M.L. Roemhildt

Effects of increased chronic loading on articular cartilage material properties in the lapine tibio-femoral joint.

Methods of producing relevant and quantifiable load alterations in vivo with which to study load-induced cartilage degeneration analogous to osteoarthritis are limited. An animal model was used to investigate the effects of increased chronic loads on articular cartilage. Mature rabbits were randomized into one of three experimentally loaded groups and a fourth unoperated control group. A mechanical-loading device was skeletally fixed to the hind limb of animals in the loaded groups. Engaging the device resulted in an additional load of 0%, +22% or +44% body weight to the medial compartment of the experimental knee, while allowing normal joint function. Following a 12-week loading protocol, a creep-indentation test and needle probe test were used to determine the biphasic material properties and thickness of the cartilage at four locations of each femoral and tibial condyle of the experimental and contralateral limbs. Analyses of covariance were performed to compare outcome measures across the treatment groups. The effect of increased load was site and load-level specific with alterations of material properties and thickness most prominent in the posterior region of the medial compartment of the tibia. At this site, permeability increased 128% and thickness increased 28% in the +44% body weight group relative to the 0% body weight group. This model of altered chronic loading initiated changes in the material properties to the articular cartilage at the sites of increased load over 12-weeks that were consistent with early degenerative changes suggesting that increased tibio-femoral loading may be responsible for the alterations. This work begins to elucidate the chronic-load threshold and the time course of cartilage degeneration at different levels of altered loading.

Chronic in vivo load alteration induces degenerative changes in the rat tibiofemoral joint

OBJECTIVE We investigated the relationship between the magnitude and duration of sustained compressive load alteration and the development of degenerative changes in the rat tibiofemoral joint. METHODS A varus loading device was attached to the left hind limb of mature rats to apply increased compression to the medial compartment and decreased compression to the lateral compartment of the tibiofemoral joint of either 0% or 100% body weight for 0, 6 or 20 weeks. Compartment-specific assessment of the tibial plateaus included biomechanical measures (articular cartilage aggregate modulus, permeability and Poissons ratio, and subchondral bone modulus) and histological assessments (articular cartilage, calcified cartilage, and subchondral bone thicknesses, degenerative scoring parameters, and articular cartilage cellularity). RESULTS Increased compression in the medial compartment produced significant degenerative changes consistent with the development of osteoarthritis (OA) including a progressive decrease in cartilage aggregate modulus (43% and 77% at 6 and 20 weeks), diminished cellularity (38% and 51% at 6 and 20 weeks), and increased histological degeneration. At 20 weeks, medial compartment articular cartilage thickness decreased 30% while subchondral bone thickness increased 32% and subchondral bone modulus increased 99%. Decreased compression in the lateral compartment increased calcified cartilage thickness, diminished region-specific subchondral bone thickness and revealed trends for reduced cellularity and decreased articular cartilage thickness at 20 weeks. CONCLUSIONS Altered chronic joint loading produced degenerative changes consistent with those observed clinically with the development of OA and may replicate the slow development of non-traumatic OA in which mechanical loads play a primary etiological role.

Mineralization of articular cartilage in the sprague-dawley rat: characterization and mechanical analysis

The formation of mineralized deposits in human articular cartilage is a common occurrence [1–4]; however, the relationship between mineral deposition and material properties of the articular cartilage is not well understood nor the relationship between mineral deposition and the development of degenerative joint disease. Several different crystalline structures have been identified in articular cartilage and synovial fluid including monosodium urate, calcium pyrophosphate dihydrate (CPPD), and basic calcium phosphates (BCPs). These distinct mineral phases are associated with specific pathologies and mechanisms of crystal formation such as the development of monosodium urate in gout and CPPD in pseudogout. Less is known regarding the deposition of BCPs, a class of compounds including carbonate-substituted hydroxyapatite (cHA), tricalcium phosphates (TCP), octacalcium phosphate (OCP), and whitlockite, in articular cartilage. The presence of BCP calcification of articular cartilage in humans has been associated with decreased joint function [1, 3], aging [2] and severity of osteoarthritis [1, 3]. Commonly used methods of crystal detection such as polarized light microscopy of synovial fluid and conventional radiography of the joint can be insensitive to the detection of BCP crystals and more sensitive techniques such as microradiography or electron microscopy of articular cartilage sections are required to detect areas of BCP mineralization [3, 5, 6]. It is not yet known how regions of mineralization may influence the tribological properties (friction, wear, and lubrication) of the articulating surfaces and the material and structural properties of articular cartilage. Animal models with which to study the mechanisms of mineralization of articular cartilage are limited.

Changes Induced by Chronic in vivo Load Alteration in the Tibiofemoral Joint of Mature Rabbits

We investigated the relationship between the magnitude and duration of chronic compressive load alteration and the development and progression of degenerative changes in the rabbit tibiofemoral joint. Varus loading devices were attached to the hind limb of mature NZW rabbits. Altered compressive loads of 0%, 50%, and 80% body weight (BW) were applied to the tibiofemoral joint for 12 h per day for 12 and 24 weeks (n = 4 animals/group). Compartment‐specific assessment of the tibial plateau included histological assessments (articular cartilage, calcified cartilage, and subchondral bone thicknesses, degeneration score, and articular cartilage cellularity) and biomechanical measures (aggregate modulus, permeability, Poissons ratio). Analyses of variance techniques were used to examine the relationship between each outcome measure with load magnitude and duration as independent variables in the model. Degenerative changes developed in the medial compartment with increased magnitude of compressive loading and included fibrillation, increased degeneration score, and reduced cellularity of the articular cartilage. Increased calcified cartilage thickness was observed in both the medial and lateral compartments following exposure to altered loading of 80% BW for 24 weeks. This work demonstrates that in vivo chronic compressive load alteration to the tibiofemoral joint can initiate progressive macroscopic and histological‐based degenerative changes analogous to the early changes occurring in OA.

Gait alterations in rats following attachment of a device and application of altered knee loading

Animal models are widely used to study cartilage degeneration. Experimental interventions to alter contact mechanics in articular joints may also affect the loads borne by the leg during gait and consequently affect the overall loading experienced in the joint. In this study, force plate analyses were utilized to measure parameters of gait in the rear legs of adult rats following application of a varus loading device that altered loading in the knee. Adult rats were assigned to Control, Sham, or Loaded groups (n ≥ 4/each). Varus loading devices were surgically attached to rats in the Sham and Loaded groups. In the Loaded group, this device applied a controlled compressive overload to the medial compartment of the knee during periods of engagement. Peak ground reaction forces during walking were recorded for each rear leg of each group. Analyses of variance were used to compare outcomes across groups (Control, Sham, and Loaded), leg (contralateral, experimental) and device status (disengaged, engaged) to determine the effects of surgically attaching the device and applying a compressive overload to the joint with the device. The mean peak vertical force in the experimental leg was reduced to 30% in the Sham group in comparison to the contralateral leg and the Control group, indicating an effect of attaching the device to the leg (p<0.01). No differences were found in ground reaction forces between the Sham and Loaded groups with application of compressive overloads with the device. The significant reduction in vertical force due to the surgical attachment of the varus loading device must be considered and accounted for in future studies.

Calcium phosphate particulates increase friction in the rat knee joint

OBJECTIVE Basic calcium phosphate (BCP) particulates are commonly found in cartilage and synovial fluid of osteoarthritis (OA) joints with the amount of BCP correlating with knee OA severity. How cartilage mineralization affects joint degeneration has yet to be determined. The objective of this study was to determine whether BCP in the synovial fluid affects the rat knee joint coefficient of friction (COF). METHODS The COFs of knees from both hind limbs of four mature male rats were measured post mortem using a pendulum apparatus with an infrared tracking system. The three conditions evaluated were (1) the naïve state, (2) after the injection of 100 μL of phosphate buffered saline (PBS) (sham) and (3) after the injection of 100 μL of a 1 mg/mL BCP suspension. The decrease in the pendulum amplitude (decay) was fit using two friction models: (1) a one parameter Stanton linear decay model and (2) a two parameters combination Stanton linear decay and viscous damping exponential decay model. RESULTS The COF increased 17.6% after injection of BCP compared to the naïve (P = 0.0012) and 16.0% compared to the saline injected (P = 0.0018) joints as derived from the one parameter model. The COF did not differ between naïve and saline injected joints. Results from the two parameters model showed a similar increase in COF after injection of BCP while the viscous damping was not significantly different between conditions. CONCLUSIONS The increased joint friction with BCP particulates suggests BCPs may play a role in articular surface degradation and OA development.

Changes in in vitro compressive contact stress in the rat tibiofemoral joint with varus loading

Increased compression of the tibiofemoral joint, due to increased body mass or malalignment, is a risk factor for the onset and progression of osteoarthritis. This work investigates compressive stresses and contact areas in the articular cartilage of the rat tibiofemoral joint during standing with different applied varus loads. Cadaver rat knees underwent loading of the extensors combined with varus loading (0%, 50% or 100% of bodyweight) of the tibiofemoral joint. Articular cartilage contact stress was evaluated using stereophotogrammetric measurements of biplanar radiographs, high-resolution micro-computed tomography and discrete element analysis. Random coefficients regression models were used to analyze the relationship between peak and spatially averaged contact stresses and contact areas as a function of increasing varus loadings. The contact stresses increased linearly in the medial compartment. Peak stress significantly increased 0.042 MPa (p=0.006) and spatially averaged stress significantly increased 0.029 MPa (p=0.045) for each 10% increase in varus loading. There was a trend for a small decrease in contact areas in the lateral compartment with varus loading. This is the first report of the contact stresses in a rat tibiofemoral joints under simulated weight bearing conditions. The 0.42 MPa increase in peak contact stress at the cartilage-cartilage interface of the medial compartment with 100% bodyweight varus load is similar to the reported change in peak contact stress associated with development of symptomatic knee osteoarthritis in humans. Determination of contact stresses in rat tibiofemoral joints allows comparison to contact stresses in humans with the development of osteoarthritis.

Tissue Modification of the Lateral Compartment of the Tibio-Femoral Joint Following In Vivo Varus Loading in the Rat

This study describes the first application of a varus loading device (VLD) to the rat hind limb to study the role of sustained altered compressive loading and its relationship to the initiation of degenerative changes to the tibio-femoral joint. The VLD applies decreased compressive load to the lateral compartment and increased compressive load to the medial compartment of the tibio-femoral joint in a controlled manner. Mature rats were randomized into one of three groups: unoperated control, 0% (sham), or 80% body weight (BW). Devices were attached to an animals leg to deliver altered loads of 0% and 80% BW to the experimental knee for 12 weeks. Compartment-specific material properties of the tibial cartilage and subchondral bone were determined using indentation tests. Articular cartilage, calcified cartilage, and subchondral bone thicknesses, articular cartilage cellularity, and degeneration score were determined histologically. Joint tissues were sensitive to 12 weeks of decreased compressive loading in the lateral compartment with articular cartilage thickness decreased in the peripheral region, subchondral bone thickness increased, and cellularity of the midline region decreased in the 80% BW group as compared to the 0% BW group. The medial compartment revealed trends for diminished cellularity and aggregate modulus with increased loading. The rat-VLD model provides a new system to evaluate altered quantified levels of chronic in vivo loading without disruption of the joint capsule while maintaining full use of the knee. These results reveal a greater sensitivity of tissue parameters to decreased loading versus increased loading of 80% BW for 12 weeks in the rat. This model will allow future mechanistic studies that focus on the initiation and progression of degenerative changes with increased exposure in both magnitude and time to altered compressive loads.

In Situ Microindentation for Determining Local Subchondral Bone Compressive Modulus

Alterations to joint tissues, including subchondral bone, occur with osteoarthritis. A microindentation technique was developed to determine the local compressive modulus of subchondral bone. This test, in conjunction with a cartilage indentation test at the same location, could evaluate changes of these material properties in both tissues. The accuracy of the technique was determined by applying it to materials of known moduli. The technique was then applied to rat tibial plateaus to characterize the local moduli of the subchondral bone. An established nanoindentation method was adopted to determine the modulus of subchondral bone following penetration of the overlying articular cartilage. Three cycles of repeated loadings were applied (2.452 N, 30 s hold). The slope of the load-displacement response during the unloading portion of the third cycle was used to measure the stiffness. Indentation tests were performed on two polyurethane foams and polymethyl-methacrylate for validation (n=15). Regression analysis was used to compare the moduli with reference values. Subchondral bone moduli of tibial plateaus from Sprague-Dawley rats (n=5) were measured for central and posterior locations of medial and lateral compartments. An analysis of variance was used to analyze the effects of compartment and test location. The measured moduli of the validation materials correlated with the reference values (R(2)=0.993, p=0.05). In rat tibial plateaus, the modulus of the posterior location was significantly greater than the center location (4.03+/-1.00 GPa and 3.35+/-1.16 GPa respectively, p=0.03). The medial compartment was not different from the lateral compartment. This method for measuring the subchondral bone in the same location as articular cartilage allows studies of the changes in these material properties with the onset and progression of osteoarthritis.

101 IN VITRO VALIDATION OF A VARUS LOADING DEVICE IN THE RABBIT KNEE

of radiolabeled anti-rat type II collagen antibody were detected in the synovial fluid 10 minutes and 24 hours post-IA administration relative to the non-specific antibody. Conclusions: Rapid clearance of a non-specific antibody from the synovial fluid and joint tissues is observed after IA dosing to a normal rat knee joint. The data highlights that if sustained drug exposure is required for efficacy, the retention time of the therapeutic needs to be increased. Sustained retention of an anticollagen II antibody in the rat joint suggests that binding to a resident protein may extend antibody retention time.