| Part Ⅰ T2 mapping and quantitative analysis of the porcine patellar osteoarthritis model in vitro by collagenaseObjective To imitate the change of collagen content in early osteoarthritis cartilage through degrading collagen of porcine patellae ex vivo by type Ⅱ collagenase. Then, to investigate the ability of T2 mapping in early detection of the change of cartilage collagen by quantitative analysis.Materials and MethodsMaterials:20 fresh porcine patellae were randomly assigned to 4 groups,3 treated groups and 1 control group. Phosphate buffered saline (PBS) solution and 100mg/100ml,150mg/100ml,200mg/100ml type Ⅱ collagenase solution (collagenase in PBS solution) were prepared.Experimental methos:The samples from 3 treated groups were respectively immersed in PBS solution with 100mg/100ml,150mg/100ml and 200mg/100ml type Ⅱ collagenase for 4 h, whereas control group samples in PBS solution for 4h.MRI scan:All treated porcine patellae were scanned by 3.0 T MR scanner (3.0 T Achieva TX, Philips). Conventional T2-weighted scan and T2 mapping with 5-echo spin echo sequence (TR/TE=1368ms/10,20,30,40,50ms) were used.Images postprocessing and measurement of T2 value:All image data of T2WI and T2 mapping were imported into Image J software. Region of interest (ROI) of superficial, deep and full layers of cartilage in the middle slice of patellae on T2WI were outlined manually and saved. Then, ROIs were copied onto corresponding T2 mapping images. T2 relaxation times of superficial, deep and full layers of cartilage were measured.Pathology and measurement of mean optical density:Cartilage was cut from central zone of patellae of treated groups and control group. Cartilage sections were stained by Van Gieson method and mean optical density (OD) after Van Gieson stain were measured.Statistical analysis:One-way analysis of variance (ANOVA) was employed to compare the T2 relaxation times among corresponding cartilage layers (superficial, deep and full layers) and OD of 4 groups, with P<0.05 was regarded significant. Pearson correlation coefficient was calculated between T2 relaxation times and mean density.ResultsGross pathology showed all articular cartilages maintained intact after treatment, while the cartilage of 150mg/100ml and 200mg/100ml treated groups turned dark slightly.T2 relaxation times of superficial and full layers and mean density increased significantly in any treated group in comparison with control group(P<0.05). Significant differences in T2 relaxation times of superficial layers and mean density were found between any two treated groups, whereas no difference of T2 relaxation time of full layers was found between 100mg/100ml and 150mg/100ml treated groups(P>0.05). Pearson correlation analysis showed a close correlation (r=-0.837) between T2 relaxation times of full layers and mean density.ConclusionThrough T2 mapping technique quantificationally analyzing cartilage treated by type Ⅱ collagenase and statistical analysis, the study results show that T2 mapping can adequately detect the change of cartilage collagen in the early stage of osteoarthritis, and shows a good clinical application prospect.Part II Establishment of porcine models of early knee osteoarthritisObjective To establish early knee osteoarthritis (KOA) animal model with miniature pigs by operation method to meet functional MRI study.Materials and MethodsSubject:12 Tibet miniature pigs,10 to 12 months,33-36 kg weight, were randomly divided into 4 groups (3 pigs per group).Establishment of Animal model:Pigs were fixed on operating table with right lateral position following general anesthesia. After opening the articular cavity of left knees through lateral approach and pushing patellae and patellar ligament aside, lateral menisci resection and anterior cruciate ligament transaction were performed when joint being bent. Right knees kept intact served as control.MRI scan:Pigs in 4 groups (referred to as 2,4,6,8 week group) were sacrificed after being fed for 2,4,6,8 weeks after surgery. Two hind legs were cut down from hips, and immediately two knees were scanned by 3.0 T MR scanner (3.0 T Achieva TX, Philips) and a 12-channel SENSE knee coil. Sagittal 3D T1-weighted fast field echo with water-selective excitation (3D WATS) scan was performed firstly, with TR/TE=20 ms/4.9 ms. Then, sagittal T1p and T2 mapping scan followed. T1p used 3D balanced turbo field echo (B-TFE) sequence with the following parameters: TR/TE=5.9 ms/3.0 ms, spin-lock pulse amplitude:500Hz, time of spin lock=1,10, 20,30,40 ms. T2 mapping used 5-echo fast spin echo sequence (TR/TE=1368ms/10, 20,30,40,50ms, flip angle=90。). Images were fitted on a pixel by pixel basis to the exponentially decaying T1p function using IDL to generate T1p relaxation map.Histopathology:Cartilage of operated knees was cut from lateral condyle of femur and lateral tibial plateau aside resected lateral menisci. Cartilage sections were stained by safranin O.Observation indexes:①Enimals’ diet, activity and wound healing after surgery.②Evaluation of cartilage’s display and measurement of cartilage thickness on the images of 3D WATS sequence, T1p relaxation map and T2 mapping.③Gross observation of operated knee articular cartilage by reference to the control④Evaluation of cartilage’s histopathology and grading according to OARSI system under optical microscope.ResultsAll incisions were primary healing without infection and animal death.Cartilage could be displayed on the every group’s images of 3D WATS sequence, T1p relaxation map and T2 mapping. The cartilage thickness was 1.0-1.3mm. MRI showed the thickness and image quality of cartilage of porcine models of KOA could adequately meet the need of funtional MRI study.Gross observation showed there was slightly faded and intact surface on the cartilage of lateral condyle of femur and lateral tibial plateau aside resected lateral menisci of operated knees of 4 week (after surgery) group.Histopathology demonstrated superficial fibrillation of cartilage and reducing or disappearing of cells in 4 week group. Safranin O stainning displayed the color loss of upper 1/3 of cartilage, and OARSI grading categorised them as grade 1.5～2.5. These revealed 4 week group best met early KOA.ConclusionThe porcine model of KOA with the cartilage of more than 1 mm thickness can meet the need of cartilage’s functional MRI in vivo better. The porcine model of early KOA established by surgery is proved stable and controllable. Histopathology demonstrated that the changes of left knee joint cartilage of the 4 week group best meet early KOA, which is a good animal model to be served for early KOA study in vivo with functional MRI.PartⅢ T1p and T2 mapping of porcine early knee osteoarthritisObjective To evalute the ability of T1p and T2 mapping in the diagnosis of cartilage degeneration of early knee osteoarthritis (KOA) through porcine models established by surgery.Materials and MethodsSubject:3 male Tibet miniature pigs with 10 to 12 months and 33-36 kg weight.Establishment of Animal model:Pigs were fixed on operating table following general anesthesia. Lateral menisci resection and anterior cruciate ligament transaction were performed on the porcine left knees, and right knees kept intact served as control.3 pigs were respectively fed for 4 weeks after surgery before being sacrificed.MRI scan:Pigs’both hind legs were cut down from hips after being sacrificed, and two knees immediately were scanned by 3.0 T MR scanner (3.0 T Achieva TX, Philips). The sequences of scanning were sagittal 3D WATS, sagittal T1p and T2 mapping. T1p used 3D balanced turbo field echo (B-TFE) sequence with time of spin lock=1,10,20,30,40 ms. T2 mapping used 5-echo fast spin echo sequence (TR/TE =1368ms/10,20,30,40,50ms, flip angle= 90°).Measurement of T1p and T2 values:All image data were imported into Image J software. ROIs of cartilage of lateral condyle of femur and lateral tibial plateau aside resected lateral menisci were manually outlined on the sagittal plane and saved. Then, ROIs were copied onto corresponding T1p relaxation maps and T2 mapping images. T1p and T2 values of cartilage were measured.Histopathology:Cartilage of operated knees was cut from lateral condyle of femur and lateral tibial plateau aside resected lateral menisci. Cartilage sections were stained by safranin O, and grading was operated according to OARSI system.Statistical analysis:Paired Sample t test was employed to compare the and the control, with P<0.05 was regarded significant.ResultsThe OARSI grade of porcine models (4 weeks after surgery) was 1.5-2.5, met the cartilage change of early KOA.Paired Sample t test revealed T1p and T2 values of operated articular cartilage were higher significantly than that of the control. By contrast with the mean T1p value of the control, the mean T1p value of operated articular cartilage was 32.94% higher, while the mean T2 value of operated articular cartilage was 17.52% higher than the control.ConclusionThe study of the ability of T1p and T2 mapping in the diagnosis of cartilage degeneration of early KOA with porcine models shows that both techniques can discover the cartilage degeneration of early KOA. By contrast, T1p is more sensitive than T2 mapping.PartIV Correlation analysis between T1p value, T2 value and proteoglycan content, collagen content of cartilage of porcine knee osteoarthritisObjective To investigate gradually degrading knee articular cartilage with T1p and T2 mapping and research the correlation between T1p value or T2 value and proteoglycan content or collagen content through porcine models of osteoarthritis in vivo established by surgery.Materials and MethodsSubject:12 Tibet miniature pigs,10 to 12 months,33-36 kg weight, were randomly divided into 4 groups (3 pigs per group).Establishment of Animal model:Pigs were fixed on operating table following general anesthesia. After opening the articular cavity of left knees through lateral approach, lateral menisci resection and anterior cruciate ligament transaction were performed. Right knees kept intact served as control. Pigs in 4 groups (referred to as 2,4,6,8 week group) were fed for 2,4,6,8 weeks after surgery.MRI scan:Pigs were sacrificed at the predetermined time. Left hind legs were cut down from hips, and immediately the knees were scanned by 3.0 T MR scanner. Sagittal 3D T1-weighted fast field echo with water-selective excitation (3D WATS) scan was performed firstly, with TR/TE= 20 ms/4.9 ms. Then, sagittal T1p and T2 mapping scan followed. T1p used 3D balanced turbo field echo (B-TFE) sequence with the time of spin lock=1,10,20,30,40 ms. T2 mapping used 5-echo fast spin echo sequence (TR/TE=1368ms/10,20,30,40,50ms, flip angle=90°).Images postprocessing and measurement of T1p and T2 values:All image data were imported into Image J software. ROIs of cartilage of lateral condyle of femur and lateral tibial plateau aside resected lateral menisci were manually outlined on the sagittal plane and saved. Then, ROIs were copied onto corresponding T1p relaxation maps and T2 mapping images. T1p and T2 values of cartilage were measured.Pathology and measurement of mean optical density:Cartilage of operated knees was cut from lateral condyle of femur and lateral tibial plateau aside resected lateral menisci. Cartilage sections were stained by safranin O and Van Gieson method with the same operating steps and conditions. Mean optical density (OD) of 3 sections per stained cartilage was measured and the mean OD was calculated. The measuring range was from cartilage surface to the boundary separating cartilage and bone. The mean OD after safranin O and Van Gieson staining respectively represent the content of proteoglycan and collagen.Statistical analysis:Scatter diagrams of T1p value or T2 value and OD of proteoglycan or collagen were plotted. The correlation between T1p value or T2 value and proteoglycan content or collagen content were analyzed by Pearson correlation analysis, with P<.0.05 was regarded significant.ResultsThe mean T1p value and T2 value of operated knee articular cartilage respectively was 71.765±8.794 ms and 52.078±9.846 ms. The mean OD of cartilage after safranin O and Van Gieson staining respectively was 0.3457±0.1165 and 0.2036±0.0955. The R2 of correlation between T1p value and OD after safranin O staining was 0.793 (P<0.001), and the R2 of correlation between T2 value and OD after safranin O staining was 0.576 (P<0.001). The R2 of correlation between Tip value and OD after Van Gieson staining was 0.689 (P<0.001), and the R2 of correlation between T2 value and OD after Van Gieson staining was 0.734 (P< 0.001).ConclusionWe investigated the gradual degrading knee articular cartilages, which are from porcine models of osteoarthritis by surgery, with T1p and T2 mapping and analyzed the correlation between T1p value or T2 value and proteoglycan content or collagen content. The research mode is closer to clinical practice and is completely different from any of former related researches. Our research results demonstrate there are correlative relationships between T1p value or T2 value and proteoglycan content or collagen content. T1p is more sensitive to the change of proteoglycan in degraded cartilage than T2 mapping, while T2 mapping is more sensitive to the change of collagen than T1p. |
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