Expansion of the sagittal suture induces proliferation of skeletal stem cells and sustains endogenous calvarial bone regeneration

Abstract

In newborn humans, and up to approximately 2 y of age, calvarial bone defects can naturally regenerate. This remarkable regeneration potential is also found in newborn mice and is absent in adult mice. Since previous studies showed that the mouse calvarial sutures are reservoirs of calvarial skeletal stem cells (cSSCs), which are the cells responsible for calvarial bone regeneration, here we hypothesized that the regenerative potential of the newborn mouse calvaria is due to a significant amount of cSSCs present in the newborn expanding sutures. Thus, we tested whether such regenerative potential can be reverse engineered in adult mice by artificially inducing an increase of the cSSCs resident within the adult calvarial sutures. First, we analyzed the cellular composition of the calvarial sutures in newborn and in older mice, up to 14-mo-old mice, showing that the sutures of the younger mice are enriched in cSSCs. Then, we demonstrated that a controlled mechanical expansion of the functionally closed sagittal sutures of adult mice induces a significant increase of the cSSCs. Finally, we showed that if a calvarial critical size bone defect is created simultaneously to the mechanical expansion of the sagittal suture, it fully regenerates without the need for additional therapeutic aids. Using a genetic blockade system, we further demonstrate that this endogenous regeneration is mediated by the canonical Wnt signaling. This study shows that controlled mechanical forces can harness the cSSCs and induce calvarial bone regeneration. Similar harnessing strategies may be used to develop novel and more effective bone regeneration autotherapies.

Keywords: Prrx1; Prx1; SSC; calvarial skeletal stem cells; calvarial sutures.

Fig. 1.

Single cell RNA-sequencing analysis of 4-d-old, 2-mo-old, 4-mo-old, and 14-mo-old calvarial sutures. (A–D) Uniform Manifold Approximation and Projection (UMAP) plot showing unbiased graph-based clusters distribution of all cell populations in sutures isolated from 4-d-old (A), 2-mo-old (B), 4-mo-old (C), and 14-mo-old (D) mice. (E) Quantification of osteogenic and non-osteogenic cell lineages (absolute numbers counted in each sample and percentages of the total cells within each sample). (F–I) UMAPs displaying the expression of Prx1/Prrx1, Ctsk, Gli1, and Axin2 in calvarial sutures of 4-d-old (F), 2-mo-old (G), 4-mo-old (H), and 14-mo-old (I) mice. (J) Quantification of cells expressing Prx1/Prrx1, Ctsk, Gli1, and Axin2 (absolute numbers counted in each sample and percentages of the total cells within each sample). 4-d-old mice: total cells 2,293 (n = 8); 2-mo-old mice: total cells 3,159 (n = 5); 4-mo-old mice: total cells 6,308 (n = 5); 14-mo-old mice; total cells 5,064 (n = 6).

Fig. 2.

Subcluster analysis of the osteogenic cells of the expanding sutures of 4-d-old mice. (A) UMAP plot showing the identity of the subclusters identified among the osteogenic lineage cells. (B) UMAP plots showing the location of cells expressing Prx1/Prrx1, Ctsk, Gli1, and Axin2. (C) Unbiased trajectory pseudotime analysis of the cells of the osteogenic lineage, from the most undifferentiated (yellow) to the most differentiated (pink). (D) Trajectory analysis identifying Prx1/Prrx1 expressing cells, Alkaline phosphatase (Alpl) expressing cells, Bone sialoprotein (Ibsp) expressing cells, Osterix (Sp7) expressing cells, Collagen type 1(Col1a1) expressing cells, and Osteocalcin (Bglap) expressing cells.

Fig. 3.

Expansion of the sagittal suture increases the number of suture resident cSSCs. (A) Expansion device. An expansion device made of an orthodontic nickel-titanium wire (0.3 mm in diameter), able to deliver an initial 0.2 N of tensile force, is inserted into two equidistant holes created 2 mm from the sagittal suture, in the parietal bones of 2-mo-old mice. After insertion, the calvarial skin is repositioned over the device and the wound is closed with resorbable sutures. In control mice of the same age, holes are created but the expansion device is not inserted (mock surgery). (B) Suture isolation. 7 d post-surgery, non-expanded, and expanded calvarial sutures are dissected and cells are isolated. (C) Histological evaluation. 7 d post-surgery, coronal (frontal) sections of the sagittal suture show that the applied tensile force increased the sagittal suture width (black arrows indicate the newly formed tissue between the two osteogenic fronts). (D) scRNA-seq analysis. Uniform Manifold Approximation and Projection (UMAP) plot showing unbiased graph-based clusters distribution of all cell populations in the non-expanded and expanded sutures isolated from 2-mo-old animals (six mice/group). (E) Quantification of osteogenic cells: scRNA-seq data is interrogated to quantify osteogenic and non-osteogenic cells of the expanded and non-expanded sutures (absolute numbers counted in each sample and percentages of the total cells within each sample). (F) Quantification of cells expressing specific genes: scRNA-seq data is interrogated to quantify cells expressing Prx1/Prrx1, Ctsk, Gli1, Axin2, Runx2, Sp7 (Osterix), or Osteocalcin (Bglap) of the expanded and non-expanded sutures (absolute numbers counted in each sample and percentages of the total cells within each sample). (G) IVM evaluated areas. IVM is utilized to image cells expressing EGFP (co-expressing Prx1/Prrx1) in three equidistantly distributed regions (A–C) along the total length of the sagittal suture of Prx1-creER-EGFP 2-mo-old mice. (H) IVM visualization of EGFP-expressing cells 7 d post-surgery. (Left) IVM (maximum intensity projection) of the sagittal suture in non-expanded control animals (coronal view). (Right) IVM maximum intensity projection of the sagittal suture in expanded animals (superior view and coronal view). Dashed lines demarcate the sagittal suture space (S.S.) (note the different scale bars in the non-expanded and expanded images). Left parietal (L.P.) and right parietal (R.P.) bone is visualized by second harmonic generation (blue). Prx1/Prrx1+ cells are visualized by expression of EGFP (green). (I) IVM quantification. 7 d post-surgery, the number of cells expressing EGFP (co-expressing Prx1/Prrx1) are quantified in control and expanded sutures (n = 4 to 5, averages of total cells counted in regions A, B, and C) (**P < 0.01).

Fig. 4.

Expansion of the sagittal suture induces expression of Birc5, Ccnd1, Espl1, or Ki67 in Prx1/Prrx1 expressing cells. (A) scRNA-seq quantification of the expression of Birc5, Ccnd1, Espl1, in Prx1/Prrx1 expressing cells of non-expanded and expanded sutures. (B) scRNA-seq quantification of the expression of Birc5, Ccnd1, Espl1, in Runx2 expressing cells of non-expanded and expanded sutures. (C) scRNA-seq quantification of the expression of Birc5, Ccnd1, Espl1, in Sp7 expressing cells of non-expanded and expanded sutures. (D) scRNA-seq quantification of the expression of Birc5, Ccnd1, Espl1, in Osteocalcin expressing cells of non-expanded and expanded sutures. Dots represent single cells and numerical values on the y axes indicate the level of expression of the Birc5, Ccnd1, Espl1, or Ki67 gene. *P < 0.05 (six mice/group).

Fig. 5.

Subcluster analysis of the osteogenic cells of the mechanically expanded sutures. (A) UMAP plot showing the identity of the subclusters identified among the osteogenic lineage cells. (B) UMAP plots showing the location of cells expressing Prx1/Prrx1, Ctsk, Gli1, and Axin2. (C) Unbiased trajectory pseudotime analysis of the cells of the osteogenic lineage, from the most undifferentiated (yellow) to the most differentiated (pink). (D) Trajectory analysis identifying Prx1/Prrx1 expressing cells, Bone sialoprotein (Ibsp) expressing cells, Collagen type 1(Col1a1) expressing cells, and Osteocalcin (Bglap) expressing cells.

Fig. 6.

Subcluster analyses of the osteogenic cells of the mechanically expanded and the naturally expanding sutures. (A) UMAP plot showing the identity of the subclusters identified among the osteogenic lineage cells of both samples. (B) UMAP plots showing the location of cells of the mechanically expanded sutures (EXPANDED, in red) and of the naturally expanding sutures (4 d old, in blue).

Fig. 7.

Expansion of the sagittal suture enhances the regeneration of a c-CSD remotely located from the suture. (A) µCT rendering (left, whole skull superior view, with cranial base bones visible through the defect; right, coronal (frontal) section of the parietal bones) and histological sections of the sagittal suture (depicted by green-dashed line) and of the defect (depicted by blue-dashed line) in non-expanded 2-mo-old Prx1-creER-EGFP^+/− mice. (B) µCT rendering (left, whole skull superior view; right, coronal (frontal) section of the parietal bones) and histological sections of the sagittal suture (depicted by green-dashed line) and of the defect (depicted by blue-dashed line) in expanded 2-mo-old Prx1-creER-EGFP^+/− mice. Samples were obtained 60 d after creation of the defects. Red arrows indicate the c-CSD, located 3 mm lateral to the sagittal suture. Blue arrows indicate the sagittal sinus. The implanted radiopaque expansion device can be seen in the coronal view. H&E and Goldner’s Trichrome were used for staining unmineralized tissue sections. (C and D) µCT quantification of bone volume (BV) and of the ratio between bone volume (BV) total volume (TV) regenerated within the c-CSDs, 60 d after surgery (n = 4 to 5, **P < 0.01).

Fig. 8.

Regeneration of c-CSDs sustained by mechanical expansion of the sagittal suture is mediated by Wnt signaling. (A) µCT rendering (whole skull superior view and coronal (frontal) section of the parietal bones) and histological sections of the sagittal suture (depicted by green-dashed line) and of the c-CSD (depicted by blue-dashed line) in suture expanded 2-mo-old Prx1-creER-EGFP^+/−; β-catenin^+/+ mice 60 d after creation of the defect. (B) µCT rendering (whole skull superior view with cranial base bones visible through the defect, and coronal (frontal) section of the parietal bones) and histological sections of the sagittal suture (depicted by green-dashed line) and of the c-CSD (depicted by blue-dashed line) in suture expanded 8-wk-old Prx1-creER-EGFP^+/−; β-catenin^−/− mice 60 d after creation of the defect. Red arrows indicate the c-CSD, located 3 mm lateral to the sagittal suture. Blue arrows indicate the sagittal sinus. (C) µCT quantification of the regenerated bone volume (BV) in c-CSDs 60 d after surgery (n = 5). (D) µCT quantification of regenerated bone volume (BV)/total volume (TV) in c-CSDs 60 d after surgery (n = 5). (E) qPCR of canonical Wnt signaling responsive genes (Axin2 and β-catenin (Ctnnb1)) in Prx1/Prrx1 expressing cells isolated from Prx1-creER-EGFP^+/−; β-catenin^+/+ and Prx1-creER-EGFP^+/−; β-catenin^−/− mice treated with tamoxifen (six pooled mice/group, two technical replicates/group). All mice were treated with tamoxifen for 10 d, starting 5 d before surgery. *P < 0.05, **P < 0.01.

Fig. 9.

PRX1/PRRX1 is expressed in human calvarial sutures. (A) In situ hybridization in the sagittal suture of human fetal calvaria (Day 80 post-conception). (Top) Negative control targeting DapB bacterial gene (DapB-AF647) demonstrates no detectable signal. (Middle) Positive control targeting POLYUBIQUITIN-C (UBC-AF647) is detected in most of the cells. (Bottom) Sections stained with the probe targeting human PRX1/PRRX1 (PRRX1-AF647) present with signal in a discrete number of suture cells (see 2× Inset). Dashed lines (Left) identify calvarial bones. (B) Quantitative RT-PCR analysis of gene expression in primary cells isolated from the parietal bone and from the sagittal suture of human fetal calvaria. Quantitative PCR was performed to assess the expression of PRX1/PRRX1 in primary cells derived from the parietal bone (P Bone) and from the sagittal suture (Sag Sut). Post-conception age and sex of the fetus are shown in parentheses.