Multiparity and Aging Impact Chondrogenic and Osteogenic Potential at Symphyseal Enthesis: New Insights into Interpubic Joint Remodeling

Abstract

Pregnancy and childbirth cause adaptations to the birth canal to allow for delivery and fast recovery. To accommodate delivery through the birth canal, the pubic symphysis undergoes changes that lead to the interpubic ligament (IpL) and enthesis formation in primiparous mice. However, successive deliveries influence joint recovery. We aimed to understand tissue morphology and chondrogenic and osteogenic potential at symphyseal enthesis during pregnancy and postpartum in primiparous and multiparous senescent female mice. Morphological and molecular differences were found at the symphyseal enthesis among the study groups. Despite the apparent incapacity to restore cartilage in multiparous senescent animals, the symphyseal enthesis cells are active. However, these cells have reduced expression of chondrogenic and osteogenic markers and are immersed in densely packed collagen fibers contiguous to the persistent IpL. These findings may indicate alterations of key molecules in the progenitor cell population maintenance of the chondrocytic and osteogenic lineages at the symphyseal enthesis in multiparous senescent animals, possibly compromising the mouse joint histoarchitecture recovery. This sheds light on the distention of the birth canal and the pelvic floor that may play a role in pubic symphysis diastasis (PSD) and pelvic organ prolapse (POP), both in orthopedic and urogynecological practice in women.

Keywords: aging; cartilage; chondrogenesis; parity; pregnancy; pubic symphysis.

Figure 1

Representative cells and extracellular matrix (ECM) morphological aspects of symphyseal enthesis during pregnancy and postpartum in primiparous (A–H) and multiparous senescent (I-P) female mice at light and transmission electron microscopy. (A) At D12, pubic symphysis (PS) architecture is composed of the fibrocartilaginous disc (FC) between hyaline cartilage (HC). (B,C) At D18 and 3dpp, between pubic bones (PB) and interpubic ligament (IpL), see demarcated areas with dotted lines that approximately correspond to two distinct regions in the symphyseal enthesis: bone proximal region (BPR) with numerous oval cells next to each other and bone distal region (BDR) with elongated and sparse cells. (D) At 21dpp, the BPR and BDR of the enthesis are indistinguishable, and a similar non-pregnant joint morphology is restored. See the organization of cells around the PB and the isogenous groups (arrow). (I–L) Symphyseal enthesis of multiparous senescent mice with persistent IpL during pregnancy and postpartum. BDR and BPR are almost indistinguishable in demarcated areas with dotted lines. (E–H,M–P) Ultrastructure of symphyseal enthesis cell types and ECM aspects during pregnancy and postpartum in primiparous and multiparous senescent female mice. Observe the presence of a developed rough endoplasmic reticulum (RER) in the cytoplasm, nucleus (N), and collagen fiber (Col) arrangement. (A–D,I–L) Masson trichrome staining, scale bar = 40 μm. (E–H,M–P) Transmission electron microscopy, scale bar = 1 μm.

Figure 2

Arrangement of collagen fibers at conventional and polarized light microscopy of symphyseal enthesis in transverse sections during pregnancy and postpartum in primiparous (A–D) and multiparous senescent (E–H) female mice. (A) At D12, observe the transition of the fibrocartilaginous disc (FC) to hyaline cartilage (HC) at the pubic symphysis (PS) architecture. Specifically in the FC, observe the collagen fiber at a perpendicular orientation related to the PS. (B,C) By interpubic ligament (IpL) formation, note collagen fiber crimps at the bone proximal region (BPR) and bone distal region (BDR) (arrows and insets). (D) At 21dpp, at the restored HC, see the birefringence around cells in their lacunae with a noticeable basophilic territorial matrix (arrow) next to the pubic bone (PB). (E–H) Compare the arrangement of collagen fibers at the symphyseal enthesis in the transverse section during pregnancy and postpartum in the multiparous senescent female mice. Note the parallel organization of the birefringent collagen fibers and the presence of crimps (arrows and insets). Sirius Red and hematoxylin staining, scale bar = 40 μm.

Figure 3

Spatiotemporal gene expression of Col2a1 (A), Runx-2 (B), and Sox-9 (C) at the interpubic joint during pregnancy and postpartum remodeling in primiparous and multiparous senescent female mice by qPCR. Two-way ANOVA with Bonferroni test. Significance p-value < 0.05 (*), < 0.01 (**), < 0.001 (***), < 0.0001 (****). A.U.= arbitrary unit. N = 3 animals per group (D18, and 3dpp); 6 animals per group (D12, and 21dpp).

Figure 4

Immunopositive cells for Runx-2 at the symphyseal enthesis during pregnancy and postpartum in primiparous and multiparous senescent female mice. (A–D) Runx-2 immunostaining at hyaline cartilage (HC), fibrocartilage (FC), bone proximal region (BPR), bone distal region (BDR), and interpubic ligament (IpL). (E–H) Runx-2 immunostaining at the symphyseal enthesis and IpL. If distinguishable, demarcated areas with dotted lines correspond approximately to BPR and BDR. Immunohistochemistry (IHC) and hematoxylin staining. Scale bar = 40 μm.

Figure 5

Immunopositive cells for Sox-9 at the symphyseal enthesis during pregnancy and postpartum in primiparous and multiparous senescent female mice. (A–D) Sox-9 immunostaining at hyaline cartilage (HC), fibrocartilage (FC), bone proximal region (BPR), bone distal region (BDR), and interpubic ligament (IpL). (E–H) Sox-9 immunostaining at the symphyseal enthesis and IpL. If distinguishable, demarcated areas with dotted lines correspond approximately to BPR and BDR. Immunohistochemistry (IHC) and hematoxylin staining. Scale bar = 40 μm.