Cranial Suture Regeneration Mitigates Skull and Neurocognitive Defects in Craniosynostosis

Abstract

Craniosynostosis results from premature fusion of the cranial suture(s), which contain mesenchymal stem cells (MSCs) that are crucial for calvarial expansion in coordination with brain growth. Infants with craniosynostosis have skull dysmorphology, increased intracranial pressure, and complications such as neurocognitive impairment that compromise quality of life. Animal models recapitulating these phenotypes are lacking, hampering development of urgently needed innovative therapies. Here, we show that Twist1^+/- mice with craniosynostosis have increased intracranial pressure and neurocognitive behavioral abnormalities, recapitulating features of human Saethre-Chotzen syndrome. Using a biodegradable material combined with MSCs, we successfully regenerated a functional cranial suture that corrects skull deformity, normalizes intracranial pressure, and rescues neurocognitive behavior deficits. The regenerated suture creates a niche into which endogenous MSCs migrated, sustaining calvarial bone homeostasis and repair. MSC-based cranial suture regeneration offers a paradigm shift in treatment to reverse skull and neurocognitive abnormalities in this devastating disease.

Keywords: Twist1; calvarial deformity; mesenchymal stem cells; neurocognitive abnormalities; suture regeneration.

Figure 1.. Twist1^+/− mice with craniosynostosis exhibit increased intracranial pressure and cognitive behavioral abnormalities

(A) Diagram depicting the intracranial pressure measurement setup. (B, C) Representative intracranial pressure (ICP) traces (B) and quantification (C) of ICP values (WT, n=8; MUT, n=6 mice). (D, F, I) Schematics of rotarod test (D), novel object test (F), and three chamber test (I). (E) Rotarod performance scored as time (seconds) on the rotarod. (G, J) Representative animal tracks in novel object test (G) and three chamber test (J). (H, K) Preference indices in novel object test (H) and sociability and social novelty tests (K). WT, wild type mice, n=20; MUT, Twist1^+/− mice with bilateral suture fusion, n=20. Data are mean ± s.e.m. (C, E, H, K). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, NS, not significant calculated by two-tailed unpaired t-test.

Figure 2.. Suture MSCs and M-GM can support the regeneration of a cranial suture

(A-F) Overview of the calvarial surgery showing the calvaria before surgery (A, C) and after surgery (B, D). MicroCT of calvaria (C, D). White arrowheads indicate the residual hallmark of the fused suture (A, C, D). The defect on one side of the calvaria was filled with pure M-GM or left empty as a control, and the defect on the other side was filled with M-GM plus Gli1⁺ cells (E, F). Scale bar, 2 mm. (G-L) MicroCT images (3-D reconstruction and slice) for controls (G1, I1, K1 for blank, and H1, J1, L1 for pure M-GM) and M-GM plus suture MSCs (M-GM+SCs; H2, J2, L2) at one, three, and six months post-surgery. Dotted lines (white) outline the original surgical defects. Scale bar, 2 mm. (M) HE staining of blank control (left), M-GM (middle) and M-GM+SC (right) groups at one, three, and six months post-surgery. Outlined areas in M-GM+SC group are separately shown in right panels. Black arrowheads indicate the positions of initial defects, and asterisk shows suture-like structure in M-GM+SCs six months post-surgery. Scale bar, 200 μm. (N) Immunofluorescence staining for M-GM+SCs at one month (left), three months (middle), and six months post-surgery (right). Red fluorescently labeled cells were harvested from one-month-old Gli1-Cre^ERT2;tdT^fl/+ mice. White arrowheads indicate donor cells (red) in surrounding tissues; white dotted lines show boundaries of bones. Scale bar, 200 μm in upper panel; 25 μm in lower panel.

Figure 3.. Dura mater cells contribute to the regenerated sutures in Twist1^+/− mice

(A-D) Qtracker FITC (green) labeled dura mater cells migrate from the dura at day 0 (A) to the regenerated suture at four weeks post-surgery (B-D). Donor cells fluorescently labeled in red were harvested from one-month-old Gli1-Cre^ERT2;tdT^fl/+ mice. Gli1⁺ cells were labeled with Alexa-647 and arrowheads indicate the co-labeled signals (C, D). Scale bar, 50 μm in inset in (A), 200 μm in (A); 100 μm in (B); 50 μm in (D). (E) Schematic of dura mater blockage surgery. (F) MicroCT images (3-D reconstruction and slice), HE and immunofluorescence staining of the defects six weeks post-dura mater blocking. Black dotted lines in (F) outline the original surgical defects. (F2, F3) Asterisks indicate the Parafilm membrane (yellow dotted lines in F3); arrowheads indicate the dura mater. Scale bars, 500 μm in (F) and (F1); 100 μm in (F2) and (F3). (G, H) 3-D reconstructed microCT images of bone defects filled with M-GM+MSCs for kidney capsule transplantation one day (G) or six weeks post-surgery (H). HE and immunofluorescence staining of the explant are shown in the right panel. Scale bars, 1 mm in (H); 200 μm in (H1) and (H2). (I) Schematic drawing shows that M-GM+SCs might provide a niche that recruits endogenous dura mater cells into the regenerated suture, while donor cells also contribute to the self-renewal of surrounding tissues. Bone boundaries are outlined by brown lines.

Figure 4.. Regenerated sutures show similar gene expression profile and function to natural cranial sutures

(A-D) RNAseq analysis of normal sutures (WT), fused sutures (MUT), and the endogenous cells from regenerated sutures (REG). Gene expression profiles are shown with heatmap (A), t-SNE visualization of color-coded regions for the three groups (B), volcano plots indicating the number of differentially expressed genes (C), and gene signatures of Gli1, Prrx1, Axin2, Msx2, and Ostn based on the relative expression levels of WT, MUT, and REG (D). (E) The osteogenic (upper panel, alizarin red staining in inset), chondrogenic (middle panel, whole mount immunostaining and section staining in inset), and adipogenic (lower panel) differentiation ability of cells in the regenerated suture. (F-K) The ability of the regenerated suture to repair bone at one day (F-H) or three months post-injury (I-K). MicroCT slices of blank (G, J) and M-GM+SCs (H, K) sides. Red fluorescently labeled cells were from donor mice (K1, K2). Asterisks indicate injuries in parietal bones (G, H, J, K); arrowheads in (H, K) indicate the regenerated sutures. Scale bars, 2 mm in (F), (I); 1 mm in (G), (H), (J) and (K); 100 μm in (J1, J2) and (K1, K2). (L-S) Visualization one day (L-N) and three months (O-Q) after transplantation of regenerated sutures or parietal bones without sutures (non-suture transplant) dissected from CAG-EGFP mice. MicroCT slices of non-suture transplant group (M, P) and transplants with sutures (N, Q). (Q1) Green fluorescently labeled tissues were from CAG-EGFP mice, while red cells were from donor Gli1-Cre^ERT2;tdT^fl/+ mice. Arrowheads indicate the regenerated sutures (L, N, O, Q). (R) White dotted lines outline the original size of the transplants, while yellow dotted lines indicate the boundary of the regenerated tissue (R). (S), Quantification of the fold change of the transplant surface area from (R). Scale bars, 2 mm in (L), (O), (R); 1 mm in (M), (N), (P) and (Q); 200 μm in (Q1). Data are mean ± s.e.m. (S). **P < 0.01, calculated by independent two-tailed Student’s t-test.

Figure 5.. Regenerated sutures rescue skull deformity in Twist1^+/− mice with craniosynostosis

(A, C) Colored dots indicate landmarks (shown in Figures S7A-S7C) of the top calvarium (T-Calvarium) and lateral calvarium (L-Calvarium) shape in all three groups: multiple skull views of normal mice (WT, n=7, blue), Twist1^+/− mice with bilateral coronal suture fusion (MUT, n=6, red), and Twist1^+/− mice with bilateral coronal suture regeneration (REG, n=5, green). (B, D) Wireframe deformations representing the shape differences between WT, MUT and REG groups for each region. (E, F) Total variation between WT, MUT and REG groups was determined by principal component analysis (PCA) for both shape regions. Results of discriminant function analysis (DFA) of three groups for each shape region: top calvarium (E1-E3) and lateral calvarium (F1-F3). Procrustes distance (PD) and P-value (*P < 0.05, **P < 0.01) were analyzed for every comparison.

Figure 6.. Suture regeneration normalizes intracranial pressure and partially restores neurocognitive function in Twist1^+/− mice with craniosynostosis

(A, B) Representative intracranial pressure (ICP) traces (A) and quantification (B) of ICP values (WT, n=7; MUT, n=5; REG, n=5 mice). (C) Rotarod performance scored as time (seconds) on the rotarod (WT, n=10; MUT, n=12; REG, n=10 mice). (D, E) Representative animal tracks (D) and preference index (E) of novel object test (WT, n=11; MUT, n=12; REG, n=17 mice). (F, G, H) Representative animal tracks (F) and preference indices of sociability (G) and social novelty (H) in the three-chamber test (WT, n=10; MUT, n=12; REG, n=12 mice). (I, J, K) ICP values plotted against preference indices in novel object test (I), sociability (J) and social novelty (K) in the three-chamber test (WT, n=7; MUT, n=6; REG, n=6 mice). Data are mean ± s.e.m. (B, C, E, G, H). *P < 0.05, **P < 0.01, ***P < 0.001, NS, not significant calculated by one-way ANOVA (B, E, G) with Tukey post hoc tests and two-tailed unpaired t-test (C).

Figure 7.. Suture regeneration surgery performed at postnatal day 14 restores brain morphology in Twist1^+/− mice with craniosynostosis

(A) Representative magnetic resonance images (MRI) of WT, MUT and REG mouse brains. Hippocampus (Hipp), cortex (Ctx), corpus callosum (Cc) and thalamus (Th) are outlined by yellow or cyan dotted lines. Scale bar, 2 mm. (B-F) Quantifications of volume of whole brain (B), cortex (C), hippocampus (D), corpus callosum (E) and thalamus (F). (WT, n=5; MUT, n=5; REG, n=5 mice). (G-I) Representative images of Cux1⁺ (G), Ctip2⁺ (H), Tbr1⁺ (I) cells in somatosensory cortex. Scale bar, 50 μm. (J-L) Quantification of Cux1⁺ (J), Ctip2⁺ (K), and Tbr1⁺ (L) cells in somatosensory cortex with 300 μm width (WT, n=4; MUT, n=4; REG, n=4 mice). *P < 0.05, **P < 0.01, ***P < 0.001, NS, not significant calculated by one-way ANOVA with Tukey post hoc tests.