Unilateral closed fractures were produced in the left femur of isoflurane-anesthesized 8-week-old male RAG1−/− mice and wild-type (WT) mice (C57BL/6N background; Bundesinstitut für Risikobewertung, Berlin, Germany) according to Bonnarens and Einhorn.26, 27 The mice were evaluated at 3, 7, 14, 21, and 28 days. All bones were assessed at surgery and at harvesting by radiography. Fractures that did not meet standard criteria were not included in the subsequent analyses.
Animal experiments were carried out in accordance with the Care and Use of Animals and the national welfare guidelines. The study was approved by the local legal representative (Landesamt für Gesundheit und Soziales, Berlin, Germany, Registration Number G 0206/08).
Micro–computed tomography (µCT)
For characterization of 3D callus properties, including size, geometry, structure, and mineralization, the newly formed mineralized callus tissue was analyzed using high-resolution µCT. At days 7, 14, 21, and 28, fractured and contralateral femurs were batch scanned with a fixed isotropic voxel size of 10.5 µm (70 kVp, 114 µA; Viva40 µCT, Scanco Medical AG, Brüttisellen, Switzerland). The scan axis coincided nominally with the diaphyseal axis of the femurs. A minimum of 630 slices was chosen such that it included the fracture callus in all dimensions. The cortical bone was excluded from the volume of interest (VOI) manually. A fixed global threshold of 190 mg hydroxyapatite (HA)/cm3 was selected that allowed the rendering of mineralized callus only. This threshold was verified by manually evaluating 10 single tomographic slices from 4 samples per group to isolate the mineralized tissue and preserve its morphology while excluding unmineralized tissues. Standard µCT measures were calculated for each sample. All analyses were performed on the digitally extracted callus tissue using 3D distance techniques (Scanco Medical AG software).28
For histologic analysis of fractures, femurs were harvested at 3, 7, 14, 21, and 28 days. All histologic analyses were conducted according to the ASBMR standards adapted to the study of bone repair.29, 30 Bones were excised with little surrounding soft tissue, fixed with 4% paraformaldehyde (PFA) at 4°C for 48 hours and decalcified in a 1:1 solution of 4% PFA and 14 % EDTA at 4°C for 3 weeks. Bones were dehydrated, embedded in paraffin, and cut into 4-µm sagittal slices. Movat-pentachrome staining was performed for histomorphometry.31 Computerized histomorphometric analysis was performed with an image-analysis system (KS400 3.0, Zeiss, Eching, Germany).
For immunofluorescent staining and confocal microscopy, histologic samples were fixed in 4% PFA and equilibrated in 30% sucrose/PBS. Cryostat sections of adult femurs were stained and mounted with fluorescent mounting medium (DakoCytomation, Glostrup, Denmark). Stainings were carried on using Cy5-labeled monoclonal antibodies against B220 (RA3.6B2) and biotinylated anti-CD3 (500A2; eBioscience, San Diego, CA, USA). For secondary reagent streptavidin-Alexa555 (Invitrogen Life Technologies, Carlsbad, CA, USA) was used. Cell nuclei were visualized by labeling with cytox green. All confocal microscopy was carried out on a DM IRE2 microscope (Leica, Wetzlar, Germany).
For osteoblast counting, Movat-pentachrome staining and osteocalcin immunohistochemistry were applied. Cells were counted as osteoblasts if they had contact with bone and displayed the typical palisade-like morphology. Three regions of the callus were selected in the lateral periosteal callus: a proximal region, a region at the level of the gap, and a distal region. For osteocalcin immunohistochemistry, paraffin sections were deparaffinized, washed, and hydrated. Antigen retrieval was performed using 0,18 % trypsin for 10 minutes at room temperature. All solutions were prepared in Tris-buffered saline (TBS). Samples were blocked with normal goat serum (S-1000, Vector Laboratories, Burlingame, CA, USA). Primary mouse monoclonal antibody to osteocalcin (Cat. no. LAX-210-333-C100, Enzo Life Sciences, Lörrach, Germany) was diluted in DAKO-Diluent (S-00809, Glostrup, Denmark) 1:4000. Samples were incubated at 4°C overnight and incubated with a secondary antibody (biotinylated antirabbit, made in goat, BA-1000) followed by incubation with AB- complex (alkaline phosphatse universal, AK-5200, Vectastatin ABC Kit). After incubation with chromogen buffer and AP substrate (Alkaline Phosphatase Substrate Kit 1, SK-5100, Vector Laboratories), sections were counterstained with methyl green.
For detection of collagen type II expression by chondrocytes, immunohistochemistry was applied. Sections were deparaffinized in xylene and rehydrated by an ascendant line of alcohol until hydration with PBS. For antigen retrieval, samples were incubated with hyaluronidase (2 hours at 37°C), followed by 0.1% pepsin (30 minutes at 37°C). Samples were blocked with normal horse serum (S-2000, Vector Laboratories) and diluted in PBS for 20 minutes at room temperature. Dilution of the primary antibody (mouse monoclonal to collagen type II, Cat. no. 031502101, Quartett, Berlin, Germany) was made in DAKO-Diluent (S-0809) 1:50. Samples were incubated with the primary antibody at 4°C over night. Samples were incubated with the secondary antibody (biotinylated antimouse IgG, made in horse, BA-2000) for 30 minutes at room temperature. Samples were washed two times in PBS followed by incubation with AB-complex (Alkaline Phosphatase Universal, AK-5200, Vectastatin ABC Kit) for 50 minutes. AP substrate (Alkaline Phosphatase Substrate Kit 1, SK-5100, Vector Laboratories) was added to the samples and incubated for 15 minutes.
In addition, collagen type X expressed by hypertrophic chondrocytes was detected by immunohistochemistry. Preparation of sections was similar to that for collagen II immunohistochemistry. For antigen retrieval, samples were incubated with pepsin (2 hours at 37°C), followed by incubation with hyaluronidase (30 minutes at 37°C). Samples were blocked with normal horse serum (S-2000, Vector Laboratories) and diluted in PBS for 20 minutes at room temperature. Dilution of the primary antibody (mouse monoclonal to collagen type X, Cat. no. 2031501005, Quartett) was made in DAKO-Diluent (S-0809) 1:100. Samples were incubated with the primary antibody at 4°C over night followed by incubation with the secondary antibody (biotinylated antimouse IgG, made in horse, BA-2000) for 30 minutes at room temperature. Samples then were incubated with AB-complex (Alkaline Phosphatase Universal, AK-5200, Vectastatin ABC Kit) for 50 minutes. After taking of the chromogen buffer, the AP substrate (Alkaline Phosphatase Substrate Kit 1, SK-5100, Vector Laboratories) was added to the samples and incubated for 15 minutes. Each step of incubation was followed by washing two times. After staining, the samples were counterstained with hematoxylin. To ensure specificity of antibodies, we performed negative controls that did not reveal any nonspecific staining.
Tatrate-resistant acid phosphatase (TRACP) staining was conducted at pH 5.0 in the presence of L(+)-tartaric acid using naphtol AS-MX phosphate (Sigma, St Louis, MO, USA) in N,N-dimetylformamide as a substrate. To meet the criteria of osteoclasts, cells had to have more than 2 nuclei, show TRACP+ staining, and be located next to the bone. The number of osteoclasts was determined.
mRNA preparation and expression analysis
Bones were excised, and the surrounding soft tissues were dissected. To standardize tissue collection, only the diaphyseal regions with or without callus were harvested. Collected tissues from 6 mice per time point were snap-frozen in liquid nitrogen and stored at −80°C. Control samples (day 0 time point) were collected from unfractured femurs of each RAG1−/− and WT mice. Tissues were pulverized with pestle and mortar under continuous cooling with liquid nitrogen. After homogenization (T10, Ultra-turrax, IKA Werke GmbH, Staufen, Germany), total RNA was isolated from each sample using TRIzol (Invitrogen Life Technologies) according to the manufacturer's protocol. DNA was eliminated with DNAse I (Invitrogen Life Technologies).
All reagents for quantitative real-time polymerase chain reaction (qRT-PCR) analysis were purchased from Bio-Rad (ie, iScrip cDNA Synthesis Kit, iQ SYBR Green Supermix; Paris, France), and plate assays were read in Icycler IQ5 optical system software, Version 2.0 (Bio-Rad). One microgram of total RNA was used for each preparation of cDNA. The methods of DNA amplification were as described previously.32 In qRT-PCR, all samples were run in duplicate. Each plate contained two negative controls and a positive control. Expression levels were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Sample threshold values were then normalized for each time-point: ΔCT = CT(exp) – CT(GAPDH). CT values of day 0 were used as a reference: ΔΔCT = ΔCT(exp dayx) - ΔCT(exp day0). The fold change in mRNA expression for each time point was plotted in a graph using day 0 as a reference: 2−ΔΔCT(day 0 = 1.