Impact of storage solution formulation during refrigerated storage upon chondrocyte viability and cartilage matrix.

Various preservation solutions have been evaluated for longer hypothermic cartilage storage for tissue transplantation; however, the results are mixed. This research was carried out to determine whether phosphate-buffered saline (PBS) or organ preservation solutions would preserve both the extracellular matrix and chondrocytes of articular cartilage better than culture medium during refrigerated storage in the time frame that cartilage is stored for clinical use. Porcine cartilage plugs were stored, without the underlying bone, in culture medium with and without fetal bovine serum (FBS), PBS, Belzer's and Unisol solutions for 1 month at 4°C. Metabolic activity was tested using a resazurin reduction method, and matrix permeability was evaluated by measuring electrical conductivity. Storage in culture medium with 10% FBS was shown to provide good cartilage metabolic function for 7 days, decreasing to about 36% after 1 month of storage. There was no significant difference between samples stored in culture medium with and without FBS after 1 month of storage (p = 0.5005). Refrigerated storage of cartilage in PBS and two different solutions (Belzer's and Unisol) designed for optimal refrigerated tissue and organ storage results in loss of chondrocyte function and retention of matrix permeability. In contrast, the opposite, namely significantly better retention of chondrocyte function and loss of matrix permeability, was observed with culture medium. Future research should be focused on combining retention of chondrocyte function and matrix permeability by storage solution formulation.

Recombinant Dendroides canadensis antifreeze proteins as potential ingredients in cryopreservation solutions.

Expanding cryopreservation methods to include a wider range of cell types, such as those sensitive to freezing, is needed for maintaining the viability of cell-based regenerative medicine products. Conventional cryopreservation protocols, which include use of cryoprotectants such as dimethylsulfoxide (Me2SO), have not prevented ice-induced damage to cell and tissue matrices during freezing. A family of antifreeze proteins (AFPs) produced in the larvae of the beetle, Dendroides canadensis allow this insect to survive subzero temperatures as low as -26°C. This study is an assessment of the effect of the four hemolymph D. canadensis AFPs (DAFPs) on the supercooling (nucleating) temperature, ice structure patterns and viability of the A10 cell line derived from the thoracic aorta of embryonic rat. Cryoprotectant solution cocktails containing combinations of DAFPs in concentrations ranging from 0 to 3mg/mL in Unisol base mixed with 1M Me2SO were first evaluated by cryomicroscopy. Combining multiple DAFPs demonstrated significant supercooling point depressing activity (∼9°C) when compared to single DAFPs and/or conventional 1M Me2SO control solutions. Concentrations of DAFPs as low as 1 µg/mL were sufficient to trigger this effect. In addition, significantly improved A10 smooth muscle cell viability was observed in cryopreservation experiments with low DAFP-6 and DAFP-2 concentrations in combination with Me2SO. No significant improvement in viability was observed with either DAFP-1 or DAFP-4. Low and effective DAFP concentrations are advantageous because they minimize concerns regarding cell cytotoxicity and manufacturing cost. These findings support the potential of incorporating DAFPs in solutions used to cryopreserve cells and tissues.

Stent overlapping and geometric curvature influence the structural integrity and surface characteristics of coronary nitinol stents.

Preliminary studies have revealed that some stents undergo corrosion and fatigue-induced fracture in vivo, with significant release of metallic ions into surrounding tissues. A direct link between corrosion and in-stent restenosis has not been clearly established; nonetheless in vitro studies have shown that relatively high concentrations of heavy metal ions can stimulate both inflammatory and fibrotic reactions, which are the main steps in the process of restenosis. To isolate the mechanical effects from the local biochemical effects, accelerated biomechanical testing was performed on single and overlapping Nickel-Titanium (NiTi) stents subjected to various degrees of curvature. Post testing, stents were evaluated using Scanning Electron Microscopy (SEM) to identify the type of surface alterations. Fretting wear was observed in overlapping cases, in both straight and curved configurations. Stent strut fractures occurred in the presence of geometric curvature. Fretting wear and fatigue fractures observed on stents following mechanical simulation were similar to those from previously reported human stent explants. It has been shown that biomechanical factors such as arterial curvature combined with stent overlapping enhance the incidence and degree of wear and fatigue fracture when compared to single stents in a straight tube configuration.

The role of vascular calcification in inducing fatigue and fracture of coronary stents.

Traditional approaches for in-vitro pulsatile and fatigue testing of endovascular stents do not take into consideration the pathologies of the stented vessel and their associated biomechanical effects. One important pathology is calcification, which may be capable of inducing changes in the vessel wall leading to inhomogeneous distribution of stresses combined with wall motion during the cardiac cycle. These local property changes in the region adjacent to stents could directly influence in-vivo stent performance. Seven cases containing a total of 18 stents were obtained from autopsy. Radiographs were evaluated and vessels were sectioned for histology and stent topographical analysis. Stents were retrieved by chemical removal of surrounding tissue and surfaces were evaluated using 3D digital optical and scanning electron microscopy for biomechanical abrasion and fracture features. Pathologic complications such as restenosis and thrombus formation were assessed from histological sections. Direct evidence of fracture was found in 6 of the 7 cases (in 12 out of 18 stents; 9 drug eluting and 3 bare metal). The degree of stent alterations was variable, where separation of segments due to fracture occurred mostly in drug-eluting stents. All fracture surfaces were representative of a high cycle fatigue mechanism. These fractures occurred in complex lesions involving the presence of diffuse calcification alone, or in combination with vessel angulations and multiple overlapping stents. Morphologic analysis of tissue at or near some fracture sites showed evidence of thrombus formation and/or neointimal tissue growth.

Clinical device-related article surface characterization of explanted endovascular stents: evidence of in vivo corrosion.

Limited information exists regarding the in vivo stability of endovascular stents. Nine excised human vascular segments with implanted stents (n = 16) manufactured from stainless steel, nickel-titanium, tantalum, and cobalt-based alloys were analyzed. The stent/tissue components were separated using an established tissue dissolution protocol and control and explanted stents were evaluated by digital optical and electron microscopy. Metallic content in surrounding tissues was measured by mass spectroscopy. Surface alterations, consistent with corrosion mediated by electrochemical and mechanical factors, were observed in 9 of the 16 explanted stents and were absent from control stents. Tissue dissolved from around corroded stents corresponded with a higher metallic content. The effect of these changes in the microtopography of stents on their mechanical properties (fatigue strength and fracture limit) in addition to the potential for released metallic debris contributing to the biological mechanisms of in-stent restenosis supports the need for further investigations.

In-vivo corrosion and local release of metallic ions from vascular stents into surrounding tissue.

OBJECTIVES: To evaluate retrieved bare metal vascular stents and surrounding tissue. BACKGROUND: Limited information is available regarding the condition of stent surfaces and their interaction with vascular tissue following implantation. Corrosion of stents presents two main risks: release of metallic ions into tissue and deterioration of the mechanical properties of stents which may contribute to fracture. Release of heavy metal ions could alter the local tissue environment leading to up-regulation of inflammatory mediators and promote in-stent restenosis. METHODS: Nineteen cases were collected from autopsy, heart explants for transplant, and vascular surgery (23 vessels containing 33 bare metal stents). A method was developed for optimal tissue dissolution and separation of the stent/tissue components without inducing stent corrosion. When available, chemical analysis was performed to assess metallic content in both the control and dissolved tissue solutions. Electron microscopy and digital optical microscopy imaging were used to evaluate stents. RESULTS: Twelve of the 33 stents showed varying degrees of corrosion. Metallic levels in the tissue surrounding the corroded stents were significantly higher (0.5-3.0 mcg/cm² stent) than in control solutions (0-0.30 mcg/cm² stent) and in tissue surrounding stents that did not undergo corrosion (0- 0.20 mcg/cm² stent). CONCLUSIONS: Corrosion of some retrieved stents is described which leads to transfer of heavy metal ions into surrounding tissue. The contribution of this metallic ion release to the mechanisms of in-stent restenosis as well as its effect on the mechanical properties of stents is unknown and requires further investigation.