Intravascular tissue reactions induced by various types of bioabsorbable polymeric materials: correlation between the degradation profiles and corresponding tissue reactions

The nine bioabsorbable polymers or copolymers, most of which are commercially used surgical sutures, were processed into sterilized short segments under the good manufacturing practice conditions. They were (1) Polysorb (US Surgical, CT, USA), 10% polyglycolic acid/90% polylactic acid with coating of glycolide and ε-caprolactone (Surgical suture material. It is also the material used in the coating of Matrix® detachable coil.); (2) 25/75 PLGA (A synthesized polymer thread specifically created for the experiment.); (3) BondekPlus (Teleflex Medical Mansfield, MA, USA; 100% PGA (Surgical suture material. It is also the same polymeric material used in the Cerecyte® coil material.)); (4) Monocryl (Ethicon, Somerville, NJ, USA) 75% PGA/25% ε-caprolactone (surgical suture material); (5) Biosyn (US Surgical, CT, USA), 60% PGA/26% trimethylene carbonate/14% p-dioxanone (surgical suture material); (6) Maxon (US Surgical, CT, USA), 67% PGA/33% trimethylene carbonate (surgical suture material); (7) 75/25 PLGA (A synthesized polymer thread specifically created for the experiment); (8) 10/90 PLGA (A synthesized polymer thread specifically created for the experiment); and (9) poly-l-lactic acid (PLLA; A synthesized polymer thread specifically created for the experiment), respectively. Thickness of the coils vary depending on each material (0.480–0.760 mm).

In vitro evaluation of these nine polymers was conducted as follows. A 20 mg of each coil was immersed in 10 ml of phosphate-buffered saline (PBS) at 37°C (pH 7.4). Samples were collected at 2, 4, 8, 12, and 16 weeks. In vitro degradation was determined by measuring mass loss on microbalance and molecular weight using gel permeation chromatography (GPC). For GPC, 0.25 wt.% of each material was dissolved in chloroform or hexafluoroisopropanol, filtered and injected into a Waters Stratagel HT6E column (Mw range 1,000–10,000,000), followed by a Stratagel HT2 column (Mw range 100–10,000), at a flow rate of 0.6 ml/min. Polystyrene standards (Aldrich Co.), with molecular weights of 760 to 2,000,000, were used to obtain GPC calibration curves. Dilute solution viscosity was conducted to confirm GPC findings according to ASTM D2857-95. Poly-(methylmethacrylate) standards were used for calibration. The Mark–Houwink equation was used to estimate molecular weight (M): where η represents the intrinsic viscosity (determined experimentally), K = 0.0003 for PLG, and a = 0.668 for PLG.

With the technical assistance of the Boston Scientific Inc., the prepared candidate materials underwent heat treatments, and one of the ends had a rounded edge to prevent the fibers from unraveling after implantations. The samples were sterilized twice by ethylene oxide gas and packed individually.

All animal experiments were conducted in accordance with policies set by the Chancellor’s Animal Research Committee and guidelines from the National Institutes of Health. Forty-four carotid arteries in 22 New Zealand white rabbits (body weight, 4–5 kg) were used in this study. Each animal was anesthetized with 35 mg/kg of intramuscularly administered ketamine and 5 mg/kg xylazine. After the induction of anesthesia, animals were placed on a heating pad to maintain their body temperatures at 37°C during the procedure. Every surgical procedure was conducted under sterilized techniques.

Forty-four surgical implantations of prepared materials were performed in bilateral common carotid arteries (CCAs) of 22 rabbits. The details of the surgical procedure have been reported previously [15]. Briefly, in supine position, a 3-cm midline incision was made in the neck of a rabbit, and bilateral CCAs were exposed. Each CCA was ligated at the distal end of the exposed segment with a 3-0 proline suture, and a 22-G Angiocath® (BD Medical Sandy, UT, USA) was inserted into the CCA in a retrograde fashion. Next, a 2-cm segment of each prepared bioabsorbable polymeric suture or platinum coil was inserted into the artery through the Angiocath. Finally, another ligature with 3-0 proline was placed near the inserting point to fix the implant and to prevent the migration of the implant into the circulation. Figure 1 shows a simplified diagram explaining anatomical relationship after the implantation.

Tissue samples were harvested at postoperative day 14. The harvesting time point was determined based on the previous study [15], in which histologic findings using the same animal model implanted with Polysorb–platinum hybrid coils (PGLA coils) and bare platinum coils were evaluated over four different postoperative time points (days 3, 7, 14, and 30), and variable tissue reactions were observed at day 14.

Animals were euthanized with an intracardiac injection of 60 mg/kg sodium pentobarbital. The previous incisions were reopened, and each arterial segment containing the implanted material was gently removed and fixed in 10% buffered formalin. All the harvested samples were sent for histologic analysis. The numerical breakdown of the materials implanted in the animals was Maxon (n = 4 arteries), Polysorb (n = 4), Monocryl (n = 4), Biosyn (n = 4), 25/75 PGLA (n = 4), 75/25 PGLA (n = 4), 90/10 PGLA (n = 4), BondekPlus (n = 4), PLLA (n = 4), and platinum control (n = 4). Four sham samples (without implantation) were also evaluated as controls.

The arterial segments containing platinum were embedded in methylmethacrylate, and the segments containing bioabsorbable polymers were embedded in paraffin. All specimens were sectioned transversely at 2-mm intervals from the distal to the proximal end. The middle third sections were selected for comparing one material to another in order to minimize variability among samples. Sections were stained with hematoxylin and eosin, Masson’s trichrome, and Elastic Van Gieson. Special attention was given to the amount, location, and quality of thrombus, inflammatory cells, and fibrous tissues.

Materials used in this study have different braiding patterns and various mechanical properties. Therefore, qualitative histologic analysis, instead of quantitative analysis, was utilized to evaluate the tissue reaction elicited by each material. Every examined sample was categorized into three different groups according to comprehensive histologic findings. “Minimal tissue reaction” was defined as follows. All four specimens showed no remarkable thrombus organization (the thickest part of fibrous tissue formation surrounding the implanted materials was less than 100 μm), and the intraluminal space was predominantly filled with either immature thrombus or fresh red blood cells. “Strong tissue reaction” was defined as follows. All four specimens presented significant fibrous tissue formation surrounding the implanted materials (the thinnest part of organized thrombus around the material is thicker than 100 μm), and the samples also presented remarkable intimal hyperplasia of the vessel wall. “Moderate tissue reaction” is designated for samples that did not completely satisfy either of the former criteria. Three investigators independently reviewed a minimum of five slides from each specimen, and then the interpretations were confirmed by an experienced neuropathologist (H.V.V.).