In vitro tissue culture model validation-the influence of tissue culture components on IPL energy output.

Intense pulsed light (IPL) has been used therapeutically in a number of clinical settings and has been shown to have a photobiomodulatory effect on connective tissue cells, such as those derived from skin and tendon. In vitro cell culture models are essential tools preclinically in investigating such treatment modalities, as they help in optimising parameters for successful treatment. However, as culture system components have been reported to absorb part of the irradiated energy, which in turn has a bearing on the amount of light reaching the cells, it is important to establish specific parameters for the particular in vitro model used. This study, therefore, investigates the effect of our tissue culture system components on the IPL energy delivered. Individual wells of multi-well plates were irradiated with IPL at different device settings and under variable culture conditions (e.g. in the absence or presence of cell culture media with or without the pH indicator dye, phenol red), and the energy lost through the culture system determined. Our data demonstrated that the IPL device delivered significantly lower outputs than those published, and energy absorption by the culture equipment would further reduce fluencies delivered to the cell monolayer. Furthermore, energy absorption by media containing phenol red was marginally greater than clear media and resulted in only a small increase in temperature, which would not be harmful to cells. The use of phenol red-containing media therefore is valid and physiologically relevant when examining light-culture system interactions.

In vitro studies to evaluate the effect of varying culture conditions and IPL fluencies on tenocyte activities.

Tendons are dense, fibrous connective tissues which carry out the essential physiological role of transmitting mechanical forces from skeletal muscle to bone. From a clinical perspective, tendinopathy is very common, both within the sporting arena and amongst the sedentary population. Studies have shown that light therapy may stimulate tendon healing, and more recently, intense pulsed light (IPL) has attracted attention as a potential treatment modality for tendinopathy; however, its mechanism of action and effect on the tendon cells (tenocytes) is poorly understood. The present study therefore investigates the influence of IPL on an in vitro bovine tendon model. Tenocytes were irradiated with IPL at different devise settings and under variable culture conditions (e.g. utilising cell culture media with or without the pH indicator dye phenol red), and changes in tenocyte viability and migration were subsequently investigated using Alamar blue and scratch assays, respectively. Our data demonstrated that IPL fluencies of up to 15.9 J/cm2 proved harmless to the tenocyte cultures (this was the case using culture media with or without phenol red) and resulted in a significant increase in cell viability under certain culture conditions. Furthermore, IPL treatment of tenocytes did not affect the rate of cell migration. This study demonstrates that irradiation with IPL is not detrimental to the tenocytes and may increase their viability under certain conditions, thus validating our in vitro model. Further studies are required to elucidate the effects of IPL application in the clinical situation.

Contrasting effects of n-3 and n-6 fatty acids on cyclooxygenase-2 in model systems for arthritis.

Cyclooxygenase-2 (COX-2) is intimately involved in symptoms of arthritis while dietary n-3 polyunsaturated fatty acids (PUFA) are thought to be beneficial. In these experiments, using both bovine and human in vitro systems that mimic features of arthritis, we show that the n-3 PUFA eicosapentaenoic acid (EPA) is able to reduce mRNA and protein levels of COX-2. Activity, as assessed through prostaglandin E(2) formation, was also reduced in a dose-dependent manner. These effects of EPA contrasted noticeably with the n-6 PUFA, arachidonic acid. The data provide direct evidence for a molecular mechanism by which dietary n-3 PUFA, such as EPA, can reduce inflammation and, hence, associated symptoms in arthritis.

Inhibition of aggrecan turnover in short-term explant cultures of bovine tendon.

The large aggregating proteoglycan, aggrecan, better known for its physiological role in articular cartilage where it serves to facilitate resistance of compressive forces during joint articulation, is also present within the distinct functional regions of tendon (i.e., compressed/fibrocartilaginous and tensional). Previous studies demonstrate that an increased turnover of aggrecan occurs in tendon, which is mediated principally by the 'aggrecanases' and, as such, these proteinases may play an important role in the normal functioning of the tissue. In the present study, utilising bovine tendon explant culture systems, we demonstrated that aggrecanase-mediated tendon aggrecan turnover may be modulated by generic metalloproteinase inhibitors (i.e., the aggrecanase inhibitor, actinonin and the broad-spectrum MMP inhibitor, marimistat). As expected, no MMP-generated aggrecan catabolites were detected in the culture system, suggesting that tendon aggrecanases may be inhibited by marimistat. Furthermore, immunohistochemical analyses revealed that aggrecan metabolites are present in the endotenon, surrounding the collagen fibre bundles, suggesting that aggrecan may provide functions of water imbibement and resistance of reversible and repeated compressive loads manifest between the collagen fibres; these functions, in turn, may be associated with increased aggrecan turnover in this tissue. Thus, inhibition of tendon aggrecanases and consequently aggrecan turnover in this tissue, may be related to some of the deleterious effects observed in the tendons of patients undergoing drug therapy with broad-spectrum MMP inhibitors for cancer and arthritis.

Catabolism of aggrecan proteoglycan aggregate components in short-term explant cultures of tendon.

Hyaluronan (HA) and link protein are essential components of the aggrecan proteoglycan aggregate, whereby HA binds multiple aggrecan monomers, an interaction which is stabilised by link protein. In this study, we have examined the turnover of the aggregate components, HA, link protein, and the N-terminal G1 domain of aggrecan, in explant cultures of tissue from compressed and tensional regions of young and mature bovine tendons. Western blot analyses revealed the release of highly processed link protein and G1-containing metabolites, in the absence of catabolic agents, indicating an increased turnover of these components in tendon. In addition, significant levels of HA were released from the tissue matrix into the media compartment. Furthermore, RT-PCR analysis showed that the mRNA expression of link protein and enzymes/proteins associated with HA metabolism may be modulated in the distinct functional regions of tendon with development. Perturbation of normal aggrecan aggregate metabolism may lead to tissue dysfunction.

Effect of serum albumin on glycosaminoglycan inhibition of hydroxyapatite formation.

The interaction of proteins with hydroxyapatite (HAP), specifically proteoglycans and their spatial arms, glycosaminoglycans (GAGs) in addition to serum proteins, play an important role in regulating biological mineralisation. In this study, seeded HAP growth experiments revealed that inhibition of HAP formation by bovine serum albumin (BSA) was significantly elevated in combination with chondroitin 4-sulphate (C4S) versus BSA with heparin. Data suggest that C4S and BSA adsorb to different calcium sites on the HAP surface. In contrast, complexes of heparin and BSA may adsorb to HAP, thus reducing the rate of inhibition due to steric effects. In addition, the amount of protein bound to HAP growth seed was significantly higher in the presence of heparin versus C4S, confirming this suggestion. Furthermore, the data indicate that a GAG-induced conformational change in BSA occurs, which affects inhibition. This work provides novel information concerning binary molecule modulation of HAP growth.

Immunolocalisation and expression of proteoglycan 4 (cartilage superficial zone proteoglycan) in tendon.

Cartilage superficial zone protein/proteoglycan (SZP) or proteoglycan 4 (PRG4), has been demonstrated to have the potential for several distinct biological functions including cytoprotection, lubrication and matrix binding. In the present study, we have examined both the immunolocalisation and the mRNA expression pattern of PRG4 in tissue harvested from the compressed and tensional regions of young and mature bovine tendons. Immunohistochemical analyses, utilizing monoclonal antibody 3-A-4 which recognizes a conformational-dependent epitope on native PRG4, demonstrated that PRG4 is present predominantly at the surface of fibrocartilaginous regions of tendon, with the intensity of immunoreactivity in this region increasing with age. RT-PCR analyses revealed that the expression of PRG4 mRNA can be modulated by exposure to cytokines and growth factors. In addition, analyses of human pathological tendon revealed that PRG4 may also be expressed as an alternatively spliced form lacking exons which encode part of the N-terminal matrix-binding and cell-proliferative domain; however, it remains to be determined whether such splice variants are a feature of human tendon, regardless of disease state. Taken together, these data indicate that PRG4 may play an important cytoprotective role by preventing cellular adhesion to the tendon surface as well as providing lubrication during normal tendon function, in a manner complimentary to cartilage PRG4. Structural modifications to SZP, together with a reduction in synthesis during tendon inflammation with injury and disease may account for the formation of tendon adhesions and contribute to the overall dysfunction of the tissue.

Effects of n-3 fatty acids on cartilage metabolism.

Although the clinical benefits of dietary supplementation with n-3 polyunsaturated fatty acids (PUFA) has been recognised for a number of years, the molecular mechanisms by which particular PUFA affect metabolism of cells within the synovial joint tissues are not understood. This study set out to investigate how n-3 PUFA and other classes of fatty acids affect both degradative and inflammatory aspects of metabolism of articular cartilage chondrocytes using an in vitro model of cartilage degradation. Using well-established culture models, cartilage explants from normal bovine and human osteoarthritic cartilage were supplemented with either n-3 or n-6 PUFA, and cultures were subsequently treated with interleukin 1 to initiate catabolic processes that mimic cartilage degradation in arthritis. Results show that supplementation specifically with n-3 PUFA, but not n-6 PUFA, causes a decrease in both degradative and inflammatory aspects of chondrocyte metabolism, whilst having no effect on the normal tissue homeostasis. Collectively, our data provide evidence supporting dietary supplementation of n-3 PUFA, which in turn may have a beneficial effect of slowing and reducing inflammation in the pathogenesis of degenerative joint diseases in man.

Pathologic indicators of degradation and inflammation in human osteoarthritic cartilage are abrogated by exposure to n-3 fatty acids.

OBJECTIVE: To determine if n-3 polyunsaturated fatty acid (PUFA) supplementation (versus treatment with n-6 polyunsaturated or other fatty acid supplements) affects the metabolism of osteoarthritic (OA) cartilage. METHODS: The metabolic profile of human OA cartilage was determined at the time of harvest and after 24-hour exposure to n-3 PUFAs or other classes of fatty acids, followed by explant culture for 4 days in the presence or absence of interleukin-1 (IL-1). Parameters measured were glycosaminoglycan release, aggrecanase and matrix metalloproteinase (MMP) activity, and the levels of expression of messenger RNA (mRNA) for mediators of inflammation, aggrecanases, MMPs, and their natural tissue inhibitors (tissue inhibitors of metalloproteinases [TIMPs]). RESULTS: Supplementation with n-3 PUFA (but not other fatty acids) reduced, in a dose-dependent manner, the endogenous and IL-1-induced release of proteoglycan metabolites from articular cartilage explants and specifically abolished endogenous aggrecanase and collagenase proteolytic activity. Similarly, expression of mRNA for ADAMTS-4, MMP-13, and MMP-3 (but not TIMP-1, -2, or -3) was also specifically abolished with n-3 PUFA supplementation. In addition, n-3 PUFA supplementation abolished the expression of mRNA for mediators of inflammation (cyclooxygenase 2, 5-lipoxygenase, 5-lipoxygenase-activating protein, tumor necrosis factor alpha, IL-1alpha, and IL-1beta) without affecting the expression of message for several other proteins involved in normal tissue homeostasis. CONCLUSION: These studies show that the pathologic indicators manifested in human OA cartilage can be significantly altered by exposure of the cartilage to n-3 PUFA, but not to other classes of fatty acids.

Inhibition of hydroxyapatite crystal growth by bone proteoglycans and proteoglycan components.

The small leucine-rich proteoglycans (SLRPs) interact with hydroxyapatite (HAP) and have been demonstrated to be important modulators of mineralisation. In the present study we have examined the effect of bone SLRPs, purified bone glycosaminoglycan (GAG) chains and core proteins as well as commercial chondroitin 4-sulphate, chondroitin 6-sulphate and desulphated chondroitin on HAP crystal growth. Seeded HAP growth experiments revealed that addition of bone GAG chains resulted in almost complete inhibition of crystal growth (93%), with addition of core proteins and intact PGs resulting in 55 and 37% inhibition, respectively. In contrast, commercial chondroitin 4-sulphate was significantly less inhibitory compared with the bone SLRPs and components, yielding only a 6% reduction in HAP-induced crystal growth at the same concentration. Significantly, chondroitin 6-sulphate was found to be noninhibitory, whilst desulphated chondroitin was inhibitory to seeded HAP growth. The data indicate that direct adsorption of SLRPs to growth sites and their ability to bind calcium are significant determinants in the inhibitory process. In addition, PG/GAG chemistry and the conformation of the macromolecules in solution have also been shown to be important. This work provides new information regarding the role of bone SLRPs and their components in the regulation of the mineralisation process.

Interaction of glucuronic acid and iduronic acid-rich glycosaminoglycans and their modified forms with hydroxyapatite.

Proteoglycans and their spatial arms, glycosaminoglycans (GAGs), are known to interact with hydroxyapatite (HAP) and have been implicated as important modulators of mineralisation. In the present study isotherm data (0.02 M sodium acetate, pH 6.8) revealed that the iduronic-rich GAGs heparan sulphate, heparin and dermatan sulphate showed greater binding onto HAP with higher adsorption maxima compared with the glucuronic acid-rich GAGs chondroitin-4-sulphate, chondroitin-6-sulphate and hyaluronan. Chemically desulphated chondroitin showed no adsorption onto HAP. With the exception of hyaluronan, the GAGs studied showed no desorbability in sodium acetate buffer only, whereas in di-sodium orthophosphate, desorption occurred much more readily. The data indicates that GAG chemistry and conformation in solution greatly influence the interaction of these molecules with HAP. The conformational flexibility of iduronic acid residues may be an important determinant in the strong binding of iduronic acid-rich GAGs to HAP, increasing the possibility of the appended anionic groups matching calcium sites on the HAP surface, compared with more rigid glucuronic acid residues. This work provides important information concerning interfacial adsorption phenomena between the organic-inorganic phases of mineralised systems.