Step one plus or 7500 fast systems

Since the tooth sockets of mouse upper molar tooth are very small, we used the left maxilla including extraction sockets for RNA isolation. In the semistabilized femoral fracture model, the whole femur including fracture site was subjected to RNA isolation as described previously (Mohan et al., 2005 (link)). Total RNA was isolated from these bones as previously described (Yang et al., 2017 (link)). The maxilla and femur samples were dissected on days 0, 3, 5, and 7 after the operation. Quantitative real-time PCR was performed by using the One-Step SYBR Prime Script PLUS RT-PCR (TAKARA, Shiga, Japan) using Step One Plus or 7500 Fast systems (Thermo Fisher Scientific). The mRNAs examined were Sp7 (Osterix), Col1a1 (type I collagen), Runx2, Sox9, Acan (Aggrecan), Col2a1 (type II collagen), and Col10a1 (type X collagen). Relative expression was determined using Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control. Expression levels were calculated as fold-change relative to control group (0 days), which were isolated immediately after the operations. The primers used for each gene are shown in Table 1 in Supplemental information.

Mouse femora or molars were homogenized in TRIzol reagent (Thermo Fisher Scientific) using Tissue Lyser (Qiagen, Hilden, Germany), and total RNA was purified using the PureLink RNA Micro kit (Thermo Fisher Scientific). Quantitative Real-Time PCR was performed by using the One Step SYBR Prime Script PLUS RT-PCR (TAKARA, Shiga, Japan) using StepOnePlus or 7500 Fast systems (Thermo Fisher Scientific). Gene expression data were normalized to Gapdh or Col1a1 expression. The primers for each gene are shown in Table 1.Table 1

Primers used for Real-time PCR.

Gene	Forward (5′–3′)	Reverse (5′–3′)
Csf-1	GAACAGCCTGTCCCATCCATC	TGAGGCCAGCTCAGTGCAA
Rankl	CATGTGCCACTGAGAACCTTGAA	CAGGTCCCAGCGCAATGTAAC
Opg	CATGAGGTTCCTGCACAGCTTC	ACAGCCCAGTGACCATTCCTAGTTA
F4/80	GAGATTGTGGAAGCATCCGAGAC	GACTGTACCCACATGGCTGATGA
Csf-1r	GGCCCAGCCTGTATTTGCAC	ACCGCTGCTTGGCAGGTTAG
Col1a1	TCAGTGCAATTGTGTTGCTGAAAG	GATACCAAACTGGGCGTGCTG
Gapdh	TGTGTCCGTCGTGGATCTGA	TTGCTGTTGAAGTCGCAGGAG