Effect of thrombin peptide 508 (TP508) on bone healing during distraction osteogenesis in rabbit tibia

Abstract

Thrombin-related peptide 508 (TP508) accelerates bone regeneration during distraction osteogenesis (DO). We have examined the effect of TP508 on bone regeneration during DO by immunolocalization of Runx2 protein, a marker of osteoblast differentiation, and of osteopontin (OPN) and bone sialoprotein (BSP), two late markers of the osteoblast lineage. Distraction was performed in tibiae of rabbits over a period of 6 days. TP508 (30 or 300 microg) or vehicle was injected into the distraction gap at the beginning and end of the distraction period. Two weeks after active distraction, tissue samples were harvested and processed for immunohistochemical analysis. We also tested the in vitro effect of TP508 on Runx2 mRNA expression in osteoblast-like (MC3T3-E1) cells by polymerase chain reaction analysis. Runx2 and OPN protein were observed in preosteoblasts, osteoblasts, osteocytes of newly formed bone, blood vessel cells and many fibroblast-like cells of the soft connective tissue. Immunostaining for BSP was more restricted to osteoblasts and osteocytes. Significantly more Runx2- and OPN-expressing cells were seen in the group treated with 300 microg TP508 than in the control group injected with saline or with 30 microg TP508. However, TP508 failed to increase Runx2 mRNA levels significantly in MC3T3-E1 cells after 2-3 days of exposure. Our data suggest that TP508 enhances bone regeneration during DO by increasing the proportion of cells of the osteoblastic lineage. Clinically, TP508 may shorten the healing time during DO; this might be of benefit when bone regeneration is slow.

Fig. 1

Immunolocalization of Runx2 and osteopontin proteins in TP508-treated groups (a-c, e 300 μg TP508 group, d 30 μg TP508 group). a Overview of immunoreactions in a biopsy taken from rabbit long bone after distraction osteogenesis Runx2 staining (FT fibrous tissue, NB new bone, P periosteum, PC periosteal callus). A white grid was used to count the number of immunopositive and negative cells in the region of interest. ×50. b Runx2 staining in the nuclei and cytoplasm of the cells. Runx2 was strongly expressed in osteoblasts (Ob). Note that young osteocytes (Ocy), which had recently embedded in the bone matrix, expressed Runx2 protein. Cells adjacent to the osteoblast layer, presumably pre-osteoblasts (Pre-Ob), also expressed Runx2, but with lower intensity (B bone). ×400. c Immunostaining for osteopontin. Positive staining was found in the cytoplasm of the cells (white arrows). Note that expression was stronger towards the newly formed bone (NB). ×200. d Runx2 immunostaining in tissue of a rabbit injected with 30 μg TP508. Expression was much lower than that after injection with 300 μg TP508 and was similar to saline-injected control tissue (not shown). Fewer fibrocartilage-like cells (FCC) were stained than after injection with 300 μg TP508 (HC hypertrophic cartilage-like cells). ×200. e Negative control stained with non-immune IgG instead of primary antibodies to Runx2. Note the lack of positive staining. Counter-staining with methyl green. ×200. Bars 20 μm (b, d), 50 μm (c, e), 300 μm (a)

Fig. 2

Runx2 (a, d, g), OPN (b, e, h) and BSP (c, f, i) expression in the distraction gap (B bone, FT fibrous tissue, FC fibrous cartilage-like tissue, asterisks central area of distraction gap, hatch layers of cells adjacent to the newly formed bone, arrows osteoblasts, arrowheads osteocytes) of the various groups (a-c 0 μg TP508 group, d-f 30 μg TP508 group, g-i 300 μg TP508 group). Inset (i): Newly formed bone in the gap. Original magnifications: a 50×; d, g 100×; b, e, h 100×; c, f, i, inset 200×. Bars 20 μm (c, f, i), 50 μm (b, d, e, g, h), 100 μm (a)

Fig. 3

Immunolocalization of Runx2, OPN and BSP in the 300 μg TP508 group. a Runx2 staining of a vessel wall (EC endothelial cells, SMC smooth muscle cells). Note that some cells near the blood vessel weakly express Runx2 (white arrows). ×400. b Osteopontin staining of a vessel wall. Note that the cells surrounding the blood vessel are immunonegative and only stain weakly for methyl green (white arrows). ×400. c BSP staining is restricted to the layer of osteoblasts (Ob) and hardly occurs in fibrous tissue × 400. d OPN staining is expressed in osteoblasts (Ob) and osteocytes of new bone (NB) and some fibrous tissue cells close to osteoblasts, presumably pre-osteoblasts (Pre-Ob). ×200. Bars 20 μm (b), 50 μm (c, d), 100 μm (a)

Fig. 4

Effect of TP508 injection on the number of immunopositive cells in the central portion of the distraction gap (black bars Runx2-positive cells, grey bars OPN-positive cells, white bars BSP-positive cells). Data are presented as percentage cells immunopositive for each of the three proteins examined (means±SD). *The percentages of Runx2-, OPN- and BSP-positive cells in the 300 μg TP508 group were significantly higher than for cells with the corresponding proteins in the saline-treated control group; the percentage of BSP-positive cells in the 300 μg group was also significantly higher than that in the 30 μg group (ANOVA, P<0.05)

Fig. 5

Quantitative reverse transcription/polymerase chain reaction analysis of Runx2 mRNA expression in mouse osteoblast-like (MC3T3-E1) cells cultured for 24, 48 and 72 h in the presence of TP508 (black bars control group with 0 μg/ml TP508, hatched bars 10 μg/ml TP508, white bars 100 μg/ml TP508). Data are presented as means±SD (n=8) and are representative of two experiments. *The 10 μg/ml TP508 group at 24 h of incubation was significantly lower than the control group (one-way ANOVA; Tukey’s multiple comparison test, P<0.01). Correlation analysis between mRNA and culture time: r²=0.94, P=0.06 for 10 μg/ml TP508; r²=0.99, P=0.06 for 100 μg/ml TP508