Adolescent females are at a higher risk of anterior cruciate ligament (ACL) injury than males. While prior studies have associated injury timing and menstrual cycle phase, these data are limited by indirect cycle tracking and lack of analysis on ACL structure, mechanics, or composition. Additionally, little is known about how female sex hormones influence the distinct ACL bundles. This exploratory study investigated associations between serum sex hormone concentrations and size, mechanics, and composition of the ACL and its bundles in a female adolescent pig model. Serum from nine adolescent female Yorkshire crossbreed pigs was collected pre-euthanasia and analyzed for levels of estradiol, progesterone, and testosterone. The ACL and its bundles were assessed for size via magnetic resonance imaging (MRI), mechanics via robotic testing, and composition via biochemical and histological analyses. While individual hormone levels and the estradiol-to-progesterone (E/P) ratio had no association with most metrics, the E/P ratio was significantly associated with ACL size and T2* relaxation time. Higher E/P ratios were negatively associated with anteromedial (AM) bundle cross-sectional area (CSA) (R2 = 0.44) and overall ACL volume (R2 = 0.49) and positively associated with posterolateral (PL) bundle T2* relaxation time (R2 = 0.69, p < 0.05). Serum E/P ratio was also positively associated with normalized ACL stiffness, but there were no associations observed for tissue composition. The results of this exploratory study indicate that the ACL may be responsive to exposure to the relative concentration of female sex hormone in a bundle-specific manner.

Female adolescent athletes are at a higher risk of tearing their anterior cruciate ligament (ACL) than male counterparts. While most work related to hormones has focused on the effects of estrogen to understand the increased risk of ACL injury, there are other understudied factors, including testosterone. The purpose of this study was to determine how surgical castration in the male porcine model influences ACL size and function across skeletal growth. Thirty-six male Yorkshire crossbreed pigs were raised to 3 (juvenile), 4.5 (early adolescent), and 6 months (adolescent) of age. Animals were either castrated (barrows) within 1-2 weeks after birth or were left intact (boars). Post-euthanasia, joint and ACL size were assessed via MRI, and biomechanics were assessed via a robotic testing system. Joint size increased throughout age, yet barrows had smaller joints than boars (p<0.001 for all measures). ACL cross-sectional area (CSA), length, volume, and stiffness increased with age (p<0.0001), as did ACL anteromedial (AM) bundle percent contribution to resisting loads (p=0.012). Boar ACL, AM bundle, and PL bundle volumes were 19% (p=0.002), 25% (p=0.003), and 15% (p=0.04) larger than barrows across ages. However, CSA, stiffness, and bundle contribution were similar between boars and barrows (p>0.05). The barrows had smaller temporal increases in AM bundle percent function than boars, but these data were highly variable. Thus, early and sustained loss in testosterone leads to subtle differences in ACL morphology, but may not influence measures associated with increased injury risk, such as CSA or bundle forces in response to applied loads.

OBJECTIVE:Anterior cruciate ligament (ACL) injuries are a major problem in the pediatric and adolescent populations. Some of these injuries are only partial; yet, there is limited data to inform clinical treatment of such partial tears. It is unknown how injury partial injury impacts long-term degenerative changes in the joint relative to complete injury. In this study, we hypothesized that partial (anteromedial (AM) or posterolateral (PL) bundle) tears would result in small levels of instability and degeneration relative to complete ACL tears and that the degree of degeneration would associate with joint instability. DESIGN:Partial (isolated AM or PL bundle) or complete ACL injury was arthroscopically created in 3-month-old juvenile pigs. The contralateral limb served as a sham-operated control. Six months after injury, joint biomechanics was assessed along with cartilage and meniscus degeneration (via magnetic resonance imaging [MRI], gross imaging, and histology). RESULTS:Joint laxity increases were minimal after PL bundle injury (difference relative to controls (confidence interval): 0.5 (-1.2-2.2) mm), minor after AM bundle injury (3.7 (2.0-5.4) mm), and major after ACL injury (15.8 (13.7-17.8) mm). Cartilage MRI T1ρ relaxation times increased minimally after PL bundle injury (-0.9 (-5.1-3.3) ms for lateral tibia), moderately after AM bundle injury (6.6 (1.7-11.4) ms), and substantially after ACL injury (10.8 (2.1-19.5) ms). Changes in meniscus volume followed a similar rank order. Degeneration was associated with the extent of joint destabilization. CONCLUSIONS:These findings suggest that cartilage and meniscus degeneration in the skeletally immature joint are associated with joint laxity after partial and complete ACL injuries.

Objectives: Pediatric anterior cruciate ligament (ACL) injuries are on the rise and result in high rates of post-traumatic osteoarthritis (PTOA). The pro-inflammatory biomarker monocyte chemoattractant protein-1 (MCP-1) is elevated in serum and synovial fluid (SF) in skeletally mature ACL injured patients as well as patients with OA, making it a potential target for treatments to prevent PTOA. Understanding the time course of MCP-1 after ACL injury and how it relates to cartilage health in skeletally immature subjects is important for identifying OA risk and developing novel treatment targets for OA prevention. Therefore, the objective of this study was to evaluate the time course of MCP-1 levels in serum and synovial fluid (SF) after ACL injury in a skeletally immature porcine model and assess how MCP-1 levels relate to T2* magnetic resonance imaging (MRI) measurements, indicative of cartilage matrix organization. We hypothesized that higher levels of MCP-1 in serum and synovial fluid post-injury would correspond to higher cartilage T2* values 12 weeks after ACL injury. Methods: All experimental protocols were approved by the NC State IACUC. Juvenile (3 month old) female Yorkshire cross-breed pigs (n=7) underwent unilateral arthroscopic ACL transection (ACLT) and a sham operation in the contralateral joint (Fig. 1A). Prior to surgery and at 2, 4, and 12 weeks post- operatively, injured stifle SF and serum samples were collected (pre-op synovial fluid only obtained from 4 animals). MRI scans were performed prior to surgery and at 12 weeks post-operatively. MCP-1 levels in serum and SF samples were evaluated longitudinally using a commercial enzyme-linked immunosorbent assay. Cartilage matrix organization was evaluated longitudinally using T2* MRI. Specifically, both knee joints were imaged using a 3T Siemens MAGNETOM Skyra MRI system (T2 SWI sequence, FA=15°, TR=50ms, TE: 3.4, 7, 13.47, 21, 29, 37.07ms, voxel size: 0.5x0.5x0.8mm). T2* relaxation maps were created in MATLAB by fitting mono-exponential curves on a voxel-by-voxel basis. Weight-bearing cartilage was segmented from the medial and lateral compartments of the femur, and the median T2* value of each compartment was calculated. Paired MCP-1 levels in the serum and injured stifle SF across all time points were compared using linear regression. Multiple paired t-tests with Holm-Sidak correction were performed to compare cartilage T2* values between ACLT and sham- operated joints at each time point. The interlimb percent difference in femoral cartilage T2* at 12 weeks was calculated and compared to MCP-1 levels in serum and SF using linear regressions and several methods to assess MCP-1 levels (Fig. 2). Statistical significance was set at α=0.05 overall for each analysis. Results: Generally, MCP-1 levels in serum and ACLT stifle SF were elevated after ACLT (Fig. 1). The serum MCP-1 response over time differed between animals. Specifically, serum MCP-1 levels peaked at 2 weeks in 2 animals, peaked at 4 weeks in 3 animals, and decreased after ACLT in 2 animals (Fig 1A). Contrastingly, ACLT stifle SF MCP-1 responses were more consistent between animals; six peaked at 2 week and returned near baseline levels by 12 weeks (Fig 1B). The linear regression between paired measurements of MCP-1 concentrations in the serum and ACLT stifle SF were associated (p=0.053), yet had high variability (r 2 =0.15, Fig. 1C). Interlimb differences in cartilage matrix organization were largest and most consistent at 12 weeks in the medial femoral cartilage compartment, where median T2* times were 8% longer in the medial femoral cartilage compartment of the ACLT limb compared to sham limb, yet this was not statistically significant due to variability between animals (p>0.05, Fig. 2). In the lateral femoral cartilage, the median T2* times were 3-4% longer at 12 weeks (p>0.05) and were more variable between subjects compared to the medial femoral cartilage. Associations between the interlimb percent difference in medial femoral cartilage T2* and serum MCP-1 fit well (r 2 =0.25-0.69), with Methods 2, 3, and 5 achieving statistical significance (p=0.02, p=0.03, p=0.03), but resulted in poor fits for associations with SF MCP-1 (r 2 =0.07-0.45) using a variety of MCP-1 measurement methods (Fig 2C). Similar associations between lateral femoral cartilage T2* and serum MCP- resulted in poor fits (r 2 =0.01-0.24), and associations with SF MCP-1 resulted in negative slopes (Methods 2-5, r 2 =0.00-0.58). Conclusions: This preliminary study showed that inflammatory biomarker MCP-1 levels in serum displayed a subject-dependent response, which correlated with later femoral cartilage compositional changes after ACLT in the skeletally immature porcine model. This partially supported our hypothesis. The poor correlation between serum and SF MCP-1 indicate different local and systemic responses to ACLT. The stronger correlations between serum MCP-1 and cartilage T2* values could point to the importance of the systemic inflammatory response to OA progression. The increases in cartilage T2* values and serum MCP-1 levels after ACL injury in this study are consistent with those reported in adult humans. This study includes several limitations, including the use of an animal model to study human disease, the small sample size, and the relatively short time-course. However, the results demonstrate feasibility of using a large animal porcine model to longitudinally study the link between early MCP-1 serum levels and long-term cartilage health after ACL injury. Establishing the pig as a preclinical model to study longitudinal inflammation and OA progression after ACL injury will enable development of effective treatments before translating to humans.

Finite element analysis is a useful tool to model growth of biological tissues and predict how growth can be impacted by stimuli. Previous work has simulated growth using node-based or element-based approaches, and this implementation choice may influence predicted growth, irrespective of the applied growth model. This study directly compared node-based and element-based approaches to understand the isolated impact of implementation method on growth predictions by simulating growth of a bone rudiment geometry, and determined what conditions produce similar results between the approaches. We used a previously reported node-based approach implemented via thermal expansion and an element-based approach implemented via osmotic swelling, and we derived a mathematical relationship to relate the growth resulting from these approaches. We found that material properties (modulus) affected growth in the element-based approach, with growth completely restricted for high modulus values relative to the growth stimulus, and no restriction for low modulus values. The node-based approach was unaffected by modulus. Node- and element-based approaches matched marginally better when the conversion coefficient to relate the approaches was optimized based on the results of initial simulations, rather than using the theoretically predicted conversion coefficient (median difference in node position 0.042 cm versus 0.052 cm, respectively). In summary, we illustrate here the importance of the choice of implementation approach for modeling growth, provide a framework for converting models between implementation approaches, and highlight important considerations for comparing results in prior work and developing new models of tissue growth.

Anterior cruciate ligament (ACL) injuries are increasingly common in adolescents, and injuries in this age-group are associated with many unique challenges. Recent large animal studies suggest that the size and function of the major bundles of the ACL change differently throughout skeletal growth. To better aid clinical treatment of pediatric partial ACL tears and better predict outcomes from age-specific treatments, there is a need to measure changes in ACL bundle size in humans during growth. As such, the objective of this study was to compare changes in the length and cross-sectional area (CSA) of the ACL and its primary bundles in adolescent human subjects. Magnetic resonance imaging (MRI) scans were analyzed to determine the visibility and integrity of the ACL and its anteromedial and posterolateral bundles. MRI scans were considered from a retrospective database of subjects ranging from 10 to 18 years of age. The ACL and its anteromedial and posterolateral bundles were segmented and reconstructed into 3D models, and length and CSA were calculated. Total ACL length and CSA were greater in males compared with females, with a statistically significant interaction between age and sex for CSA. Sex had a significant effect on the CSA of both bundles. These sex-dependent differences emerge with moderate to large effect sizes (range: d = 0.50 to d = 1.23) beginning around 13 years of age. Along with ACL bundle structure-function relationships previously established in preclinical animal models, these findings may point toward biomechanical changes in the adolescent human ACL.

Anterior cruciate ligament (ACL) injuries are a major problem in the pediatric and adolescent populations. Some of these injuries extend only partially through the tissue cross-section; yet, there is limited data to inform clinical treatment of such partial tears. In particular, it is unknown how injury severity impacts long-term degenerative changes in the joint. Here, we leverage a skeletally immature preclinical porcine model to evaluate joint biomechanics and degeneration after partial (isolated anteromedial (AM) or posterolateral (PL) bundle) or complete ACL injury. Six months after injury, joint laxity increases were minimal after PL bundle injury, minor after AM bundle injury, and major after ACL injury. Joint degeneration (evaluated in the cartilage and meniscus) was minimal after PL bundle injury, moderate after AM bundle injury, and substantial after ACL injury. With subjects grouped by clinical Lachman grade (indicating the extent of joint destabilization), degeneration was associated with increasing grade, irrespective of injury type. These findings point to the importance of considering joint laxity as a factor when treating young patients, particularly those with partial ACL injuries.

CONTEXT Pediatric anterior cruciate ligament (ACL) injury rates are increasing and are highest in adolescent females. Complete ACL tears are typically surgically reconstructed, but there are few guidelines and very limited data regarding the need for surgical reconstruction or rehabilitation for partial ACL tears in skeletally immature patients. OBJECTIVE To evaluate the impact of partial (anteromedial bundle) and complete ACL transection on joint laxity and tissue forces under anterior and rotational loads in male and female stifle joints throughout skeletal growth in the porcine model. DESIGN Descriptive Laboratory Study. SETTING Laboratory. PATIENTS OR OTHER PARTICIPANTS Sixty male and female Yorkshire cross-breed pigs aged 1.5, 3, 4.5, 6, and 18 months (n=6/age/sex). MAIN OUTCOME MEASURE(S) Joint laxity was measured in intact, anteromedial bundle-transected, and ACL-transected joints under applied anterior-posterior drawer and varus-valgus torque using a robotic testing system. The loading of the soft tissues in the stifle joint was measured under each condition. RESULTS Anterior-posterior joint laxity increased by 13-50% (p<0.05) after anteromedial bundle transection and 75-178% (p<0.05) after ACL transection. Destabilizations after anteromedial bundle transection increased with age (p<0.05) and were greater in late adolescent females than late adolescent males (p<0.05). In anteromedial bundle-transected joints, the posterolateral bundle resisted the anterior load. In ACL-transected joints, the medial collateral ligament (MCL) contribution was largest, followed by the medial meniscus. MCL contribution was larger while medial meniscus contribution was smaller in males versus females. CONCLUSIONS Partial ACL transection resulted in moderate increases in joint laxity, while the remaining bundle performed the primary ACL function. Destabilizations due to partial ACL transection were largest in late adolescent joints, indicating that operative treatment should be considered in active, late adolescent patients. Increased forces in the MCL and medial meniscus after ACL transection suggest that rehabilitation protocols may need to focus on protecting these tissues.

Pediatric anterior cruciate ligament (ACL) injuries are on the rise, and females experience higher ACL injury risk than males during adolescence. Studies in skeletally immature patients indicate differences in ACL size and joint laxity between males and females after the onset of adolescence. However, functional data regarding the ACL and its anteromedial and posterolateral bundles in the pediatric population remain rare. Therefore, this study uses a porcine model to investigate the sex-specific morphology and function of the ACL and its bundles throughout skeletal growth. Hind limbs from male and female Yorkshire pigs aged early youth to late adolescence were imaged using magnetic resonance imaging to measure the size and orientation of the ACL and its bundles, then biomechanically tested under anterior-posterior drawer using a robotic testing system. Joint laxity decreased (p<0.001) while joint stiffness increased (p<0.001) throughout skeletal growth in both sexes. The ACL was the primary stabilizer against anterior tibial loading in all specimens, while the functional role of the anteromedial bundle increased with age (p<0.001), with an earlier shift in males. ACL and posterolateral bundle cross-sectional area and ACL and anteromedial bundle length were larger in males than females during adolescence (p<0.01 for all), while ACL and bundle sagittal angle remained similar between sexes. Additionally, in situ ACL stiffness correlated with cross-sectional area across skeletal growth (r 2 =0.75, p<0.001 in males and r 2 =0.64, p<0.001 in females), but not within age groups. This study has implications for age and sex-specific surgical intervention strategies and suggests the need for human studies.

Context Pediatric anterior cruciate ligament (ACL) injury rates are increasing and are highest in female adolescents. Complete ACL tears are typically surgically reconstructed, but few guidelines and very limited data exist regarding the need for surgical reconstruction or rehabilitation for partial ACL tears in skeletally immature patients. Objective To evaluate the effects of partial (anteromedial bundle) and complete ACL transection on joint laxity and tissue forces under anterior and rotational loads in male and female stifle joints throughout skeletal growth in the porcine model. Design Descriptive laboratory study. Setting Laboratory. Patients or Other Participants We studied 60 male and female Yorkshire crossbreed pigs aged 1.5, 3, 4.5, 6, and 18 months (n = 6 pigs per age per sex). Main Outcome Measure(s) Joint laxity was measured in intact, anteromedial bundle-transected, and ACL-transected joints under applied anterior-posterior drawer and varus-valgus torque using a robotic testing system. Loading of the soft tissues in the stifle joint was measured under each condition. Results Anterior-posterior joint laxity increased by 13% to 50% (P < .05) after anteromedial bundle transection and 75% to 178% (P < .05) after ACL transection. Destabilization after anteromedial bundle transection increased with age (P < .05) and was greater in late female than late male adolescents (P < .05). In anteromedial bundle-transected joints, the posterolateral bundle resisted the anterior load. In ACL-transected joints, the medial collateral ligament (MCL) contribution was largest, followed by the medial meniscus. The MCL contribution was larger and the medial meniscus contribution was smaller in male versus female specimens. Conclusions Partial ACL transection resulted in moderate increases in joint laxity, with the remaining bundle performing the primary ACL function. Destabilization due to partial ACL transection (anteromedial bundle) was largest in late adolescent joints, indicating that operative treatment should be considered in active, late-adolescent patients with this injury. Increased forces on the MCL and medial meniscus after ACL transection suggested that rehabilitation protocols may need to focus on protecting these tissues.

Background: High rates of anterior cruciate ligament (ACL) injury and surgical reconstruction in both skeletally immature and mature populations have led to many studies investigating the size and shape of the healthy ligament. The purposes of the present study were to compile existing quantitative measurements of the geometry of the ACL, its bundles, and its insertion sites and to describe effects of common covariates such as sex and age. Methods: A search of the Web of Science was conducted for studies published from January 1, 1900, to April 11, 2018, describing length, cross-sectional area, volume, orientation, and insertion sites of the ACL. Two reviewers independently screened and reviewed the articles to collect quantitative data for each parameter. Results: Quantitative data were collected from 92 articles in this systematic review. In studies of adults, reports of average ACL length, cross-sectional area, and volume ranged from 26 to 38 mm, 30 to 53 mm 2 , and 854 to 1,858 mm 3 , respectively. Reported values were commonly found to vary according to sex and skeletal maturity as well as measurement technique. Conclusions: Although the geometry of the ACL has been described widely in the literature, quantitative measurements can depend on sex, age, and measurement modality, contributing to variability between studies. As such, care must be taken to account for these factors. The present study condenses measurements describing the geometry of the ACL, its individual bundles, and its insertion sites, accounting for common covariates when possible, to provide a resource to the clinical and scientific communities. Clinical Relevance: Quantitative measures of ACL geometry are informative for developing clinical treatments such as ACL reconstruction. Age and sex can impact these parameters.

Chronic kidney disease (CKD) is a common disease of aging and increases fracture risk over advanced age alone. Aging and CKD differently impair bone turnover and mineralization. We thus hypothesize that the loss of bone quality would be greatest with the combination of advanced age and CKD. We evaluated bone from young adult (6 mo.), middle-age (18 mo.), and old (24 mo.) male C57Bl/6 mice three months following either 5/6th nephrectomy, to induce CKD, or Sham procedures. CKD exacerbated losses of cortical and trabecular microarchitecture associated with aging. Aging and CKD each resulted in thinner, more porous cortices and fewer and thinner trabeculae. Bone material quality was also reduced with CKD, and these changes to bone material were distinct from those due to age. Aging reduced whole-bone flexural strength and modulus, micrometer-scale nanoindentation modulus, and nanometer-scale tissue and collagen strain (small-angle x-ray scattering [SAXS]. By contrast, CKD reduced work to fracture and variation in bone tissue modulus and composition (Raman spectroscopy), and increased percent collagen strain. The increased collagen strain burden was associated with loss of toughness in CKD. In addition, osteocyte lacunae became smaller, sparser, and more disordered with age for Sham mice, yet these age-related changes were not clearly observed in CKD. However, for CKD, larger lacunae positively correlated with increased serum phosphate levels, suggesting that osteocytes play a role in systemic mineral homeostasis. This work demonstrates that CKD reduces bone quality, including microarchitecture and bone material properties, and that loss of bone quality with age is compounded by CKD. These findings may help reconcile why bone mass does not consistently predict fracture in the CKD population, as well as why older individuals with CKD are at high risk of fragility.