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Osteoarthritis (OA) is a degenerative disease resulting in irreversible, progressive destruction of articular cartilage(1). The etiology of OA is complex and involves a variety of factors, including genetic predisposition, acute injury and chronic inflammation(2–4). Here we investigate the ability of resident skeletal stem-cell (SSC) populations to regenerate cartilage in relation to age, a possible contributor to the development of osteoarthritis(5–7). We demonstrate that aging is associated with progressive loss of SSCs and diminished chondrogenesis in the joints of both mice and humans. However, a local expansion of SSCs could still be triggered in the chondral surface of adult limb joints in mice by stimulating a regenerative response using microfracture (MF) surgery. Although MF-activated SSCs tended to form fibrous tissues, localized co-delivery of BMP2 and soluble VEGFR1 (sVEGFR1), a VEGF receptor antagonist, in a hydrogel skewed differentiation of MF-activated SSCs toward articular cartilage. These data indicate that following MF, a resident stem-cell population can be induced to generate cartilage for treatment of localized chondral disease in OA.

This preliminary study investigates the differences between experimental periodontitis and periimplantitis in a dog model, with a focus on the histopathology, the inflammatory responses and specific immunoregulatory activities, driven by Th1/Th2 positive cells. Twelve dental implants were inserted into the edentulated posterior mandibles of six Beagle dogs and were given twelve weeks time for osseointegration. Experimental periimplantitis and periodontitis (first mandible molar) was then induced using cotton-floss ligatures. Twelve weeks later, alveolar bones were quantitated by cone beam-computer tomography. Histopathological analysis of the inflamed gingiva and of the periodontal tissues was performed by light microscopy, and the Th1/ Th2 cell populations were investigated by flow cytometry. Periimplantitis as well as periodontitis were both found to be associated with pronounced bone resorption effects, both to a similar degree vertically, but with a differential bone resorption pattern mesio-distally, and with a significantly higher and consistent bone resorption result in periimplantitis; however, with a higher variance of bone resorption in periodontitis. The histological appearances of the inflammatory tissues were identical. The percentages of Th1/ Th2 cells in the inflamed gingival tissues of both experimental periimplantitis and periodontitis were also found to be similar. Experimental periodontitis and periimplantitis in the dog model show essentially the same cellular pathology of inflammation. However, bone resorption was found to be significantly higher in periimplantitis; the histopathological changes in the periodontal tissues were similar in both groups, but showed a higher inter-individual variation in periodontitis, and appeared more uniform in periimplantitis. This preliminary study indicates that more focused experimental in-vivo inflammation models need to be developed to better simulate the human pathology in the two different diseases, and in order to have a valuable tool to investigate more specifically how novel treatments/prevention approaches may heal the differential adverse effects on bone tissue and on periodontium in periodontitis and in periimplantitis.

The objectives of this study were to: (1) examine patterns of short- and long-term metal exposure within the otoliths of six offshore fish species in varying states of health, as indicated by the presence of external skin lesions, and (2) determine if there was a change in otolith metal concentrations concurrent with the Deepwater Horizon (DWH) oil spill. Otoliths collected from 2011 to 2013 in the Gulf of Mexico (GOM) were analyzed for a suite of trace metals known to be associated with DWH oil. We found that lesioned fish often had elevated levels of otolith 60Ni and 64Zn before, during, and after the DWH oil spill. In addition, metal exposure varied according to species-specific life history patterns. These findings indicate that lesioned individuals were exposed to a persistent source of trace-metals in the GoM prior to the oil spill.

Microchemical analyses of fish otoliths have revolutionized fisheries science. Molecules deposited within otoliths may originate from ambient water and diet, with molecular concentrations being subject to subsequent physiological alteration after exposure. Analyses of otolith microstructure and incorporation of inorganic elements have led to major advances in stock assessment and fisheries ecology. However, the use of otoliths for microchemical analyses has drawbacks. Specifically, otolith removal from live specimens requires specimen sacrifice, which may be forbidden in the case of protected species. In addition, otoliths rarely contain sufficient concentrations of organic matter to allow reconstruction of food-web relationships via multiple stable isotopes, and otolith microstructure can be difficult to interpret in some species. Here, we review alternatives to otoliths that can provide microchemical analytes for life-history studies in fishes. Our focus is to describe advantages and disadvantages to the use of each alternative structure, with particular attention paid to trace-element analysis for inorganic matrices and stable-isotope analysis for organic ones. In general, the chronological analysis of elemental and isotopic values within each structure depends on the inert nature (or lack of molecular turnover) of the tissue. Structures with high turnover rates or those that are metabolically active will not effectively record elemental or isotopic compositions over time. Here, we provide an assessment of the use of bony endoskeleton, fin spines, fin rays, scales, and eye lenses as alternatives or complements to fish otolith analysis.

Implantation of allograft tissues has massively grown over the last years, especially in the fields related to sports medicine. Beside the fact that often no autograft option exists, autograft related disadvantages as donor-site morbidity and prolonged operative time are drastically reduced with allograft tissues. Despite the well documented clinical success for bone allograft procedures, advances in tissue engineering raised the interest in meniscus, osteochondral and ligament/tendon allografts. Notably, their overall success rates are constantly higher than 80%, making them a valuable treatment option in orthopaedics, especially in knee surgery. Complications reported for allografting procedures are a small risk of disease transmission, immunologic rejection, and decreased biologic incorporation together with nonunion at the graft-host juncture and, rarely, massive allograft resorption. Although allografting is a successful procedure, improved techniques and biological knowledge to limit these pitfalls and maximize graft incorporation are needed. A basic understanding of the biologic processes that affect the donor-host interactions and eventual incorporation and remodelling of various allograft tissues is a fundamental prerequisite for their successful clinical use. Further, the importance of the interaction of immunologic factors with the biologic processes involved in allograft incorporation has yet to be fully dissected. Finally, new tissue engineering techniques and use of adjunctive growth factors, cell based and focused gene therapies may improve the quality and uniformity of clinical outcomes. The aim of this review is to shed light on the biology of meniscus, osteochondral and ligament/tendon allograft incorporation and how collection and storage techniques may affect graft stability and embodiment.Level of evidence V.

Meniscal injuries can occur secondary to trauma or be instigated by the changes in knee-joint function that are associated with aging, osteo- and rheumatoid arthritis, disturbances in gait, and obesity. Sixty percent of persons over 50 years of age manifest signs of meniscal pathology. The surgical and arthroscopic measures that are currently implemented to treat meniscal deficiencies bring only transient relief from pain and effect but a temporary improvement in joint function. Although tissue-engineering-based approaches to meniscal repair are now being pursued, an appropriate in-vitro model has not been conceived. The aim of this study was to develop an organ-slice culturing system to simulate the repair of human meniscal lesions in vitro. The model consists of a ring of bovine meniscus enclosing a chamber that represents the defect and reproduces its sequestered physiological microenvironment. The defect, which is closed with a porous membrane, is filled with fragments of synovial tissue, as a source of meniscoprogenitor cells, and a fibrin-embedded, calcium-phosphate-entrapped depot of the meniscogenic agents BMP-2 and TGF-β1. After culturing for 2 to 6 weeks, the constructs were evaluated histochemically and histomorphometrically, as well as immunohistochemically, for the apoptotic marker caspase 3 and collagen types I and II. Under the defined conditions, the fragments of synovium underwent differentiation into meniscal tissue, which bonded with the parent meniscal wall. Both the parent and the neoformed meniscal tissue survived the duration of the culturing period without significant cell losses. The concept on which the in-vitro system is based was thus validated. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1588-1596, 2016.

Background: Two strains of guinea pig develop spontaneous osteoarthritis of the knee. Although the disease evolves at different rates in the two strains, it is not known whether these differences are reflected in the structure of the cartilage and cancellous bone. Questions/purposes: We determined whether the three-dimensional structure of the tibial-plateau cartilage and femoral cancellous bone differed between the two strains. Methods: Six Dunkin-Hartley and six GOHI/SPF guinea pigs were evaluated. The animals were sacrificed at 11months of age. The 24 proximal tibias were used for a stereologic histomorphometric analysis of the tibial-plateau cartilage. The 24 femurs were used for a site-specific, three-dimensional quantitative analysis of the cancellous bone by micro-CT. Results: Compared to the GOHI/SPF guinea pigs, the tibial-plateau cartilage of the Dunkin-Hartley strain had a larger lesion volume (3.8% versus 1.5%) and a thicker uncalcified cartilage layer (0.042 versus 0.035mm), but a thinner calcified cartilage zone (0.008 versus 0.01mm) and a thinner subchondral cortical bone plate (0.035 versus 0.039mm). The femoral cancellous bone in the Dunkin-Hartley strain had a lower bone mineral density (477 versus 509mg/cm3). However, the trabeculae were thicker (3.91 versus 3.53 pixels) and farther apart (7.8 versus 5.6 pixels). The osteoarthritic changes in the cartilage were topographically mirrored in the subchondral bone. They were most severe on the medial side of the joint, particularly in the anterior region. Conclusions: Spontaneous osteoarthritis in the guinea pig is associated with site-specific changes in the articular cartilage layer, which are topographically mirrored in the underlying subchondral bone. Clinical Relevance: Three-dimensional structural information not revealed by two-dimensional radiography may help characterize the stages of osteoarthritis

Background Phacomatosis pigmentovascularis (PPV) has previously almost exclusively been reported in Asians and in dermatological journals. In this report, we present a case of a young black South African adolescent boy referred to our eye clinic. Case report A 15-year-old male patient presented with gradual, painless loss of vision in his right eye. Clinical examination revealed a port-wine stain with ocular involvement on the right as well as a nevus of Ota. Fundoscopy on the right revealed an advanced glaucomatous optic disc and raised intraocular pressure. A diagnosis of glaucoma associated with PPV was made. Conclusion We present the first case of PPV in an African male individual with associated glaucoma seen at our tertiary hospital.

To determine the optimal rearing temperature for Centropomus undecimalis larvae during the yolk resorption period, changes in larval development were measured at four different temperatures (23, 25, 28 and 31°C). Yolk and oil-globule volume was recorded for 25 larvae at four different times. This involved an initial measurement at hatch and at 24, 48 and 72 h posthatch (hph). Additional morphological measurements included standard length, body height and eye diameter. On average, at the end of the three trials, larvae reared at 25°C had a longer mean standard length than larvae reared at 23, 28 and 31°C. Larvae reared at 25°C also had more yolk and oil globule reserves than larvae raised at 28 and 31°C. The body height:length residuals were also the highest at 25°C (i.e. larvae had deeper or stockier bodies). The yolk sac was present up to 72 hph at 23 and 25°C, while it was entirely consumed after 48 hph in larvae held at 28 and 31°C. Larvae showed the fastest growth during the first 24 hph in all temperature treatments; this period corresponded to the highest energy consumption as determined by the decrease in yolk sac and oil-globule volume. Eye diameter did not vary significantly with time during yolk-resorption. We conclude that a temperature near 25°C is optimal for raising snook larvae during the yolk-resorption period.

Osteoarthritis (OA) is a degenerative joint condition characterized by painful cartilage lesions that impair joint mobility. Current treatments such as lavage, microfracture, and osteochondral implantation fail to integrate newly formed tissue with host tissues and establish a stable transition to subchondral bone. Similarly, tissue-engineered grafts that facilitate cartilage and bone regeneration are challenged by how to integrate the graft seamlessly with surrounding host cartilage and/or bone. This review centers on current approaches to promote cartilage graft integration. It begins with an overview of articular cartilage structure and function, as well as degenerative changes to this relationship attributed to aging, disease, and trauma. A discussion of the current progress in integrative cartilage repair follows, focusing on graft or scaffold design strategies targeting cartilage-cartilage and/or cartilage-bone integration. It is emphasized that integrative repair is required to ensure long-term success of the cartilage graft and preserve the integrity of the newly engineered articular cartilage. Studies involving the use of enzymes, choice of cell source, biomaterial selection, growth factor incorporation, and stratified versus gradient scaffolds are therefore highlighted. Moreover, models that accurately evaluate the ability of cartilage grafts to enhance tissue integrity and prevent ectopic calcification are also discussed. A summary and future directions section concludes the review.

The aim of this study was to analyze gait function and muscular strength on donor site after harvesting of a vascularized fibula osteoseptocutaneous flap. Nine patients with a mean follow-up of 33 months (range, 7-59) and a mean resection length of the middle portion of the fibula of 18.0 cm (range, 14.0-23.0) underwent an instrumented three-dimensional gait analysis to evaluate gait function. Furthermore, CYBEX II extremity system was used for muscular strength measurements. Subjective muscle strength measurements were performed according to Kendall et al. and were classified according to the British Medical Research Council. Intraindividual comparison between the operated and the nonoperated leg revealed no significant differences for gait function parameters (cadence, velocity, and stride length, P > 1.00) and for muscular strength measurements for flexion (knee: P = 0.93, ankle: P = 0.54) and extension (knee: P = 0.97, ankle: P= 0.21), respectively. In conclusion, intraindividual comparison of the operated and nonoperated sides after harvesting of the middle portion of the fibula for gaining a free fibula osteoseptocutaneous flap has no adverse affect on gait function or muscular flexion and extension strength on donor site at a mean follow-up of 33 months.

Mesenchymal stromal cells (MSCs), which reside within various tissues, are utilized in the engineering of cartilage tissue. Dexamethasone (DEX)--a synthetic glucocorticoid--is almost invariably applied to potentiate the growth-factor-induced chondrogenesis of MSCs in vitro, albeit that this effect has been experimentally demonstrated only for transforming-growth-factor-beta (TGF-β)-stimulated bone-marrow-derived MSCs. Clinically, systemic glucocorticoid therapy is associated with untoward side effects (e.g., bone loss and increased susceptibility to infection). Hence, the use of these agents should be avoided or limited. We hypothesize that the influence of DEX on the chondrogenesis of MSCs depends upon their tissue origin and microenvironment [absence or presence of an extracellular matrix (ECM)], as well as upon the nature of the growth factor. We investigated its effects upon the TGF-β1- and bone-morphogenetic-protein 2 (BMP-2)-induced chondrogenesis of MSCs as a function of tissue source (bone marrow vs. synovium) and microenvironment [cell aggregates (no ECM) vs. explants (presence of a natural ECM)]. In aggregates of bone-marrow-derived MSCs, DEX enhanced TGF-β1-induced chondrogenesis by an up-regulation of cartilaginous genes, but had little influence on the BMP-2-induced response. In aggregates of synovial MSCs, DEX exerted no remarkable effect on either TGF-β1- or BMP-2-induced chondrogenesis. In synovial explants, DEX inhibited BMP-2-induced chondrogenesis almost completely, but had little impact on the TGF-β1-induced response. Our data reveal that steroids are not indispensable for the chondrogenesis of MSCs in vitro. Their influence is context dependent (tissue source of the MSCs, their microenvironment and the nature of the growth-factor). This finding has important implications for MSC based approaches to cartilage repair.

Bradykinesia represents one of the cardinal and most incapacitating features of Parkinson's disease (PD). In this context, investigating the cerebral control mechanisms for limb movements and defining the associated functional neuroanatomy is important for understanding the impaired motor activity in PD. So far, most studies have focused on motor control of upper limb movements in PD. Ankle movement functional MRI (fMRI) paradigms have been used to non-invasively investigate supraspinal control mechanisms relevant for lower limb movements in healthy subjects, patients with Multiple sclerosis, and stroke. Using such an active and passive paradigm in 20 PD patients off medication (mean age 66.8 ± 7.2 years) and 20 healthy controls (HC; mean age 62.3 ± 6.9 years), we here wished to probe for possible activation differences between PD and HC and define functional correlates of lower limb function in PD. Active ankle movement versus rest was associated with a robust activation pattern in expected somatotopy involving key motor areas both in PD and HC. However, contrasting activation patterns in patients versus controls revealed excess activation in the patients in frontal regions comprising pre-supplementary motor areas (pre-SMA) and SMA proper. The extent of SMA activation did not correlate with behavioural parameters related to gait or motor function, and no differences were seen with the passive paradigm. This finding might be indicative of higher demand and increased effort in PD patients to ensure adequate motor function despite existing deficits. The missing correlation with behavioural variables and lack of differences with the passive paradigm suggests that this excess activation is not exclusively compensatory and also not hard-wired.

Investigational devices for articular cartilage repair or replacement are considered to be significant risk devices by regulatory bodies. Therefore animal models are needed to provide proof of efficacy and safety prior to clinical testing. The financial commitment and regulatory steps needed to bring a new technology to clinical use can be major obstacles, so the implementation of highly predictive animal models is a pressing issue. Until recently, a reductionist approach using acute chondral defects in immature laboratory species, particularly the rabbit, was considered adequate; however, if successful and timely translation from animal models to regulatory approval and clinical use is the goal, a step-wise development using laboratory animals for screening and early development work followed by larger species such as the goat, sheep and horse for late development and pivotal studies is recommended. Such animals must have fully organized and mature cartilage. Both acute and chronic chondral defects can be used but the later are more like the lesions found in patients and may be more predictive. Quantitative and qualitative outcome measures such as macroscopic appearance, histology, biochemistry, functional imaging, and biomechanical testing of cartilage, provide reliable data to support investment decisions and subsequent applications to regulatory bodies for clinical trials. No one model or species can be considered ideal for pivotal studies, but the larger animal species are recommended for pivotal studies. Larger species such as the horse, goat and pig also allow arthroscopic delivery, and press-fit or sutured implant fixation in thick cartilage as well as second look arthroscopies and biopsy procedures.

Svehlik M, Zwick EB, Steinwender G, Kraus T, Linhart WE. Dynamic versus fixed equinus deformity in children with cerebral palsy: how does the triceps surae muscle work? Objectives: To detect outcome measures that could help differentiate between dynamic and fixed equinus (FEQ) deformities in children with cerebral palsy, and secondary, to describe the function of the gastrocnemius and soleus (SOL) muscles when either dynamic triceps surae tightness or FEQ contracture is present. Design: A group-comparison study. Setting: Gait analysis laboratory. Participants: Children (N=23; 31 limbs) with cerebral palsy; 12 limbs showed a fixed contracture (FEQ group) and 19 limbs showed dynamic tightness of the triceps muscle (dynamic equinus group). Healthy children (N=12) without a neurologic or orthopedic disorder served as the control group. Interventions: Not applicable. Main Outcome Measures: Time-distance, kinematic and kinetic gait variables, muscle-tendon length, and velocity parameters. Results: Maximal ankle dorsiflexion angles were decreased in both equinus groups compared with the control group. Ankle range of motion, maximal power generation of the plantar flexors, and its timing during the gait cycle were different among groups. The ankle slope parameter showed substantial differences among groups. Muscle-tendon length parameters for the SOL and the medial (MGAC) and lateral gastrocnemius muscles were abnormal in both equinus groups compared with the control group. Maximal muscle lengths of the MGAC and SOL were longer in the dynamic equinus than FEQ group. Peak lengthening velocity of the triceps surae muscle was significantly slower for all triceps surae muscles in the FEQ group than in the dynamic equinus group and occurred in the early swing phase. Conclusions: The presented results indicate that peak lengthening velocity of the triceps surae muscle might be one of the discriminating factors between FEQ and dynamic equinus deformity in children with cerebral palsy. This could help clinical decision making for treatment of an equinus gait pattern.

Summary Aim The primary goal of this body of work is to suggest a standardized system for histopathological assessment of experimental surgical instability models of osteoarthritis (OA) in rabbits, building on past experience, to achieve comparability of studies from different centres. An additional objective is to review methodologies that have been employed in the past for assessing OA in rabbits with particular reference to the surgical anterior cruciate ligament transection (ACLT) model. Methods A panel of scientists and clinician-scientists with recognized expertise in assessing rabbit models of OA reviewed the literature to provide a critical appraisal of the methods that have been employed to assess both macroscopic and microscopic changes occurring in rabbit joint tissues in experimental OA. In addition, a validation of the proposed histologic histochemical grading system was performed. Results The ACLT variant of the surgical instability model in skeletally mature rabbits is the variation most capable of reproducing the entire range of cartilage, synovial and bone lesions recognized to be associated with OA. These lesions can be semiquantitatively graded using macroscopic and microscopic techniques. Further, as well as cartilage lesions, this ACLT model can produce synovial and bone lesions similar to that of human OA. Conclusions The ACLT variant of the surgical instability model in rabbits is a reproducible and effective model of OA. The cartilage lesions in this model and their response to therapy can be graded according to an adapted histological and histochemical grading system, though also this system is to some extent subjective and, thus, neither objective nor entirely reproducible.

Summary Objective This review focuses on histomorphometry for assessing the pathological changes in various compartments of the joint including cartilage, bone and synovium in animal models of osteoarthritis (OA). Methods Different methodological approaches are presented concerning sampling, embedding, sectioning, staining, mounting of stained sections and measurement of histomorphometric parameters using automated and semi-automated methods. Notes are provided describing some methods in greater detail. Results Histomorphometry allows a significant gain of objectivity, accuracy and reproducibility in the quantification of the main histological parameters which best characterize OA in the affected joint (cartilage thickness (CT), chondrocyte size and density, cartilage fissure, proteoglycan (PG) content, subchondral bone plate thickness (SBPT), thickness of synovial living cell layer) in animal models. Conclusion Use of histomorphometry could contribute to a better quantification of histological differences between control and OA animals. Contributing also to the introduction of normative data, it is a major advantage for therapeutic assessments in experimental OA and particularly for the analytical comparison of the efficacy of disease modifying OA drugs (DMOAD).

The insulin-like growth factor (IGF) is a major anabolic regulator in articular cartilage. The IGF-binding proteins (IGFBPs) are increased during osteoarthritis (OA), but the function of the later proteins remains unknown. In general, the IGFBPs are pluripotential effectors capable of IGF regulation and of acting on their own to control key cell functions, including survival and proliferation. The independent functions are often associated with their cell location, and therefore this study explores the distribution of IGFBP-2 and IGFBP-3 in articular chondrocytes. Immunohistochemistry was used to localize IGFBP-2 in normal human articular cartilage. Bovine chondrocytes were used for subcellular fractionation (hypotonic cell lysis) under nonreducing conditions and nuclear purification (centrifugation on sucrose cushions). Cell fraction markers and IGFBPs were assayed in the subcellular fractions by Western immunoblot. The IHC results showed association of IGFBP-2 with chondrocytes, but not with the nuclei. Subcellular fractionation of isolated chondrocytes yielded intact nuclei as assessed at the light microscopic level; the nuclear marker histone H1 was exclusively associated with this fraction. More than 90% of the cytoplasmic marker GAPDH and all the detectable IGFBP-2 were in the cytoplasmic fraction. Immunoreactive IGFBP-3 was found in the cytoplasmic and peri-nuclear/nuclear fractions. Chondrocytes contain intracellular IGFBP-2 and IGFBP-3 but only IGFBP-3 is associated with nuclei. This suggests the hypothesis that the actions of these IGFBPs in articular cartilage extend beyond the classic modulation of IGF receptor action.

Matrilins are oligomeric extracellular matrix adaptor proteins mediating interactions between collagen fibrils and other matrix constituents. All four matrilins are expressed in cartilage and mutations in the human gene encoding matrilin-3 (MATN3) are associated with different forms of chondrodysplasia. Surprisingly, however, Matn3-null as well as Matn1- and Matn2-null mice do not show an overt skeletal phenotype, suggesting a dominant negative pathomechanism for the human disorders and redundancy/compensation among the family members in the knock-out situation. Here, we show that mice lacking both matrilin-1 and matrilin-3 develop an apparently normal skeleton, but exhibit biochemical and ultrastructural abnormalities of the knee joint cartilage. At the protein level, an altered SDS-PAGE band pattern and a clear up-regulation of the homotrimeric form of matrilin-4 were evident in newborn Matn1/Matn3 and Matn1 knock-out mice, but not in Matn3-null mice. The ultrastructure of the cartilage matrix after conventional chemical fixation was grossly normal; however, electron microscopy of high pressure frozen and freeze-substituted samples, revealed two consistent observations: 1) moderately increased collagen fibril diameters throughout the epiphysis and the growth plate in both single and double mutants; and 2) increased collagen volume density in Matn1(-/-)/Matn3(-/-) and Matn3(-/-) mice. Taken together, our results demonstrate that matrilin-1 and matrilin-3 modulate collagen fibrillogenesis in cartilage and provide evidence that biochemical compensation might exist between matrilins.

Summary Objective During postnatal development, mammalian articular cartilage acts as a surface growth plate for the underlying epiphyseal bone. Concomitantly, it undergoes a fundamental process of structural reorganization from an immature isotropic to a mature (adult) anisotropic architecture. However, the mechanism underlying this structural transformation is unknown. It could involve either an internal remodelling process, or complete resorption followed by tissue neoformation. The aim of this study was to establish which of these two alternative tissue reorganization mechanisms is physiologically operative. We also wished to pinpoint the articular cartilage source of the stem cells for clonal expansion and the zonal location of the chondrocyte pool with high proliferative activity. Methods The New Zealand white rabbit served as our animal model. The analysis was confined to the high-weight-bearing (central) areas of the medial and lateral femoral condyles. After birth, the articular cartilage layer was evaluated morphologically at monthly intervals from the first to the eighth postnatal month, when this species attains skeletal maturity. The overall height of the articular cartilage layer at each juncture was measured. The growth performance of the articular cartilage layer was assessed by calcein labelling, which permitted an estimation of the daily growth rate of the epiphyseal bone and its monthly length-gain. The slowly proliferating stem-cell pool was identified immunohistochemically (after labelling with bromodeoxyuridine), and the rapidly proliferating chondrocyte population by autoradiography (after labelling with 3 H-thymidine). Results The growth activity of the articular cartilage layer was highest 1 month after birth. It declined precipitously between the first and third months, and ceased between the third and fourth months, when the animal enters puberty. The structural maturation of the articular cartilage layer followed a corresponding temporal trend. During the first 3 months, when the articular cartilage layer is undergoing structural reorganization, the net length-gain in the epiphyseal bone exceeded the height of the articular cartilage layer. This finding indicates that the postnatal reorganization of articular cartilage from an immature isotropic to a mature anisotropic structure is not achieved by a process of internal remodelling, but by the resorption and neoformation of all zones except the most superficial (stem-cell) one. The superficial zone was found to consist of slowly dividing stem cells with bidirectional mitotic activity. In the horizontal direction, this zone furnishes new stem cells that replenish the pool and effect a lateral expansion of the articular cartilage layer. In the vertical direction, the superficial zone supplies the rapidly dividing, transit-amplifying daughter-cell pool that feeds the transitional and upper radial zones during the postnatal growth phase of the articular cartilage layer. Conclusions During postnatal development, mammalian articular cartilage fulfils a dual function, viz., it acts not only as an articulating layer but also as a surface growth plate. In the lapine model, this growth activity ceases at puberty (3–4 months of age), whereas that of the true (metaphyseal) growth plate continues until the time of skeletal maturity (8 months). Hence, the two structures are regulated independently. The structural maturation of the articular cartilage layer coincides temporally with the cessation of its growth activity – for the radial expansion and remodelling of the epiphyseal bone – and with sexual maturation. That articular cartilage is physiologically reorganized by a process of tissue resorption and neoformation, rather than by one of internal remodelling, has important implications for the functional engineering and repair of articular cartilage tissue.

Objective To compare the potential of bone morphogenetic proteins 2 and 7 (BMP-2 and BMP-7) and transforming growth factor β1 (TGFβ1) to effect the chondrogenic differentiation of synovial explants by analyzing the histologic, biochemical, and gene expression characteristics of the cartilaginous tissues formed. Methods Synovial explants derived from the metacarpal joints of calves were cultured in agarose. Initially, BMP-2 was used to evaluate the chondrogenic potential of the synovial explants under different culturing conditions. Under appropriate conditions, the chondrogenic effects of BMP-2, BMP-7, and TGFβ1 were then compared. The differentiated tissue was characterized histologically, histomorphometrically, immunohistochemically, biochemically, and at the gene expression level. Results BMP-2 induced the chondrogenic differentiation of synovial explants in a dose- and time-dependent manner under serum- and dexamethasone-free conditions. The expression levels of cartilage-related genes increased in a time-dependent manner. BMP-7 was more potent than BMP-2 in inducing chondrogenesis, but the properties of the differentiated tissue were similar in each case. The type of cartilaginous tissue formed under the influence of TGFβ1 differed in terms of both cell phenotype and gene expression profiles. Conclusion The 3 tested members of the TGFβ superfamily have different chondrogenic potentials and induce the formation of different types of cartilaginous tissue. To effect the full differentiation of synovial explants into a typically hyaline type of articular cartilage, further refinement of the stimulation conditions is required. This might be achieved by the simultaneous application of several growth factors.

The articular cartilage layer of synovial joints is commonly lesioned by trauma or by a degenerative joint disease. Attempts to repair the damage frequently involve the performance of autologous chondrocyte implantation (ACI). Healthy cartilage must be first removed from the joint, and then, on a separate occasion, following the isolation of the chondrocytes and their expansion in vitro, implanted within the lesion. The disadvantages of this therapeutic approach include the destruction of healthy cartilage-which may predispose the joint to osteoarthritic degeneration-the necessarily restricted availability of healthy tissue, the limited proliferative capacity of the donor cells-which declines with age-and the need for two surgical interventions. We postulated that it should be possible to induce synovial stem cells, which are characterized by high, age-independent, proliferative and chondrogenic differentiation capacities, to lay down cartilage within the outer juxtasynovial space after the transcutaneous implantation of a carrier bearing BMP-2 in a slow-release system. The chondrocytes could be isolated on-site and immediately used for ACI. To test this hypothesis, Chinchilla rabbits were used as an experimental model. A collagenous patch bearing BMP-2 in a slow-delivery vehicle was sutured to the inner face of the synovial membrane. The neoformed tissue was excised 5, 8, 11 and 14 days postimplantation for histological and histomorphometric analyses. Neoformed tissue was observed within the outer juxtasynovial space already on the 5th postimplantation day. It contained connective and adipose tissues, and a central nugget of growing cartilage. Between days 5 and 14, the absolute volume of cartilage increased, attaining a value of 12 mm(3) at the latter juncture. Bone was deposited in measurable quantities from the 11th day onwards, but owing to resorption, the net volume did not exceed 1.5 mm(3) (14th day). The findings confirm our hypothesis. The quantity of neoformed cartilage that is deposited after only 1 week within the outer juxtasynovial space would yield sufficient cells for ACI. Since the BMP-2-bearing patches would be implanted transcutaneously in humans, only one surgical or arthroscopic intervention would be called for. Moreover, most importantly, sufficient numbers of cells could be generated in patients of all ages.

Eleven children with hemiplegic or an asymmetric diplegic cerebral palsy who had a preoperative leg length discrepancy of more than 2.5 cm underwent gait improvement surgery which included leg length equalisation. Sagittal plane kinematics and kinetics before and about 3 years after surgery for the lengthened limb and contralateral limb were evaluated. Preoperatively the unaffected limb had excessive stance phase flexion at the hip and knee, and dorsal flexion at the ankle joint. These changes could be partially reversed to produce a kinematic gait pattern comparable to age matched normal controls on the uninvolved side after equalisation of leg length.

Summary Objective According to recent reports, the synovial membrane may contain mesenchymal stem cells with the potential to differentiate into chondrocytes under appropriate conditions. In order to assess the usefulness of synovium-derived progenitor cells for the purposes of cartilage tissue engineering, we explored their requirements for the expression of chondrocyte-specific genes after expansion in vitro . Design Mesenchymal progenitor cells were isolated from the synovial membranes of bovine shoulder joints and expanded in two-dimensions on plastic surfaces. They were then seeded either as micromass cultures or as single cells within alginate gels, which were cultured in serum-free medium. Under these three-dimensional conditions, chondrogenesis is known to be supported and maintained. Cell cultures were exposed either to bone morphogenetic protein-2 (BMP-2) or to isoforms of transforming growth factor-β (TGF-β). The levels of mRNA for Sox9, collagen types I and II and aggrecan were determined by RT-PCR. Results When transferred to alginate gel cultures, the fibroblast-like synovial cells assumed a rounded form. BMP-2, but not isoforms of TGF-β, stimulated, in a dose-dependent manner, the production of messenger RNAs (mRNAs) for Sox9, type II collagen and aggrecan. Under optimal conditions, the expression levels of cartilage-specific genes were comparable to those within cultured articular cartilage chondrocytes. However, in contrast to cultured articular cartilage chondrocytes, synovial cells exposed to BMP-2 continued to express the mRNA for α1(I) collagen. Conclusions This study demonstrates that bovine synovium-derived mesenchymal progenitor cells can be induced to express chondrocyte-specific genes. However, the differentiation process is not complete under the chosen conditions. The stimulation conditions required for full transformation must now be delineated.

Integrin alpha10beta1 is a collagen-binding integrin expressed on chondrocytes. In order to unravel the role of the alpha10 integrin during development, we generated mice carrying a constitutive deletion of the alpha10 integrin gene. The mutant mice had a normal lifespan and were fertile but developed a growth retardation of the long bones. Analysis of the skeleton revealed defects in the growth plate after birth characterized by a disturbed columnar arrangement of chondrocytes, abnormal chondrocyte shape and reduced chondrocyte proliferation. Electron microscopy of growth plates from newborn mice revealed an increased number of apoptotic chondrocytes and reduced density of the collagen fibrillar network compared to these structures in control mice. These results demonstrate that integrin alpha10beta1 plays a specific role in growth plate morphogenesis and function.

Recently, two missense mutations (N88S, S90L) in the Berardinelli-Seip congenital lipodystrophy gene have been identified in autosomal dominant distal hereditary motor neuropathy and Silver syndrome. We report the phenotypic consequences of the N88S mutation in 90 patients of 1 large Austrian family and two unrelated German families. Variation in the clinical and electrophysiological phenotype enabled us to distinguish six subtypes. In 4.4%, the disorder was not penetrant. Twenty percent of the patients were subclinically affected; some of these patients could only be detected by pathological nerve conduction studies. A distal hereditary motor neuropathy type V phenotype characterized by predominant hand muscle involvement was found in 31.1%, whereas 14.5% showed typical Silver syndrome with amyotrophy of the small hand muscles and spasticity of the lower extremities. Moreover, the phenotype present in 20% was compatible with Charcot-Marie-Tooth disease. In 10%, the clinical diagnosis of pure or complicated hereditary spastic paraparesis was made. Electrophysiological studies showed an axonal neuropathy but also chronodispersion of compound motor action potentials and conduction blocks. Sensory nerve conduction studies were rarely pathological. Our study indicates that the dominant N88S mutation in the Berardinelli-Seip congenital lipodystrophy gene 2 leads to a broad spectrum of motor neuron disorders.

In this study a generalised dynamic neural network (GDNN) was designed to process gait analysis parameters to evaluate equinus deformity in ambulatory children with cerebral palsy. The aim was to differentiate dynamic calf muscle tightness from fixed muscle contracture. Patients underwent clinical examination and had instrumented gait analysis before evaluating their equinus under anaesthesia and muscle relaxation at the time of surgery to improve gait. The performance of the clinical examination, the subjective interpretation of gait analysis results, and the application of the neural network to assess ankle function were compared to the examination under anaesthesia. Evaluation of equinus by a Neural Network showed high sensitivity and specificity values with a likelihood ratio of +14.63. The results indicate that dynamic calf muscle tightness can be differentiated from fixed calf muscle contracture with considerable precision that might facilitate clinical decision-making.

A poly(ethylene glycol) (PEG)-based hydrogel was used as a scaffold for chondrocyte culture. Branched PEG-vinylsulfone macromers were end-linked with thiol-bearing matrix metalloproteinase (MMP)-sensitive peptides (GCRDGPQGIWGQDRCG) to form a three-dimensional network in situ under physiologic conditions. Both four- and eight-armed PEG macromer building blocks were examined. Increasing the number of PEG arms increased the elastic modulus of the hydrogels from 4.5 to 13.5 kPa. PEG-dithiol was used to prepare hydrogels that were not sensitive to degradation by cell-derived MMPs. Primary bovine calf chondrocytes were cultured in both MMP-sensitive and MMP-insensitive hydrogels, formed from either four- or eight-armed PEG. Most (>90%) of the cells inside the gels were viable after 1 month of culture and formed cell clusters. Gel matrices with lower elastic modulus and sensitivity to MMP-based matrix remodeling demonstrated larger clusters and more diffuse, less cell surface-constrained cell-derived matrix in the chondron, as determined by light and electron microscopy. Gene expression experiments by real-time RT-PCR showed that the expression of type II collagen and aggrecan was increased in the MMP-sensitive hydrogels, whereas the expression level of MMP-13 was increased in the MMP-insensitive hydrogels. These results indicate that cellular activity can be modulated by the composition of the hydrogel. This study represents one of the first examples of chondrocyte culture in a bioactive synthetic material that can be remodeled by cellular protease activity.

Objective: It has been suggested that chondrocyte death by apoptosis may play a role in the pathogenesis of cartilage destruction in osteoarthritis, but the results of in-vivo and in-vitro investigations have been conflicting. To investigate further the cell death in our in-vitro model for traumatic joint injury, we performed a quantitative analysis by electron microscopy (EM) of cell morphology after injurious compression. For comparison, the TUNEL assay was also performed. Design: Articular cartilage explant disks were harvested from newborn calf femoropatellar groove. The disks were subjected to injurious compression (50% strain at a strain rate of 100%/s), incubated for 3 days, and then fixed for quantitative morphological analysis. Results: By TUNEL, the cell apoptosis rate increased from 7±2% in unloaded controls to 33±6% after injury ( P =0.01; N =8 animals). By EM, the apoptosis rate increased from 5±1% in unloaded controls to 62±10% in injured cartilage ( P =0.02, N =5 animals). Analysis by EM also identified that of the dead cells in injured disks, 97% were apoptotic by morphology. Conclusions: These results confirm a significant increase in cell death after injurious compression and suggest that most cell death observed here was by an apoptotic process.

Objective To explore the role that implanted mesenchymal stem cells may play in tissue repair or regeneration of the injured joint, by delivery of an autologous preparation of stem cells to caprine knee joints following induction of osteoarthritis (OA). Methods Adult stem cells were isolated from caprine bone marrow, expanded in culture, and transduced to express green fluorescent protein. OA was induced unilaterally in the knee joint of donor animals by complete excision of the medial meniscus and resection of the anterior cruciate ligament. After 6 weeks, a single dose of 10 million autologous cells suspended in a dilute solution of sodium hyaluronan was delivered to the injured knee by direct intraarticular injection. Control animals received sodium hyaluronan alone. Results In cell-treated joints, there was evidence of marked regeneration of the medial meniscus, and implanted cells were detected in the newly formed tissue. Degeneration of the articular cartilage, osteophytic remodeling, and subchondral sclerosis were reduced in cell-treated joints compared with joints treated with vehicle alone without cells. There was no evidence of repair of the ligament in any of the joints. Conclusion Local delivery of adult mesenchymal stem cells to injured joints stimulates regeneration of meniscal tissue and retards the progressive destruction normally seen in this model of OA.

β1 integrins are highly expressed on chondrocytes, where they mediate adhesion to cartilage matrix proteins. To assess the functions of β1 integrin during skeletogenesis, we inactivated the β1 integrin gene in chondrocytes. We show here that these mutant mice develop a chondrodysplasia of various severity. β1-deficient chondrocytes had an abnormal shape and failed to arrange into columns in the growth plate. This is caused by a lack of motility, which is in turn caused by a loss of adhesion to collagen type II, reduced binding to and impaired spreading on fibronectin, and an abnormal F-actin organization. In addition, mutant chondrocytes show decreased proliferation caused by a defect in G1/S transition and cytokinesis. The G1/S defect is, at least partially, caused by overexpression of Fgfr3, nuclear translocation of Stat1/Stat5a, and up-regulation of the cell cycle inhibitors p16 and p21. Altogether these findings establish that β1-integrin-dependent motility and proliferation of chondrocytes are mandatory events for endochondral bone formation to occur.

The aim of this study was to examine the role of one of the growth/differentiation factors, GDF-5, in the process of tendon healing. Specifically, we tested the hypothesis that GDF-5 deficiency in mice would result in delayed Achilles tendon repair. Using histologic, biochemical, and ultrastructural analyses, we demonstrate that Achilles tendons from 8-week-old male GDF-5 -/- mice exhibit a short-term delay of 1-2 weeks in the healing process compared to phenotypically normal control littermates. Mutant animals took longer to achieve peak cell density, glycosaminoglycan content, and collagen content in the repair tissue, and the time course of changes in collagen fibril size was also delayed. Revascularization was delayed in the mutant mice by 1 week. GDF-5 deficient Achilles tendons also contained significantly more fat within the repair tissue at all time points examined, and was significantly weaker than control tissue at 5 weeks after surgery, but strength differences were no longer detectable by 12-weeks. Together, these data support the hypothesis that GDF-5 may play an important role in modulating tendon repair, and are consistent with previously posited roles for GDF-5 in cell recruitment, migration/adhesion, differentiation, proliferation, and angiogenesis.

The cartilage extracellular matrix is composed of a dense collagen network that entraps a range of other specialized proteins important for the proper formation and function of the tissue. Loss of two abundant cartilage components, type II collagen and perlecan, has drastic effects on skeletal development. Both collagen II and perlecan mutants have severe and lethal chondrodysplasia characterized by disorganized growth plate, lack of collagen network, defective endochondral bone formation, and abnormal intervertebral disk development. To test whether the reduced collagen density in the perlecan-null cartilage is due to enhanced activity of collagen-degrading proteinases, we have analyzed gelatinase expression and activity in the mutant tissue. Immunohistochemical analysis revealed a weak, but clear, expansion of MMP-9 deposition into the hypertrophic zone of the perlecan-null growth plate. However, in situ and SDS-PAGE zymography showed that the activity of gelatinases (MMP-2 and MMP-9) is not altered in perlecan-null cartilage, suggesting that they are not primarily linked to the reduced fibrillar network observed in the mutant. Likewise, intercrossing of perlecan mutants onto an MMP-9-null background could not rescue the ultrastructural abnormalities of the perlecan-deficient cartilage.

Objective : Induction of growth-factor-based repair in full-thickness articular cartilage defects can be impaired by the upgrowth of blood vessels and new bone into the cartilaginous compartment. We postulated that if an antiangiogenic factor (suramin) is included in the chondrogenic matrix applied to the cartilaginous compartment of a full-thickness defect, vascular upgrowth and therefore bone formation will be inhibited (functional barrier principle). Design : Full-thickness defects were created in miniature pigs and the bony portion filled with a chondrogenic matrix. The cartilaginous compartment was filled with the same matrix which additionally contained suramin, either in a free form or in free and liposome-encapsulated forms. Animals were sacrificed 8 weeks after surgery and the extent to which bone tissue had encroached on the cartilaginous compartment was graded semiquantitatively using light microscopy. Results : In 63% of the control defects, bone represented more than 50% of the repair tissue present. In 10% of the defects treated with free suramin, bone upgrowth was completely inhibited; in 55%, osseous tissue occupied 1–10% of the cartilaginous space and in the other 35%, it represented 11–50% of the repair tissue present in this compartment. In 69% of the defects treated with free and liposome-encapsulated suramin, bone upgrowth into the cartilaginous compartment was completely inhibited; in the remaining 31%, osseous tissue occupied no more than 1–10% of this space. Conclusions : To be effective, an antiangiogenic factor needs to be present at a sustained level throughout the chondrogenic treatment course.

The interaction of chondrocytes with the extracellular-matrix environment is mediated mainly by integrins. Ligated integrins are recruited to focal adhesions (FAs) together with scaffolding proteins and kinases, such as integrin-linked kinase (Ilk). Ilk binds the cytoplasmic domain of beta1-, beta2- and beta3-integrins and recruits adaptors and kinases, and is thought to stimulate downstream signalling events through phosphorylation of protein kinase B/Akt (Pkb/Akt) and glycogen synthase kinase 3-beta (GSK3-beta). Here, we show that mice with a chondrocyte-specific disruption of the gene encoding Ilk develop chondrodysplasia, and die at birth due to respiratory distress. The chondrodysplasia was characterized by abnormal chondrocyte shape and decreased chondrocyte proliferation. In addition, Ilk-deficient chondrocytes showed adhesion defects, failed to spread and formed fewer FAs and actin stress fibres. Surprisingly, phosphorylation of Pkb/Akt and GSK3-beta is unaffected in Ilk-deficient chondrocytes. These findings suggest that Ilk regulates actin reorganization in chondrocytes and modulates chondrocyte growth independently of phosphorylation of Pkb/Akt and GSK3-beta.

Articular cartilage exhibits anisotropic mechanical properties when subjected to tension. However, mechanical anisotropy of mature cartilage in compression is poorly known. In this study, both confined and unconfined compression tests of cylindrical cartilage discs, taken from the adult human patello-femoral groove and cut either perpendicular (normal disc) or parallel (tangential disc) to the articular surface, were utilized to determine possible anisotropy in Young's modulus, E, aggregate modulus, Ha, Poisson's ratio, v and hydraulic permeability, k, of articular cartilage. The results indicated that Ha was significantly higher in the direction parallel to the articular surface as compared with the direction perpendicular to the surface ( Ha = 1.237 ± 0.486 MPa versus Ha = 0.845 ± 0.383 MPa, p = 0.017, n = 10). The values of Poisson's ratio were similar, 0.158 ± 0.148 for normal discs compared with 0.180 ± 0.046 for tangential discs. Analysis using the linear biphasic model revealed that the decrease of permeability during the offset compression of 0–20 per cent was higher ( p = 0.015, n = 10) in normal (from 25.5 × 10− 15 to 1.8 × 10−15 m4/N s) than in tangential (from 12.3 × 10− 15 to 1.3 × 10− 15 m4/N s) discs. Based on the results, it is concluded that the mechanical characteristics of adult femoral groove articular cartilage are anisotropic also during compression. Anisotropy during compression may be essential for normal cartilage function. This property has to be considered when developing advanced theoretical models for cartilage biomechanics.

The mechanisms by which tendon strength is established during growth and development and restored following injury are not completely understood and are likely to be complex, multifactorial processes. Several studies examining the relationship between mechanical behavior and ultrastructural characteristics of tendons and ligaments during growth and maturation suggest that collagen fibril diameter is strongly correlated with tendon strength. Because of the similarities between development and repair processes of musculoskeletal tissues, increases in tendon strength during healing may be related to increases in fibril ultrastructural parameters such as fibril size, numerical density, and area fraction. In this study, we compared murine Achilles tendons at various time points after tenotomy with sham-operated controls in tensile tests to failure and examined tendons using electron microscopy to assess collagen fibril ultrastructure. We found that in the 6-week period following Achilles tenotomy, fibril mean diameter remained significantly smaller than sham-side diameter by a factor of 2‐3. Despite the persistently small fibril size, increasing numerical density resulted in a gradual increase in fibril area fraction. Biomechanical strength did not reach that of intact tendons until some time between 5 and 7 weeks, approximately the same time period when fibril area fraction began to approach sham values. These data suggest that parameters other than collagen fibril size are most responsible for increased tendon strength during healing.

This study evaluated the effects of medial hamstring lengthening on gait in patients with diplegic cerebral palsy. A group of patients who underwent medial hamstring lengthening and distal rectus transfer was compared to a group of patients who underwent intramuscular psoas lengthening as well. Patients who underwent a psoas lengthening showed an increased anterior pelvic tilt and a tendency towards genu recurvatum, features that could indicate postoperative hamstring weakness. These results underline the importance of medial hamstrings for pelvis and knee motion in the sagittal plane during walking. In additional the limitations of the popliteal angle as a clinical measure to evaluate hamstring length in the presence of hip flexor tightness are discussed.

Objective Data pertaining to the quantitative structural features and organization of normal articular cartilage are of great importance in understanding its biomechanical properties and in attempting to establish this tissue's counterpart by engineering in vitro . A comprehensive set of such baseline data is, however, not available for humans. It was the purpose of the present study to furnish the necessary information. Design The articular cartilage layer covering the medial femoral condyle of deceased persons aged between 23 and 49 years was chosen for the morphometric analysis of cell parameters using confocal microscopy in conjunction with unbiased stereological methods. The height of the hyaline articular cartilage layer, as well as that of the calcified cartilage layer and the subchondral bone plate, were also measured. Results The mean height of the hyaline articular cartilage layer was found to be 2.4mm, the volume density of chondrocytes therein being 1.65%, the number of cells per mm 3 of tissue 9626 and the mean cell diameter 13μm. Other estimators (including matrix mass per cell and cell profile density) were also determined. Conclusions A comparison of these normal human quantitative data with those published for experimental animals commonly used in orthopaedic research reveals substantial differences, consideration of which in tissue engineering strategies destined for human application are of paramount importance for successful repair. Copyright 2002 OsteoArthritis Research Society International. Published by Elsevier Science Ltd. All rights reserved .

The purpose of this study was to evaluate hip and pelvis rotations in groups of hemiplegic and diplegic children before and after surgical correction of fixed internal rotation deformity of the hip. Twenty-two children with cerebral palsy (eight diplegia, 14 hemiplegia) having fixed internal rotation deformity at the hip were treated by multilevel surgery which included derotation osteotomy of the femur. Evaluation was done before and at a mean of 3.1 years after surgery using three-dimensional computerized gait analysis. Preoperatively, the patients in the hemiplegia group had a significantly greater compensatory external rotation of the pelvis than those in the diplegic group. Post-operatively there were no significant differences between the two groups. In the hemiplegia group the external rotation of the pelvis was corrected after correction of hip rotation by derotation osteotomy. Patients in the diplegia group showed significant improvements in the hip rotation with no significant change in the pelvis rotation after multilevel surgery.

Indentation testing is a widely used technique for nondestructive mechanical analysis of articular cartilage. Although cartilage shows an inhomogeneous, layered structure with anisotropic mechanical properties, most theoretical indentation models assume material homogeneity and isotropy. In the present study, quantitative polarized light microscopy (PLM) measurements from canine cartilage were utilized to characterize thickness and structure of the superficial, collageneous tissue layer as well as to reveal its relation to experimental indentation measurements. In addition to experimental analyses, a layered, transversely isotropic finite element (FE) model was developed and the effect of superficial (tangential) tissue layer with high elastic modulus in the direction parallel to articular surface on the indentation response was studied. The experimental indentation stiffness was positively correlated with the relative thickness of the superficial cartilage layer. Also the optical retardation, which reflects the degree of parallel organization of collagen fibrils as well as collagen content, was related to indentation stiffness. FE results indicated effective stiffening of articular cartilage under indentation due to high transverse modulus of the superficial layer. The present results suggest that indentation testing is an efficient technique for the characterization of the superficial degeneration of articular cartilage.

Fragestellung: Ziel der Untersuchung war die prospektive kontrollierte Untersuchung der Ergebnisse von Mehretageneingriffen zur Gangverbesserung bei Kindern mit spastischer Diplegie. Methode: Bei 20 Kindern mit Diplegie wurden dreidimensionale Ganganalysen vorgenommen. Eine Gruppe von zehn Kindern wurde mit operativen Mehretageneingriffen zur Gangverbesserung behandelt, wobei die Indikationen zu den einzelnen operativen Eingriffen definierten Kriterien folgten. Zehn weitere Kinder, die ihre Physiotherapie fortsetzten, stellten eine Kontrollgruppe dar. Eineinhalb Jahre nach der ersten Untersuchung wurde die Ganganalyse wiederholt. Zielparameter waren Zeit-Weg-Parameter und kinematische Parameter des Beckens, der Hufte, des Knies und des Sprunggelenkes in der Sagittalebene. Ergebnisse: In der operierten Gruppe erhohte sich die Ganggeschwindigkeit durch eine verlangerte Schrittlange im Vergleich zur Kontrollgruppe. Mehretageneingriffe fuhrten zu einer geringen Zunahme der Beckenkippung. Der Bewegungsumfang am Kniegelenk konnte deutlich verbessert werden, die Beweglichkeit am Sprunggelenk blieb in beiden Gruppen unverandert. Durch die postoperativ verbesserte Kniestreckung wurde die Stabilitat des Standbeins verbessert. Zusatzlich wurde dadurch das freie Schwingen des kontralateralen Schwungbeins beim Gehen erleichtert. Schlussfolgerung: Die vorliegende prospektive kontrollierte Untersuchung belegt Gangverbesserungen nach operativen Mehretageneingriffen bei Kindern mit spastischer Diplegie. Aim: To evaluate prospectively the outcome of gait-improvement surgery in children with spastic diplegia. Method: Three-dimensional gait analysis was performed in twenty children with spastic diplegia. Ten children underwent single event multilevel surgery for gait improvement. Indications for individual procedures followed a fixed set of selection criteria. The other ten children continued with their physiotherapy programme and served as a control group. A second gait analysis was performed in all children after 1.5 years. Time-distance parameters and kinematics of the pelvis, hip, knee and ankle joints in the sagittal plane served as main outcome measures Results: The patients walked faster with an increased stride length after surgery in comparison to the conservatively treated controls. The average pelvic tilt increased slightly and the range of motion of the knee joint increased considerably after multilevel surgery. The motion at the ankle remained unchanged over the study period in both the groups. An improved knee extension during the stance phase of gait served to improve stance limb stability and facilitated an unhindered swing phase of the opposite limb. Conclusion: This prospective trial showed favourable changes in gait function after multilevel surgery in spastic diplegic children.

Chondrocytes are known to sense and respond to mechanical and physicochemical stimuli by multiple regulatory pathways, including upstream signaling, transcription, translation, posttranslational modifications, and vesicular transport. Due to the complexity of identifying the biophysical phenomena that occur during cartilage loading in vivo, the regulatory mechanisms that govern chondrocyte mechanotransduction are not fully understood. Recent studies have shown that fluid flow during dynamic compression of cartilage explants can stimulate proteoglycan and protein synthesis. In this study, we examined the effect of deformations of cell and extracellular matrix on chondrocyte biosynthesis. We used tissue shear loading, since tissue shear causes little volumetric deformation and can thereby decouple fluid flow from cell and matrix deformation. Shear loading was applied over a wide range of frequencies, 0.01-1.0 Hz, using 1-3% sinusoidal shear strain amplitudes, and the resulting proteoglycan and protein syntheses were measured using radiolabel incorporation. In addition, quantitative autoradiography was used to investigate spatial variations in matrix biosynthesis and to correlate these variations with the spatial profiles of biophysical stimuli. Our data show that tissue shear loading at 1-3% strain amplitude stimulated the synthesis of protein by approximately 50% and proteoglycans by approximately 25% at frequencies between 0.01 and 1.0 Hz. The relatively uniform patterns of biosynthesis in the radial and vertical directions within cylindrical explants revealed by autoradiography suggest that the stimulatory effect was associated with the relatively uniform deformation caused by simple shear loading. These results suggest that chondrocytes can respond to tissue shear stress-initiated pathways for the production of collagen and proteoglycan, which include deformation of cells and pericellular matrix, even in the absence of macroscopic tissue-level fluid flow.

Multilevel surgery for gait improvement was performed on twelve ambulatory children with diplegic type of cerebral palsy and dynamic equinus deformity. Dynamic equinus deformities were defined as those who had an equinus at initial contact during preoperative gait analysis, and where the equinus deformity was correctable passively during physical examination. Ankle function was evaluated by clinical examination and gait analysis before surgery, and at least 3 years after surgery. The ankle showed an increase in dorsal flexion at initial contact, at single stance and in swing. There was an increase in dorsal flexion at the beginning of push-off, without a decrease in the range of motion of the ankle during push-off. Ankle moments demonstrated significant improvement in the maximum flexor moment in the second half of single stance. There was a change from abnormal generation of the energy in mid-stance to the normal pattern of energy absorption. Positive work during push-off was significantly increased. Conservative management of dynamic equinus deformities combined with multilevel surgery to correct other deformities of the locomotion system resulted in significant improvements in ankle function during gait.

Seventeen patients with diplegic cerebral palsy were assessed by clinical examination and three-dimensional gait analysis before and after surgery to improve gait. Selection of surgical procedures was according to a fixed set of selection criteria. The average postoperative follow-up was 3.8 years (range, 2.6-5.7 years). Clinical examination revealed an improved range of motion for the ankle and no reduction in the power grade at the hip, knee and ankle after surgery. Kinematic parameters showed improved knee extension in stance and significant changes towards a normal ankle motion pattern postoperatively. Kinetic evaluation demonstrated that most of the total power during walking was generated at the hip, with the ankle contributing a small part. After surgery, patients walked faster with an increased power generation at the hip during first double support and at the ankle during push off. Power generation at the hip in stance is pointed out to be an important mechanism for propulsion during walking.

Mechanical overloading of cartilage has been implicated in the initiation and progression of osteoarthrosis. Our objectives were to identify threshold levels of strain rate and peak stress at which sub-impact loads could induce cartilage matrix damage and chondrocyte injury in bovine osteochondral explants and to explore relationships between matrix damage, spatial patterns of cell injury, and applied loads. Single sub-impact loads characterized by a constant strain rate between 3×10−5 and 0.7 s −1 to a peak stress between 3.5 and 14 MPa were applied, after which explants were maintained in culture for four days. At the higher strain rates, matrix mechanical failure (tissue cracks) and cell deactivation were most severe near the cartilage superficial zone and were associated with sustained increased release of proteoglycan from explants. In contrast, low strain rate loading was associated with cell deactivation in the absence of visible matrix damage. Furthermore, cell activity and proteoglycan synthesis were suppressed throughout the cartilage depth, but in a radially dependent manner with the most severe effects at the center of cylindrical explants. Results highlight spatial patterns of matrix damage and cell injury which depend upon the nature of injurious loading applied. These patterns of injury may also differ in terms of their long-term implications for progression of degradative disease and possibilities for cartilage repair.

Conflicting data exist as to whether insulin-like growth factor I (IGF-I) messenger RNA (mRNA) and peptide are expressed within chondrocytes. This question is pertinent to the mode of GH action on longitudinal bone growth. We have, therefore, investigated this issue in normal rats and in hypophysectomized rats treated for 24 h with GH or IGF-I using in situ hybridization and immunohistochemistry. Serum IGF-I, body weight, and tibial growth plate, but not articular cartilage, height increased with both treatments. Both IGF-I mRNA and IGF-I immunoreactivity occurred in all chondrocyte layers of growth plate and articular cartilage. The percentage of cells with IGF-I mRNA correlated well with IGF-I immunoreactivity under all experimental conditions. In normal rats, IGF-I expression was highest in the upper hypertrophic zone in growth plate (68–71%) and articular cartilage (32–34%). Hypophysectomy, GH, or IGF-I did not significantly affect this percentage. In the stem cell and proliferative and lower hypertro...

The objective of this study was to investigate how temporal and spatial patterns of characteristic extracellular matrix molecules are altered in the absence of normal functional skeletal muscle contractions during avian synovial joint development. By using in situ detection of protein and mRNA expression in developing avian feet and femorotibial joints from a wide range of developmental stages, we demonstrate that the morphological abnormalities that result from embryonic immobilization are associated with altered patterns of tenascin-C and collagen-XII expression within developing joint structures. As the joints fuse in immobilized embryos, the cells of the presumptive articular surface differentiate from flattened fibroblasts to more rounded chondrocytes and collagens XII and I are no longer detected at sites of complete joint fusion. Although the expression of collagen XII persists at normal levels elsewhere within the immobilized joint, tenascin-C expression is diminished within the chondroepiphysis, synovium, and tendons, as well as within the remains of the fibrous articular surface. This effect is most notable for the shortest tenascin variant (Tn 190) within the chondroepiphysis and the largest variant (Tn230) within tendons, synovium, and the fibrous surface layer of the joint. This study thus provides in vivo support of previous in vitro work that suggests that tenascin expression is sensitive to external changes in mechanical loading environment. However, these data do not support a similar conclusion for collagen XII during early development.

Matrilin 1, or cartilage matrix protein, is a member of a novel family of extracellular matrix proteins. To date, four members of the family have been identified, but their biological role is unknown. Matrilin 1 and matrilin 3 are expressed in cartilage, while matrilin 2 and matrilin 4 are present in many tissues. Here we describe the generation and analysis of mice carrying a null mutation in the Crtm gene encoding matrilin 1. Anatomical and histological studies demonstrated normal development of homozygous mutant mice. Northern blot and biochemical analyses show no compensatory up-regulation of matrilin 2 or 3 in the cartilage of knockout mice. Although matrilin 1 interacts with the collagen II and aggrecan networks of cartilage, suggesting that it may play a role in cartilage tissue organization, studies of collagen extractability indicated that collagen fibril maturation and covalent cross-linking were unaffected by the absence of matrilin 1. Ultrastructural analysis did not reveal any abnormalities of matrix organization. These data suggest that matrilin 1 is not critically required for cartilage structure and function and that matrilin 1 and matrilin 3 may have functionally redundant roles.

The rate of longitudinal bone growth is regulated primarily by modulations in the activity of epiphyseal plate hypertrophic chondrocytes, these being manifested as changes in cell and matrix volume. It was the purpose of this study to ascertain whether the cytoplasmic organelles representing the cellular production apparatus, i.e. rough endoplasmic reticulum, Golgi apparatus and mitochondria, contribute to these changes by modulating their rate of activity or by increasing/decreasing the surface area and/or volume of their membranes. Using rats at different stages of growth, the surface areas and volumes of the three organellar systems were quantified in epiphyseal plate chondrocytes at the onset and termination of hypertrophy by ultrastructural stereology. Matrix synthesis during the same span was assessed by monitoring the production of its principal components, namely, fibrillar collagen (ultrastructural morphometry) and glycosaminoglycans (quantitative (35)S-autoradiography). Each organelle adapts to increases (21- to 35-day-old rats) and decreases (35- to 80-day-old rats) in growth rate by its own individual combination of the two alternative mechanisms, but modulations in the level of activity predominate over alterations in the surface area or volume of their membranes. These findings point to the danger of relying solely on data gleaned from a quantitative ultrastructural analysis of organellar parameters and emphasise the necessity of conducting functional assays in parallel, as performed here.