Early Changes of Articular Cartilage and Subchondral Bone in The DMM Mouse Model of Osteoarthritis methods
Aim. Evidence-backed execution summary for Early Changes of Articular Cartilage and Subchondral Bone in The DMM Mouse Model of Osteoarthritis methods from Early Changes of Articular Cartilage and Subchondral Bone in The DMM Mouse Model of Osteoarthritis.
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mouse
Subject model for the experiment.
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- confirm full cohort details in the source paper
Histology
reagent used in the protocol.
- Use
- Both hind limbs were then put in 5% ethylenediaminetetraacetic acid (EDTA) in phosphate buffered saline (pH 7.0) for decalcification for 10-12 days after the µCT scan. Decalcified limbs were further processed and embedded in paraffin in frontal orientation, with serial sections taken at a thickness of 4&#...
Histological analyses of joint changes after DMM surgery
Osteoarthritis Research Society International (OARSI) histopathology scoring system was applied to evaluate OA severity (Fig. ). On the medial compartments of the joint, the tibial plateau developed earlier (starting at 2 weeks) and more severe OA compared to femoral condyle (starting at 5 weeks) (Fig. )...
- Use
- Osteoarthritis Research Society International (OARSI) histopathology scoring system was applied to evaluate OA severity (Fig. ). On the medial compartments of the joint, the tibial plateau developed earlier (starting at 2 weeks) and more severe OA compared to femoral condyle (starting at 5 weeks) (Fig. )...
Gait analysis
Mice were weighed and transferred to the Behavior Study Suite at Robarts Research Institute at Western University two days prior to gait acquisitions. Gait patterns and relative parameters were captured by the Catwalk ® gait analysis system (Noldus Inc.) as described. Briefly, mice were placed on a glass...
- Use
- Mice were weighed and transferred to the Behavior Study Suite at Robarts Research Institute at Western University two days prior to gait acquisitions. Gait patterns and relative parameters were captured by the Catwalk ® gait analysis system (Noldus Inc.) as described. Briefly, mice were placed on a glass...
Limb harvest and µCT
Mouse hind limbs were then scanned using an eXplore RS in vivo x-ray µCT scanner (GE Healthcare Biosciences, London, ON, Canada). Calibrating standards for air, water and a cortical bone mimic, SB3 (Gammex RMI), were included with the specimens. The scanning protocol consisted of 900 xy-ray projection images, s...
- Use
- Mouse hind limbs were then scanned using an eXplore RS in vivo x-ray µCT scanner (GE Healthcare Biosciences, London, ON, Canada). Calibrating standards for air, water and a cortical bone mimic, SB3 (Gammex RMI), were included with the specimens. The scanning protocol consisted of 900 xy-ray projection images, s...
Histology
Both hind limbs were then put in 5% ethylenediaminetetraacetic acid (EDTA) in phosphate buffered saline (pH 7.0) for decalcification for 10-12 days after the µCT scan. Decalcified limbs were further processed and embedded in paraffin in frontal orientation, with serial sections taken at a thickness of 4&#...
- Use
- Both hind limbs were then put in 5% ethylenediaminetetraacetic acid (EDTA) in phosphate buffered saline (pH 7.0) for decalcification for 10-12 days after the µCT scan. Decalcified limbs were further processed and embedded in paraffin in frontal orientation, with serial sections taken at a thickness of 4&#...
OARSI scoring
OARSI scoring system was used to evaluate OA severity in three sections with different depth after TB staining. Sections were blinded and scored by three different experienced scientists. Average scores were used in statistical analyses.
- Use
- OARSI scoring system was used to evaluate OA severity in three sections with different depth after TB staining. Sections were blinded and scored by three different experienced scientists. Average scores were used in statistical analyses.
Gait analysis
Software used for acquisition, scoring, statistics, or reporting.
- Use
- Mice were weighed and transferred to the Behavior Study Suite at Robarts Research Institute at Western University two days prior to gait acquisitions. Gait patterns and relative parameters were captured by the Catwalk ® gait analysis system (Noldus Inc.) as described. Briefly, mice were placed on a glass...
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Histological analyses of joint changes after DMM surgery
OA phenotype after DMM surgery was confirmed by histology using toluidine blue (TB) staining. Regional proteoglycan loss and chondrocyte clustering occurred on the medial compartments of the joint at 2 weeks post-surgery, especially the medial tibial plateau (MTP) (Fig., white arrow). Collagen disorganization was also shown in this small region in adjacent sections by picrosirius red (PR) staining (Fig., yellow arrow). Cartilaginous tissue formed on the medial edge of tibia and stained strongly with TB at 2-week time-point in DMM mice (Fig., black arrow), with collagen fibril organization similar to that of articular cartilage (Fig., white arrow). 5 weeks post-surgery, mild to moderate cartilage damage can be found in the surgical joints of DMM mice. Cartilage erosion and fibrillation, as well as large osteophytes were noticed on the MTP. Proteoglycan loss...
Histological analyses of joint changes after DMM surgery
Osteoarthritis Research Society International (OARSI) histopathology scoring system was applied to evaluate OA severity (Fig. ). On the medial compartments of the joint, the tibial plateau developed earlier (starting at 2 weeks) and more severe OA compared to femoral condyle (starting at 5 weeks) (Fig. ) at the same time-points. Although not statistically significant and much milder, lateral compartments showed a similar trend (Fig. ).
TRAP staining
TRAP positive cells were found in the subchondral bone of DMM mice starting at 5 weeks post-surgery, with most of these cells located at the site of osteophyte formation (Fig. ). Very little positive staining was found in the subchondral bone of the SHAM group, although the expected staining at growth plate was observed (Fig. ). Figure 3 Tartrate-resistant acid phosphatase (TRAP) staining of osteoclast activity after DMM surgery. Osteoclast activity in the subchondral bone appeared increased at 5- and 10-week following DMM surgery, mostly within the osteophyte (red rectangle), but not at 2-weeks. The expected staining at the growth plate was observed (black rectangle). Scale bar: 200 µm.
Subchondral bone changes after DMM surgery
3D reconstruction of joints from micro-CT analyses is shown in Fig.. Irregular bone surface and abnormal patellar shape (osteophyte formations) was noticed in DMM mice as early as 2 weeks post-surgery (Fig., hollow arrow) and became more obvious at later time-points (Fig., hollow arrow). Osteophyte formation of the MTP was noticed at 5 and 10 weeks post-surgery, with those at 10 weeks being more severe (Fig., white arrow). In the coronal plane in the middle of the knee joint, subchondral bone of the MTP didn't show difference at 2 weeks in DMM Group compared to SHAM (Fig. ), but became denser at later time-points (Fig., circled). BMDs of all compartments are shown in Fig.. As seen in the coronal plane images, BMDs of DMM animals showed no statistical differences comparing to their SHAM controls at 2 weeks, with DMM animals displayi...
Subchondral bone changes after DMM surgery
Bone mineral density (BMD) of subchondral bone in the DMM model. At 2 weeks pot-surgery, BMDs of the MTP in DMM mice didn't showed statistical significant differences comparing to their SHAM controls. However, DMM mice showed a larger spread of BMD at 2 weeks post-surgery ( A ). Clearer differences were detected between DMM and SHAM at both 5 and 10 weeks post-surgery with higher BMDs in DMM mice. ( A ). BMDs of the MFC were higher in DMM mice 10 weeks after surgery ( B ). There was no significant differences found in the lateral compartments between DMM and SHAM at all time-points ( C, D ). Error bar stands for the 95% confidence interval.
Subluxation/dislocation of patella after DMM
In a few of our mice (5 of 27, 2 mice of 2-week time-point and 3 of 10-week time-point) that underwent DMM surgery, subluxation/dislocation of the patella was noted as early as 2 weeks post-surgery by micro-CT as well as histological staining (Fig. ). In these mice, joint condition deteriorated faster as shown by 3D reconstruction and histology. The LTP seemed to have more severe OA than the medial side (Fig. ), which might be due to abnormal mechanical force on the lateral side of the knee caused by the subluxated/dislocated patella. Osteophyte formation can be observed on the lateral side of the femoral condyle and TRAP staining showed highly increased osteoclast activity within the osteophyte which indicating increased bone remodeling (Fig. ). Of note, mice with tibia dislocation were not included in this study. Figure 6 Possible subluxation/dislocation of the pat...
Materials and Methods
All mice were housed on a 12-hour light/dark cycle with unrestricted access to standard mouse food and water. C57/Bl6 wild type mice were randomly designated into 2 groups: DMM and SHAM, and three termination time-points (2, 5 and 10 weeks post-surgery). DMM or sham surgery was performed when mice were 12 weeks of age. All animals were housed and used in accordance with Western University's Animal Care and Use Guidelines. The protocol was approved by the Animal Care and Veterinary Services of Western University (protocol# 2015-031). Surgery was performed on the left knee joint of mice as previously described,. A single dose of Amoxicillin (Novopharm, Toronto, Ontario, Canada) was injected subcutaneously right after the surgery to prevent joint infection (20 mg/kg). Buprenorphine (Schering-Plough, Herfordshire, UK) was administered twice (0 and 4 hours after surgery...
Histology
Both hind limbs were then put in 5% ethylenediaminetetraacetic acid (EDTA) in phosphate buffered saline (pH 7.0) for decalcification for 10-12 days after the µCT scan. Decalcified limbs were further processed and embedded in paraffin in frontal orientation, with serial sections taken at a thickness of 4 µm and dried in a 37 °C oven for 24 hours, then stored in room temperature. Histology was generally performed as described,. All sections for staining were deparaffinized in two changes of xylene, rehydrated in ethanol with decreasing concentration ending in water, and stained as follows. General histology of cartilage and bone was assessed in toluidine blue (TB, 0.04% toluidine blue in 0.2 M acetate buffer, pH 3.75-4.25, 5 min), Safranin-O and fast-green (0.02% fast green for 30 minutes, 1% acetic acid for 10 secon...
Measurement outputs
What raw and processed outputs should exist?
OA phenotype after DMM surgery was confirmed by histology using toluidine blue (TB) staining. Regional proteoglycan loss and chondrocyte clustering occurred on the medial compar...
- Raw artifact
- Per-run gait capture with paw placement, timing, and stride features for each animal
- Processed artifact
- Cleaned gait metrics table and recovery trend summary across timepoints
- Reported as
- Group comparisons of gait indices, stride metrics, or recovery curves
Mice were weighed and transferred to the Behavior Study Suite at Robarts Research Institute at Western University two days prior to gait acquisitions. Gait patterns and rel...
- Raw artifact
- Per-run gait capture with paw placement, timing, and stride features for each animal
- Processed artifact
- Cleaned gait metrics table and recovery trend summary across timepoints
- Reported as
- Group comparisons of gait indices, stride metrics, or recovery curves
Statistical analysis was performed in Graph Pad Prism 7. Median and 2.5-97.5 percentile were calculated for OARSI score, BMD (error bar means 95% confidence interval). Gai...
- Raw artifact
- Per-run gait capture with paw placement, timing, and stride features for each animal
- Processed artifact
- Cleaned gait metrics table and recovery trend summary across timepoints
- Reported as
- Group comparisons of gait indices, stride metrics, or recovery curves
Analysis plan
How should the outputs become interpretable results?
Acquisition
Capture run-level gait data for each animal and preserve the timepoint or treatment labeling.
inferred from protocolPreprocessing / cleaning
OA phenotype after DMM surgery was confirmed by histology using toluidine blue (TB) staining.
from paperScoring or quantification
Quantify the primary readouts for this experiment: OA phenotype after DMM surgery was confirmed by histology using toluidine blue (TB) staining. Regional proteoglycan loss and chondrocyte clustering occurred on the medial compar...; Mice were weighed and transferred to the Behavior Study Suite at Robarts Research Institute at Western University two days prior to gait acquisitions. Gait patterns and rel...; Statistical analysis was performed in Graph Pad Prism 7. Median and 2.5-97.5 percentile were calculated for OARSI score, BMD (error bar means 95% confidence interval). Gai....
from paperStatistical comparison
OA phenotype after DMM surgery was confirmed by histology using toluidine blue (TB) staining. Regional proteoglycan loss and chondrocyte clustering occurred on the medial compar...; Osteoarthritis Research Society International (OARSI) histopathology scoring system was applied to evaluate OA severity (Fig. ). On the medial compartments of the joint, t...; 3D reconstruction of joints from micro-CT analyses is shown in Fig.. Irregular bone surface and abnormal patellar shape (osteophyte formations) was noticed in DMM mice as...; Bone mineral density (BMD) of subchondral bone in the DMM model. At 2 weeks pot-surgery, BMDs of the MTP in DMM mice didn't showed statistical significant differences comp...
from paperReporting output
Report representative outputs alongside summary comparisons for OA phenotype after DMM surgery was confirmed by histology using toluidine blue (TB) staining. Regional proteoglycan loss and chondrocyte clustering occurred on the medial compar..., Mice were weighed and transferred to the Behavior Study Suite at Robarts Research Institute at Western University two days prior to gait acquisitions. Gait patterns and rel..., Statistical analysis was performed in Graph Pad Prism 7. Median and 2.5-97.5 percentile were calculated for OARSI score, BMD (error bar means 95% confidence interval). Gai....
inferred from protocolStructured statistical methods
OA phenotype after DMM surgery was confirmed by histology using toluidine blue (TB) staining. Regional proteoglycan loss and chondrocyte clustering occurred on the medial compar...; Osteoarthritis Research Society International (OARSI) histopathology scoring system was applied to evaluate OA severity (Fig. ). On the medial compartments of the joint, t...; 3D reconstruction of joints from micro-CT analyses is shown in Fig.. Irregular bone surface and abnormal patellar shape (osteophyte formations) was noticed in DMM mice as...; Bone mineral density (BMD) of subchondral bone in the DMM model. At 2 weeks pot-surgery, BMDs of the MTP in DMM mice didn't showed statistical significant differences comp...
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Evidence quotes (8)
OA phenotype after DMM surgery was confirmed by histology using toluidine blue (TB) staining. Regional proteoglycan loss and chondrocyte clustering occurred on the medial compartments of the joint at 2 weeks post-surgery, especially the medial tibial plateau (MTP) (Fig., white arrow). Collagen disorganization was also shown in this small region in adjacent sections by picrosirius red (PR) staining (Fig., yellow arrow). Cartilaginous tissue formed on the medial edge of tibia and stained strongly with TB at 2-week time-point in DMM mice (Fig., black arrow), with collagen fibril organization similar to that of articular cartilage (Fig., white arrow). 5 weeks post-surgery, mild to moderate cartilage damage can be found in the surgical joints of DMM mice. Cartilage erosion and fibrillation, as well as large osteophytes were noticed on the MTP. Proteoglycan loss and fibrillation were also present on the medial femoral condyle (MFC) (Fig., white arrow). Cartilage lesion on the MTP was deep into the calcified zone 10 weeks post-surgery(Fig. ). Endochondral ossification can be observed in the osteophyte with bone marrow formed within the osteophyt...
Osteoarthritis Research Society International (OARSI) histopathology scoring system was applied to evaluate OA severity (Fig. ). On the medial compartments of the joint, the tibial plateau developed earlier (starting at 2 weeks) and more severe OA compared to femoral condyle (starting at 5 weeks) (Fig. ) at the same time-points. Although not statistically significant and much milder, lateral compartments showed a similar trend (Fig. ).
TRAP positive cells were found in the subchondral bone of DMM mice starting at 5 weeks post-surgery, with most of these cells located at the site of osteophyte formation (Fig. ). Very little positive staining was found in the subchondral bone of the SHAM group, although the expected staining at growth plate was observed (Fig. ). Figure 3 Tartrate-resistant acid phosphatase (TRAP) staining of osteoclast activity after DMM surgery. Osteoclast activity in the subchondral bone appeared increased at 5- and 10-week following DMM surgery, mostly within the osteophyte (red rectangle), but not at 2-weeks. The expected staining at the growth plate was observed (black rectangle). Scale bar: 200 µm.
3D reconstruction of joints from micro-CT analyses is shown in Fig.. Irregular bone surface and abnormal patellar shape (osteophyte formations) was noticed in DMM mice as early as 2 weeks post-surgery (Fig., hollow arrow) and became more obvious at later time-points (Fig., hollow arrow). Osteophyte formation of the MTP was noticed at 5 and 10 weeks post-surgery, with those at 10 weeks being more severe (Fig., white arrow). In the coronal plane in the middle of the knee joint, subchondral bone of the MTP didn't show difference at 2 weeks in DMM Group compared to SHAM (Fig. ), but became denser at later time-points (Fig., circled). BMDs of all compartments are shown in Fig.. As seen in the coronal plane images, BMDs of DMM animals showed no statistical differences comparing to their SHAM controls at 2 weeks, with DMM animals displaying a larger spread of their BMDs (Fig. ). Clearer differences were detected between DMM and SHAM at both 5 and 10 weeks post-surgery, with increased BMDs in DMM animals at MTP (5 and 10 weeks) and MFC (10 weeks only) (Fig. ). While the lateral compartments showed no difference between tw...
Bone mineral density (BMD) of subchondral bone in the DMM model. At 2 weeks pot-surgery, BMDs of the MTP in DMM mice didn't showed statistical significant differences comparing to their SHAM controls. However, DMM mice showed a larger spread of BMD at 2 weeks post-surgery ( A ). Clearer differences were detected between DMM and SHAM at both 5 and 10 weeks post-surgery with higher BMDs in DMM mice. ( A ). BMDs of the MFC were higher in DMM mice 10 weeks after surgery ( B ). There was no significant differences found in the lateral compartments between DMM and SHAM at all time-points ( C, D ). Error bar stands for the 95% confidence interval.
In a few of our mice (5 of 27, 2 mice of 2-week time-point and 3 of 10-week time-point) that underwent DMM surgery, subluxation/dislocation of the patella was noted as early as 2 weeks post-surgery by micro-CT as well as histological staining (Fig. ). In these mice, joint condition deteriorated faster as shown by 3D reconstruction and histology. The LTP seemed to have more severe OA than the medial side (Fig. ), which might be due to abnormal mechanical force on the lateral side of the knee caused by the subluxated/dislocated patella. Osteophyte formation can be observed on the lateral side of the femoral condyle and TRAP staining showed highly increased osteoclast activity within the osteophyte which indicating increased bone remodeling (Fig. ). Of note, mice with tibia dislocation were not included in this study. Figure 6 Possible subluxation/dislocation of the patella after DMM surgery. Safranin-O staining showed the out-grown osteophyte stained strongly for GAG ( A ). TRAP staining demonstrated increased number and activity of osteoclasts ( B, D ). µCT showed out-grown osteophyte formation ( C, arrow) and deteriorated joint condition ( C ). Toluidine...
All mice were housed on a 12-hour light/dark cycle with unrestricted access to standard mouse food and water. C57/Bl6 wild type mice were randomly designated into 2 groups: DMM and SHAM, and three termination time-points (2, 5 and 10 weeks post-surgery). DMM or sham surgery was performed when mice were 12 weeks of age. All animals were housed and used in accordance with Western University's Animal Care and Use Guidelines. The protocol was approved by the Animal Care and Veterinary Services of Western University (protocol# 2015-031). Surgery was performed on the left knee joint of mice as previously described,. A single dose of Amoxicillin (Novopharm, Toronto, Ontario, Canada) was injected subcutaneously right after the surgery to prevent joint infection (20 mg/kg). Buprenorphine (Schering-Plough, Herfordshire, UK) was administered twice (0 and 4 hours after surgery) subcutaneously to minimized pain (0.05 mg/kg). Mice were housed in colony cage after the surgery (up to 6 mice in one cage) and running wheels were put in two days after the surgery to encourage exercise. Excluding the mice that had dislocated patella after surgery (discussed in Results and...
Both hind limbs were then put in 5% ethylenediaminetetraacetic acid (EDTA) in phosphate buffered saline (pH 7.0) for decalcification for 10-12 days after the µCT scan. Decalcified limbs were further processed and embedded in paraffin in frontal orientation, with serial sections taken at a thickness of 4 µm and dried in a 37 °C oven for 24 hours, then stored in room temperature. Histology was generally performed as described,. All sections for staining were deparaffinized in two changes of xylene, rehydrated in ethanol with decreasing concentration ending in water, and stained as follows. General histology of cartilage and bone was assessed in toluidine blue (TB, 0.04% toluidine blue in 0.2 M acetate buffer, pH 3.75-4.25, 5 min), Safranin-O and fast-green (0.02% fast green for 30 minutes, 1% acetic acid for 10 seconds, and 1.5% Safranin O for 3 minutes). Collagen was stained with picrosirius red (PR, 0.1% Sirius red in saturated picric acid solution, 60 min; 0.5% acetic acid washes) and polarized microscope was used to evaluate the size and organization of collagen fibrils of articular cartilage an...
Machine-readable layer
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"text": "OA phenotype after DMM surgery was confirmed by histology using toluidine blue (TB) staining. Regional proteoglycan loss and chondrocyte clustering occurred on the medial compartments of the joint at 2 weeks post-surgery, especially the medial tibial plateau (MTP) (Fig., white arrow). Collagen disorganization was also shown in this small region in adjacent sections by picrosirius red (PR) staining (Fig., yellow arrow). Cartilaginous tissue formed on the medial edge of tibia and stained strongly with TB at 2-week time-point in DMM mice (Fig., black arrow), with collagen fibril organization similar to that of articular cartilage (Fig., white arrow). 5 weeks post-surgery, mild to moderate cartilage damage can be found in the surgical joints of DMM mice. Cartilage erosion and fibrillation, as well as large osteophytes were noticed on the MTP. Proteoglycan loss..."
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"name": "Histological analyses of joint changes after DMM surgery",
"text": "Osteoarthritis Research Society International (OARSI) histopathology scoring system was applied to evaluate OA severity (Fig. ). On the medial compartments of the joint, the tibial plateau developed earlier (starting at 2 weeks) and more severe OA compared to femoral condyle (starting at 5 weeks) (Fig. ) at the same time-points. Although not statistically significant and much milder, lateral compartments showed a similar trend (Fig. )."
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"text": "TRAP positive cells were found in the subchondral bone of DMM mice starting at 5 weeks post-surgery, with most of these cells located at the site of osteophyte formation (Fig. ). Very little positive staining was found in the subchondral bone of the SHAM group, although the expected staining at growth plate was observed (Fig. ). Figure 3 Tartrate-resistant acid phosphatase (TRAP) staining of osteoclast activity after DMM surgery. Osteoclast activity in the subchondral bone appeared increased at 5- and 10-week following DMM surgery, mostly within the osteophyte (red rectangle), but not at 2-weeks. The expected staining at the growth plate was observed (black rectangle). Scale bar: 200 µm."
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"name": "Subchondral bone changes after DMM surgery",
"text": "3D reconstruction of joints from micro-CT analyses is shown in Fig.. Irregular bone surface and abnormal patellar shape (osteophyte formations) was noticed in DMM mice as early as 2 weeks post-surgery (Fig., hollow arrow) and became more obvious at later time-points (Fig., hollow arrow). Osteophyte formation of the MTP was noticed at 5 and 10 weeks post-surgery, with those at 10 weeks being more severe (Fig., white arrow). In the coronal plane in the middle of the knee joint, subchondral bone of the MTP didn't show difference at 2 weeks in DMM Group compared to SHAM (Fig. ), but became denser at later time-points (Fig., circled). BMDs of all compartments are shown in Fig.. As seen in the coronal plane images, BMDs of DMM animals showed no statistical differences comparing to their SHAM controls at 2 weeks, with DMM animals displayi..."
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"name": "Subchondral bone changes after DMM surgery",
"text": "Bone mineral density (BMD) of subchondral bone in the DMM model. At 2 weeks pot-surgery, BMDs of the MTP in DMM mice didn't showed statistical significant differences comparing to their SHAM controls. However, DMM mice showed a larger spread of BMD at 2 weeks post-surgery ( A ). Clearer differences were detected between DMM and SHAM at both 5 and 10 weeks post-surgery with higher BMDs in DMM mice. ( A ). BMDs of the MFC were higher in DMM mice 10 weeks after surgery ( B ). There was no significant differences found in the lateral compartments between DMM and SHAM at all time-points ( C, D ). Error bar stands for the 95% confidence interval."
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"name": "Subluxation/dislocation of patella after DMM",
"text": "In a few of our mice (5 of 27, 2 mice of 2-week time-point and 3 of 10-week time-point) that underwent DMM surgery, subluxation/dislocation of the patella was noted as early as 2 weeks post-surgery by micro-CT as well as histological staining (Fig. ). In these mice, joint condition deteriorated faster as shown by 3D reconstruction and histology. The LTP seemed to have more severe OA than the medial side (Fig. ), which might be due to abnormal mechanical force on the lateral side of the knee caused by the subluxated/dislocated patella. Osteophyte formation can be observed on the lateral side of the femoral condyle and TRAP staining showed highly increased osteoclast activity within the osteophyte which indicating increased bone remodeling (Fig. ). Of note, mice with tibia dislocation were not included in this study. Figure 6 Possible subluxation/dislocation of the pat..."
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"text": "All mice were housed on a 12-hour light/dark cycle with unrestricted access to standard mouse food and water. C57/Bl6 wild type mice were randomly designated into 2 groups: DMM and SHAM, and three termination time-points (2, 5 and 10 weeks post-surgery). DMM or sham surgery was performed when mice were 12 weeks of age. All animals were housed and used in accordance with Western University's Animal Care and Use Guidelines. The protocol was approved by the Animal Care and Veterinary Services of Western University (protocol# 2015-031). Surgery was performed on the left knee joint of mice as previously described,. A single dose of Amoxicillin (Novopharm, Toronto, Ontario, Canada) was injected subcutaneously right after the surgery to prevent joint infection (20 mg/kg). Buprenorphine (Schering-Plough, Herfordshire, UK) was administered twice (0 and 4 hours after surgery..."
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"name": "Histology",
"text": "Both hind limbs were then put in 5% ethylenediaminetetraacetic acid (EDTA) in phosphate buffered saline (pH 7.0) for decalcification for 10-12 days after the µCT scan. Decalcified limbs were further processed and embedded in paraffin in frontal orientation, with serial sections taken at a thickness of 4 µm and dried in a 37 °C oven for 24 hours, then stored in room temperature. Histology was generally performed as described,. All sections for staining were deparaffinized in two changes of xylene, rehydrated in ethanol with decreasing concentration ending in water, and stained as follows. General histology of cartilage and bone was assessed in toluidine blue (TB, 0.04% toluidine blue in 0.2 M acetate buffer, pH 3.75-4.25, 5 min), Safranin-O and fast-green (0.02% fast green for 30 minutes, 1% acetic acid for 10 secon..."
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"datePublished": "2018",
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"name": "Hang Fang"
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"name": "Frank Beier"
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"name": "Daozhang Cai"
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"identifier": "10.1038/s41598-018-21184-5"
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