Eric Breitbart

Mesenchymal stem cells accelerate bone allograft incorporation in the presence of diabetes mellitus

Allograft (Allo) incorporation in the presence of a systemic disease like diabetes mellitus (DM) is becoming a major issue in the orthopedic community. Mesenchymal stem cells (MSC) are multipotent stem cells that may be derived from adult, whole bone marrow and have been shown to induce bone formation in segmental defects when combined with the appropriate carrier/scaffold. The objectives of this study were to analyze the effect of DM upon Allo incorporation in a segmental rat femoral defect and to also investigate MSC augmentation of Allo incorporation. Segmental (5 mm) femoral defects were created in non‐DM and DM rats and treated with Allo containing demineralized bone matrix (DBM) or DBM with MSC augmentation. Histological scoring at 4 weeks demonstrated less mature bone in the DM/DBM group compared to its non‐DM counterpart (p < 0.001). However, there was significantly more mature bone in the DM/MSC group when compared to the DM/DBM group at both 4 and 8 weeks (p < 0.001 and p = 0.004). Furthermore, significantly more bone formation was observed in the DM/MSC group compared to the DM/DBM group at the 4‐week time point (p < 0.001). The results of this study suggest that MSC are a potential adjunct for bone regeneration when implanted in an orthotopic site in the presence of DM.

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.

rhBMP-2 enhances the bone healing response in a diabetic rat segmental defect model.

Objective: Recombinant human bone morphogenetic protein-2 (rhBMP-2) has been shown to enhance new bone formation in fracture and bone defect models in both normal and diabetic rats. Effects of rhBMP-2 in a segmental femoral defect model in diabetes mellitus (DM) BB Wistar rats have not been reported. Methods: Collagen sponge soaked with either buffer or rhBMP-2 was inserted in a mid-diaphyseal 3.0-mm defect fixed with polyimide plate and stainless steel screws, in 62 DM BB Wistar rats. Progress of new bone formation in the defect was monitored with serial radiographs every 2 weeks. Histomorphometric analysis of the new bone formation was done on undecalcified sections of the extracted femurs at 3 and 6 weeks post surgery. Further analysis of the new bone was done by assessment of neoangiogenesis using immunohistochemical staining for Platelet endothelial cell adhesion molecule-1. Mechanical testing was performed at 9 weeks to assess the new bone with respect to 4 different parameters of mechanical and structural properties of bone. Results: Radiographs assessed over a 6-point grading system showed statistically significant improvement in scores in rhBMP-2-treated rats at 6 weeks (P < 0.001). Histomorphometric analysis showed statistically significant increase in area of new bone formation between rats treated with rhBMP-2 compared with buffer at both 3 and 6 weeks (P < 0.001). On Platelet endothelial cell adhesion molecule-1 staining at 3 weeks, the mean number of vessels in rhBMP-2-treated DM rats was 12.76 ± 5.43/mm2 compared with 4.49 ± 1.89/mm2 in buffer treated DM rats (P = 0.034). On mechanical testing, all 4 DM/buffer rats had nonunion. In DM/rhBMP-2 rats, the torque to failure and torsional rigidity values were 393.57 ± 233.3 (P < 0.03) and 29,711 ± 6224 (P < 0.002), respectively. Conclusions: Clearly, although DM has a known impact on osseous healing, its negative effects are ameliorated with the application of the rhBMP-2-collagen carrier and demonstrates the potential clinical role of this adjunct in the clinical arena.

Role of local insulin augmentation upon allograft incorporation in a rat femoral defect model

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.

Effects of local insulin delivery on subperiosteal angiogenesis and mineralized tissue formation during fracture healing.

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 (Col1α2, osteopontin) was significantly enhanced with UL treatment when compared with saline controls (p < 0.05). Significant differences in VEGF + 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.

The effects of local vanadium treatment on angiogenesis and chondrogenesis during fracture healing

This study quantified the effects of local intramedullary delivery of an organic vanadium salt, which may act as an insulin‐mimetic on fracture healing. Using a BB Wistar rat femoral fracture model, local vanadyl acetylacetonate (VAC) was delivered to the fracture site and histomorphometry, mechanical testing, and immunohistochemistry were performed. Callus percent cartilage was 200% higher at day 7 (p < 0.05) and 88% higher at day 10 (p < 0.05) in the animals treated with 1.5 mg/kg of VAC. Callus percent mineralized tissue was 37% higher at day 14 (p < 0.05) and 31% higher at day 21 (p < 0.05) in the animals treated with 1.5 mg/kg of VAC. Maximum torque to failure was 104% and 154% higher at 4 weeks post‐fracture (p < 0.05) for the healing femurs from the VAC‐treated (1.5 and 3.0 mg/kg) animals. Animals treated with other VAC doses demonstrated increased mechanical parameters at 4 weeks (p < 0.05). Immunohistochemistry detected 62% more proliferating cells at days 7 (p < 0.05) and 94% more at day 10 (p < 0.05) in the animals treated with 1.5 mg/kg VAC. Results showed 100% more vascular endothelial growth factor‐C (VEGF‐C) positive cells and 80% more blood vessels at day 7 (p < 0.05) within the callus subperiosteal region of VAC‐treated animals (1.5 mg/kg) compared to controls. The results suggest that local VAC treatment affects chondrogenesis and angiogenesis within the first 7–10 days post‐fracture, which leads to enhanced mineralized tissue formation and accelerated fracture repair as early as 3–4 weeks post‐fracture.

Bone and wound healing in the diabetic patient.

Impaired soft tissue regeneration and delayed osseous healing are known complications associated with diabetes mellitus with regard to orthopedic surgery, making the management and treatment of diabetic patients undergoing foot and ankle surgery more complex and difficult. At the moment several options are available to address the known issues that complicate the clinical outcomes in these high-risk patients. Using a multifaceted approach, with close attention to intraoperative and perioperative considerations including modification of surgical technique to supplement fixation, local application of orthobiologics, tight glycemic control, administration of supplementary oxygen, and biophysical stimulation via low-intensity pulsed ultrasound and electrical bone stimulation, the impediments associated with diabetic healing can potentially be overcome, to yield improved clinical results for diabetic patients after acute or elective foot and ankle surgery.

Correlation of growth factor levels at the fusion site of diabetic patients undergoing hindfoot arthrodesis and clinical outcome

BackgroundThe purpose of this study was to quantify the levels of growth factors (platelet-derived growth factor [PDGF] AB, vascular endothelial growth factor [VEGF], insulin-like growth factor [IFG] 1, and transforming growth factor [TGF]- &bgr;1) within bone samples at the fusion site of diabetic patients undergoing hindfoot arthrodesis to determine any correlation with successful fusion. MethodsThe study included 10 adult diabetic patients from one US center, with an average age of 57.9 years (range 49–71 years). Interventions included hindfoot arthrodeses with a fixator (n=8) and pantalar arthrodeses with fixator (n=2). During each procedure, a bony bed sample was taken from the fusion site and frozen. This local bone bed was analyzed for growth factors using ELISA kits. A platelet-rich plasma (PRP) concentration was applied to each fusion site during the procedure. ResultsThree of the ten patients had a nonunion. After normalizing to bicinchoninic acid assay (BCA) total protein levels, a 70% decrease in PDGF-AB (0.044 pg/&mgr;g non-union to 0.149 pg/&mgr;g union; P=0.016) and a 44% decrease in VEGF (0.522 pg/&mgr;g non-union to 0.924 pg/&mgr;g union; P=0.031) were observed in the three nonunion bone samples compared with the arthrodesis group. No difference existed in the levels of IGF-I between the groups. Insignificantly higher levels of TGF-&bgr;1 were detected in the nonunion group (0.199 ng/&mgr;g non-union to 0.142 ng/&mgr;g union; P=0.544). ConclusionsSignificant differences in local growth factor (PDGF, VEGF) levels exist between diabetic patients who achieve union and non-union. Potentially, growth factor levels, PDGF-AB and VEGF, in bone may affect the outcome of successful arthrodesis in diabetic patients. This study supports the concept that growth factor levels within the fusion site of a diabetic patient may affect the outcome of a successful hindfoot arthrodesis.