Histochemical examination of adipose derived stem cells combined with ß-TCP for bone defects restoration under systemic administration of 1α,25(OH)2D3.

The purpose of this study was to evaluate the effects of osteogenic differentiated adipose-derived stem cell (ADSC) loaded beta-tricalcium phosphate (ß-TCP) in the restoration of bone defects under intraperitoneal administration of 1α,25-dihydroxyvitamin D3(1α,25(OH)2D3). ADSCs were isolated from the fat tissue of 8 week old Wister rats and co-cultured with ß-TCP for 21 days under osteogenic induction. Then the ADSC-ß-TCP complexes were implanted into bone defects in the femora of rats. 1α,25(OH)2D3 (VD) or normal saline (NS) was administrated intraperitoneally every other day after the surgery. Femora were harvested at day 7, day 14 and day 28 post-surgery. There were 4 groups for all specimens: ß-TCP-NS group; ß-TCP-ADSC-NS group; ß-TCP-VD group and ß-TCP-ADSC-VD group. Alkaline phosphatase (ALP) was up-regulated obviously in ADSC groups compared with non-ADSC groups at day 7, day 14 and day 28, although high expression of runt-related transcription factor 2 (RUNX2) was only seen at day 7. Furthermore, the number of TRAP-positive osteoclasts and the expression of cathepsin K (CK) were significantly decreased in VD groups compared with non-VD groups at day 7 and day 14. As a most significant finding, the ß-TCP-ADSC-VD group showed the highest BV/TV ratio compared with the other three groups at day 28. Taken together, ADSC-loaded ß-TCP under the administration of 1α,25(OH)2D3 made a promising therapy for bone defects restoration.

Local administration of calcitriol positively influences bone remodeling and maturation during restoration of mandibular bone defects in rats.

The aim of this study was to investigate the influence of calcitriol on osteoinduction following local administration into mandibular bone defects. Calcitriol-loaded absorbable collagen membrane scaffolds were prepared using the polydopamine coating method and characterized by scanning electron microscopy. Composite scaffolds were implanted into rat mandibular bone defects in the following groups: no graft material (control), bare collagen membrane (CM group), collagen membrane bearing polydopamine coating (DOP/CM group), and collagen membrane bearing polydopamine coating absorbed with calcitriol (CAL/DOP/CM group). At 1, 2, 4 and 8weeks post-surgery, the osteogenic potential of calcitriol was examined by histological and immunohistochemical methods. Following in vivo implantation, calcitriol-loaded composite scaffolds underwent rapid degradation with pronounced replacement by new bone and induced reunion of the bone marrow cavity. Calcitriol showed strong potential in inhibiting osteoclastogenesis and promotion of osteogenic differentiation at weeks 1, and 2. Furthermore, statistical analysis revealed that the newly formed bone volume in the CAL/DOP/CM group was significantly higher than other groups at weeks 1, and 2. At weeks 4, and 8, the CAL/DOP/CM group showed more mineralized bone and uniform collagen structure. These data suggest that local administration of calcitriol is promising in promoting osteogenesis and mineralization for restoration of mandibular bone defects.

Expression of HMGB1 in the periodontal tissue subjected to orthodontic force application by Waldo's method in mice.

Recent studies indicate that high mobility group box protein 1 (HMGB1) originating from periodontal ligament (PDL) cells can be a potential regulator in the process of orthodontic tooth movement and periodontal tissue remodeling. The aim of this study is to investigate HMGB1 expression in periodontal tissue during orthodontic tooth movement in mice according to Waldo's method. Six 7-week-old C57BL6 mice were used in these experiments. The elastic band was inserted into the teeth space between the right first and second maxillary molars. After 3 days of mechanical loading, mice were fixed with transcardial perfusion of 4 % paraformaldehyde in 0.1 M phosphate buffer (pH 7.4), and the maxillary was extracted for histochemical analyses. The histological examination revealed local PDL tear at the tension side and the formation of extensive cell-free hyaline zones at the compression side. The immunolocalization of HMGB1 was significantly presented at tension side of PDL, apical area and dental pulp, whereas at the compression side of PDL, the labeling of HMGB1 was almost undetectable as the presence of hyaline zone. Taken together, we concluded that the orthodontic tooth movement by Waldo's method leads to histological changes and HMGB1 expression pattern that differ from those of coil spring method, including PDL tear and extensive hyaline zone which may severely destroy periodontal tissue and in turn impede tooth movement.

Expression of matrix Gla protein and osteocalcin in the developing tibial epiphysis of mice.

This study aimed to investigate the expression of matrix Gla protein (MGP) and osteocalcin (OCN) in the tibial epiphysis of developing mice. At 1, 2, 3, and 4 weeks after birth, tibiae were removed and processed for histochemical observations and western blot analyses under anesthesia. To evaluate bone volume, the specimens were scanned with Micro CT Scanner from the articular cartilage through the growth plate, along the long axis of tibia. At 1 week after birth, OCN reactivity was faint in the region of vascular invasion, while hardly any MGP reactivity was discernible. Subsequently, MGP reactivity was seen on the cartilaginous lacunar walls of hypertrophic chondrocytes, while OCN reactivity was evenly found not only in the bone matrix, but also in the cartilaginous lacunar walls and on the bone surfaces. Furthermore, double-immunostaining clearly showed that MGP reactivity appeared closer to the cartilage matrix than OCN reactivity until postnatal week 3. Interestingly, the immunoreactivities for MGP and OCN both showed tidemarks in the articular cartilage at postnatal week 4, and MGP reactivity was more intense than OCN reactivity. Statistical analyses showed an overall upward trend in MGP and OCN expression levels during tibial epiphysis development, even though OCN was more abundant than MGP at every time-point. Taken together, our findings suggest that the expression of MGP and OCN increased gradually in the murine developing tibial epiphysis, and the two mineral-associated proteins may occur at the same location during a particular period, but at different levels.

Histological Evidence of Increased Osteoclast Cell Number and Asymmetric Bone Resorption Activity in the Tibiae of Interleukin-6-Deficient Mice.

Interleukin-6 (IL-6) is a multifunctional cytokine considered to modulate bone homeostasis. Based on previous contradictory studies, we aimed to verify the influence of IL-6 deficiency on bone remodeling using an IL-6 knockout (IL-6-/-) murine model. Eight-month-old male mice, homozygous for the disrupted IL-6 gene, and their wild type (WT) littermates (control), were used. After transcardiac perfusion, tibiae were removed for histochemical analysis. Compared with the control group, IL-6 deficiency increased tartrate resistant acid phosphatase (TRAP)-positive osteoclast numbers and up-regulated the alkaline phosphatase (ALP) activity of osteoblasts in the metaphysis of the tibia. However, further analysis of serial histological sections from IL-6-/- mice found a significant discrepancy in osteoclast number, with the higher number of TRAP-positive osteoclasts conflicting with the lower number of cathepsin K-positive osteoclasts. Moreover, TUNEL staining identified a significantly higher rate of osteoclast apoptosis in IL-6-/- mice as compared with their WT controls. IL-6 deficiency induced abundant TRAP-positive osteoclasts but delayed bone remodeling by significantly inhibiting the bone resorption activity of osteoclasts and promoting osteoclast apoptosis.

Histochemical examination of cathepsin K, MMP1 and MMP2 in compressed periodontal ligament during orthodontic tooth movement in periostin deficient mice.

The purpose of this study was to investigate immunolocalization of collagenolytic enzymes including cathepsin K, matrix metalloproteinase (MMP) 1 and 2 in the compressed periodontal ligament (PDL) during orthodontic tooth movement using a periostin deficient (Pn-/-) mouse model. Twelve-week-old male mice homozygous for the disrupted periostin gene and their wild type (WT) littermates were used in these experiments. The tooth movement was performed according to Waldo's method, in which elastic bands of 0.5 mm thickness were inserted between the first and second upper molars of mice under anesthesia. At 1 and 3 days after orthodontic force application, mice were fixed with transcardial perfusion of 4 % paraformaldehyde in 0.1 M phosphate buffer (pH 7.4), and the first molars and peripheral alveolar bones were extracted for histochemical analyses. Compared with WT mice, immunolocalization of cathepsin K, MMP1 and MMP2 was significantly decreased at 1 and 3 days after orthodontic tooth movement in the compressed PDL of Pn-/- mice, although MMP1-reactivity and MMP2-reactivity decreased at different amounts. Very little cathepsin K-immunoreactivity was observed in the assessed regions of Pn-/- mice, both before and after orthodontic force application. Furthermore, Pn-/- mice showed a much wider residual PDL than WT mice. Taken together, we concluded that periostin plays an essential role in the function of collagenolytic enzymes like cathepsin K, MMP1 and MMP2 in the compressed PDL after orthodontic force application.