Optimization of 3D autologous chondrocyte-seeded polyglycolic acid scaffolds to mimic human ear cartilage.

Auricular reconstruction in children with microtia is one of the more complex procedures in plastic surgery. Obtaining sufficient native material to build an ear requires harvesting large fragments of rib cartilage in children. Herein, we investigated how to optimize autologous chondrocyte isolation, expansion and re-implantation using polyglycolic acid (PGA) scaffolds for generating enough cartilage to recapitulate a whole ear starting from a small ear biopsy. Ear chondrocytes isolated from human microtia subjects grew slower than microtia rib or healthy ear chondrocytes and displayed a phenotypic shift due to the passage number. Rabbit ear chondrocytes co-cultured with mesenchymal stem cells (MSC) at a 50 : 50 ratio recapitulated the cartilage biological properties in vitro. However, PGA scaffolds with different proportions of rabbit chondrocytes and MSC did not grow substantially in two months when subcutaneously implanted in immunosuppressed mice. In contrast, rabbit chondrocyte-seeded PGA scaffolds implanted in immunocompetent rabbits formed a cartilage 10 times larger than the original PGA scaffold. This cartilage mimicked the biofunctional and mechanical properties of an ear cartilage. These results indicate that autologous chondrocyte-seeded PGA scaffolds fabricated following our optimized procedure have immense potential as a solution for obtaining enough cartilage for auricular reconstruction and opens new avenues to redefine autologous cartilage replacement.

Morphometric analysis of the human common hepatic artery reveals a rich and accessible target for sympathetic liver denervation.

This study quantified the distribution of nerves and adjacent anatomies surrounding human common hepatic artery (CHA) as guidance for catheter based denervation. CHA collected from cadaveric human donors (n = 20) were histologically evaluated and periarterial dimensions and distributions of nerves, lymph nodes, pancreas and blood vessels quantified by digital morphometry. Nerve abundance decreased significantly with distance from the aortic ostium (P < 0.0001) and was higher in the Superior/Inferior compared to the Anterior/Posterior quadrants (P = 0.014). In each locational group, nerves were absent from the artery wall, and starting 0.5-1.0 mm from the lumen exhibited a first order dependence on radial distance, fully defined by the median distance. Median subject-averaged nerve distance to the lumen was 2.75 mm, ranging from 2.1-3.1 mm in different arterial segments and quadrants and 2.0-3.5 mm in individuals. Inter-individual variance was high, with certain individuals exhibiting 50th and 75th nerve distances of, respectively, 3.5 and 6.5 mm The pancreas rarely approached within 4 mm of the lumen proximally and 2.5 mm more distally. The data indicate that the CHA is a rich and accessible target for sympathetic denervation regardless of sex and diabetes, with efficacy and safety most optimally balanced proximally.

Balloon-based drug coating delivery to the artery wall is dictated by coating micro-morphology and angioplasty pressure gradients.

Paclitaxel coated balloon catheters (PCB) were developed as a polymer-free non-implantable alternative to drug eluting stents, delivering similar drug payloads in a matter of minutes. While PCB have shown efficacy in treating peripheral arterial disease in certain patient groups, restenosis rates remain high and there is no class effect. To help further optimize these devices, we developed a scanning electron microscopy (SEM) imaging technique and computational modeling approach that provide insights into the coating micromorphology dependence of in vivo drug transfer and retention. PCBs coated with amorphous/flaky or microneedle coatings were inflated for 60 sec in porcine femoral arteries. Animals were euthanized at 0.5, 24 and 72 h and treated arteries processed for SEM to image endoluminal coating distribution followed by paclitaxel quantification by mass spectrometry (MS). Endoluminal surfaces exhibited sparse coating patches at 0.5 h, predominantly protruding (13.71 vs 0.59%, P < 0.001), with similar micro-morphologies to nominal PCB surfaces. Microneedle coating covered a 1.5-fold endoluminal area (16.1 vs 10.7%, P = 0.0035) owing to higher proximal and distal delivery, and achieved 1.5-fold tissue concentrations by MS (1933 vs 1298 µg/g, P = 0.1745) compared to amorphous/flaky coating. Acute longitudinal coating distribution tracked computationally predicted microindentation pressure gradients (r = 0.9, P < 0.001), with superior transfer of the microneedle coatings attributed to their amplification of angioplasty contact pressures. By 24 h, paclitaxel concentration and coated tissue areas both declined by >93% even as nonprotruding coating levels were stable between 0.5 and 72 h, and 2.7-fold higher for microneedle vs flaky coating (0.64 vs 0.24%, P = 0.0195). Tissue retained paclitaxel concentrations at 24-72 h trended 1.7-fold higher post treatment with microneedle coating compared to the amorphous/flaky coating (69.9 vs 39.9 µg/g, P = 0.066). Thus, balloon based drug delivery is critically dependent on coating micromorphologies, with superior performance exhibited by micromorphologies that amplify angioplasty pressures.

Splenic artery denervation: target micro-anatomy, feasibility, and early preclinical experience.

This study sought to evaluate perisplenic artery nerve distribution and the feasibility of splenic artery denervation (SDN). The NEXION radiofrequency catheter was used to perform SDN in healthy and inflammatory arthritis pigs. Splenic artery anatomy, nerve distribution, and splenic norepinephrine (NEPI) levels were evaluated before and after SDN. Perisplenic artery nerves were primarily distributed within 2.5 mm of the arterial lumen and were largely sympathetic on the basis of tyrosine hydroxylase expression. The pancreas, tended to be circumferentially positioned around the proximal splenic artery, typically >2.5 mm from the lumen, ensuring that most of the nerves could be targeted without affecting this sensitive organ. The mid segment of the splenic artery was relatively free of contact with the adjacent pancreas. Splenic NEPI levels and nerve abundance followed a decreasing gradient from the proximal to distal splenic artery. SDN resulted in significant reductions in splenic NEPI levels at day 14 (60.7%, P = 0.024) in naïve pigs and day 45 (100%, P = 0.001) in inflammatory arthritis pigs. There was no significant effect of SDN on joint soft tissue injury or circulating inflammatory markers in the inflammatory arthritis model. The majority of perisplenic arterial nerves are within close proximity of the lumen and are primarily sympathetic efferent fibers. Nerves in the mid-segment may be the preferred SDN target given their proximity to the artery and paucity of periarterial off-target organs. SDN appears safe and effective at reducing splenic NEPI levels.

Preclinical Evaluation of Mesh Implants: The Pathologist's Perspective.

Surgical and laparoscopic implantation of mesh devices is on the rise for a variety of applications. The complexity and range of evolving mesh designs calls for consistent and detailed pathologic evaluation in determining host responses and assessing overall safety. This review addresses the components of evaluation of mesh implants in animal models, with emphasis on histologic parameters, semiquantitative scoring matrices, and morphometric analyses that have been specifically adapted to this class of implants. Necropsy assessment should include implant persistence, architecture, and associated host responses such as exudation and adhesions. Microscopic evaluation should focus on primary relevant responses such as bioresorption, integration/tissue ingrowth, neovascularization, and inflammation. Selection of the best means of processing and evaluation can be complicated, as meshes may include one or more biologic components (e.g., collagen), synthetic polymer fibers, coatings, and other molecules. The architecture of some meshes can influence tissue responses and complicate sampling, sectioning, and evaluation. Recognition of specific study objectives and knowledge of anticipated responses helps to determine the appropriate histologic or immunochemical stains, while understanding of mesh composition and anticipated persistence in tissue determines the suitability of paraffin or resin embedding, and both guide the evaluation of mesh devices in the preclinical setting.

Defining drug and target protein distributions after stent-based drug release: Durable versus deployable coatings.

BACKGROUND: Innovations in drug eluting stent designs make it increasingly important to develop models for differentiating performance through spatial definition of drug, receptor binding and cell state. METHODS: Two designs of sirolimus analog eluting stents were implanted into porcine coronary arteries for 28, 60 or 90 days (n = 9/time point), durable coating (Xience) and deployable absorbable coating (MiStent). Explanted arteries were evaluated for drug content (n = 3/time point) by LC-MS/MS and for drug and target protein (mTOR) distributions by immunofluorescence (IF, n = 6/time point). A computational model was developed to predict drug release and arterial distribution maps. RESULTS: Both stents released the majority of drug load by 28 days, with different tissue retention efficiencies (91.4 ±â€¯4.9% MiStent versus 21.5 ±â€¯1.9% Xience, P < 0.001). Computational modeling of MiStent coating deployment and microcrystal dissolution recapitulated in vivo drug release and net tissue content and predicted that >98.5% of deployed drug remains crystalline through 90 days. Immunofluorescence and computational modeling showed peristrut drug localization for both stents, with similar peaks, but high interstrut levels only at sites of coating deployment from the absorbable coating. Co-localization of mTOR-IF with drug-IF for both devices showed persistent drug effects, though with differential drug-receptor pharmacokinetics. CONCLUSIONS: Immunofluorescence and computational modeling provide insights into drug distribution and binding status that can help differentiate drug delivery technologies. Herein we found that tissue deployment of slow dissolving crystalline drug particles results in temporally and spatially more uniform drug delivery to interstrut zones that might otherwise be under-dosed without excess peristrut drug.

Calcified plaque modification alters local drug delivery in the treatment of peripheral atherosclerosis.

BACKGROUND: Calcific atherosclerosis is a major challenge to intraluminal drug delivery in peripheral artery disease (PAD). OBJECTIVES: We evaluated the effects of orbital atherectomy on intraluminal paclitaxel delivery to human peripheral arteries with substantial calcified plaque. METHODS: Diagnostic angiography and 3-D rotational imaging of five fresh human lower limbs revealed calcification in all main arteries. The proximal or distal segment of each artery was treated using an orbital atherectomy system (OAS) under simulated blood flow and fluoroscopy. Explanted arterial segments underwent either histomorphometric assessment of effect or tracking of 14C-labeled or fluorescent-labeled paclitaxel. Radiolabeled drug quantified bulk delivery and fluorescent label established penetration of drug over finer spatial domain in serial microscopic sections. Results were interpreted using a mathematical model of binding-diffusion mediated arterial drug distribution. RESULTS: Lesion composition affected paclitaxel absorption and distribution in cadaveric human peripheral arteries. Pretreatment imaging calcium scores in control femoropopliteal arterial segments correlated with a log-linear decline in the bulk absorption rate-constant of 14C-labeled, declining 5.5-fold per calcified quadrant (p=0.05, n=7). Compared to controls, OAS-treated femoropopliteal segments exhibited 180µm thinner intima (p<0.001), 45% less plaque calcification, and 2 log orders higher paclitaxel bulk absorption rate-constants. Correspondingly, fluorescent paclitaxel penetrated deeper in OAS-treated femoropopliteal segments compared to controls, due to a 70% increase in diffusivity (p<0.001). CONCLUSIONS: These data illustrate that calcified plaque limited intravascular drug delivery, and controlled OAS treatment of calcific plaques resulted in greater drug permeability and improved adjunct drug delivery to diseased arteries.

Pulsatility and high shear stress deteriorate barrier phenotype in brain microvascular endothelium.

Microvascular endothelial cells at the blood-brain barrier exhibit a protective phenotype, which is highly induced by biochemical and biomechanical stimuli. Amongst them, shear stress enhances junctional tightness and limits transport at capillary-like levels. Abnormal flow patterns can reduce functional features of macrovascular endothelium. We now examine if this is true in brain microvascular endothelial cells. We suggest in this paper a complex response of endothelial cells to aberrant forces under different flow domains. Human brain microvascular endothelial cells were exposed to physiological or abnormal flow patterns. Physiologic shear (10-20 dyn/cm2) upregulates expression of tight junction markers Zona Occludens 1 (1.7-fold) and Claudin-5 (more than 2-fold). High shear stress (40 dyn/cm2) and/or pulsatility decreased their expression to basal levels and altered junctional morphology. We exposed cells to pathological shear stress patterns followed by capillary-like conditions. Results showed reversible recovery on the expression of tight junction markers. Flow protection of barrier phenotype commensurate with junctional signaling pathways decrease (Src, 0.25-fold, ERK, 0.77-fold) when compared to static conditions. This decrease was lost under high shear and pulsatile flow. In conclusion, abnormal shear stress inherent to systemic vascular disease leads to barrier impairment, which could be reverted by hemodynamic interventions.

Treatment with chondroitin sulfate to modulate inflammation and atherogenesis in obesity.

BACKGROUND AND AIMS: Osteoarthritic patients treated with high doses of chondroitin sulfate (CS) have a lower incidence of coronary heart disease--but the mechanistic aspects of these beneficial effects of CS remain undefined. We examined how CS treatment affects the formation of atheroma via interaction with endothelial cells and monocytes. METHODS: We characterized arterial atheromatous plaques by multiphoton microscopy and serum pro-inflammatory cytokines by immunoenzymatic techniques in obese mice receiving CS (1 g/kg/day, i.p.) or vehicle for 6 days. Effects of CS on signaling pathways, cytokine secretion and macrophage migration were evaluated in cultures of human coronary endothelial cells and in a monocyte cell line stimulated with TNF-α by Western blot, immunoenzymatic techniques and transwell migration assays. RESULTS: Treatment of obese mice with CS reduced the extension of foam cell coverage in atheromatous plaques of arterial bifurcations by 62.5%, the serum concentration of IL1ß by 70%, TNF-α by 82% and selected chemokines by 25-35%. Cultures of coronary endothelial cells and monocytes stimulated with TNF-α secreted less pro-inflammatory cytokines in the presence of CS (P < 0.01). CS reduced the activation of the TNF-α signaling pathway in endothelial cells (pErk 36% of reduction, and NFκB 33% of reduction), and the migration of activated monocytes to inflamed endothelial cells in transwells (81 ± 6 vs. 13 ± 2, P < 0.001). CONCLUSIONS: CS interferes with the pro-inflammatory activation of monocytes and endothelial cells driven by TNF-α thus reducing the propagation of inflammation and preventing the formation of atherosclerotic plaques.