Characterization of heterogeneous skin constructs for full thickness skin regeneration in murine wound models.

An autologous heterogeneous skin construct (AHSC) has been developed and used clinically as an alternative to traditional skin grafting techniques for treatment of cutaneous defects. AHSC is manufactured from a small piece of healthy skin in a manner that preserves endogenous regenerative cellular populations. To date however, specific cellular and non-cellular contributions of AHSC to the epidermal and dermal layers of closed wounds have not been well characterized given limited clinical opportunity for graft biopsy following wound closure. To address this limitation, a three-part mouse full-thickness excisional wound model was developed for histologic and macroscopic graft tracing. First, fluorescent mouse-derived AHSC (mHSC) was allografted onto non-fluorescent recipient mice to enable macroscopic and histologic time course evaluation of wound closure. Next, mHSC-derived from haired pigmented mice was allografted onto gender- and major histocompatibility complex (MHC)-mismatched athymic nude mouse recipients. Resulting grafts were distinguished from recipient murine skin via immunohistochemistry. Finally, human-derived AHSC (hHSC) was xenografted onto athymic nude mice to evaluate engraftment and hHSC contribution to wound closure. Experiments demonstrated that mHSC and hHSC facilitated wound closure through production of viable, proliferative cellular material and promoted full-thickness skin regeneration, including hair follicles and glands in dermal compartments. This combined macroscopic and histologic approach to tracing AHSC-treated wounds from engraftment to closure enabled robust profiling of regenerated architecture and further understanding of processes underlying AHSC mechanism of action. These models may be applied to a variety of wound care investigations, including those requiring longitudinal assessments of healing and targeted identification of donor and recipient tissue contributions.

Evaluation in a porcine wound model and long-term clinical assessment of an autologous heterogeneous skin construct used to close full-thickness wounds.

Acute and chronic wounds involving deeper layers of the skin are often not adequately healed by dressings alone and require therapies such as skin grafting, skin substitutes, or growth factors. Here we report the development of an autologous heterogeneous skin construct (AHSC) that aids wound closure. AHSC is manufactured from a piece of healthy full-thickness skin. The manufacturing process creates multicellular segments, which contain endogenous skin cell populations present within hair follicles. These segments are physically optimized for engraftment within the wound bed. The ability of AHSC to facilitate closure of full thickness wounds of the skin was evaluated in a swine model and clinically in 4 patients with wounds of different etiologies. Transcriptional analysis demonstrated high concordance of gene expression between AHSC and native tissues for extracellular matrix and stem cell gene expression panels. Swine wounds demonstrated complete wound epithelialization and mature stable skin by 4 months, with hair follicle development in AHSC-treated wounds evident by 15 weeks. Biomechanical, histomorphological, and compositional analysis of the resultant swine and human skin wound biopsies demonstrated the presence of epidermal and dermal architecture with follicular and glandular structures that are similar to native skin. These data suggest that treatment with AHSC can facilitate wound closure.

Imaging the pancreatic vasculature in diabetes models.

BACKGROUND: Vascular parameters, such as vascular volume, flow, and permeability, are important disease biomarkers for both type 1 and type 2 diabetes. Therefore, it is essential to develop approaches to monitor the changes in pancreatic microvasculature non-invasively. METHODS: Here, we describe the application of the long-circulating, paramagnetic T1 contrast agent, protected Graft Copolymer bearing covalently linked gadolinium diethylenetriaminepentaacetic acid residues and labelled with fluorescein (PGC-GdDTPA-F) for the non-invasive semi-quantitative evaluation of vascular changes in diabetic models using magnetic resonance imaging. RESULTS: We observed a significantly higher accumulation of protected graft copolymer bearing covalently linked gadolinium diethylenetriaminepentaacetic acid residues and labelled with fluorescein in the pancreata of BBDR rats induced to develop diabetes, as compared to non-diabetic controls at 1 h post-injection. No differences were seen in the blood pool, kidney, or muscle, indicating that the effect is specific to the diabetic pancreas. Fluorescence microscopy revealed a marked increase in contrast agent availability in the pancreas with the development of the pathology. Similar changes were noted in the homozygous Leprdb mouse model of type 2 diabetes. This effect appeared to result both from the increase of vascular volume and permeability. CONCLUSIONS: High-molecular weight paramagnetic blood volume contrast agents are valuable for the in vivo definition of pancreatic microvasculature dynamics by magnetic resonance imaging. The increase in vascular volume and permeability, associated with diabetic inflammation, can be monitored non-invasively and semi-quantitatively by magnetic resonance imaging in diabetic BBDR rats. This imaging strategy represents a valuable research tool for better understanding of the pathologic process.

siRNA delivery to CNS cells using a membrane translocation peptide.

RNA interference (RNAi) is a sequence-specific gene silencing technique that has been applied to multiple pathological conditions. In this report, we describe the generation and in vitro characterization of an RNAi-based fluorescent probe for use as a therapeutic in the setting of ischemic stroke. Probe delivery to bEnd.3 brain endothelial cells and primary cortical neurons and astrocytes was promoted by incorporating small interfering RNA (siRNA) into complexes with fluorescently labeled myristoylated polyarginine peptides. The resulting probe was partially protected from serum nuclease degradation and was efficiently internalized by cells as confirmed by flow cytometry and confocal microscopy. In addition, application of the siRNA probe directed against c-Src, a protein implicated in stroke pathology, led to statistically significant reduction of endogenous c-src mRNA levels in all cell types tested. Results demonstrate the proof-of-principle that functionalized peptide--siRNA probes can be used as potential tools for dual imaging and therapeutic applications.

Biomechanics of horizontal canal benign paroxysmal positional vertigo.

Horizontal canal (HC) benign paroxysmal positional vertigo (HC-BPPV) is a vestibular disorder characterized by bouts of horizontal ocular nystagmus induced during reorientation of the head relative to gravity. The present report addresses the application of a morphologically descriptive 3-canal biomechanical model of the human membranous labyrinth to study gravity-dependent semicircular canal responses during this condition. The model estimates dynamic cupular and endolymph displacements elicited during HC-BPPV provocative diagnostic maneuvers and canalith repositioning procedures (CRPs). The activation latencies in response to an HC-BPPV provocative diagnostic test were predicted to vary depending upon the initial location of the canalith debris (e.g. within the HC lumen vs. in the ampulla). Results may explain why the onset latency of ocular nystagmus evoked by the Dix-Hallpike provocative maneuver for posterior canal BPPV are typically longer than the latencies evoked by analogous tests for HC-BPPV. The model was further applied to assess the efficacy of a 360 degrees -rotation CRP for the treatment of canalithiasis HC-BPPV.

Combination treatment with theranostic nanoparticles for glioblastoma sensitization to TMZ.

PURPOSE: Tumor resistance to chemotherapeutic drugs is one of the major obstacles in the treatment of glioblastoma multiforme (GBM). In this study, we attempted to modulate tumor response to chemotherapy by combination treatment that included experimental (small interference RNA (siRNA), chlorotoxin) and conventional (temozolomide, TMZ) therapeutics. PROCEDURES: siRNA therapy was used to silence O(6)-methylguanine methyltransferase (MGMT), a key factor in brain tumor resistance to TMZ. For targeting of tumor cells, we used chlorotoxin (CTX), a peptide with antitumoral properties. siRNA and CTX were conjugated to iron oxide nanoparticles (NP) that served as the drug carrier and allowed the means to monitor the changes in tumor volume by magnetic resonance imaging (MRI). RESULTS: Theranostic nanoparticles (termed CTX-NP-siMGMT) were internalized by T98G glioblastoma cells in vitro leading to enhancement of TMZ toxicity. Combination treatment of mice bearing orthotopic tumors with CTX-NP-siMGMT and TMZ led to significant retardation of tumor growth, which was monitored by MRI. CONCLUSIONS: While our results demonstrate that siRNA delivery by targeted nanoparticles resulted in modulating tumor response to chemotherapy in GBM, they also point to a significant contribution of CTX to tumor cell death.

In vivo imaging of the systemic delivery of small interfering RNA.

Short interfering RNAs (siRNAs) have emerged as a potent new class of therapeutics, which regulate gene expression through sequence-specific inhibition of mRNA translation. Human trials of siRNAs have highlighted the need for robust delivery and detection techniques that will enable the application of these therapeutics to increasingly complex disease and organ systems. Efforts to monitor the in vivo trafficking and efficacy of siRNAs have routinely involved bioluminescence imaging of naked siRNA molecules. More recently, siRNAs have been incorporated into a variety of molecular imaging probes to promote their detection with clinically relevant imaging modalities. Lipid-, polymer-, and nanoparticle-based siRNA delivery vehicles have proven effective in improving the stability, bioavailability, and target specificity of siRNAs following systemic administration in vivo. Additionally, these methods provide a platform to modify siRNAs with a variety of contrast agents and have enabled nuclear and magnetic resonance imaging of siRNA delivery in preclinical studies. These image-guided delivery approaches represent a crucial step in the transition of siRNA therapeutics to the clinic.

Noninvasive MRI-SERS imaging in living mice using an innately bimodal nanomaterial.

We report a novel nanomaterial (AuMN-DTTC) that can be used as a bimodal contrast agent for in vivo magnetic resonance imaging (MRI) and Raman spectroscopy. The probe consists of MRI-active superparamagnetic iron oxide nanoparticles, stably complexed with gold nanostructures. The gold component serves as a substrate for a Raman active dye molecule to generate a surface-enhanced Raman scattering (SERS) effect. The synthesized probe produces T2 weighted contrast and can be used as a SERS active material both in silico (in aqueous solution) and in vivo. A quantitative assessment of T2 relaxation times was obtained using multiecho MRI analysis. The T2 relaxation times of AuMN-DTTC and MN (dextran-coated iron oxide nanoparticles) were 29.23 + 1.45 and 31.58 + 1.7 ms, respectively. The SERS signature of AuMN-DTTC revealed peaks at 508, 629, 782, 844, 1080, 1108, 1135, and 1242 cm(-1). Intramuscular administration of the probe resulted in a decrease of the T2 relaxation time of muscle from 33.4 + 2.5 to 20.3 + 2.2 ms. SERS peaks were observed at 508, 629, 782, 844, 1080, 1108, 1135, and 1242 cm(-1), consistent with the in silico results. Our studies illustrate for the first time the design and in vivo application of a contrast agent, whose component modalities include MRI and SERS. The value of this agent lies in its innately bimodal nature and its application in vivo for molecular imaging applications.

The role of 3-canal biomechanics in angular motion transduction by the human vestibular labyrinth.

The present work examines the role of the complex geometry of the human vestibular membranous labyrinth in the process of angular motion transduction by the semicircular canals. A morphologically descriptive mathematical model was constructed to address the biomechanical origins of temporal signal processing and directional coding in determining the inputs to the brain. The geometrical model was developed based on shrinkage-corrected temporal bone sections using a segmentation/data-fitting procedure. Endolymph fluid dynamics within the 3-canal labyrinth was modeled using an asymptotic form of the Navier-Stokes equations and solved to estimate endolymph and cupulae volume displacements. The geometrical model was manipulated to study the role of major morphological features on directional and temporal coding. Anatomical results show that the bony osseous canals provide reasonable estimates of the orientation of the delicate membranous canals--the two differed by only 3.48 +/- 1.89 degrees . Biomechanical results show that the maximal response directions are distinct from the anatomical canal planes, but can be closely approximated by fitting a flat plane to the centerline of the canal of interest and weighting each location along the centerline with the inverse of the cross-sectional area squared. Vector cross-products of these maximal response directions, in turn, determine the null planes and prime directions that transmit the direction of angular motion to the brain as three independent directional channels associated with the nerve bundles. Finally, parameter studies indicate that changes in canal cross-sectional area and shape only moderately affect canal temporal and directional coding, while three-canal orientation is critical to directional coding.

Three-dimensional biomechanical model of benign paroxysmal positional vertigo.

A morphologically descriptive 3-canal mathematical model was developed to quantify the biomechanical origins of gravity-dependent semicircular canal responses under pathological conditions of canalithiasis and cupulolithiasis--conditions associated with the vestibular disorder benign paroxysmal positional vertigo (BPPV). The model describes the influence of displaced calcium carbonate debris (particles) located within the labyrinth on the time-dependent responses of the ampullary organs. The particles were modeled as spheres free to move in the canal lumen (canalithiasis) or adhered to a cupula (cupulolithiasis). The model predicts canal responses to the diagnostic Dix-Hallpike maneuver, and to a modified Epley canalith repositioning (CRP) treatment. Results for canalithiasis predict activation latencies and response magnitudes consistent with clinical observations during the Dix-Hallpike maneuver. The magnitude of the response evoked by the Dix-Hallpike test was primarily due to the total weight of the particles while the latency to peak response was due to the time required for the stone to move from the ampulla to the posterior apex of the canal. Results further illustrate the effectiveness of the Epley CRP in repositioning the particles and relieving the symptoms of the canalithiasis type of BPPV.