Diabetic Fracture Healing Research Articles

Heterochronic Parabiosis Rejuvenates Aged Bones

INTRODUCTION: Diabetes mellitus is associated with an increased incidence of non-union, delayed union and mal-union of fractures. The mechanism(s) leading to impaired fracture healing remain to be elucidated. Recruitment of tissue stem and progenitor cells is vital to restoring injured bones. Our laboratory has recently identified a clonal precursor of bone, cartilage and stroma (BCSP) that generates mature skeletal tissue through a series of functionally characterized cellular intermediates. We hypothesize that impaired recruitment of this BCSP and its downstream progenitors contributes to poor fracture healing in diabetes.

Diminished Recruitment of Resident Skeletal Progenitor Cells in Diabetic Fracture Healing

Tevlin, Ruth MB, BAO, BCh, MRCSI; Seo, Elly MA; McArdle, Adrian MB, BCh, MRCSI; Marecic, Owen BA; Wearda, Taylor BS; Senarath-Yapa, Kshemendra MBBChir, MRCS; Zielins, Elizabeth R. MD; Chan, Charles K. PhD; Weissman, Irving L. MD; Longaker, Michael T. MD, MBA, FACS Author Information

Leptin receptor deficient diabetic (db/db) mice are compromised in postnatal bone regeneration

Increased fragility fracture risk with improper healing is a frequent and severe complication of insulin resistance (IR). The mechanisms impairing bone health in IR are still not fully appreciated, which gives importance to studies on bone pathologies in animal models of diabetes. Mice deficient in leptin signaling are widely used models of IR and its comorbidities. Leptin was first recognized as a hormone, regulating appetite and energy balance; however, recent studies have expanded its role showing that leptin is a link between insulin-dependent metabolism and bone homeostasis. In the light of these findings, it is intriguing to consider the role of leptin resistance in bone regeneration. In this study, we show that obese diabetic mice lacking leptin receptor (db/db) are deficient in postnatal regenerative osteogenesis. We apply an ectopic osteogenesis and a fracture healing model, both showing that db/db mice display compromised bone acquisition and regeneration capacity. The underlying mechanisms include delayed periosteal mesenchymatic osteogenesis, premature apoptosis of the cartilage callus and impaired microvascular invasion of the healing tissue. Our study supports the use of the db/db mouse as a model of IR associated bone-healing deficits and can aid further studies of mesenchymatic cell homing and differentiation, microvascular invasion, cartilage to bone transition and callus remodeling in diabetic fracture healing.

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<0.05). TNF-α significantly increased mRNA levels of several chemokines in vitro which were knocked down with FOXO1 siRNA (P<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.

Review: The Diabetic Bone: A Cellular and Molecular Perspective

With the increasing worldwide prevalence of diabetes the resulting complications, their consequences and treatment will lead to a greater social and financial burden on society. One of the many organs to be affected is bone. Loss of bone is observed in type 1 diabetes, in extreme cases mirroring osteoporosis, thus a greater risk of fracture. In the case of type 2 diabetes, both a loss and an increase of bone has been observed, although in both cases the quality of the bone overall was poorer, again leading to a greater risk of fracture. Once a fracture has occurred, healing is delayed in diabetes, including nonunion. The reasons leading to such changes in the state of the bone and fracture healing in diabetes is under investigation, including at the cellular and the molecular levels. In comparison with our knowledge of events in normal bone homeostasis and fracture healing, that for diabetes is much more limited, particularly in patients. However, progress is being made, especially with the use of animal models for both diabetes types. Identifying the molecular and cellular changes in the bone in diabetes and understanding how they arise will allow for targeted intervention to improve diabetic bone, thus helping to counter conditions such as Charcot foot as well as preventing fracture and accelerating healing when a fracture does occur.

The effects of low‐intensity pulsed ultrasound upon diabetic fracture healing

In the United States, over 17 million people are diagnosed with type 1 diabetes mellitus (DM) with its inherent morbidity of delayed bone healing and nonunion. Recent studies demonstrate the utility of pulsed low-intensity ultrasound (LIPUS) to facilitate fracture healing. The current study evaluated the effects of daily application of LIPUS on mid-diaphyseal femoral fracture growth factor expression, cartilage formation, and neovascularization in DM and non-DM BB Wistar rats. Polymerase chain reaction (PCR) and ELISA assays were used to measure and quantify growth factor expression. Histomorphometry assessed cartilage formation while immunohistochemical staining for PECAM evaluated neovascularization at the fracture site. In accordance with previous studies, LIPUS was shown to increase growth factor expression and cartilage formation. Our study also demonstrated an increase in fracture callus neovascularization with the addition of LIPUS. The DM group showed impaired growth factor expression, cartilage formation, and neovascularization. However, the addition of LIPUS significantly increased all parameters so that the DM group resembled that of the non-DM group. These findings suggest a potential role of LIPUS as an adjunct for DM fracture treatment.

Inflammation as death or life signal in diabetic fracture healing

Increased apoptosis of chondrocytes and osteoblasts and prolonged survival of osteoclasts lead to early destruction of callus tissue and impair bone remodeling in fracture healing of diabetic patients. Diabetes is accompanied by an increased inflammatory state, reactive oxygen species (ROS) generation and accumulation of advanced glycation end products (AGEs), a heterogenous group of toxic metabolites that can induce inflammation. Prolonged hyperglycemia and insulin resistance correlate with increased apoptosis rate and, accordingly, the proapoptotic role of several inflammatory mediators, ROS and AGEs has been also documented. In this review we summarize the most recent reports supporting the idea that inflammatory signaling increases chondrocyte and osteoblast death and prolongs osteoclast survival, resulting in impaired bone regeneration in diabetes. Antagonising inflammatory signal pathways and solution of inflammation may deserve greater attention in the management of diabetic fracture healing.

High Levels of Tumor Necrosis Factor-α Contribute to Accelerated Loss of Cartilage in Diabetic Fracture Healing

Diabetes interferes with fracture repair; therefore, we investigated mechanisms of impaired fracture healing in a model of multiple low-dose streptozotocin-induced diabetes. Microarray and gene set enrichment analysis revealed an up-regulation of gene sets related to inflammation, including tumor necrosis factor (TNF) signaling in the diabetic group, when cartilage is being replaced by bone on day 16, but not on days 12 or 22. This change coincided with elevated osteoclast numbers and accelerated removal of cartilage in the diabetic group (P < 0.05), which was reflected by smaller callus size. When diabetic mice were treated with the TNF-specific inhibitor, pegsunercept, the number of osteoclasts, cartilage loss, and number of TNF-alpha and receptor activator for nuclear factor kB ligand positive chondrocytes were significantly reduced (P < 0.05). The transcription factor forkhead box 01 (FOXO1) was tested for mediating TNF stimulation of osteoclastogenic and inflammatory factors in bone morphogenetic protein 2 pretreated ATDC5 and C3H10T1/2 chondrogenic cells. FOXO1 knockdown by small-interfering RNA significantly reduced TNF-alpha, receptor activator for nuclear factor kB ligand, macrophage colony-stimulating factor, interleukin-1alpha, and interleukin-6 mRNA compared with scrambled small-interfering RNA. An association between FOXO1 and the TNF-alpha promoter was demonstrated by chromatin immunoprecipitation assay. Moreover, diabetes increased FOXO1 nuclear translocation in chondrocytes in vivo and increased FOXO1 DNA binding activity in diabetic fracture calluses (P < 0.05). These results suggest that diabetes-enhanced TNF-alpha increases the expression of resorptive factors in chondrocytes through a process that involves activation of FOXO1 and that TNF-alpha dysregulation leads to enhanced osteoclast formation and accelerated loss of cartilage.

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. 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. 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 microCT. 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. 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 < 0.05). Analysis of microCT 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 < 0.05) on day 16, consistent with the reduction in cartilage. Real-time PCR showed significantly elevated levels of mRNA expression for TNF-alpha, 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. 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.

The effects of local platelet rich plasma delivery on diabetic fracture healing

Several studies have documented that diabetes impairs bone healing clinically and experimentally. The percutaneous delivery of platelet rich plasma (PRP) was used in the diabetic BB Wistar femur fracture model to investigate the use of PRP as a concentrated source of critical early growth factors on bone healing. PRP delivery at the fracture site normalized the early (cellular proliferation and chondrogenesis) parameters while improving the late (mechanical strength) parameters of diabetic fracture healing. These results suggest a role for PRP in mediating diabetic fracture healing and potentially other high risk fractures.

The effects of local insulin delivery on diabetic fracture healing

Several studies have documented that diabetes impairs bone healing clinically and experimentally. Systemic insulin treatment has been shown to ameliorate impaired diabetic bone healing. However, these studies failed to distinguish between a direct and a systemic effect of insulin upon bone healing. A novel intramedullary insulin delivery system was used in the diabetic BB Wistar femur fracture model to investigate the potential direct effects of insulin on bone healing. Insulin delivery at the fracture site normalized the early (cellular proliferation and chondrogenesis) and late (mineralized tissue, cartilage content and mechanical strength) parameters of diabetic fracture healing without affecting the systemic parameters of blood glucose. These results suggest a critical role for insulin in directly mediating fracture healing and that decreased systemic insulin levels in the diabetic state lead to reduced localized insulin levels at fracture site with concomitant increases in diabetic fracture healing time.

Decreased platelet derived growth factor expression during fracture healing in diabetic animals.

Animal model experiments have suggested that diabetes inhibits cell proliferation during fracture healing. Immunohistochemical analysis of proliferating cell nuclear antigen revealed significant reductions in cellular proliferation rates in the fracture callus of spontaneously diabetic BB Wistar rats as compared with healthy BB Wistar rats. Because platelet derived growth factor is associated with the early stage of fracture healing, it was hypothesized that diabetes causes decreased platelet derived growth factor expression during the early phase of fracture healing with a concomitant decrease in cell proliferation. Midshaft femur fractures were created in healthy and spontaneously diabetic BB Wistar rats and analyzed at Days 2, 4, and 7 after fracture for expression of platelet derived growth factor. Immunohistochemistry showed decreased localization of platelet derived growth factor in early diabetic fracture callus compared with healthy controls. Platelet derived growth factor messenger ribonucleic acid levels, as determined by reverse transcription and polymerase chain reaction, also were decreased in early diabetic fractures compared with healthy controls. Therefore the decreased cell proliferation rates associated with diabetic fracture healing are consistent with decreased platelet derived growth factor levels and suggest a causal relationship. These results suggest that diabetes is affecting the early phase of fracture healing by inhibiting cell proliferation through decreasing expression of platelet derived growth factor.

Alterations of Cartilage and Collagen Expression during Fracture Healing in Experimental Diabetes

We investigated alterations in the expression of mRNA for type II and type X collagen in fracture callus of experimentally induced diabetic animals compared with controls and performed radiographic, histological, immunocytochemical and biomechanical studies.Experimentally induced diabetic rats exhibited an alteration in the temporal expression of type II and type X collagen mRNA and a decrease in type X mRNA expression as compared to controls. Radiographs showed a more intense periosteal reaction and a more rapid reconstitution of cortices in control versus diabetic animals. Histologically there was a delay in chondrocyte maturation and hypertrophy seen in diabetics. Immunolocalization of type X collagen demonstrated a delay in type X collagen expression around the hypertrophic chondrocytes. Biomechanical analysis showed a decrease in the strength of healing fractures in diabetic animals.Fracture healing in diabetic patients is compromised and may lead to delays in bone union. Though the exact mechanisms are unknown, we present evidence of decreased mechanical strength of the fracture and suggest that associated changes in collagen expression and chondrocyte maturation are mechanisms leading to delayed healing in untreated and poorly controlled diabetes.

Type X Collagen in Fracture Callus and the Effects of Experimental Diabetes

Studies of fracture repair in diabetes have shown decreased mechanical strength and total collagen in the callus. Type I and Type II collagen are synthesized by bone and cartilage cells, respectively, while Type X collagen is synthesized by hypertrophic chondrocytes in endochondral ossification. A standardized fracture model was chosen to investigate extracellular matrix changes during fracture healing of normal and diabetic rats. Histological examination of the fracture callus in both normal and diabetic animals showed progression from a fibrous tissue to cartilage to bone. An antiserum to Type X collagen was prepared, and immunostaining was observed in the matrix surrounding the hypertrophic chondrocytes. Fracture calluses from streptozotocin induced diabetic rats had similar histology and immunostaining to controls. Radiolabelled proteins were extracted from the calluses of normal and diabetic rats to measure Type X collagen. Type X collagen expression in the fracture callus of normal rats reached a maximum at day fourteen and was decreased by between 54% and 70% in the fracture callus of diabetic rats. The decrease in Type X collagen synthesis may have a role in the defect of fracture healing in diabetes.

Retardation of fracture healing in experimental diabetes

Fracture healing is retarded in rats with uncontrolled alloxan diabetes, as determined by tensile-strength and histochemical evaluation of the fracture callus. Treated alloxan diabetes produces histochemical evidence of a less severe delay of fracture healing but no adverse effect on gain in callus tensile strength.