Diabetes Causes the Accelerated Loss of Cartilage During Fracture Repair Which Is Reversed by Insulin Treatment

Fracture healing in diabetic individuals and in animal models of diabetes is impaired. To investigate mechanisms by which diabetes may affect fracture healing we focused on the transition from cartilage to bone, a midpoint in the fracture healing process. Femoral fractures were induced in mice rendered diabetic by multiple low dose streptozotocin treatment and compared to matching normoglycemic mice. One group of diabetic animals was treated with slow release insulin to maintain normal serum glucose levels. The results indicate that there was relatively little difference in the initial formation of the fracture callus on day 10. However, on day 16 the diabetic group had significantly smaller callus, greater loss of cartilage and enhanced osteoclastogenesis that was normalized by treatment with insulin when assessed by histomorphometric analysis. Chondrocyte apoptosis was significantly higher in diabetic mice and this increase was blocked by insulin. These changes were accompanied by diabetes-increased mRNA levels of RANKL, TNF-α, and ADAMTS-4 and -5 measured by real-time PCR, which was reversed by insulin treatment. On days 16 and 22 bone formation within the callus of diabetic mice was significantly less than the normoglycemic and brought to normal levels by insulin treatment. These results suggest that a significant effect of diabetes on fracture healing is increased chondrocyte apoptosis and osteoclastogenesis that accelerates the loss of cartilage and reduces the anlage for endochondral bone formation during fracture repair. That insulin reverses these effects demonstrates that they are directly related to the diabetic condition

Chemokine Expression Is Upregulated in Chondrocytes in Diabetic Fracture Healing

Chemokines are thought to play an important role in several aspects of bone metabolism including the recruitment of leukocytes and the formation of osteoclasts. We investigated the impact of diabetes on chemokine expression in normal and diabetic fracture healing. Fracture of the femur was performed in streptozotocin-induced diabetic and matched normoglycemic control mice. Microarray analysis was carried out and chemokine mRNA levels in vivo were assessed. CCL4 were examined in fracture calluses by immunohistochemistry and the role of TNF in diabetes-enhanced expression was investigated by treatment of animals with the TNF-specific inhibitor, pegsunercept. In vitro studies were conducted with ATDC5 chondrocytes. Diabetes significantly upregulated mRNA levels of several chemokines in vivo including CCL4, CCL8, CCL6, CCL11, CCL20, CCL24, CXCL2, CXCL5 and chemokine receptors CCR5 and CXCR4. Chondrocytes were identified as a significant source of CCL4 and its expression in diabetic fractures was dependent on TNF (P \u3c 0.05). TNF-α significantly increased mRNA levels of several chemokines in vitro which were knocked down with FOXO1 siRNA (P \u3c 0.05). CCL4 expression at the mRNA and proteins levels was induced by FOXO1 over-expression and reduced by FOXO1 knockdown. The current studies point to the importance of TNF-α as a mechanism for diabetes enhanced chemokine expression by chondrocytes, which may contribute to the accelerated loss of cartilage observed in diabetic fracture healing. Moreover, in vitro results point to FOXO1 as a potentially important transcription factor in mediating this effect

Diabetes Reduces Mesenchymal Stem Cells in Fracture Healing Through a TNFα-Mediated Mechanism

Aims/hypothesis Diabetes interferes with bone formation and impairs fracture healing, an important complication in humans and animal models. The aim of this study was to examine the impact of diabetes on mesenchymal stem cells (MSCs) during fracture repair. Methods Fracture of the long bones was induced in a streptozotocin-induced type 1 diabetic mouse model with or without insulin or a specific TNFα inhibitor, pegsunercept. MSCs were detected with cluster designation-271 (also known as p75 neurotrophin receptor) or stem cell antigen-1 (Sca-1) antibodies in areas of new endochondral bone formation in the calluses. MSC apoptosis was measured by TUNEL assay and proliferation was measured by Ki67 antibody. In vitro apoptosis and proliferation were examined in C3H10T1/2 and human-bone-marrow-derived MSCs following transfection with FOXO1 small interfering (si)RNA. Results Diabetes significantly increased TNFα levels and reduced MSC numbers in new bone area. MSC numbers were restored to normal levels with insulin or pegsunercept treatment. Inhibition of TNFα significantly reduced MSC loss by increasing MSC proliferation and decreasing MSC apoptosis in diabetic animals, but had no effect on MSCs in normoglycaemic animals. In vitro experiments established that TNFα alone was sufficient to induce apoptosis and inhibit proliferation of MSCs. Furthermore, silencing forkhead box protein O1 (FOXO1) prevented TNFα-induced MSC apoptosis and reduced proliferation by regulating apoptotic and cell cycle genes. Conclusions/interpretation Diabetes-enhanced TNFα significantly reduced MSC numbers in new bone areas during fracture healing. Mechanistically, diabetes-enhanced TNFα reduced MSC proliferation and increased MSC apoptosis. Reducing the activity of TNFα in vivo may help to preserve endogenous MSCs and maximise regenerative potential in diabetic patients

Diminished Bone Formation During Diabetic Fracture Healing Is Related to the Premature Resorption of Cartilage Associated with Increased Osteoclast Activity

Histological and molecular analysis of fracture healing in normal and diabetic animals showed significantly enhanced removal of cartilage in diabetic animals. Increased cartilage turnover was associated with elevated osteoclast numbers, a higher expression of genes that promote osteoclastogenesis, and diminished primary bone formation. Introduction Diminished bone formation, an increased incidence of nonunions, and delayed fracture healing have been observed in animal models and in patients with diabetes. Fracture healing is characterized by the formation of a stabilizing callus in which cartilage is formed and then resorbed and replaced by bone. To gain insight into how diabetes affects fracture healing, studies were carried out focusing on the impact of diabetes on the transition from cartilage to bone. Materials and Methods A low-dose treatment protocol of streptozotocin in CD-1 mice was used to induce a type 1 diabetic condition. After mice were hyperglycemic for 3 weeks, controlled closed simple transverse fractures of the tibia were induced and fixed by intramedullary pins. Histomorphometric analysis of the tibias obtained 12, 16, and 22 days after fracture was performed across the fracture callus at 0.5 mm proximal and distal increments using computer-assisted image analysis. Another group of 16-day samples were examined by μCT. RNA was isolated from a separate set of animals, and the expression of genes that reflect the formation and removal of cartilage and bone was measured by real-time PCR. Results Molecular analysis of collagen types II and X mRNA expression showed that cartilage formation was the same during the initial period of callus formation. Histomorphometric analysis of day 12 fracture calluses showed that callus size and cartilage area were also similar in normoglycemic and diabetic mice. In contrast, on day 16, callus size, cartilage tissue, and new bone area were 2.0-, 4.4-, and 1.5-fold larger, respectively, in the normoglycemic compared with the diabetic group (p \u3c 0.05). Analysis of μCT images indicated that the bone volume in the normoglycemic animals was 38% larger than in diabetic animals. There were 78% more osteoclasts in the diabetic group compared with the normoglycemic group (p \u3c 0.05) on day 16, consistent with the reduction in cartilage. Real-time PCR showed significantly elevated levels of mRNA expression for TNF-α, macrophage-colony stimulating factor, RANKL, and vascular endothelial growth factor-A in the diabetic group. Similarly, the mRNA encoding ADAMTS 4 and 5, major aggrecanases that degrade cartilage, was also elevated in diabetic animals. Conclusions These results suggest that impaired fracture healing in diabetes is characterized by increased rates of cartilage resorption. This premature loss of cartilage leads to a reduction in callus size and contributes to decreased bone formation and mechanical strength frequently reported in diabetic fracture healing

TNFα Contributes to Diabetes Impaired Angiogenesis in Fracture Healing

Diabetes increases the likelihood of fracture, interferes with fracture healing and impairs angiogenesis. The latter may be significant due to the critical nature of angiogenesis in fracture healing. Although it is known that diabetes interferes with angiogenesis the mechanisms remain poorly defined. We examined fracture healing in normoglycemic and streptozotocin-induced diabetic mice and quantified the degree of angiogenesis with antibodies to three different vascular markers, CD34, CD31 and Factor VIII. The role of diabetes-enhanced inflammation was investigated by treatment of the TNFα-specific inhibitor, pegsunercept starting 10 days after induction of fractures. Diabetes decreased both angiogenesis and VEGFA expression by chondrocytes. The reduced angiogenesis and VEGFA expression in diabetic fractures was rescued by specific inhibition of TNF in vivo. In addition, the TNF inhibitor rescued the negative effect of diabetes on endothelial cell proliferation and endothelial cell apoptosis. The effect of TNFα in vitro was enhanced by high glucose and an advanced glycation endproduct to impair microvascular endothelial cell proliferation and tube formation and to stimulate apoptosis. The effect of TNF, high glucose and an AGE was mediated by the transcription factor FOXO1, which increased expression of p21 and caspase-3. These studies indicate that inflammation plays a major role in diabetes-impaired angiogenesis in endochondral bone formation through its effect on microvascular endothelial cells and FOXO1

A novel method of sampling gingival crevicular fluid from a mouse model of periodontitis

Using a mouse model of silk ligature-induced periodontal disease (PD), we report a novel method of sampling mouse gingival crevicular fluid (GCF) to evaluate the time-dependent secretion patterns of bone resorption-related cytokines. GCF is a serum transudate containing host-derived biomarkers which can represent cellular response in the periodontium. As such, human clinical evaluations of PD status rely on sampling this critical secretion. At the same time, a method of sampling GCF from mice is absent, hindering the translational value of mouse models of PD. Therefore, we herein report a novel method of sampling GCF from a mouse model of periodontitis, involving a series of easy steps. First, the original ligature used for induction of PD was removed, and a fresh ligature for sampling GCF was placed in the gingival crevice for ten minutes. Immediately afterwards, the volume of GCF collected in the sampling ligature was measured using a high precision weighing balance. The sampling ligature containing GCF was then immersed in a solution of PBS-Tween 20 and subjected to ELISA. This enabled us to monitor the volume of GCF and detect time-dependent changes in the expression of such cytokines as IL-1b, TNF-α, IL-6, RANKL, and OPG associated with the levels of alveolar bone loss, as reflected in GCF collected from a mouse model of PD. Therefore, this novel GCF sampling method can be used to measure various cytokines in GCF relative to the dynamic changes in periodontal bone loss induced in a mouse model of PD. Correspondence: Toshihisa Kawai, DDS, PhD, Department of Immunology and Infectious diseases, The Forsyth Institute, 245 First Street, Cambridge, MA 02142, Tel: 617-892-8317, Fax: 617-892-8437, [email protected]. # Contributed equally to this work HHS Public Access Author manuscript J Immunol Methods. Author manuscript; available in PMC 2017 November 01. Published in final edited form as: J Immunol Methods. 2016 November ; 438: 21–25. doi:10.1016/j.jim.2016.08.008. Author Manuscript Author Manuscript Author Manuscript Autho

Diminished bone formation during diabetic fracture healing is related to premature resorption of cartilage associated with increased osteoclast activity

Thesis (DSc) --Boston University, Goldman School of Dental Medicine, 2007 (Department of Oral Biology and Periodontology).Includes bibliographic references: leaves 115-133.Introduction: Diminished bone formation, an increased incidence of non unions and delayed fracture healing have been observed in animal models and in patients with diabetes. Fracture healing is characterized by the formation of a stabilizing callus in which cartilage is formed then resorbed and replaced by bone. To gain insight into how diabetes affects fracture healing, studies were carried out focusing on the impact of diabetes on the transition from cartilage to bone. Methods: A low-dose treatment protocol of streptozotocin in CD-1 mice was used to induce type I diabetes. After mice were hyperglycemic for three weeks, controlled closed simple transverse fractures of the tibia or femur were induced and fixed by intramedullary pins. Histomorphometric analysis of the tibias obtained 12, 16 and 22 days after fracture was performed across the fracture ca11us at O.5 mm proximal and distal increments using computer assisted image analysis. Another group of 16-day samples were examined by micro-computed tomography (micro CT). RNA was isolated from a separate set of animals and the expression of genes that reflect the formation and removal of cartilage and bone was measured by real time PCR. In addition, RNA was used for genetic profiling using microarray and data obtained was analyzed using gene set enrichment analysis (GSEA). Another study was performed on femur fractures and involved an additional group of animals which received insulin treatment through subcutaneous implants. Similar histological analysis was performed lO, 16 and 22 days after fracture. RNA was also isolated from a separate set of animals for real time PCR on genes that regulate cartilage, bone formation and bone remodeling. Results: Molecular analysis of collagen types II and X mRNA expression showed that expression of matrix genes was the same during the initial period of callus formation. Histomorphometric analysis of day 12 fracture calluses showed that callus size and cartilage area were also similar in normoglycemic and diabetic mice. In cotrast, on day 16 ca11us size, cartilage tissue and new bone area were 2.0, 4.4 and 1.5 fold larger respectively in the normoglycemic compared to the diabetic group (P[less than]0.05). Analysis of micro CT images indicated that the bone volume in the normoglycemic animals was 38% 1arger than in diabetics. There were 78% more osteoclasts in the diabetic compared to the normoglycemic group (P=0.05) on day 16 which is consistent with the reduction in cartilage. Real time PCR showed significantly elevated levels of mRNA expression for TNF-[alpha], MCSF and RANKL in the diabetic group. Similarly, mRNAs encoding ADAMTS 4 and 5, major aggrecanases that degrade cartilage, were also elevated in diabetic animals. There was a 4.5 fold increase in apoptotic chondrocytes in the diabetic group compared to the normoglycemic. Gene set enrichment analysis showed that the 16 day diabetic animals exhibited up regulation of gene sets related to matrix degradation, inflammation and apoptosis. Insulin treatment resulted in normalization of the diabetic effect on fracture healing. Conclusion: These results suggest that impaired fracture healing in diabetes is characterized by increased rates of cartilage resorption. This premature loss of cartilage leads to a reduction in callus size and may contribute to decreased bone formation and mechanical strength frequently reported in diabetic fracture healing

Management of Concomitant Intrusion and Complicated Crown-Root Fracture Injury of Maxillary Central Incisors in a Child

Dental intrusions are a severe type of injury because they impact the neurovascular supply of the tooth as well as the supporting tissues which predispose the tooth to necrosis and root resorption. Management of these injuries requires repositioning of the teeth under close monitoring to avoid complications. The management becomes more comprehensive when an intrusion is combined with other injuries, such as a crown-root fracture. This case report represents a 4-year follow-up of a child who suffered from a concomitant injury of intrusion and complicated crown-root fracture to the maxillary immature permanent central incisors. The management involved a multidisciplinary approach including endodontics, pedodontics, orthodontics, periodontics, and prosthodontics. Given the guidelines of dental trauma and the circumstances of the case, the fractured teeth were root canal treated, filled with a bioceramic plug and gutta-percha, and then restored with posts/cores and temporary crowns. The intrusion was managed initially by passive eruption followed by an active orthodontic eruption, after which the teeth were restored with permanent ceramic crowns. Throughout the course of treatment, the teeth showed no complications of root resorption or ankylosis, although one tooth developed a periapical infection which was managed by apical surgery. At the 4-year follow-up, the teeth revealed healthy periodontium and good esthetics