Insulin, Not the Preservative m-cresol, Instigates Loss of Infusion Site Patency Over Extended Durations of CSII in Diabetic Swine.

Insulin infusion sets worn for more than 4-5 days have been associated with a greater risk of unexplained hyperglycemia, a phenomenon that has been hypothesized to be caused by an inflammatory response to preservatives such as m-cresol and phenol. In this cross-over study in diabetic swine, we examined the role of the preservative m-cresol in inflammation and changes in infusion site patency. Insulin pharmacokinetics (PK) and glucose pharmacodynamics (PD) were measured on delivery of a bolus of regular human insulin U-100 (U-100R), formulated with or without 2.5 mg/mL m-cresol, to fasted swine following 0, 3, 5, 7, and 10 days of continuous subcutaneous insulin infusion (CSII). In a subsequent study with the same animals, biopsies were evaluated from swine wearing infusion sets infusing nothing, saline, or U-100R either with or without 2.5 mg/mL m-cresol, following 3, 7, and 10 days of CSII. Exposure to m-cresol did not impact any PK or PD endpoints. PK and PD responses dropped markedly from Days 7-10, regardless of the presence of m-cresol. Histopathology results suggest an additive inflammatory response to both the infusion set and the insulin protein itself, peaking at Day 7 and remaining stable beyond.

An Overview on the Considerations for the Planning of Nonclinical Necropsies for Medical Device Studies.

The terminal collection and histological processing of medical devices is an expensive, labor-, and material-intensive endeavor, which requires adequate experience, innovation, and preparation for success. It is also an exciting endeavor that continually challenges, intellectually engages, and improves the skills and knowledge of the pathologist. Awareness of the importance of the medical device pathologist's involvement, communication, and oversight throughout the development, implementation, and execution of a nonclinical assessment of a medical device is in the best interest of the test facility, the histopathology laboratory, the pathologist, the sponsor, and, ultimately, the patients. This article serves to present as a primer of key considerations for the approach and conduct of "nontoxicological" studies, defined as studies involving animal models of deployment or implantation of medical devices as well as surgical animal models.

Biocompatibility, bone healing, and safety evaluation in rabbits with an IlluminOss bone stabilization system.

Bone healing, biocompatibility, and safety employing the IlluminOss System (IS), comprised of an inflatable balloon filled with photopolymerizable liquid monomer, was evaluated in New Zealand white rabbits. Successful bone healing and callus remodeling over 6 months was demonstrated radiologically and histologically with IS implants in fenestrated femoral cortices. Biocompatibility was demonstrated with IS implants in brushed, flushed femoral intramedullary spaces, eliciting no adverse, local, or systemic responses and with similar biocompatibility to K-wires in contralateral femurs up to 1 year post-implant. Lastly simulated clinical failures demonstrated the safety of IS implants up to 1 year in the presence of liquid or polymerized polymer within the intramedullary space. Polymerized material displayed cortical bone and vasculature effects comparable to mechanical disruption of the endosteum. In the clinically unlikely scenario with no remediation or polymerization, a high dose monomer injection resulted in marked necrosis of cortical bone, as well as associated vasculature, endosteum, and bone marrow. Overall, when polymerized and hardened within bone intramedullary spaces, this light curable monomer system may provide a safe and effective method for fracture stabilization. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2181-2190, 2017.

Ultra-hydrophilic stent platforms promote early vascular healing and minimise late tissue response: a potential alternative to second-generation drug-eluting stents.

AIMS: Simple surface modifications can enhance coronary stent performance. Ultra-hydrophilic surface (UHS) treatment of contemporary bare metal stents (BMS) was assessed in vivo to verify whether such stents can provide long-term efficacy comparable to second-generation drug-eluting stents (DES) while promoting healing comparably to BMS. METHODS AND RESULTS: UHS-treated BMS, untreated BMS and corresponding DES were tested for three commercial platforms. A thirty-day and a 90-day porcine coronary model were used to characterise late tissue response. Three-day porcine coronary and seven-day rabbit iliac models were used for early healing assessment. In porcine coronary arteries, hydrophilic treatment reduced intimal hyperplasia relative to the BMS and corresponding DES platforms (1.5-fold to threefold reduction in 30-day angiographic and histological stenosis; p<0.04). Endothelialisation was similar on UHS-treated BMS and untreated BMS, both in swine and rabbit models, and lower on DES. Elevation in thrombotic indices was infrequent (never observed with UHS, rare with BMS, most often with DES), but, when present, correlated with reduced endothelialisation (p<0.01). CONCLUSIONS: Ultra-hydrophilic surface treatment of contemporary stents conferred good healing while moderating neointimal and thrombotic responses. Such surfaces may offer safe alternatives to DES, particularly when rapid healing and short dual antiplatelet therapy (DAPT) are crucial.

Particulates from hydrophilic-coated guiding sheaths embolise to the brain.

AIMS: We sought to evaluate the incidence of embolic material in porcine brains following vascular interventions using hydrophilic-coated sheaths. METHODS AND RESULTS: A new self-expanding stent and delivery system (SDS) was deployed through a hydrophilic-coated (Flexor Ansel; Cook Medical, Bloomington, IN, USA) guiding sheath into the iliac and/or carotid arteries of 23 anaesthetised Yucatan mini swine. The animals were euthanised at three, 30, 90 and 180 days and their brains were removed for histological analysis. In an additional single control animal, the guiding sheath was advanced but no SDS was deployed. Advancement of the coated guiding sheath with or without the SDS was associated with frequent foreign material in the arterioles of the brain. The embolic material was amorphous, non-refractile, non-crystalline, non-birefringent and typically lightly basophilic with a slightly stippled appearance on haematoxylin and eosin (H&E) stain. Material was observed at all time points involving 54% of all study animals (i.e., test and control) and in vitro after incubation in 0.9% saline. CONCLUSIONS: The hydrophilic coating on a clinically used guiding sheath readily avulses and embolises to the brain during deployment in a porcine model. Further documentation of this effect and monitoring in clinical scenarios are warranted.

Evaluation of an intramedullary bone stabilization system using a light-curable monomer in sheep.

Percutaneous intramedullary fixation may provide an ideal method for stabilization of bone fractures, while avoiding the need for large tissue dissections. Tibiae in 18 sheep were treated with an intramedullary photodynamic bone stabilization system (PBSS) that comprised a polyethylene terephthalate (Dacron) balloon filled with a monomer, cured with visible light in situ, and then harvested at 30, 90, or 180 days. In additional 40 sheep, a midshaft tibial osteotomy was performed and stabilized with external fixators or external fixators combined with the PBSS and evaluated at 8, 12, and 26 weeks. Healing and biocompatibility were evaluated by radiographic analysis, micro-computed tomography, and histopathology. In nonfractured sheep tibiae, PBSS implants conformably filled the medullary canal, while active cortical bone remodeling and apposition of new periosteal and/or endosteal bone was observed with no significant macroscopic or microscopic observations. Fractured sheep tibiae exhibited increased bone formation inside the osteotomy gap, with no significant difference when fixation was augmented by PBSS implants. Periosteal callus size gradually decreased over time and was similar in both treatment groups. No inhibition of endosteal bone remodeling or vascularization was observed with PBSS implants. Intramedullary application of a light-curable PBSS is a biocompatible, feasible method for fracture fixation.

Tuning of collagen scaffold properties modulates embedded endothelial cell regulatory phenotype in repair of vascular injuries in vivo.

Perivascularly implanted matrix embedded endothelial cells (MEECs) are potent regulators of inflammation and intimal hyperplasia following vascular injuries. Endothelial cells (ECs) in collagen scaffolds adopt a reparative phenotype with significant therapeutic potential. Although the biology of MEECs is increasingly understood, tuning of scaffold properties to control cell-substrate interactions is less well-studied. It is hypothesized that modulating scaffold degradation would change EC phenotype. Scaffolds with differential degradation are prepared by cross-linking and predegradation. Vascular injury increases degradation and the presence of MEECs retards injury-mediated degradation. MEECs respond to differential scaffold properties with altered viability in vivo, suppressed smooth muscle cell (SMC) proliferation in vitro, and altered interleukin-6 and matrix metalloproteinase-9 expression. When implanted perivascularly to a murine carotid wire injury, tuned scaffolds change MEEC effects on vascular repair and inflammation. Live animal imaging enables real-time tracking of cell viability, inflammation, and scaffold degradation, affording an unprecedented understanding of interactions between cells, substrate, and tissue. MEEC-treated injuries improve endothelialization and reduce SMC hyperplasia over 14 d. These data demonstrate the potent role material design plays in tuning MEEC efficacy in vivo, with implications for the design of clinical therapies.

Arterial microanatomy determines the success of energy-based renal denervation in controlling hypertension.

Renal denervation (RDN) is a treatment option for patients with hypertension resistant to conventional therapy. Clinical trials have demonstrated variable benefit. To understand the determinants of successful clinical response to this treatment, we integrated porcine and computational models of intravascular radiofrequency RDN. Controlled single-electrode denervation resulted in ablation zone geometries that varied in arc, area, and depth, depending on the composition of the adjacent tissue substructure. Computational simulations predicted that delivered power density was influenced by tissue substructure, and peaked at the conductivity discontinuities between soft fatty adventitia and water-rich tissues (media, lymph nodes, etc.), not at the electrode-tissue interface. Electrode irrigation protected arterial wall tissue adjacent to the electrode by clearing heat that diffuses from within the tissue, without altering periarterial ablation. Seven days after multielectrode treatments, renal norepinephrine and blood pressure were reduced. Blood pressure reductions were correlated with the size-weighted number of degenerative nerves, implying that the effectiveness of the treatment in decreasing hypertension depends on the extent of nerve injury and ablation, which in turn are determined by the tissue microanatomy at the electrode site. These results may explain the variable patient response to RDN and suggest a path to more robust outcomes.

Osteopetrorickets due to Snx10 deficiency in mice results from both failed osteoclast activity and loss of gastric acid-dependent calcium absorption.

Mutations in sorting nexin 10 (Snx10) have recently been found to account for roughly 4% of all human malignant osteopetrosis, some of them fatal. To study the disease pathogenesis, we investigated the expression of Snx10 and created mouse models in which Snx10 was knocked down globally or knocked out in osteoclasts. Endocytosis is severely defective in Snx10-deficient osteoclasts, as is extracellular acidification, ruffled border formation, and bone resorption. We also discovered that Snx10 is highly expressed in stomach epithelium, with mutations leading to high stomach pH and low calcium solubilization. Global Snx10-deficiency in mice results in a combined phenotype: osteopetrosis (due to osteoclast defect) and rickets (due to high stomach pH and low calcium availability, resulting in impaired bone mineralization). Osteopetrorickets, the paradoxical association of insufficient mineralization in the context of a positive total body calcium balance, is thought to occur due to the inability of the osteoclasts to maintain normal calcium-phosphorus homeostasis. However, osteoclast-specific Snx10 knockout had no effect on calcium balance, and therefore led to severe osteopetrosis without rickets. Moreover, supplementation with calcium gluconate rescued mice from the rachitic phenotype and dramatically extended life span in global Snx10-deficient mice, suggesting that this may be a life-saving component of the clinical approach to Snx10-dependent human osteopetrosis that has previously gone unrecognized. We conclude that tissue-specific effects of Snx10 mutation need to be considered in clinical approaches to this disease entity. Reliance solely on hematopoietic stem cell transplantation can leave hypocalcemia uncorrected with sometimes fatal consequences. These studies established an essential role for Snx10 in bone homeostasis and underscore the importance of gastric acidification in calcium uptake.

Methodological standardization for the pre-clinical evaluation of renal sympathetic denervation.

Transcatheter ablation of renal autonomic nerves is a viable option for the treatment of resistant arterial hypertension; however, structured pre-clinical evaluation with standardization of analytical procedures remains a clear gap in this field. Here we discuss the topics relevant to the pre-clinical model for the evaluation of renal denervation (RDN) devices and report methodologies and criteria toward standardization of the safety and efficacy assessment, including histopathological evaluations of the renal artery, periarterial nerves, and associated periadventitial tissues. The pre-clinical swine renal artery model can be used effectively to assess both the safety and efficacy of RDN technologies. Assessment of the efficacy of RDN modalities primarily focuses on the determination of the depth of penetration of treatment-related injury (e.g., necrosis) of the periarterial tissues and its relationship (i.e., location and distance) and the effect on the associated renal nerves and the correlation thereof with proxy biomarkers including renal norepinephrine concentrations and nerve-specific immunohistochemical stains (e.g., tyrosine hydroxylase). The safety evaluation of RDN technologies involves assessing for adverse effects on tissues local to the site of treatment (i.e., on the arterial wall) as well as tissues at a distance (e.g., soft tissue, veins, arterial branches, skeletal muscle, adrenal gland, ureters). Increasing experience will help to create a standardized means of examining all arterial beds subject to ablative energy and in doing so enable us to proceed to optimize the development and assessment of these emerging technologies.

Evaluation of renal nerve morphological changes and norepinephrine levels following treatment with novel bipolar radiofrequency delivery systems in a porcine model.

OBJECTIVE: To evaluate the safety and effectiveness of different bipolar radiofrequency system algorithms in interrupting the renal sympathetic nerves and reducing renal norepinephrine in a healthy porcine model. METHODS: A porcine model (N = 46) was used to investigate renal norepinephrine levels and changes to renal artery tissues and nerves following percutaneous renal denervation with radiofrequency bipolar electrodes mounted on a balloon catheter. Parameters of the radiofrequency system (i.e. electrode length and energy delivery algorithm), and the effects of single and longitudinal treatments along the artery were studied with a 7-day model in which swine received unilateral radiofrequency treatments. Additional sets of animals were used to examine norepinephrine and histological changes 28 days following bilateral percutaneous radiofrequency treatment or surgical denervation; untreated swine were used for comparison of renal norepinephrine levels. RESULTS: Seven days postprocedure, norepinephrine concentrations decreased proportionally to electrode length, with 81, 60 and 38% reductions (vs. contralateral control) using 16, 4 and 2-mm electrodes, respectively. Applying a temperature-control algorithm with the 4-mm electrodes increased efficacy, with a mean 89.5% norepinephrine reduction following a 30-s treatment at 68°C. Applying this treatment along the entire artery length affected more nerves vs. a single treatment, resulting in superior norepinephrine reduction 28 days following bilateral treatment. CONCLUSION: Percutaneous renal artery application of bipolar radiofrequency energy demonstrated safety and resulted in a significant renal norepinephrine content reduction and renal nerve injury compared with untreated controls in porcine models.

Preclinical evaluation of the InCraft" aortic endograft in a sheep model.

Animal models remain the gold standard for the preclinical evaluation of tissue response, sealing and integrity of aortic endografts. Preclinical testing of the InCraft® device was performed to evaluate these attributes. Through the femoral arteries of eight male crossbred sheep, 22 mm diameter InCraft® Aortic Bifurcate devices were deployed in the abdominal aortas, and shortened 13 mm diameter iliac limbs were deployed in the right iliac arteries. Vessels were excised for radiographic and histopathologic assessment at six months. There were no instances of graft thrombosis, type I endoleak or endograft migration. No fractures of the stents or fixation barbs were observed. There were minimal inflammatory changes on histology, characterized by histiocytes and multinucleated giant cells located along the fabric. The InCraft® device has favorable tissue compatibility and functions well in a sheep model, maintaining patency and sealing without migration, stent fracture or abnormal histologic changes.

Serial Evaluation of Vascular Response After Implantation of a New Sirolimus-Eluting Stent With Bioabsorbable Polymer (MISTENT): an optical coherence tomography and histopathological study.

BACKGROUND: Novel vascular scaffolds aim at equipoise between safety and efficacy. Intravascular optical coherence tomography (OCT) allows in-vivo serial assessment of stent-vessel interactions with high resolution and frequent sampling and may complement histology assessment. We investigated the vascular response to a novel absorbable coating sirolimus-eluting stent (AC-SES) by means of serial OCT and histology evaluation in a porcine model. METHODS: One AC-SES and one bare-metal stent (BMS) were implanted in separate coronary arteries of three Yucatan mini-swine. Serial OCT was performed post procedure and at 3-, 28-, 90-, and 180-day follow-up. Normalized optical density (NOD) was used for the assessment of tissue response over time. Histological evaluation was performed at day 180. RESULTS: A total of 6408 stent struts were analyzed. OCT revealed 100% of struts covered at 28 days, and a significant difference in NOD from 3 to 28 days (0.64 ± 0.07 vs 0.71 ± 0.05, respectively; P<.001) in the AC-SES group. Neointimal thickness was 0.14 ± 0.08 mm, 0.17 ± 0.11 mm, and 0.16 ± 0.09 mm in the AC-SES group and 0.18 ± 0.10 mm, 0.14 ± 0.09 mm, and 0.10 ± 0.08 mm in the BMS group, while rates of uncovered struts were 0%, 0%, and 3.1% and 1.4%, 7.8%, and 21.5%, respectively, at 28, 90, and 180 days. Minimal inflammation and a mature endothelialization were demonstrated in both groups by histology. CONCLUSION: OCT serial assessment of vascular response suggested NIH maturation 28 days following AC-SES implantation in pigs. These findings, coupled with histological demonstration of low inflammation scores and complete endothelial coverage as measured at 180 days, suggest a satisfactory healing response to AC-SES.

Enhanced drug delivery capabilities from stents coated with absorbable polymer and crystalline drug.

Current drug eluting stent (DES) technology is not optimized with regard to the pharmacokinetics of drug delivery. A novel, absorbable-coating sirolimus-eluting stent (AC-SES) was evaluated for its capacity to deliver drug more evenly within the intimal area rather than concentrating drug around the stent struts and for its ability to match coating erosion with drug release. The coating consisted of absorbable poly-lactide-co-glycolic acid (PLGA) and crystalline sirolimus deposited by a dry-powder electrostatic process. The AC-SES demonstrated enhanced drug stability under simulated use conditions and consistent drug delivery balanced with coating erosion in a porcine coronary implant model. The initial drug burst was eliminated and drug release was sustained after implantation. The coating was absorbed within 90 days. Following implantation into porcine coronary arteries the AC-SES coating is distributed in the surrounding intimal tissue over the course of several weeks. Computational modeling of drug delivery characteristics demonstrates how distributed coating optimizes the load of drug immediately around each stent strut and extends drug delivery between stent struts. The result was a highly efficient arterial uptake of drug with superior performance to a clinical bare metal stent (BMS). Neointimal thickness (0.17±0.07 mm vs. 0.28±0.11 mm) and area percent stenosis (22±9% vs. 35±12%) were significantly reduced (p<0.05) by the AC-SES compared to the BMS 30 days after stent implantation in an overlap configuration in porcine coronary arteries. Inflammation was significantly reduced in the AC-SES compared to the BMS at both 30 and 90 days after implantation. Biocompatible, rapidly absorbable stent coatings enable the matching of drug release with coating erosion and provide for the controlled migration of coating material into tissue to reduce vicissitudes in drug tissue levels, optimizing efficacy and reducing potential toxicity.

Engraftment and tumorigenesis of HTLV-1 transformed T cell lines in SCID/bg and NOD/SCID mice.

Human T cell leukemia/lymphoma virus type-1 (HTLV-1) is recognized as the etiological agent of adult T cell leukemia (ATL). Although HTLV-1 can immortalize human lymphocytes in culture, identification of molecular events leading to tumorigenesis after HTLV-1 infection remain elusive. SCID/bg and NOD/SCID mice have reduced natural killer (NK) cell activity and were inoculated intraperitoneally with HTLV-1 transformed cells to refine and characterize the SCID mouse as a small animal model for investigation of HTLV-1 tumorigenesis. HTLV-1 transformed cell lines originally derived by cocultivation of uninfected peripheral blood mononuclear cells (PBMC) with lethally irradiated leukemic cells from patient samples (SLB-1, MT-2 and HT-1-RV) were lymphomagenic when inoculated into NOD/SCID mice. In contrast, immortalized cell lines generated by transfection PBMC with an infectious molecular clone of HTLV-1 (ACH or ACH.p12) were not tumorigenic. The differing behaviors of HTLV-1 infected cell lines in NOD/SCID mice indicates that viral infection and immortalization of human PBMC for growth in culture is not sufficient for induction of a tumorigenic phenotype. The higher level of engraftment of HTLV-1 transformed cell lines in NOD/SCID mice suggests that this is an effective animal model to investigate molecular determinants of HTLV-1 lymphomagenesis.