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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.

Objectives (1) To determine whether the biocompatibility of coralline hydroxyapatite (CHA) granules could be improved by using an octacalcium phosphate (OCP) coating layer, and/or functionalized with bone morphogenetic protein 2 (BMP-2), and (2) to investigate if BMP-2 incorporated into this coating is able to enhance its osteoinductive efficiency, in comparison to its surface-adsorbed delivery mode. Methods CHA granules (0.25 g per sample) bearing a coating-incorporated depot of BMP-2 (20 μg/sample) together with the controls (CHA bearing an adsorbed depot of BMP-2; CHA granules with an OCP coating without BMP-2; pure CHA granules) were implanted subcutaneously in rats (n = 6 animals per group). Five weeks later, the implants were retrieved for histomorphometric analysis to quantify the volume of newly generated bone, bone marrow, fibrous tissue and foreign body giant cells (FBGCs). The osteoinductive efficiency of BMP-2 and the rates of CHA degradation were also determined. Results The group with an OCP coating-incorporated depot of BMP-2 showed the highest volume and quality or bone, and the highest osteoinductive efficacy. OCP coating was able to reduce inflammatory responses (improve biocompatibility), and also simple adsorption of BMP-2 to CHA achieved this. Conclusions The biocompatibility of CHA granules (reduction of inflammation) was significantly improved by coating with a layer of OCP. Pure surface adsorption of BMP-2 to CHA also reduced inflammation. Incorporation of BMP-2 into the OCP coatings was associated with the highest volume and quality of bone, and the highest biocompatibility degree of the CHA granules. Clinical significance Higher osteoinductivity and improved biocompatibility of CHA can be obtained when a layer of BMP-2 functionalized OCP is deposited on the surfaces of CHA granules.

© 2021, AO Research Institute Davos. All rights reserved.The aim of the present study was to investigate in vivo whether bone morphogenetic protein-7 (BMP-7) was able to promote and accelerate dental implant healing at a low dose in an osteopenic environment by using a delayed drug-release system. Skeletally mature Chinese goats, having physiologically osteopenic (osteoporotic-like) facial bones, served as an animal model. Dental implants were provided with a delayed-release drug-delivery system and BMP-7 was applied at three different dosages. The implants, inserted into healed extraction sockets, were removed 1, 2 and 3 weeks after surgery. Quantification of osseointegration and formation of new bone in the peri-implant space were measured histomorphometrically. Data revealed no evidence of any adverse drug effect at or near the implantation sites. After the first postoperative week, bone neoformation was minimal; after the second week, peri-implant bone formation appeared, particularly in the groups with low dosages of BMP-7. After 3 weeks, new-bone volume was the largest in the group with the lowest (near-physiological) dosage of BMP-7, also showing the highest efficacy of BMP-7. Other dosage or release modes were found to be significantly less effective. BMP-7 was highly efficacious in promoting and accelerating bone formation in the peri-implant space in a hostile osteopenic environment if released by a slow-mode mechanism over time at near physiological activities. Therefore, biological functionalisation of dental implants by a high-power osteogenic factor may improve their healing success in hostile bony environments (osteopenia, osteoporosis, bone atrophy etc.).

© 2021, Mary Ann Liebert, Inc., publishers 2021.In dental clinical practice, systemic steroids are often applied at the end of implant surgeries to reduce postsurgical inflammation (tissue swelling, etc.) and to reduce patient discomfort. However, the use of systemic steroids is associated with generalized catabolic effects and with a temporarily reduced immunological competence. We hypothesize that by applying locally anticytokine antibodies (antitumor necrosis factor alpha and anti-interleukin-1 beta) together with a bioactive osteogenic implant at the time of the surgical intervention for the placement of a construct, we will be able to achieve the same beneficial effects as those using systemic steroids but are able to avoid the generalized antianabolic effects and the reduced immunocompetence effects, associated with the systemic use of steroids. In an adult rat model, a collagen sponge, soaked with the osteogenic agent bone morphogenetic protein-2, was used as an example for a bioactive implant material and was surgically placed subcutaneously. In the acute inflammatory phase after implantation (2 days after surgery) we investigated the local inflammatory tissue response, and 18 days postsurgically the efficiency of local osteogenesis (to assess possible antianabolic effects). We found that the negative control groups, treated postsurgically with systemic steroids, showed a significant suppression of both the inflammatory response and the osteogenetic activity, that is, they were associated with significant general antianabolic effects, even when steroids were used only at a low dose level. The local anticytokine treatment, however, was able to significantly enhance new bone formation activity, that is, the anabolic activity, over positive control values with BMP-2 only. However, the anticytokine treatment was unable to reduce the local inflammatory and swelling responses.

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.

PURPOSE To test the hypothesis if a novel single-chamber experimental dental implant allows in vivo the quantitative assessment of osseointegration over time and as a function of different surface properties (physical, chemical, geometric, biologic [osteoconductive or osteoinductive]) in a biologically unfavorable environment (local osteoporosis). MATERIALS AND METHODS Three prototypes of a novel experimental implant with different chamber sizes (small, medium, and large) were compared with each other to find out the minimum size of bone chambers needed to allow a discriminative quantification of osseointegration over time. For the comparison of low and high surface osteoconductivity properties, conventional sandblasted, acid-etched chamber surfaces (low surface osteoconductivity) were compared with biomimetically (calcium phosphate) coated ones (high surface osteoconductivity). The implants (4 implants per animal; 88 implants per time point) were inserted into the edentulous maxillae of a total of 66 adult goats with a physiologically osteoporotic masticatory apparatus. Two, 4, and 8 weeks later, they were excised and prepared for a histomorphometric analysis of the volume of neoformed bone within the chamber space and of the bone-to-implant contact (BIC) area. RESULTS The implants with small chambers did not show significant differences in bone coverage (BIC) nor bone volume (relative and absolute volume), neither as a function of time nor as of implant surface property (low versus high surface osteoconductivity). However, medium and large chambers revealed significant differences respecting both of these parameters over the 8-week postoperative time period. CONCLUSION The new implant model permits a discriminative quantification of osseointegration in vivo in an osteoporotic bone environment for implants with medium-sized and large-sized chambers. Quantitative assessment of osseointegration is possible, both over time and as a function of low and high surface osteoconductivity properties.

The disk of hyaline cartilage that is interposed between the epiphysis and the metaphysis of each of the long bones is responsible for its elongation, and, thus, when the lower limbs are concerned, for increases in bodily height. This so-called growth plate is avascular, aneural, and alymphatic. It consists solely of chondrocytes and an extracellular matrix which the cells elaborate. The growth plate is architectonically striking in so far as the chondrocytes are aligned in strictly vertical columns, which represent the functional units of longitudinal bone growth. The growth process begins with the slow division of chondrocytes in the resting ("stem cell") zone and proceeds with their rapid proliferation in the adjacent zone. These cells then undergo a process of progressive enlargement, which culminates in the zone of terminal hypertrophy. The life history of any given cell is recapitulated in a vertical column. The neoformation of cartilage in the axial direction is synchronized with its destruction at the vascular invasion front of the metaphysis and results in an elongation of the bony trabeculae. The mechanism that governs the highly coordinated sequence of events that underlies the growth of the long bones is complex; it is subject to influence by genetic, hormonal, nutritional, environmental, and pathological factors.

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

Biomimetically deposited calcium phosphate-based coatings of prostheses can serve as a vehicle for the targeted delivery of growth factors to the local implant environment. Based on indirect evidence in previous studies we hypothesize that such agents are liberated gradually from the coating via a cell-mediated degradation. In the present study, we tested this hypothesis by investigating the release mechanism and its kinetics by use of a radiolabeled osteogenic agent (

Absorbed collagen sponge (ACS)/bone morphogenetic protein-2 (BMP-2) are widely used in clinical practise for bone regeneration. However, the application of this product was found to be associated with a significant pro-inflammatory response, particularly in the early phase after implantation. This study aimed to clarify if the pro-inflammatory activities, associated with BMP-2 added to ACS, were related to the physical state of the carrier itself, i.e., a wet or a highly dehydrated state of the ACS, to the local degree of vascularisation and/or to local biomechanical factors. ACS (0.8 cm diameter)/BMP-2 were implanted subcutaneously in the back of 12 eight-week-old Sprague Dawley rats. Two days after surgery, the implanted materials were retrieved and analysed histologically and histomorphometrically. The acute inflammatory response following implantation of ACS was dependent of neither the presence or absence of BMP-2 nor the degree of vascularization in the surrounding tissue nor the hydration state (wet versus dry) of the ACS material at the time of implantation. Differential micro biomechanical factors operating at the implantation site appeared to have an influence on the thickness of inflammation. We conclude that the degree of the early inflammatory response of the ACS/BMP-2 may be associated with the physical and chemical properties of the carrier material itself.

No single processing technique is capable of optimally preserving each and all of the structural entities of cartilaginous tissue. Hence, the choice of methodology must necessarily be governed by the nature of the component that is targeted for analysis, for example, fibrillar collagens or proteoglycans within the extracellular matrix, or the chondrocytes themselves. This article affords an insight into the pitfalls that are to be encountered when implementing the available techniques and how best to circumvent them. Adult articular cartilage is taken as a representative pars pro toto of the different bodily types. In mammals, this layer of tissue is a component of the synovial joints, wherein it fulfills crucial and diverse biomechanical functions. The biomechanical functions of articular cartilage have their structural and molecular correlates. During the natural course of postnatal development and after the onset of pathological disease processes, such as osteoarthritis, the tissue undergoes structural changes which are intimately reflected in biomechanical modulations. The fine structural intricacies that subserve the changes in tissue function can be accurately assessed only if they are faithfully preserved at the molecular level. For this reason, a careful consideration of the tissue-processing technique is indispensable. Since, as aforementioned, no single methodological tool is capable of optimally preserving all constituents, the approach must be pre-selected with a targeted structure in view. Guidance in this choice is offered.

In clinical orthopaedics, total joint replacements and spinal fusions are routine undertakings. Many of the implicated patients suffer from osteoporosis, severe arthrosis or osteopaenia. In individuals thus afflicted, the bony bed lacks the mechanical stability that is a requisite for a firm anchorage of the implant and its functional competence. To promote the bony bondage of an implant it is necessary to induce neo-ossification by the introduction of an osteogenic agent, such as bone morphogenetic protein 2 (BMP-2). Since this growth factor is generally applied in a free form and at high dosages to maximise its osteogenicity, untoward side effects frequently ensue. We hypothesise that the administration of BMP-2 using a suitable delivery vehicle, and its gradual, low dose release therefrom in a cell-mediated manner, would avert the triggering of undesired side effects and enhance its efficacy. To test this postulate, implants of porous titanium were coated with a layer of calcium phosphate into which BMP-2 was biomimetically incorporated at dosages ranging from 0.8 to 500 µg/g of coating material (delivery system) prior to their surgical placement in the tibiae of adult sheep. The volume and the surface area of newly-formed bone were evaluated histomorphometrically after 3 and 6 weeks. The highest values were achieved using BMP-2 dosages of 20 to 100 µg/g of coating: The deposition of bone was confined to the immediate vicinity of the implant and was observed deep within the interstices of its meshwork, to the walls of which it bonded well. The findings of the study attest to the validity of our hypothesis.

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.

Although the placement of dental and orthopedic implants is now generally a safe, reliable and successful undertaking, the functional outcome is less assured in patients whose bone-healing capacity is compromised. To enhance peri-implant osteogenesis in these individuals, BMP-2 could be locally administered. However, neither a free suspension nor an implant-adsorbed depot of the agent is capable of triggering sustained bone formation. We hypothesize that this end could be achieved by incorporating BMP-2 into the three-dimensional crystalline latticework of a bone-mineral like, calcium-phosphate implant coating, wherefrom it would be liberated gradually - as the inorganic layer undergoes osteoclast-mediated degradation - not rapidly, as from an implant-adsorbed (two-dimensional) depot. To test this postulate, we compared the osteoinductive efficacies of implant coatings bearing either an incorporated, an adorbed, or an incorporated and an adsorbed depot of BMP-2 at a maxillary site in miniature pigs. The implants were retrieved 1, 2 and 3 weeks after surgery for the histomorphometric analysis of bone formation within a defined ‘osteoinductive’ space.At each juncture, the volume of newly-formed bone within the osteoinductive space was greatest around implants that bore a coating-incorporated depot of BMP-2, peak osteogenic activity being attained during the first week and sustained thereafter. In the other groups, the temporal course of bone formation was variable, and the peak levels were not sustained.The findings of this study confirm our hypothesis: they demonstrate that we now have at our disposal a means of efficaciously augmenting and expediting peri-implant bone formation. Clinically, this possibility would render the process of implant placement a safer and a more reliable undertaking in patients whose bone-healing capacity is compromised, and would also permit a curtailment of the postoperative recovery period by a forestallment of the mechanical-loading phase.

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.

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).

Polymers that are used in clinical practice as bone-defect-filling materials possess many essential qualities, such as moldability, mechanical strength and biodegradability, but they are neither osteoconductive nor osteoinductive. Osteoconductivity can be conferred by coating the material with a layer of calcium phosphate, which can be rendered osteoinductive by functionalizing it with an osteogenic agent. We wished to ascertain whether the morphological and physicochemical characteristics of unfunctionalized and bovine-serum-albumin (BSA)-functionalized calcium-phosphate coatings were influenced by the surface properties of polymeric carriers. The release kinetics of the protein were also investigated. Two sponge-like materials (Helistat® and Polyactive®) and two fibrous ones (Ethisorb™ and poly[lactic-co-glycolic acid]) were tested. The coating characteristics were evaluated using state-of-the-art methodologies. The release kinetics of BSA were monitored spectrophotometrically. The characteristics of the amorphous and the crystalline phases of the coatings were not influenced by either the surface chemistry or the surface geometry of the underlying polymer. The mechanism whereby BSA was incorporated into the crystalline layer and the rate of release of the truly incorporated depot were likewise unaffected by the nature of the polymeric carrier. Our biomimetic coating technique could be applied to either spongy or fibrous bone-defect-filling organic polymers, with a view to rendering them osteoconductive and osteoinductive.

We investigated the inflammatory response to, and the osteoinductive efficacies of, four polymers (collagen, Ethisorb™, PLGA and Polyactive®) that bore either an adsorbed (fast-release kinetics) or a calcium-phosphate-coating-incorporated (slow-release kinetics) depot of BMP-2. Titanium-plate-supported discs of each polymer (n = 6 per group) were implanted at an ectopic (subcutaneous) ossification site in rats (n = 48). Five weeks later, they were retrieved for a histomorphometric analysis of the volumes of ectopic bone and foreign-body giant cells (a gauge of inflammatory reactivity), and the degree of polymer degradation. For each polymer, the osteoinductive efficacy of BMP-2 was higher when it was incorporated into a coating than when it was directly adsorbed onto the material. This mode of BMP-2 carriage was consistently associated with an attenuation of the inflammatory response. For coated materials, the volume density of foreign-body giant cells was inversely correlated with the volume density of bone (r2 = 0.96), and the volume density of bone was directly proportional to the surface-area density of the polymer (r2 = 0.97). Following coating degradation, other competitive factors, such as the biocompatibility and the biodegradability of the polymer itself, came into play.

A novel biphasic calcium phosphate (CaP) granulate consisting of hydroxyapatite (HA) and beta-tricalciumphosphate (TCP) was compared with pure HA and pure TCP and with autograft as positive control.Four standardized bone defects were prepared in both mandibular angles of 16 minipigs and grafted with autogenous bone chips, HA, HA/TCP (60% : 40%), or TCP. Histologic and histomorphometric analysis of bone formation and graft degradation followed healing periods of 2, 4, 8, and 24 weeks.2 weeks: more bone formation in defects filled with autograft than with the three CaP materials (P0.05). 4 weeks: bone formation differed significantly (P0.05) between all four materials (autograftTCPHA/TCPHA). 8 weeks: more bone formation in defects with autograft and TCP than with HA/TCP (P0.05), and HA/TCP had more bone formation than HA (P0.05). 24 weeks: no difference in bone formation between the groups. Autograft and TCP resorbed quickly and almost completely over 8 weeks, whereas HA/TCP and HA showed limited degradation over 24 weeks.All defects healed with mature lamellar bone and intimate contact between bone and the remaining graft material. The rate of bone formation corresponded to the content of TCP in the CaP materials.

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.

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.

INTRODUCTION The transcription factor activating enhancer binding protein 2 epsilon (AP-2ε) was recently shown to be expressed during chondrogenesis as well as in articular chondrocytes of humans and mice. Furthermore, expression of AP-2ε was found to be upregulated in affected cartilage of patients with osteoarthritis (OA). Despite these findings, adult mice deficient for AP-2ε (Tfap2e(-/-)) do not exhibit an obviously abnormal cartilaginous phenotype. We therefore analyzed embryogenesis of Tfap2e(-/-) mice to elucidate potential transient abnormalities that provide information on the influence of AP-2ε on skeletal development. In a second part, we aimed to define potential influences of AP-2ε on articular cartilage function and gene expression, as well as on OA progression, in adult mice. METHODS Murine embryonic development was accessed via in situ hybridization, measurement of skeletal parameters and micromass differentiation of mesenchymal cells. To reveal discrepancies in articular cartilage of adult wild-type (WT) and Tfap2e(-/-) mice, light and electron microscopy, in vitro culture of cartilage explants, and quantification of gene expression via real-time PCR were performed. OA was induced via surgical destabilization of the medial meniscus in both genotypes, and disease progression was monitored on histological and molecular levels. RESULTS Only minor differences between WT and embryos deficient for AP-2ε were observed, suggesting that redundancy mechanisms effectively compensate for the loss of AP-2ε during skeletal development. Surprisingly, though, we found matrix metalloproteinase 13 (Mmp13), a major mediator of cartilage destruction, to be significantly upregulated in articular cartilage of adult Tfap2e(-/-) mice. This finding was further confirmed by increased Mmp13 activity and extracellular matrix degradation in Tfap2e(-/-) cartilage explants. OA progression was significantly enhanced in the Tfap2e(-/-) mice, which provided evidence for in vivo relevance. This finding is most likely attributable to the increased basal Mmp13 expression level in Tfap2e(-/-) articular chondrocytes that results in a significantly higher total Mmp13 expression rate during OA as compared with the WT. CONCLUSIONS We reveal a novel role of AP-2ε in the regulation of gene expression in articular chondrocytes, as well as in OA development, through modulation of Mmp13 expression and activity.

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.

The article summarizes the collective views expressed at the fourth session of the workshop Tissue Engineering--the Next Generation, which was devoted to the translation of results of tissue engineering research into applications. Ernst Hunziker described the paradigm of a dual translational approach, and argued that tissue engineering should be guided by the dimensions and physiological setting of the bodily compartment to be repaired. Myron Spector discussed collagen-glycosaminoglycan (GAG) scaffolds for musculoskeletal tissue engineering. Jeanette Libera focused on the biological and clinical aspects of cartilage tissue engineering, and described a completely autologous procedure for engineering cartilage using the patient's own chondrocytes and blood serum. Arthur Gertzman reviewed the applications of allograft tissues in orthopedic surgery, and outlined the potential of allograft tissues as models for biological and medical studies. Savio Woo discussed a list of functional tissue engineering approaches designed to restore the biochemical and biomechanical properties of injured ligaments and tendons to be closer to that of the normal tissues. Specific examples of using biological scaffolds that have chemoattractants as well as growth factors with unique contact guidance properties to improve their healing process were shown. Anthony Ratcliffe discussed the translation of the results of research into products that are profitable and meet regulatory requirements. Michael Lysaght challenged the proposition that commercial and clinical failures of early tissue engineering products demonstrate a need for more focus on basic research. Arthur Coury described the evolution of tissue engineering products based on the example of Genzyme, and how various definitions of success and failure can affect perceptions and policies relative to the status and advancement of the field of tissue engineering.

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.

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.

In a previous investigation, we demonstrated that when bovine serum albumin (BSA) is biomimetically co-precipitated with Ca 2+ and PO 4 3− ions upon titanium-alloy implants, it becomes incorporated into the crystal lattice and is not merely deposited on its surface. Moreover, the protein elicited a change in crystal structure from an octacalcium phosphate type to a carbonated apatite one, which bears a closer resemblance to natural bone mineral. In the present study, we investigated the dissolution rate and mechanical strength of such BSA-containing coatings as a function of protein concentration within the bathing medium (10 ng/ml to 1.0 mg/ml). BSA-containing coatings released Ca 2+ ions more slowly (5 ppm/min) than did non-BSA-containing ones (10 ppm/min), but this rate did not change as a function of protein concentration within the bathing medium. In contrast, the strength of coatings increased almost linearly as a function of protein concentration within the bathing medium, indicating that BSA incorporated into the crystal lattice enhances its mechanical strength in a concentration-dependent manner.

IGF-I mediates growth-promoting actions of GH. In the present study we investigated whether the somatostatin analog octreotide blunts the stimulatory effects of GH and/or IGF-I on bone growth in hypophysectomized rats infused for 6 d with vehicle, GH, or IGF-I. We found that octreotide significantly suppressed the GH-induced rise in liver IGF-I mRNA (−27%) and peptide (−32%) and the serum IGF-I level (−26%) and concomitantly inhibited GH-stimulated, but not IGF-I-stimulated, body weight gain (−31%), tibial epiphyseal width (−14%), and bone growth rate (−24%). Furthermore, octreotide significantly reduced the GH-induced increase in the number of IGF-I immunoreactive chondrocytes in all layers (except in the upper hypertrophic zone) of the tibial growth plate cartilage (P < 0.0001 for stem cell and proliferative zone; P < 0.0005 for lower hypertrophic zone). These findings demonstrate that octreotide does not interfere with IGF-I action, but does interfere with local GH-stimulated IGF-I production in the growth plate. Thus, besides inhibiting pituitary GH secretion, octreotide exerts inhibitory peripheral effects on GH-stimulated longitudinal bone growth.

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.

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.

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.

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.

The development of clinical strategies for cartilage repair and inhibition of matrix degradation may be facilitated by a better understanding of (1) the chondrocyte phenotype in the context of a damaged extracellular matrix, and (2) the roles of biochemical and biomechanical pathways by which matrix metabolism is mediated. Using methods of quantitative autoradiography, we examined the cell-length scale patterns of proteoglycan deposition and turnover in the cell-associated matrices of chondrocytes in adult bovine and calf cartilage explants. Results highlight a rapid turnover in the pericellular matrix, which may indicate spatial organization of PG metabolic pools, and specific biomechanical roles for different matrix regions. Subsequent to injurious compression of calf explants, which resulted in grossly visible tissue cracks and caused a decrease in the number of viable chondrocytes within explants, cell-mediated matrix catabolic processes appeared to increase, resulting in apparently increased rates of proteoglycan turnover around active cells. Furthermore, the influences of cell-stimulatory factors such as IL-1 beta appeared to be delayed in their effects subsequent to injurious compression, suggesting interactions between biomechanical and biochemical pathways of PG degradation. These results may provide a useful reference point in the development of in vitro models for cartilage injury and disease, and hint at possible new approaches in the development of cartilage repair strategies.

Perlecan is a heparan sulfate proteoglycan that is expressed in all basement membranes (BMs), in cartilage, and several other mesenchymal tissues during development. Perlecan binds growth factors and interacts with various extracellular matrix proteins and cell adhesion molecules. Homozygous mice with a null mutation in the perlecan gene exhibit normal formation of BMs. However, BMs deteriorate in regions with increased mechanical stress such as the contracting myocardium and the expanding brain vesicles showing that perlecan is crucial for maintaining BM integrity. As a consequence, small clefts are formed in the cardiac muscle leading to blood leakage into the pericardial cavity and an arrest of heart function. The defects in the BM separating the brain from the adjacent mesenchyme caused invasion of brain tissue into the overlaying ectoderm leading to abnormal expansion of neuroepithelium, neuronal ectopias, and exencephaly. Finally, homozygotes developed a severe defect in cartilage, a tissue that lacks BMs. The chondrodysplasia is characterized by a reduction of the fibrillar collagen network, shortened collagen fibers, and elevated expression of cartilage extracellular matrix genes, suggesting that perlecan protects cartilage extracellular matrix from degradation.

Skeletal elements are formed either by replacing a preformed cartilagenous matrix template in a process called endochondral ossification or directly from mesenchyme by a process known as membranous ossification. Longitudinal growth of bones is achieved by growth plates where calcified cartilage is converted into bone. To investigate the role of extracellular matrix as well as intracellular signaling pathways in the formation and growth of bone, the genes coding for type II collagen and cyclic guanosine 3′,5′-monophosphate (cGMP)-dependent protein kinase (cGK) II, were disrupted. It is demonstrated that loss of Col2a1 or cGKII led to abnormal endochondral ossification and skeletal development. In cGKII −/− mice, bones derived by membranous ossification developed normally while bones derived by endochondral ossification were shortened. This growth defect was not associated with a general metabolic disturbance. In Col2a1 knockout mice, endochondral ossification was completely absent, whereas membraneous ossification was not affected. Despite the defects in bone formation, invasion of blood vessels into bone cavities and formation of bone marrow occurred in Col2a1-null mice. Taken together, the phenotypes of these two knockout mice show that chondrocytes need a well-functioning extracellular matrix scaffold and a normal cGMP-signaling system for endochondral ossification to form a normal skeleton.

The effects of mechanical injury on the metabolism of cartilage matrix are of interest for understanding the pathogenesis of osteoarthrosis and the development of strategies for cartilage repair. The purpose of the present study was to examine the effects of injury on matrix turnover in a calf articular cartilage explant system for which the effects of mechanical loading on cell activity and the cell-mediated pathways of matrix metabolism are already well characterized. New methods of quantitative autoradiography were used in combination with established biochemical and biomechanical techniques for the analysis of cell and matrix responses to acute mechanical injury, with particular attention to the processes of localized matrix turnover in the cell-associated matrices of individual chondrocytes. Matrix deposition and turnover around cells in control explants was spatially dependent, with the highest rates of proteoglycan deposition and tunrtover arid the lowest rates of collagen deposition (as indicated by [3H]proline autoradiography) occurring in the pericellular matrix. Injurious compression was associated with (a) an abrupt decrease in the tensile load-carrying capacity of the collagen matrix, apparently associated with mechanical failure of the tissue, (b) a considerable but subtotal decrease in cell viability, marked by the emergence of an apparently inactive cell population interspersed within catabolically active but abnormally large cells, and (c) sustained, elevated rates of protcoglycan turnover, particularly in the cell-associated matrices of apparently viable cells, which involved the increased release of aggregating species in addition to a spectrum of degradation fragments that were also in controls. These results may represent an in vitro model for the responses of chondrocytes and the cartilage extracellular matrix to mechanical injury.