Patients with diabetes are at increased risk for delayed union or nonunion following skeletal trauma or elective orthopedic surgery, due to impaired osseous regeneration. Researchers are investigating the underlying etiologies in an effort to find therapeutic solutions.
By David N. Paglia MS, Siddhant K. Mehta BS, Kristen Mason BS, Eric A. Breitbart MD, Aaron Wey BS, Andrew Park MS, Swaroopa Vaidya MS, Ravi Verma BS, Dana Graves PhD, J. Patrick O’Connor PhD, and Sheldon S. Lin MD
In the United States, more than 21 million people suffer from diabetes mellitus (DM), a systemic disease resulting in impaired glucose homeostasis. With a better understanding of its underlying pathophysiology, additional therapeutic regimens have been developed that optimize blood glucose control, resulting in a longer life span in the diabetic patient. As such, other secondary issues linked to DM are now surfacing, including the association between DM and impaired osseous healing.
Several retrospective studies have evaluated complications following elective arthrodesis in DM patients.1-3 Although the patients in these studies represented a sub-population with diabetic neuropathy, the noted increase in incidence of delayed union, nonunion, and pseudoarthrosis was significant in DM patients.1-3 Perlman and Thordarson4 compared the results of ankle fusion in several nonunion risk groups. They found a higher incidence of nonunion after attempted arthrodesis in DM patients compared to non-DM patients, with three of eight attempted arthrodeses in non-neuropathic DM patients resulting in nonunion compared to a 28% nonunion rate in the overall study population. Another study analyzed the predisposing factors contributing to nonunion after ankle arthrodesis. Non-neuropathic patients with major medical problems including renal failure, DM, and alcohol abuse (11 of 13, 85%), were noted to have a significantly higher risk of nonunion (p<0.04) than healthy patients.5
Impaired fracture healing has also been described in several cohort studies of acute fractures in patients with DM.6,7 Loder demonstrated a significant delay in fracture healing in DM patients without neuropathy. Although the value of this study was compromised by variability in patient demographics, fracture pattern, and fracture location, diabetic patients experienced a significant delay in time to union, uniting at an average of 187% of the time required for fractures to heal in patients without DM.7 Cozen, in an 18-patient comparative cohort study, noted more than double the healing time for DM vs. non-DM patients with lower extremity fractures (8.2 vs. 3.6 months, with three of nine DM patients having partial unions).6
Similar findings have been obtained in animal experiments that have measured a reduction in the biomechanical strength of the DM fracture callus.8-11 Wray and Stunkle showed that the breaking strength of a healing fracture in alloxan-induced diabetic animals was significantly less than that of control animals.11 Herbsman et al demonstrated a significant reduction in the tensile strength of a fibular fracture in an alloxan-induced DM rat model, at four weeks after fracture.9 Macey et al showed that the fracture callus from untreated streptozocin-induced DM rats had a 29% decrease in tensile strength and a 50% decrease in stiffness compared to non-DM animals two weeks after the production of a closed fracture.10 Beam et al showed that at six and eight weeks after fracture, the callus from insulin-dependent DM rats had decreased torsional rigidity (70% and 58% respectively) and decreased callus stiffness (78% and 71% respectively) compared to non-DM rats.12
Many inferences about fracture healing can be drawn from data regarding the effect of DM on collagen synthesis and cellular proliferation.13-15 The synthesis of type X collagen by chondrocytes undergoing hypertrophy is a critical step in the process of endochondral ossification.14,15 Previous studies have demonstrated a reduction in the synthesis of collagen by articular cartilage and bone cells from diabetic rats.13-15 Topping et al showed that type X collagen synthesis was 54% to 70% lower in the fracture callus of DM rats than non-DM rats.
Macey et al hypothesized that the decreased mechanical strength in the fracture callus of DM animals during the early stages of repair results from diminished synthesis of collagen secondary to impaired cellular proliferation and/or migration. Between days four and 11 post-fracture, a significant difference in collagen content was observed between the untreated DM animal fracture calluses (50%) and the control group (55%). Treatment of DM animals with insulin resulted in restored tensile strength and callus stiffness similar to the corresponding controls. The DNA content, an indicator of callus cellularity, was decreased 40% in the untreated DM group, suggesting retarded cellular proliferation. Moreover, a decreased collagen-to-DNA ratio (representative of collagen synthesis) was documented during the 14 day healing period in DM animals. In comparison, the control animals demonstrated a rapid increase in the collagen-to-DNA ratio as well as a rapid increase in the collagen content of the callus between days four and seven. The correlation of decreased mechanical strength and decreased or abnormal collagen synthesis suggests that early events play an important, persistent, and deleterious role in DM fracture healing.10
Additional experimental studies have supported the hypothesis that DM alters the early stages of fracture healing. Beam et al showed that insulin-dependent DM rats treated with a low insulin dose to maintain hyperglycemia without ketoacidosis had a decrease in fracture site cell proliferation at 2, 4, and 7 days after fracture compared to non-DM rats.12 These results parallel those of a study which demonstrated that an insulin-deficient environment, mimicking Type I diabetes, yields delayed cartilage synthesis and reduced ossification in a cultured organ explant system.16 Additionally, DM rats have decreased early vascularity at the fracture site compared to control rats at 10 days after fracture.17 These alterations in the early parameters of fracture healing—namely decreased cell proliferation, collagen synthesis, and angiogenesis—ultimately translate to reduced biomechanical properties of the DM fracture callus.
Using chemically-induced and spontaneous DM animal models, our lab has investigated several complementary mechanistic concepts to characterize diabetic fracture healing using a standard, closed, femur fracture model. With insight from our recent research, we present several distinct theories that can potentially explain the underlying etiology of the impaired osseous healing that is observed in diabetic populations, namely local growth factor deficiencies, excessive osteoclastogenesis yielding accelerated cartilage resorption, and elevated advanced glycation end products (AGEs). Furthermore, our scientific data support the hypothesis that adequate blood glucose control can potentially overcome the deleterious effects of diabetes mellitus on osseous healing.
Local growth factor deficiencies
Various growth factors including platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), insulin-like growth factor-I (IGF-I), and transforming growth factor-beta (TGF-β) have been identified as playing critical roles in the early stages of the musculoskeletal healing process.18,19 PDGF is released by the alpha granules of platelets and aids in the migration, proliferation, and differentiation of osteoprogenitor cells. PDGF also up-regulates VEGF and stabilizes newly formed capillaries at the site of injury. VEGF is secreted from platelets, osteoblasts, and chondrocytes, and promotes angiogenesis. VEGF also plays a role in the conversion of cartilage into bone and in osteoblast proliferation and differentiation. The principal source of systemic IGF-I is the liver. However, IGF-I can be made locally by a number of cell types, including osteoblasts, chondrocytes, and endothelial cells. IGF-I acts to promote bone matrix synthesis, cell proliferation and differentiation, and resorption during the early stages of fracture healing. TGF-β is released by platelets during the inflammation stage of fracture healing, but affects all stages of the healing process. TGF-β can stimulate undifferentiated mesenchymal stem cells to proliferate during the early stages of healing, and has also been shown to recruit osteoclast precursor cells.18,19
Impaired bone formation in diabetic fracture healing has been linked to deficient production of growth factors during the healing process. The number of cells that express basic fibroblast growth factor (bFGF), a growth factor that acts as mitogen to bone cells in vitro, is high in rats at one and three weeks after fracture. Immunohistochemical staining showed that less bFGF positive staining was detected in diabetic rats in both the soft callus and periosteum, and this deficit is restored by insulin treatment.20 Similarly, the number of cells that express platelet derived growth factor and mRNA that encodes PDGF is reduced in early diabetic fracture calluses compared with healthy controls.21 Interestingly a reduced rate of cellular proliferation in the diabetic animals correlates with a deficit in PDGF. Diabetes-induced deficits in IGF-1 production have been linked to osteopenia in humans and to impaired vertebral fracture repair.22,23 We have observed a reduction in the synthesis of critical growth factors, including PDGF-β, TGF-β, IGF-I, and VEGF, at the fracture site during the early phases of diabetic fracture healing. This reduction in local levels of critical growth factors within the fracture callus has been associated with decreased cell proliferation and cell differentiation.21,24 These observations demonstrate that DM impairs the production of critical growth factors, which ultimately influences bone healing.
Similar to our work, Street et al studied the angiogenic effects of fracture hematoma by examining the levels of VEGF and PDGF in fracture hematoma and peripheral blood samples of human patients.25 A significant increase in the levels of VEGF and PDGF existed in fracture hematoma compared to plasma. One critical observation was a significant reduction of PDGF levels in the fracture hematoma of patients over 65 years old. Giannoudis et al26 theorized that the early local increase of growth factors observed after reaming may have led to new bone formation through increased cell proliferation, differentiation, chemotaxis, and migration of osteoblasts, and sustained angiogenesis. These previous data correspond to the measured growth factor levels and mRNA expression levels gathered from our recent unpublished experimental in vivo study. These studies of normal elderly subjects who exhibit impaired healing, associated with decreased local growth factor levels also seen in patients with diabetes, exemplify the importance of maintaining appropriate intrinsic growth factor levels for both the healing of the elderly and diabetic individuals. One recent unpublished study by Verma et al27 reported a correlation of decreased growth factors within the fusion bed site of DM patients with Charcot arthropathy undergoing elective hindfoot fusion. Patients who did not achieve union had significantly lower levels of PDGF and VEGF in the fusion bed than patients who did achieve union. Our observed reduction of growth factors in DM fracture hematoma compared to non-DM levels may support this observation28,29 and may explain the high incidence of complications, such as nonunion and delayed union, reported in DM fracture and elective arthrodesis series.2,3,6,7,30
The clinical implication of our studies is clearly demonstrated by focusing on the association between decreased expression of PDGF and DM during early fracture healing. Tyndall et al specifically examined the effects of DM on the expression of PDGF during fracture healing and found that at three early time points, DM animals expressed less PDGF than non-DM animals.21 Furthermore, our gene expression data show that the mRNA levels for PDGF along with the other growth factors are significantly decreased in DM animals. Experimentally, Al-Zube et al treated femur fractures in diabetic rats with rhPDGF-BB and found that the rhPDGF-BB ameliorated the diabetic induced reduction of PDGF.31 The treated animals showed improved early cell proliferation rates and mechanical properties when compared to the diabetic controls.31 Together these studies suggest that DM fracture healing is characterized by decreased cell proliferation and subsequently decreased mechanical properties due to reduced PDGF expression.
If impaired fracture healing due to DM is attributed to decreases in local growth factors, it may be possible then to improve healing by focusing treatment modalities on increasing growth factors in the fracture callus. Treatment with platelet rich plasma (PRP), a bioactive component derived from autologous blood containing a high concentration of platelets, has been shown to ameliorate the deleterious effects of DM on fracture healing both experimentally and clinically. Gandhi et al studied the effect of PRP on early and late parameters of diabetic fracture healing.24 They showed that local percutaneous delivery of PRP, loaded with critical growth factors expressed during early bone healing, had a positive effect on the early parameters of cell proliferation and chondrogenesis, and affected the later, mechanical properties of the healing bone 24 (Figures 1 and 2). Clinically, Gandhi et al used PRP in high risk foot and ankle surgery patients. Many of the high risk patients were diabetic, and the clinical use of PRP supported the experimental findings with improved union rates compared to diabetic patients who did not receive PRP treatment.32
In summary, DM leads to delayed or non-unions, but this effect is improved by growth factor augmentation with PRP or rhPDGF-BB. Understanding the effect of diabetes on growth factor levels during fracture healing will yield future therapies that will compensate for the effect of diabetes by improving local and systemic growth factor levels.
Osteoclastogenesis and cartilage resorption
Impaired osteoclastogenesis is one facet of diabetic osteopathology that may be a critical limiting factor of fracture remodeling in diabetic individuals. Impaired osteoclastogenic regulation at the end of the cartilaginous phase, specifically the accelerated resorption of calcified cartilage, sets the stage for inappropriate healing and a frail diabetic fracture callus.
Insight into the influence of DM on the extent of osteoclastogenesis can be obtained from several studies analyzing the effect of DM upon release and expression of several inflammatory cytokines critical for osteoclastogenesis.33,34 Kayal et al demonstrated that elevated levels of Receptor Activator for Nuclear Factor κ B Ligand (RANKL), Tumor Necrosis Factor-alpha (TNF-α), VEGFa and c as well as Aggrecanase-1 and -2 (ADAMTS-4 and -5) were present in fracture calluses at 12 and 16 days after fracture in streptozotocin-induced diabetic mice.33 High levels of TNF-α and RANKL present in the diabetic mice likely enhanced chondrocyte apoptosis and accelerated loss of callus cartilage. ADAMTS-4 and -5, in particular, are essential in degrading the proteoglycan matrix component of cartilage. Insulin treatment significantly increased bone formation at 16 and 22 days after fracture and decreased levels of ADAMTS-4 and -5 in this mouse model (Figure 3).
Utilizing a similar mouse model, Alblowi et al34 demonstrated that the cytokine dysregulation caused by diabetes, particularly TNF-a, plays a mechanistic role in the accelerated loss of cartilage. Alblowi demonstrated that increased TNF-α expression levels at day 16 after fracture, when cartilage is being replaced by bone, coincided with accelerated cartilage resorption, elevated osteoclast number, and a smaller callus. When diabetic mice were treated with the TNF-specific inhibitor pegsunercept, the elevated osteoclast number, cartilage resorption, and number of chondrocytes that tested positive for TNF-α were significantly reduced. Alblowi et al found that diabetes-enhanced TNF-α levels increases expression of resorptive factors in chondrocytes through a process that involves activation of transcription factor forkhead box O1 (FOXO1). They also concluded that TNF-α dysregulation observed in the systemic disease of DM leads to enhanced osteoclast formation and accelerated loss of callus cartilage.
Although animal models give an interesting insight into the mechanisms behind excessive osteoclastogenesis yielding accelerated cartilage resorption in diabetic fracture healing, it is important to understand the clinical implications of this concept. Suzuki et al measured the bone mineral content and fasting levels of serum intact parathyroid hormone (i-PTH), intact osteocalcin (i-OC), tartrate-resistant acid phosphatase (TRAP), and osteoclastogenesis inhibitory factor/osteoprotogerin (OCIF/OPG) among male type 2 diabetic subjects and their age-matched non-diabetic controls.35 This set of proteins is essential in both bone homeostasis and fracture healing, and all are potentially affected by diabetes. Suzuki et al found that serum levels of i-PTH and i-OC were significantly lower in DM patients than in non-diabetic controls. Although serum levels of TRAP were significantly higher in diabetic patients than those of non-diabetics, Suzuki et al found no definitive correlation when comparing i-OC and OPG serum levels within the same group. TRAP and OCIF/OPG were both shown to have a negative correlation with bone mineral density.
Potential therapies to ameliorate excessive osteoclastogenesis present in diabetic individuals have been examined by Jeffcoate et al in relation to neuropathic osteoarthropathy (Charcot Foot).36 The pathogenesis of acute Charcot Foot has been associated with abnormal foot biomechanics and a pronounced inflammatory reaction. This inflammatory Charcot reaction leads to osteoclastogenesis and bone lysis, making bone prone to fracture. Jeffcoate et al suggest that inhibitors of RANKL, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and interleukin 1β attenuate osteolysis and inflammatory arthritis associated with diabetes and hold potential for future treatment of diabetes. The investigators suggest short term use of high doses of glucocorticoids, known to decrease NF-κB expression, and TNFα agonists (infliximab and etanercept) hold therapeutic potential in the clinical arena. Jeffcoate et al acknowledge that patients with ulcerations of the skin or other risks of infections would need to carefully consider these therapies before treatment because diabetic individuals are more prone to infection and possible tissue necrosis than non-diabetics. Each of these therapies holds the potential to ameliorate the accelerated cartilage resorption and bone lysis that leads to diabetic bone fractures. Further research may yield a treatment that effectively manages this facet of the diabetic pathology.
Advanced glycation end products
Advanced glycation end products (AGEs) have been implicated in contributing to diminished bone healing in patients with DM.37 When reducing sugars, such as glucose, react with amino groups in proteins, lipids, and nucleic acids through a series of reactions forming Schiff bases and Amadori products, AGEs are produced.38 The accumulation of AGEs leads to tissue damage through structural modification of proteins,39 stimulation of cellular responses via receptors specific for AGE proteins,40 and generation of reactive oxygen intermediates.41,42 In patients with DM, AGE formation is markedly accelerated because of the increased availability of glucose and increased oxidative stress. AGEs have been found to accumulate in bone tissue and affect its structural and mechanical properties.43
Most notable is the accumulation of pentosidine during aging in cortical bone of the human femur. Pentosidine is a well-defined biomarker for AGEs.44 Accumulation of AGEs in bone collagen matrix has been shown to alter the mechanical properties of bone by decreasing toughness, which could contribute to skeletal fragility.45-47 AGEs modify cellular behavior by interacting with specific receptors, such as RAGE (receptor for AGEs). Santana et al37 assessed the presence of RAGE by immunohistochemistry in healing craniotomy defects in diabetic animals. They found that craniotomy defects in diabetic animals had a 60% impairment in healing, compared to non-diabetic animals. RAGE was expressed at higher levels in healing bone tissues in diabetic animals compared to control animals. In non-diabetic animals, application of AGE-modified bovine serum albumin to the cranial defect reduced bone healing by 40-60%.37
Another mechanism through which advanced glycation endproducts could inhibit bone formation is by stimulating apoptosis.48 If the number of osteoblasts or pre-osteoblasts is reduced by apoptosis, fewer cells would be available to produce bone matrix. These results suggest that AGEs found in the DM patient may play a role in the pathogenesis of osteopenia and age-related decreased bone strength and increased susceptibility to fracture.
While several clinical studies have described an increased fracture risk associated with AGEs in diabetic patients secondary to an osteopenic state,49,50 a clinical investigation to evaluate the impact of AGEs upon osseous healing after skeletal injury or elective orthopaedic surgery has yet to be performed.
Animal data from our laboratory by Beam et al12 have demonstrated that upon achieving adequate blood glucose control, it is possible to overcome the deleterious effects of DM on osseous healing. This study showed a normalization of various early and late parameters of the bone healing process in diabetic animals to that of non-diabetic animals (Figures 4 and 5). Such findings suggest that adequate glucose control can potentially allow for significant improvement of clinical outcomes and patient functioning. The effect of glucose control in patients undergoing orthopedic surgery has also been evaluated.51-60 However, these studies do not focus on osseous healing, but rather on the association of DM and impaired wound healing and infection, and a subsequent reduction in these events by achieving adequate glucose control. The need still exists for clinical investigation to validate our scientific data that support the hypothesis that adequate glucose control improves osseous healing in diabetic patients sustaining skeletal fractures or undergoing orthopaedic elective surgery.
The clinical significance of DM’s effect on fracture healing is clear. With an impaired osseous regenerative capacity, patients with DM sustaining skeletal trauma or undergoing elective orthopaedic surgery are at greater risk for delayed union and nonunion. Advancements in biotechnology have provided improved means for the investigation of diabetic fracture healing in an effort to better understand the underlying etiology and further guide the development of therapeutic protocols. Our research supports several pathomechanistic theories including local growth factor deficiencies, excessive osteoclastogenesis yielding accelerated cartilage resorption, and an abnormal elevation of advanced glycation end products (AGEs). From a therapeutic perspective, our research shows that adequate blood glucose control yields improved outcomes with respect to the early and late parameters of fracture healing. However, additional investigation through animal and clinical studies is needed to further validate these theories and better define a standard approach to this common problem of impaired osseous healing in the diabetic patient.
David N. Paglia, MS, is a PhD student in biomedical engineering at the University of Medicine and Dentistry of New Jersey and the New Jersey Institute of Technology, both in Newark. Siddhant K. Mehta, BS, is a medical student at St. George’s Medical University in Grenada. Kristen Mason, BS, is a graduate of Lehigh University and is currently pursuing admission to dental college after a period of orthopaedic research. Eric Breitbart, MD, is a second year orthopaedic resident at UMDNJ, Aaron Wey, BS, is a first year medical student, and Andrew Park, MS, is a second year medical student, and Swaroopa Vaidya, MS, is a research staff member of the orthopaedics department at UMDNJ. Ravi Verma, BS is obtaining his MBA from Duke University in Durham, NC, prior to his fourth year of medical school at UMDNJ. Dana Graves, DDS, was previously chair of the department of periodontics at UMDNJ and is currently associate dean of translational research at the University of Pennsylvania in Philadelphia, focusing on the affects of diabetes on bone. James Patrick O’Connor, PhD, is a prominent faculty member in the biochemistry department and Sheldon S. Lin, MD is a prominent orthopaedic surgeon at UMDNJ.
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