Protential Effects And Its Mechanism Of PTH(1-34) On Knee Joints Of DH Guinea Pigs With Spontaneous Osteoarthritis.

Part one Experimental study on guinea pigs with spontaneous kneeosteoarthritisObjective: To investigate the characteristics of Dunkin Hartley guineapigs with spontaneous knee osteoarthritis, and observe age-related changes inarticular cartilage, subchondral bone mineral density and estradiol levels inblood serum in this model over the entire course of osteoarthritis progression,and provide a scientific model of primary osteoarthritis.Methods: We studied spontaneous knee osteoarthritis in32femaleDunkin Hartley guinea pigs. Animals were randomly sacrificed at1,3,6,9, or12months of age (eight animals at each time point). Knee joints spaces wereopened. Then, cartilage surfaces of femur and tibia were observed andrecorded by digital camera. After disarticulation, femurs were fixed for72hours in70%ethanol and decalcified for6weeks with15%EDTA-2Na(pH7.4, at4°C). Tissues were regularly dehydrated, embedded in paraffin, andcut into6-8um-thick sections. The sections were stained with Masson. Usingwith light microscope (Olympus BX61, Japan), three color digital imageswere recorded to analysis articular cartilage lesions from each section indifferent regions. Mankin’s score system was properly adjusted and applied tomeasure morphological changes of cartilage. The expressions ofglycosaminoglycans and matrix metalloproteinase-3antibody were detectedusing immunohistochemistry method. Positive expression was quantified byoptical density method, using with Image pro-Plus (IPP) software. Bloodsamples were harvested before sacrifice for enzyme-linked immunosorbentassay (ELISA) analysis of estradiol concentration.Results:1As the animals’ age increased, they gained significantly more weight. The weight changes in each group were compared, and there weresignificantly different from each group animals(P<0.05).2The articular cartilage in1-month old animals was smooth and withoutany evidence of degeneration. At3months of age, mild discontinuous fibrosisoccasionally appeared. By6months of age, cartilage ulcerations and matrixloss were obvious and osteophytes had begun to develop when animalsreached9months of age.3As seen by Masson’s trichrome staining, articular cartilage in1-monthold animals had normal cellularity and extracellular matrix. Early histologicalchanges were observed in3-month old animals, including focal proteoglycanloss and fibrillation. At6months old, animals displayed chondrocytehypertrophy,“cloning” and surface cartilage lesions, and more obviouschondrocyte death/loss. Fibrillation and proteoglycan loss were observed in9-month old animals, and these degenerative changes extended into deeperzones.4All of the aforementioned changes were semi-quantitatively confirmedby age-dependent increased histological score according to the Mankinscoring system. There was a significant difference of Mankin scores betweenin each groups (P <0.05).5The cartilage surface of normal group was smooth as if it was coveredwith some amorphous substance, and the stria structure was intact, and nocollagen was exposed. The collagenous fibers on the cartilage surface of3-month group were exposed, and they arranged irregularly as if they wereeroded by running water, and in6month group some collagenous fiber retiabecame coarse, broken, exfoliative and even were turned over. In9monthgroup, there were some scattering on the surface of chondrocyte, collagenousfiber retia became coarser than6month group.6TEM revealed more details regarding cellular degenerations during OAprogression in this model: more chondrocytes were observed in1-month oldanimals and no apoptotic or necrotic chondrocytes were found; cell nucleiand membranes were integrated and rich in cytoplasmic organoids, chromatin was distributed uniformity, and few abnormal chondrocytes were detected in3-month old animals, with irregular shapes and loss of cytoplasmic organoids.Age-related increase in apoptotic or necrotic chondrocytes were detected, asshown in Figure5, the cavity around the cell became smaller with cellular andnuclear contraction, cytoplasmic organoid loss or disappearance, and loss ofnormal chondrocytes occurred. For each age group, normal and degeneratingcell numbers were calculated, as shown in Figure6, and the percentage ofdegenerating cells increased in an age-related manner.7We used immunohistochemistry to examine the distribution of MMP-3and GAG expression in knee joint cartilage from guinea pigs from each agegroup. The IOD values in each group showed that expression of GAG wasintense in younger animals, with a significant age-related decrease in GAGexpression beginning from6months of age(P<0.05). In contrast, an increasein MMP-3expression was detected with increasing age of the animals(P<0.05).8From months1to6, serum estradiol levels increased markedly(P<0.05), before remaining stable with no significant differences between6-month old and9-month old animals.Conclusion: Age-related articular cartilage degeneration occurred inDunkin Hartley guinea pigs beginning at3months of age in parallel withactivate cellular degeneration and matrix catabolism, while no directlypositive or negative correlation between osteoarthritis progression andestradiol serum level. Dunkin Hartley guinea pigs is a scientific model ofprimary osteoarthritis.Part two The protential effects of PTH(1-34) on knee joints of DH guineapigs with spontaneous osteoarthritis.Objective: To investigate the effect of PTH(1-34) on articular cartilagedegeneration in knee joints of DH guinea pigs with spontaneous osteoarthritis.Methods: Forty-eight1-month-old guinea pigs were divided into sixgroups: four groups were untreated and sacrificed at1,3,6and9months ofage; the other two groups received PTH (1-34)(15ug/kg/day,5days weekly) from3months of age, and were sacrificed at6and9months. Knee jointsspaces were opened. Then, cartilage surfaces of femur and tibia were observedand recorded by digital camera. After disarticulation, femurs were fixed for72hours in70%ethanol and decalcified for6weeks with15%EDTA-2Na(pH7.4, at4°C). Tissues were regularly dehydrated, embedded in paraffin, andcut into6-8um-thick sections. The sections were stained with Masson. Usingwith light microscope (Olympus BX61, Japan), three color digital imageswere recorded to analysis articular cartilage lesions from each section indifferent regions. Mankin’s score system was properly adjusted and applied tomeasure morphological changes of cartilage. The expressions of Type-IICollagen, matrix metalloproteinases-13and sclerostin antibody were detectedusing immunohistochemistry method. Positive expression was quantified byoptical density method, using with Image pro-Plus (IPP) software.Results:1The gross morphology at1month of age showed that the guinea pigs’articular surfaces were smooth, with an absence of osteophyte formation in thefemoral condyles and tibial plateaus. By3months of age, mild discontinuousfibrosis could be seen at the femoral condyles and tibial plateaus. The animalsdisplayed greater severity of OA with increasing age,6-month-old guinea pigsshowed obvious cartilage ulceration, and at9months of age, cartilage loss andtypical osteophyte were developed, the degeneration was particularlypronounced in the medial condyle. Although animals in the PTH(1-34)-treated group had rougher surfaces, ulceration and osteophytes, thesewere not as serious as those in the control groups.2With the Masson stain, the articular cartilage in1-month-old animalshad a smooth surface and normal cellularity, and no abnormalities were notedin the chondrocytes or extracellular matrix. Early histological changes wereobserved in3-month-old animals, including focal proteoglycan loss andfibrillation. With increasing age,6-month-old animals displayed more obviouschondrocyte death/loss, fibrillation and proteoglycan loss, and thesedegenerative changes extended into deeper zone and cell cloning is prominent in9-month-old animals. The cartilage samples in the PTH (1-34)-treatedgroups showed marked differences when compared with the control groups,with slight articular cartilage degeneration.3The Mankin score reflected pathological changes to the cartilage. Forthe age-matched groups, the Mankin scores increased with age (P <0.05).PTH (1-34) treatment reduced the Mankin score at6and9months of ageseparately, in contrast to the control group (P <0.05).4The analysis found a significant decrease in type-II collagen expressionand an increase in the expression of MMP-13and SOST in the control groupsas the age of the animals increased (P <0.05). Guinea pigs treated with PTH(1-34) showed a increase in type-II collagen expression, and a decrease inMMP-13and SOST expression at6and9months of age respectively, ascompared with the control groups(P <0.05).Conclusion: Early intervention with PTH (1-34) stimulated typeⅡc ollagen synthesis and inhibited expressions of SOST and MMP-13, andprevention of further aggravation of cartilage degradation in a naturallyoccurring OA modelPart three The mechanism of PTH(1-34) on knee joints of DH guinea pigswith spontaneous osteoarthritis.Objective: PTH (1-34) was a potent bone-turnover agent and appliedinto treatments of bone metabolic diseases. This study aimed to study themechanism of parathyroid hormone (1-34) on the micro-structure ofsubchondral bone, and protect against cartilage degradation in an animalmodel of naturally occurring OA.Methods: Forty-eight1-month-old guinea pigs were divided into sixgroups: four groups were untreated and sacrificed at1,3,6and9months ofage; the other two groups received PTH (1-34)(15ug/kg/day,5days weekly)from3months of age, and were sacrificed at6and9months. Knee jointsspaces were opened. Then, cartilage surfaces of femur and tibia were observedand recorded by digital camera. After soft tissues removed, femurs wereplaced under the probe and followed same position. The distal femur was scanned by micro computed tomography(Micro-CT). Static parameters anddynamic parameters, including bone mineral density(BMD), bone volume(BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), Platethickness(Pl.Th) and Structure Model Index(SMI). After Micro-CT test,femurs were fixed for72hours in70%ethanol and decalcified for6weekswith15%EDTA-2Na (pH7.4, at4°C). Tissues were regularly dehydrated,embedded in paraffin, and cut into6-8um-thick sections. The expressions ofosteoprotegerin, receptor activator of nuclear factor-κB ligand and Parathyroidhormone-related peptide antibody in cartilage and subchondral bone weredetected using immunohistochemistry method. Positive expression wasquantified by optical density method, using with Image pro-Plus (IPP)software.Results:1The expressions of OPG, RANKL and PTH1R in cartilage: Theanalysis found a significant decrease in OPG and PTH1R expression and anincrease in the expression of RANKL in the control groups as the age of theanimals increased (P <0.05). Guinea pigs treated with PTH (1-34) showed aincrease in OPG and PTH1R expression, and a decrease in RANKLexpression at6and9months of age respectively, as compared with the controlgroups (P <0.05).2The expressions of OPG, RANKL and PTH1R in subchondral bone:The analysis found a significant decrease in OPG and PTH1R expression andan increase in the expression of RANKL in the control groups as the age of theanimals increased (P <0.05). Guinea pigs treated with PTH (1-34) showed aincrease in OPG and PTH1R expression, and a decrease in RANKLexpression at6and9months of age respectively, as compared with the controlgroups (P <0.05).3With increasing age, the control group had a gradually decreasing ratioof OPG/RANKL in both the cartilage and subchondral bone. Compared withthe control animals, increases in the OPG/RANKL ratio for cartilage andsubchondral bone in the PTH (1-34)-treated groups (P <0.05). 4Bone mineral density increased with age and reached constant levelsafter6months. PTH (1-34)-treated animals showed higher values comparedwith those of the control groups, with a significant difference between animalsaged9months. Tb.Th increased with age, and BV/TV was highest at3months,and then remained constant. With advanced age, the SMI were markedlydecreased. Compared with the control groups, PTH (1-34) treatment increasedBV/TV at both6months and9months, and SMI at9months of age. From1to6months of age, subchondral plate thickness of control group wereincreased markedly (P<0.05), and remained fairly constant thereafter. ThoughPTH treated group has slightly increased subchondral plate thickness, there isno difference when compared with control group.Conclusion: PTH (1-34) can increase OPG and PTH1R expression,OPG/RANKL ratio in both cartilage and subchondral bone, decrease RANKLexpression in both cartilage and subchondral bone, and retard the deteriorationof subchondral trabecular bone.