| Blunt trauma of solid visceral organs(such as spleen and liver) leads to severe hemorrhage, which is often life-threatening to patients. Laparotomy is a traditional therapy for such emergencies. With the rapid development of interventional techniques in recent years, transcatheter embolization provides an alternative for patients with spleen or liver hemorrhage. In particular, for patients with stable hemodynamics, transcatheter arterial embolization(TAE) has become the best non-laparotomy choice for management of bleeding caused by spleen or liver rupture. Embolic agents employed in TAE procedure block blood flow and reduce tissue perfusion, thus serve as a key determinant for successful hemostatic embolization.Embolic agents commonly used at present for TAE include coils, gelfoam, cyanoacrylate, and Onyx, etc. Coils, as a permanent embolic agent, have a variety of shapes and sizes, and are made from precious metal(platinum, tungsten, etc.) with complex craftsmanship. These characters lead to inconvenience of employing different size of coils according to diameter of target vessel as well as expensiveness. In addition, disadvantages of coils include incomplete occlusion, low rate of thrombus formation, and chronic inflammation or even damage of visceral organs caused by permanent coils embolization. Gelfoam is derived from animal protein composed of various amino acids and can be absorbed by human tissues. The advantages of low price and easy usability make gelfoam the first homeostatic agents employed in surgery. In the interventional field, gelfoam is the only commercially biodegradable embolic material approved to be applied in vivo until now. It is used mainly to either stop bleeding temporarily or to devascularize a tumor prior for surgical removal. Degradation of gelfoam is influenced by many factors including diameter of particles and dosage, resulting in complete degradation from days to months. So increased arterial rebleeding may occur if gelfoam is absorbed prior to compl ete hemostasis.The α-n-butyl cyanoacrylate(α-NBCA) is a commonly used liquid embolic agent for hemostasis of solid viscera. Due to its powerful adhesion and rapid rate of polymerization, α-NBCA is prone to polymerize within the catheter. Moreover, heat released during polymerization may cause injury of artery intima, or even severe inflammation, pain, or tissue ischemia. Onyx, another liquid embolic agent developed recently, is mainly used to embolize brain aneurysm or arteriovenous malformation. Although Onyx can be used in hemostasis of solid viscera, disadvantages of rapid coagulation, requirements of special catheter, demands of skillfulness and severe artery spasm make it an inferior choice.Taken together, there is no ideal embolic material for interventional management of patients with bleeding of solid visceral organs.Development of alginate microspheres loaded with therapeutic drugs as a novel transcatheter embolic material has become a hot research spot in this filed. Alginate microspheres are widely used in medical and pharmaceutical research owing to its advantages of ability of being loaded with drugs, targeted delivery, safety, non-toxicity, controllable degradation, broad adaptability and convenient usability. Thrombin, an effective hemostatic drug, catalyzes conversion of plasma fibrinogen to fibrin, resulting in rapid thrombus formation. It can be used to stop bleeding in different body parts, especially irregular wound surfaces. Unfortunately, autologous thrombus formed by thrombin catalysis is soft, weak, prone to exfoliate or autolysis, and resulting in inadequate hemostasis. In this study, we firstly demonstrated proof-of-concept of a novel embolic microspheres(Thrombin-loaded Alginate-Calcium Microspheres, TACMs), which merged the advantages of conventional embolic microspheres and thrombin for treating blunt trauma and hemorrhage of solid viscera by TAE. Then the physical, pharmacological and biological characteristics of TACMs were investigated. Finally, in vivo model of kidney and spleen injury was employed to explore the optimal protocol for TACMs use and further evaluate its embolic effects. This study may provide promising novel embolic material for blunt trauma of solid viscera with high efficiency, safety, broad indication, conv enience, and biocompatibility.The followings are the methods and results of this study.1. Preparation of Thrombin-loaded Alginate-Calcium Microspheres(TACMs)Based on physical characters of alginate as well as pharmaceutical characters of thrombin, electrostatic droplet technique was adopted to make TACMs. By using an electrostatic droplet generator, TACMs were successfully prepared. Alginate solution(3% w/v) was made by dissolving sodium alginate in normal saline(NS). Then the thrombin was resolved into the alginate solution with final concentration of 10mg/m L. The mixed solution would be used as raw material. Solidifying solution, another indispensable component, was composed of 30g/L Ca Cl2, 5g/L Na Cl, 1g/L Na H2PO4 and 5g/L Tween-20. Under the force of electric field of electrostatic droplet generator(YD-06) and gravity, droplet from raw material solution overcame the attachment of needle tip and surface tension of fluid to quickly drop into the solidifying solution. Then TACMs would be formed by reaction of carboxyl group in alginate molecule with calcium ions in solidifying solution. By elaborate parameters set up, two TACMs with different diameters(350μm and 600μm) were made. The results showed that diameter of TACMs was positively correlated with voltage of electrostatic field, while negatively correlated with gauge of the needle, distance between needle tip and surface of solidifying solution, and outflow velocity of raw material solution. Under light and scanning electron microscope, TACMs presented with even size distribution, inwardly or superficially scattered thrombin, porous structure, and rough surface with many micropores. Such structural characteristics were in favor of fast release of thrombin upon contact of TACMs with blood. In addition, the rough surface of TACMs was prone to entrap platelets and red blood cells, activate coagulation, and rapidly form mixed thrombus.The successful preparation of TACMs will lay the foundation for further investigation of pharmaceutical, physical, and biological characteristics of TACMs, as well as the assessment of feasibility of embolic homeostasis.2. Pharmaceutical and physical characteristics of TACMs(1) Capacity of drug loading(DL) and entrapment efficiency(EE)TACMs were firstly dissolved in 1m L ethylene diamine tetraacetic acid(EDTA) solution. Then entrapment efficiency(EE) and drug loading(DL) were calculated by measuring the thrombin concentrations by the ultraviolet spectrophotometer. The rate of EE and the capacity of DL were 72.4±8.6% and 20±4.1mg/ml in 350μm TACMs, and 70.5 ±6.1% and 13±5.2mg/m L in 600μm TACMs, respectively. Although rate of EE in both groups reached as high as 70% with no significant difference(P>0.05), the DL capacity of 350μm TACMs was higher than that of 600μm TACMs(P<0.05). The difference may result from smaller particle size of 350μm TACMs. Smaller microspheres would take more effective space in the given volume, thus could be loaded more drugs.(2) Mode of thrombin release and activity retention in TACMsTACMs were dissolved in NS and stirred at 100 rpm at 4°C. At predetermined time points, drug releasing curve was plotted and drug releasing pattern was established by detecting thrombin concentration with ultraviolet spectrophotometer. From 0h to 2h, there was a burst releasing phase in 350μm TACMs group with a 34.4±4.9% of the cumulative releasing percent of thrombin, followed by a slow releasing phase with a 90.23% releasing percent at 192 h. In contrast, there was mainly a slow releasing phase in the 600μm TACMs group with a 67.7% releasing percent at 192 h. The 350μm TACMs group had both burst and slow releasing phase, and higher cumulative releasing percent than 600μm TACMs group. The burst releasing effect was essential for mixed thrombus formation, while the slow drug release could maintain thrombin concentration at the target sites. Therefore, 350μm TACMs were more suitable for clinical application.According to the method described in Chinese Pharmacopoeia-Part II, residual activity retention of thrombin was measured. The results showed that the average activity retention of thrombin in the 1h, 24 h, 72 h and 120 h groups was 93.78%, 95.74%, 90.66% and 92.72%, respectively(93.23±2.12%). Therefore, the activity of thrombin loaded in TACMs prepared by electrostatic droplet technique was well retained.(3) Modulus of elasticity of TACMs and its effect on strength and stress resistance of mixed thrombusTo investigate the physical characteristics of TACMs and the effect of thrombin on the microsphere structure, modulus of elasticity in both TACMs and empty alginate microspheres were detected by atomic force microscope(AFM, Dimesion 3100).We found that TACMs was a soft material underwent plastic deformation. The modulus of elasticity in TACMs group was smaller than that of empty alginate microspheres prepared in the same way, suggesting that loaded thrombin might exert effect on chelation of sodium alginate and calcium ions in the solidifying solution. The result of such effect converted the internal structure of TACMs from tight to loose, thus leading to consequent changes in mechanical characteristics.To investigate the effect of TACMs on thrombus strength, the thrombelastography(TEG) and high sensitivity biomechanical measurement system were applied to assess strength and stress of mixed thrombus with different content of microspheres. TEG showed that strength of TACMs mixed thrombus was significantly higher than simple autologous thrombus. And the strength of mixed thrombus was related to content of microspheres. In addition, results of stress test, consistent with TEG measurement, further established an optimized ratio of 2:3 of TACMs to whole blood, under which mixed thrombus has the strongest stress resistance.The above data suggested that TACMs prepared by electrostatic droplet t echnique had the advantages of high rate of drug utility, ideal drug releasing pattern, well retained drug activity, which made it possible to accomplish the aims of rapid mixed thrombus formation with blood and TACMs in vitro and continuous drug releasing after embolization in the blood vessels in vitro. Moreover, as a soft material with plastic deformation ability, TACMs mixed with blood could form firm thrombus with higher strength and stress resistance in contrast to soft and fragile simple autologous thrombus. Thus, pharmaceutical characteristics of TACMs and mechanical characteristics of mixed thrombus formed by microspheres might meet the needs of effective embolization of target arteries in clinical practice.3. Biocompatibility of TACMs(1) Cytotoxicity test in vitroAccording to the basic requirement for cytotoxicity and the translational medical needs of TACMs, mouse fibroblast cell line L929, human vascular endothelial cells(VECs) and human red blood cells(RBC) were applied to indirect(extract liquid) and direct contact cytotoxicity test. Extracts from TACMs showed grade 1 toxicity(low cytotoxicity)in contrast to grade 0 in negative control(normal culture medium) group and grade 4 in positive control(phenol) group respectively. The experimental system was stable and the grading was reasonable and reliable.As an embolic material, TACMs would inevitably directly contact with arterial endothelial cells and red blood cells in blood vessels. Co-culture of VECs with TACMs showed no difference in cell survival rate compared with control group, suggesting that TACMs had no cytotoxicity for VECs. In addition, gradient dosage of TACMs did not cause hemolysis when co-cultured with RBC from human peripheral blood.The above data suggested that TACMs had good cytocompatibility and hemocompatibility when serving as novel embolic agent used in blood vessels.(2) Animal study in vivoPreclinical evaluation of novel medical material was mainly based on animal study. So we adopted C57 BL/6J mice, SD rats and New Zealand white rabbits in this study. Tissue compatibility of TACMs including potential irritation, systemic acute toxicity, genetic toxicity, tissue inflammation, and degradation rate in vivo were evaluated.Dorsal skin of New Zealand white rabbits was very sensitive to serve as an ideal model for irritation test. During 72 hours observation after intradermal injection of extract fluid of TACMs, there were no pathological skin signs including erythema, rash, blister or edema, etc. The irritation score in TACMs extract fluid group was 0, which is the same to that in the NS control group. These results suggested that TACMs was non-toxic and non-irritating, thus was safe when applied in vivo.High concentration of extract liquid from TACMs was injected into abdominal cavity of C57 mouse to observe the systemic acute toxic reaction, using NS as control group. During 28 days of observation, general condition of mice in two groups was normal. No death or abnormal behaviors were found in both groups. In addition, there was no significant difference in gain of body weight between two groups. Histological examination showed no obvious pathological changes in heart, liver, spleen, lung and kidney in mice injected with extract liquid of TACMs.Genetic toxicity of TACMs was evaluated by micronucleus test of bone marrow of C57 mouse. After Formation rate of micronucleus in TACMs extract fluid group was similar to that in negative control group. While formation rate of micronucleus in positive control(cyclophosphamide injection) was significantly higher than that in the former two groups. These results suggested that TACMs induced neither systemic acute toxic reaction, nor genetic toxicity with broken DNA and aneuploidy.The SD rats were used to investigate the degradation of TACMs in vivo. TACMs were subcutaneously implanted at dorsal skin. Then implanted microspheres and their surrounding tissues were collected at the 2, 4, 6, 8, 10 and 12 week. Finally histological analysis was performed to detect diameter of microspheres and inflammatory reaction in the surrounding tissues. We found that TCAMs were gradually degraded and absorbed. The time for complete degradation was 10 weeks. During the whole period, inflammatory infiltration mainly from lymphocytes could be seen in tissues around the microspheres, but the numbers of infiltrated cells decreased with degradation of TACMs.The above data suggested that TACMs did not induce systemic acute toxic reaction, had no genetic toxicity or tissue irritation, and were biodegradable material, although it triggered mild to moderate chronic local inflammation. The duration of the chronic local inflammation was dependent on degradation rate of TACMs in vivo.Taken together, cytotoxic test in vitro and animal study in vivo demonstrated that TACMs met the biosafety requirements for embolic material with excellent cyto-, hemo-, and tissue compatibility and fully degradability, which laid a solid theoretical foundation for further study of embolic hemostasis in vivo.4. Method exploration and embolic effect evaluation of TACMs(1) The establishment of TACMs application methodTo evaluate the feasibility, efficiency and safety TACMs mixed thrombus in treating blunt trauma and hemorrhage of solid viscera, we performed studies on assessing the feasibility of transcatheter delivery of embolic agent, the embolization effect and post-procedural complications.Contrast agents are the most commonly used medium in cardiovascular intervention. It is mainly applied to enhance the visibility of blood vessels and organs when being exposed under X-rays. The majority of contrast agents used nowadays contains iodine. Because TACMs mixed thrombus was invisible under X-rays, we firstly evaluate the feasibility of adding contrast agents to mixed thrombus during its preparation. And we found that all the four commonly used iodine containing contrast agents decreased the activity of thrombin, which was concentration dependent. So it was reasonable to avoid contact of TACMs with contrast agents as far as possible during interventional embolization. To meet the needs of reserving thrombin activity in microspheres and tracing delivered embolic material simultaneously, we developed a novel delivery method named ―sandwich‖ method. When applying this method, we firstly filled the catheter with contrast agent followed by injection of certain amount of TACMs mixed thrombus, and finally re-filled the catheter with contrast agent. The ―sandwich‖ method could not only clearly trace the track of mixed thrombus, but also separate TACMs from contrast agents, thus effectively reserve activity of thrombin in TACMs.(2) The embolic effect for transcatheter application of TACMs mixed thrombusTo clarify the embolic effect and underling mechanism of TACMs mixed thrombus, renal injury model of rabbits was established. Rabbits were divided in to 4 groups: TACMs mixed thrombus group, empty alginate microsphere group, simple thrombus group and NS control group. The results showed that TACMs mixed thrombus could be clearly traced and completely shut down blood flow after being delivered to the renal artery without reflux of embolic agent. The time from accomplishment of embolic agent delivery to complete hemostasis of damaged kidney was short and the amount of mixed thrombus needed was small. Most importantly, post-procedural survival rate of rabbits at 1 week was 100%. While in simple thrombus group, larger amount of thrombus was needed to achieve total embolization than that of TACMs mixed thrombus. During embolization, due to autolysis of autologous thrombus prior to complete hemostasis, re-bleeding occurred and resulting in post-procedural mortality rate at 6 h reached as high as 50%. In contrast, transcatheter arterial embolization failed in both empty alginate microsphere group and N S control group. Further histological analysis of kidneys in each group showed that large amount of fibrin bundles encapsulated around the microsphere in the TACMs mixed thrombus, while no scattered microspheres were found in renal parenchyma; in simple au tologous thrombus group, only scattered thin and small fragments of fibrin could be seen; in empty alginate group, not only no microsphere retention or thrombus formation were found at target embolic site in renal artery, but also microsphere embolization appeared in multiple untargeted terminal arteries in kidney. Although simple thrombus or microsphere alone could not stop bleeding effectively and reliably, mixed thrombus formed by combining both of them(TACMs) was an effective, safe, and reliable embolic agent. The mechanism underling this novel embolic agent might be as the following:(1) in vitro, contact of TACMs with blood led to formation of mixed thrombus. During this process, TACMs not only participate into thrombus formation as an component, but also released thrombin in a burst way to rapidly convert fibrinogen to cross-linked fibrin bundles, which built up firm mixed thrombus;(2) in vivo, when being used to embolize injured arteries, TACMs not only exerted its physical embolization effect on arteries, but also released thrombin sustainably to form new fibrin bundles to solidify the mixed thrombus, thus refrained from crush or dislodgement against impact of blood flow and compression of arterial wall.To investigate the embolic effect and post-procedural complications of TACMs mixed thrombus in blunt trauma of spleen, spleen injury and hemorrhage model of Beagle was established by an open surgery. Then embolization of proximal artery of spleen was performed by using TACMs mixed thrombus. The results showed that preparation of TACMs mixed thrombus in vitro was convenient and time-saving. By applying ―sandwich‖ method, movement track of embolic material and its localization in target vessels could be clearly displayed. Because the TACMs mixed thrombus contained no organic solvent, there was neither any need for catheters made of special material, nor chemical stimulated spasm of vessel. By using commercially available catheters, mixed thrombus could be quickly, easily, and conveniently delivered to target site without occlusion and stickiness of catheter or reflux of embolic agent. The successful rate of embolization using TACMs mixed thrombus was 100%. After TAE, follow up angiography were performed to observe morphological changes of target artery and spleen. Meanwhile, general conditions including body weight, body temperature and hemodynamics were also monitored. The result of angiography showed that reperfusion of spleen parenchyma might occur due to reopening of totally occluded spleen artery. Severe post-procedural complications including re-bleeding, pseudoaneurysm or total spleen infarction, which must be treated by open surgery, were not found. And spleen and its functional were well reserved. There was no significant difference between experimental group and blank control group in body weight, body temperature and hemodynamic parameters. The above operation observations and post-operative results suggested that TACMs mixed thrombus could be prepared easily and were readily to be used. The procedure was easy in terms of operation and the embolic effect of the procedure was reliable without severe complications requiring surgery management. All these results made mixed TACMs thrombus an ideal embolic material for transcatheter homeostasis.Taken together, absence of ideal embolic agent for treatment of blunt trauma of solid visceral organs calls for researches of novel embolic materials. In this study, we successfully developed a novel macromolecular complex embolic material(TACMs), and thoroughly assessed pharmaceutical characteristics, biocompatibility and clinical applicability of this material. We found that the novel embolic material had the advantages of rapid and reliable embolic hemostasis, generalized applicability, readily operation and superior biocompatibility, which made it an ideal embolic material for transcatheter hemostasis of blunt trauma of solid viscera. The development and application of this novel material are promising in improving effect and prognosis of transcatheter hemost asis of solid visceral hemorrhage. In addition, our study may shed lights on directions and offer new ideas for future researches of natural biological material. |
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