Table 1.
Scoring system for collagen type II immunohistochemistry.
Table 2.
Scoring system for MMP-13 immunohistochemistry.
Table 3.
Scoring system for ICAM-1 and DIPEN immunohistochemistry.
Figure 1.
Histomorphometric comparison of knee joint morphology between non-operated Mc1re/e and WT mice.
A) Overview of Safranin O/Fast green stained frontal section of a right mouse knee joint from a non-operated 11 week old Mc1re/e mouse (40× magnification). Lateral femoral condyle (LFC), lateral tibial plateau (LTP) and lateral meniscus (LM) as well as medial femoral condyle (MFC), medial tibial plateau (MTP) and medial meniscus (MM) are labeled. For osteoarthritis induction with DMM, the medial meniscotibial ligament is transected. B–E) Histological evaluation of cartilage area was performed with Safranin O/Fast green stained frontal sections of right knee joints from non-operated 11 weeks old and 6 months old WT and Mc1re/e mice. B+D) Medial, tibial (dotted line) and femoral (broken line) Safranin O stained cartilage was circuited with Bamboo tablet. C+E) Cartilage area of Mc1re/e mice was significantly smaller in 11 weeks (p = 0.0159) and 6 months old (p = 0.0079) animals compared to cartilage area of WT mice. For statistical analyses mean values of tibial and femoral cartilage areas of 5–6 section per knee joint was determined. Data are presented as boxplots reflecting the 25th and 75th percentile as boxes, the median as horizontal line and minimum and maximum values as whiskers. Bars = 500 µm, * p<0.05 and ** p<0.01 wild type vs. Mc1re/e.
Figure 2.
Assessment of cartilage degradation 4 and 8 weeks after osteoarthritis induction.
A-E) Frontal sections of knee joints from WT and Mc1re/e mice of non-operated (non-op) 11 week old animals, and of animals 4 and 8 weeks post operation (p.o.) by DMM were stained with Safranin O/Fast green. A) Representative images of the medial area from sections of right knee joints from non-operated 11 weeks old animals and from mice 4 and 8 weeks after osteoarthritis induction (p.o.) show disease progression over the time. B–E) 5–6 sections in 60–80 µm intervals of right knees (DMM, B, C) and left knees (Sham, D, E) were scored histological according to Glasson et al. [21] (B, D) and morphometrically (C, E). B) We observed OA-progression over time in WT (p = 0.0043) and Mc1re/e (p = 0.0095) mice compared to non-operated controls. Mc1re/e mice had higher scores 4 weeks (p = 0.0411) and 8 weeks (p = 0.0649) after OA-induction. C) Percentages of degraded cartilage area also indicated an OA-progression over time in WT (p = 0.0043) and Mc1re/e (p = 0.0095) compared to non-operated controls. Mc1re/e mice had more degraded cartilage 4 weeks (p = 0.0649) and 8 weeks (p = 0.0411) after OA-induction compared to WT animals. D+E) Sham operated knee joints showed similar scores (D) and area of degraded cartilage (E) compared to non-operated controls. Mean scores of medial tibia and femur were included in statistical analysis. Data are presented as box plots reflecting the 25th and 75th percentile as boxes, the median as horizontal line and minimum and maximum values as whiskers. White bars indicate wild type and grey bars indicate mutant group. Bars = 500 µm, §§ p<0.01 4/8 weeks post-surgery vs. non-operated, * p<0.05 wild type vs. Mc1re/e.
Figure 3.
Localization of MC1, collagen II, MMP-13 and ICAM-1 in knee joints of Mc1re/e and wild type mice.
A+C) Frontal sections of right knee joints from WT and Mc1re/e mice of non-operated (non-op) 11 weeks old and 6 months old animals, and of mice 4 and 8 weeks after osteoarthritis induction (post operation (p.o.)) were stained with antibodies against MC1, collagen II, MMP-13 and ICAM-1 as described in material and methods. Representative pictures of medial parts of the knee joints from each time point are shown. Bars = 200 µm.
Figure 4.
Quantitative scoring of collagen II, MMP-13 and ICAM-1 immunohistochemistry.
Medial parts of frontal sections of right knee joints from WT and Mc1re/e mice of non-operated (non-op) 11 weeks old and 6 months old animals, and of mice 4 and 8 weeks after osteoarthritis induction (post operation (p.o.)) were scored after staining. 1-3 sections of right knees (DMM) were scored histological according to the scoring system illustrated in Tab. 1 for collagen II, in Tab. 2 for MMP-13 and in Tab. 3 for ICAM-1 staining. A) Compared to non-operated controls collagen II stained cartilage area of WT knee joints is decreased 4 weeks (p = 0.0079) and 8 weeks (p = 0.0043) after OA-induction as well as in non-operated 6 month old animals (p = 0.0159). Sections of Mc1re/e knee joints showed no difference over time. Collagen II stained cartilage area of non-operated 11 weeks old Mc1re/e mice (p = 0.0159) and Mc1re/e mice 4 weeks (p = 0.0159) after OA induction is decreased compared to WT mice. B) Compared to non-operated controls number of MMP-13 positive non-hypertrophic articular chondrocytes of WT knee joints is increased 8 weeks (p = 0.0022) after OA-induction as well as in non-operated 6 month old animals (p = 0.0076). Sections of Mc1re/e knee joints showed no difference over time. MMP-13 positive chondrocytes of non-operated 11 weeks old Mc1re/e mice (p = 0.0079) and Mc1re/e mice 4 weeks (p = 0.035) after OA induction are increased compared to WT mice. C) Compared to non-operated controls number of ICAM-1 positive articular chondrocytes of Mc1re/e and WT knee joints are decreased 4 weeks (Mc1re/e: p = 0.0159; WT: p = 0.0381) and 8 weeks (Mc1re/e: p = 0.0159; WT: p = 0.0159) after OA-induction as well as in non-operated 6 month old animals (Mc1re/e: p = 0.0317; WT: p = 0.0159). There is no difference between WT and Mc1re/e regarding the number of ICAM-1 positive chondrocytes in non-operated controls whereas Mc1re/e mice 4 (p = 0.0303) and 8 weeks (p = 0.0079) after OA-induction showed lower numbers of ICAM-1 positive cells. Mean scores of medial tibia and femur were included in statistical analysis. Data are presented as box plots reflecting the 25th and 75th percentile as boxes, the median as horizontal line and minimum and maximum values as whiskers. §§ p<0.01 4/8 weeks post-surgery vs. non-operated, § p<0.05 4/8 weeks post-surgery vs. non-operated, ** p<0.01 wild type vs. Mc1re/e, * p<0.05 wild type vs. Mc1re/e.
Figure 5.
Comparison of bone architecture between Mc1re/e and WT mice at the day of DMM surgery (day 0) using µCT.
A–F) µCT 3D analysis of bone parameters was performed within a defined VOI including the tibia epiphysis of both knees respectively. A) Frontal view on 3D model of the proximal tibia of the right leg showing the VOI marked in purple (L: lateral, M: medial). B) µCT analysis of mean grayscale within the VOI as an indicator for bone density indicated no difference between WT and Mc1re/e mice. C) 3D calculation of BV/TV within the VOI as an indicator of bone mass also revealed no difference between both groups. In Trabecular number (Tb.N, D) and trabecular thickness (Tb.Th, E) no difference was observed whereas trabecular separation (Tb.Sp, F) was increased in Mc1re/e compared to controls (p = 0.028). Values of left and right knees were combined. Data are presented as boxplots reflecting the 25th and 75th percentile as boxes, the median as horizontal line and minimum and maximum values as whiskers, stars indicating extreme outliers. * p<0.05 wild type vs. Mc1re/e.
Figure 6.
Radiographic evaluation of osteophytes at 4 and 8 weeks past OA induction.
A–C) Knees were oriented as described in material and methods section and were analyzed for osteophytes (white arrow) in transaxial (A), sagittal (B) and coronal (C) plane. F: femur, T: tibia, Fib: fibula, L: lateral and M: medial. D) At 4 and 8 weeks the number and size of osteophytes was calculated for the right and the left knee of each animal, respectively. 4 of 5 Mc1re/e mice developed osteophytes whereas only 2 from 7 WT mice developed one osteophyte after OA-induction.
Figure 7.
Alteration of bone density and bone volume at 4 and 8 weeks past OA induction.
A–B) Reconstructed coronal image of Mc1re/e knee joints at the day of OA-surgery (day 0) (A) and 8 weeks after OA-surgery (B). White arrow marks an area of increased mineral deposition (L: lateral, M: medial). C) µCT analysis of mean grayscale within the VOI of the right knees as an indicator for bone density revealed an increase in bone densitiy 4 (p = 0.043) and 8 weeks (p = 0.043) after OA-indution in Mc1re/e mice and 8 weeks (p = 0.018) after OA-induction in WT animals. Mc1re/e mice showed a higher bone density 4 (p = 0.007) and 8 weeks (p = 0.042) after OA-induction compared to WT mice. D) In Mc1re/e mice but not in WT animals, BV/TV within the VOI of the right knee as an indicator of bone mass was increased at both time points after OA-induction compared to day 0 (p = 0.043). There was a higher bone mass in the epiphysis of Mc1re/e mice 4 (p = 0.007) and 8 weeks (p = 0.004) after OA-induction compared to WT animals. Data were normalized to the values at day 0 (day of surgery, (set as 1, grey dotted line). Data are presented as boxplots reflecting the 25th and 75th percentile as boxes, the median as horizontal line and minimum and maximum values as whiskers with dots indicating outliers and stars indicating extreme outliers. § p<0.05 4/8 weeks post-surgery vs. day 0, * p<0.05 and ** p<0.01 wild type vs. mutant.
Figure 8.
Alteration of bone architecture after OA induction.
µCT 3D-analysis of trabecular parameters in the right knee joint (DMM, A–C) and the left knee joint (Sham, D–F) depicted as trabecular number (Tb.N, A,D), trabecular thickness (Tb.Th, B,E) and trabecular separation (Tb.Sp, C,F). A) Tb.N declined in WT (p = 0.018) and Mc1re/e (p = 0.043) at both time points after OA-induction compared to day 0 with no differences observed between both groups. B) Compared to day 0 animals Tb.Th was increased in Mc1re/e mice at both time points (p = 0.043) after OA-induction whereas WT mice showed an increase only 8 weeks after operation (p = 0.018). Mc1re/e mice had thicker trabeculae at both time points compared to WT animals (p = 0.004) C) Th.Sp was decreased in Mc1re/e animals reaching significance 8 weeks after OA-induction compared to day 0 mice (p = 0.043). At both time points, Mc1re/e animals developed less Th.Sp compared to WT group (p = 0.019). Mild alterations in trabecular structure also occurred in sham operated contra-lateral knees in both groups but to a significantly lesser extend compared to destabilized knees. Data were normalized to the values at day 0 (day of surgery, set as 1, grey dotted line). Data are presented as box plots reflecting the 25th and 75th percentile as boxes, the median as horizontal line and minimum and maximum values as whiskers with dots indicating outliers and stars indicating extreme outliers. White bars indicate wild type and grey bars indicate Mc1re/e group. § p<0.05 4/8 weeks post-surgery vs. day 0, * p<0.05 and ** p<0.01 wild type vs. mutant.