Introduction 1988 2006 1994 1999 2000 2006 2000 2002 2002 2002 2005 2000 2002 2002 2002 1999 2000 2004 2005a b 2005 1999 2000 1992 1975 1992 1999 1999 2000 2004 2000 2004 2005a b 2005 2000 2005a b 1997 1999 1997 1999 Materials and methods Animal model of DO 2005a b 1997 2005a b All animal experimental procedures were approved and performed following the guidelines for animal scientific procedures (Animal Scientific Procedures Act 1986, British Home Office). Antibodies 2001 2005 2000 2005 2005 Immunohistochemical procedures 2 2 Scoring 1 Fig. 1 a-c e d a FT NB P PC white grid b Ob Ocy Pre-Ob B c white arrows NB d FCC HC e Bars b  d c e a  Cell cultures 2 1998 2 g 4 4 3 RNA analysis and quantitative real-time polymerase chain reaction Total RNA from cultured cells was isolated by using TRIZOL reagent (Gibco) according to the manufacturer’s instructions. The RNA content was determined by measuring the absorbance in water at 260 nm by means of an Ultrospec III spectrophotometer (Amersham, Buckinghamshire, England). cDNA synthesis was performed by using 750 ng total RNA in a final reaction volume of 20 μl containing 5 U transcriptor (Roche Applied Science), 5× polymerase chain reaction (PCR) buffer, 4 U random primers (Roche), 20 U protector RNase inhibitor (Roche), 1 mmol each dNTP and 10 μl template. The reverse transcription step was performed on a Gene Amp 9700 Thermocycler (Applied Biosystems, Foster City, Calif., USA) at 55°C for 30 min followed by 85°C for 5 min. 1999 Runx2 PBGD sample calibrator -(ΔΔ Ct) 2001 Statistical analysis P P Results 1 1 2 3 1 2 3 1 Fig. 2 a d g b e h c f i B FT FC asterisks hatch arrows arrowheads a-c d-f g-i Inset i a d g b e h c f i inset Bars c f i b d e g h a  Fig. 3 a EC SMC white arrows b white arrows c Ob d Ob NB Pre-Ob Bars b c d a  Table 1 ± + ++ +++ Marker Group with 0 μg TP508 Group with 30 μg TP508 Group with 300 μg TP508 RUNX2 + to ++ + to ++ ++ to +++ OPN ± to ++ ± to + +++ BSP ± to +++ ± + to +++ 1 2 1 2 3 1 2 1 1 3 3 2 2 1 2 In the group injected with 300 μg TP508, immunostaining for Runx2, OPN and BSP was distributed throughout the regenerating areas. The immunostaining for Runx2, OPN and BSP in the connective tissue in the gap (endosteal area) was relatively high and as intense as in the periosteal callus tissue. In the 30 μg TP508 group and in the control group, staining was less intense in the central gap area than in the periosteal callus area. P 4 P 4 P P Fig. 4 black bars grey bars white bars P  P 5 Fig. 5 black bars hatched bars white bars n P 2 P 2 P  Discussion 2005a b 2005 2003 1999 2000 2003 2004 2000 2005a b 2005 2005 2000 2002 2005 2005 1994 2004 2001 2000 2002 2005 2000 2001 1995 1998 2002 2003 2005b 2005a 2005a 1997 1999 1999 2001 2001 In conclusion, we have demonstrated that the increase of bone regeneration by thrombin-related peptide TP508 is associated with an increase in the immunostaining for Runx2, an essential transcription factor of the osteoblastic lineage, and for the bone matrix proteins BSP and OPN. TP508 may thus be a candidate for enhancing bone regeneration when bone regeneration is slow, as occurs in elderly patients.