Friction reducing ability of a poly-l-lysine and dopamine modified hyaluronan coating for polycaprolactone cartilage resurfacing implants.

Frictional properties of cartilage resurfacing implants should be sufficiently low to limit damaging of the opposing cartilage during articulation. The present study determines if native lubricious molecule proteoglycan 4 (PRG4) can adsorb onto a layer-by-layer bioinspired coating composed of poly-l-lysine (PLL) and dopamine modified hyaluronic acid (HADN) and thereby can reduce the friction between implant and articular cartilage. An ELISA was developed to quantify the amount of immobilized human recombinant (rh)PRG4 after exposure to the PLL-HADN coating. The effect on lubrication was evaluated by comparing the coefficient of friction (CoF) of bare polycaprolactone (PCL) disks to that of PLL-HADN coated PCL disks while articulated against cartilage using a ring-on-disk geometry and a lubricant solution consisting of native synovial fluid components including rhPRG4. The PLL-HADN coating effectively immobilized rhPRG4. The surface roughness of PCL disks significantly increased while the water contact angle significantly decreased after application of the coating. The average CoF measured during the first minute of bare PCL against cartilage exceeded twice the CoF of the PLL-HADN coated PCL against cartilage. After 60 min, the CoF reached equilibrium values which were still significantly higher for bare PCL compared to coated PCL. The present study demonstrated that PCL can effectively be coated with PLL-HADN. Additionally, this coating reduces the friction between PCL and cartilage when a PRG4-rich lubricant is used, similar to the lubricating surface of native cartilage. This makes PLL-HADN coating a promising application to improve the clinical success of PCL-based cartilage resurfacing implants.

Synovial and cartilage responsiveness to peri-operative hyaluronic acid ± dexamethasone administration following a limited injury to the rabbit stifle joint.

Posttraumatic osteoarthritis (PTOA) can develop after an injury to the knee. Previous studies have indicated that an intra-articular (IA) injection of the potent glucocorticoid dexamethasone (DEX) may significantly prevent induction of PTOA. The aim of the present study was to investigate the effectiveness of a single IA injection of hyaluronic acid (HA), alone and in combination with DEX following a localized intra-articular injury as a PTOA-preventing treatment option. An established rabbit model of surgical injury consisting of dual intra-articular (IA) drill holes in a non-cartilaginous area of the femoral notch near the origin of the anterior cruciate ligament (ACL) to allow for bleeding into the joint space was used. Immediately following surgery, subjects were treated with HA, HA + DEX, or received no treatment. An uninjured control group was used for comparison (N = 5/group). Rabbits were sacrificed and investigated at 9 weeks post-injury. At 9 weeks post-injury, there was a significant protective capacity of the single IA treatment of DEX + HA on the histological grade of the synovial tissue, and some variable location-specific effects of HA alone and HA + DEX interactions on cartilage damage. Thus, it is possible that co-treatment with HA may interfere with the effectiveness of the DEX. In vitro friction testing indicated that DEX did not interfere with the lubricating ability of HA or synovial fluid on cartilage. These results suggest that a single IA administration of HA in combination with DEX following an IA injury is not recommended for inhibition of PTOA progression in this model.

Proteoglycan 4 reduces friction more than other synovial fluid components for both cartilage-cartilage and cartilage-metal articulation.

OBJECTIVE: The clinical success of focal metallic resurfacing implants depends largely on the friction between implant and opposing cartilage. Therefore, the present study determines the lubricating ability of the synovial fluid components hyaluronic acid (HA), proteoglycan 4 (PRG4) and a surface-active phospholipid (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, POPC), on the articulation between cartilage and a Cobalt Chromium Molybdenum (CoCrMo) implant surface, compared with two cartilage surfaces. METHODS: A ring-on-disk geometry was used to perform repeated friction measurements at physiologically relevant velocities (6 and 60 mm/s) using lubricants with an increasing number of components present. Shear measurements were performed in order to evaluate the viscosity. To ensure that it is clinically relevant to explore the effect of these components, the presence of PRG4 in synovial fluid obtained from primary and revision knee and hip implant surgeries was examined. RESULTS: PRG4 in the presence of HA was found to significantly reduce the coefficient of friction for both cartilage-cartilage and cartilage-CoCrMo interface. This is relevant, as it was also demonstrated that PRG4 is still present at the time of revision surgeries. The addition of POPC had no effect for either configurations. HA increased the viscosity of the lubricating fluid by one order of magnitude, while PRG4 and POPC had no effect. CONCLUSION: The present study demonstrates the importance of selecting the appropriate lubrication solution to evaluate implant materials with biotribology tests. Because PRG4 is a key component for reducing friction between cartilage and an opposing surface, developing coatings which bind PRG4 is recommended for cartilage resurfacing implants.

YAP/TAZ regulates the expression of proteoglycan 4 and tenascin C in superficial-zone chondrocytes.

The roles of cell division control protein 42 homologue (CDC42) and actin polymerisation in regulating the phenotype of superficial-zone chondrocytes (SZCs) have been demonstrated in vitro; however, the signalling pathway(s) downstream have yet to be fully elucidated. The study hypothesis was that Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ) act downstream to regulate proteoglycan 4 (PRG4) and tenascin C (TNC). Bovine SZCs grown in monolayer were treated with ML141 (CDC42 inhibitor) or the actin depolymerising agents, latrunculin B and cytochalasin D, to determine the effect on YAP/TAZ. Verteporfin (YAP/TAZ inhibitor) and YAP/TAZ siRNA-mediated knockdown were used to determine their role in regulating PRG4 and TNC. ML141 treatment reduced total YAP/TAZ protein, nuclear TAZ levels and the YAP/TAZ target gene, connective tissue growth factor (CTGF) mRNA levels. Latrunculin B decreased nuclear TAZ, while cytochalasin D treatment trended towards increased nuclear TAZ (p = 0.06), correlating with decreased and increased CTGF mRNA levels, respectively. Verteporfin treatment decreased PRG4 and TNC expression, with no effect on actin polymerisation. siRNA-mediated knockdown of YAP/TAZ revealed that PRG4 was regulated by YAP/TAZ while TNC was regulated by TAZ only. As cytochalasin D can activate myocardin-related transcription factor-A (MRTF-A), siRNA-mediated knockdown was performed to determine the role of MRTF-A in regulating YAP/TAZ. Although nuclear TAZ decreased, no significant changes in total protein levels were observed. Findings suggested that CDC42 and actin polymerisation regulated SZCs through multiple actin-regulated pathways. Understanding the regulation of these chondroprotective molecules may have important implications for prevention/treatment of osteoarthritis.

Cellular electrophysiological principles that modulate secretion from synovial fibroblasts.

Rheumatoid arthritis (RA) is a progressive disease that affects both pediatric and adult populations. The cellular basis for RA has been investigated extensively using animal models, human tissues and isolated cells in culture. However, many aspects of its aetiology and molecular mechanisms remain unknown. Some of the electrophysiological principles that regulate secretion of essential lubricants (hyaluronan and lubricin) and cytokines from synovial fibroblasts have been identified. Data sets describing the main types of ion channels that are expressed in human synovial fibroblast preparations have begun to provide important new insights into the interplay among: (i) ion fluxes, (ii) Ca2+ release from the endoplasmic reticulum, (iii) intercellular coupling, and (iv) both transient and longer duration changes in synovial fibroblast membrane potential. A combination of this information, knowledge of similar patterns of responses in cells that regulate the immune system, and the availability of adult human synovial fibroblasts are likely to provide new pathophysiological insights.

Rheological effects of macromolecular interactions in synovial fluid.

The rheological properties of synovial fluid (SF) are largely attributed to the presence of high molecular weight hyaluronan (HA). However, rheological differences between SF and pure HA solutions suggest that SF proteins actively contribute towards the bulk viscoelasticity of this biological fluid. Due to various experimental challenges involved with the rheometry of low-viscosity biological fluids, the macromolecular interactions in SF and their relative rheological importance are still a matter of active discussion. Interestingly however, recent evidence suggests that the concentration and structure of proteoglycan 4 (PRG4, also known as lubricin) can directly modulate the viscoelastic properties of HA-PRG4 solutions. The objective of this review is to highlight recent rheological studies that examine the macromolecular interactions between HA and proteins in SF. First, a general overview of the chemical composition of SF and the molecular structure of its key constituents HA and PRG4 is provided. Subsequently, diverse rheological experimental techniques that have been developed to characterize HA solutions are discussed. Finally, rheological investigations of macromolecular interactions between HA, serum proteins, and PRG4 are examined. This review illustrates how diverse rheological techniques can expand our understanding of the composition-structure-function relationships in SF.

Lubricin/Proteoglycan 4 binds to and regulates the activity of Toll-Like Receptors In Vitro.

Proteoglycan 4 (PRG4/lubricin) is secreted by cells that reside in articular cartilage and line the synovial joint. Lubricin may play a role in modulating inflammatory responses through interaction with CD44. This led us to examine if lubricin could be playing a larger role in the modulation of inflammation/immunity through interaction with Toll-like receptors (TLRs). Human Embryonic Kidney (HEK) cells overexpressing TLRs 2, 4 or 5 and surface plasmon resonance were employed to determine if full length recombinant human lubricin was able to bind to and activate TLRs. Primary human synovial fibroblasts were also examined using flow cytometry and Luminex multiplex ELISA. A rat destabilization model of osteoarthritis (OA) was used to determine if lubricin injections were able to regulate pain and/or inflammation in vivo. Lubricin can bind to and regulate the activity of TLRs, leading to downstream changes in inflammatory signalling independent of HA. We confirmed these findings in vivo through intra-articular injections of lubricin in a rat OA model where the inhibition of systemic inflammatory signaling and reduction in pain were observed. Lubricin plays an important role in regulating the inflammatory environment under both homeostatic and tissue injury states.

[Memorandum on sustainable reinforcement of prevention and health promotion: challenges at the federal, state and local level]. / Memorandum--Prävention und Gesundheitsförderung nachhaltig stärken: Herausforderungen auf Bundes-, Landes- und kommunaler Ebene.

Research-based evidence and practice-based experience are core requirements for the effective implementation of preventive interventions. The knowledge gained in the Prevention Research Funding Initiative of the German Federal Ministry of Education and Research (2004-2013) was therefore amalgamated, reflected and consolidated in the Cooperation for Sustainable Prevention Research (KNP) meta-project. In annual strategy meetings, researchers and practitioners from the field and other experts developed 3 memoranda providing recommendations for the further development of research and practice in the field of prevention and health promotion. Memorandum III is primarily aimed at decision-makers in politics and administration at the federal, state and local level, in civil society and in the workplace. Its recommendations show that structuring efforts are urgently needed to achieve sustainable policy, particularly in the fields of health, education, employment and social affairs. Memorandum III brings together the knowledge extracted and problems identified in research projects. More so than its 2 predecessors, Memorandum III abstracts knowledge from the individual projects and attempts to derive guidance for action and decision-making, as shown by the 7 recommendations that appear to useful for consensus-building in practice and research. Value judgments are inevitable. Prevention and health promotion are an investment in the future: of social health, social capital and social peace. Improvement of the framework conditions is needed to achieve the harmonized awareness and the sustained effectiveness of these structure-building efforts in different policy areas, spheres of life, fields of action, and groups of actors. This includes the implementation of an overall national strategy as well as the expansion of sources of funding, extension of the legal framework, overarching coordination, and the establishment of a National Center of Excellence to develop and safeguard prevention and health promotion. The memorandum is intended to stimulate a discourse resulting in structure-building and stabilizing measures designed to ensure the sustainability of prevention and health promotion.

Cartilage boundary lubrication of ovine synovial fluid following anterior cruciate ligament transection: a longitudinal study.

OBJECTIVE: To assess ovine synovial fluid (oSF) from different post-injury time points for (1) proteoglycan-4 (PRG4) and hyaluronan (HA) concentration, (2) HA molecular weight (MW) distribution, (3) cartilage boundary lubrication function, and (4) lubricant composition-function relationships. The association between cartilage boundary lubrication and gross cartilage changes after injury was also examined. METHODS: oSF was collected 2, 4, 10, and 20 weeks post anterior cruciate ligament (ACL) transection in five skeletally mature sheep. PRG4 and HA concentrations were measured using sandwich enzyme-linked immunosorbent assay, and HA MW distribution by agarose gel electrophoresis. Cartilage boundary lubrication of oSF was assessed using a cartilage-cartilage friction test. Gross damage to articular cartilage was also quantified at 20 weeks using modified Drez scoring protocol. RESULTS: Early (2-4 weeks) after ACL injury, PRG4 concentrations were significantly higher (P = 0.045, P = 0.037), and HA concentrations were substantially lower (P = 0.005, P = 0.005) compared to 20 weeks. The HA MW distribution also shifted towards lower ranges in the early post-injury stage. The kinetic friction coefficients were significantly higher 2-4 weeks post injury (P = 0.008 and P = 0.049) compared to 20 weeks. Poor cartilage boundary lubricating ability early after injury was associated with cartilage damage at 20 weeks. CONCLUSION: Altered composition and diminished boundary lubrication of oSF early after ACL transection may pre-dispose the articular cartilage to degenerative changes and initiate osteoarthritis (OA). These observations also provide potential motivation for biotherapeutic interventions at earlier time points post injury.

The impact of early intra-articular administration of interleukin-1 receptor antagonist on lubricin metabolism and cartilage degeneration in an anterior cruciate ligament transection model.

OBJECTIVE: Study the impact of intra-articular interleukin-1 receptor antagonist (IL-1 ra) treatment on lubricin biosynthesis following anterior cruciate ligament transection (ACLT) in the rat and evaluate the effect of combined IL-1 ra and recombinant human lubricin (rhPRG4) treatments on chondrocyte apoptosis. METHODS: ACLT was performed in male Lewis rats. Treatments included IL-1 ra or vehicle (n = 36 in each group). IL-1 ra intra-articular dosing was performed on days 1, 3, 5 and 7 following ACLT using Anakinra (150 mg/ml; 40 µl). At 3 and 5 weeks, animals were sacrificed and RNA was isolated. Histological analyses included Safranin O and H&E. Lubricin synovial fluid (SF) lavage concentrations were determined at 5 weeks. ACLT animals were treated with a single injection of vehicle, IL-1 ra (75 mg/ml; 40 µl), rhPRG4 (200 µg/ml; 40 µl), or IL-1 ra + rhPRG4 (75 mg/ml + 200 µg/ml; 40 µl) (n = 6 in each group) on day 7 following ACLT and cartilage was probed for cleaved caspase-3 at 5 weeks. RESULTS: IL-1 ra treatment improved lubricin expression (P < 0.001) and lubricin SF lavage concentrations in the IL-1 ra group was higher (P = 0.005) than the vehicle. IL-1 ra treatment reduced cartilage and synovial scores (P < 0.001) compared to vehicle. IL-1 ra and rhPRG4 acted synergistically to reduce caspase-3 positive chondrocytes (P < 0.001) compared to individual treatments. CONCLUSION: IL-1 ra treatment preserved lubricin following ACLT and a combined treatment of IL-1 ra + rhPRG4 may act synergistically to reduce cartilage catabolism.

Molecular weight characterization of PRG4 proteins using multi-angle laser light scattering (MALLS).

OBJECTIVES: Alternative splicing and variable post-translational modifications result in proteoglycan 4 (PRG4) proteins with historically reported apparent molecular weights (Ma) ranging from 150 to 400 kDa. The objectives of this study were to (1) identify and determine the weight averaged molecular weights (M(W)'s) of PRG4 proteins purified from medium with transforming growth factor-beta 1 (TGF-ß1) conditioned by mature bovine articular cartilage explants and (2) to examine the effect of reduction and alkylation (RA) on PRG4. METHODS: Non-reduced (NR) and RA preparations of PRG4 were separated using high performance liquid chromatography-size-exclusion chromatography with an in-line multi-angle laser light scattering (MALLS) detector, which was used for absolute determination of PRG4 M(W). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), immunoblotting, and tandem mass spectrometry (MS/MS) analysis were used to confirm the identity of separated proteins. RESULTS: Three putative PRG4 monomers, one with previously uncharacterized M(W), were identified in NR and RA PRG4 preparations of 239 (223,255), 379 (369,389), and 467 (433,501) kDa. Additionally ∼1 MDa putative PRG4 dimer was identified. Release of a ∼90 kDa PRG4 fragment was also observed on SDS-PAGE after RA. Western Blotting with anti-PRG4 antibodies detected immunoreactive bands with Ma similar to M(W) for all species and excised bands were confirmed to be PRG4 by MS/MS. CONCLUSIONS: A variety of monomeric PRG4 proteins and a disulfide-bonded dimer/multimer are secreted by chondrocytes in bovine cartilage explants. The observed decrease in M(W)'s of monomeric PRG4 species upon RA may be due to the release of post-translationally cleaved fragments. Further study of these species will provide insight into the PRG4 molecular structure and function relationship.

The effect of molecular weight on hyaluronan's cartilage boundary lubricating ability--alone and in combination with proteoglycan 4.

OBJECTIVES: (1) assess the molecular weight dependence of hyaluronan's (HA) cartilage boundary lubricating ability, alone and in combination with proteoglycan 4 (PRG4), at physiological concentrations; (2) determine if HA and PRG4 interact in solution via electrophoretic mobility shift assay (EMSA). METHODS: The cartilage boundary lubricating ability of a broad range of MW HA (20 kDa, 132 kDa, 780 kDa, 1.5 MDa, and 5 MDa) at 3.33 mg/ml, both alone and in combination with PRG4 at 450 µg/ml, was assessed using a previously described cartilage-on-cartilage friction test. Static, µ(static, Neq), and kinetic, <µ(kinetic, Neq)>, were calculated. An EMSA was conducted with PRG4 and monodisperse 150 kDa and 1,000 kDa HA. RESULTS: Friction coefficients were reduced by HA, in a MW-dependent manner. Values of <µ(kinetic, Neq)> in 20 kDa HA, 0.098 (0.089, 0.108), were significantly greater compared to both 780 kDa, 0.080 (0.072, 0.088), and 5 MDa, 0.079 (0.070, 0.089). Linear regression showed a significant correlation between both µ(static, Neq) and <µ(kinetic, Neq)>, and log HA MW. Friction coefficients were also reduced by PRG4, and with subsequent addition of HA; however the synergistic effect was not dependent on HA MW. Values of <µ(kinetic, Neq)> in PRG4, 0.080 (0.047, 0.113), were significantly greater than values of PRG4+various MW HA (similar in value, averaging 0.040 (0.033, 0.047)). EMSA indicated that migration of 150 kDa and 1,000 kDa HA was retarded when combined with PRG4 at high PRG4:HA ratios. CONCLUSIONS: These results suggest alterations in HA MW could significantly affect synovial fluid's cartilage boundary lubricating ability, yet this diminishment in function could be circumvented by physiological levels of PRG4 forming a complex, potentially in solution, with HA.

Differential regulation of proteoglycan 4 metabolism in cartilage by IL-1alpha, IGF-I, and TGF-beta1.

OBJECTIVES: To determine (1) if interleukin-1 alpha (IL-1alpha), insulin like growth factor I (IGF-I), and transforming growth factor-beta 1 (TGF-beta1) regulate proteoglycan 4 (PRG4) metabolism in articular cartilage, in terms of chondrocytes expressing PRG4 and PRG4 bound at the articular surface, and (2) if these features of cartilage PRG4 metabolism correlate with its secretion. METHODS: Articular cartilage explants were harvested and cultured for 6 days with or without 10% fetal bovine serum (FBS), alone, or with the addition of 10ng/ml IL-1alpha, 300ng/ml IGF-I, or 10ng/ml TGF-beta1. PRG4 expression by chondrocytes in the cartilage disks was assessed by immunohistochemistry (IHC). PRG4 bound to the articular surface of disks was quantified by extraction and enzyme-linked immunosorbent assay (ELISA). PRG4 secreted into culture medium was quantified by ELISA and characterized by Western Blot. RESULTS: PRG4 expression by chondrocytes near the articular surface was markedly decreased by IL-1alpha, stimulated by TGF-beta1, and not affected by IGF-I. The level of PRG4 accumulation in the culture medium was correlated with the number of chondrocytes expressing PRG4. The amount of PRG4 bound at the articular surface was modulated by incubation in medium including FBS, but did not correlate with levels of PRG4 secretion. CONCLUSIONS: Cartilage secretion of PRG4 is highly regulated by certain cytokines and growth factors, in part through alteration of the number of PRG4-secreting chondrocytes near the articular surface. The biochemical milieu may regulate the PRG4 content of synovial fluid during cartilage injury or repair.

A model of synovial fluid lubricant composition in normal and injured joints.

The synovial fluid (SF) of joints normally functions as a biological lubricant, providing low-friction and low-wear properties to articulating cartilage surfaces through the putative contributions of proteoglycan 4 (PRG4), hyaluronic acid (HA), and surface active phospholipids (SAPL). These lubricants are secreted by chondrocytes in articular cartilage and synoviocytes in synovium, and concentrated in the synovial space by the semi-permeable synovial lining. A deficiency in this lubricating system may contribute to the erosion of articulating cartilage surfaces in conditions of arthritis. A quantitative intercompartmental model was developed to predict in vivo SF lubricant concentration in the human knee joint. The model consists of a SF compartment that (a) is lined by cells of appropriate types, (b) is bound by a semi-permeable membrane, and (c) contains factors that regulate lubricant secretion. Lubricant concentration was predicted with different chemical regulators of chondrocyte and synoviocyte secretion, and also with therapeutic interventions of joint lavage and HA injection. The model predicted steady-state lubricant concentrations that were within physiologically observed ranges, and which were markedly altered with chemical regulation. The model also predicted that when starting from a zero lubricant concentration after joint lavage, PRG4 reaches steady-state concentration approximately 10-40 times faster than HA. Additionally, analysis of the clearance rate of HA after therapeutic injection into SF predicted that the majority of HA leaves the joint after approximately 1-2 days. This quantitative intercompartmental model allows integration of biophysical processes to identify both environmental factors and clinical therapies that affect SF lubricant composition in whole joints.

Effect of synovial fluid on boundary lubrication of articular cartilage.

OBJECTIVES: The lubrication of articulating cartilage surfaces in joints occurs through several distinct modes. In the boundary mode of lubrication, load is supported by surface-to-surface contact, a feature that makes this mode particularly important for maintenance of the normally pristine articular surface. A boundary mode of lubrication is indicated by a kinetic friction coefficient being invariant with factors that influence formation of a fluid film, including sliding velocity and axial load. The objectives of this study were to (1) implement and extend an in vitro articular cartilage-on-cartilage lubrication test to elucidate the dependence of the friction properties on sliding velocity, axial load, and time, and establish conditions where a boundary mode of lubrication is dominant, and (2) determine the effects of synovial fluid (SF) on boundary lubrication using this test. METHODS: Fresh bovine osteochondral samples were analyzed in an annulus-on-disk rotational configuration, maintaining apposed articular surfaces in contact, to determine static (mu(static) and mu(static),(N(eq)) and kinetic ([mu(kinetic)] and [mu(kinetic),(N(eq))]) friction coefficients, each normalized to the instantaneous and equilibrium (N(eq)) normal loads, respectively. RESULTS: With increasing pre-sliding durations, mu(static) and mu(static),(N(eq)) were similar, and increased up to 0.43 +/- 0.03 in phosphate buffered saline (PBS) and 0.19 +/- 0.01 in SF, whereas [mu(kinetic)] and [mu(kinetic),(N(eq))] were steady. Over a range of sliding velocities of 0.1-1 mm/s and compression levels of 18% and 24%, [mu(kinetic)] was 0.072 +/- 0.010 in PBS and 0.014 +/- 0.003 in SF, and [mu(kinetic),(N(eq))] was 0.093 +/- 0.005 in PBS and 0.018 +/- 0.002 in SF. CONCLUSIONS: A boundary mode of lubrication was achieved in a cartilage-on-cartilage test configuration. SF functioned as an effective friction-lowering boundary lubricant for native articular cartilage surfaces.

Static and dynamic compression regulate cartilage metabolism of PRoteoGlycan 4 (PRG4).

The boundary lubrication function of articular cartilage is mediated in part by molecules at the articular surface and in synovial fluid, encoded by Prg4. The objective of this study was to determine whether static and dynamic compression regulate PRG4 biosynthesis by cartilage explants. Articular cartilage disks were harvested to include the articular surface from immature bovines. Some disks were subjected to 24 h (day 1) of loading, followed by 72 h (days 2-4) of free-swelling culture to assess chondrocyte responses following unloading. Loading consisted of 6 or 100 kPa of static compression, with or without superimposed dynamic compression (10 or 300 kPa peak amplitude, 0.01 Hz). Other disks were cultured free-swelling as controls. PRG4 secretion into culture medium was inhibited by all compression protocols during day 1. Following unloading, cartilage previously subjected to dynamic compression to 300 kPa exhibited a rebound effect, secreting more PRG4 than did controls, while cartilage previously subjected to 100 kPa static loading secreted less PRG4. Immunohistochemistry revealed that all compression protocols also affected the number of cells expressing PRG4. The paradigm that mechanical stimuli regulate biosynthesis in cartilage appears operative not only for load bearing matrix constituents, but also for PRG4 molecules mediating lubrication.

Tissue engineering of stratified articular cartilage from chondrocyte subpopulations.

OBJECTIVE: To test if subpopulations of chondrocytes from different cartilage zones could be used to engineer cartilage constructs with features of normal stratification. ESIGN: Chondrocytes from the superficial and middle zones of immature bovine cartilage were cultured in alginate, released, and seeded either separately or sequentially to form cartilage constructs. Constructs were cultured for 1 or 2 weeks and were assessed for growth, compressive properties, and deposition, and localization of matrix molecules and superficial zone protein (SZP). RESULTS: The cartilaginous constructs formed from superficial zone chondrocytes exhibited less matrix growth and lower compressive properties than constructs from middle zone chondrocytes, with the stratified superficial-middle constructs exhibiting intermediate properties. Expression of SZP was highest at the construct surfaces, with the localization of SZP in superficial-middle constructs being concentrated at the superficial surface. CONCLUSIONS: Manipulation of subpopulations of chondrocytes can be useful in engineering cartilage tissue with a biomimetic approach, and in fabricating constructs that exhibit stratified features of normal articular cartilage.

Sexual growth dimorphism affects birth sex ratio in house mice.

We present the first empirical evidence that mammalian sex-ratio deviations result from variation in adult-weight sexual dimorphism via correlated effects on blastocyst development. Two selection lines of mice exhibiting high and low sexual dimorphism in adult weight showed correlated sexual weight differences at birth and at weaning, caused by relatively decelerated growth of males in the low line from before birth. The sex ratio at birth was significantly female-biased in the low line, and significantly lower than in the highly dimorphic line. Concomitantly, blastomere numbers were at significantly higher variance in the low than in the highly dimorphic line, owing to an increased frequency of slowly growing blastocysts. Since low-dimorphism mice produced more corpora lutea and more female pups than the high-dimorphism mice, but not more males, birth sex-ratio bias most parsimoniously resulted from the loss of slowly growing male blastocysts. This is in agreement with the observation that sex-ratio skews in mammals arise when timing of uterine responsiveness (i.e. its temporally limited capacity for implantation) varies in relation to sex-specific embryonic growth rates. Hence, natural mammalian sex-ratio variation that stems from developmental asynchrony might be a by-product of natural selection for sexual dimorphism in adult weight.