Macrophage-lineage TRAP+ cells recruit periosteum-derived cells for periosteal osteogenesis and regeneration

Abstract

The periosteum, a thin tissue that covers almost the entire bone surface, accounts for more than 80% of human bone mass and is essential for bone regeneration. Its osteogenic and bone regenerative abilities are well studied, but much is unknown about the periosteum. In this study, we found that macrophage-lineage cells recruit periosteum-derived cells (PDCs) for cortical bone formation. Knockout of colony stimulating factor-1 eliminated macrophage-lineage cells and resulted in loss of PDCs with impaired periosteal bone formation. Moreover, macrophage-lineage TRAP+ cells induced transcriptional expression of periostin and recruitment of PDCs to the periosteal surface through secretion of platelet-derived growth factor-BB (PDGF-BB), where the recruited PDCs underwent osteoblast differentiation coupled with type H vessel formation. We also found that subsets of Nestin+ and LepR+ PDCs possess multipotent and self-renewal abilities and contribute to cortical bone formation. Nestin+ PDCs are found primarily during bone development, whereas LepR+ PDCs are essential for bone homeostasis in adult mice. Importantly, conditional knockout of Pdgfrβ (platelet-derived growth factor receptor beta) in LepR+ cells impaired periosteal bone formation and regeneration. These findings uncover the essential role of periosteal macrophage-lineage cells in regulating periosteum homeostasis and regeneration.

Keywords: Bone Biology; Bone development.

Figure 1. Nestin⁺ and LepR⁺ cells are located in the outer layer of the periosteum.

(A–D) Representative images of coronal tibia diaphyseal periosteum sections from 0.5-, 1-, 3-, and 12-month-old male mice stained for TRAP and Nestin (A), TRAP and LepR (B), periostin (C), Emcn and CD31 (D), and representative H&E staining images (E). (F–N) Quantification of the periosteal (F), endosteal TRAP⁺ mononuclear cells (G), periosteal TRAP⁺ multinuclear cells (H), Nestin⁺ cells (I and J), LepR⁺ cells (K and L), periostin⁺ cells (M), and CD31^hiEmcn^hi vessels (N) in the inner layer of periosteum (no. cells/P.BS) and/or whole periosteum (no. cells/periosteum) (n = 5 mice/group). (O and P) Quantification of the thickness (O) and cellularity (P) of the inner layer of periosteum. Scale bars: 100 μm (n = 5 mice/group). Dashed lines in A–D indicate the limit between the inner layer and outer layer of periosteum and cortical bone. Dashed lines in E indicate the limit between the inner layer and outer layer of periosteum. The double-headed arrows indicate the width of the inner layer of periosteum. Data are presented as mean ± SEM. *P < 0.05; **P < 0.01. C, cortical bone; CL, cambium layer (inner layer) of periosteum; FL, fibrous layer (outer layer) of periosteum; NS, not significant as determined by ANOVA with Bonferroni’s post hoc analysis.

Figure 2. Subsets of periosteal Nestin⁺ and LepR⁺ cells possess self-renewal capacity and commit to osteogenic lineage cells.

In vivo serial transplantation of Nestin-GFP⁺PDGFR-α⁺ CD45^–Ter119^–CD31^– (A) and LepR-YFP⁺CD45^–Ter119^–CD31^– PDCs (B). Single cell–derived colonies from donors’ PDCs were expanded to generate 5 × 10⁶ cells and injected into the femora of five 1-month-old NOD SCID mice at a density of 1 × 10⁶ per injection. GFP⁺ or YFP⁺ cells were sorted from the primary recipients’ bone marrow at 8 weeks after injection and harvested for forming CFU-Fs. The colonies were then transplanted into the secondary recipient mice. FAC analysis of CFU-Fs showed the percentages of GFP⁺ or YFP⁺ cells expressing MSC markers (CD90 and CD105) (bottom panels in A and B). (C–J) Lineage-tracing of periosteal Nestin⁺ cells in Nes-creERT2 R26R-EYFP mice. Samples were collected 2, 7, and 14 days or 2, 14, and 30 days after tamoxifen (100 mg/kg i.p.) administration at P14 or P60, respectively (C, G). Representative images of coronal tibia diaphyseal periosteum sections stained for YFP and Osx (D and H), YFP and CD31 (E and I). Quantification of Nestin⁺ lineage cells’ contribution to periosteal Osx⁺ osteoblasts (F and J, left panel), CD31⁺ endothelial cells (F and J, right panel) (n = 5 mice/group). Representative images from 1- or 3-month-old LepR-cre R26R-EYFP mice stained for YFP and Osx (L), YFP and CD31 (M). Scale bars: 20 μm. (N) Quantification of LepR⁺ lineage cells’ contribution to periosteal Osx⁺ osteoblasts (left panel) and CD31⁺ endothelial cells (right panel). Data are presented as mean ± SEM. *P < 0.05; **P < 0.01. C, periosteal cortical bone; NS, not significant as determined by ANOVA with Bonferroni’s post hoc analysis; P, periosteum.

Figure 3. Deficiency in macrophage-lineage cells impairs cortical bone formation and periosteum homeostasis.

(A–C). Upper panels, representative images of coronal tibia diaphyseal periosteum sections from Csf1^–/– mice and their control littermates (Csf1^+/+) stained for TRAP and Nestin (A), TRAP and LepR (B), and periostin (C). Lower panels, high-power magnification images of the boxed area of the upper panels. (D–F) Quantification of TRAP⁺ mononuclear cells (D), Nestin⁺ cells and LepR⁺ cells (E), and periostin⁺ cells (F) in the inner layer of periosteum (no. cells/P.BS) and/or whole periosteum (no. cells/periosteum) (n = 5 mice/group). Dashed lines in A–C indicate the limit between periosteum and cortical bone. Scale bars: 100 μm (upper panels), 20 μm (lower panels). Data are presented as mean ± SEM. *P < 0.05; **P < 0.01. C, periosteal cortical bone; NS, not significant as determined by 2-tailed Student t test; P, periosteum.

Figure 4. Ablation of TRAP⁺ cells impairs recruitment of PDCs for periosteal bone formation.

(A) Diphtheria toxin treatment in iDTR and Trap-cre iDTR mice. (B) Representative TRAP staining images of coronal trabecular bone (TB) and cortical bone sections. Black arrows indicate TRAP⁺ cells. Scale bars: 300 μm (n = 5 mice/group). (C) Representative microcomputed tomography (μCT) images. Scale bars: 1 mm (n = 5 mice/group). (D–G) Representative images of coronal tibia diaphyseal periosteum sections stained for TRAP and Nestin (D, top), TRAP and LepR (D, middle), periostin (D, bottom), and quantification of the TRAP⁺ mononuclear cells (E), Nestin⁺ cells (F), and LepR⁺ cells (G) in the inner layer of periosteum (no. cells/P.BS) and/or whole periosteum (no. cells/periosteum). (H and I) Percentage of Ki-67⁺ (H) or Brdu⁺ (I) cells in Nestin⁺ or LepR⁺ cells on periosteum. (J) Quantification of periostin⁺ cells in the inner layer of periosteum (no. cells/P.BS). Scale bars: 20 μm (n = 5 mice/group). Dashed line in D indicates the line between periosteum and cortical bone. Data are presented as mean ± SEM. *P < 0.05; **P < 0.01. BM, bone marrow; C, cortical bone; NS, not significant as determined by 2-tailed Student t test; P, periosteum.

Figure 5. PDGF-BB secreted by TRAP⁺ cells recruits PDCs to the periosteal surface for cortical bone formation.

(A) Representative images of coronal tibia diaphyseal periosteum sections from Trap-cre Pdgfb^fl/fl mice and Pdgfb^fl/fl mice stained for TRAP and Nestin (top) and TRAP and LepR (bottom). (B and C) Quantification of Nestin⁺ cells (B) and LepR⁺ cells (C) in the inner layer of periosteum (no. cells/P.BS) and/or whole periosteum (no. cells/periosteum). Scale bars: 20 μm (n = 5 mice/group). (D and E) Percentage of Ki-67⁺ (D) or Brdu⁺ (E) cells in Nestin⁺ or LepR⁺ cells on periosteum (n = 5 mice/group). (F) Tibiae 14 days after cortical bone defect surgery. Representative μCT images of tibial cortex and quantification of the newly formed bone volume (BV/TV). Scale bars: 1 mm (n = 5 mice/group). (G) H&E staining of defect site sections. Red boxes indicate the defect sites. Scale bars: 200 μm (n = 5 mice/group). (H–J). Representative images of defect site sections stained for Nestin and LepR. Quantification of Nestin⁺ cells (I) and LepR⁺ cells (J) in the defect sites. Scale bars: 100 μm (n = 5 mice/group). Dashed lines indicate the limit between periosteum and cortical bone. Data are presented as mean ± SEM. *P < 0.05; **P < 0.01. C, cortical bone; P, periosteum; NS, not significant as determined by 2-tailed Student t test.

Figure 6. PDGF-BB induces periostin expression via the PDGFR-β/PI3K/AKT/-CREB signaling pathway.

(A and B) Representative images of coronal tibia diaphyseal periosteum sections from Trap-cre Pdgfb^fl/fl mice (A, left panels) and Ctsk^–/– mice (B, left panels) with their control littermates stained for periostin. Quantification of the periostin⁺ cells in periosteum (no. cells/periosteum). Scale bars: 20 μm (n = 5 mice/group). (C) Nestin-GFP⁺PDGFR-α⁺CD45^–Ter119^–CD31^– PDCs and PDC-derived osteoblasts were treated with 10 ng/ml PDGF-BB or vehicle. Western blot (left panel) and qRT-PCR analysis (right panel) of periostin expression level (n = 5 mice). (D and E) Western blot (left panel) and qRT-PCR analysis (right panel) of periostin expression level in PDCs. Cells were treated with 10 ng/ml PDGF-BB or vehicle for the indicated times (D) or with the indicated doses of PDGF-BB or vehicle for 6 hours (E) (n = 5 mice). (F) Western blot analysis of the phosphorylation of PDGFR-β, PI3K, AKT, and CREB (n = 5 mice). (G) Representative images stained for p-CREB and α-Tubulin in PDC-derived osteoblasts. Scale bars: 10 μm (n = 5 mice). (H) Western blot (left panel) and qRT-PCR analysis (right panel) of periostin expression levels in PDC-derived osteoblasts. Cells were treated with 10 ng/ml PDGF-BB or vehicle in the presence or absence of various inhibitors, as indicated (n = 5 mice). (I) p-CREB binding sites on periostin promoter. (J) ChIP analysis of p-CREB on specific periostin promoter regions in the cells with PDGF-BB or vehicle treatment (n = 3 mice). Data are presented as mean ± SEM. *P < 0.05; **P < 0.01; NS, not significant as determined by 2-tailed Student t test.

Figure 7. Knockout of Pdgfr-β in Nestin⁺ cells impairs recruitment of PDCs and periosteal bone formation in young mice.

(A and B) Representative images of coronal tibia diaphyseal periosteum sections from Nestin-creERT2 Pdgfr-β^fl/fl mice and their control littermates (Pdgfr-β^fl/fl) stained for TRAP and Nestin (A) and TRAP and LepR (B). (C and D) Quantification of Nestin⁺ cells (C) and LepR⁺ cells (D) in the inner layer of periosteum (no. cells/P.BS) and/or whole periosteum (no. cells/periosteum). Scale bars: 20 μm (n = 5 mice/group). (E and F) Percentage of Ki-67⁺ (E) or Brdu⁺ (F) cells in Nestin⁺ or LepR⁺ cells on periosteum (n = 5 mice/group). (G) Representative μCT images. Dashed line indicates the line between periosteum and cortical bone. Data are presented as mean ± SEM. *P < 0.05; **P < 0.01. Scale bars: 1 mm (n = 5 mice/group). 1 M, 1-month-old; 3 M, 3-month-old; C, cortical bone; P, periosteum; NS, not significant as determined by 2-tailed Student t test.

Figure 8. Knockout of Pdgfr-β in LepR⁺ cells impairs recruitment of PDCs and periosteal bone formation in adult mice.

(A and B) Representative images of coronal tibia diaphyseal periosteum sections from LepR-cre Pdgfr-β^fl/fl mice and Pdgfr-β^fl/fl mice stained for TRAP and Nestin (A) and TRAP and LepR (B). (C and D) Quantification of Nestin⁺ cells (C) and LepR⁺ cells (D) in the inner layer of periosteum (no. cells/P.BS) and/or whole periosteum (no. cells/periosteum). Scale bars: 20 μm (n = 5 mice/group). (E and F) Percentage of Ki-67⁺ (E) or Brdu⁺ (F) cells in Nestin⁺ or LepR⁺ cells on periosteum (n = 5 mice/group). (G) Representative μCT images. Scale bars: 1 mm (n = 5 mice/group). 1 M, 1-month-old; 3 M, 3-month-old. (H) Tibiae 14 days after cortical bone defect surgery. Representative μCT images of tibial cortex and quantification of newly formed bone volume (BV/TV). Scale bars: 1 mm (n = 5 mice/group). (I) H&E staining of defect site sections. Red boxes indicate the defect sites. Scale bars: 200 μm (n = 5 mice/group). (J and K) Representative images of defect site sections stained for Nestin and LepR. Quantification of Nestin⁺ cells (K, left panel) and LepR⁺ cells (K, right panel) in the defect sites. Scale bars: 100 μm (n = 5 mice/group). Dashed lines indicate the limit between periosteum and cortical bone. Data are presented as mean ± SEM. *P < 0.05; **P < 0.01. C, cortical bone; P, periosteum; NS, not significant as determined by 2-tailed Student t test.