The Anterior Cruciate Ligament (ACL) plays a vital role in maintaining function and stability in the knee. Over the last several decades, much research has been focused on elucidating the anatomy, structural properties, biomechanics, pathology, and optimal treatments for the ACL. Through careful and objective study, the ACL can be understood to be a dynamic structure, rich in neurovascular supply. Although it is referred to as one ligament, it is comprised of two dis-tinct bundles which function synergistically to facilitate normal knee kinematics. The bony morphology of the knee defines normal knee kinematics, as well as the nature of the soft-tissue structures about the knee. Characterized by individual uniqueness, bony morphology varies from patient to patient. The ACL, which is a reflection of each patient's unique bony morphol-ogy, is inherently subject to both anatomic and morphologic variation as well. Furthermore, the ACL is subject to physiologic aging, which can affect the anatomic and structural properties of the ligament over time. A successful anatomic ACL Reconstruction, which may be considered the functional restoration of the ACL to its native dimensions, collagen orientation, and inser-tion sites according to individual anatomy, considers all these principles. It is vital to respect the nature we observe, rather than to “create” nature to fit a one-size-fits-all surgery. Double bundle ACL Reconstruction may therefore be thought of more as a concept rather than a specific technique, one that respects the individual unique anatomy of each patient to provide a truly indi-vidualized, anatomic, and value-based ACL Reconstruction

The anterior cruciate ligament (ACL) is the connective tissue located at the end of long bones providing stability to the knee joint. After tear or rupture clinical reconstruction of the tissue remains a challenge due to the particular mechanical properties required for proper functioning of the tissue. The outstanding mechanical properties of the ACL are characterized by a viscoelastic behavior responsible of the dissipation of the loads that are transmitted to the bone. These mechanical properties are the result of a very specialized graded extracellular matrix that transitions smoothly between the heterotypic cells, stiffness and composition of the ACL and the adjacent bone. Thus, mimicking the zonal biochemical composition, cellular phenotype and organization are key to reset the proper functioning of the ACL. We have previously shown how the biochemical composition presented to cells in electrospun scaffolds results in haptokinesis, reverting contact-guidance effects. [1]. Here, we demonstrate that contact guidance can also be disrupted by structural parameters in aligned wavy scaffolds. The presentation of a wavy fiber arrangement affected the cell organization and the deposition of a specific ECM characteristic of fibrocartilage. Cells cultured in wavy scaffolds grew in aggregates, deposited an abundant ECM rich in fibronectin and collagen II, and expressed higher amounts of collagen II, X and tenomodulin as compared to aligned scaffolds. In-vivo implantation in rabbits of triphasic scaffolds accounting for aligned-wavy-aligned zones showed a high cellular infiltration and the formation of an oriented ECM, as compared to traditional aligned scaffolds. [2]

Introduction. Simultaneous correction of knee varus malalignment with medial open wedge high tibial osteotomy (MOWHTO) combined with anterior cruciate ligament (ACL) surgery aims to address symptomatic unicompartmental osteoarthritis in addition to restore knee stability in order to improve outcomes. The aim of this study is to present at least 5 years results of 32 patients who underwent simultaneous knee realignment osteotomy with ACL surgery. Methods. Patients with symptomatic instability due to chronic ACL deficiency or failed previous ACL surgery together with a varus malalignment of ≥6°, previous medial meniscectomy and symptomatic medial compartment pain who were treated with MOWHTO combined with ACL surgery were enrolled. ACL surgery was performed with the anatomical single bundle all-inside technique using TightRope. ®. RT (Arthrex, Naples, FL, USA) and MOWHTO using TomoFix. ®. medial high tibia plate (DePuy Synthes, Raynham, MA, USA) in all cases. Patients were evaluated preoperatively and at 6 months, 12 months and annually postoperatively using the Knee Injury and Osteoarthritis Outcome Score (KOOS), Oxford Knee Score (OKS) and Euroqol's Visual Analogue Score (VAS) for pain. Results. 32 patients (22 men and 10 women) with a mean age of 41.2 years and mean BMI of 28.6 kg/m. 2. , underwent the combined procedures. Tibiofemoral neutral re-alignment was achieved in all patients with HTO. Complete subjective and objective scores have been obtained in 84.4% of patients with at least 5 years of follow-up (mean 8.7 years). An improvement in total KOOS of 27.1 points (p<0.003), OKS of 15.1 (p<0.003) and VAS for pain of 24.7 points (p<0.001) were detected. No ACL reconstruction failure was noted. Complications consisted of one superficial wound infection and one delayed union. Plate removal was needed in 20 (62.5%) patients due to pes anserinus pain. Conclusions. Simultaneous restoration of coronal knee axis by applying HTO and stability by ACL reconstruction/revision were reported to offer excellent improvement in early outcomes in patients with ACL rupture and symptomatic unicompartmental osteoarthritis. The combined procedure requires careful pre-operative planning and is therefore technically challenging. However, by restoring the neutral axis and providing stability, it represents a good joint preserving alternative to arthroplasty for active middle-aged patients

Successful anterior cruciate ligament (ACL) reconstructions strive a firm ligament-bone integration. Therefore, the aim of this study was to address in more detail the enthesis as the thriphasic bone attachment of the ACL using a tissue engineering approach. To establish a tissue-engineered enthesis-like construct, triphasic scaffolds embroidered from poly(L-lactide-co-caprolactone) and polylactic acid functionalized with collagen foam were colonized with osteogenically differentiated human mesenchymal stromal cells (hMSCs) and lapine (L) ACL fibroblasts. These triphasic scaffolds with a bone-, a fibrocartilage transition- and a ligament phase were seeded directly after spheroid assembly or with 14 days precultured LACL fibroblast spheroids and 14 days osteogenically differentiated hMSCs spheroids (=longer preculture) and cultured for further 14 days. Cell survival was tested. Collagen type I and vimentin were immunolabeled and the content of DNA and sulfated glycosaminoglycan (sGAG) was quantified. The relative gene expression of tenascin C, type I and X collagens, Mohawk and Runx2 was analyzed. Compared to the LACL spheroids the hMSC spheroids adhered better to the scaffold surface with faster cell outgrowth on the fibers. Collagen type I and vimentin were mainly detected in the hMSCs colonizing the bone zone. The DNA content was generally higher in the bone (hMSCs) than in the ligament zones and after short spheroid preculture higher than after longer preculture whereas the sGAG content was greater after longer preculture for both cell types. The longer precultivated hMSCs expressed more type I collagen in comparison to those only shortly precultured before scaffold seeding. Type I collagen and tenascin C were higher expressed in scaffolds directly colonized with LACL compared to those seeded after longer spheroid preculture. The gene expression of ECM components and transcription factors depended on cell type and preculturing condition. Zonal colonization of triphasic scaffolds using the spheroid method is possible offering a novel approach for enthesis tissue engineering

Objectives. In this study, we compared the pain behaviour and osteoarthritis (OA) progression between anterior cruciate ligament transection (ACLT) and osteochondral injury in surgically-induced OA rat models. Methods. OA was induced in the knee joints of male Wistar rats using transection of the ACL or induction of osteochondral injury. Changes in the percentage of high limb weight distribution (%HLWD) on the operated hind limb were used to determine the pain behaviour in these models. The development of OA was assessed and compared using a histological evaluation based on the Osteoarthritis Research Society International (OARSI) cartilage OA histopathology score. Results. Both models showed an increase in joint pain as indicated by a significant (p < 0.05) decrease in the values of %HLWD at one week post-surgery. In the osteochondral injury model, the %HLWD returned to normal within three weeks, while in the ACLT model, a significant decrease in the %HLWD was persistent over an eight-week period. In addition, OA progression was more advanced in the ACLT model than in the osteochondral injury model. Furthermore, the ACLT model exhibited a higher mean OA score than that of the osteochondral injury model at 12 weeks. Conclusion. The development of pain patterns in the ACLT and osteochondral injury models is different in that the OA progression was significant in the ACLT model. Although both can be used as models for a post-traumatic injury of the knee, the selection of appropriate models for OA in preclinical studies should be specified and relevant to the clinical scenario. Cite this article: T. Tawonsawatruk, O. Sriwatananukulkit, W. Himakhun, W. Hemstapat. Comparison of pain behaviour and osteoarthritis progression between anterior cruciate ligament transection and osteochondral injury in rat models. Bone Joint Res 2018;7:244–251. DOI: 10.1302/2046-3758.73.BJR-2017-0121.R2

We report the effects of local administration of osteogenic protein-1 on the biomechanical properties of the overstretched anterior cruciate ligament in an animal model. An injury in the anterior cruciate ligament was created in 45 rabbits. They were divided into three equal groups. In group 1, no treatment was applied, in group II, phosphate-buffered saline was applied around the injured ligament, and in group III, 12.5 μg of osteogenic protein-1 mixed with phosphate-buffered saline was applied around the injured ligament. A control group of 15 rabbits was assembled from randomly-selected injured knees from among the first three groups. Each rabbit was killed at 12 weeks. The maximum load and stiffness of the anterior cruciate ligament was found to be significantly greater in group III than either group 1 (p = 0.002, p = 0.014) or group II (p = 0.032, p = 0.025). The tensile strength and the tangent modulus of fascicles from the ligament were also significantly greater in group III than either group I (p = 0.002, p = 0.0174) or II (p = 0.005, p = 0.022). The application of osteogenic protein-1 enhanced the healing in the injured anterior cruciate ligament, but compared with the control group the treated ligament remained lengthened. The administration of osteogenic protein-1 may have a therapeutic role in treating the overstretched anterior cruciate ligament

Recent findings have identified the importance of previously undiagnosed or neglected meniscus lesions in association with anterior cruciate ligament (ACL) injuries (e.g. medial meniscus ramp lesions and posterior root tears of the lateral meniscus). There is increasing biomechanical evidence that they bear the potential to alter both anteroposterior and rotational laxity patterns in ACL injured knees. Few data exist with respect to the presence of these specific tear entities in large series of ACL injured patients. The purpose of the study was to analyze the meniscus tear pattern in a series of ACL injured knees with a special focus on ramp lesions of the medial meniscus and posterior root lesions of the lateral meniscus. The hypothesis was that a significant number of ACL injured patients would display these types of lesions. Data from 358 patients undergoing an ACL reconstruction (227 males /131 females, age: 28±10) were extracted from a center-based registry. The type of ACL tear (partial versus complete) as well as the presence of associated meniscus lesions were documented. Meniscus lesions were classified into the following categories: medial ramp lesions, lateral root lesions, medial ramp and lateral root lesion, other medial meniscus injuries, other lateral meniscus injuries, other bimeniscal injuries. Chi-square tests were used to determine whether the percentage of meniscal lesions differed between types of ACL tear, gender and age (below 21, 21–35, above 35). Significance was set at p < 0.05. Isolated ACL tears were present in 107 (30%) of the operated knees (31 partial; 327 complete). Complete ACL lesions were more likely to present an associated meniscus injury (321 out of 327, 71%) than partial tears (13 out of 31, 42%). The incidence of meniscus injuries which are associated with ACL tears is very high (70%). Previously undiagnosed or neglected meniscus injuries like medial ramp or lateral root tears could be identified in 35% of patients. As such, the hypothesis was confirmed that an important amount of ACL injured knees display this specific intraarticular soft tissue damage. A systematic evaluation of these lesions under arthroscopy should thus be performed and specific repair needs to be evaluated

Abstract

The concept of decellularised xenografts as a basis for anterior cruciate ligament (ACL) reconstruction was introduced to overcome limitations in alternative graft sources such as substantial remodelling delaying recovery and donor site morbidity. This study aimed to measure the biomechanical properties of decellularised porcine super flexor tendon (pSFT) processed to create ACL grafts of varying diameters, with a view to facilitating production of stratified ‘off the shelf’ products with specified functional properties for use in ACL reconstructive surgery. Decellularisation was carried out using a previously established procedure, including antibiotic washes, low concentration detergent (0.1% sodium dodecyl sulphate) washes and nuclease treatments. Decellularised pSFTs were prepared to create double-bundle grafts of 7, 8 and 9mm diameter (n=6 in each group). Femoral and tibial fixations were simulated utilising Arthrex suspension devices (Tightrope®) and interference screws in bovine bone respectively. Dynamic stiffness and creep were measured under cyclic loading between 50–250N for 1000 cycles at 1Hz. This was followed by ramp to failure at 200mm/min from which linear stiffness and load at failure were measured. Data were analysed using either 1- or 2-way ANOVA as appropriate with Tukey post-hoc analysis (p<0.05). Significant differences were found between all groups for dynamic stiffness and between 7 & 9mm and 8 & 9mm groups for dynamic creep. Significant differences were also found between 7, 8 & 9mm groups for linear stiffness (167.8±4.9, 186.9±16.6 & 216.3±12.4N/mm respectively), but no significant differences were found between groups for load at failure (531.5±58.9, 604.1±183.3 & 627.9±72.4N respectively). This study demonstrated that decellularised pSFTs possess comparable biomechanical properties to other ACL graft options (autografts and allografts). Furthermore, grafts can be stratified by their diameter to provide varying biomechanical profiles depending on the anatomy and individual needs of the recipient

More than 250,000 people are suffering from Anterior Cruciate Ligament (ACL) related injuries each year in the US, with a cost of $17–25K/patient. There is an unmet clinical demand for improving grafts/scaffolds to provide biological integration in addition to mechanical support. Currently, no data is available for the utilization of fibrous scaffolds with bimodal distribution for ACL regeneration. The novelty in this study is that it proposes for the first time to investigate the collagen fibril diameter distribution in healthy and injured bovine ACL tissue, and utilization of such structure for scaffold design. Objectives are 1) developing a bovine ACL tear model and measuring the collagen fibril diameter distribution of both healthy and injured ACL tissues, and 2) fabricating scaffolds to mimic the structural properties of healthy and injured ACL tissue. Bovine ACL tissues (1–3 years old) were harvested and characterized for their fibril diameter distribution using Transmission Electron Microscopy (TEM) and biomechanical properties under tension. The electrospun polycaprolactone (PCL) scaffolds were characterized using SEM and mechanical testing. Healthy and injured ACL fibril diameter, and that of PCL scaffolds representing healthy and injured ACL are compared using unpaired student t-test. The proposed fibrous scaffold design represents a significant departure from the conventional unimodal approach, and is expected to have significant contribution to ACL regeneration. These discoveries will serve as the foundation for the development of biomimetic tissue engineering substrates aimed at promoting biological graft fixation

We have investigated whether cells derived from haemarthrosis caused by injury to the anterior cruciate ligament could differentiate into the osteoblast lineage in vitro. Haemarthroses associated with anterior cruciate ligament injuries were aspirated and cultured. After treatment with β-glycerophosphate, ascorbic acid and dexamethasone or 1,25 (OH). 2. D. 3. , a significant increase in the activity of alkaline phosphatase was observed. Matrix mineralisation was demonstrated after 28 days and mRNA levels in osteoblast-related genes were enhanced. Our results suggest that the haemarthrosis induced by injury to the anterior cruciate ligament contains osteoprogenitor cells and is a potential alternative source for cell-based treatment in such injury

Anterior cruciate ligament injury is the most common and economically costly sport injuries, frequently requiring expensive surgery and rehabilitation. Post-operative knee septic arthritis represents a serious complication with an incidence rate between 0.14% and 1.7%. A common practice to avoid septic arthritis is the “vancomycin wrap”, consisting in the soaking of the graft for 10–15 minutes within a sterile gauze swab previously saturated with 5 mg/mL vancomycin. Even though several studies have been conducted to investigate vancomycin toxicity on different musculoskeletal tissues or cells, little is known about the effect of such antimicrobial on tendon-derived cells.

The aim of this study was to determine the in vitro toxicity of different concentrations of vancomycin at different time points on human primary tenocytes (hTCs).

hTCs were isolated from hamstring grafts of patients undergoing anterior cruciate ligament reconstruction. After expansion, cells were treated with different concentrations of vancomycin (2.5, 5, 10, 25, 50 and 100 mg/mL) for 10, 15, 30 and 60 minutes. In vitro toxicity was evaluated measuring: metabolic activity through the reduction of 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT Assay); cytotoxicity (Live/Dead assay); and cell apoptosis (Annexin V apoptosis kit).

The metabolic activity of hTCs was affected by vancomycin treatment starting from 10 mg/mL at all time points (p < 0.05) and dropped down at 100 mg/mL at all time points (0.05 < p < 0.001). Cells viability resulted to be unaffected only by 2.5 mg/mL vancomycin at all time points. Vancomycin resulted to be cytotoxic starting from 10 mg/mL after 15 minutes of treatment and at all higher concentrations under study at all time points. Cells died when treated with vancomycin concentrations higher than 5 mg/mL but not through apoptosis, as confirmed by negative staining for Annexin V.

In our experimental conditions, vancomycin resulted to be toxic on hTCs at concentrations higher than 5 mg/mL. The use of this antibiotic on tendons to prevent infections could be useful and safe for resident cells if used at a concentration of 2.5 mg/mL up to 1 hour of treatment.

We studied bone-tendon healing using immunohistochemical methods in a rabbit model. Reconstruction of the anterior cruciate ligament was undertaken using semitendinosus tendon in 20 rabbits. Immunohistochemical evaluations were performed at one, two, four and eight weeks after the operation. The expression of CD31, RAM-11, VEGF, b-FGF, S-100 protein and collagen I, II and III in the bone-tendon interface was very similar to that in the endochondral ossification. Some of the type-III collagen in the outer layer of the graft, which was deposited at a very early phase after the operation, was believed to have matured into Sharpey-like fibres. However, remodelling of the tendon grafted into the bone tunnel was significantly delayed when compared with this ossification process. To promote healing, we believe that it is necessary to accelerate remodelling of the tendon, simultaneously with the augmentation of the ossification

Abstract

We examined whether somatosensory evoked potentials (SEPs) were detectable after direct electrical stimulation of injured, reconstructed and normal anterior cruciate ligaments (ACL) during arthroscopy under general anaesthesia. We investigated the position sense of the knee before and after reconstruction and the correlation between the SEP and instability. We found detectable SEPs in all ligaments which had been reconstructed with autogenous semitendinosus and gracilis tendons over the past 18 months as well as in all cases of the normal group. The SEP was detectable in only 15 out of 32 cases in the injured group, although the voltages in the injured group were significantly lower than those of the controls. This was not the case in the reconstructed group. The postoperative position sense in 17 knees improved significantly, but there was no correlation between it and the voltage. The voltage of stable knees was significantly higher than that of the unstable joints. Our findings showed that sensory reinnervation occurred in the reconstructed human ACL and was closely related to the function of the knee

Our objectives were to establish the envelope of passive movement and to demonstrate the kinematic behaviour of the knee during standard clinical tests before and after reconstruction of the anterior cruciate ligament (ACL). An electromagnetic device was used to measure movement of the joint during surgery. Reconstruction of the ACL significantly reduced the overall envelope of tibial rotation (10° to 90° flexion), moved this envelope into external rotation from 0° to 20° flexion, and reduced the anterior position of the tibial plateau (5° to 30° flexion) (p < 0.05 for all). During the pivot-shift test in early flexion there was progressive anterior tibial subluxation with internal rotation. These subluxations reversed suddenly around a mean position of 36 ± 9° of flexion of the knee and consisted of an external tibial rotation of 13 ± 8° combined with a posterior tibial translation of 12 ± 8 mm. This abnormal movement was abolished after reconstruction of the ACL

Cell sheets are manufactured from a high-density cell layer stabilized by its own freshly produced extracellular matrix (ECM). They could serve as versatile scaffolds for tissue repair. Unfortunately, their production often remains time-consuming requiring weeks of culturing. Ligament cell sheets are so far barely available. Regarding musculoskeletal tissues exposed to high repetitive biomechanical forces, the stability of cell sheets is insufficient. It could help to combine them with a biomechanical competent scaffold e.g. produced by an embroidering technique. Hence, we wanted to (1) develop a very rapid strategy to produce ACL ligamentocyte sheets within 24 h by using a thermoresponsive polymer surface, (2) use the sheets for scaffold seeding and (3) reflect the fibrocartilaginous transition zone of an ACL enthesis by combining sheets of ligamentocytes with chondrocytes or chondrogenic precursor cells as a strategy for directed seeding of two cell types on topologically different scaffold areas.

Different cell numbers of lapine ACL ligamentocytes (L-ACLs), lapine articular chondrocytes (L-ACs) and human mesenchymal stromal cells (H-MSCs) were used for sheet formation. Experiments were performed with novel, self-assembled poly(glycidyl ether) (PGE) brushes based on random glycidyl methyl ether and ethyl glycidyl ether copolymers on polystyrene 12-well cell culture plates, which allow rapid sheet formation within 24 h. Uncoated plates served as controls. Temperature-triggered detachment was performed by 10 min incubation with PBS at ambient temperature before treatment with fresh warm PBS for 5 min at 37 degrees Celsius. Harvested cell sheets were transferred on polyglycolic acid (PGA) or embroidered poly-lactic acid / poly-co-caprolactone (PLA/P[LA-CL]) scaffolds, functionalized with collagen foam and fluorine gas treatment (prepared at the IPF in Dresden and the FILK in Freiberg). Cell distribution, growth, vitality and synthesis of ECM components were monitored up to 7 days. Cell numbers required for sheet preparation (3.9 cm2) depended strongly on the cell type (L-ACLs: 0.395 mio/cm2, L-AC: 0.342 mio/cm2, H-MSCs: 0.131 mio/cm2) and was highest for L-ACLs. The majority of cells survived sheet assembly, detachment, transfer onto the scaffolds and culturing. Cells migrated from the sheets into the scaffolds and spread through the scaffolds. L-ACLs and L-ACs produced ECM and maintained their phenotypes (type II collagen and sulfated glycosaminoglycans in L-AC sheets, decorin and tenascin C in L-ACL sheets). The presence and distribution of two cell types in scaffold cocultures (L-ACLs and H-MSCs) was proven by anti-human vimentin labeling. Hence, the PGE brush surface allows rapid formation (24 h) of cell sheets.

Evidence suggests that anterior cruciate ligament (ACL) injured individuals do not use the same movement strategies as healthy individuals. It is unknown how this may affect them in more challenging activities of daily living and sport. The aim of this study is to evaluate how ACL injured patients perform a single leg squat (SLS) compared to healthy controls. SLS was evaluated as it is more challenging than gait and therefore more relevant to clinical decision making about progressing to sporting maneuvers. To date, 6 ACL deficient (ACLD) (5 males, 1 female; mass=88±22 kg; height=1.78±0.11 m; age=35±11 years), 5 ACL reconstructed (ACLR) (5 males; mass= 83±12 kg; height=1.74±0.07 m; age=29±10 years) and 5 controls (3 males, 2 females; mass= 72±13 kg; height=1.70±0.09 m; age=30±3 years) performed a SLS on the injured leg for the ACL injured participants and the dominant leg for the control group. Motion analysis was performed using a Vicon Nexus system and a Kistler force platform. Knee extension moments and angles were calculated using Vicon Nexus software. The ACLD group had reduced peak flexion angles compared to ACLR and control groups (65±5, 77±7 and 82±9 degrees respectively). Peak extension moments were similar across all groups (ACLD= 0.94±0.26 Nm/kg, ACLR=1.06±0.37 Nm/kg, control=1.04±0.36 Nm/kg). Peak knee moments occurred just after peak flexion and therefore at a smaller flexion angle for the ACLD group compared to the ACLR and control group (59±13, 75±7 and 80±6 degrees). Extension moments were similar when evaluated at a consistent angle of 50 degrees (ACLD=0.70±0.30Nm/kg, ACLR=0.63±0.34Nm/kg control=0.61±0.32Nm/kg). In this sample, the controls squatted deepest followed by the ACLR group, with the ACLD group squatting least deep. This did not translate to an identical pattern for the knee extensor moments. Performance of ACL injured individuals needs to be evaluated on more challenging tasks to fully assess recovery. Further research, with more subjects, will clarify if ACLD individuals are using a strategy to protect their knee or if others factors are preventing them from squatting deeper. This would suggest that these individuals may not have fully recovered and will not be able to perform more challenging activities

In a study combining tissue mechanics and fracture morphology for the first time, we examined the ruptured surfaces of anterior cruciate ligaments of rabbits and related their appearance to the initial loading conditions. Sixteen specimens were stretched to failure at rates of displacement of 10 and 500 mm/min. We used video images to study the changes which occurred during the fracture process and SEM to examine the appearance of the ruptured surfaces. The surfaces of ligaments tested at 10 mm/min had more pulled-out collagen fibres and the fibres had more pronounced waviness compared with those tested at 500 mm/min. We have shown that the macroscopic appearance of ruptured ligaments can be related to their microscopic appearance and that it is possible to deduce whether failure was by gradual tearing of the fibres or catastrophic failure

Summary. The presence or absence of crimp within the anterior cruciate ligament (ACL) sub-bundle anatomy was correlated with knee flexion angle changes and provided a measure of differential loading within its sub-bundle microstructure. Introduction. Previous studies have shown that macroscopically the anteromedial (AM) and posterolateral (PL) bundles of the ACL tighten/slacken differently with knee flexion angle. This research used fibre crimp morphology, revealed following in situ fixation of the intact ligament structures, to investigate patterns of differential fibre recruitment across each ACL sub-bundle. Methods. Twelve mature ovine knees were divided into four test-groups of three: control, hyper-extension (8°), neutral position (50°), and deep flexion (170°). For the control group, ligament-bone (tibia) samples were isolated and chemically fixed, unconstrained, in 10% formalin. For the flexed groups the whole joints were first positioned in the relevant angle of flexion using a custom-built rig and formalin-fixed in-situ before undergoing dissection. All ligament-bone samples were decalcified using 10% formic acid and cryo-sectioned in the sagittal plane to obtain 20μm serial slices. These were then examined in their fully hydrated state using differential inference contrast (DIC) microscopy. The degree of crimping, taken as an approximate indicator of elongation and thus loading, was classified as either substantial, intermediate or minimal. Crimp coarseness or wavelength, crimp angle and number of visible apices per unit fibre length were measured and analysed statistically using both ANOVA and post hoc analysis (significance, p < 0.05). Results. Control group. Substantial crimping was present in all regions of both AM and PL bundles. However two distinct crimp morphologies were discerned, coarse and fine. Coarse crimp dominated both the small anterior region of the AM bundle proximal to the tibial plateau and the entire PL bundle. Fine crimp was present in the remaining part of the AM bundle. The wavelengths and maximum angles of the coarse and fine crimp were significantly different at 44.8 ± 5.9 µm/45° and 16.2 ± 3.1 μm/15° respectively. This difference in crimp morphology was consistent along the serial sections of the ACL. From the serial sections, a three dimensional interpretation of the degree of crimping was obtained for the three different loading positions. A summary of the main findings is presented as follows: Hyper-extension. A small antero-medial portion of the AM bundle was substantially crimped (indicating an unloaded state) and the remaining region exhibiting only intermediate crimping; the bulk of the PL bundle was largely free of crimp (indicating a relatively loaded state) except for a substantially crimped region in its postero-lateral aspect. Neutral position. The anterior portion of the AM bundle exhibited minimal crimp but elsewhere was intermediate. The entire PL bundle was substantially crimped. Deep flexion. The substantially crimped medial portion of the AM bundle transformed progressively into an intermediate crimp morphology as the lateral aspect of the bundle was approached. Both the postero-medial and antero-lateral aspects of the PL bundle were largely free of crimp whereas the intervening region was intermediately crimped. Conclusion. Through serial sectioning, this study has revealed differential patterns of fibre recruitment, following loading, in all four quadrants of the sub-bundles of the ovine ACL at different knee flexion angles