Intra-articular anaesthesia mitigates established pain in experimental osteoarthritis: a preliminary study of gait impulse redistribution as a biomarker of analgesia pharmacodynamics.

OBJECTIVE: Develop a sensitive, functional biomarker of persistent joint pain in a large animal model of experimental osteoarthritis. Evaluate Impulse Ratio as a measure of weight distribution among supporting limbs throughout the early natural history of osteoarthritis and with local anaesthesia and analgesia. DESIGN: The distribution of weight bearing in the trot of 11 skeletally-mature dogs was analyzed before and after unilateral surgical intervention (cranial cruciate transection or distal femoral focal impact). The short-term effects of two analgesic treatments (intra-articular lidocaine and intra-dermal meloxicam) were then evaluated as an index of pain relief based on the redistribution of weight-bearing impulse between normal and injured limbs. RESULTS: Impulse Ratio was able to resolve weight redistribution between limbs in both long-term (weekly for over 400 days) and short-term (15 min intervals) joint evaluations. Joint pain relief from lidocaine administration could be reliably tracked over its brief acting time course. Meloxicam administration resulted in ambiguous results, where average weight bearing in the injured limb did not increase, but the variability of limb use changed transiently and reversibly. CONCLUSION: Joint function and the role of persistent joint pain in the development of osteoarthritis can be investigated effectively and efficiently in a large animal model through the use of Impulse Ratio. Impulse Ratio can be a functionally relevant and sensitive biomarker of locomotion-related joint pain.

Viscoelasticity of the articular cartilage surface in early osteoarthritis.

OBJECTIVE: Structural and biochemical changes in articular cartilage occur throughout the pathogenesis of osteoarthritis (OA). Early changes include proteoglycan loss and collagen network disorganization at or near the articular surface. These changes accompany reductions in mechanical properties of cartilage, yet the relationships between mechanics and structure in early OA are poorly defined. Thus, the overall goal of this work was to measure changes in the microscale mechanics and structure of the articular surface in an in vivo model of OA to better understand the early pathogenesis of cartilage degeneration in this disease. DESIGN: A canine cranial cruciate ligament transection (CCL(x)) model was used. The contralateral joint served as an internal control (Ctl). The frequency dependence of the dynamic indentation modulus (E(∗)) was evaluated, and creep behavior was measured to estimate the instantaneous (E(i,inst)) and equilibrium (E(i,eq)) indentation moduli and longest creep time-constant (τ). These functional parameters were related to microscopic metrics of cartilage structure and biochemistry, measured by polarized light microscopy and digital densitometry of proteoglycan staining by safranin-O. RESULTS: CCL(x) and Ctl cartilage exhibited frequency sensitivity. E(i,inst), E(i,eq), and τ were lower in CCL(x) vs Ctl cartilage. These mechanical changes were accompanied by a reduction in superficial zone thickness and changes in superficial zone collagen organization, as well as a non-significant reduction in superficial zone proteoglycan staining. CONCLUSIONS: Changes in the microscale viscoelastic behavior of the cartilage surface are a functional hallmark of early OA that accompany significant changes to the microstructural organization of the collagenous extracellular matrix.

New understanding of the complex structure of knee menisci: implications for injury risk and repair potential for athletes.

Menisci help maintain the structural integrity of the knee. However, the poor healing potential of the meniscus following a knee injury can not only end a career in sports but lead to osteoarthritis later in life. Complete understanding of meniscal structure is essential for evaluating its risk for injury and subsequent successful repair. This study used novel approaches to elucidate meniscal architecture. The radial and circumferential collagen fibrils in the meniscus were investigated using novel tissue-preparative techniques for light and electron microscopic studies. The results demonstrate a unique architecture based on differences in the packaging of the fundamental collagen fibrils. For radial arrays, the collagen fibrils are arranged in parallel into ∼10 µm bundles, which associate laterally to form flat sheets of varying dimensions that bifurcate and come together to form a honeycomb network within the body of the meniscus. In contrast, the circumferential arrays display a complex network of collagen fibrils arranged into ∼5 µm bundles. Interestingly, both types of architectural organization of collagen fibrils in meniscus are conserved across mammalian species and are age and sex independent. These findings imply that disruptions in meniscal architecture following an injury contribute to poor prognosis for functional repair.

Non-uniform strain distribution within rat cartilaginous growth plate under uniaxial compression.

Growth plates are highly inhomogeneous in morphology and composition. Mechanical loading can modulate longitudinal bone growth, though the mechanisms underlying this mechanobiology are poorly understood. The proximal tibial growth plates of six rats were tested in vitro under uniaxial compression to 5% strain, and confocal microscopy was used to track and capture images of fluorescently labeled cell nuclei with increasing applied strains. The local strain patterns through the growth plate thickness were quantified using texture correlation analysis. The technique of texture correlation analysis was first validated by comparing theoretical simulated strain maps generated from numerically distorted images. The texture correlation algorithm was sensitive to the grid size superimposed on the original image, but remained insensitive to parameters related to the size of the final image mask, which was searched by the correlation algorithm for each grid point of the original image. Within the growth plate, experimental strain distributions were non-uniform in all six specimens. Growth plates were mostly under compression strains. The strain distributions differed among the histomorphological zones of the growth plate, which was most obvious in specimens with regular growth plate shape: higher compressive strains (4-8 times higher than the applied 5% strain) were located mainly in regions overlapping the reserve and hypertrophic zones with lower compressive strains in the proliferative zone. This study documents the non-uniform mechanical behavior of growth plate across its three histological zones when exposed to compression. Further investigation is required to establish the significance of non-uniform strain fields during growth in vivo.

Intra-articular injection of synovial mesenchymal stem cells improves cartilage repair in a mouse injury model.

Controversy remains whether articular cartilage has an endogenous stem/progenitor cell population, since its poor healing capacity after injury can lead to diseases such as osteoarthritis. In the joint environment there are mesenchymal stem/progenitor cells (MSCs) in the synovial membrane and synovial fluid that can differentiate into cartilage, but it is still under debate if these cells contribute to cartilage repair in vivo. In this study, we isolated a Sca-1 positive, chondrogenesis capable population of mouse synovial MSCs from C57BL6 and MRL/MpJ "super-healer" strains. Intra-articular injection of Sca-1 + GFP + synovial cells from C57BL6 or MRL/MpJ into C57BL6 mice following cartilage injury led to increased cartilage repair by 4 weeks after injury. GFP expression was detected in the injury site at 2 weeks, but not 4 weeks after injury. These results suggest that synovial stem/progenitor cells, regardless of strain background, have beneficial effects when injected into an injured joint. MSCs derived from MRL/MpJ mice did not promote an increased repair capacity compared to MSCs derived from non-healing C57BL6 controls; however, MRL/MpJ MSCs were observed within the defect area at the time points examined, while C57BL6 MSCs were not.

The notochordal cell in the nucleus pulposus: a review in the context of tissue engineering.

An understanding of developmental biology can provide useful insights into how different tissue-engineered repairs might be designed. During embryogenesis of the intervertebral disk, the cells of the notochord play a critical role in initiating tissue formation, and may be responsible for development of the nucleus pulposus. In some species, including humans, these notochordal cells may eventually be lost, either through apoptosis or terminal differentiation, and are replaced by chondrocyte-like cells. However, there is some evidence that the notochordal cells may persist in at least some humans. This review discusses some of the potential applications of notochordal cells in tissue engineering of the nucleus pulposus.

Tissue transfers: substrates for cytology and cytochemistry of animal tissues.

The Tissue Transfer Technique (TTT) is a novel method of sampling animal tissue that can be used to study tissue morphology, chemistry and physiology. This review provides an overview of the technique and demonstrates its use to detect the tissue distribution of specific epitopes, lectin binding sites and nucleic acids as well as its application as an organ monolayer in culture. These applications are compared and contrasted with standard histological techniques including the "Tissue Printing Technique" developed to sample plant tissue.

Periarticular cancellous bone changes following anterior cruciate ligament injury.

To understand more fully the early bone changes in an experimental model of osteoarthrosis, we quantified periarticular bone mineral density and bone mechanical properties in anterior cruciate ligament transected (ACLX) knee joints (4, 10, 32, and 39 wk post-ACLX) compared with contralateral joints and unoperated normal joints of skeletally mature animals. Maximal stress and energy were significantly reduced in ACLX cancellous bone from the medial femoral condyles at 4 wk postinjury. All mechanical properties (e.g., yield stress and elastic modulus) declined after 4 wk and were significantly reduced at 10 wk. ACLX bone mineral density was significantly reduced at all measured time points. Ash content was significantly reduced at 10 and 32 wk. Changes in the lateral condyles were similar but less pronounced than in the medial condyles. These bony changes accompanied the earliest articular cartilage molecular changes and preceded changes in the articular cartilage gross morphology. We suggest that these early changes in bone mechanical behavior contribute to the progression of osteoarthrosis and pathogenic changes in the joint.

Early regional adaptation of periarticular bone mineral density after anterior cruciate ligament injury.

The present study measured early-stage adaptation of bone mineral (BMD) in the periarticular cancellous bone of the canine knee (stifle) joint after anterior cruciate ligament (ACL) transection (ACLX). Regional changes in BMD in the tibia and femur were analyzed by using quantitative computed tomography (qCT) at 3 wk and 12 wk after unilateral ACLX to determine whether there were focal points for BMD changes and whether these changes occurred early after the induced knee injury. BMD decreased rapidly after ACLX, and the more pronounced response was in the femur. In the 3-wk group, there were decreases in BMD in the tibia and the femur, and these changes were significant in the posterior-medial region of the femur, which showed a decrease of BMD in the ACLX limb (-0.048 +/- 0.011 g/cm(3)). In the 12-wk group, all regions in the tibia and femur exhibited significant decreases in BMD, and the average decrease was greatest in the posterior-medial region of the femur (-0.142 +/- 0.021 g/cm(3)). The regions of pronounced periarticular cancellous BMD adaptation corresponded to observed focal cartilage defects. Early decreases in BMD in the injured knee may be related to altered loading and kinematics in the knee and may be an important link in the pathogenesis of posttraumatic osteoarthritis.

The developmental morphology of a "periosteal" ligament insertion: growth and maturation of the tibial insertion of the rabbit medial collateral ligament.

The structural properties of ligament insertions change dramatically during growth and maturation, but little is known about their developmental anatomy. This study describes and quantifies changes in the gross and microscopic anatomy of the tibial insertion of the rabbit medial collateral ligament (MCL) during development and at skeletal maturity. Eighty animals were used for growth and descriptive studies. From this group, 27 animals, ranging in age from 1 to 24 months, were injected with fluorescent bone markers and their tibial insertions were processed undecalcified for histology. Sections were examined by polarized light and fluorescence microscopy to identify matrix and cells and to quantify mineral formation. Results showed that animals achieved histological skeletal maturity between 9 and 12 months of age. Body weights were a poor index of skeletal maturity. The tibial insertion was composed of five tissue layers, which changed proportions during growth and maturation. In immature animals, MCL fibers entered the periosteum; in older animals, MCL fibers were cemented to the tibia by advancing mineral. The tibial attachment of the MCL was thus transferred from the periosteum to the cortex during growth, suggesting that the term "periosteal insertion" is imprecise in adults. The hypothesis is put forward that these structural changes account for the reported increase in tensile failure of this insertion near skeletal maturity.

Absolute concentrations of mRNA for type I and type VI collagen in the canine meniscus in normal and ACL-deficient knee joints obtained by RNase protection assay.

Relatively little is known about the cellular and molecular responses of the knee joint meniscus to joint injury, despite the functional importance of the tissue. We investigated how meniscus cells respond to joint injury in the early stages of post-traumatic osteoarthritis by characterizing the changes in matrix gene expression in menisci at 3 and 12 weeks post-surgery in dogs in which the anterior cruciate ligament (ACL) in one joint was transected and the other unoperated joint served as a control. Changes in the total RNA and DNA concentrations of the menisci were determined. Absolute concentrations of the mRNA of the COL1A1 gene of type 1 collagen, the major fibrillar collagen of the meniscus, and the COL6A3 gene of type VI collagen, a major repair molecule, were determined by quantitative ribonuclease (RNase) protection assay. The concentration of total RNA in medial and lateral menisci increased from 40 to 60 microg RNA/g wet wt in unoperated, control joints to 200-350 microg RNA/g wet wt in ACL-deficient joints. No significant changes were detected in the concentration of DNA (900-1200 microg DNA/g wet wt). Low concentrations of COL1A1 (2-3 pmol mRNA/g DNA) and COL6A3 (0.3-0.6 pmol mRNA/g DNA) mRNA transcripts were measured in normal menisci. ACL-deficiency induced a 20-38 fold increase in COL1A1 and COL6A3 mRNA concentration at 3 weeks, and an 11-19 fold increase at 12 weeks post-surgery. In general, the increase in COL1A1 and COL6A3 mRNA concentrations was greater in medial menisci than in lateral menisci. These results demonstrate that the menisci initiate a vigorous biosynthetic response to transection of the ACL.

In situ chondrocyte deformation with physiological compression of the feline patellofemoral joint.

The mechanical environment is an important factor affecting the maintenance and adaptation of articular cartilage, and thus the function of the joint and the progression of joint degeneration. Recent evidence suggests that cartilage deformation caused by mechanical loading is directly associated with deformation and volume changes of chondrocytes. Furthermore, in vitro experiments have shown that these changes in the mechanical states of chondrocytes correlate with a change in the biosynthetic activity of cartilage cells. The purpose of this study was to apply our knowledge of contact forces within the feline patellofemoral joint to quantify chondrocyte deformation in situ under loads of physiological magnitude. A uniform, static load of physiological magnitude was applied to healthy articular cartilage still fully intact and attached to its native bone. The compressed cartilage was then chemically fixed to enable the evaluation of cartilage strain, chondrocyte deformation and chondrocyte volumetric fraction. Patella and femoral groove articular cartilages differ in thickness, chondrocyte aspect ratio, and chondrocyte volumetric fraction in both magnitude and depth distribution. Furthermore, when subjected to the same compressive loads, changes to all of these parameters differ in magnitude and depth distribution between patellar and femoral groove articular cartilage. This evidence suggests that significant chondrocyte deformation likely occurs during in vivo joint loading, and may influence chondrocyte biosynthetic activity. Furthermore, we hypothesise that the contrasts between patella and femoral groove cartilages may explain, in part, the site-specific progression of osteoarthritis in the patellofemoral joint of the feline anterior cruciate ligament transected knee.

Stress governs tissue phenotype at the femoral insertion of the rabbit MCL.

The cells in the midsubstance portion of skeletal ligaments typically have elongated shapes, but where ligaments insert into bone the cells appear very rounded and the tissue phenotype is that of fibrocartilage. Between the midsubstance and the insertions there is a gradient in cell shape and tissue phenotype that has been hypothesized to reflect a gradient of mechanical stresses. To test this hypothesis, cell shapes (an index of tissue phenotype) were quantified in the central part of the femoral insertion of the rabbit medial collateral ligament by computer-assisted histomorphometry. Morphometric measurements were correlated with the mechanical stresses and strains in the central part of the insertion as predicted by finite element analysis. Throughout the ligament the direction of the predicted principal tensile stresses coincides with the direction of the collagen fibers which curve from the midsubstance to meet the femur at nearly right angles. Principal compressive stresses also occur within the ligament: the highest are localized near the bone; the lowest in the midsubstance. The areas with the roundest cells correspond to the areas with the highest principal compressive stresses in the model; the areas with the flattest cells correspond to the areas with the lowest compressive stresses in the model. A correlation between cell shape and mechanical stresses suggests that physiological loading of the MCL is important for the maintenance of tissue phenotype throughout this insertion. We theorize that the cells in ligament insertions adapt to the prevailing local mechanical environment.

The effects of increased tension on healing medical collateral ligaments.

The effects of motion and increased levels of stress on the biomechanical, biochemical, and morphological properties of healing medial collateral ligaments were assessed in a rabbit model. In one group, the medial collateral ligament of the left hindlimb was transected and allowed to heal with cage activity for either 6 or 12 weeks. In another group, the transected ligaments were permitted to heal for 4 weeks and then were placed under increased stress by inserting a stainless steel pin perpendicularly underneath the healing medial collateral ligament. The animals were allowed cage activity for an additional 2 or 8 weeks. The varus-valgus joint laxity and the stress-strain properties of the medical collateral ligament substance were obtained. Further, the quantity of total collagen, amount and ratio of the collagen cross-links, dihydroxylysinonorleucine and hydroxylysinonorleucine, and the histologic appearance of the healing medical collateral ligaments were evaluated for all groups. At 6 weeks, knees with a transected medial collateral ligament were twice as lax as the controls. However, joints with the stainless steel tension pin had varus-valgus values approximately 1.5 times those of the controls. At 12 weeks, joints with increased stress were not statistically different from the controls. The group that had healing with increased stress for 12 weeks produced the highest stress for a given strain compared to any other group. Also, the total collagen levels and the ratio of dihydroxylysinonorleucine/hydroxylysinonorleucine were the closest to normal of any transected group. Finally, qualitative histologic improvements were seen, including a more longitudinal arrangement of collagen fibers and decreased cellularity.

Whole joint embedding in polymethylmethacrylate: a method for preparing intact musculoskeletal organ systems for histomorphology and 3-D reconstruction.

Large and medium size undecalcified joints were embedded in methylmethacrylate resin. Sections of 600 microns prepared from polymethylmethacrylate blocks show minimal distortion and are suitable for surface staining and three-dimensional reconstruction. The 5 microns sections prepared from these slabs retain good cytological detail. This method permits the examination of musculoskeletal organ systems at both macroscopic and microscopic levels.

Early morphometric and anisotropic change in periarticular cancellous bone in a model of experimental knee osteoarthritis quantified using microcomputed tomography.

OBJECTIVE: To quantify early stage microstructural changes of periarticular cancellous bone in a canine anterior cruciate ligament transection model for experimental osteoarthritis. DESIGN: Unilateral transection of the anterior cruciate ligament was performed in 10 animals. Bone structure changes were quantified in five animals at 3-week post-transection and five animals at 12-week post-transection. An additional two non-operated animals were used as controls. BACKGROUND: Changes in trabecular architecture of the periarticular cancellous bone in early stage post-traumatic osteoarthritis is not well understood. Previous studies have found alterations in bone mineral density in experimental osteoarthritis suggesting adaptation of the trabecular structure. Early change of the periarticular bone following a ligament injury may contribute to the long-term development of osteoarthritis. METHODS: ++. Bone cores from the medial condyles of the femoral and tibial pairs were scanned with a three-dimensional microtomographic system. Structural indices were quantified including bone volume ratio, bone surface ratio, trabecular thickness, trabecular separation, trabecular number, as well as structural anisotropy determined by the mean-intercept-length method.Results. Significant structural changes were observed at 3-week post-transection, and were more prominent at 12-week post-transection. These changes were accompanied by decreasing anisotropy. CONCLUSIONS: Periarticular cancellous bone microstructure is significantly altered in experimental osteoarthritis. These changes occurred as early as 3-week post-transection, and were large at 12-week post-transection. RELEVANCE: The pathogenesis of post-traumatic osteoarthritis is poorly understood, but it is clear that this disease involves the entire organ system of the joint, including the cartilages, synovium, ligaments, and bones. This study focuses on the changes that occur in the bones during the early stages following a joint injury, and contributes to a better overall understanding of the disease aetiology.

Analysing nuclear shape as a function of relative spatial position in the femoral insertion of the medial collateral ligament.

Quantification of biological structure by morphometry facilitates the correlation of structure to biological function. To test a hypothesis concerning the correlation of a gradient in cell and nuclear shape and a corresponding gradient in mechanical stress within a ligament insertion into bone, a computerized approach was developed for quantifying mean nuclear shape as a function of position within this insertion. Three femoral insertions of the medial collateral ligament of the rabbit were prepared for histology. Commercially available software was used to measure nuclear perimeter and area by video-based planimetry on microscopic images, then custom software was used to define an overlying mesh of polygons on each insertion, to normalize the geometry of the insertion, and to calculate the mean nuclear roundness (a function of area and perimeter) for each polygon in the mesh. This approach allows the comparison of mean nuclear roundness of different polygons and among different animals that would be extremely difficult to do manually. In addition, these morphometry results can be used to correlate with functional data.

The effects of mechanical loading on the mRNA expression of growth-plate cells.

Bone growth is a complex process involving proliferation, maturation and hypertrophy of chondrocytes in the growth plates. Mechanical forces applied to growing bones alter their longitudinal growth. However, the mechanisms by which chondrocytes modulate longitudinal bone growth are not well understood. This in vitro study investigated the effects of mechanical loading on the mRNA expression pattern of key molecular components of the growth-plate. Short-term static loading was applied to rat proximal tibial growth-plate explants. Various age groups at specific developmental stages were investigated. In situ hybridization was used to assess the mRNA expression of the cells in different zones of the growth-plate. Four key components were investigated: 18s (basic cell metabolism), type II collagen (major extracellular matrix component), type X collagen (matrix component in hypertrophic zone) and PTH-PTHrP receptors (pre-hypertrophic chondrocytes). The spatial variation in the mRNA expression between loaded explants and their contralateral controls was compared to establish: -the sensitivity of the different growth-plate zones to mechanical loading; -the sensitivity of the different developmental stages to loading. Preliminary results indicated that static loading on the growth plate of 80 d.o. rats affects type II and X collagen gene expressions while PTH-PTHrP remains insensitive to static loading. Improved understanding of growth-plate mechanics and the underlying biology is required to provide a scientific basis for the treatment of progressive deformities.

Expression of proteoglycans and collagen in the hypertrophic phase of experimental osteoarthritis.

These studies seek to define the gene expression of proteoglycans and collagens in the developing hypertrophic phase of an experimental model of osteoarthritis (OA). Total RNA was extracted from articular cartilage of nonoperated and operated dog knees 10 weeks after induction of OA by transection of the anterior cruciate ligament. The relative amounts of mRNA for type II collagen and the core proteins for the aggrecan, biglycan, decorin, and fibromodulin were analyzed by Northern blotting. Total RNA in OA vs nonoperated knees was statistically significantly elevated 2.5x, the mRNA for type II collagen was elevated 8x, aggrecan 2x, biglycan 4x, fibromodulin 2x. The level for decorin was increased 1.6x, but this difference was not statistically significant. Chondrocytes respond actively to joint injury. Gene expression of proteoglycans and type II collagen is discoordinate in early experimental OA, and may contribute to the development of cartilage abnormalities.

Embryonic stem cell therapy improves bone quality in a model of impaired fracture healing in the mouse; tracked temporally using in vivo micro-CT.

In the current study, we used an estrogen-deficient mouse model of osteoporosis to test the efficacy of a cell-generated bone tissue construct for bone augmentation of an impaired healing fracture. A reduction in new bone formation at the defect site was observed in ovariectomized fractures compared to the control group using repeated measures in vivo micro-computed tomography (µCT) imaging over 4 weeks. A significant increase in the bone mineral density (BMD), trabecular bone volume ratio, and trabecular number, thickness and connectivity were associated with fracture repair in the control group, whereas the fractured bones of the ovariectomized mice exhibited a loss in all of these parameters (p<0.001). In a separate group, ovariectomized fractures were treated with murine embryonic stem (ES) cell-derived osteoblasts loaded in a three-dimensional collagen I gel and recovery of the bone at the defect site was observed. A significant increase in the trabecular bone volume ratio (p<0.001) and trabecular number (p<0.01) was observed by 4 weeks in the fractures treated with cell-loaded collagen matrix compared to those treated with collagen I alone. The stem cell-derived osteoblasts were identified at the fracture site at 4 weeks post-implantation through in situ hybridization histochemistry. Although this cell tracking method was effective, the formation of an ectopic cellular nodule adjacent to the knee joints of two mice suggested that alternative in vivo cell tracking methods should be employed in order to definitively assess migration of the implanted cells. To our knowledge, this study is the first of its kind to examine the efficacy of stem cell therapy for fracture repair in an osteoporosis-related fracture model in vivo. The findings presented provide novel insight into the use of stem cell therapies for bone injuries.