Atom transfer radical polymerization (ATRP) was used to produce a versatile drug delivery system capable of encapsulating a range of molecules. Inverse miniemulsion ATRP permitted the synthesis of biocompatible and uniformly cross-linked poly(ethylene oxide)-based nanogels entrapping gold nanoparticles, bovine serum albumin, rhodamine B isothiocyanate-dextran, or fluoresceine isothiocyanate-dextran. These moieties were entrapped to validate several biological outcomes and to model delivery of range of molecules. Cellular uptake of nanogels was verified by transmission electron microscopy, gel electrophoresis, Western blotting, confocal microscopy, and flow cytometry. Fluorescent co-localization of nanogels with a fluorophore-conjugated antibody for clathrin indicated clathrin-mediated endocytosis. Further, internalization of nanogels either with or without GRGDS cell attachment-mediating peptides was quantified using flow cytometry. After 45 minutes of incubation, the uptake of unmodified FITC-Dx-loaded nanogels was 62%, whereas cellular uptake increased to >95% with the same concentration of GRGDS-modified FITC-Dx- nanogels. In addition, a co-culture of human umbilical vascular endothelial cells (HUVECs) and human mesenchymal stem cells (hMSCs) validated cell endocytosis. Application of ATRP enabled the synthesis of a functionalized drug delivery system with a uniform network that is capable of encapsulating and delivering inorganic, organic, and biological molecules.
Local insulin delivery has been shown to improve osseous healing in diabetic animals. The purpose of this study was to quantify the effects of local intramedullary delivery of saline or Ultralente insulin (UL) on various fracture healing parameters using an in vivo non-diabetic BB Wistar rat model. Quantitation of local insulin levels showed a rapid release of insulin from the fractured femora, demonstrating complete release at 2 days. RT-PCR analysis revealed that the expression of early osteogenic markers (Col1a2, osteopontin) was significantly enhanced with UL treatment when compared with saline controls (p < 0.05). Significant differences in VEGF þ cells and vascularity were evident between the treatment and control groups at day 7 (p < 0.05). At day 21, histomorphometric analysis demonstrated a significant increase in percent mineralized tissue in the UL-treated animals compared with controls (p < 0.05), particularly within the subperiosteal region of the fracture callus. Mechanical testing at 4 weeks showed significantly greater mechanical strength for UL-treated animals (p < 0.05), but healing in control animals caught up at 6 weeks post-fracture. These results suggest that the primary osteogenic effect of UL during the early stages of fracture healing (1-3 weeks) is through an increase in osteogenic gene expression, subperiosteal angiogenesis, and mineralized tissue formation. ß
Each year, over one million orthopedic operations are performed which a bony defect is presence, requiring the use of further augmentation in addition to bony fixation. Application of autogenous bone graft is the standard of care to promote healing of these defects, but several determents exist in using autogenous bone graft exist including limited supply and donor site morbidity. Prior work has demonstrated that local insulin application to fracture sites promote fracture healing, but no work has been performed to date in its effects upon defect healing/allograft incorporation. The goal of this study was to examine the potential role of local insulin application upon allograft incorporation. Microradiographic, histologic, and histomorphometric analysis outcome parameters showed that local insulin significantly accelerated new bone formation. Histological comparisons using predetermined scoring systems demonstrated significantly greater healing in femora treated with insulin compared to control femora (p < 0.001). Quantitatively more bone production was also observed, specifically in areas of endosteal (p = 0.010) and defect (p = 0.041) bone in femora treated with local insulin, compared to control femora, 6 weeks after implantation. This study demonstrates the potential of local insulin as an adjunct for the treatment of segmental defect and allograft incorporation.
A significant number of lower extremity fractures result in mal-union necessitating effective treatments to restore ambulation. Prior research in diabetic animal fracture models demonstrated improved healing following local insulin application to the fracture site and indicated that local insulin therapy can aid bone regeneration, at least within an insulin-dependent diabetic animal model. This study tested whether local insulin therapy could accelerate femur fracture repair in normal, non-diabetic rats. High (20 units) and low (10 units) doses of insulin were delivered in a calcium sulfate carrier which provided sustained release of the exogenous insulin for 7 days after fracture. Histomorphometry, radiographic scoring, and torsional mechanical testing were used to measure fracture healing. The fracture calluses from rats treated with high-dose insulin had significantly more cartilage than untreated rats after 7 and 14 days of healing. After 4 weeks of healing, femurs from rats treated with low-dose insulin had significantly higher radiographic scores and mechanical strength (p < 0.05), compared to the no treatment control groups. 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. The effect of insulin on diabetic fracture healing has been well documented.1-3 Diabetes leads to reduced cellular proliferation in the early callus, reduced collagen synthesis and content compared to non-diabetic control animals, and reduced biomechanical properties of the healing fracture.1 Administration of systemic insulin to regulate blood glucose within normal levels ameliorates impaired fracture healing in an insulin-dependent diabetic rat model.2 Remarkably though, local insulin treatment at the fracture site in insulin-dependent diabetic rats that were maintained in a severe hyperglycemic state also ameliorates impaired fracture healing associated with diabetes.1,2 Local insulin therapy improved fracture site cell proliferation, cartilage formation, new bone content, and callus strength in hyperglycemic, insulin-dependent, diabetic rats. 1,4The experiments performed in diabetic animals indicate that insulin acts to positively regulate fracture healing at the systemic and local levels. Use of insulin to augment fracture healing or other bone regeneration processes in normal animal models of bone regeneration has not been investigated. Elevating systemic insulin levels would cause hypoglycemia in normal mammals and thus is not a therapeutic option. However, local application of insulin to a fracture site that would provide locally high yet systemically near normal insulin levels could be a therapeutic strategy to enhance fracture healing.The effects of local insulin therapy on femur fracture healing were measured using a non-diabetic rat model. We hypothesize that in a dose dependent manner, local insulin com...
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