Calcified Cartilage Layer Research Articles

Combined degenerative and regenerative remodeling responses of the mandibular condyle to experimentally induced disordered occlusion

The purposes of this research were to investigate the long-term responses of mandibular condylar cartilage to experimentally induced disordered occlusion and to evaluate changes in the expression of the SDF-1/CXCR4 axis. Experimentally induced disordered occlusions were created in 8-week-old female Sprague-Dawley rats by orthodontic methods. After 24 weeks, remodeling of the mandibular condylar cartilage was assessed by hematoxylin and eosin staining. Protein and mRNA expression of SDF-1, CXCR4, MMP9, IL6, OPG, and RANKL were investigated by means of immunohistochemical staining and real-time polymerase chain reaction. Obvious cartilage degenerative remodeling responses were observed; they appeared as uneven distributions of cellular disposition, loss of cartilage surface integrity, and cell-free areas. Regenerative responses presenting as thickening of the whole and the calcified cartilage layers in the experimental group were also observed. Compared with the age-matched controls, the protein and mRNA levels of SDF-1, CXCR4, MMP9, IL6, and OPG, but not RANKL, were increased in the experimental group (all, P<0.05). In addition, the mRNA level of RANKL/OPG showed a decreasing trend in the experimental group compared with the age-matched controls (P= 0.052). This study demonstrated that long-term experimentally induced disordered occlusion leads to a combined response in degeneration and regeneration of mandibular cartilage, accompanied by active interaction of the SDF-1/CXCR4 axis and local upregulation of MMP9, IL6, and OPG.

The Cartilage-Bone Interface

In the knee joint, the purpose of the cartilage-bone interface is to maintain structural integrity of the osteochondral unit during walking, kneeling, pivoting, and jumping--during which tensile, compressive, and shear forces are transmitted from the viscoelastic articular cartilage layer to the much stiffer mineralized end of the long bone. Mature articular cartilage is integrated with subchondral bone through a approximately 20 to approximately 250 microm thick layer of calcified cartilage. Inside the calcified cartilage layer, perpendicular chondrocyte-derived collagen type II fibers become structurally cemented to collagen type I osteoid deposited by osteoblasts. The mature mineralization front is delineated by a thin approximately 5 microm undulating tidemark structure that forms at the base of articular cartilage. Growth plate cartilage is anchored to epiphyseal bone, sometimes via a thin layer of calcified cartilage and tidemark, while the hypertrophic edge does not form a tidemark and undergoes continual vascular invasion and endochondral ossification (EO) until skeletal maturity upon which the growth plates are fully resorbed and replaced by bone. In this review, the formation of the cartilage-bone interface during skeletal development and cartilage repair, and its structure and composition are presented. Animal models and human anatomical studies show that the tidemark is a dynamic structure that forms within a purely collagen type II-positive and collagen type I-negative hyaline cartilage matrix. Cartilage repair strategies that elicit fibrocartilage, a mixture of collagen type I and type II, are predicted to show little tidemark/calcified cartilage regeneration and to develop a less stable repair tissue-bone interface. The tidemark can be regenerated through a bone marrow-driven growth process of EO near the articular surface.

Does prior sustained compression make cartilage-on-bone more vulnerable to trauma?

BackgroundThis study investigated how varying levels of prior creep deformation in cartilage-on-bone samples influences their mechanical response and vulnerability to structural damage following a single traumatic impact. MethodsBovine patellae were subjected to varying intervals of prior creep loading at a constant stress of 4MPa. Immediately following removal of this stress the samples were impacted with a pendulum indenter system at a fixed energy of 2.2J. FindingsWith increasing prior creep, the peak force on impact rose, the duration of impact and time to reach peak force both decreased, and both the energy dissipated during impact and the magnitude of impulse were both unchanged by the level of prior creep. With increasing prior creep, the severity of impact-induced osteochondral damage increased: articular cartilage cracks penetrated to a greater depth, extending to the calcified cartilage layer resulting in hairline fractures or articular cartilage delamination and associated secondary damage to the vascular channels in the subchondral bone. InterpretationThe study shows that exposure of the cartilage-on-bone system to prior creep can significantly influence its response to subsequent impact, namely force attenuation and severity of damage to the articular cartilage, calcified cartilage and vascular channel network in the subchondral bone.

Microfracture for knee chondral defects: a survey of surgical practice among Canadian orthopedic surgeons

The purpose of this study was to describe the practice of microfracture surgery for knee chondral defects among Canadian orthopedic surgeons. All orthopedic surgeon members of the Canadian Orthopaedic Association were invited to participate in a survey, designed to explore the microfracture technique used by orthopedic surgeons in the treatment for knee chondral defects The primary outcome measure was an emailed 26-item questionnaire, which explored indications for microfracture surgery, surgical techniques, types of postoperative rehabilitation regimes used and assessment of outcome. In addition, responses were compared between orthopedic surgeons with a sports medicine practice to surgeons with a non-sports medicine practice. The survey response rate was 24.6% (299/1,216), with 131 regularly performing microfracture. 41% of surgeons indicated that they had no upper limit for age at the time of surgery, and 87% indicated no upper limit for body mass index. The majority of respondents (97%) resected cartilage back to a stable margin, while 69% of respondents removed the calcified cartilage layer prior to creating holes. Only 11% of respondents used continuous passive motion (CPM) postoperatively, and 39% did not restrict weight bearing. Sports surgeons were more likely than non-sports surgeons to remove the calcified cartilage layer, use a 45° pick, use CPM and restrict weight bearing postoperatively (all P values < 0.05). This survey on microfracture for knee chondral defects revealed widespread variation among surgeons regarding the indications for surgery, surgical technique, postoperative rehabilitation and assessment of outcome. Sports surgeons demonstrate better evidence-based practice than non-sports surgeons for a few important parameters. Cross-sectional survey, Level II.

Biochemical-based MRI in diagnosis of early osteoarthritis

Cartilage anatomy Each biochemical MRI technique targets a specific part of the cartilage anatomy; thus, understanding its structure becomes paramount. A majority of the articular cartilage comprises water, type II collagen and proteoglycans, particularly glucosaminoglycan (GAG) chains [3]. Each GAG chain has considerable negative charge, which attracts water at times of unloading. Here, swelling is prevented by a strict organization of collagen fibers [2]. As described by Ulrich-Vinther et al., normal cartilage can be divided into three zones [3]. A superficial zone contains the highest water content, but the lowest proteoglycan concentration. Collagen fibers present here are in parallel orientation to the articular surface. An intermediate zone consists of fibers in a more oblique fashion. Finally, the deep zone has a dense array of collagen fibers oriented perpendicular to the articular surface. It contains the highest proteoglycan concentration and the lowest water content, an inverse relationship to its superficial counterpart. Underneath these zones is a small layer of calcified cartilage, consisting of radially oriented collagen fibers embedded in a calcified matrix. This separates overlying deep zone from underlying subchondral bone [3].

Elevated cross-talk between subchondral bone and cartilage in osteoarthritic joints

Osteoarthritis (OA) is a degenerative joint disease and one of the leading causes of disability in the United States and across the world. As a disease of the whole joint, OA exhibits a complicated etiology with risk factors including, but not limited to, ageing, altered joint loading, and injury. Subchondral bone is hypothesized to be involved in OA development. However, direct evidence supporting this is lacking. We previously detected measurable transport of solute across the mineralized calcified cartilage in normal joints, suggesting a potential cross-talk between subchondral bone and cartilage. Whether this cross-talk exists in OA has not been established yet. Using two models that induced OA by either ageing or surgery (destabilization of medial meniscus, DMM), we tested the hypothesis that increased cross-talk occurs in OA. We quantified the diffusivity of sodium fluorescein (mol. wt. 376Da), a marker of small-sized signaling molecules, within calcified joint matrix using our newly developed fluorescence loss induced by photobleaching (FLIP) method. Tracer diffusivity was found to be 0.30±0.17 and 0.33±0.20μm2/s within the calcified cartilage and 0.12±0.04 and 0.07±0.03μm2/s across the osteochondral interface in the aged (20–24-month-old, n=4) and DMM OA joints (5-month-old, n=5), respectively, which were comparable to the control values for the contralateral non-operated joints in the DMM mice (0.48±0.13 and 0.12±0.06μm2/s). Although we did not detect significant changes in tissue matrix permeability in OA joints, we found i) an increased number of vessels invading the calcified cartilage (and sometimes approaching the tidemark) in the aged (+100%) and DMM (+50%) joints relative to the normal age controls; and ii) a 60% thinning of the subchondral bone and calcified cartilage layers in the aged joints (with no significant changes detected in the DMM joints). These results suggested that the capacity for cross-talk between subchondral bone and articular cartilage could be elevated in OA. Further studies are needed to identify the direction of the cross-talk, the signaling molecules involved, and to test whether subchondral bone initiates OA development and could serve as a pharmaceutical target for OA treatment.This article is part of a Special Issue entitled “Osteoarthritis”.

Microdrilled Cartilage Defects Treated with Thrombin-Solidified Chitosan/Blood Implant Regenerate a More Hyaline, Stable, and Structurally Integrated Osteochondral Unit Compared to Drilled Controls

This study analyzed the long-term cartilage and subchondral bone repair of microdrilled defects treated with chitosan glycerol-phosphate/blood implant, using thrombin (Factor IIa) to accelerate in situ solidification. We also evaluated the cartilage repair response to six smaller microdrill holes compared with two larger holes. Bilateral knee trochlear cartilage defects were created in n=8 skeletally mature rabbits, drilled with six proximal 0.5 mm and two distal 0.9 mm holes, then covered with in situ-solidified IIa-implants (treated) or with IIa-alone (control). After 6.5 months of repair, cartilage repair tissues were analyzed by histological scoring and histomorphometry for hyaline matrix characteristics and osseous integration. Subchondral repair bone was analyzed by 3D microcomputed tomography and compared to acute defects (n=6) and intact trochlea (n=8). Implant-treated cartilage repair tissues had higher structural integrity through the entire defect (p=0.02), twofold higher percent staining for glycosaminoglycan (p=0.0004), and ~24% more collagen type II staining over the smaller drill holes (p=0.008) compared with controls. Otherwise, hole diameter had no specific effect on cartilage repair. The subchondral bone plate was partially restored in treated and control defects but less dense than intact trochlea, with evidence of incomplete regeneration of the calcified cartilage layer. More residual drill holes (p=0.054) were detected in control versus treated defects, and control defects with more than 40% residual holes presented abnormally thicker trabeculae compared with treated defects. Low osteoclast numbers after 6.5 months repair suggested that bone was no longer remodeling. The subchondral bone plate surrounding the defects exhibited a significant thickening compared with age-matched intact trochlea. These data suggest that debridement and drilling can lead to long-term subchondral bone changes outside the cartilage defect. Compared with drilled controls, chitosan implants solidified with thrombin elicited a more hyaline and structurally integrated osteochondral unit, features needed for long-term durability.

Equine Models of Articular Cartilage Repair.

Articular cartilage injuries of the knee and ankle are common, and a number of different methods have been developed in an attempt to improve their repair. Clinically, there are 2 distinct aims of cartilage repair: 1) restoration of joint function and 2) prevention or at least delay of the onset of osteoarthritis. These goals can potentially be achieved through replacement of damaged or lost articular cartilage with tissue capable of functioning under normal physiological environments for an extended period, but limitations of the final repair product have long been recognized and still exist today. Screening of potential procedures for human clinical use is done by preclinical studies using animal models. This article reviews equine chondral defect models that have been recently recognized to have specific advantages for translation into human articular cartilage regeneration. Defect models in the femoropatellar, femorotibial, and tibiotalar joints have been developed. The horse provides the closest approximation to humans in terms of articular cartilage and subchondral bone thickness, and it is possible to selectively leave the entire calcified cartilage layer or completely remove it. The defect on the equine medial femoral condyle emulates medial femoral condylar lesions in humans. Other advantages of the equine model include an ability to use an arthroscope to create lesions and perform second-look arthroscopies, the large lesion size allowing for more tissue for evaluation, and the ability to have controlled exercise and test the ability of the repair to cope with athletic exercise as well as institute rehabilitation regimens.

Microfracture: Basic Science Studies in the Horse.

The therapeutic value of microfracture has been demonstrated in clinical patients. The rationale is that focal penetration of the dense subchondral plate exposes cartilage defects to the benefits of cellular and growth factor influx in addition to improving anchorage of the new tissue to the underlying subchondral bone and, to some extent, the surrounding cartilage. While functional outcomes have been reported, there is a paucity of data on the histological, biochemical, and molecular changes in human patients. This paper reviews 4 basic science studies of microfracture using an equine chondral defect model that gave some insight into possible mechanisms of action and also how the microfracture response could be augmented. In study I, microfracture of full-thickness chondral defects in exercised horses significantly increased the repair tissue volume in the defects at both 4 and 12 months. No adverse effects were seen. In study II, an investigation of the healing of full-thickness chondral defects during the first 8 weeks with or without microfracture demonstrated that microfracture significantly increased type II collagen expression as early as 8 weeks after treatment compared to controls; aggrecan expression was progressively increased during the first 8 weeks but was not significantly enhanced by microfracture. In study III, it was demonstrated that removal of the calcified cartilage layer provided optimal amount and attachment of repair tissue, emphasizing that careful removal of calcified layer is critical during debridement prior to microfracture. Study IV assessed the ability of IL-1ra/IGF-1 combination gene therapy to further modulate repair of microfractured chondral defects. The repair tissue in gene therapy–treated joints demonstrated increased proteoglycan and type II collagen content compared to microfracture alone.

Modulation of cartilage differentiation by melanoma inhibiting activity/cartilage-derived retinoic acid-sensitive protein (MIA/CD-RAP)

Melanoma inhibiting activity/cartilage-derived retinoic acid-sensitive protein (MIA/CD-RAP) is a small soluble protein secreted from malignant melanoma cells and from chondrocytes. Recently, we revealed that MIA/CD-RAP can modulate bone morphogenetic protein (BMP)2-induced osteogenic differentiation into a chondrogenic direction. In the current study we aimed to find the molecular details of this MIA/CD-RAP function. Direct influence of MIA on BMP2 by protein-protein-interaction or modulating SMAD signaling was ruled out experimentally. Instead, we revealed inhibition of ERK signaling by MIA/CD-RAP. This inhibition is regulated via binding of MIA/CD-RAP to integrin alpha5 and abolishing its activity. Active ERK signaling is known to block chondrogenic differentiation and we revealed induction of aggrecan expression in chondrocytes by treatment with MIA/CD-RAP or PD098059, an ERK inhibitor. In in vivo models we could support the role of MIA/CD-RAP in influencing osteogenic differentiation negatively. Further, MIA/CD-RAP-deficient mice revealed an enhanced calcified cartilage layer of the articular cartilage of the knee joint and disordered arrangement of chondrocytes. Taken together, our data indicate that MIA/CD-RAP stabilizes cartilage differentiation and inhibits differentiation into bone potentially by regulating signaling processes during differentiation.

Joint Curettage Arthrodesis Technique in the Foot: A Histological Analysis

Arthrodesis via joint contour preservation using the curettage method has become popular in foot and ankle surgery to avoid segmental shortening and the need to bone graft. Despite its popularity, the effect of joint curettage has never been histologically evaluated. Knowledge of the histological appearance after joint curettage would be helpful to the foot and ankle surgeon to better understand the function of joint surface preparation for arthrodesis. Five cadaver specimens were used to harvest the first metatarsocuneiform and subtalar joints for routine histological analysis after performing joint curettage technique. One specimen was used as a reference, whereas the remaining specimens were processed after joint surface preparation. Results show a residual layer of calcified cartilage overlying the subchondral plate interface on all osteochondral specimens after joint curettage. This suggests there is a natural histological barrier that may interfere with arthrodesis consolidation. 5.

Increased hydraulic conductance of human articular cartilage and subchondral bone plate with progression of osteoarthritis

Osteoarthritis (OA) is characterized by progressive degeneration of articular cartilage and remodeling of the subchondral bone plate, comprising calcified cartilage and underlying subchondral bone. Calcified cartilage remodeling due to upward invasion by vascular canals or to calcified cartilage erosion may contribute to biomechanical alteration of the osteochondral tissue and its subchondral bone plate component. The study hypothesis was that hydraulic conductance of osteochondral tissue and subchondral bone plate increases with structural changes indicative of increasing stages of OA. Osteochondral cores were harvested from the knees of cadaveric tissue donors and from discarded fragments from patients with OA undergoing knee surgery. The osteochondral cores from tissue donors were macroscopically normal, and the cores from patients with OA had partial-thickness or full-thickness erosion to bone. The cores were perfusion-tested to determine the hydraulic conductance, or ease of fluid flow, in their native state and after enzymatic removal of cartilage. Adjacent portions were analyzed by 3-dimensional histology for calcified cartilage, subchondral bone, and subchondral bone plate thickness and vascular canal density. Hydraulic conductance of native osteochondral tissue and subchondral bone plate was higher (2,700-fold and 3-fold, respectively) in fully eroded samples than in normal samples. The calcified cartilage layer was thicker (1.5-fold) in partially eroded samples than in normal samples but thinner and incomplete in fully eroded samples. Subchondral bone plate vascularity was altered with increasing stages of OA. During joint loading, increased hydraulic conductance of the osteochondral tissue and subchondral bone plate could have deleterious biomechanical consequences for cartilage. Increased fluid exudation from overlying and opposing cartilage, increased fluid depressurization, and increased cartilage tissue strains could lead to chondrocyte death and cartilage damage.

Microfracture for Osteochondritis Dissecans Lesions

Microfracture is a marrow stimulating technique used in the treatment of full-thickness chondral defects. In the case of osteochondritis dissecans lesions, which are unstable and require extraction, microfracture is a viable treatment option. It is a relatively simple procedure that can be done in conjunction with other arthroscopic procedures and require minimal equipment. Careful surgical technique, including removal of the calcified cartilage layer as well as adherence to a stringent postoperative regimen, is critical to the long-term success of this procedure.

Thoracic ossification of the human ligamentum flavum: histopathological and immunohistochemical findings around the ossified lesion

The object of this study was to histopathologically and immunohistochemically characterize ossification of the ligamentum flavum (OLF) in samples of the thoracic spine harvested en bloc during surgery and to enhance the understanding of the ossifying process, particularly calcification and ossification. Samples of OLF plaque were obtained en bloc from 43 patients who underwent posterior decompression. The histopathological findings were correlated with radiological subtypes using computed tomography. The expression of type I and type II collagens, vascular endothelial growth factor (VEGF), transforming growth factor (TGF)beta, and bone morphogenetic protein (BMP)-2 was investigated. Surgical decompression using the posterior floating and en bloc resection technique resulted in neurological improvement in 40 of 43 patients. Progression of the OLF lesion longitudinally and medially was associated with significant degeneration of elastic fibers, fiber bundle derangement, decrements in fiber diameter, and fragmentation. Calcification and ossification paralleled the degeneration of the elastic fibers, extended more medially, and fused in the central area. Expression of BMP-2, TGFbeta, and VEGF was significant in chondrocytes in the calcified cartilage and fibrocartilage layers, especially around the calcified front. Histopathologically, the progress of calcification and ossification was closely associated with the degeneration of elastic fibers and with significant expression of BMP-2, TGFbeta, and VEGF in the ossification front.

Effects of Calcified Cartilage on Healing of Chondral Defects Treated with Microfracture in Horses

Background Microfracture of full-thickness articular defects has been shown to significantly enhance the amount of repair tissue. However, there is a suggestion that leaving calcified cartilage inhibits this repair response. Hypothesis Removal of the calcified cartilage with retention of subchondral bone enhances the amount of attachment of the repair tissue compared with retention of the calcified cartilage layer. Study Design Controlled laboratory study. Methods There were 1-cm2 articular cartilage defects made in 12 skeletally mature horses on the axial weightbearing portion of both medial femoral condyles. Using a custom measuring device and direct arthroscopic observation of the subchondral bone beneath the calcified cartilage layer, the authors removed the calcified cartilage from 1 defect of each horse. The repair was assessed with arthroscopy, clinical examination, radiographic and magnetic resonance imaging examinations, biopsy at 4 months, gross and histopathologic examinations at 12 months, as well as mRNA and immunohistochemical evaluations. Results Removal of calcified cartilage with retention of the subchondral bone plate increased the overall repair tissue as assessed by arthroscopic (4 months) and gross evaluation (12 months). An increase in the level of the subchondral bone was also observed with removal of the calcified cartilage layer. The clinical pain, radiographic examinations, magnetic resonance imaging evaluations, histologic character, matrix proteins, or mRNA expression do not appear to differ based on level of defect debridement. Clinical Relevance Removal of the calcified cartilage layer appears to provide optimal amount and attachment of repair tissue. Therefore, close arthroscopic visualization is recommended for debridement of clinical lesions to ensure removal of the calcified cartilage layer.

The normal human chondro-osseous junctional region: evidence for contact of uncalcified cartilage with subchondral bone and marrow spaces

BackgroundThe chondro-osseous junctional region of diarthrodial joints is peculiarly complex and may be considered to consist of the deepest layer of non-calcified cartilage, the tidemark, the layer of calcified cartilage, a thin cement line (between the calcified cartilage and the subchondral bone) and the subchondral bone. A detailed knowledge of the structure, function and pathophysiology of the normal chondro-osseous junction is essential for an understanding of the pathogenesis of osteoarthrosis.MethodsFull thickness samples from human knee joints were processed and embedded in paraffin wax. One hundred serial sections (10 μm thick) were taken from the chondro-osseous junctional region of a block from the medial tibial plateau of a normal joint. They were stained with haematoxylin and eosin and photographed. For a simple physical reconstruction images of each 10th sequential tissue section were printed and the areas of the photomicrographs containing the chondro-osseous junctional region were cut out and then overlaid so as to create a three-dimensional (3D) model of this region. A 3D reconstruction was also made using computer modelling.ResultsHistochemical staining revealed some instances where prolongations of uncalcified cartilage, delineated by the tidemark, dipped into the calcified cartilage and, in places, abutted onto subchondral bone and marrow spaces. Small areas of uncalcified cartilage containing chondrocytes (virtual islands) were seen, in two-dimensional (2D) sections, to be apparently entombed in calcified matrix. The simple physical 3D reconstruction confirmed that these prolongations of uncalcified cartilage were continuous with the cartilage of zone IV and demonstrated that the virtual islands of uncalcified cartilage were cross-sections of these prolongations. The computer-generated 3D reconstructions clearly demonstrated that the uncalcified prolongations ran through the calcified cartilage to touch bone and marrow spaces and confirmed that the apparent entombment of chondrocytes was a 2D artefact.ConclusionThis study demonstrates that the chondro-osseous junctional region is more complex than previously described. The tidemark is a clearly defined boundary delineating uncalcified from calcified cartilage. It is not a straight line across a joint, but a complex three-dimensional structure that follows uncalcified cartilage prolongations dipping down through the calcified cartilage to abut onto subjacent bone or marrow spaces.

A comparative study of articular cartilage thickness in the stifle of animal species used in human pre-clinical studies compared to articular cartilage thickness in the human knee

Histological measurements of the thickness of non-calcified and calcified cartilage, as well as the subchondral bone plate in five locations on the femoral trochlea and medial femoral condyles of species were used in preclinical studies of articular cartilage and compared to those of the human knee. Cadaver specimens were obtained of six human knees, as well as six equine, six goat, six dog, six sheep and six rabbit stifle joints (the animal equivalent of the human knee). Specimens were taken from the lateral trochlear ridge, medial trochlear ridge and medial femoral condyle. After histopathological processing, the thickness of non-calcified and calcified cartilage layers, as well as the subchondral bone plate, was measured. Average articular cartilage thickness over five locations were 2.2-2.5 mm for human, 0.3 mm for rabbit, 0.4-0.5 mm for sheep, 0.6-1.3 mm for dog, 0.7-1.5 mm for goat and 1.5-2 mm for horse. The horse provides the closest approximation to humans in terms of articular cartilage thickness, and this approximation is considered relevant in pre-clinical studies of cartilage healing.

The histology and affinities of sinacanthid fishes: primitive gnathostomes from the Silurian of China

Craniofacial Development, Dental Institute, Guy’s Tower, London, SE1 9RT, UKReceived October 2003; accepted for publication March 2005On the basis of well preserved specimens from the Lower Silurian of the Tarim Basin, Xinjiang Uygur AutonomousRegion and Shiqian County, Guizhou Province, People’s Republic of China we describe in detail the histologicalstructure of sinacanthid spines, the only known remains of a group of fishes common in Silurian strata from China.The sinacanthids have previously been assigned either to the acanthodians or to the chondrichthyans. The spinestructure is composed of an outer layer of atubular dentine and an inner layer of globular calcified cartilage, and thenature and distribution of these tissues indicates that the spines were formed as a result of interaction between theendoskeleton and dermoskeleton. The tissue distribution and style of growth described herein places the sina-canthids crownwards of the placoderms, and possibly within the total group Chondrichthyes. However, before theycan be firmly placed within a phylogenetic scheme, further evidence is required both on the general anatomy ofsinacanthids and on the nature of chondrichthyan apomorphies. © 2005 The Linnean Society of London, ZoologicalJournal of the Linnean Society, 2005, 144, 379–386.

Expression patterns of hedgehog signaling peptides in naturally acquired equine osteochondrosis

Hypertrophic differentiation and endochondral ossification of growth cartilage are regulated by a complex array of signaling peptides, including parathyroid hormone-related protein (PTH-rP), Indian hedgehog (Ihh), and bone morphogenetic proteins (BMPs). This study investigated the expression of Ihh, Patched1 and 2 (Ptc1, Ptc2), Smoothened (Smo), Gli1, and Gli3, in naturally acquired articular osteochondrosis, using an equine model. Cartilage was harvested from osteochondrosis (OC) affected femoropatellar or scapulohumeral joints from immature horses and normal control horses of similar age. Ihh, Ptc1, Smo, Gli1, and Gli3 mRNA expression levels were evaluated by real-time quantitative PCR. Spatial tissue expression was determined by in situ hybridization for Ihh and Smo and immunohistochemistry for Ptc1 and Ptc2. The expression of Ihh was significantly increased in OC cartilage compared to normal control cartilage and was localized mainly to the deep layer of articular cartilage, just above the calcified zone, with some mild expression also present in the middle cartilage layer. The expression of Gli1 was significantly decreased in OC samples, but there was no significant difference in expression of Gli3, Ptc1 and Smo in OC cartilage compared to normal cartilage. The expression of Ptc1 protein was present at the junction of deep and calcified layers, while Ptc2 protein was expressed throughout the middle, deep, and calcified cartilage layers. Spatial expression of Smo was variable between animals and confined mainly to the middle and deep layers when present. Half of the OC samples displayed areas of moderate to strong Smo expression compared to mild or minimal expression in normal controls. The increased Ihh expression in OC suggests a role of Ihh in diseased cartilage, although it is not known if a PTH-rP/Ihh feedback cycle exists in articular cartilage. The disparity between increased Ihh expression and decreased Gli1 expression in OC cartilage suggests a different primary transcription factor for Ihh or the presence of an elevated Ihh inhibitor in these tissues.

Tetrapolar Measurement of Electrical Conductivity and Thickness of Articular Cartilage

A tetrapolar method to measure electrical conductivity of cartilage and bone, and to estimate the thickness of articular cartilage attached to bone, was developed. We determined the electrical conductivity of humeral head bovine articular cartilage and subchondral bone from a 1- to 2-year-old steer to be 1.14+/-0.11 S/m (mean+/-sd, n =11) and 0.306+/-0.034 S/m, (mean+/-sd, n =3), respectively. For a 4-year-old cow, articular cartilage and subchondral bone electrical conductivity were 0.88+/-0.08 S/m (mean+/-sd, n =9) and 0.179+/-0.046 S/m (mean+/-sd, n =3), respectively. Measurements on slices of cartilage taken from different distances from the articular surface of the steer did not reveal significant depth-dependence of electrical conductivity. We were able to estimate the thickness of articular cartilage with reasonable precision (<20% error) by injecting current from multiple electrode pairs with different inter-electrode distances. Requirements for the precision of this method to measure cartilage thickness include the presence of a distinct layer of calcified cartilage or bone with a much lower electrical conductivity than that of uncalcified articular cartilage, and the use of inter-electrode distances of the current injecting electrodes that are on the order of the cartilage thickness. These or similar methods present an attractive approach to the non-destructive determination of cartilage thickness, a parameter that is required in order to estimate functional properties of cartilage attached to bone, and evaluate the need for therapeutic interventions in arthritis.