Figure 6. Tissue-Specific Deletion of Ngf in LysM-Expressing Cells but Not Pdgfrα-Expressing Cells Blunts Calvarial Bone Defect Repair.

Tissue-specific deletion of Ngf was performed using LysM-Cre (Ngf^LysM) or Pdgfrα-CreER^T2 lines (Ngf^Pdgfrα), and bone healing was compared with Cre⁻ animals (Ngf^fl/fl). Healing was assessed using a 1.8 mm, circular, full-thickness frontal bone defect.

(A–D) Location of PDGFRα or LysM reporter activity before and after injury. (A and B) PDGFRα reporter activity (PDGFRα-CreER^T2-mGFP, recolored red) within the uninjured frontal bone and at d3 post-injury. White scale bar, 50 μm. (C and D) LysM reporter activity (LysM-Cre-tdTomato) within the uninjured frontal bone and at d3 post-injury. White dashed lines indicate bone edges. White scale bar, 50 μm.

(E–G) Validation of Ngf deletion in either PDGFRα-expressing stromal cells (Ngf^Pdgfrα) or LysM-expressing monocytes/macrophages (Ngf^LysM) in comparison with Ngf^fl/fl mice, d3 post-injury. NGF immunohistochemistry among (E) Ngf^fl/fl control bone defects, (F) Ngf^Pdgfrα bone defects, or (G) Ngf^LysM bone defects. NGF immunostaining appears green, while reporter activity appears red. White scale bar, 50 μm.

(H–J) μCT reconstructions of the defect site in a top-down view (above) and coronal cross-sectional images (below) among (H) Ngf^fl/fl, (I) Ngf^Pdgfrα, and (J) Ngf^LysM animals. Margins of original defect are indicated by dashed black or red lines. Black scale bar, 500 μm; white scale bar, 200 μm.

(K–O) μCT quantification of bone healing among Ngf^fl/fl, Ngf^Pdgfrα, and Ngf^LysM mice including (K) bone volume (BV), (L) bone volume/tissue volume (BV/TV), (M) bone formation area (BFA), (N) bone healing score (score), and (O) defect diameter.

(P–R) H&E stain of coronal cross-section of the healing defect site from (P) Ngf^fl/fl, (Q) Ngf^Pdgfrα, and (R) Ngf^LysM mice. Black arrowheads indicate span of initial defect. Black scale bar, 50 μm.

(S–V) Immunohistochemical staining of TUBB3⁺ (beta III tubulin) nerve fibers at the defect edge from (S) Ngf^fl/fl, (T) Ngf^Pdgfrα, and (U) Ngf^LysM mice, appearing green, and (V) quantification of TUBB3 immunoreactivity within the calvarial defect. Dashed white lines indicate bone edge. White scale bar, 50 μm.

In graphs, each dot represents a single animal. n = 9–11 per group for (K)–(O) and N = 7 per group for (V). †p < 0.05 and †††p < 0.001 in comparison with Ngf^fl/fl and Ngf^Pdgfrα. Data are represented as mean ± SD. See also Figure S5.