Plain Language Summary of Work
This work tested whether local insulin therapy could accelerate femur fracture repair in normal, non-diabetic rats.Histomorphometry, radiographic scoring, and torsional mechanical testing were used to measure fracture healing. Using a closed mid-diaphyseal fracture created in the right femur in normal, non-diabetic rats treated with 2 doses of insulin (10 and 20 units) delivered in a calcium sulfate carrier, we demonstrated that low dose insulin (10 units) had a positive effect on biomechanical properties, supported with higher qualitative radiographic scoring, but did not appear to have a significant effect on any of the histological parameters measured. On the other hand, we demonstrated that the high dose (20 units) treatment had significantly increased the amount of cartilage in the fracture callus at 7- and 14-days post-fracture, consistent with enhanced chondrogenesis, but this did not result in better healing outcomes as determined by mechanical testing. Further, our study results also indicate that insulin dose or duration of insulin treatment are critical variables in controlling the fracture healing process since the high dose did not have a positive effect upon the biomechanical parameters at 4 weeks after fracture. The results of this study suggest that locally delivered insulin is a potential therapeutic agent for treating bone fractures. Further studies are necessary, such as large animal proof of concepts, prior to the clinical use of insulin for bone fracture treatment.
We investigated further to quantify the effects of local, early delivery of Ultralente insulin (UL) on various fracture healing parameters in non-diabetic BB Wistar rats. Quantification of insulin levels showed a rapid release of insulin from the fractured femora, demonstrating complete release at 2 days. We demonstrated that this release significantly enhanced mineralization-related markers (Col1a2, osteopontin) with UL insulin treatment 4- and 7- days post fracture. Our work also demonstrated significant differences in vascularity (75% increase in the insulin group) and vessels-forming cells between the treatment and control groups at day 7, with more evident in the insulin-treated group. These early changes resulted in a significant increase in percent-mineralized tissue in the UL-treated animals compared with controls 21 days post-fracture, and in a significantly greater mechanical strength 4 weeks post-fracture. Our study demonstrated that acute, local insulin treatment immediately after fracture promoted healing in non-diabetic rats.
Although local UL insulin results suggest a promising new therapeutic strategy for treating bone fractures and other skeletal injuries, additional studies are needed to understand how dose and duration of treatment affect bone regeneration as well as to characterize insulin’s mechanism of action. Faced with the challenge of finding possible delivery vehicles for insulin to enhance bone fracture healing, 2 calcium salts carriers, Calcium sulfate (CaSO4) and β-tricalcium phosphate (TCP), were investigated as possible delivery vehicles. Our findings showed that; in vitro, beta tricalcium phosphate sustained a prolonged insulin release compared to calcium sulfate. Also, our results showed the strong solidification tendency of calcium sulfate as compared to the paste-like state of beta tri-calcium phosphate carrier in a saline solution at 37°C temperature.
Resulting Publications
Publications (Ph.D. Dissertation):
- Rutgers University (Formerly: University of Medicine and Dentistry of New Jersey). Newark, NJ, USA. Biomedical Engineering, April 2008 Ph.D. Dissertation: The Role of Local Growth Factor Delivery on BoneFracture Healing: Recombinant Human Platelet Derived Growth Factor and Insulin.
Publications (Journal articles):
- Park AG, Paglia DN, Al-Zube L, Hreha J, Vaidya S, Breitbart E, Benevenia J,O’Connor JP, Lin SS. Local Insulin Therapy Enhances Fracture Healing in a Rat Model. Journal of Orthopaedic Research. 2013 May; 31(5): 776-782.
- David N. Paglia, Aaron Wey, Eric A. Breitbart, Jonathan Faiwiszewski, Siddhant K. Mehta, Loay Al-Zube, Swaroopa Vaidya, Jessica A. Cottrell, Dana Graves, Joseph Benevenia, J. Patrick O’Connor, Sheldon S. Lin. Effects of Local Insulin Delivery on Subperiosteal Angiogenesis and Mineralized Tissue Formation during Fracture Healing. Journal of Orthopaedic Research. 2013 May; 31(5): 783-791.
- Loay A. Al-Zu’be, Thakir D. Al-Momani, Osama M. Al-Bataineh, and Lubna H. In vitro Characterization of Calcium Salts as Delivery Vehicles for Insulin. Journal of Biomimetics Biomaterials and Tissue Engineering. 2013 June; 17: 53-58.
Publications (Patents):
- Sheldon Suton Lin, Ankur Gandhi, James Patrick O’Connor, Loay A. Al-Zube, Joseph Benevenia, Russell Parsons. Localized insulin delivery for bone healing. US Patent no. US7763582, assigned to the University of Medicine and Dentistry of New Jersey, Newark, NJ, USA.
Technical Summary of Work
This work tested whether local insulin therapy could accelerate femur fracture repair in normal, non-diabetic rats.Histomorphometry, radiographic scoring, and normalized torsional mechanical testing were used to measure fracture healing at multiple time points. Using a closed mid-diaphyseal fracture created in the right femur in normal, non-diabetic rats treated with 2 doses of insulin delivered in a calcium sulfate carrier, we demonstrated that low dose insulin (10 units) treatment resulted in significantly greater percent-torque to failure as compared to all other treatment groups (p < 0.05) and greater percent-shear stress as compared to the negative control groups (p < 0.05), supported with higher qualitative radiographic scoring, but did not appear to have a significant effect on any of the histological parameters measured. Also, we demonstrated that at 7- and 14-days post-fracture the high dose (20 units) treatment resulted in significant increase in percent-cartilage in the fracture callus as compared to the low dose treatment and negative control groups (p < 0.05), consistent with enhanced chondrogenesis, but this did not result in better healing outcomes as determined by mechanical testing. Further, we demonstrated that when local UL insulin was delivered with a calcium sulfate carrier, tissue insulin levels were significantly greater (p < 0.05) in the fractured femora than the unfractured/untreated femora of the rats treated with high dose within the first 4 days. No statistically significant difference in plasma insulin levels was detected. Although local UL insulin results suggest a promising new therapeutic strategy for treating bone fractures and other skeletal injuries, additional studies are needed to understand how dose and duration of treatment affect bone regeneration as well as to characterize insulin’s mechanism of action.
We also quantified the effects of local intramedullary delivery of Ultralente insulin (UL) on subperiosteal angiogenesis and mineralized tissue formation during fracture healing in a non-diabetic rat model. Histomorphometry, protein qualification, normalized torsional mechanical testing, gene expression analysis, and immunohistochemistry were used and quantified at multiple time points. Using a closed mid-diaphyseal fracture created in the right femur in normal, non-diabetic rats treated with either saline (control group) or UL insulin (experimental group) administrated in the intramedullary canal, we demonstrated that percent-mineralized tissue in insulin-treated animals was significantly greater than controls (p = 0.021) at day 21 post-fracture, however, this effect was not sustained at later time points. UL insulin treatment resulted in a 39% increase in the maximum torque to failure (p= 0.008) and a 58% increase in maximum shear stress (p=0.018) as compared to the control group 4 weeks post fracture.
Our study also demonstrated a significant increase in early osteogenic gene expression, but not chondrogenic gene expression, in animals treated with insulin. Specifically osteopontin mRNA level at 4 day following fracture (p=0.015) and Colla2 mRNA level at 7 days following fracture (p=0.001).
Further, our study revealed significant increase in the average blood vessel (BV) density (number of BVs/mm2 callus area) and in the number of VEGF-C+ cells within the subperiosteal region in the insulin-treated group compared with controls (p = 0.008 and p < 0.001, respectively). In this project differences in local insulin levels were detected between the right, fractured femora and left, intact femora of the rats treated with insulin within the first 12–48 h (significantly higher at 48 h [p = 0.045]), but were largely depleted by 96 h. Overall, our work demonstrated that acute, local insulin treatment immediately after fracture promoted healing in non-diabetic rats through an increase in osteogenic gene expression, subperiosteal angiogenesis, and mineralized tissue formation. While the current study did find significant increases in early osteogenic gene expression, no such changes were evident for chondrogenic gene expression. This finding is possibly due to the rapid release of insulin from the fractured femora.
In a subsequent study we investigated release kinetics of insulin from two different osteoconductive carriers, calcium sulfate (CaSO4) and β-tricalcium phosphate (TCP), in vitro at multiple time points. Our results demonstrated an early burst in insulin release when delivered via the calcium sulfate carrier and a continuous and rapid release within the 1st 12 hours, whereas, β-tricalcium phosphate carrier caused significantly slower release kinetics within the same period (p < 0.05). Further, our study demonstrated the solidification tendency of the calcium sulfate carrier as opposed to the paste-like state of the β-tricalcium phosphate carrier 48 hours after being placed in a saline solution at 37°C. Our solidification observation indicates that CaSO4 might serve as a stabilizing material around the fracture site due to its high solidification tendency; however, the solidification of CaSO4 might lead to microvasculature obstruction during revascularization in the healing process. Overall, calcium sulfate (CaSO4) caused the release of a large, early-burst of insulin immediately after its placement in the saline solution followed by a rapid release of its content of insulin within 12 hours, while beta tri-calcium phosphate (TCP) resulted in a smaller early-burst of insulin followed by a continuous and sustainable release projected linearly for up to ~12 hours.
Summary of the Significance of the Work
Over 10 million surgical procedures are performed annually in the United States to treat musculoskeletal injuries (Young CS et. al. 2009). Musculoskeletal conditions are among the most disabling and costly conditions suffered by Americans. Aggregate total Musculoskeletal medical care expenditures in the United States reached $796.3 billion in 2011, from which $190.6 billion are the annual cost of injuries and fractures (The burden of musculoskeletal diseases in the united states, 2014). Approximately 10% of the 7.9 million annual fracture patients in the United States experience nonunion and/or delayed unions, which have a substantial economic and quality of life impact (The burden of musculoskeletal diseases in the united states, 2014). Although many orthopaedic problems existed in the past now have practical solutions because of the collaborative basic and clinical research, impaired fracture healing represents an ongoing failure of initial fracture management (Hayda RA et. al. 1998). Therefore, any research that helps to mitigate health outcomes associated with fracture nonunion and/or delayed unions is very valuable to the US in particular. Our research is especially valuable because we demonstrated that local delivery of insulin, which is widely available and relatively cheap, can be utilized as a therapeutic agent to significantly promote fracture healing in health and compromised conditions such as diabetes and osteoporosis. We also demonstrated the dose dependent effect of local insulin delivery on early and late fracture healing stages. We also identified possible research avenues needed to understand how dose and duration of treatment affect bone regeneration as well as to characterize insulin’s mechanism of action. We also identified possible methods to deliver insulin via a carrier that would avoid burst dosing effects and early dissipation of insulin that will increase the effectiveness and safety. Overall, our studies promote the use of insulin to promote bone healing in animal models and strongly support continued research in the use of insulin. Insulin is much cheaper than many other pharmaceutical agents, so further verification of its clinical value is also relevant to the reduction of healthcare costs.
Summary of Implementation/Influence of the Work
My patent was licensed to CreOsso, LLC, Montclair, NJ, USA (http://www.creosso.com/index.html). CreOsso, LLC. seeks to leverage close to 10 years of research into the effects of local delivery of insulin and insulin-mimetics on bone regeneration in long bones, extremities and the spine. My 3 papers and 1 patent related to this project have been widely cited, with a total of 69 citations. For specific citation details, please refer to my google scholar profile. A few notable citations include:
- Yuasa, N.A. Mignemi, J.V. Barnett et al. 2014. “The temporal and spatial development of vascularity in a healing displaced fracture”. Bone. 2014 October; 67:208-221. – In this study, the researchers combining novel techniques of bone angiography and a reproducible murine femur fracture model to demonstrate for the first time the complete temporal and spatial pattern of revascularization in a displaced/stabilized fracture. These researchers from Vanderbilt University Medical Center (Nashville, TN) cited 2 of my papers and specifically mentioned our studies in the context of demonstrating the significant of addressing vascular dysfunction in reducing fracture healing complications.
- O. Malekzadeh, M. Ransjo et al. 2016. ‘Insulin released from titanium discs with insulin coatings – kinetics and biological activity’. Journal of Biomedical Materials Research Part B. 2016 May; 105(7):1847-1854. – These researchers ran an in vitro study where human recombinant insulin was immobilized onto titanium discs, and the insulin release kinetics was evaluated using Electro‐chemiluminescence immunoassay, furthermore, they evaluated the biological effects of the released insulin on human osteoblast‐like MG‐63 cells. These researchers from the University of Gothenburg (Gothenburg, Sweden) cited 2 of my papers in the context of demonstrating the anabolic significance of insulin in bone healing and the different methods we used to locally administrate insulin around the fracture site via injection or in combination with carriers.
- B. Clifton, D.N. Paglia et. al. 2014. ‘Effects of Wnt5a Haploinsufficiency on Bone Repair’. Journal of Orthopaedic Trauma’. 2014 August; 28(8); e191-e197. – These researchers, to better understand the effect of the Wnt5a on bone repair, carried out Femoral fracture experiments on Wnt5a+/+ and Wnt5a+/− mice. These researchers from the University of Connecticut Health Center (CT, USA) cited 2 of my papers in the context of demonstrating the testing methodologies used.
- Zhu, Y. Zhu, B. Ni et. al. 2014. ‘Mesoporous Silica Nanoparticles/Hydroxyapatite Composite Coated Implants to Locally Inhibit Osteoclastic Activity’. 2014; 6(8); 5456-5466. – These researchers developed a composite coating drug delivery system, on the surface of stainless Kirschner wires, composed of HA and mesoporous silica nanoparticles (MSNs) to provide functions of drug delivery for zoledronic acid molecules to resist unwanted bone resorption and accelerate fracture healing. These researchers from the Second Military Medical University (Shanghai, P. R. China) cited one of my papers in the context of demonstrating that biological coated-internal fixation implants have shown great potentials in accelerating fracture healing through incorporating and locally release bioactive components.