Reduced bone loss in a murine model of postmenopausal osteoporosis lacking complement component 3

Abstract

The growing field of osteoimmunology seeks to unravel the complex interdependence of the skeletal and immune systems. Notably, we and others have demonstrated that complement signaling influences the differentiation of osteoblasts and osteoclasts, the two primary cell types responsible for maintaining bone homeostasis. However, the net effect of complement on bone homeostasis in vivo was unknown. Our published in vitro mechanistic work led us to hypothesize that absence of complement component 3 (C3), a central protein in the complement activation cascade, protects against bone loss in the ovariectomy-based model of postmenopausal osteoporosis. Indeed, we report here that, when compared to their C57BL/6J (WT) counterparts, ovariectomized C3 deficient mice experienced reduced bone loss at multiple sites and increased stiffness at the femoral neck, the latter potentially improving mechanical function. WT and B6;129S4-C3^tm1Crr /J (C3^-/- ) mice were either ovariectomized or sham-operated at 6 weeks of age and euthanized at 12 weeks. MicroCT on harvested bones revealed that the trabecular bone volume fraction in the metaphyses of both the proximal tibiae and distal femora of ovariectomized C3^-/- mice is significantly greater than that of their WT counterparts. Lumbar vertebrae showed significantly greater osteoid content and mineral apposition rates. Mechanical testing demonstrated significantly greater stiffness in the femoral necks of ovariectomized C3^-/- mice. These results demonstrate that C3 deficiency reduces bone loss at ovariectomy and may improve mechanical properties. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:118-128, 2018.

Keywords: bone histomorphometry; bone microCT; complement cascade; osteoimmunology; osteoporosis.

Figure 1

Femoral necks were tested by monotonic loading to failure in a direction parallel to the femoral diaphysis.

Figure 2. Trabecular bone loss at the distal femur was greater in WT OVX

(A) BV/TV at the distal femur was significantly reduced in WT OVX relative to WT sham; however, BV/TV was not significantly different between C3^−/− cohorts. When presented relative to sham values, BV/TV was significantly greater in C3^−/− mice relative to WT mice, with a delta of 15%. (B, D) Tb.N and Tb.Sp were significantly different from sham in WT OVX cohorts; in C3^−/− OVX, significance was only achieved in Tb.N. Relative to sham values, Tb.N and Tb.Sp in the distal femora of C3^−/− mice were significantly greater and significantly decreased, respectively. (C) Tb.Th values of OVX cohorts did not differ significantly from those of sham cohorts. Left of the solid vertical line, data points represents the value determined for a given animal with the group mean and standard deviation shown. p values indicate only significant differences between treatment groups, as determined by 2-way ANOVA. Data points right of the solid vertical line reflect OVX data scaled to the mean sham value, with the delta indicating the difference between means of these two groups. p values indicate only significant differences as determined by t-test or nonparametric test. (E) 3-D reconstructions were made from scans of distal femora from samples nearest the mean.

Figure 3. Trabecular bone loss at the proximal tibia was slightly greater in WT OVX

Bone loss at the proximal tibia was significantly greater than sham in both ovariectomized cohorts as determined by BV/TV, Tb.N, Tb.Th, and Tb.Sp. (A) When presented relative to sham values, BV/TV was significantly lower, indicating greater bone loss, in WT OVX mice than in their C3^−/− OVX counterparts. (B-D) Scaled OVX values for Tb.N, Tb.Th, and Tb.Sp in the proximal tibia were not significantly different between groups. Left of the solid vertical line, data points represent the value determined for a given animal with group mean and standard deviation shown. p values indicate only significant differences between treatment groups, as determined by 2-way ANOVA. Data points right of the solid vertical line reflect OVX data scaled to the mean sham value, with the delta indicating the difference between means of these two groups. p values indicate only significant differences as determined by t-test or nonparametric test.

Figure 4. Endocortical erosion was increased in WT OVX but unchanged in C3^−/− OVX

(A) Bone loss at the endocortical surface was significantly greater in WT OVX, both relative to sham-operated counterparts and relative to C3^−/− OVX when scaled. (B) PV of the OVX cohorts were not significantly different from sham values; however, trends were in opposite directions. When scaled to sham, PV at the tibial midshaft was significantly greater in WT OVX when compared to C3^−/− OVX. Left of the solid vertical line, data points represent the value determined for a given animal with the group mean and standard deviation shown. p values indicate only significant differences between treatment groups, as determined by 2-way ANOVA. Data points right of the solid vertical line reflect OVX data scaled to the mean sham value, with the delta indicating the difference between means of these two groups. p values indicate only significant differences as determined by t-test or nonparametric test.

Figure 5. Femoral neck stiffness is greater in C3^−/− OVX

Mechanical testing demonstrated that stiffness at the femoral neck, when presented relative to sham values, was significantly greater in C3^−/− OVX, with a delta of 36%. No significant differences in stiffness were observed between sham and OVX cohorts; however, values of OVX cohorts trended in opposite directions relative to sham, and scaled calculations show a significant increase in stiffness for C3^−/− OVX over that of WT OVX, with a p value of 0.002. Left of the solid vertical line, data points represent the value determined for a given animal with group mean and standard deviation shown. p values indicate only significant differences between treatment groups, as determined by 2-way ANOVA. Data points right of the solid vertical line reflect OVX data scaled to the mean sham value, with the delta indicating the difference between means of these two groups. p values indicate only significant differences as determined by t-test or nonparametric test.

Figure 6. Osteoblast activity increases in C3^−/− OVX

(A–B) Relative to sham-operated mice, neither of the ovariectomized cohorts demonstrated a significant difference in either OS/BS or MS/BS. Because trends were opposite in direction, when presented relative to sham, MS/BS was significantly greater in C3^−/− OVX when compared to WT OVX. (C) MAR was significantly greater in C3^−/− OVX, while MAR trended down in WT OVX relative to sham-operated counterparts; the delta between % sham groups was 17%. (D) Despite this increased activity, N.Ob/T.Ar trended down in both OVX groups relative to their sham-operated counterparts. Left of the solid vertical line, data points represent the value determined for a given animal with group mean and standard deviation shown. p values indicate only significant differences between treatment groups, as determined by 2‐way ANOVA. Data points right of the solid vertical line reflect OVX data scaled to the mean sham value, with the delta indicating the difference between means of these two groups. p values indicate only significant differences as determined by t-test or nonparametric test. (E-H) Images of sections stained with Hematoxylin, Acid Fuchsin-Ponceau, and Toluidine blue show the variable levels of osteoid per bone surface between cohorts. (I-L) The difference in MAR between cohorts is qualitatively evident in epifluorescence microscopy of sections of representative L4 vertebrae.

Figure 7. Osteoclast number and activity are reduced in OVX cohorts

(A) At 6 weeks post-surgery, ES/BS of L4 vertebrae was significantly reduced in both OVX cohorts relative to their sham-operated counterparts. When scaled to sham, average ES/BS values for WT OVX and C3^−/− OVX are similar with a delta of only 2%. (B) L4 vertebral N.Oc/T.Ar is also significantly reduced in the OVX cohorts relative to their sham-operated counterparts. Scaled values yield a delta of only 6% between groups. (C, D) In distal femora, at 6 weeks post-surgery, both ES/BS and N.Oc/T.Ar were greater in the sham cohorts relative to their OVX counterparts; these trends were significant in all cases but in N.Oc/T.Ar of WT cohorts. Scaled values were not significantly different for either parameter. The delta for ES/BS was 1%, and the delta for N.Oc/T.Ar was 24%. Left of the solid vertical line, data points represent the value determined for a given animal with group mean and standard deviation shown. p values indicate only significant differences between treatment groups, as determined by 2‐way ANOVA. Data points right of the solid vertical line reflect OVX data scaled to the mean sham value, with the delta indicating the difference between means of these two groups. p values indicate only significant differences as determined by t-test or nonparametric test. (E-H) As indicated in 7B, representative images of TRAP stained sections of L4 vertebrae reveal far fewer osteoclasts in OVX cohorts than in sham cohorts. Black arrowheads highlight some of the red-stained osteoclasts in each field.