Davood Varghai

Mechanical analysis of resorbable plates for long-term soft-tissue molding

Background: Resorbable plates are used with increasing frequency in pediatric craniofacial surgery. A recent innovation has been the use of these plates in long-term soft-tissue molding. However, these plates were not designed for these applications, and an unanswered question remains the mechanical rigidity and stability of the plate when placed in a prolonged out-of-plane bend. Methods: Ten varieties of resorbable plates were folded to a right angle and subjected to a standardized 80-g load at body temperature over a duration of 2 weeks. Angle height was measured as an index of the ability of the plate to maintain its out-of-plane bend against an axial force. Each plate was subjected to 3 successive tests, after bending back to its original configuration. Results: All plates showed a progressive decrease in angle height over 2 weeks. Thickness of the plate appeared to correlate with mechanical rigidity and ability to maintain out-of-plane bend. The plates that performed the best were the Synthes PLGA (85/15) 1.2-mm and BioGeneral PLA (poly-D,L-lactic acid) 1.0-mm plates. Decrease in angle height occurred over shorter intervals in successive tests. Conclusions: Of all the plates tested, none were able to maintain initial out-of-plane bend over 2 weeks of testing. Further research and development by industry are required to optimize mechanical properties of resorbable plates for applications in soft-tissue molding.

Fluorescence of Pc 4 in U87 cells following photodynamic therapy

Introduction: Given the length of procedures and the brightness of operating room lights, there is concern that photosensitizers used to locate brain tumors and treat them with photodynamic therapy (PDT) may photobleach before they can be fully utilized. The phthalocyanine photosensitizer Pc 4 is resistant to photobleaching. In this study, we tested the hypothesis that exposure of Pc 4-loaded glioma cells to photoactivating light will result in continuing fluorescence of Pc 4. Methods: U87 human glioma cells were cultured in MEM with 5% penicillin/streptomycin, 5% sodium pyruvate, 10% fetal bovine serum, and 25 mM HEPES. These cultures were given 0 or 125 nM Pc 4, followed 2 hours later by three separate exposures of 200 J/cm2 of red light (&lgr;max = 675 nm). Confocal fluorescence images were collected before and after each exposure. Results: Pc 4 fluorescence was localized to cytoplasmic membranes of the U87 glioma cells, as previously seen in other types of cells. After exposure to PDT, Pc 4 fluorescence was not reduced and even increased. Discussion: Pc 4 may be useful for the intra-operative detection of glioma by fluorescence and for PDT, since neither Pc 4 level nor its fluorescence is likely to decrease during exposure to operating room lights.

Augmentation of intraorbital volume with fat injection.

Background: Enophthalmos is a challenging surgical problem to correct. Standard techniques to adjust orbital volume require invasive maneuvers such as osteotomies. Fat injection may provide a simple and less-invasive way of augmenting orbital volume to correct enophthalmos. Methods: The right eye orbital volume of 10 New Zealand White rabbits was augmented with fat. Autologous fat was diced and injected into the retrobulbar space. Computed tomographic scans were evaluated for changes in globe position and retrobulbar volume. Visually evoked potentials were conducted to test the integrity of the optic tract. Rabbits were killed at 12 weeks after surgery. Orbital exenterations were performed to allow for gross and histologic evaluation. Results: Right globe position showed a mean increase in eye proptosis of 3.4 mm at postoperative day 1 and 0.9 mm at 11 weeks postoperatively in comparison with the left globe position. No significant change was noted in the left globe position. Retrobulbar volume demonstrated an initial mean increase of 31 percent and a final mean increase of 9.8 percent at 11 weeks in the right eye compared with the left eye. Visually evoked potentials revealed intact optic pathways in all animals. Gross anatomical evaluation showed deposition of fat grafts. Histologic analysis showed both revascularized and necrotic areas of fat. No retinal or optic nerve damage was identified. Conclusions: Fat injection can augment orbital volume in an animal model and preserve visual function. Further investigation is necessary to document the clinical safety and value of this technique in humans.

RNA interfering molecule delivery from in situ forming biodegradable hydrogels for enhancement of bone formation in rat calvarial bone defects

RNA interference (RNAi) may be an effective and valuable tool for promoting the growth of functional tissue, as short interfering RNA (siRNA) and microRNA (miRNA) can block the expression of genes that have negative effects on tissue regeneration. Our group has recently reported that the localized and sustained presentation of siRNA against noggin (siNoggin) and miRNA-20a from in situ forming poly(ethylene glycol) (PEG) hydrogels enhanced osteogenic differentiation of encapsulated human bone marrow-derived mesenchymal stem cells (hMSCs). Here, the capacity of the hydrogel system to accelerate bone formation in a rat calvarial bone defect model is presented. After 12 weeks post-implantation, the hydrogels containing encapsulated hMSCs and miRNA-20a resulted in more bone formation in the defects than the hydrogels containing hMSCs without siRNA or with negative control siRNA. This localized and sustained RNA interfering molecule delivery system may provide an excellent platform for healing bony defects and other tissues. STATEMENT OF SIGNIFICANCE Delivery of RNAi molecules may be a valuable strategy to guide cell behavior for tissue engineering applications, but to date there have been no reports of a biomaterial system capable of both encapsulation of cells and controlled delivery of incorporated RNA. Here, we present PEG hydrogels that form in situ via Michael type reaction, and that permit encapsulation of hMSCs and the concomitant controlled delivery of siNoggin and/or miRNA-20a. These RNAs were chosen to suppress noggin, a BMP-2 antagonist, and/or PPAR-γ, a negative regulator of BMP-2-mediated osteogenesis, and therefore promote osteogenic differentiation of hMSCs and subsequent bone repair in critical-sized rat calvarial defects. Simultaneous delivery of hMSCs and miRNA-20a enhanced repair of these defects compared to hydrogels containing hMSCs without siRNA or with negative control siRNA. This in situ forming PEG hydrogel system offers an exciting platform for healing critical-sized bone defects by localized, controlled delivery of RNAi molecules to encapsulated hMSCs and surrounding cells.

Optimal gadolinium dose level for magnetic resonance imaging (MRI) contrast enhancement of U87-derived tumors in athymic nude rats for the assessment of photodynamic therapy

This study aims to determine the effect of varying gadopentetate dimeglumine (Gd-DTPA) dose on Dynamic Contrast Enhanced-Magnetic Resonance Imaging (DCE-MRI) tracking of brain tumor photodynamic therapy (PDT) outcome. Methods: We injected 2.5 x 105 U87 cells (derived from human malignant glioma) into the brains of six athymic nude rats. After 9, 12, and 13 days DCE-MRI images were acquired on a 9.4 T micro-MRI scanner before and after administration of 100, 150, or 200 μL of Gd-DTPA. Results: Tumor region normalized DCE-MRI scan enhancement at peak was: 1.217 over baseline (0.018 Standard Error [SE]) at the 100 μL dose, 1.339 (0.013 SE) at the 150 μL dose, and 1.287 (0.014 SE) at the 200 μL dose. DCE-MRI peak tumor enhancement at the 150 μL dose was significantly greater than both the 100 μL dose (p < 3.323E-08) and 200 μL dose (p < 0.0007396). Discussion: In this preliminary study, the 150 μL Gd-DTPA dose provided the greatest T1 weighted contrast enhancement, while minimizing negative T2* effects, in DCE-MRI scans of U87-derived tumors. Maximizing Gd-DTPA enhancement in DCE-MRI scans may assist development of a clinically robust (i.e., unambiguous) technique for PDT outcome assessment.

Monitoring Pc 4-mediated Photodynamic Therapy of U87 Tumors with Dynamic Contrast Enhanced-Magnetic Resonance Imaging (DCE-MRI) in the Athymic Nude Rat

Post-operative verification of the specificity and sensitivity of photodynamic therapy (PDT) is most pressing for deeply placed lesions such as brain tumors. We wish to determine whether Dynamic Contrast Enhanced-Magnetic Resonance Imaging (DCE-MRI) can provide a non-invasive and unambiguous quantitative measure of the specificity and sensitivity of brain tumor PDT. Methods: 2.5 x 105 U87 cells were injected into the brains of six athymic nude rats. After 5-6 days, the animals received 0.5 mg/kg b.w. of the phthalocyanine photosensitizer Pc 4 via tail-vein injection. On day 7 peri-tumor DCE-MRI images were acquired on a 7T microMRI scanner before and after tail-vein administration of 100 μL gadolinium and 400 μL saline. After this scan the animals received a 30 J/cm2 dose of 672-nm light from a diode laser (i.e., PDT). The DCE-MRI scan protocol was repeated on day 13. Next, the animals were euthanized and their brains were explanted for Hematoxylin and Eosin (H&E) histology. Results: No tumor was found in one animal. The DCE-MRI images of the other five animals demonstrated significant tumor enhancement increase (p < 0.053 two-sided t-test and p < 0.026 one-sided t-test) following PDT. H&E histology presented moderate to severe tumor necrosis. Discussion: The change in signal detected by DCE-MRI appears to be due to PDT-induced tumor necrosis. This DCE-MRI signal appears to provide a quantitative, non-invasive measure of the outcome of PDT in this animal model and may be useful for determining the safety and effectiveness of PDT in deeply placed tumors (e.g., glioma).

The Impact of Age Upon Healing: Absolute Quantification of Osteogenic Genes in Calvarial Critical-Sized Defects.

Background:The current study was performed to elucidate changes in growth factor expression over time in critical-sized calvarial defects in rats from infancy to skeletal maturity. Materials and Methods:Critical-sized parietal defects of 5, 6, and 8 mm were created in postnatal day 6 (P6), postnatal day (P20), and postnatal day (P84) adult rats, respectively. Dura was harvested at 3, 7, or 14 days after surgery, and serial micro–computed tomography imaging was performed through 12 weeks postoperatively. Absolute quantitative polymerase chain reaction was performed for Bone Morphogenic Protein-2 (BMP-2), Fibroblast Growth Factor-2 (FGF-2), Insulin-like Growth Factor-1 (IGF-1), and Transforming Growth Factor-&bgr;1 (TGF-&bgr;). Results:The P6 (6-d-old) rats showed the greatest difference in gene expression between the dura derived from the defect side and the dura derived from the control side, demonstrating significant differences in TGF-&bgr;1, BMP-2, IGF-1, and FGF-2 at various time intervals. Absolute gene expression in the defect dura was highest in the P6 rats and declined with age. Significant differences were noted at limited time points in the P20 rats for TGF-&bgr;1 and BMP-2 as well as in the P84 rats for TGF-&bgr;1. TGF-&bgr;1 was the only gene studied that showed significant differences at postoperative days 3, 7, and 14 in varying age groups. Conclusions:The P6 rats have a higher osteogenic potential accompanied by a more vigorous alteration in growth factor expression compared with the P20 or P84 rats. Decrease in BMP-2 and FGF-2 as well as relative increase in TGF&bgr;-1 messenger RNA were observed in healing defects. These data provide valuable insight into the mechanism of healing of critical-sized defects and may be of use to engineer factor-releasing implants to correct skull defects.

Functional Measures of Therapy Based on Radiological Imaging

We seek a noninvasive, functional, radiological imaging method to determine the specificity and sensitivity of an ablative treatment for deeply placed lesions that cannot be directly visualized, in our case phthalocyanine-4 photodynamic therapy (Pc 4-PDT) of brain tumors. In a preliminary study we had expected that micro-positron emission tomography (μPET) would show dramatically reduced if not negligible 18F-FDG activity following Pc 4-PDT; however, our study has not found a statistically significant difference between the imaging of brain tumors in animals that underwent Pc 4-PDT and those that did not. While several magnetic resonance imaging (MRI) pulse sequences also did not discriminate tumors that had received treatment, our study of dynamic contrast enhancement magnetic resonance imaging (DCE-MRI) was able to discriminate tumors that had undergone necrosis following Pc 4-PDT. We expect that in addition to imaging therapeutic necrosis, it will also be possible to utilize other noninvasive, radiological imaging techniques to track apoptosis and/or autophagy in deeply placed lesions following treatments such as Pc 4-PDT.

Abstract 85: COMPARING THE EFFICACY OF DEFEROXAMINE OR AMIFOSTINE PRE-TREATMENT IN THE PREVENTION OF OSTEORADIONECROSIS OF THE MURINE MANDIBLE

Introduction: Osteoradionecrosis (ORN) of the mandible and facial bones is a signi cant problem for patients who are treated for head and neck cancer. Evidence suggests that deferoxamine (DFO) and amifostine (AMFO) may have bene cial roles in the prevention and treatment of ORN. DFO has been shown to have a vasculogenic effect while Amifostine has been shown to prevent cytotoxicity and apoptosis. We hypothesize that DFO and AMFO will have bene cial effects in the prevention and treatment of ORN in a rat model, especially when used in combination.

Hardware, software, and scanning issues encountered during small animal imaging of photodynamic therapy in the athymic nude rat

Small animal imaging devices are now commonly used to study gene activation and model the effects of potential therapies. We are attempting to develop a protocol that non-invasively tracks the affect of Pc 4-mediated photodynamic therapy (PDT) in a human glioma model using structural image data from micro-CT and/or micro-MR scanning and functional data from 18F-fluorodeoxy-glucose (18F-FDG) micro-PET imaging. Methods: Athymic nude rat U87-derived glioma was imaged by micro-PET and either micro-CT or micro-MR prior to Pc 4-PDT. Difficulty insuring animal anesthesia and anatomic position during the micro-PET, micro-CT, and micro-MR scans required adaptation of the scanning bed hardware. Following Pc 4-PDT the animals were again 18F-FDG micro-PET scanned, euthanized one day later, and their brains were explanted and prepared for H&E histology. Histology provided the gold standard for tumor location and necrosis. The tumor and surrounding brain functional and structural image data were then isolated and coregistered. Results: Surprisingly, both the non-PDT and PDT groups showed an increase in tumor functional activity when we expected this signal to disappear in the group receiving PDT. Co-registration of the functional and structural image data was done manually. Discussion: As expected, micro-MR imaging provided better structural discrimination of the brain tumor than micro-CT. Contrary to expectations, in our preliminary analysis 18F-FDG micro-PET imaging does not readily discriminate the U87 tumors that received Pc 4-PDT. We continue to investigate the utility of micro-PET and other methods of functional imaging to remotely detect the specificity and sensitivity of Pc 4-PDT in deeply placed tumors.