| Fluid and solute transport is essential for bone and cartilage not only to performtheir regulatory function but also to maintain their viability. My studies are trying tobetter understand the roles of solute transport in bone and joint system by investigatingthe extracellular solute transport in bone, bone to cartilage and intracellular calciumresponse in in situ articular chondrocytes.My first study aimed to investigate solute transport altered by loading andphysiological conditions in bone Lacunar-canalicular system (LCS).Using the novelexperimental approach based on fluorescence recovery after photobleaching (FRAP)and synchronized mechanical loading imaging, we previously quantified the diffusionand convection of a small tracer in LCS for an intermittent cyclic loading. In the presentstudy, we will focus on the solute diffusion and convection altered by loadingconditions and physiological environment (bone diffuse microdamage, Perlecan/Hspg2deficiency genotype) in bone LCS. The solute-matrix interaction, quantified in terms ofthe reflection coefficient through the osteocyte PCM would be measured andtheoretically modeled. The present studies mainly include:①. We expanded the investigation of solute transport using a larger tracer, whichis comparable in size to some signaling proteins secreted by osteocytes. Murine tibiaewere subjected to sequential FRAP tests under rest-inserted cyclic loading while theloading magnitude (0,2.8or4.8N) and frequency （0.5,1or2Hz）were varied. Thecharacteristic transport rate k and the transport enhancement relative to diffusion (k/k0)were measured under each loading condition, from which the peak solute velocity in theLCS ware derived using our LCS transport model.1) The transport enchantmentlinearly increased with loading magnitude (0-5N).2) The transport enhancementdecreased when the loading frequency increased from0.5Hz,1Hz to2Hz, respectively.Significance differences in transport enhancement were detected between the cases of0.5Hz vs.1Hz and0.5Hz vs.2Hz.3) Using the measured LCS parameters, thesimulated transport enhancement at various solute velocities were obtained, showing anoverall power relationship. The mean velocity of parvalbumin convection through theLCS canaliculi under different loading magnitudes and frequencies was found. For the4.8N loading at0.5Hz, the mean velocity was55.8μm/s. As the loading magnitudedecreased to2.8N, the solute velocity was reduced to43.1μm/s. Further reduction in the solute velocity was seen when the loading frequency was increased from0.5Hz to1and2Hz.4) The fluid velocity estimated previously in16-week-old male murine tibiaeis58.9μm/s under477micron strain. Comparing to the previously study, the transportenhancement under477με axial compressive loading was calculated for20-22week-old murine tibiae in this study. And the solute velocity (53.9μm/s) was derived,yielding a reflection coefficient of parvalbumin through the osteocyte PCM of σ=1-vs/vf=0.084.②. Recent experiment point to activation of bone remodeling after fatiguedifferentially response to linear microcracks and diffusion microdamge, there was noactivation of resorption in response to diffuse microdamge alone. Despite its criticalrole in damage repair, how solute transport in the osteocyte lacunar-canalicular system(LCS) is altered after tissue damage is not fully understood. In chapter3, weinvestigated the effects of matrix-damage on diffusive transport through the LCS instatic or reloaded conditions.1) For static condition, the mean diffusivity of sodiumfluorescein within the LCS increased at the damaged regions compared with the controlregion.2) With increasing tensile loading, tracer diffusivity remained unchanged in theundamaged regions and tended to increase in the damaged zones. The transportenhancement increased significantly at successively higher tensile loads.③. Perlecan/Hspg2(PLN) deficient bone showed decreased canalicular densityand a reduced number of transverse tethering elements in canaliculi. The goal of chapter4was to investigate the alteration of solute diffusion and convection in PLN deficientmurine cortical bones.1) Solute diffusivity and convection transport enhancementincreased in perlecan/hspg2deficient murine (Hypo) osteocyte LCS compared withwild type (CTL).2) The mean solute velocity of parvalbumin in CTL and Hypo was48.2μm/s and68.4μm/s, respectively. The reflection coefficient of parvalbumin inCTL and Hypo was0.057and0.039, relatively. The sieve property of osteocyte PCMreduced in perlecan/hspg2deficient tibiae. The results proved our hypothesis that PLNdeficiency alters solute transport in LCS, which may have consequences on bone’smaintenance and mechanical adaptation.Taken together with the three different effects on solute transport in bone LCS, ourdata indicate the dynamic strain in vivo, the different physiological or pathologicalenvirment alter solute transport in bone LCS.My second study focused on the interactions (transport) between subchondral boneand cartilage in osteoarthritic joints. Subchondral bone is hypothesized to be involved in OA development. However, direct evidence supporting this is lacking. We previouslydetected measurable transport of solute across the mineralized calcified cartilage innormal joints, suggesting a potential cross-talk between subchondral bone and cartilage.Whether this cross-talk exists in OA has not been established yet. Using two modelsthat induced OA by either ageing or surgery (destabilization of medial meniscus. DMM),we tested the hypothesis that increased cross-talk occurs in OA.Although we did notdetect significant changes in tissue matrix permeability in OA joints in preliminarystdudy, In charpter5, we found1) an increased number of vessels invading the calcifiedcartilage (and sometimes approaching the tidemark) in the aged (+100%) and DMM(+50%) joints relative to the normal age controls; and2) a60%thinning of thesubchondral bone and calcified cartilage layers in the aged joints (with no significantchanges detected in the DMM joints). These results suggested that the capacity forcross-talk between subchondral bone and articular cartilage could be elevated in OA.My third project investigated the effects of Prostaglandin E2(PGE2) onintracellular calcium ([Ca2+]i) response of in situ porcine articular chondrocytes. Incharpter6, we determined the changes of PGE2-induced [Ca2+]iresponse of in situchondrocytes in superficial zone, middle zon and deep zone of articular cartilage. Lowconcentration PGE2(2.5×10-8mg/ml)(PGE2-L) enlarged [Ca2+]iresponse of bothcultured porcine chondrocytes and in situ articular chondrocytes in porcine cartilageexplants. Relative to cultured chondrocytes, the frequency of [Ca2+]iresponse of in situchondrocytes significantly increased. Among the superficial, middle and deep zone,PGE2-induced [Ca2+]ioscillation has been elevated dramatically in superficial zone.Together these initial observations reveal the importance of very low concentraions ofPGE2in signaling pathway of chondrocytes.In conclusion, the studies suggest the powerful effect of transport in bone andcartilage function. |
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