The effects of periosteum removal on the osteocytes in mouse calvaria

Objectives: To investigate the early influence of periosteum removal on osteocytes in mouse calvaria. Material and methods: Fifteen C57BL/6 male mice were used in this study. Under anesthesia, a 2X3mm rectangular shape periosteum, 2mm behind the coronal suture and 1mm beside the sagittal suture, was surgically removed from the left side of calvaria whereas the right side was intact as control. Five mice were sacrificed at 1 day, 3 days and 7 days after the operation and the calvaria together with the surrounding tissues were histologically examined. Results: At 3 days and 7 days, the number of empty lacunae significantly increased at the periosteum stripped side compared to the intact control side. The empty lacunae were mainly restricted to the outer half of the calvaria under the periosteum stripped area. Sclerostin distribution in lacunae and canaliculi also dramatically decreased at the periosteum stripped side at all the time points. Conclusion: Periosteum removal would induce early apoptosis of the osteocytes in the outer layer of the bone and decrease sclerostin within this area. Correspondence to: Songtao Wu, Department of Oral Implantology and Regenerative Dental Medicine, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo113-8549, Japan, Tel: +81 3 5803 4664; E-mail: [email protected]; [email protected] Received: September 27, 2015; Accepted: October 20, 2015; Published: October 26, 2015 Introduction Periosteum, the stiff envelope bounds the skeleton, plays an essential role in offering protection and nutrition to the cortical bone [1]. Furthermore, periosteum also severs as a reservoir of progenitor cells and preserves a remarkable bone regeneration capacity, which is crucial for both prenatal bone development and postnatal fracture healing [2]. For immature skeletal system, periosteum-derived ossification makes long bones grow radially [3] and in fracture healing process, periosteum is also inevitable [4]. The formation of cartilage and bone within the callus owes largely to the mesenchymal progenitor cell from the periosteum [5]. When the periosteum is irritated by trauma, infection, neoplasia, or sometimes surgical procedure, new bone would be rapidly formed, which is favorable or unfavorable [6-9]. A previous study showed that the elevation of periosteum can induce new bone formation underneath [10]. In spite of the importance of periosteum, in some surgical circumstances, it has to be completely removed from the bone surface, such as in proper open reduction and in plate applications for treating bone fractures [11], and surgical removal of soft tissue sarcomas (STS) to ensure the adequate surgical margins [12]. In dental implant surgeries, clinicians often apply tension releasing incisions, cutting the periosteum at the base of the muco-periosteal flap to complete a primary closure, especially in ridge expansion procedures [13]. This incision into the periosteal layer leaves a hole, resulting in direct contact of the mucosa to the cortical bone, which can be considered as another kind of the periosteum stripping. The apoptosis of osteocyte is another highlighted topic, which is already known to be a result of many physiological or pathological changes, such as rapid modeling process [14], age-associated oxidative stress [15], estrogen deficiency [16], overwhelming of glucocorticoid [17]. Also from a mechanical view, unloading of bone which weakens the canaliculiar fluid shear stress will result in osteocyte apoptosis [18]. Conversely, excessive loading which produces microdamage disrupting the canaliculiar system integrity also leads to osteocyte apoptosis [19]. Although the studies concerning periosteum stripping or osteocyte apoptosis have been often reported respectively, the reaction of osteocytes to the periosteum stripping has not been clarified yet. In this study, we histologically examined the effects of periosteum removal on the osteocytes in mouse calvaria. Materials and methods Animal models Fifteen male C57BL/6 mice of 12 weeks old, weighing 25-30g, were used in this study. All the experimental procedures had been approved by the Animal Experiment Committee at Tokyo Medical and Dental University. (Approval number: 0140174A). Wu S (2015) The effects of periosteum removal on the osteocytes in mouse calvaria Volume 1(5): 146-149 Dent Oral Craniofac Res, 2015 doi: 10.15761/DOCR.1000134 Surgical procedure General anesthesia was performed by an intraperitoneal injection of a combination of ketamine and xylazine (75 mg and 0.5 mg per kg body weight, respectively). The hair of calvarial zone was shaved and the skin over the skull was cleaned with 70% ethanol. Incision of 20mm length was made in the central line of head and partial thickness flap was raised. A 2 X 3mm rectangular (2mm behind the coronal suture and 1mm beside the sagittal suture) area was marked with ink on left side of calvarial periosteum (Figure 1A). Then, the stripping was performed with scalpel and dental excavator to remove the periosteum completely (Figure 1B and 1C). The right side of calvaria was intact as control. Finally the surgical area was closed with suturing. Five mice were sacrificed for each time point at 1, 3 and 7 days after the operation. Following the anesthesia mentioned above, a perfusion fixation with 4% paraformaldehyde was performed to secure the bone tissue. Then, the whole calvarial bone together with the skin was harvested and fixed in the 4% paraformaldehyde. Procedure for histological analysis After fixation in 4% paraformaldehyde at 4°C for 2 days and washing in phosphate buffered saline (PBS) at 4°C for 1 days, samples were decalcified in 20% ethylenediaminetetraacetic acid (EDTA) at 4°C for 14 days and embedded in paraffin. Serial sections of 4 μm thickness were prepared and subjected to hematoxylin and eosin (HE) staining and immunohistochemical (IHC) staining. Images of stained specimens were acquired with an upright microscope (AxioSkop2, Carl Zeiss, Oberkochen, Germany). Immunohistochemical (IHC) staining Immunohistochemistry analysis of markers for sclerostin was carried out in the paraffin embedded sections. Briefly, deparaffinized and hydrated sections were incubated with 0.1% trypsin solution (DifcoTM Trypsin 250, BD Biosciences) for 20 minutes in water bath at 37°C. After cooling, the sections were rinsed with Tris-buffered solution (50mM Tris, 150mM NaCl and 0.05% Tween20, TBST) and incubated with 3% hydrogen peroxide in methanol for 30 minutes at room temperature to remove the internal peroxidase. After rinsing the sections with TBST, sections were incubated with the primary antibody (goat-anti-mouse sclerostin 1:200, R&D Systems, Minneapolis, MN) diluted in TBST overnight at 4°C in a humidifying chamber. After washes with TBST, secondary antibody (Histofine RR Simple StainTM Mouse MAX-PO(G), Nichirei, Tokyo, Japan) was applied and incubated with the sections for 1 hour at room temperature. Sections were developed with 3,3N-diaminobenzidine tertrahydrochloride (DAB) for 2.5 minutes and, after washing with tap water, counterstained with 1% methyl green for 20 minutes. Slides were mounted using permount mounting media (Fisher Scientifics). Images of stained specimens were acquired in the same way as HE staining. Histomorphometry analysis For histomorphometric analysis of HE stain slides, high resolution digital images (at 10 times magnification) were acquired on the left side of calvaria within bone area 1mm away from the sagittal suture, and the same area of the control side. For the images of both sides, the number of empty lacunae and total lacunae was counted using the enlarged images. In the same area of HE stain slide, the images of sclerostin immunohistochemical stain slides were also obtained. Statistical analysis Statistical analyses were performed with standard Student’s t-tests for comparisons between different sides of within the same time point. The one-way analysis of variance (ANOVA) was employed for longitudinal comparisons between different time points of the same side. Results Histological findings In the HE stain slides, the periosteum stripping side showed slightly increased number of empty lacunae than control side at day 1. The number of empty lacunae increased significantly on periosteum stripping side than the control side at day 3 and 7. Interestingly, the number of empty lacunae also increased at day 7 in control side (Figures 2 and 3). Notably, almost all the empty lacunae were found in the outer half of the calvaria, whereas the inner half layer was less affected. Immunohistochemical (IHC) stain In all 3 time points, the periosteum removal sides showed less staining for sclerostin whereas the control sides showed more sclerostin distribution among osteocytes. Also in control side, a clear network of canaliculi radius, which was positively stained, spread out from the lacunae in the middle or inner layer of the bone could be often observed. However in the periosteum removed side, even in the 1day group, the canaliculi existing sclerostin were seldom observed around the lacunae (Figure 4).