors were passed Dissection technique around each bundle to identify them when it was possible. A meticulous dissection of the ACL fibers was performed Prior to dissection, the specimens were submitted to a 10% up to their osseous insertion in the lateral femoral condyle. formaldehyde fixative solution and stored in a mixture of The insertional fibers were carefully separated from the 54% ethylene glycol, 40% ethanol and 6% formaldehyde surrounding synovial tissue while registering the presence with colorant (CTR ­Scientific®, Monterrey, Mexico). Sub- of the indirect ACL insertion fibers or “fan like” exten- sequently, the specimens were kept for 60 days in liquid sion fibers by identifying a macroscopically visible fold glycerin [2]. between the direct and indirect insertion of the ACL [39]. The dissection technique was standardized and performed At the same time, the relationship of the femoral inser- in the same way by all the team members to ensure consist- tion to the lateral intercondylar ridge was analyzed using ency, and was based on our own experience and the dissec- a microsurgical microscope with 10 × magnification (Carl tion protocols reported in previous studies [3, 16, 19, 26, 34, Zeiss ­OPMI® Pico, Berlin, Germany). To properly identify 37, 39, 40]. The team consisted of a knee surgeon (FVC), the footprint without damaging it during dissection, an an anatomist (REEO), and a senior orthopedic surgery and ink stain was applied on the bone immediately around the traumatology resident (JRPM). periphery of the entire femoral insertion site (direct + indi- The limbs were amputated at two sites, the first at the rect portion) by one of the authors who did not participate level of the thigh at 30 cm from the knee joint line and the in the dissections or in the morphological classification of other at the leg at 20 cm from the knee joint line. During the femoral footprint (VMPM) [16]. Then, the ACL was the dissections, the knees were placed in a mechanical press delicately excised as closely as possible to the intercon- that kept the knees in full extension and made flexion at 30°, dylar wall of the lateral condyle in 90° of flexion using 60° and 90° possible without any translational or rotational a No. 11 scalpel blade. After that step, the previous ink forces. mark was removed. The remnant of the femoral insertion All superficial soft tissues on the anterior, posterior, medial and lateral surfaces of the knee were removed up to the joint capsular plane. The entire extensor apparatus was resected distally from the anterior tibial tuberosity and subsequently removed. After exposing the anterior side of the knee joint, the synovial tissue and the Hoffa fat pad were carefully dissected and separated from the joint soft tissue structures (meniscus and transverse ligament). The entire joint capsule, tibial and fibular collateral ligaments, menisco-femoral ligaments, posterior cruciate ligament and synovial tissues were carefully excised. Starting from the uppermost point of the intercondylar notch, the femur was separated along the sagittal plane with an oscillating saw (Precision 7, S ­ tryker®, Madrid, Spain). This was done to observe the full medial wall of the lateral femoral condyle and the lateral intercondylar ridge (resident’s ridge). Care was taken to avoid injury to the ACL or its bony insertion. Exposure after the medial condylectomy made for an excellent view of the femoral Fig. 1  Different areas and zones of the femoral insertion of the ACL attachment of the ACL. The anteromedial and postero- in a cadaveric knee from the young group. The whole femoral ACL footprint (red), direct insertion (green), indirect insertion (yellow), the lateral fascicles (AM and PL) were identified by observ- direct insertion area of AM (A) and PL bundles (B), fan-like exten- ing the orientation of the fibers and the differences in the sion fibers area of AM (C) and PL bundles (D) 13 Knee Surgery, Sports Traumatology, Arthroscopy Fig. 2  Morphometries performed on each knee like this with the mil- attachment (E), Border of posterior articular cartilage in a horizontal limeter digital vernier and then were performed a second time using line with the posterior extent of articular cartilage (F), Border of pos- the image J computer program. Proximal border of the direct ACL terior articular cartilage in a horizontal line with the distal border of attachment (A), Distal border of the direct ACL attachment (B), the direct ACL attachment (G), Border of the anterior articular car- Anterior extent of the direct ACL attachment (C), Posterior extent of tilage in a horizontal line with the anterior extent of the direct ACL the direct ACL attachment (D), Border of the posterior articular car- attachment (H), Border of the inferior articular cartilage in a vertical tilage in a horizontal line with the proximal border of the direct ACL line with the distal border of the direct ACL attachment (I) after its transection as well as the previous division of its 3. Horizontal distance between the proximal border of the fascicles served for its later classification. direct ACL attachment and the adjacent margin of the Following a method previously described, all the differ- articular cartilage (A–E) ent ACL footprint portions were marked with ink: (1) the 4. Horizontal distance between the distal border of the whole femoral ACL footprint, (2) the direct insertion, (3) direct ACL attachment and the adjacent margin of the the indirect insertion (fan-like extension), (4) the whole articular cartilage (B–G) AM and PL area, (5) the direct insertion area of AM and 5. Horizontal distance between the posterior extent of the PL bundles and (6) the fan-like extension fibers area of direct ACL attachment and the posterior articular sur- AM and PL bundles (Fig. 1) [16]. The points 4–5–6 were face (D–G) marked only when the AM and PL bundles were clearly 6. Horizontal distance between the anterior extent portion identifiable. of the direct ACL attachment and the anterior articular Subsequently, the team of surgeons proceeded to meas- surface (C–H) ure the following morphometric parameters using a mil- 7. Vertical distance between the distal border of the direct limeter digital caliper with a precision of 0.01 mm (Mitu- ACL attachment and the inferior articular surface (B–I). toyo Digimatic w/Absolute Encoders-Series 500, Tokyo, Japan). Figure 2 details each of the landmarks used. The Afterwards, the ink was again completely removed, and landmarks were defined by consensus in the dissection a reference marker was used as the specimens were pho- team when it was done. tographed using an 18.7-megapixel high-resolution digital camera (model EOS 1300D, Nikon Corp., Tokyo, Japan) 1. Proximodistal diameter of the direct ACL attachment positioned on a professional tripod (Canon Inc., Tokyo, area (long axis) (A–B) Japan). The photograph consisted of an accurate view of 2. Anteroposterior diameter of the direct ACL attachment the medial wall of the lateral femoral condyle perpendicular area (short axis) (C–D) to the femoral insertion with the knees placed at 0° and 90° 13 Knee Surgery, Sports Traumatology, Arthroscopy of knee flexion. The camera lens was placed 25 cm parallel Table 1  Distribution of the sample by gender and age of the knees to the articular surface, and the ACL femoral footprint was at included in the study the center of the image. In this way, a high-resolution image Category Semicircular Ribbon-like of each sample was recorded in case of deterioration and to Male > 50 (n = 25) 1 (4%) 24 (96%) be used to resolve differences between observers in the post- Male < 50 (n = 20) 18 (90%) 2 (10%) consensus meeting. The same images were used to calculate Female > 50 (n = 20) 1 (5%) 19 (95%) the area of the ACL femoral footprint and to repeat the same Female < 50 (n = 16) 15 (93.75%) 1 (6.25%) morphometric analyses carried out manually. All dissections and photographs were evaluated by three independent observers. There were three knee surgeons (RMA, TACE and JCM) blind to the age and gender of the to calculate the Bonferroni-corrected alpha: pre-specified specimens. It was carried out just after the dissection and alpha/number of comparisons. A p value of less than 0.05 in a second revision after 2 weeks. The specimens were was considered significant and all tests were two-tailed. An again evaluated in a different order from the first evaluation. a priori sample size estimation was not done as the decision Moreover, the 2nd round of evaluations was blinded to the was taken to use all the samples that were available in our results of the first evaluation. During the interval, the speci- laboratory, regardless of the result. Post-hoc power analyses mens were stored in a cold room for preservation. After the were performed based on the main outcome of finding differ- evaluations were concluded, the differences were reviewed ences between different age groups. The asymptotic relative in a consensus meeting using a nominal group technique efficiency method was performed for the Mann–Whitney U without a hierarchy. The shape of the femoral footprint was tests. The results are presented as median observed power described as ribbon-like (or flattened), oval/elliptical, round/ and median sample required for achieving 80% power on an circular, semicircular, small-semicircular or undetermined in alfa level of 0.05 based on observed differences. accordance with the classifications previously proposed [3, 18, 19, 26, 34, 35, 37]. The images were downloaded to a personal computer. The Results Image J software version 1.49 (National Institute of Health, Bethesda, USA, http://​rsbweb.​nih.​gov/​ij/) was used to per- In the final analysis, 81 knees were included (45 male and form the previously described measurements and compare 36 female). The distribution of the sample by gender and them with those made at the time of dissection. Moreover, age is shown in Table 1. From the original sample of 88 the software was used to estimate the footprint area after knees 7 were eliminated. Advanced osteoarthritis of the adjusting the computer images to the actual knee size. The tibio-femoral or patello-femoral joint was present in 3 of whole ACL area and the whole direct and indirect insertion them, 2 had scars that were probably the result of previous area were always calculated. In cases where a precise defini- knee surgery, and 2 had a complete ACL tear. The mean age tion of the two bundles was possible, the whole area and the of the specimens was 87 years ± 8.3 for the group of males direct insertion area as well as the fan-like extension fibers over 50 yearsold, 28 years ± 6.2 for the group of males under area of the AM and PL bundles were also calculated [16]. 50 years of age, 82 years ± 9.2 for the females over 50 years The accuracy of the area measurement was less than 0.1 mm old and 26 years ± 7.4 for the females under 50. Significant and 0.1 ­mm2. differences in the ages of the specimens from different gen- ders were not observed. The mean weight and height of the Statistical analysis cadaveric specimens were 79.5 kg ± 13.6 and 1.66 ± 0.08, respectively. The results of interobserver analysis were con- Statistical analyses were performed using SPSS software cordant (Kappa = 0.93) and the results of the intraobserver package (version 19.0 for Windows XP). Intra and inter-rate analysis were always the same (Kappa = 1). No difference reliability was calculated using the Cohen’s kappa and Fleiss was detected between the morphometric values obtained Kappa coefficients, respectively. The Mann–Whitney U test from the cadavers and from the images. was used to determine the significance of the differences between the ages of the different groups. The Chi-square test ACL femoral footprint shape was used to compare genders within the same age group and to compare age groups between specimens of the same gen- The knees from the cadavers of males younger than 50 years der. Because we performed multiple comparisons, we used of age presented a “semicircular” morphology in 90% the Bonferroni correction method to calculate the appropri- (n = 18/20) of the cases (Fig. 3A, B). In the group of males ate alpha value for which associations can be considered over 50 years old, a ribbon-like appearance was founded in statistically significant. The following formula was used 96% (n = 24/25) of the cases (Fig. 3C, D). In the group of 13 Knee Surgery, Sports Traumatology, Arthroscopy morphologies. When independently comparing the group of men < 50 years vs males > 50 years, females < 50 years vs females > 50 years and the totality of the knees < 50 years all the knees vs > 50 years (regardless of gender), the value of p was < 0.001. However, no differences were observed between males and females of the same age group (Table 2). Macroscopic anatomy In young cadavers of both genders, the most prevalent mor- phology was semicircular, which had a straight anterior border that contacts the lateral intercondylar ridge and its posterior side convex that almost contacts the posterior articular car- tilage. The indirect fibers in this group were scarce and they occupied a small area in the portion closest to the posterior articular cartilage. It was possible to identify the two ACL bundles separately in 100% of the cases. In all knees, the direct insertion site and the fanlike extensions were successfully divided. In the cadavers of people of advanced age, the direct femoral insertion moves away from the posterior edge of the articular cartilage and is kept in bounded anteriorly by the lat- eral intercondylar ridge, having a continuity with the posterior cortex of the femur. The insertion morphology is thinner and has the shape of a flattened ellipse, simulating a ribbon. It is smaller compared to its younger counterpart and the indirect insertion fibers are more abundant while occupying a larger Fig. 3  Representative images of the different ACL morphologies area and almost come into contact with the posterior edge of observed in this study before and after cutting the ACL near its femo- the articular cartilage. In some cases (8/45), it was not possible ral insertion. A Semicircular morphology. B Oval/elliptical morphol- ogy. C Semicircular morphology without the ligament. D Oval/ellip- to identify the two bundles of the ligament, individually. tical morphology without the ligament. The asterisks (*) indicate the femoral insertion sites of the ACL in the specimens before and after Morphometries and areas the ligament is cut The average measurements of the morphometries and areas of the study groups are shown in Tables 3 and 4. Relative Table 2  Values of p obtained by comparing the different study groups to gender, there were no significant differences for substan- relative to age and gender tially few variables. One exception was the entire area of Comparison p value the ACL (127 ­mm2 ± 23 in Women vs. 145 ­mm2 ± 21 in men, p = 0.01 and 111 m ­ m2 ± 18 vs 123 m ­ m2 ± 19, p = 0.03 males < 50 years vs. males > 50 years < 0.001 for < 50 and > 50 years old, respectively). However, a pattern females < 50 years vs. females > 50 years < 0.001 of greater morphometries and areas of the ACL in men than total knees < 50 years vs. total knees > 50 years < 0.001 in women was observed. Moreover, the distance from the males < 50 years vs. females < 50 years n.s. posterior edge of the insertion to the articular cartilage was males > 50 years vs. females > 50 years n.s. always greater in women. Nonetheless, all these results were Total male knees vs. total female knees n.s. not significant (Table 3). Most of the distances between the articular cartilage and the direct femoral insertion of the ACL (the distances up to women less than 50 years old, the semicircular morphology the posterior articular cartilage) were significantly greater was observed in 93.7% (n = 15/16) of the cases. In women in the older group. At the same time, it was noted that the over 50 years, the ribbon-like morphology was found in 95% anteroposterior diameter of the direct insertion of the ACL (n = 19/20) of the cases (Table 1). No other types of mor- significantly decreased in both genders with the passage of phologies previously described in the literature were found. time. The ACL as well as all the areas (direct and indirect) The chi-square test was applied to compare the of the AM and PL fascicles do not present significant varia- results of the prevalence rates of the different observed tions with respect to age. However, the proportion between 13 Knee Surgery, Sports Traumatology, Arthroscopy Table 3  Comparison between the average values obtained in each gender for the same age group Category Age Female (mean ± SD) Male (mean ± SD) Female vs. range male (p (years) value) Proximodistal diameter of the direct ACL attachment area (Long axis) < 50 16.55 mm ± 2.42 17.02 mm ± 2.24 n.s. > 50 13.22 mm ± 1.85 15.44 mm ± 1.95 n.s. Anteroposterior diameter of the direct ACL attachment area (Short axis) < 50 9.32 mm ± 1.13 11.22 mm ± 1.4 n.s. > 50 3.12 mm ± 0.72 4.92 mm ± 0.85 n.s. Horizontal distance between the proximal border of the direct ACL attach- < 50 2.2 mm ± 0.55 2.0 mm ± 0.48 n.s. ment and the adjacent margin of the articular cartilage > 50 7.3 mm ± 2.4 6.7 mm ± 1.93 n.s. Horizontal distance between the distal border of the direct ACL attachment < 50 2.5 mm ± 0.42 2.3 mm ± 0.66 n.s. and the adjacent margin of the articular cartilage > 50 8.8 mm ± 1.76 8.5 mm ± 2.12 n.s. Horizontal distance between the posterior extent of the direct ACL attach- < 50 2.3 mm ± 0.61 2.1 mm ± 0.59 n.s. ment and the posterior articular surface > 50 8.2 mm ± 2.02 7.9 mm ± 1.75 n.s. Horizontal distance between the anterior extent portion of the direct ACL < 50 13.21 mm ± 1.25 14.32 mm ± 1.66 n.s. attachment and the anterior articular surface > 50 14.55 mm ± 1.87 15.02 mm ± 2.04 n.s. Vertical distance between the distal border of the direct ACL attachment and < 50 2.02 mm ± 0.88 1.55 mm ± 0.72 n.s. the inferior articular surface > 50 2.97 mm ± 1.22 2.43 mm ± 1.07 n.s. Whole femoral footprint area < 50 127 ­mm2 ± 23 145 ­mm2 ± 21 0.012* > 50 111 ­mm2 ± 18 123 ­mm2 ± 19 0.03 Whole direct insertion area < 50 104 ­mm2 ± 17 122 ­mm2 ± 20 n.s. > 50 57 ­mm2 ± 11 61 ­mm2 ± 15 n.s. Whole indirect insertion area < 50 19 ­mm2 ± 6 17 ­mm2 ± 4 n.s. > 50 47 ­mm2 ± 14 56 ­mm2 ± 15 n.s. Whole AM footprint area < 50 67 ­mm2 ± 15 78 ­mm2 ± 13 n.s. > 50 55 ­mm2 ± 13 62 ­mm2 ± 14 n.s. Direct insertion area of AM bundle < 50 52 ­mm2 ± 10 63 ­mm2 ± 11 n.s. > 50 26 ­mm2 ± 7 32 ­mm2 ± 8 n.s. Indirect insertion area of AM bundle < 50 10 ­mm2 ± 3 12 ­mm2 ± 3 n.s. > 50 25 ­mm2 ± 5 34 ­mm2 ± 8 n.s. Whole PL footprint area < 50 59 ­mm2 ± 10 65 ­mm2 ± 11 n.s. > 50 53 ­mm2 ± 8 58 ­mm2 ± 9 n.s. Direct insertion area of PL bundle < 50 48 ­mm2 ± 5 51 ­mm2 ± 6 n.s. > 50 24 ­mm2 ± 3 29 ­mm2 ± 5 n.s. Indirect insertion area of PL bundle < 50 10 ­mm2 ± 1 12 ­mm2 ± 2 n.s. > 50 22 ­mm2 ± 5 28 ­mm2 ± 7 n.s. We used the Bonferroni correction method to determine the p value at which the comparisons would be considered statistically significant due to multiple comparisons. The Bonferroni-corrected alpha value in these comparisons is 0.0125 instead of the traditional 0.05. Significant p values at this threshold are marked with an asterisk the direct and indirect areas between the younger and older Discussion knees does show significant variations over time. The whole area of the ACL does not show significant variations but The main finding of the present study was that the femoral the proportion between their direct and indirect insertion insertion of the ACL presents variations in its morphology areas changed with aging, the direct areas being significantly over time. It goes from being large and semicircular in shape higher in the under 50 groups and the indirect insertion areas in young subjects to a smaller and flattened ribbon-like significantly greater in the over 50 groups (Table 4). shape in older subjects, which confirms the initial hypoth- Relative to the result from the post-hoc power analyses, esis. In the same way, significant differences were detected the median power achieved across comparisons between in most of the morphometric parameters and areas analyzed age groups was 0.99 (0.81–0.99), with median sample size between the different age groups of the same gender. Addi- requirements of 4 (3–10) cadavers per group. This confirms tionally, there were no differences between genders in the the validity of the acquired sample for this study. same age group. 13 Knee Surgery, Sports Traumatology, Arthroscopy Table 4  Comparison between the average values obtained in each age group for the same gender Category Gender < 50 years (mean ± SD) > 50 years (mean ± SD) < 50 years vs > 50 years (p value) Proximodistal diameter of the direct ACL attachment area Female 16.55 mm ± 2.4 13.22 mm ± 1.8 n.s. Male 17.0 mm ± 2.2 15.44 mm ± 1.9 n.s. Anteroposterior diameter of the direct ACL attachment area Female 9.32 mm ± 1.1 3.12 mm ± 0.72 < 0.0001* Male 11.22 mm ± 1.4 4.92 mm ± 0.85 < 0.0001* Horizontal distance between the proximal border of the direct Female 2.2 mm ± 0.55 7.3 mm ± 2.4 < 0.0001* ACL attachment and the adjacent margin of the articular Male 2.0 mm ± 0.48 6.7 mm ± 1.93 < 0.0001* cartilage Horizontal distance between the distal border of the direct ACL Female 2.5 mm ± 0.42 8.8 mm ± 1.76 < 0.0001* attachment and the adjacent margin of the articular cartilage Male 2.3 mm ± 0.66 8.5 mm ± 2.12 < 0.0001* Horizontal distance between the posterior extent of the direct Female 2.3 mm ± 0.61 8.2 mm ± 2.02 < 0.0001* ACL attachment and the posterior articular surface Male 2.1 mm ± 0.59 7.9 mm ± 1.75 < 0.0001* Horizontal distance between the anterior extent portion of the Female 13.21 mm ± 1.25 14.55 mm ± 1.87 n.s. direct ACL attachment and the anterior articular surface Male 14.32 mm ± 1.66 15.02 mm ± 2.04 n.s. Vertical distance between the distal border of the direct ACL Female 2.02 mm ± 0.88 2.97 mm ± 1.22 n.s. attachment and the inferior articular surface Male 1.55 mm ± 0.72 2.43 mm ± 1.07 n.s. Whole femoral footprint area Female 127 ­mm2 ± 23 111 ­mm2 ± 18 0.02 Male 145 ­mm2 ± 21 123 ­mm2 ± 19 0.0006* Whole direct insertion area Female 104 ­mm2 ± 17 57 ­mm2 ± 11 < 0.0001* Male 122 ­mm2 ± 20 61 ­mm2 ± 15 < 0.0001* Whole indirect insertion area Female 19 ­mm2 ± 6 47 ­mm2 ± 14 < 0.0001* Male 17 ­mm2 ± 4 56 ­mm2 ± 15 < 0.0001* Whole AM footprint area Female 67 ­mm2 ± 15 55 ­mm2 ± 13 n.s. Male 78 ­mm2 ± 13 62 ­mm2 ± 14 n.s. Direct insertion area of AM bundle Female 52 ­mm2 ± 10 26 ­mm2 ± 7 < 0.0001* Male 63 ­mm2 ± 11 32 ­mm2 ± 8 < 0.0001* Indirect insertion area of AM bundle Female 10 ­mm2 ± 3 25 ­mm2 ± 5 < 0.0001* Male 12 ­mm2 ± 3 34 ­mm2 ± 8 < 0.0001* Whole PL footprint area Female 59 ­mm2 ± 10 53 ­mm2 ± 8 n.s. Male 65 ­mm2 ± 11 58 ­mm2 ± 9 n.s. Direct insertion area of PL bundle Female 48 ­mm2 ± 5 24 ­mm2 ± 3 < 0.0001* Male 51 ­mm2 ± 6 29 ­mm2 ± 5 < 0.0001* Indirect insertion area of PL bundle Female 10 ­mm2 ± 1 22 ­mm2 ± 5 < 0.0001* Male 12 ­mm2 ± 2 28 ­mm2 ± 7 < 0.0001* We used the Bonferroni correction method to determine the p value at which the comparisons would be considered statistically significant due to multiple comparisons. The Bonferroni-corrected alpha value in these comparisons is 0.0125 instead of the traditional 0.05. Significant p values at this threshold are marked with an asterisk One type of tissue at the femoral attachment was this study, a significant difference between the morphology described as dense mid substance fibers that are attached to was seen between cadavers of those younger and older than a narrow oval area on the lateral condyle (direct insertion). 50 years. In the older than 50, a thin structure with its greater Another was a thin, membranous, peripheral attachment of diameter from proximal to distal in the shape of a ribbon-like fibers that spread out towards the posterolateral condyle, was observed, similar to the one described by Smigielski which increases the area of the insertion site (a fan-like [37] in a study with cadavers with a mean age of 67 years. extension or indirect insertion site) [33]. In people younger than 50, a large semicircular structure, The shape of the femoral footprint was described as rib- similar to the one described by Ferretti [7], was found. The bon-like (or flattened) [37], oval/elliptical, [3, 18, 19, 21, 26, latter study was conducted on specimens with a mean age of 34, 35, 38] round/circular [10, 12], or semicircular [7]. In 75, and the shape differs from the one in the present study 13 Knee Surgery, Sports Traumatology, Arthroscopy given that an anterior border straight and a posterior border 61 ­mm2,[19] 50.8 m ­ m2 [25] and 67 ­mm2 [39]). This is con- convex have been described. Conversely, most authors have sistent with the fact that all previous cadaver studies where described an ovoid or elliptical insertion area [3, 18, 19, 21, the femoral footprint was measured have presented a mean 26, 34, 35, 38]. All those studies were conducted on cadavers age that was greater than 50. The reported age ranges from of people with a mean age that was greater than 50 years. 57 to 83 years of age. Araki et al. reported a mean age of Thus, more studies on the cadavers of younger people need 62.0 years [1], Colombet et al. 75 years [3], Fujimaki et al. to be done to confirm the observed morphology. 57.5 years [8], Gali et al. 74 years [9], Hart et al. 82.5 years Due to the decrease in the size of the direct insertion with [13], Iriuchisima et al. 82.5 years [16] and 83 years [17], increasing age, the mean distance from the insertion of the Iwama et al. 80 years [19], Mochizuki et al. 61.3 years [25], ACL to the posterior cartilage of the lateral femoral condyle Sasaki et al. 69.8 years [34], Siebold et al. 82 years, [36] increases from 2.3 to 8.2 mm (p =  < 0.0001) in women, and Suruga et al. 83 years, [39 years and 78.8 years [38]], Kim from 2.1 to 7.9 mm in men (p =  < 0.0001). On the contrary, et al. 70 years, [20] and Tampere et al. 81.5 years [41]. These the morphometries towards the lower and anterior cartilage results are evidently lower than what we observed in patients do not suffer significant changes. In this study, the direct under 50 years of age, where they presented a larger direct insertion of the ACL decreases at its anteroposterior axis, femoral insertion area; 114 m ­ m2 (SD ± 17) in females and 2 which causes this structure to become thinner or flattened in 122 ­mm (SD ± 20) in males. This was a statistically signifi- patients older than 50, similar to previous studies [37, 39]. cant difference compared to patients older than 50 years of On the other hand, the difference between the different age. No studies were found with a large direct insertion area age groups in the areas corresponding to the direct and similar to the results of this study. indirect insertions of the ACL is great. We hypothesize Here, a significant increase in the indirect insertion area that this indirect insertion occurs as a consequence of the was observed in patients over 50 years of age, presenting 50 degeneration of the fibers of direct insertion of the ACL ­mm2 (SD ± 14) in women and 56 ­mm2 (SD ± 15) in men, over time. Additionally, our findings indicate that both similar to that observed in previous studies. Suruga et al. fascicles of the ACL contribute to the decrease in size of reported an area of 59 m ­ m2 in patients with a mean age of the direct insertion area. In this regard, the AM bundle 83 years [39], and Iriuchishima et al. reported 45 ­mm2 with a in female was 52 m ­ m 2 in cadavers under 50 years and mean age of 82.5 years [16]. In addition, it was observed that 2 30 ­mm in cadavers older than 50 years old, while it was indirect fibers have a smaller insertional area in the cadavers 59 ­mm2 vs 32 ­mm2 in males. The same significant effect of those under 50 years of age when compared to the over-50 was observed in the posterolateral fascicle. Similarly, an cadavers, showing that indirect fibers increase with age. This increase in the area of indirect insertion was observed in effect may be due to a degeneration of the direct fibers into both fascicles in older cadavers with the AM bundle being fan-like fibers as age increases. 10 ­mm2 in females younger than 50 and 29 ­mm2 in females There are previous studies that showed degenerative his- older over 50. The males presented 11 m ­ m2 in the cadav- topathologic changes in the ACL bundles that occur with 2 ers of those under 50 vs. 34 m ­ m in the over 50-year-old aging in osteoarthritic knees, presenting a weaker and thin- group. ner ligament in those patients [4, 14, 28] that may explain Otherwise, unlike the changes that occur between direct the degeneration of the mid substance insertions on the fem- and indirect insertion as they age, there is no statistically oral condyle. A recent study demonstrated that both fascicles significant difference between the total size of the femoral of the ACL presented histologic changes. The AM bundle insertion of both fascicles between age groups. It was 145 showed degenerative changes in 53% of the specimens and ­mm2 and 123 ­mm2, respectively in males and 127 ­mm2 and the PL bundle showed degeneration in 78% of the knees. The 111 ­mm2 in females. Contrarily, a different proportion was PL fascicle also presented more severe degenerative changes found between the direct and indirect portion in both age [41], suggesting that intercondylar notch stenosis and inter- groups. The direct insertion of the whole area goes from condylar notch osteophytes led to ACL destruction [15, 22]. 84% in males under 50 years of age to 49% in those over For this reason, describing the correct size and morphol- 50 years of age, while it goes from 90 to 51% in females, ogy of the ACL footprint is critical to reproducing the native respectively. The hypothesis put forward in this work is that anatomy as closely as possible. It is important to consider the direct insertion suffers a degeneration in both fascicles this change in the configuration of the ACL morphology and the indirect insertion area increases with aging. as age advances for carrying out femoral tunnels in ACL Therefore, an area of direct femoral insertion similar to reconstruction. Subsequent studies with cadavers divided that reported in the literature was observed in the cadavers into decadal subgroups, for example, are needed to confirm of those over 50-year-old. In the present study, it was 57 our results [5, 30]. ­mm2 (SD ± 11) in females and 61 ­mm2 (SD ± 15) in males, These findings should be considered to avoid employing similar to those reported by previous studies (56 m ­ m2,[16] reconstruction techniques in which femoral tunnels with oval 13 Knee Surgery, Sports Traumatology, Arthroscopy or rectangular shapes are used in patients under 50 years of the dissection protocol, drafting the manuscript, approval of the of age. They do not fit with the morphology of the femoral final version, search for current and ancient literature as well as an agreement that all of the manuscript's minors were reviewed and it has insertion of the ACL in this age group. validity and integrity. VMPM; intellectual contribution and revision Despite the rigorous methodology followed, certain of the manuscript from the orthopedic point of view, methodological limitations of the study must be acknowledged. One is that support for the design of the study, acquisition, analysis and interpre- the use of cadavers belonging to a single population does tation of the data, writing of part of the manuscript, approval of the final version of the manuscript, realization of the covenants for the not allow us to generalize our results for all populations. obtaining cadaveric samples, reviewer of the entire manuscript. SGL; Moreover, the lack of a histological analysis to precisely substantial contribution to the manuscript from the anatomical point delimit the limit between the direct and indirect fibers of the of view, proposal of the research idea, contribution of cadavers for the ACL is another limitation. There is also the use of embalmed study, approval of the final version of the manuscript interpretation of the data, supervision of the study. REEO; substantial contribution cadavers, which has not previously been associated with to the manuscript from the anatomical point of view, proposal of the alterations in the morphological patterns of bones and liga- research idea, contribution of cadavers for the study, approval of the ments. However, the use of fresh-frozen specimens would final version of the manuscript interpretation of the data, supervision be ideal. Finally, another limitation is that we did not per- of the study. JRPM; conception of the research idea, protocol design and drafting, obtaining, analyzing and interpreting the data, carrying form a sample size estimation a priori because we worked out the dissections, drafting the manuscript, approval of the final ver- with all the samples available in our laboratory. Nonethe- sion. JCM; one of the two main investigators of the study, editing of less, post-hoc power analyses indicated that our sample was the manuscript before its final version, orthopedic adviser, proposal of adequate. Moreover, we divided our initial sample size into the idea, proposal of the clinical relevance of the study, implementation of the protocol methodology, interpretation of results, approval of the four groups to perform hypotheses testing, which further final version, writing of part of the manuscript. reduced our power. However, to mitigate this type of error, we used a Bonferroni-corrected alpha value to denote statis- Funding Open Access Funding provided by Universitat Autonoma de tical significance when we performed multiple comparisons. Barcelona. None. A decision was also taken to use non-parametric statistical tests for a more conservative statistical approach. Declarations Conflict of interest Each author certifies that neither he or she, nor any member of his or her immediate family, has funding or commercial Conclusions associations (consultancies, stock ownership, equity interest, patent/ licensing arrangements, etc.) that might pose a conflict of interest in The femoral insertion of the ACL presents variations in its connection with the submitted article. Each author certifies that his morphology, area and morphometric characteristics over or her institution approved the human protocol for this investigation and that all investigations were conducted in conformity with ethical time. It goes from a large semicircular shape that almost con- principles of research. tacts the posterior articular cartilage in the younger group to a smaller, flattened and ribbon-like shape that moves away Ethical approval The present protocol was approved by the Institutional from the edge of the articular cartilage and is kept in bound review board of the Faculty of Medicine and University Hospital Dr. José Eleuterio Gonzalez of the Universidad Autonoma de Nuevo Leon anteriorly by the lateral intercondylar ridge in the older age (OR16-00007). group. Acknowledgements The authors would like to thank all the technical Open Access This article is licensed under a Creative Commons Attri- staff of the Macroscopic Anatomy Laboratory of the Department of bution 4.0 International License, which permits use, sharing, adapta- Human Anatomy of the U.A.N.L. School of Medicine. Also to Mr. Eric tion, distribution and reproduction in any medium or format, as long Goode for his help in reviewing the English language. as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are Author contributions RMA; conception of the research idea, protocol included in the article's Creative Commons licence, unless indicated design and drafting, obtaining, analyzing and interpreting the data, otherwise in a credit line to the material. If material is not included in drafting the manuscript and approval of its final version as well as the article's Creative Commons licence and your intended use is not the agreement with the other authors that all doubts or aspects of the permitted by statutory regulation or exceeds the permitted use, you will manuscript were meticulously reviewed before being sent to publica- need to obtain permission directly from the copyright holder. To view a tion. SP; acquisition and analysis of data, realization of the statistical copy of this licence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. analysis, intellectual advisor in the area of human anatomy, analysis of the data and interpretation of them, support of the final version of the manuscript. 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