Phase I study of multi-gene cell therapy in patients with peripheral artery disease.

UNLABELLED: Alternative treatment strategies for claudication are needed and cell-based therapies designed to induce angiogenesis are promising. The purpose of this report was to conduct a Phase I safety, dose-escalating, non-randomized, open-label study of autologous, fully differentiated venous endothelial and smooth muscle cells called MultiGeneAngio (MGA) for claudication due to peripheral artery disease. Twelve subjects, at two centers, received a single intra-arterial infusion of a suspension of equal amounts of transduced autologous venous smooth muscle cells expressing vascular endothelial growth factor (VEGF165) and endothelial cells expressing angiopoietin-1 (Ang-1) (Cohort 1: 1 × 10(7), Cohort 2: 2 × 10(7), Cohort 3: 5 × 10(7), Cohort 4: 7 × 10(7)). The treatment was given unblinded and in the more symptomatic lower extremity. Transduced cells were tested for in vitro doubling time, telomerase activity, and gene expression. The main outcomes were clinical safety and tolerability. Other safety measures included ankle-brachial index (ABI) and walking time on a treadmill. All subjects were male (mean age 60 ± 5 years) including 25% with diabetes mellitus. At 1-year follow-up, there was one serious adverse event possibly related to MGA. Safety endpoints including VEGF and Ang-1 plasma protein levels were within normal ranges in all subjects. The mean maximal walking time increased from baseline to 1 year and the index limb ABI was unchanged, indicating no safety concerns. MGA, an autologous, transduced, cell-based therapy was well tolerated and safe in this Phase I study. Further evaluation is warranted in randomized human studies. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT00390767.

The MICHR Genomic DNA BioLibrary: An Empirical Study of the Ethics of Biorepository Development.

In this article, we report on an effort to study the development and usefulness of a large, broad-use, opt-in biorepository for genomic research, focusing on three ethical issues: providing appropriate understanding, recruiting in ways that do not comprise autonomous decisions, and assessing costs versus benefits. We conclude the following: (a) Understanding can be improved by separating the task of informing subjects from documenting informed consent (Common Rule) and permission to use personal health information and samples for research (Health Insurance Portability and Accountability Act [HIPAA]); however, regulations might have to be changed to accommodate this approach. (b) Changing recruiting methods increases efficiency but can interfere with subject autonomy. (c) Finally, we propose a framework for the objective evaluation of the utility of biorepositories and suggest that more attention needs to be paid to use and sustainability.

Characterization of biofluids prepared by sessile drop formation.

Sessile drop formation, also called drop deposition, has been studied as a potential medical diagnostic, but the effects of complex biofluid rheology on the final deposition pattern are not well understood. We studied two model biofluids, blood plasma and synovial fluid, when deposited onto slightly hydrophilic substrates forming a contact angle of 50-90°. Drops were imaged during the evaporation process and geometric properties of the drop, such as contact angle and drop height, were calculated from the images. The resulting dried biofluid drops were then examined using light microscopy and Raman spectroscopy to assess morphological and chemical composition of the dried drop. The effect of substrate contact angle (surface wetting) and fluid concentration was examined. We found that when biofluids are deposited onto slightly hydrophilic surfaces, with a contact angle of 50-90°, a ring-shaped deposit was formed. Analysis of the drying drop's geometric properties indicates that biofluid dynamics follow the piling model of drop formation, as proposed by Deegan et al. The final deposition pattern varied with substrate surface and concentration, as shown by light microscopy photos of dried drops. The chemical composition of the outer ring was minimally affected by substrate surface, but the spatial heterogeneity of protein distribution within the ring varied with concentration. These results indicate that biofluid drop deposition produces ring-shaped deposits which can be examined by multiple analytical techniques.

Alterations to bone mineral composition as an early indication of osteomyelitis in the diabetic foot.

OBJECTIVE: Osteomyelitis in the diabetic foot is a major risk factor for amputation, but there is a limited understanding of early-stage infection, impeding limb-preserving diagnoses. We hypothesized that bone composition measurements provide insight into the early pathophysiology of diabetic osteomyelitis. RESEARCH DESIGN AND METHODS: Compositional analysis by Raman spectroscopy was performed on bone specimens from patients with a clinical diagnosis of osteomyelitis in the foot requiring surgical intervention as either a biopsy (n = 6) or an amputation (n = 11). RESULTS: An unexpected result was the discovery of pathological calcium phosphate minerals in addition to normal bone mineral. Dicalcium phosphate dihydrate, also called brushite, and uncarbonated apatite were found to be exclusively associated with infected bone. CONCLUSIONS: Compositional measurements provided a unique insight into the pathophysiology of osteomyelitis in diabetic foot ulcers. At-patient identification of pathological minerals by Raman spectroscopy may serve as an early-stage diagnostic approach.

Label-free Raman monitoring of extracellular matrix formation in three-dimensional polymeric scaffolds.

Monitoring extracellular matrix (ECM) components is one of the key methods used to determine tissue quality in three-dimensional scaffolds for regenerative medicine and clinical purposes. Raman spectroscopy can be used for non-invasive sensing of cellular and ECM biochemistry. We have investigated the use of conventional (confocal and semiconfocal) Raman microspectroscopy and fibre-optic Raman spectroscopy for in vitro monitoring of ECM formation in three-dimensional poly(ethylene oxide terephthalate)-poly(butylene terephthalate) (PEOT/PBT) scaffolds. Chondrocyte-seeded PEOT/PBT scaffolds were analysed for ECM formation by Raman microspectroscopy, biochemical analysis, histology and scanning electron microscopy. ECM deposition in these scaffolds was successfully detected by biochemical and histological analysis and by label-free non-destructive Raman microspectroscopy. In the spectra collected by the conventional Raman set-ups, the Raman bands at 937 and at 1062 cm(-1) which, respectively, correspond to collagen and sulfated glycosaminoglycans could be used as Raman markers for ECM formation in scaffolds. Collagen synthesis was found to be different in single chondrocyte-seeded scaffolds when compared with microaggregate-seeded samples. Normalized band-area ratios for collagen content of single cell-seeded samples gradually decreased during a 21-day culture period, whereas collagen content of the microaggregate-seeded samples significantly increased during this period. Moreover, a fibre-optic Raman set-up allowed for the collection of Raman spectra from multiple pores inside scaffolds in parallel. These fibre-optic measurements could give a representative average of the ECM Raman signal present in tissue-engineered constructs. Results in this study provide proof-of-principle that Raman microspectroscopy is a promising non-invasive tool to monitor ECM production and remodelling in three-dimensional porous cartilage tissue-engineered constructs.

Effects of pathology dyes on Raman bone spectra.

We report an overlooked source of artifacts for clinical specimens, where unexpected and normally negligible contaminants can skew the interpretation of results. During an ongoing study of bone fragments from diabetic osteomyelitis, strong Raman signatures were found, which did not correspond with normal bone mineral or matrix. In a bone biopsy from the calcaneus of a patient affected by diabetic osteomyelitis, Raman microspectroscopic analysis revealed regions with both abnormal mineral and degraded collagen in addition to normal bone. Additional bands indicated a pathological material. Stenotrophomonas maltophilia was identified in the wound culture by independent microbiologic examination. We initially assigned the unusual bands to xanthomonadin, a bacterial pigment from S. maltophilia. However, the same bands were also found more than a year later on a second specimen that had been noticeably contaminated with pathology marking dye. Drop deposition/Raman spectroscopy of commonly used pathology dyes revealed that a blue tissue-marking dye was responsible for the unusual bands in both specimens, even in the first specimen where there was no visible evidence of contamination.

Preclinical evaluation of a novel implant for treatment of a full-thickness distal femoral focal cartilage defect.

A novel, nonresorbable, monolithic composite structure ceramic, developed using a partially stabilized zirconia ceramic common to implantable devices, was used in a cementless weight-bearing articular implant to test the feasibility of replacing a region of degenerated or damaged articular cartilage in the knee as part of a preclinical study using male mongrel dogs lasting up to 24 weeks. Gross/histological cartilage observations showed no differences among control, 12-week and 24-week groups, while pull-out tests showed an increase in maximum pull-out load over time relative to controls. Hence, the use of a novel ceramic implant as a replacement for a focal cartilage defect leads to effective implant fixation within 12 weeks and does not cause significant degradation in opposing articular cartilage in the time frame evaluated.

Biomedical tissue phantoms with controlled geometric and optical properties for Raman spectroscopy and tomography.

To support the translation of Raman spectroscopy into clinical applications, synthetic models are needed to accurately test, optimize and validate prototype fiber optic instrumentation. Synthetic models (also called tissue phantoms) are widely used for developing and testing optical instrumentation for diffuse reflectance, fluorescence, and Raman spectroscopies. While existing tissue phantoms accurately model tissue optical scattering and absorption, they do not typically model the anatomic shapes and chemical composition of tissue. Because Raman spectroscopy is sensitive to molecular composition, Raman tissue phantoms should also approximate the bulk tissue composition. We describe the fabrication and characterization of tissue phantoms for Raman tomography and spectroscopy. These phantoms have controlled chemical and optical properties, and also multilayer morphologies which approximate the appropriate anatomic shapes. Tissue phantoms were fabricated to support on-going Raman studies by simulating the human wrist and rat leg. Surface meshes (triangle patch models) were generated from computed tomography (CT) images of a human arm and rat leg. Rapid prototyping was used to print mold templates with complex geometric patterns. Plastic casting techniques used for movie special effects were adapted to fabricate molds from the rapid prototypes, and finally to cast multilayer gelatin tissue phantoms. The gelatin base was enriched with additives to model the approximate chemistry and optical properties of individual tissue layers. Additional studies were performed to determine optimal casting conditions, phantom stability, layer delamination and chemical diffusion between layers. Recovery of diffuse reflectance and Raman spectra in tissue phantoms varied with probe placement. These phantoms enable optimization of probe placement for human or rat studies. These multilayer tissue phantoms with complex geometries are shown to be stable, with minimal layer delamination and chemical diffusion.

Fiber-optic Raman spectroscopy of joint tissues.

In this study, we report adaptation of Raman spectroscopy for arthroscopy of joint tissues using a custom-built fiber-optic probe. Differentiation of healthy and damaged tissue or examination of subsurface tissue, such as subchondral bone, is a challenge in arthroscopy because visual inspection may not provide sufficient contrast. Discrimination of healthy versus damaged tissue may be improved by incorporating point spectroscopy or hyperspectral imaging into arthroscopy where the contrast is based on the molecular structure or chemical composition. Articular joint surfaces of knee cadaveric human tissue and tissue phantoms were examined using a custom-designed Raman fiber-optic probe. Fiber-optic Raman spectra were compared against reference spectra of cartilage, subchondral bone and cancellous bone collected using Raman microspectroscopy. In fiber-optic Raman spectra of the articular surface, there was an effect of cartilage thickness on recovery of signal from subchondral bone. At sites with intact cartilage, the bone mineralization ratio decreased but there was a minimal effect in the bone mineral chemistry ratios. Tissue phantoms were prepared as experimental models of the osteochondral interface. Raman spectra of tissue phantoms suggested that optical scattering of cartilage has a large effect on the relative cartilage and bone signal. Finite element analysis modeling of light fluence in the osteochondral interface confirmed experimental findings in human cadaveric tissue and tissue phantoms. These first studies demonstrate the proof of principle for Raman arthroscopic measurement of joint tissues and provide a basis for future clinical or animal model studies.

Raman spectroscopy of synovial fluid as a tool for diagnosing osteoarthritis.

For many years, viscosity has been the primary method used by researchers in rheumatology to assess the physiochemical properties of synovial fluid in both normal and osteoarthritic patients. However, progress has been limited by the lack of methods that provide multiple layers of information, use small sample volumes, and are rapid. Raman spectroscopy was used to assess the biochemical composition of synovial fluid collected from 40 patients with clinical evidence of knee osteoarthritis (OA) at the time of elective surgical treatment. Severity of knee osteoarthritis was assessed by a radiologist using Kellgren/Lawrence (K/L) scores from knee joint x rays, while light microscopy and Raman spectroscopy were used to examine synovial fluid (SF) aspirates (2 to 10 microL), deposited on fused silica slides. We show that Raman bands used to describe protein secondary structure and content can be used to detect changes in synovial fluid from osteoarthritic patients. Several Raman band intensity ratios increased significantly in spectra collected from synovial fluid in patients with radiological evidence of moderate-to-severe osteoarthritis damage. These ratios can be used to provide a "yes/no" damage assessment. These studies provide evidence that Raman spectroscopy would be a suitable candidate in the evaluation of joint damage in knee osteoarthritis patients.

Effect of conformation and drop properties on surface-enhanced Raman spectroscopy of dried biopolymer drops.

Biofluids are complex solutions consisting of small ions and large biopolymers such as DNA, proteins, or proteoglycans. Biopolymers affect fluid properties but their effect on drop deposition has not been examined. Hyaluronic acid (HA), an important component in synovial fluid, was chosen as a model biopolymer, and examined using surface-enhanced Raman spectroscopy (SERS). Nanoliter volumes of HA solutions were dried onto a patterned SERS substrate and spectra were collected from the dried hyaluronic acid drops with a near-infrared Raman microscope. Characteristic hyaluronic acid bands were examined. Capillary viscometry measured properties of HA solutions, and entanglement behavior was also modeled using scaling theory principles. Viscosity measurements were incorporated into models of suspended particle droplets to account for the effect of inter-chain attraction on droplet formation. Microscope images were used to evaluate the shape of the dried drop. Relative drop thickness was estimated from concentric rings found at drop edges using established models of light interference by thin films. We found SERS spectra were sensitive not only to polymer conformation, but also to type of deposition (ring versus uniform), and the thickness of the resulting deposition. These data suggest an approach to elucidate the effects of biopolymers and dehydrated biofluids on SERS analysis.

Photoacoustic tomography of carrageenan-induced arthritis in a rat model.

Laser-based photoacoustic tomography (PAT), a novel, nonionizing, noninvasive, laser-based technology, has been adapted to the diagnosis and imaging of inflammatory arthritis. A commonly used adjuvant induced arthritis model using carrageenan was employed to simulate acute rheumatoid arthritis in rat tail joints. Cross-sectional photoacoustic images of joints affected by acute inflammation were compared to those of the control. The diameter of the periosteum and the optical absorption of intra-articular tissue were measured on each joint image. Significant differences were found on PAT imaging between the affected joints and the control for both variables measured, including enlarged periosteum diameter and enhanced intra-articular optical absorption occurring in the joints affected with carrageenan-induced arthritis. Anatomical correlation with histological sections of imaged joints and microMRI results verified the findings of PAT. This suggests that PAT has the potential for highly sensitive diagnosis and evaluation of pathologic hallmarks of acute inflammatory arthritis.

Bile acid-oligopeptide conjugates interact with DNA and facilitate transfection.

Bile acids conjugated to oligoarginine-containing peptides (BACs) form complexes with DNA based on the electrostatic interactions between negatively charged phosphate groups of the nucleic acid and the positively charged side chain guanidinium groups of the oligoarginine in the BACs. Charge neutralization of both components and subsequent increases of the net positive charge of the complex combined with the water-soluble lipophilic nature of the bile acid results in changes in the physicochemistry and biological properties of the complexes. We have examined the relationship of a series of 13 BACs on their interaction with circular plasmid DNA (pDNA). The formation of soluble, low-density and insoluble, high-density complexes was analyzed using several methods. The formation of high-density complexes was dependent on the DNA concentration, and was enhanced by increasing the BAC to pDNA charge ratio. Several of the BAC:pDNA complexes demonstrated exclusion of the DNA-intercalator Hoechst 33258 from pDNA, and were also protected from DNase activity. Several BAC conjugates interacted with pDNA to form nanometer-sized particles suitable for cell transfection in vitro. Five of the 13 BACs were transfection competent as single agents, and 11 of the 13 BACs showed enhancement of transfection in combination with DOPE containing liposomes or silica nanoparticles.

Identifying chemical changes in subchondral bone taken from murine knee joints using Raman spectroscopy.

Application of Raman spectroscopy to analysis of subchondral bone is described. The effect of cartilage health on subchondral bone has been widely studied using radiological and histological methods; however, there is no method to directly assay mineral components. We present Raman spectra of femur condyles and observe mineral bands that arise from the subchondral bone. In two separate experiments, transgenic mouse models of early-onset osteoarthritis (OA) and lipoatrophy were compared to tissue from wild-type mice. Raman spectroscopy was used to identify chemical changes in the mineral of subchondral bone that may accompany or precede morphological changes that can be observed by histology. The transgenic mice were compared to age-matched wild-type mice. Subtle alterations in the mineral or collagen matrix were observed by Raman spectroscopy using established Raman markers such as the carbonate-to-phosphate ratio, mineral-to-matrix ratio (MTMR), and amide I ratio. The Raman microscope configuration enabled rapid collection of Raman spectra from the mineralized layer that lies under an intact layer of non-mineralized articular cartilage. The effect of the cartilage layer on collection of spectra is discussed. The technique proposed is capable of providing insight into the chemical changes that occur in subchondral bone on a molecular level.

Imaging of joints with laser-based photoacoustic tomography: an animal study.

Photoacoustic tomography (PAT), a nonionizing, noninvasive, laser-based technology was adapted to joint imaging for the first time. Pulsed laser light in the near-infrared region was directed toward a joint with resultant ultrasonic signals recorded and used to reconstruct images that present the optical properties in subsurface joint tissues. The feasibility of this joint imaging system was validated on a Sprague Dawley rat tail model and verified through comparison with histology. With sufficient penetration depth, PAT realized tomographic imaging of a joint as a whole organ noninvasively. Based on the optical contrast, various intra- and extra-articular tissues, including skin, fat, muscle, blood vessels, synovium and bone, were presented successfully in images with satisfactory spatial resolution that was primarily limited by the bandwidth of detected photoacoustic signals rather than optical diffusion as occurs in traditional optical imaging. PAT, with its intrinsic advantages, may provide a unique opportunity to enable the early diagnosis of inflammatory joint disorders, e.g., rheumatoid arthritis, and to monitor therapeutic outcomes with high sensitivity and accuracy.

Correlating changes in collagen secondary structure with aging and defective type II collagen by Raman spectroscopy.

A novel application of Raman spectroscopy for monitoring damage to ocular collagen in wild-type mice and Del1 (+/-) transgenic mice, a murine animal model of osteoarthritis, is described. In order to understand the progression of diseases of collagen, it is necessary to use methods that can recognize alterations in affected tissue due to chemical and/or genetic modifications. The heterozygous Del1 (+/-) transgenic mouse is established as a model for early-onset osteoarthritis caused by modifications to the type II collagen gene (COL2A1) that result in a truncated collagen fiber. We expect that abnormal type II collagen is expressed in articular cartilage and eye tissue of the Del1 (+/-) mouse. Eyes excised from a subset of specimens from another study using Del1 (+/-) mice were examined by Raman spectroscopy for evidence of defective collagen. Spectral contributions from the collagen protein were readily observed. The amide III envelope (1220-1280 cm-1) was used to characterize changes in collagen secondary structure. Raman spectra of the sclera component of eyes taken from transgenic and older wild-type mice show an increased collagen disorder, as expected. These preliminary results suggest that Raman is capable of recognizing and measuring abnormality in eye collagen and may have potential as a diagnostic tool for ocular collagen damage.

Lipopolysaccharide recognition protein, MD-2, facilitates cellular uptake of E. coli-derived plasmid DNA in synovium.

BACKGROUND: Several cell types are susceptible to transfection in vivo using naked plasmid DNA. The mechanisms involved in mediating in vivo transfection are incompletely known, but evidence suggests that receptor-mediated endocytosis is important for specific types of cells. In this study we tested the hypothesis that residual Escherichia coli lipopolysaccharide (LPS) forms a non-covalent complex with expression plasmid DNA, and host-cell-derived soluble LPS-binding proteins bind to the DNA-LPS complexes in order to facilitate receptor-mediated endocytosis. METHODS: Cells from the murine synovial lining were used as an in vivo model system and in vivo luciferase imaging was used to quantify levels of transgene expression. Using a series of gene-deleted mice, the roles of LPS recognition complex proteins, lipopolysaccharide-binding protein (LBP), CD14 and MD-2, in the process of in vivo transfection were determined. RESULTS: Luciferase expression assays revealed that mice lacking LBP or CD14 had increased luciferase expression (p < 0.023 and < 0.165, respectively), while mice deleted of MD-2 had significant reductions in luciferase expression (p < 0.001). Gene deletion of hyaluronic acid binding protein CD44 was used as a control and had no statistically significant effect on transgene expression in vivo. In muscle tissue, where neither cell surface nor soluble MD-2 is expressed, no MD-2 dependence of plasmid transfection was identified, suggesting the role of MD-2 is tissue or cell type specific. Additionally, depleting mice of macrophages showed that luciferase expression is occurring within fibroblast-like synoviocytes. CONCLUSIONS: Our data support a physical association between LPS and E. coli-derived plasmid DNA, and that in vivo transfection of fibroblast-like synoviocytes is dependent on the soluble form of the LPS-binding protein MD-2.

Adenovirus binding to cultured synoviocytes triggers signaling through MAPK pathways and induces expression of cyclooxygenase-2.

BACKGROUND: Recombinant adenovirus can be administered in vivo to achieve transduction of a number of cell types including human synoviocytes. Immunogenicity of adenoviruses has limited their utility as vectors for gene delivery; however, specific mechanisms underlying the acute inflammatory response to adenovirus are not well understood. Activation of a number of signal transduction pathways occurs rapidly upon adenovirus binding to cell-surface receptors. We investigated stimulated expression of mitogen-activated protein kinases (MAPKs), cyclooxygenase-2 (COX-2) and prostaglandin E(2) (PGE(2)) in human primary synovial fibroblasts to adenovirus expressing the E. coli beta-galactosidase gene. METHODS: Cultured rheumatoid synoviocytes were exposed to transduction-competent Ad/RSVlacZ recombinant adenovirus or transduction-incompetent (psoralen/UV-irradiated) Ad/RSVlacZ. The effects on COX-2 expression, PGE(2) levels and MAPK signaling in synoviocytes were assessed using a combination of reverse-transcription polymerase chain reaction amplification and immunoblotting. RESULTS: Adenovirus treatment of synoviocytes increased levels of COX-2 mRNA and protein as well as PGE(2). Psoralen-treated transcriptionally inactive adenovirus was equivalent to untreated adenovirus for early COX-2 induction suggesting that viral genes were not required. Adenovirus treatment stimulated phosphorylation of ERK-1/-2, p38 MAPK, and JNK. Inhibition of the ERK and p38 MAPK pathways inhibited COX-2 expression and PGE(2) production. CONCLUSIONS: Taken together, these data demonstrate that a MAPK-dependent increase in COX-2 results in local prostaglandin production. These findings have clinical implications for use of adenovirus as vectors for in vivo gene delivery.