Immunolocalization of sibling and RUNX2 proteins during vertical distraction osteogenesis in the human mandible

Abstract

We tested the hypothesis that mechanical loading of human bone increases expression of the transcription factor RUNX2 and bone matrix proteins osteopontin (OPN), bone sialoprotein (BSP), dentin matrix protein-1 (DMP1), and matrix extracellular phosphoglycoprotein (MEPE). We examined this in tissue sections of atrophic mandibular bone taken from edentulous patients who had undergone distraction osteogenesis. In undistracted bone, weak to moderate staining for OPN and BSP was found in osteoblasts and bone matrix of immature woven bone. RUNX2 was also detectable in osteoblasts and in cells of the periosteum. In woven bone, but not in lamellar bone, a small number of osteocytes stained for all proteins tested. After distraction, staining intensity had increased in the existing old bone and staining was seen in more bone cells than before distraction. We also found a high expression of DMP1 and MEPE in many osteocytes embedded in woven bone and in some osteocytes of lamellar bone not seen before distraction. New bone trabeculae were forming in the fibrous tissue of the distraction gap containing all stages of intramembranous bone formation. Moderate to strong staining was seen for all five proteins tested in osteocytes located in woven bone of these trabeculae and for RUNX2, OPN, and BSP in osteoblasts lining the trabecular surfaces. We conclude that loading of atrophic human jawbone by distraction activates matrix synthesis of bone cells in and around existing bone. Increased staining of DMP1 and MEPE in osteocytes after loading is in line with the concept that these proteins may be involved in signaling the effector cells to adapt the bone structure to its mechanical demands.

Figure 1

Vertical distraction osteogenesis in the human mandible (A) and area of interest (B). (A) An osteotomy is made to separate the upper (or transport) bone segment from the main body. The distractor, positioned underneath the mucosa with the activation part exposed to the oral cavity, is fastened with screws to the upper and lower bone segments. The screw of the distractor is turned daily. This allows the upper bone segment to move upward and puts tension on the soft connective tissue in the gap. At the end of the distraction period, the distractor is left in place to keep the segments in fixed position for various weeks to close the gap with bone. At the end of healing, bone biopsies are taken parallel to the location of the distractor screw and contain (old) bone of the upper segment, the distraction gap with new bone and, occasionally, some (old) bone of the lower segment. (B) Schematic drawing of the areas of interest. The osteotomy line separates the old bone from the new bone formed in the gap.

Figure 2

Immunolocalization of SIBLING proteins in undistracted bone (baseline levels). (A) Osteopontin (OPN) immunostaining was localized in cement lines (CL), osteoid (O), and osteocytes (OCY) of recently deposited bone. P, periosteum. (B) Dentin matrix protein-1 (DMP1) (LFMb-31) expression in osteocytes (arrows) adjacent to periosteum and in the area close to the Haversian channel (HC). (C) High magnification of boxed area in B. Note that DMP1 expression is located primarily in the lacunar wall. (D) Matrix extracellular phosphoglycoprotein (MEPE) protein (LFMb-33) was found in the osteocytes (arrow). Bars: A = 100 μm; B = 20 μm; C, D = 10 μm.

Figure 3

Immunolocalization of SIBLING proteins in the postdistracted old bone. (A) OPN immunostaining was localized in newly formed bone (NB) near bone marrow spaces (arrow). BM, bone marrow. (B) Strong expression of OPN was found in the osteotomy line (OL). Note that new OPN-positive woven bone (NB) is replacing a lamellar bone area with many empty osteocyte lacunae (EL, arrowhead). (C) High magnification of boxed area in A. A multinucleated OPN-positive osteoclast (OC) resorbed lamellar bone. Some bone contains empty osteocyte lacunae (EL); osteoblasts (OB) deposit new bone matrix in Howship's lacuna (asterisks). Note that OPN staining is also located in the canaliculi of osteocytes. (D) Bone sialoprotein (BSP) was present in osteoblasts (OB) and osteocytes (arrows, OCY) but was absent in marrow stromal cells (MSC) (inset: low-power view). (E) MEPE expression in osteocytes (OCY) in lamellar (vertical arrow) and woven bone (horizontal arrow). BM, bone marrow. (F) RUNX2 was located in numerous marrow stromal cells (MSC), osteoblasts (OB), and young osteocytes (OCY). (G) High magnification of boxed area in F. RUNX2 was located in numerous marrow stromal cells (MSC), osteoblasts, and young osteocytes (OCY) near the bone surface. (H) Negative control stained with a control IgG. Note the lack of positive staining (brown). Bars: A, B, D = 100 μm; C, F, H = 50 μm; D, G = 20 μm; Inset in D = 100 μm.

Figure 4

Immunolocalization of SIBLING proteins in bone trabeculae formed in the distraction gap. (A) BSP immunostaining in the oldest bone area adjacent the osteotomy line (OL). Its expression is observed in the osteotomy line (OL) and in the central area of new trabecular bone (bars indicate thickness of BSP-positive bone). Peripheral layers of trabeculae (asterisk) stain weakly or not. (B) Polarized light microscopy of A reveals this peripheral bone as lamellar bone (birefringe bright area, arrows) and the central (dark) area as woven bone (WB). (C) BSP immunostaining in the younger bone area. (D) High magnification of boxed area in C. BSP staining is present in the matrix and osteocytes of woven bone (WB), but not in the lamellar bone (LB). SCT, soft connective tissue. (E) OPN-positive osteoclasts (OC) are found in the oldest bone area (LB, lamellar bone). Note the OPN-positive cells (presumably pre-osteoblasts, pre-OB) adjacent to the osteoblastic layer (OB) close to young woven bone (WB). (F) Staining of DMP1 monoclonal (MC) antibody. Strong expression is observed in young osteocytes (rounded shape, arrow) and decreases in mature osteocytes (flattened shape) in lamellar bone (LB). Note that the protein is prominent in the lacunar wall and in canaliculi. (G) Staining of DMP1 polyclonal antibody shows the same pattern as the monoclonal antibody. (H) MEPE staining reveals a similar distribution as DMP1. (I) High magnification of boxed area of H. Strong MEPE staining is present in the lacunar wall of osteocytes of woven bone (WB). (J) RUNX2 localizes in almost all fibrous tissue cells around new trabecular bone (TB). Strong expression is seen in osteoblasts (OB) and in cells adjacent to the osteoblastic layer. (K) High magnification of boxed area in J, pre-ob, preosteoblasts; TB, trabecular bone. (L) Control section stained with control IgG. Bars: A, B, H = 100 μm; C = 50 μm; D—G, I—L = 20 μm.