Intravitreal Pharmacokinetic Study of the Antiangiogenic Glycoprotein Opticin.

Opticin is an endogenous vitreous glycoprotein that may have therapeutic potential as it has been shown that supranormal concentrations suppress preretinal neovascularization. Herein we investigated the pharmacokinetics of opticin following intravitreal injection in rabbits. To measure simultaneously concentrations of human and rabbit opticin, a selected reaction monitoring mass spectrometry assay was developed. The mean concentration of endogenous rabbit opticin in 7 uninjected eyes was measured and found to be 19.2 nM or 0.62 µg/mL. When the vitreous was separated by centrifugation into a supernatant and collagen-containing pellet, 94% of the rabbit opticin was in the supernatant. Intravitreal injection of human opticin (40 µg) into both eyes of rabbits was followed by enucleation at 5, 24, and 72 h and 7, 14, and 28 days postinjection (n = 6 at each time point) and measurement of vitreous human and rabbit opticin concentrations in the supernatant and collagen-containing pellet following centrifugation. The volume of distribution of human opticin was calculated to be 3.31 mL, and the vitreous half-life was 4.2 days. Assuming that rabbit and human opticin are cleared from rabbit vitreous at the same rate, opticin is secreted into the vitreous at a rate of 0.14 µg/day. We conclude that intravitreally injected opticin has a vitreous half-life that is similar to currently available antiangiogenic therapeutics. While opticin was first identified bound to vitreous collagen fibrils, here we demonstrate that >90% of endogenous opticin is not bound to collagen. Endogenous opticin is secreted by the nonpigmented ciliary epithelium into the rabbit vitreous at a remarkably high rate, and the turnover in vitreous is approximately 15% per day.

Biomimetic proteoglycans diffuse throughout articular cartilage and localize within the pericellular matrix.

Biomimetic proteoglycan (BPG) diffusion into articular cartilage has the potential to restore the lost proteoglycan content in osteoarthritic cartilage given these molecules mimic the structure and properties of natural proteoglycans. We examined the diffusion characteristics of BPGs through cartilage with the use of a custom-made in vitro cartilage diffusion model in both normal bovine and human osteoarthritic cartilage explants. BPGs were introduced into the cartilage through essentially one-dimensional diffusion using osteochondral plugs. The molecular diffusion was shown to be size and concentration dependent. Diffusion profiles were found over different diffusion time intervals and the profiles were fit to a nonlinear Fickian diffusion model. Steady state 011012-7diffusion coefficients for BPGs were found to be 4.01 and 3.53 µm2 /s for 180 and 1600 kDa BPGs, respectfully, and these values are similar to other large molecule diffusion in cartilage. In both bovine and osteoarthritic human cartilage, BPGs were found localized around the chondrocytes. BPG localization was examined by labeling collagen type VI and soaking 5 µm thick sections of cartilage with BPG solutions demonstrating that the BPGs diffused into the cartilage and preferentially localized alongside collagen type VI in the pericellular matrix.

Two compartment pharmacokinetic model describes the intra-articular delivery and retention of rhprg4 following ACL transection in the Yucatan mini pig.

Treatment of the injured joint with rhPRG4 is based on recent observations that inflammation diminishes expression of native PRG4. Re-establishing lubrication between pressurized and sliding cartilage surfaces during locomotion promotes the nascent expression of PRG4 and thus intra-articular (IA) treatment strategies should be supported by pharmacokinetic evidence establishing the residence time of rhPRG4. A total of 21 Yucatan minipigs weighing ∼55 kg each received 4 mg of 131 I-rhPRG4 delivered by IA injection 5 days following surgical ACL transection. Animals were sequentially euthanized following IA rhPRG4 at 10 min (time zero), 24, 72 h, 6, 13 and 20 days later. The decay of the 131 I-rhPRG4 was measured relative to a non-injected aliquot and normalized to the weight of cartilage samples, menisci and synovium, and known cartilage volumes from each compartment surface obtained from representative Yucatan minipig knees. Decay of 131 I-rhPRG4 from joint tissues best fit a two-compartment model with an α half-life (t1/2α ) of 11.28 h and ß half-life (t1/2ß ) of 4.81 days. The tibial and femoral cartilage, meniscii, and synovium retained 7.7% of dose at 24 h. High concentrations of rhPRG4 were found in synovial fluid (SF) that was non-aspiratable and resided on the articular surfaces, removable by irrigation, at 10 min following 131 I-rhPRG4 injection. Synovial fluid K21 exceeded K12 and SF t1/2ß was 28 days indicating SF is the reservoir for rhPRG4 following IA injection. Clinical Significance: rhPRG4 following IA delivery in a traumatized joint populates articular surfaces for a considerable period and may promote the native expression of PRG4. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:386-396, 2019.

Endocan, sepsis, pneumonia, and acute respiratory distress syndrome.

Acute respiratory distress syndrome (ARDS) and hospital-acquired pneumonia (HAP) are major problems of public health in intensive care units (ICUs), occurring in 15% of critically ill patients. Among the factors explaining ARDS development, sepsis is known as a frequent cause. Sepsis, ARDS, and HAP increase morbidity, mortality, length of stay in the ICU, and the overall costs of healthcare. The major challenge remains to identify accurately among critically ill patients those at risk of poor outcomes who could benefit from novel therapies. Endocan is released by the pulmonary endothelium in response to local or systemic injury. It inhibits mainly leukocyte diapedesis rather than leukocyte rolling or adhesion to the endothelial cells both in vitro and in vivo. Endocan was evaluated in 25 clinical reports, including 2454 critically ill patients and 452 healthy controls. The diagnostic value of endocan for sepsis or sepsis severity was equal to procalcitonin but its prognostic value was better. A predictive value for postoperative pneumonia was evidenced in two studies, and a predictive value for ARDS in four studies from three independent centers. This review presents an overview of the structure, expression, and functions of endocan. We also hereby summarize the potential applications of endocan in the prediction and prognosis of ARDS and HAP, as well as in the prognosis of sepsis.

SEMA3A suspended in matrigel improves titanium implant fixation in ovariectomized rats.

The aim of this study was to evaluate the effect of SEMA3A released from matrigel on implant fixation in ovariectomized (OVX) rats. Sixty female rats were subjected to bilateral ovariectomy. Twelve weeks later, rats were randomly divided into three groups according to implants they accepted: (1) Control, implants with distilled water; (2) Matrigel, implants with matrigel coating; (3) Matrigel + SEMA3A, implants with coating of SEMA3A suspended in matrigel. Implants were inserted in metaphysis of proximal tibiae in all animas bilaterally. In vitro release of SEMA3A was tested using enzyme linked immunosorbent assay. In vitro release of SEMA3A was detectable during the first 10 days, and a burst release of was observed during the first 3 days. No significant difference was observed between Control and Matrigel group. The protective effects of SEMA3A in matrigel on peri-implant bone, implant osseointegration and fixation was confirmed. Compared to matrigel alone, SEMA3A suspended in matrigel increased percent bone volume by 88.7% and 83.3% (p < 0.01), bone-to-implant contact ratio by 148.9% (p < 0.01), and 24.8% (p < 0.05), the maximal push-out force by 149.3% and 209.2% (p < 0.01) at 4 and 8 weeks after implant insertion, respectively. Surface modification with SEMA3A suspended in matrigel improved implant osseointegration and fixation in the proximal tibiae of OVX rats. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 2060-2065, 2017.

Dual antiplatelet and anticoagulant APAC prevents experimental ischemia-reperfusion-induced acute kidney injury.

BACKGROUND: Renal ischemia-reperfusion predisposes to acute kidney injury (AKI) and mortality. APAC, mast cell heparin proteoglycan mimetic is a potent dual antiplatelet and anticoagulant inhibiting thrombosis in several vascular models. METHODS: Clinically relevant (0.06 and 0.13 mg/kg) and high (0.32 and 7.3 mg/kg) heparin doses of APAC and unfractionated heparin (UFH) were administered i.v. in pharmacological studies. Antithrombotic action of APAC and UFH was assessed with platelet aggregation to collagen, activated partial thromboplastin (APTT) and prothrombin (PT) times. Pharmacodynamics of [64Cu]-APAC or -UFH were monitored by PET/CT. Next, APAC and UFH doses (0.06 and 0.13 mg/kg) were i.v. administered 10 min prior to renal ischemia-reperfusion injury (IRI) in rats. RESULTS: APAC in contrast to UFH inhibited platelet aggregation. During 0.06 and 0.13 mg/kg dose regimens APTT and PT remained at baseline, but at the high APTT prolonged fourfold to sixfold. Overall bio-distribution and clearance of APAC and UFH were similar. After bilateral 30-min renal artery clamping, creatinine, urea nitrogen and neutrophil gelatinase-associated lipocalin concentrations and histopathology indicated faster renal recovery by APAC (0.13 mg/kg). APAC, unlike UFH, prevented expression of innate immune ligand hyaluronan and tubulointerstitial injury marker Kim-1. Moreover, in severe bilateral 1-h renal artery clamping, APAC (0.13 mg/kg) prevented AKI, as demonstrated both by biomarkers and survival. Compatible with kidney protection APAC reduced the circulating levels of vascular destabilizing and pro-inflammatory angiopoietin-2 and syndecan-1. No tissue bleeding ensued. CONCLUSION: APAC and UFH were similarly eliminated via kidneys and liver. In contrast to UFH, APAC (0.13 mg/kg) was reno-protective in moderate and even severe IRI by attenuating vascular injury and innate immune activation.

Controllable production of transplantable adult human high-passage dermal papilla spheroids using 3D matrigel culture.

We have succeeded in culturing human dermal papilla (DP) cell spheroids and developed a three-dimensional (3D) Matrigel (basement membrane matrix) culture technique that can enhance and restore DP cells unique characteristics in vitro. When 1 × 10(4) DP cells were cultured on the 96-well plates precoated with Matrigel for 5 days, both passage 2 and passage 8 DP cells formed spheroidal microtissues with a diameter of 150-250 µm in an aggregative and proliferative manner. We transferred and recultured these DP spheroids onto commercial plates. Cells within DP spheres could disaggregate and migrate out, which was similar to primary DP. Moreover, we examined the expression of several genes and proteins associated with hair follicle inductivity of DP cells, such as NCAM, Versican, and α-smooth muscle actin, and confirmed that their expression level was elevated in the spheres compared with the dissociated DP cells. To examine the hair-inducing ability of DP spheres, hair germinal matrix cells (HGMCs) and DP spheres were mixed and cultured on Matrigel. Unlike the dissociated DP cells and HGMCs cocultured in two dimensions, HGMCs can differentiate into hair-like fibers under the induction of the DP spheres made from the high-passage cells (passage 8) in vitro. We are the first to show that passage 3 human HGMCs differentiate into hair-like fibers in the presence of human DP spheroids. These results suggest that the 3D Matrigel culture technique is an ideal culture model for forming DP spheroids and that sphere formation partially models the intact DP, resulting in hair induction, even by high-passage DP cells.

In vivo mouse

The aim of this in vivo study was to evaluate the feasibility of ^{99m}Tc-labeled cartilage link protein (CLP) probe for the single-photon emission computed tomography (SPECT) of lung cancer. Xenograft mouse model were established from a luciferase expressing cell line derived from a human lung cancer. Bioluminescence imaging (BLI) was carried out prior to ^{99m}Tc-CLP and ^{99m}Tc-methoxyisobutyl isonitrile (MIBI) SPECT scans. The image quality of ^{99m}Tc-CLP scan was validated with BLI and compared with well established ^{99m}Tc-MIBI scan. Results of multimodal imaging analyses suggested that ^{99m}Tc-CLP was a sensitive and reliable SPECT agent for lung cancer imaging.

Bases teóricas para la propuesta de un ensayo clínicoaleatorizado, controlado y abierto, para evaluarla eficacia de la adición de bemiparina a la solución de icodextrina en pacientes en diálisis peritoneal con trastornos del transporte peritoneal[FRIAT-BEM-2005-01] / No disponible

Considerando todas las investigaciones realizadas hasta la fecha sobre las causas que contribuyen al deterioro de la membrana peritoneal y su fisiopatología, se puede concluir que es de gran interés investigar si la administración de heparinaintraperitoneal puede aportar un beneficio sobre el mantenimiento de la función peritoneal en los enfermos tratados con diálisis peritoneal (DP). Las acciones descritas a favor de esta idea son:1) La inflamación crónica del peritoneo es una causa de alteración de la función peritoneal y la heparina tiene acciónantiinflamatoria.2) La fibrosis peritoneal debida a diálisis peritoneal o a traumatismo puede ser evitada o mejorada con heparina ip.3) La heparina (HNF y HBPM, incluida bemiparina) indúcela síntesis de tPA por las células mesoteliales, lo que supone una acción fibrinolítica.4) La heparina, más la HBPM que la HNF, inhibe la angiogénesis.5) La heparina intraperitone al favorece la eliminación delos AGE en la DP.6) Modelos animales y estudios clínicos de corta extensión han demostrado una mejora de la función peritoneal con heparina.7) Por el momento, no se han encontrado problemas de seguridad en su administración intraperitoneal. Es por tanto una hipótesis verosímil que el uso de heparinaintraperitoneal puede modificar favorablemente la función peritoneal de pacientes en diálisis peritoneal (AU)

Multiple investigations performed on peritoneal pathophysiology during peritoneal dialysis (PD) suggest that intraperitonealheparin might modify most of the causes of membrane deterioration. The actions described favouring this idea are:1) Peritoneal Chronic inflammation alters peritoneal function and heprain has anti-inflammatory properties.2) Peritoneal fibrosis related to peritoneal dialysis or traumaticinjury may be avoided or limited with heparin.3) Heparine induces tPA síntesis by mesothelial cells, which represents a potentiation of fibrinolytic action.4) Heparine, sècifically low-molecular weight heparin, inhibitsangiogenesis.5) Intraperitoneal heparin favors the removal of advanced glycosilation end products in PD.6) Animal models and clinical studies with small series of patients have demonstrated an improvement of peritoneal function with intraperitoneal heparine use.7) Until now, no adverse effects of the intraperitoneal heparinuse have been found. In consequence, it is a plausible hipothesis to consider that intraperitonealheparin may favourably modify peritoneal function inpatients under peritoneal dialysis (AU)

Biomechanics of articular cartilage and determination of material properties.

Descriptions of the mechanical behaviors of articular cartilage and their correlations with collagen, proteoglycan, water, and ions are summarized, with particular emphasis on understanding the osmotic effect inside the tissue. First, a descriptive explanation is presented of the biphasic theory required to understand how interstitial water contributes toward the viscoelastic behavior of any hydrated soft tissue. Then, the famous osmotic effect in charged, hydrated soft tissue is interpreted in light of the triphasic mixture theory framework. In the introduction of mechanical testing methods, our emphasis is on the popular indentation technique, which can determine the material properties of cartilage in situ or in vivo. The widely accepted indentation analysis solutions in cartilage biomechanics history are summarized and evaluated. At the end of this paper, a new generalized correspondence principle between charged, hydrated soft tissue and linear, isotropic, elastic material (i.e., elasticity theory) is introduced. This principle makes the employment of triphasic theory as straightforward as using an elasticity theory to solve any equilibrium problem where the elasticity theory can be used to model the material. By using this generalized correspondence principle, the fixed charge density of bovine cartilage has been simply and conveniently calculated from the indentation testing data. The results of proteoglycan content from this mechanical test are remarkably consistent with those from standard biochemical assay. This new correspondence principle significantly improves the power of indentation tests in the determination of mechanoelectrochemical properties of articular cartilage.

Atherogenic remnant lipoproteins: role for proteoglycans in trapping, transferring, and internalizing.

Unraveling the mechanisms controlling remnant lipoprotein clearance is important, as these lipoproteins are highly atherogenic. The most critical molecule in this process is apoE, which mediates high-affinity binding of remnant lipoproteins to members of the LDL receptor (LDLR) family and cell-surface heparan sulfate proteoglycans (HSPGs), which have been shown to play major independent as well as cooperative roles in remnant lipoprotein clearance. While all the players may have been identified, our understanding of how they interact and function together continues to evolve. In this issue of the JCI, MacArthur et al. (see the related article beginning on page 153) demonstrated that HSPGs under normal physiological conditions are critically important in the clearance of remnant lipoproteins, independent of LDLR family members. The complexity of VLDL and chylomicron remnant clearance was exemplified by the studies of Jones et al., also in this issue (see the related article beginning on page 165). Despite defective clearance of LDL in mice with a deficiency in the adaptor protein controlling internalization of the LDLR, called autosomal recessive hypercholesterolemia (ARH), remnant lipoprotein clearance was not grossly abnormal. A likely explanation is that the abnormal LDLRs bind the remnants and then transfer them to another acceptor for internalization. While the studies clearly demonstrate that the LDLR-related protein 1 is not involved and suggest a role for an additional unidentified receptor, it remains a possibility that HSPGs are responsible for remnant uptake by hepatocytes in the presence of defective LDLR internalization.

G1 domain of aggrecan cointernalizes with hyaluronan via a CD44-mediated mechanism in bovine articular chondrocytes.

OBJECTIVE: To determine whether aggrecan fragments bound to hyaluronan (HA) can be retained and internalized by articular chondrocytes and whether these events are dependent on HA and its receptor, CD44. An additional objective was to determine whether partial degradation of aggrecan is a prerequisite for internalization. METHODS: Binding and internalization of a variety of fluorescein isothiocyanate (FITC)- or biotin-labeled HA/proteoglycan probes were investigated on normal bovine articular cartilage chondrocytes, bovine articular chondrocytes transfected with a dominant-negative construct of CD44, or COS-7 cells transfected with wild-type CD44. The probes were defined as being internalized by the presence of label associated with the cells following extensive trypsinization of the cell surface. RESULTS: Biotinylated aggrecan fragments bound to FITC-HA were cointernalized in bovine articular chondrocytes or COS-7 cells transfected with CD44. Intracellular vesicles containing FITC-HA colocalized with a fluorescent probe for lysosomes. The internalization of the aggrecan fragments was dependent on the presence of HA as well as the presence of functional CD44. Intact aggrecan/FITC-HA complexes bound to the cell surface but were not internalized. However, following brief trypsin digestion of the aggrecan/HA complex, the remaining proteoglycan fragments were bound and internalized. CONCLUSION: Partially degraded aggrecan fragments (e.g., aggrecan G1 domains bound to HA) can be internalized by articular chondrocytes via a mechanism involving HA/CD44-mediated endocytosis. Further, the presence of an intact aggrecan monomer bound to HA inhibits the internalization of HA as well as HA-bound fragments.

Serglycin secreted by leukocytes is efficiently eliminated from the circulation by sinusoidal scavenger endothelial cells in the liver.

This study was undertaken to determine the fate of the circulating chondroitin sulfate proteoglycan serglycin. The human monocytic cell line THP-1 was cultured under serum-free conditions in the presence of [35S]sulfate. The conditioned medium was harvested and 35S-macromolecules were purified by Q-Sepharose anion-exchange chromatography and Superose 6 gel chromatography. After labeling with 125I, the purified material was treated with chondroitinase ABC and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. A major band with mr of approximately 14 kDa appeared, consistent with the core protein of serglycin. The identity of the proteoglycan was confirmed by amino-terminal amino acid sequencing. Purified serglycin, labeled either with [35S]sulfate or 125I and fluorescein isothiocyanate, was injected intravenously into rats. The blood content of radiolabeled serglycin fell by 50% from 1 to 2.4 min after injection, indicating an initial t1/2 of 1.4 min or shorter. Approximately 90% of the recovered radioactivity was localized in the liver, 5% in the blood, and 5% altogether in urine, kidneys, and spleen about 30 min after injection. Isolation of liver cells at the same time point showed that 70% of the radioactivity was taken up by the sinusoidal scavenger endothelial cells, and 23 and 7% by the hepatocytes and Kupffer cells, respectively. When excess amounts of unlabeled hyaluronan was coinjected with radiolabeled serglycin, the elimination of serglycin was significantly inhibited, indicating that the hyaluronan receptor on the sinusoidal scavenger endothelial cells is responsible for the elimination of serglycin.

A beta and perlecan in rat brain: glial activation, gradual clearance and limited neurotoxicity.

A beta1-40 and perlecan (A beta + perlecan) were infused into rat hippocampus for 1 week via osmotic pumps. At the end of the infusion a deposit of A beta immunoreactive material was found surrounding the infusion site. No neurons could be identified within this A beta deposit. The neuron-free area resulting from A beta + perlecan was significantly larger than that found after infusions of A beta40-1 and perlecan (reverse A beta + perlecan), perlecan alone or phosphate-buffered saline vehicle. Following infusion of A beta + perlecan, the glial cells segregated in a manner similar to that associated with compacted amyloid plaques in Alzheimer's disease (AD). Activated microglia/macrophages were prevalent within the A beta deposit while the perimeter of the deposit was delimited by reactive astrocytes. Thioflavin S and Congo red staining indicated a beta-pleated sheet conformation of the A beta deposits, implying formation of fibrils. Intact, apparently healthy neurons were found immediately adjacent to the A beta + perlecan deposit. In contrast, reverse A beta peptide did not form congophilic deposits despite the presence of perlecan. Apoptotic profiles visualized with bisbenzamide or TUNEL staining of fragmented DNA were not seen at any of the infusion sites, yet were readily seen in hippocampal sections from animals treated with kainic acid. At 8 weeks, A beta immunoreactivity, Thioflavin S and Congo red staining was reduced, indicating that A beta was being cleared. There also was no evidence of neuron loss by Nissl or TUNEL staining. The zone of apparent necrosis did not expand between 1 and 8 weeks, and in some instances appeared to contract. The consistency of the A beta + perlecan infusion method in producing reliable A beta amyloid deposits permits estimates of the rate at which fibrillar A beta amyloid can be removed from the brain, and may provide a useful model to study this process in vivo. However, the absence of clearly identifiable degenerating/dying neurons at the 1 or 8 week survival times suggests that either fibrillar A beta + perlecan slowly displaced the brain parenchyma during infusion, or neurons were killed very gradually during the process of clearing the A beta.

Pharmacokinetics and disposition characteristics of recombinant decorin after intravenous injection into mice.

The pharmacokinetics and disposition characteristics of recombinant decorin after intravenous administration were investigated in mice. Following bolus injection of 111In-labeled decorin at doses of 0.02 and 0.1 mg/kg, radioactivity rapidly disappeared from the circulation and approximately 70% of the dose accumulated in liver within 10 min. 111In-labeled decorin was preferentially localized in hepatic nonparenchymal cells. At a higher dose of 1 mg/kg, clearance from the circulation and hepatic uptake of [111In]decorin were slower than at lower doses. Both the accumulation in other tissues and urinary excretion of [111In]decorin were 5% or less. Pharmacokinetic analysis demonstrated that hepatic uptake clearance was large and accounted almost completely for total body clearance; in addition the clearance values decreased as the dose increased, suggesting that the hepatic uptake of decorin is mediated by a specific mechanism which becomes saturated at higher doses. In competitive inhibition experiments, hepatic uptake of 111In-labeled decorin was partially inhibited (about 20-30%) by several sulfated glycans such as glycosaminoglycans and dextran sulfate and by mannosylated bovine serum albumin (BSA), mannan and mannose to a lesser extent (about 10%). On the other hand, polyinosinic acid, polycytidylic acid and succinylated BSA were ineffective, suggesting that the scavenger receptor for polyanions in the liver is not involved in the hepatic uptake of decorin. A basic protein, protamine, and a ligand of the apoE receptor, lactoferrin, also had no effect. Taken together, the present results have demonstrated that recombinant decorin is rapidly eliminated from the blood circulation through extensive uptake by the liver, primarily by the nonparenchymal cells, following systemic administration. The sugar structure and mannose residue in decorin have also been suggested to play an important role in the hepatic uptake of decorin. These findings provide useful information for the development of decorin as a therapeutic agent.

Variation in proteoglycan metabolism by articular chondrocytes in different joint regions is determined by post-natal mechanical loading.

In this study we investigated the hypothesis that cartilage from defined regions of ovine stifle joints, which were subjected to differing mechanical stresses, contained phenotypically distinct chondrocyte populations. Chondrocyte phenotypes were identified by the relative biosynthesis of the proteoglycans (PGs) aggrecan, biglycan and decorin. Articular cartilage (AC) from adult and neonatal ovine stifle joints were examined. Cells were cultured as both full-depth AC explants and in alginate beads after their isolation from the AC matrix. When chondrocytes from the various topographical regions of adult ovine knee joints were cultured as explants they demonstrated a consistent difference with regard to the metabolism of aggrecan and decorin. Significantly, this topographically-dependent phenotypic expression of PGs was preserved when the chondrocytes were cultured in alginate beads. In adult joints, chondrocytes from the central region of the tibial plateau not covered by the meniscus, which is subjected to high mechanical loads in-vivo, synthesized less aggrecan but more decorin than cells from regions covered by the meniscus. When chondrocytes from identical AC regions of neonatal ovine joints were cultured as explants, no topographical difference in aggrecan nor decorin metabolism could be detected. The results of this study, in association with the existing literature, lead us to propose that post-natal mechanical loading of AC could select for chondrocyte clones or induce a lasting modulation of chondrocyte phenotypic expression in different joint regions. Such cellular changes could result in the synthesis of PG populations that confer properties to AC most suited to resist the variable mechanical stresses in the different joint regions. This study serves to emphasize the importance of using cartilage from identical joint areas when examining PG metabolism by chondrocytes. Further investigation into the relationship between mechanical loading, regional chondrocyte phenotype selection and the response of these cells to anabolic and catabolic factors may provide important insights into the focal nature of AC degeneration in osteoarthritis.

Matrigel: a useful tool to study endothelial differentiation.

Matrigel is a matrix of a mouse basement membrane neoplasm. It represents a complex mixture of basement membrane proteins including laminin, type IV collagen, entactin/nitrogen and proteoheparan sulfate, but it also contains growth factors. Matrigel induces endothelial cells to differentiate as evidenced by both the morphologic changes and by the reduction in proliferation and, therefore, offers a convenient model to study biochemical and molecular events associated with angiogenesis. Further, Matrigel permits to study the roles of the extracellular matrix in angiogenesis.

Insulin-like growth factor stimulation of articular chondrocyte proteoglycan synthesis. Availability and responses at different ages.

It was found that recovery of articular chondrocyte proteoglycan (PG) synthesis was retarded in old mice after in vivo exposure to both IL-1 or hydrogen peroxide. We examined whether this could be related to diminished serum levels of insulin-like growth factor 1 (IGF-1), the main anabolic factor, or to changes in cartilage IGF responsiveness with age. A small decline of IGF-1 concentration was observed in serum of old mice, but the level still appeared to be supra-optimal to maintain normal cartilage PG synthesis over a culture period of 1 to 3 days. Moreover, PG synthesis was at least equally stimulated in patellar cartilage from 18-month-old mice compared to 3-month-old mice over a wide range of IGF-1 concentrations, and similar findings were obtained after stimulation with serum. In addition, we studied the capacity of IGF-1 or serum to induce recovery of PG synthesis in vitro after IL-1 exposure in vivo. In a 3-day culture period normal cartilage PG synthesis was stimulated to the same extent with serum or IGF-1, but recovery from IL-1 mediated suppression of PG synthesis was more pronounced with serum. This latter capacity was similar for serum of mice aged 3 or 18 months and was noted for both young and old cartilage. Our data show that retarded recovery of chondrocyte PG synthesis in old mice cannot be explained by age-related changes in IGF-1 availability and cartilage responses to IGF. They also indicate that serum factors other than IGF-1 are important for recovery, either alone or in combination with IGF-1.