Animals

A total number of 34 male and female CD-1 mice with a body weight of 3545g and an age of 1820 months were used. The age of 1820 months was chosen according to reports of others, demonstrating age-associated physiological alterations and tumor development after 1618 months in male and 18 months in female CD-1 mice [16]. The animals were bred at the Institute for Clinical and Experimental Surgery, Saarland University, Germany, and housed at a regular light and dark cycle with free access to tap water and standard pellet food (Altromin, Lage, Germany).

All experiments were performed according to the German legislation on the protection of animals and the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, National Research Council, Washington DC, USA). The experiments were approved by the local governmental animal protection committee (permit number: 04/2019).

Mice were anesthetized by intraperitoneal (i.p.) injection of ketamine (75mg/kg body weight, Ursotamin, Serumwerke Bernburg, Bernburg, Germany) and xylazine (15mg/kg body weight, Rompun, Bayer, Leverkusen, Germany). The pin-clip model using a segmental defect served as control and was performed as described previously [15]. Under aseptic conditions, a ~4mm medial parapatellar incision was created at the right knee and the patella was dislocated laterally. After drilling a hole (diameter of 0.50mm) into the intracondylar notch, a distally flattened pressfit 24 Gauge needle (diameter of 0.55mm) was implanted intramedullary and the wound was closed. The pin was flattened at the distal end to avoid secondary dislocation. After insertion of the pin, the diaphysis of the femur was exposed by a lateral approach. Subsequently, a custom-made clip of 6mm length was implanted ventrodorsally into the femur and lateral of the already implanted pin. A gap size of 1.8mm was created by means of a spherical trephine under permanent saline solution cooling. Moreover, the periosteum was stripped 2mm proximally and distally of the gap along the longitudinal axis of the femoral bone. The implant position was confirmed by radiography (MX-20, Faxitron X-ray Corporation, Wheelin, IL, USA). All procedures were done under an operating microscope, guaranteeing a high level of precision. For analgesia the mice received tramadol-hydrochloride (Grnenthal, Aachen, Germany) in the drinking water (1mg/mL) 1 day prior to surgery until 3 days after surgery.

Seventeen mice were daily treated with 200mg/kg body weight PTH 134 (Bachem AG, Budendorf, Switzerland) dissolved in 100 L saline, subcutaneously (PTH group). Control animals (n=17) received an equal amount of saline (control group), subcutaneously. The used PTH dosage corresponds to other experimental studies investigating the effects of PTH on fracture healing in mice [13]. At 2 weeks [n=5 each group (3 male; 2 female)] and 10 weeks [n=9 each group (5 male; 4 female)] the animals were euthanized by an overdose of anesthetics and the femora were excised for further CT and histological analyses. Additional animals were euthanized accordingly at 2 weeks [n=3 each group (2 male; 1 female)] and tissue was harvested for Western blot analyses.

At 2 and 10 weeks after surgery the animals were anesthetized and lateral radiographs of the osteotomized femora were performed. Bone healing was analyzed according to the Goldberg score with stage 0 indicating radiological non-union, stage 1 indicating possible union and stage 2 indicating radiological union [17].

The specimens were scanned (Skyscan 1176, Bruker, Billerica, MA) at a spatial resolution of 9m with a standardized setup (tube voltage: 50kV; current: 200 A; intervals: 0.4; exposure time: 3500 ms; filter: 0.5mm aluminum). Images were stored in three-dimensional arrays. To express gray values as mineral content (bone mineral density; BMD), calcium hydroxyapatite (CaHA) phantom rods with known BMD values (0.250 and 0.750g CaHA/cm3) were employed for calibration. The region of interest (ROI) defining the novel bone was contoured manually excluding any original cortical bone. The thresholding allowed the differentiation between poorly and highly mineralized bone. The threshold to distinguish between poorly and highly mineralized bone was based upon visual inspection of the images, qualitative comparison with histological sections and other studies investigating bone repair and callus tissue by CT [18, 19]. A BMD with more than 0.642g/cm3, resulting in gray values of 98255, was defined as highly mineralized bone. Poorly mineralized bone was assumed to have a BMD value between 0.410g/cm3 and 0.642g/cm3, resulting in gray values of 6897.

The following parameters were calculated from the callus region of interest for each specimen: poorly mineralized bone volume (PM), highly mineralized bone volume (HM), bone volume fraction of tissue volume (BV/TV), bone surface (BS) density (BS/TV), trabecular thickness, trabecular separation and trabecular number.

After removal of the soft tissue and the implants, the bending stiffness of the isolated femora was measured by a 3-point-bending device using a non-destructive approach. This allowed the subsequent use of the specimens for CT as well as histological and immunohistochemical analyses and, thus, a reduction of the number of laboratory animals. Due to the different stages of healing, the loads, which had to be applied, markedly varied between individual animals. Loading was stopped individually in every case when the actual load-displacement curve deviated more than 1% from linearity. Bending stiffness (N/mm) was calculated from the linear elastic part of the load-displacement diagram [20].

After biomechanical testing and CT analysis, bones were fixed in paraformaldehyde for 24h. Subsequently, the specimens were embedded in a 30% sucrose solution for another 24h and then frozen at 80 C. Longitudinal sections through the femoral axis with a thickness of 4m were cut by the Kawamotos film method [21, 22] for histomorphometric analyses and stained with Safranin-O. At a magnification of 12.5 (Olympus BX60 Microscope, Olympus, Shinjuku, Japan; Zeiss Axio Cam and Axio Vision 3.1, Zeiss) structural indices were calculated according to the recommendations of Gerstenfeld et al. [23]. The following histomorphometric parameters of the bone defects were evaluated: (i) total callus area, (ii) bone callus area, (iii) cartilaginous callus area and (iv) fibrous callus area. The total callus area was defined as the entire osseous, cartilaginous and fibrous callus tissue between the two drilling holes of the clip outside of the cortices. Pre-existing cortical bone of the proximal and distal fragment, however, was excluded. Each area was marked and calculated using the ImageJ analysis system (NIH, Bethesda, USA).

In addition, tartrate-resistant acid phosphate (TRAP) activity was analyzed in the callus tissue at 2 and 10 weeks after surgery. For this purpose, longitudinal sections of 4m were incubated in a mixture of 5mg naphotol AS-MX phosphate and 11mg fast red TR salt in 10 mL 0.2M sodium acetate buffer (pH 5.0) for 1h at 37C. Sections were counterstained with methyl green and covered with glycerin gelatin. TRAP-positive multinucleated cells (three or more nuclei each cell) were counted. In the specimens, one high-power field (HPF, 400 magnification) was placed in a standardized manner in the central region of the callus, while three additional HPFs were placed on each site of the periosteal callus.

To analyze the cellular composition within the callus tissue of atrophic non-unions at 2 and 10 weeks after surgery, longitudinal sections with a thickness of 4m were cut. For the immunohistochemical detection of microvessels, sections were stained with a monoclonal rat anti-mouse antibody against the endothelial cell marker CD31 (1:100; Abcam, Cambridge, UK). A goat anti-rat IgG-Alexa555 antibody served as secondary antibody (1:100; Life Technology, Eugene, USA). Cell nuclei were stained with Hoechst 33342 (2g/mL; Sigma-Aldrich, Taufkirchen Germany). To detect the neutrophilic granulocyte marker myeloperoxidase (MPO) and the macrophage marker CD68, sections were stained with a polyclonal rabbit anti-mouse antibody against MPO (1:100; Abcam) and a polyclonal rabbit anti-mouse antibody against CD68 (1:100; Abcam). A goat anti-rabbit IgG-antibody (1:200; Dianova, Hamburg, Germany) served as corresponding secondary antibody.

In the specimens, the number of CD31-positive microvessels as well as MPO- and CD68-positive cells was counted. For this purpose, one HPF was placed in a standardized manner in the central region of the callus, while three additional HPFs were placed on each site of the periosteal callus.

Protein expression within the callus tissue was determined by Western blot analysis, including the expression of vascular endothelial growth factor (VEGF), cyclooxygenase (COX)-2 and phosphoinositide 3-kinase (PI3K). The callus tissue was frozen and stored at 80C until required. Analyses were performed from callus tissue at 2 weeks after surgery (n=3 each group). After saving the whole protein fraction, analysis was performed using the following antibodies: rabbit anti-mouse VEGF (1:300, Abcam, Cambridge, UK), COX-2 (1:30, Abcam) and mouse anti-mouse PI3K (1:100, Santa Cruz Biotechnology, Heidelberg, Germany). Primary antibodies were followed by corresponding horseradish peroxidase-conjugated secondary antibodies (1:1000, R&D Systems). Protein expression was visualized by means of luminol-enhanced chemiluminescence after exposure of the membrane to the Intas ECL Chemocam Imager (Intas Science Imaging Instrument GmbH, Gttingen, Germany) and normalized to -actin signals (1:1000, mouse anti-mouse -actin, Santa Cruz Biotechnology) to correct for unequal loading.

All data are given as meansSEM. After testing the data for normal distribution (KolmogorovSmirnov test) and equal variance (F-test), comparisons between the two groups were performed by an unpaired Students ttest. For nonparametrical data, a MannWhitney Utest was used. All statistics were performed using the SigmaPlot 13.0 software (Jandel Corporation, San Rafael, CA, USA). A pvalue of <0.05 was considered to indicate significant differences.

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Parathyroid hormone stimulates bone regeneration in an atrophic ... - Journal of Translational Medicine