Epac-2 ameliorates spontaneous colitis in Il-10-/- mice by protecting the intestinal barrier and suppressing NF-κB/MAPK signalling.

Intestinal barrier dysfunction and intestinal inflammation interact in the progression of Crohn's disease (CD). A recent study indicated that Epac-2 protected the intestinal barrier and had anti-inflammatory effects. The present study examined the function of Epac-2 in CD-like colitis. Interleukin-10 gene knockout (Il-10-/- ) mice exhibit significant spontaneous enteritis and were used as the CD model. These mice were treated with Epac-2 agonists (Me-cAMP) or Epac-2 antagonists (HJC-0350) or were fed normally (control), and colitis and intestinal barrier structure and function were compared. A Caco-2 and RAW 264.7 cell co-culture system were used to analyse the effects of Epac-2 on the cross-talk between intestinal epithelial cells and inflammatory cells. Epac-2 activation significantly ameliorated colitis in mice, which was indicated by reductions in the colitis inflammation score, the expression of inflammatory factors and intestinal permeability. Epac-2 activation also decreased Caco-2 cell permeability in an LPS-induced cell co-culture system. Epac-2 activation significantly suppressed nuclear factor (NF)-κB/mitogen-activated protein kinase (MAPK) signalling in vivo and in vitro. Epac-2 may be a therapeutic target for CD based on its anti-inflammatory functions and protective effects on the intestinal barrier.

The influence of locally applied granulocyte-colony stimulating factor on osteoporotic bone.

OBJECTIVES: The compromised capacity of bone healing in osteoporotic population renders a serious concern of patients and clinicians. This study aimed to investigate the influence of G-CSF on bone reconstruction using an osteoporotic animal model. MATERIALS AND METHODS: Sixty skeletal mature female Spraque-Dawley rats underwent bilateral ovariectomy (OVX) and were assigned into three groups (n = 20). Three months after OVX, defects of 5 mm in cranial and 2 mm in femur were surgically created on all the animals. The defects were left unfilled, filled with gelatin sponge (GS), or filled with granulocyte-colony stimulating factor (G-CSF) infused GS. Specimens were retrieved for histomorphometric and micro-CT analyses at weeks 1, 4, 8, and 12 after surgery. RESULTS: At early stage of week 1 to week 8, the histomorphometric and micro-CT analysis demonstrated more advanced bone formation in femur in the control group; by week 12, all groups achieved cortical closure. In cranial bone, more advanced bone formation was exhibited in G-CSF-treated group at both early and late stages, although this observation was not statistically significant. CONCLUSIONS: The results indicated that in osteoporotic bone, G-CSF may advance bone healing in cranial bone where spontaneous bone formation was insufficient.

Ruscogenins Improve CD-Like Enteritis by Inhibiting Apoptosis of Intestinal Epithelial Cells and Activating Nrf2/NQO1 Pathway.

Interaction of intestinal barrier dysfunction and intestinal inflammation promotes the progression of Crohn's disease (CD). A more recent study has suggested that ruscogenins (RUS) can exert anti-inflammatory effects through activation of the Nrf2/NQO1 pathway. The current study is aimed at determining the functionalization of RUS on CD-like colitis. Wild-type (WT) mice induced with trinitrobenzene sulfonic acid (TNBS) exhibit a significant inflammation in their colon and are hence widely used for CD models. In the current study, the mice were treated with the Nrf-2 antagonist (ML385) or ruscogenin (RUS) whereas normal WT mice were kept as the negative control. Comparative analysis was then performed on the inflammation and barrier function of the colons. In vitro analysis of mouse colonic organoid systems revealed the influence of RUS on LPS-induced apoptosis, cytokine, and chemokine expressions in the intestinal epithelium. It was found that RUS ameliorates murine colitis through activation of the Nrf2/NQO1 pathway which was presented as a decrease in inflammation score and downregulated levels of cytokine and chemokine synthesis, as well as increased intestinal permeability. Further, it was noted that RUS alleviated LPS-induced apoptosis in the intestinal epithelium cells through upregulation of the Nrf2/NQO1 signaling pathway in the mouse colonic organoids. In addition, ruscogenin (RUS) attenuated the levels of Bax and C-caspase-3 through activation of the Nrf2/HO1 signaling pathway both in vivo and in vitro. Therefore, it was evident that RUS can be applied as a potential alternative therapeutic agent in CD based on its protective effects on the barrier function and anti-inflammatory activity.

Effects of Bisphosphonates on Osseointegration of Dental Implants in Rabbit Model.

This study is to investigate the effect of bisphosphonates on the osseointegration of dental implants in a rabbit model. Twenty female New Zealand White rabbits were equally assigned into control and experiment groups which received saline or zoledronic acid treatment 4 weeks prior to surgery. Titanium dental implant was placed on the calvarial bone. Zoledronic acid or saline treatment continued after surgery for 4 weeks (short-term subgroup) or 8 weeks (long-term subgroup) until sacrifice. Three different fluorochrome labeling solutions were administrated for assessing bone growth rates. Samples of the calvarial bone and mandible were subjected to microcomputed tomography (micro-CT), confocal microscope, and histology analysis. Zoledronic acid treatment significantly reduced bone growth rates in the calvarial bone, but had no significant influence in bone mineral density and trabecular microarchitecture. Significantly lower bone-to-implant contact ratios were found in zoledronic acid-treated animals compared to controls at week 4 but not at week 8. Oncologic dose zoledronic acid suppresses the bone growth rates of the calvarial bone; ZA may have an adverse effect on osseointegration of dental implant in short term, but this effect tends to diminish in long term.

Comparison of Vascular Morphometry in Jawbones and Long Bones: Micro-CT Study in a Rat Model Treated with Zoledronic Acid.

The study was aimed at investigating the effect of zoledronic acid on vascular morphometry in jawbones and long bones on a rat model. Twenty-four skeletal mature Sprague-Dawley female rats were administered oncologic dose of zoledronic acid (ZA) or normal saline for 4 weeks and then subjected to tooth extraction on the mandible and maxilla and a bone defect creation on the femur. After the surgical procedures, ZA or saline treatment was continued until sacrifice at week 2, week 4, and week 8 postoperatively. Vascular perfusion with MICROFIL was performed on all the animals. Micro-CT analysis demonstrated a tendency of decreased vessel density and vessel number in ZA-treated groups but no statistical difference. In conclusion, the neovessel formation is suppressed but not significantly by ZA treatment, indicating that angiogenesis inhibition may contribute to the development of MRONJ but does not play a key role.

Effect of Periapical Diseases in Development of MRONJ in Immunocompromised Mouse Model.

OBJECTIVES: This study aimed to assess the effect of zoledronic acid on an immunocompromised mice model with periapical disease. MATERIALS AND METHODS: Thirty C57BL/6N mice were randomly divided into three groups (N = 10). All animals were subjected to bilateral ovariectomy (OVX) and then treated with saline (Veh), zoledronic acid (ZA), or concomitant zoledronic acid and dexamethasone (ZA/Dx) for 12 weeks. Eight weeks after starting drug administration, pulpal exposure was conducted on the lower left first molar. Four weeks after pulpal exposure, all mice were sacrificed and the mandibles were collected for radiological and histological examinations. RESULTS: Microcomputed tomography (µ-CT) examination showed significantly reduced periapical bone resorption in the ZA/Dx group and decreased periodontal bone resorption in both ZA and ZA/Dx groups. Higher bone mineral density (BMD) and strengthened microstructure were found in ZA and ZA/Dx groups. More empty lacunae were found in ZA and ZA/Dx groups. CONCLUSIONS: Apical periodontitis aggravates MRONJ under immunocompromised circumstances. Concurrent use of ZA and steroids inhibits alveolar bone resorption but increases the risk of developing MRONJ.

Skeletal Site-Specific Response of Jawbones and Long Bones to Surgical Interventions in Rats Treated with Zoledronic Acid.

Bisphosphonates (BPs) have been extensively used for management of bone diseases with pathologically high resorption. Despite the great clinical benefits, a severe complication known as medication-related osteonecrosis of the jaw (MRONJ) has been reported. It is found that most of the reported MRONJ cases were limited in the jawbones/craniofacial bones instead of long bones. The present study aims to investigate the differential bone response to surgical procedures between jawbones and long bones exposed to BPs. Forty-eight skeletal mature Sprague Dawley female rats were administered oncologic dose of zoledronic acid (ZA) or normal saline for 4 weeks and then subjected to tooth extraction on the mandible and maxilla, and a bone defect creation on the femur. After surgical procedures, ZA or saline treatment were continued until sacrifice at week 2, week 4, and week 8, post-operatively. The samples were subjected to micro-computerized tomography (micro-CT) and histological assessment. Osteonecrosis was only found in jawbones in ZA-treated rats. ZA-treated rats showed significantly higher bone mineral density with greater bone volume in all surgical sites than that in the controls. The length of exposure of ZA did not seem to affect trabecular microstructure, and it only showed higher bone volume and BMD with longer healing time which is expected in the healing process.

Role of Periapical Diseases in Medication-Related Osteonecrosis of the Jaws.

OBJECTIVE: The present study aimed to investigate the role of periapical diseases in inducing medication-related osteonecrosis of the jaws (MRONJ) using an ovariectomized (OVX) mice model. MATERIALS AND METHODS: Twenty C57BL/6N female mice were randomly assigned to two groups. All mice were subjected to bilateral ovariectomy and then treated with oncologic dose of zoledronic acid (ZA) or vehicle for twelve weeks. Eight weeks after commence of drug administration, a pulpal exposure (PE) operation was performed on the first right lower molar to induce periapical periodontitis; the contralateral non-PE tooth was used as control. All animals were sacrificed four weeks after pulpal exposure, and the mandibles were harvested for radiological and histomorphometrical analysis. RESULTS: Micro computed tomography (µ-CT) examination demonstrated that periapical diseases significantly increased alveolar bone resorption, and the resorption was greatly attenuated by ZA treatment. Concurrent ZA therapy significantly increased bone density and histological osteocyte necrosis in the presence of periapical lesions. CONCLUSION: ZA treatment reduced bone absorption resulting from periapical disease but increased the risk of developing MRONJ in the ovariectomized mouse model.

Enhanced bone regeneration with sequential delivery of basic fibroblast growth factor and sonic hedgehog.

BACKGROUND: Bone regeneration approaches that mimic the natural processes of bone repair have generated significant attention. We hypothesized that early delivery of an angiogenic factor combined with sustained exposure to an osteogenic factor would recapitulate the critical aspects of natural bone repair. MATERIALS AND METHODS: Basic fibroblast growth factor (bFGF) and sonic hedgehog (Shh) were constructed to the recombinant adeno-associated virus, respectively (rAAV2-tet-off-bFGF and rAAV2-Shh). The previous viral vector allowed for regulation of the bFGF expression by the addition of doxycycline, a tetracycline analogue. These two viral vectors were used to cotransduce bone marrow-derived mesenchymal stem cells (BMSCs). Several osteogenic markers such as core-binding factor a-1, alkaline phosphatase and osteocalcin were detected by quantitative real-time reverse transcriptase polymerase chain reaction. Meanwhile, protein expressions of transgenes were measured by western blot. Furthermore, these cotransduced BMSCs were seeded on ß-tricalcium phosphate (ß-TCP) granules and then were implanted into the calvarium defect in a rat model. A sample of 30 Sprague-Dawley rats was divided into six groups (n=5); an 8-mm critical-sized bone defect was made in calvarium of all subjects. Each group was treated with various transgenic BMSCs and ß-TCP composites; and the sixth group is the negative control which was implanted with nothing. At 4 weeks after treatment, the samples were evaluated with histological staining. RESULTS: The expression of osteogenic marker mRNA had an increased tendency after two genes transduction (p<0.05). In addition, dramatically enhanced regeneration of critical-sized calvarial defects was observed in the groups which were implanted with two transgenic BMSCs and ß-TCP composites. And in these experimental groups, bone areas and vascular densities were increased significantly (p<0.05) than other groups. CONCLUSION: Sequential delivery of angiogenic and osteogenic factors likely has a synergistic effect, mimicking the molecular events of natural bone regeneration.