Experiment Studies On Biocompatibility And Embolization Of A New Kind Of Embolic Material: PU-BaFe₁₂O₁₉

Granule materials were first used in endovascular embolization for curing diseases. With the affluxion of flood, the materials reached and gathered together in vessel beds, slowing down the blood stream and blocked the nutrition artery and other diseased branches. Granule materials embolization is widely used in clinic especially for pre-operation of tumors and endovascular treatment of cerebral arteriovenous malformations (AVM). However, with the appearance and application of liquid materials, the development, of Granule materials appears slowly relatively. Even though they are rarely used alone, and usually used together with liquid embolic materials. However, granule materials shouldn't be ignored in clinic for so many advantages, such as ideal biocompatibility, safe and easily control in operation and suitable diameter particles can be used to cure various diseased vessals.This innovative BaFe₁₂O₁₉ particles can be seen in X-ray, having spherical shape and good suspension quality, thus they wouldn't block the catheter easily as some liquid materials. The author carried out some experiments to examine the biocompatibility and embolization effect of BaFe₁₂O₁₉ and polyurethane-BaFe₁₂O₁₉ (PU-BaFe₁₂O₁₉) particles as a new kind of embolic materials. Embolization experiments were evaluated in carotid artery of Wista rats, renal artery of New Zealand rabbits and rete mirabile (RMB) of pigs, providing some positive evidences for further clinical studies. Part 1: Study on Biocompatibility of BaFe₁₂O₁₉ andPU-BaFe₁₂O₁₉ ParticlesThe experiment of biocompatibility for BaFe₁₂O₁₉ and PU-BaFe₁₂O₁₉ particles was evaluated including Ames test, bone marrow micronucleus test, acute and subacute systemic toxicity test, hemolysis test, hemopexis test, bleeding and clotting time measurement test.1. Ames test is a vitro mutagenesis test. The indicator strains used in this test were Salmonella typhimurium TA₉₈ and TA₁₀₀ with or without S9 The positive control objects were mutagens of Dexon and diaminofluorene (2-AF), while the negative control was sterile distilled water. The results was evaluated by mutation rate (MR=mutagenesis colony number/ spontaneous colony number). Standards (WHO): the test is positive if MR>2. The result showed that MR value of all concentration of leaching liquors of BaFe₁₂O₁₉ and polyurethane-BaFe₁₂O₁₉ was smaller than 2 while bigger than 2 for both Dexon and 2-AF groups. This results Indicates these two experimental materials have no mutagenesis effection.2. Bone marrow micronucleus test in mice is a vivo mutagenesis test. After intraperitoneal injection of leaching liquor for 30h, cavum ossis suspension was taken and smeared, then the number of polychronic cells (PCE) containing micronucleus was counted and the frequency calculated (‰). The positive and negative control was cyclophosphamide and saline, tested in parallel controlled trial. The results showed that negative results in three groups of leaching liquor of both materials. The frequency of micronucleated cell for normal saline group was 1.93±0.56‰while for cyclophosphamide was 19.53±2.14‰. By statistical analysis of the data, this results indicates that three experiment groups had no significant difference compare to normal saline group(P>0.05) , and the cyclophosphamide group showed significant difference compare to the experiment and normal saline groups(P<0.001). 3. Vitro cytotoxicity test. L929 (fibroblast of mice) cells were cultivated with leaching liquor so as to evaluate its toxicity. The negative control was culture fluid while the positive was Phenol (64g/L), and the three groups were tested in parallel controlled trial. The samples were tested by MTT assay and optical density values (OD) were measured at 24h, 48h, 72h respectively. The OD value and relative growth rate (RGR) was calculated and the cytotoxicity was evaluated. Standars: 0 Ord (RGR=100%) I Ord (RGR 75%-99%) II Ord (RGR 50%-74%) IIIOrd (RGR 25%-49%) IVOrd (RGR 1%-24%) V Ord (RGR=0); 0& I (no cytotoxicity) II (slight cytotoxicity) III&IV(middling cytotoxicity) V (manifest cytotoxicity). The results showed that L929 cells growth condition in experiment groups similarly to negative group, no obvious growth inhibition. Cytotoxicity of different concentration of leaching liquor of both experiment materials to the L929 cells was I Ord, the NS group 0 Ord and the Phenol was V Ord. So we can draw a conclusion that both materials have no cytotoxic effect.4. Acute, subacute and chronic systemic toxicity test. NIH rats were Intraperitoneal injection of experiment materials suspension so as to observe the toxicity in mice. With sterile saline BaFe₁₂O₁₉ and PU-BaFe₁₂O₁₉ particulate materials were prepared in 10 mg / ml and 5 mg / ml suspension. Each mouse of every group underwent intraperitoneal injection of 0.5 ml suspension while the control group injection of NS 0.5mL during the same period in parallel control. The general condition of mice was observed including appetite, activity, weight change, toxic reactions and even the number of dead animals. According to different groups, the mice were sacrificed in differently period respectively. Their brain, heart, liver, spleen, lung, kidney, pancreas and peritoneum were taken respectively prepared for histology analysis. The results showed that general condition of all experimental animals were well and no one death, no convulsions, no paralysis, no respiratory depression, no diarrhea, no activity reduce or weight loss. In all specimens there was no cell degeneration or necrosis, no exudation and infiltration of inflammatory cells. The experiments showed the two materials had no systemic toxicity.5. Hemolysis test. To determine whether the materials have hemolysis effect by means of adding the leaching liquor into the erythrocyte suspensions. 5ml of fresh blood were collected, heparin added, glass rod to be used stirred to remove fibrinogen. Take 4ml of above blood, add 20ml NS to dilute, shake even, centrifuge and make RBC sediment. RBC was diluted into 2% suspension by adding NS, and add 2ml leaching liquor into it, shake even, bathe in 37℃water and centrifuge observed. After 4h, 1 ml of the supernatant was taken, 4 ml of 0.1% Na2CO3 solution were added, and then optical density was measured on spectrophotometer at 540 nm, then calculated the hemolysis rate. The results showed hemolysis rate of BaFe₁₂O₁₉ and PU- BaFe₁₂O₁₉ particulate were 2.08% and 2.43% respectively, it achieve the standard of not exceeding 5% according to ISO. This experiment indicates that the materials do not cause hemolysis.6. Bleeding and clotting time test. To observe the impact on the bleeding and clotting time after intraperitoneal injection of materials suspension. Materials suspension was intraperitoneal injected into each experimental mouse, and 1 ml NS for each mouse in control group. Bleeding and clotting time was detected after 7d. The results showed that there was no significant difference (p>0.05) between experimental groups and control group, indicating these two kinds of materials have no obvious impact on mice bleeding and clotting time.7. Blood hemopexis function test. The leaching liquor and blood of Wistar rat were directly added together to determine whether the materials have impact on blood hemopexis function. 1ml of each rat tail vein blood was respectively added to 20 anticoagulant tubes. Every experiment tube was added with 1 ml of leaching liquor while the control group 1 ml of NS. Blood coagulation time was measured after 30 mins. Statistical analysis of experimental data showed that there was no significant difference in prothrombin time, Fibrinogen assay, activated partial thromboplastin time and thrombin time between experimental group and control group.Part 2 Experiment Study on Embolization of Carotid Artery Brancheswith BaFe₁₂O₁₉ and PU-BaFe₁₂O₁₉ Particles in Wistar RatsObjective: To evaluate the embolization effect on carotid artery branches with BaFe₁₂O₁₉ and PU-BaFe₁₂O₁₉ particles in Wistar rats, to observe the changes in neural function and survival status, and to get a better understanding of the impact on pathology on vascular endothelium and brain tissues.Method: 1. Experiment groups: (1) BaFe₁₂O₁₉ embolization group; (2) PU-BaFe₁₂O₁₉ embolization group; (3) BaFe₁₂O₁₉ and PU-BaFe₁₂O₁₉ co-embolization group; (4) NS control group, 8 rats for each group. 2. Methods: Puncture trocars were used to puncture the carotid artery then finish embolization. With the concentration of 2.5mg/ml, 5mg/ml of the PU-BaFe₁₂O₁₉ suspension and 6mg/ml, 12mg/ml of BaFe₁₂O₁₉, distal branches of ECA and ICA were embolized successfully according to the principle of "low concentration first" "small size particles first". Embolization consisted of two steps. Low concentration of small particles was first injected, and then the high concentrations of bigger particles were injected subsequently. Angiographic was made to observe the occlusion situation of the artery branches. Not every branch to be embolized imperatively. To keep observation on the survival, behavior and physiological changes in nerve function of animals, and to recheck angiographic of carotid arteries of survival animals, and then make macroscopic and microscopic examination at 1w, 4w, 8w after the operation.Results: 1. The endovascular operation was successful performed and the particle injection resistance was well-distributed without blocking the catheters; 2. Some branches of ECA and ICA were embolized successfully in the operation, but the survival and neurological condition were better for using little particle of BaFe₁₂O₁₉ and PU-BaFe₁₂O₁₉, while much worse for using BaFe₁₂O₁₉ and PU-BaFe₁₂O₁₉ co-embolized. DSA for survived animals showed there weren't recanalization of previous occluded vessels. 3. Pathological detection showed some sporadic lamellar ischemia regions on brain surface observed in early phase, while infarction and introcession of cortex formed in late phase. Histological examination showed vessels were stuffed with these embolic materials, no obvious damage to the wall of vessels, and inflammatory cells gathered around the Embolization particles in early phase, while forein-body giant cells were observed in late phase. The embolic agents stayed well in vessels, some vascular walls thinningz, and varying degrees of damage, varying degrees of inflammatory cells was found around blood vessels.Conclusion: BaFe₁₂O₁₉ and PU-BaFe₁₂O₁₉ as embolic materials can be used to embolize branches of ECA and ICA in Wistar rats, and the embolization effect is duration without artery recanalization, only mild vessal endomembrane inflammatory reaction in pathological examination.Part 3 Experiment Study on Embolization of Renal Artery withBaFe₁₂O₁₉ and PU-BaFe₁₂O₁₉ Particles in NewZealand RabbitObjective: To evaluate the long-term and short-term Embolization effects of rabbit renal artery embolization with BaFe₁₂O₁₉ and PU- BaFe₁₂O₁₉ particles and to investigate the pathological changes in kidney afer Embolization.Method: 1. According to the kinds of particles, 24 rabbits were divided averagely into three groups: Experimental groups: BaFe₁₂O₁₉ embolization group, BaFe₁₂O₁₉ embolization group and BaFe₁₂O₁₉ and BaFe₁₂O₁₉ co-embolization group, 8 rabbits for each group. 2. Methods: Use the operation of slitting femoral artery to cannulate 4F introducer sheath, and the micro-catheter was inserted into the renal artery of one side. With the concentration of 2.5mg/ml, 5mg/ml of the PU-BaFe₁₂O₁₉ suspension and 6mg/ml, 12mg/ml of BaFe₁₂O₁₉, the distal terminal branches of renal artery were embolized, according to the principle of "low concentration first", "small size particles first". The target vessels were second grade renal artery branches or the third grade artery branches. Angiography was taken immediately after embolized to review the target vessels whether or not visualization, if necessary, additional dose injected until embolization completely. Color Doppler ultrasound examination was taken at the middle period of survived animals to observe the blood stream of the embolized renal artery. Angiographic was rechecked for the embolized artery afer operation of 1w, 2w, 4w, 8w respectively, then the animals were killed, kidney specimens to be observed and for histological examination.Results: 1. The endovascular operation was successful and the injection resistance was smooth without blocking the tubes; 2. The target vessels were successfully embolized and DSA of PU-BaFe₁₂O₁₉ embolization group, BaFe₁₂O₁₉ and BaFe₁₂O₁₉ co-embolization showed no collateral circulation, no recanalization. But 2 of 18 renal artery branches underwent recanalization in the simple BaFe₁₂O₁₉ group, the recanalization rate was 11%. 3. Kidney specimen examination showed flake infarction on the surface of early renal, atrophy in late renal and cortical depression; pathological examination showed vessels stuffed with embolization agents, mild inflammatory reaction in early renal interstitial, no destruction to vessel wall. The pathological specimen of late renal showed that the structure was sparse, the structure of glomerulus and renal tubules mostly disappeared and kidney showed diffuse granulation degeneration, mesenchymal small vessels wall hyalinized, perivascular infiltration of inflammatory cells visible.Conclusion: PU-BaFe₁₂O₁₉ alone or combined with BaFe₁₂O₁₉ can be used to embolize the New Zealand rabbit renal artery effectively, while a separate application of BaFe₁₂O₁₉ particles alone were lower effect than above mentioned. Some arteries have the possibility of recanalization and the toxic histopathologic for vessal response to the material is mild.Part4 Modeling and Embolization for Different Flows ofArteriovenous Malformation (AVM) of Brain in SwineObjective: 1. To establish three flows of brain AVM animal models with high, medium and low flows by microvascular anastomosis on the base of pig's rete mirabile (RMB). 2. To research the technical feasibility, effectiveness and safety of embolization by using these two particle materials to embolize AVM of animal model. 3. To evaluate the impact of the experiment materials on vessels and brain tissues by making macroscopic and microscopic examination.Method: 1. Animal models: 8 animals were divided into three groups randomly-2 for low-flow group, 3 for medial-flow group and 3 for high-flow group. The normal RMB is used as low-flow of AVM model. The medial flow model is established by ligation of common carotid artery of one side, which increases the blood flow through bilateral RMB. The high flow model is built on by end-side or end-end vascular anastomosis between right carotid artery proximal end and right jugular vein distal end, which increases greatly the blood flow of RMB because the direct arteriovenous fistula. In high flow model, left and right ascending pharyngeal (AP) artery acted as afferent artery and draining vein respectively, the bilateral RMB as AVM nidus. 2. Embolization of cerebral AVM model: BaFe₁₂O₁₉, PU-BaFe₁₂O₁₉ and mixture of the two materials embolized the three groups respectively. All animals adopted femoral artery incision intubation, micro-catheter being placed in the left ascending pharyngeal artery where injection of embolic materials can embolize RMB entirely. Animals in the high and medial-flow groups were sacrificed on 1 -3d. while animals in low flow group were taken angiography after 2 weeks and 2 months respectively, then the swine were sacrificed and their bilateral RMB including some surrounding brain tissues were taken for macroscopic and microscopic examination.Results: 1. Animal models: The vascular anastomosis operation was successful and medial-flow, high-flow cerebral AVM models were established ideally, only one animal in high-flow group undergoing anastomotic stenosis. 2. All RMB of experimental animals were embolized successfully and angiography rechecked showed no visualization of RMB. No catheter blocked and therefore the catheter wasn't replaced in the embolization process. Acute pathological examination showed that the embolic material was well dispersed and located in the mold RMB. Inflammatory cells Infiltrated and no obvious damage to the endothelium. Chronic pathological examination showed the vessels still blocked by experiment material with foreign body giant cells surround, some vessel walls underwent varying degrees of thinning and damage. All specimens showed no leak of embolic material.Conclusions: 1. Different cerebral AVM animal models can be formed by one carotid artery ligation as well as the anastomosis of carotid artery and jugular vein changing the blood supply and pathway. 2. Granular materials of BaFe₁₂O₁₉ and PU-BaFe₁₂O₁₉ can be embolized AVM models of different flow and have many advantages such as operation convenient, safe embolization and so on. So BaFe₁₂O₁₉ maybe become a new type of embolization material for cerebral AVM and has great potential as a therapeutic embolic agent.