胶原海绵人工硬脑膜结合臭氧修复颅脑损伤

doi:10.3969/j.issn.2095-4344.2015.21.005

摘要/Abstract

摘要：

背景：硬膜的完整性对于颅脑损伤患者手术的预后十分重要。人工硬脑膜是目前常见的硬脑膜修补材料，寻找理想的人工硬脑膜是神经外科探索的方向。
目的：观察并分析胶原海绵人工硬脑膜和臭氧治疗颅脑损伤患者的临床资料，探讨并评价其使用价值。
方法：回顾性分析60例颅脑损伤患者应用胶原海绵人工硬脑膜修补和臭氧治疗后的疗效及并发症随访结果。
结果与结论：患者中2例死于重型颅脑损伤术后弥漫性脑肿胀，1例死于脑损伤合并多器官衰竭，2例因广泛脑挫裂伤合并脑疝术后植物生存，其余55例患者进入结果分析。术后2例出现切口局部皮下积液，予穿刺抽吸及弹性绷带加压包扎后好转；1例术后出现术侧少量硬膜下积液，未行特殊处理，动态复查头颅CT显示积液逐步吸收减少。头颅CT 检查未见与人工脑膜有关的异常反应和表现。28例患者3-6个月进行颅骨修补时发现人工硬脑膜与正常硬脑膜融合恢复较好，无粘连及炎症反应发生。表明胶原海绵人工硬脑膜和臭氧在颅脑损伤患者治疗中充分发挥减压作用，维持脑功能，缩短手术时间，并发症少，相容性好，能够较好的与正常硬脑膜融合，保护脑皮质，为后期颅骨修补创造有利条件。

关键词: 生物材料, 材料相容性, 胶原海绵, 人工硬脑膜, 组织相容性, 生物医学材料, 臭氧治疗, 颅脑损伤, 围术期处理, 并发症

Abstract:

BACKGROUND: The integrity of the dura mater is very important for prognosis of patients with brain injury prognosis. Artificial dura mater is a commonly repair material, and to look for an ideal artificial dura mater is the exploration direction in the neurosurgery field.
OBJECTIVE: To observe and analyze the clinical data of brain injury patients undergoing repair of collagen sponge as dura mater substitute material with ozone therapy, and to explore and evaluate its clinical value.
METHODS: Follow-up results of therapeutic efficacy and complications in 60 cases of brain injury following repair with collagen sponge artificial dura mater and ozone treatment were retrospectively analyzed.
RESULTS AND CONCLUSION: Two patients died of postoperative diffuse brain swelling, one died of brain injury with multiple organ failures, and two cases of extensive brain injury accompanied by cerebral herniation were in vegetative state. The rest 55 patients were enrolled in the final analysis. After surgery, two patients appeared to have postoperative subcutaneous fluid and their conditions improved following puncture aspiration and pressure dressing with elastic bandage; another patient showed a small amount of subdural effusion, but did not undergo special treatment, and dynamic head CT showed the effusion was gradually reduced. Cranial CT examination showed no abnormalities associated with the artificial dura mater. At 3-6 months after surgery, the artificial dura mater was fused well with the normal dura mater in 28 cases undergoing skull patching, and there was no adhesion and inflammatory reaction. Taken together, the collagen sponge artificial dura mater with ozone can give full play to the decompression treatment of traumatic brain injury, which can maintain the brain function, shorter operative time, result in fewer complications, and have good compatibility, and moreover, the artificial dura mater can be fused well with the normal dura to protect the brain cortex, thereby providing favorable conditions for the latter skull repair.

Key words: Biocompatible Materials, Histocompatibility, Collagen, Ozone

中图分类号:

R318

曲虹，赵明光，梁英，赵丽萍，李晓红，王莹. 胶原海绵人工硬脑膜结合臭氧修复颅脑损伤[J]. 中国组织工程研究, 2015, 19(21): 3302-3308.

Qu Hong, Zhao Ming-guang, Liang Ying, Zhao Li-ping, Li Xiao-hong, Wang Ying. Collagen sponge as an artificial dura mater combined with ozone to repair brain injury[J]. Chinese Journal of Tissue Engineering Research, 2015, 19(21): 3302-3308.

图/表（结果） 4

Quantitative analysis of participants
Two cases died of severe brain injury after diffuse brain swelling, one died of brain injury with multiple organ failure, and two cases of extensive brain injury accompanied by cerebral herniation were in vegetative state. Patients were followed for 1 year, and the remaining 55 patients were enrolled in the final analysis (Table 1).

Meningeal repair with collagen sponge as the artificial dura mater material
There were no cerebrospinal fluid leakage from the incision, subcutaneous fluid, intracranial infections, epilepsy and other complications, and skin incision healed well in all patients followed by regular removal of stitches. Before discharge, head CT examinations showed no obvious abnormalities at the repair site. Patients recovered well postoperatively with normal body temperature and normal results of blood and biochemical tests (Table 1). Of the 60 patients, 2 patients died of postoperative diffuse brain swelling, one died of brain injury with multiple organ failures, and two cases of extensive brain injury accompanied by cerebral herniation were in vegetative state after surgery. Another 55 cases showed obvious midline shift on CT images at 1, 7, 14 days after surgery (P < 0.05). There was also a significant difference in the intracranial pressure at 1, 7, 14 days after surgery (P < 0.05; Table 2), indicating the treatment achieved the purpose of decompression.

Head CT results
Head CT results showed that there were no abnormal radiographic changes at 1 week after surgery and at discharge (Figure 1).

Follow-up results
According to Glasgow coma scale score, complete recovery was in 12 cases (20%), mild disability in 16 cases (27%), moderate disability in 21 cases (35%), severe disability in 7 cases (12%), vegetative state in 2 cases (3%), and death in 2 cases (3%). At 3-6 months after dura repair, 28 cases were subject to skull repair, and during the skull repair, the anatomical structures of the dura mater were found basically clear and less wound bleeding. There was shorter operative time. The dura mater separated was intact with no damage to brain tissues, and the artificial meninges and normal dura mater were fully integrated into the “pseudomembrane” that had a complete smooth surface and medial surface. No meningeal adhesions and severe inflammatory reactions in the repair area were found (Figure 2).

Observation and processing results of subcutaneous fluid
After surgery, two patients appeared with postoperative subcutaneous fluid and their conditions improved following puncture aspiration and pressure dressing with elastic bandage; another patient showed a small amount of subdural effusion, but with no special treatment, and dynamic head CT showed the effusion was gradually reduced.

Adverse reactions and complications
During the follow-up, there was no advanced incision leakage of cerebrospinal fluid, brain incarceration and epilepsy. All skin incisions were healed at stage I, with regular removal of stitches. Repair materials had no response to the host in the blood, tissue, and immunological fields.

参考文献

[1] Tsuji M, Inoue Y, Sugaya C, et al. Higher toxicity of dibutyltin and poly-L-lactide with a large amount of tin but lower toxicity of poly-L-lactide of synthetic artificial dura mater exhibited on murine astrocyte cell line. Yakugaku Zasshi. 2010;130(6):847-855.

[2] Zhang YL, Xin GH, Zeng YL. Pig peritonium as a donor transplant to repair the defect in human cerebral dura mater. Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu. 2008;12(23):4489-4492.

[3] Chen J, Shi SS, Zhang GL, et al. Bio-artificial dura mater in repair of dural defects. Zhongguo Zuzhi Gongheng Yanjiu. 2013;17(51):8914-8919.

[4] Esposito F, Grimod G, Cavallo LM, et al. Collagen-only biomatrix as dural substitute: What happened after a 5-year observational follow-up study. Clin Neurol Neurosurg. 2013;115(9):1735-1737.

[5] Parlato C, di Nuzzo G, Luongo M, et al. Use of a collagen biomatrix (TissuDura) for dura repair: a long-term neuroradiological and neuropathological evaluation. Acta Neurochir (Wien). 2011;153(1):142-147.

[6] Papakostas JC, Avgos S, Arnaoutoglou E, et al. Use of the vascu-guard bovine pericardium patch for arteriotomy closure in carotid endarterectomy. Early and long-term results. Ann Vasc Surg. 2014;28(5):1213-1218.

[7] Coleman S, Kerr H, Krishnamurthi V, et al. The use of bovine pericardium for complex urologic venous reconstruction. Urology. 2014;83(2):495-497.

[8] Costa BS, Cavalcanti-Mendes Gde A, Abreu MS, et al. Clinical experience with a novel bovine collagen dura mater substitute. Arq Neuropsiquiatr. 2011;69(2A):217-220.

[9] Chen X, Liu J, Yang GY.The clinical application of collagen dressing. Linchuang yu Shiyan Yixue Zazhi. 2010;17(23):1078-1079．

[10] Sartori C, Lepori M, Scherrer U. Interaction between nitric oxide and the cholinergic and sympathetic nervous system in cardiovascular control in humans. Pharmacol Ther. 2005;106(2):209-220.

[11] Bocci V, Paulesu L. Studies on the biological effects of ozone 1. Induction of interferon gamma on human leucocytes. Haematologica. 1990;75(6):510-515.

[12] Larini A, Bocci V. Effects of ozone on isolated peripheral blood mononuclear cells. Toxicol In Vitro. 2005;19(1): 55-61.

[13] Zhou NB, Fu ZJ, Sun T. Effects of different concentrations of oxygen-ozone on rats' astrocytes in vitro. Neurosci Lett. 2008;441(2):178-182.

[14] Parízek J, Husek Z, M?ricka P, et al. Ovine pericardium: a new material for duraplasty. J Neurosurg. 1996;84(3): 508-513.

[15] Chen YG, Zhao MM, Ning TY, et al. The application of artificial dura in emergency craniocerebral surgery. Hebei Yiyao. 2010;16(6):688-689.

[16] Roberts I, Sydenham E. Barbiturates for acute traumatic brain injury. Cochrane Database Syst Rev. 2012;12: CD000033.

[17] Timofeev I, Carpenter KL, Nortje J, et al. Cerebral extracellular chemistry and outcome following traumatic brain injury: a microdialysis study of 223 patients. Brain. 2011;134(Pt 2):484-494.

[18] Wakai A, McCabe A, Roberts I, et al. Mannitol for acute traumatic brain injury. Cochrane Database Syst Rev. 2013;8:CD001049.

[19] Tong WS, Zheng P, Xu JF, et al. Early CT signs of progressive hemorrhagic injury following acute traumatic brain injury. Neuroradiology. 2011;53(5):305-309.

[20] Walcott BP, Kahle KT, Simard JM. The DECRA trial and decompressive craniectomy in diffuse traumatic brain injury: is decompression really ineffective? World Neurosurg. 2013;79(1):80-81.

[21] Bor-Seng-Shu E, Figueiredo EG, Fonoff ET, et al. Decompressive craniectomy and head injury: brain morphometry, ICP, cerebral hemodynamics, cerebral microvascular reactivity, and neurochemistry. Neurosurg Rev. 2013;36(3):361-370.

[22] Timofeev I, Santarius T, Kolias AG, et al. Decompressive craniectomy - operative technique and perioperative care. Adv Tech Stand Neurosurg. 2012;38:115-136.

[23] Wen L, Li QC, Wang SC, et al. Contralateral haematoma secondary to decompressive craniectomy performed for severe head trauma: a descriptive study of 15 cases. Brain Inj. 2013;27(3):286-292.

[24] Sakas DE, Charnvises K, Borges LF, et al. Biologically inert synthetic dural substitutes. Appraisal of a medical-grade aliphatic polyurethane and a polysiloxane-carbonate block copolymer. J Neurosurg. 1990;73(6):936-941.

[25] Andreula CF, Simonetti L, De Santis F, et al. Minimally invasive oxygen-ozone therapy for lumbar disk herniation. AJNR Am J Neuroradiol. 2003;24(5):996-1000.

[26] Bocci V. Ozone as a bioregulator. Pharmacology and toxicology of ozonetherapy today. J Biol Regul Homeost Agents. 1996;10(2-3):31-53.

[27] Leone S, Noera G, Bertolini A. Melanocortins as innovative drugs for ischemic diseases and neurodegenerative disorders: established data and perspectives. Curr Med Chem. 2013;20(6):735-750.

[28] Dzieran J, Fabian J, Feng T, et al. Comparative analysis of TGF-β/Smad signaling dependent cytostasis in human hepatocellular carcinoma cell lines. PLoS One. 2013;8(8): e72252.

[29] Timmermann J, Hahn M, Krueger K. Short-term follow-up: micro-invasive therapy of the cervical herniated disk by percutaneous nucleotomy. J Back Musculoskelet Rehabil. 2011;24(2):89-93.

[30] Steppan J, Meaders T, Muto M, et al. A metaanalysis of the effectiveness and safety of ozone treatments for herniated lumbar discs. J Vasc Interv Radiol. 2010;21(4):534-548.

[31] Zhang Y, Ma Y, Jiang J, et al. Treatment of the lumbar disc herniation with intradiscal and intraforaminal injection of oxygen-ozone. J Back Musculoskelet Rehabil. 2013;26(3):317-322.

[32] Zhou JQ, Chen H, Chen ZY, et al. Effects of ozone oxidative preconditioning on apoptosis induced by renal ischemia/reperfusion injury in rats. Zhonghua Qiguan Yizhi Zazhi. 2012;33(2):113-117.

[33] Xin ZG, Liu B, Gao Y, et al. Small doses of triamcinolone aacetonide for heel pain. Zhongguo Xiandai Yisheng. 2011;49(1):130-131.

[34] Zeng Y, Xiong M, Yu HL, et al. Efficacy comparison of medical ozone with sodium hyaluronate in the treatment of knee osteoarthritis. Linchuang Waike Zazhi. 2010;18(8):565-567.

[35] Gautam S, Rastogi V, Jain A, et al. Comparative evaluation of oxygen-ozone therapy and combined use of oxygen-ozone therapy with percutaneous intradiscal radiofrequency thermocoagulation for the treatment of lumbar disc herniation. Pain Pract. 2011;11(2):160-166.

[36] Zamora ZB, Borrego A, López OY, et al. Effects of ozone oxidative preconditioning on TNF-alpha release and antioxidant-prooxidant intracellular balance in mice during endotoxic shock. Mediators Inflamm. 2005;2005(1):16-22.

[37] Lehnert T, Naguib NN, Wutzler S, et al. Analysis of disk volume before and after CT-guided intradiscal and periganglionic ozone-oxygen injection for the treatment of lumbar disk herniation. J Vasc Interv Radiol. 2012;23(11): 1430-1436.

[38] Torossian A, Ruehlmann S, Eberhart L, et al. Pre-treatment with ozonized oxygen (O3) aggravates inflammation in septic rats. Inflamm Res. 2004;53 Suppl 2:S122-125.

引言

Dura mater is an important anatomical structure of the central nervous system tissue, whose integrity is very important to protect the brain function, maintain nerve electrophysiological activity, structure and functional activity of the nervous system of patients undergoing surgery for brain injury^[1]. In recent years, the incidence of brain injury further increases in China, and about 30% of patients undergoing craniotomy require meningeal repair. Biological substitute materials are used for repair of the dura mater, so as to maintain the integrity of the anatomical structure, but there are many substitute materials for the dura mater with varying efficacy^[2-3]. With the development of medicine and tissue engineering technology, a variety of biological and artificial dura materials have emerged, including meningeal patches from bovine pericardium and tendon, absorbable polymer dura mater (such as collagen sponge dural patch). These materials that have been treated specially have more stable chemical properties, higher mechanical strength, better biocompatibility, milder rejection and allergic reaction, and they cannot interfere with CT and MRI re-examinations following repair^[4-8]. Collagen sponge artificial dura mater is a new type of bio-artificial dura mater material that can be completely absorbed. In China, it is first reported in the treatment of traumatic epilepsy patients, and the collagen sponge substitute can prevent cerebrospinal fluid leakage, have good hemostatic effect, and cannot trigger the onset of epilepsy in patients. There are also no postoperative infection and rejection, community, and the ozone therapy is rapidly being accepted and used in clinic because it is more secure than hyperbaric oxygen therapy that can be used in a wider population and
have indicating it has good histocompatibility^[9-10]. Ozone efficacy in the treatment of brain trauma and cerebrovascular diseases has been gradually recognized by the medical mobile features^[11-13].

In this study, the collagen sponge as a dural substitute was subjected to dural defects, and ozone-enriched autologous blood transfusion for brain injury achieved satisfactory results.

材料方法

Design
A retrospective case analysis.

Time and setting
The case analysis was completed at the Department of Neurosurgery, General Hospital of Shenyang Military Region from June 2012 to June 2014.

Subjects
A retrospective analysis was done in patients with brain injury undergoing dura mater repair.

Diagnostic criteria: (1) History of skull trauma; (2) confirmation by skull X-ray plain films, CT and other imaging examinations; (3) classification and diagnosis according to different levels of brain injury.

Inclusive criteria: (1) Brain injury due to a car accident, work injury, and falls. (2) Relaxation suture of the artificial dura mater due to dural defects or deficiency. (3) Brain injury patients who had Glasgow Coma Scale (GCS) score < 15 points and underwent craniotomy.

Exclusion criteria: (1) Patients without dural exposure or relaxation suture during craniotomy. (2) Patients with mild traumatic brain injury who dispensed with surgical treatment. (3) Bilateral mydriasis, unstable vital signs, intracranial lesions, spinal cord injury, preoperative cardiac arrest > 30 minutes, severe limb fractures or chest and abdominal injuries.

Sixty patients with brain injury undergoing the repair of artificial dura mater were enrolled, including 37 males and 23 females, with a mean age of 38.8 years (18-74). Injuries: 34 cases of traffic injury, 18 cases of fall injury and falls, 8 cases of combat injuries. At admission, Glasgow coma scale scores were 13-15 points in 11 cases, 9-12 points in 32 cases, < 8 points in 17 cases, including 2 cases of bilateral mydriasis and 15 cases of unilateral mydriasis. Preoperative CT scan showed that there were 37 cases of brain contusion with intracranial substance hematoma, 13 cases of epidural hematoma, 10 cases of subdural hematoma, 2 cases of diffuse brain swelling; 25 cases of open brain injury with depressed fracture of the skull, 12 cases of anterior skull base fracture with cerebrospinal fluid rhinorrhea. CT results showed 26 cases of midline shift. Temporal, frontal and occipital regions were major repair parts. The study was approved by the Ethics Committee of General Hospital of Shenyang Military Region, in line with the requirements of medical ethics, and all patients and their family members gave an informed consent.

Materials
Collagen sponge artificial dura mater was selected and used intraoperatively according to dural defect size and shape, which stuck to the dural defect and was fixed with biomedical glue without sutures. Collagen sponge artificial dura mater was purchased from Beijing TianXinFu Medical Appliance Co., Ltd. The artificial dura mater was mainly made of bovine tendon as a raw material in China, which was collected from state-run large-scale meat production and processing enterprises using aseptic technique to ensure the material source in line with national standards. After collection, Achilles tendon that was healthy, fresh and safe was processed into sponge-like collagen biofilm scaffolds for repair of dura mater, nerve sheath, skin, cartilage as well as prevention of various postoperative adhesions. The product consisted of collagen foils with biocompatibility, toughness, various sizes which were soft and could be completed absorbed. Biomedical glue was purchased from BIOSEAL BIOTECH, Guangzhou, China, which was developed from bovine blood source^[14-15].

Methods
Dural defect repair
Under general anesthesia, all patients underwent tracheal intubation. A large (reversed) question mark-shaped skin incision based at the ear (1 cm anterior to the antilobium) was made to expose the frontal, temporal and parietal region. Then, five or six holes were drilled on the skull, and a bone flap including the frontal, temporal squama, and parietal bone was removed. When the bone window was above the middle cranial fossa, the parts from the temporal squama to the base of the middle cranial fossa was cut using a rongeur to remove epidural hematoma, subdural hematoma, intracerebral hematoma, brain contusion lesions. The dura could be cut in radial, arc-shaped, cruciform manners, which was determined according to surgical sites and requirements. The dura edge was cut as smooth as possible to clear inactive brain tissues and intracranial hematoma. The dura should be fulgurized as little as possible to avoid dural retraction. The smooth surface of the artificial dura mater faced to tissue defects. After repair, the dura mater could even be lifted via four points at a height of 2.5-3.0 cm. Generally, the brain tissue was allowed to bulge outwards in a length of at least 2.5 cm, so there was a certain region allowing the floating of the dura, which could help to buffer the intracranial pressure. If there was no high intracranial pressure and no encephalocele, the bone flap was fixed; otherwise, the bone flap was removed to reduce the intracranial pressure. Subdural catheter was connected to a drainage bag, and the epidural catheter was attached to negative pressure. Temporalis muscle and subgaleal were tightly sutured.

Postoperative process
All patients undergoing dural repair were enrolled at the neurosurgical intensive care unit for postoperative care and treatment. As appropriate, mechanical ventilation and tracheotomy were performed to maintain the airway patency as well as dehydration and reduing intracranial pressure. Routine treatment against infection and gastrointestinal bleeding was done, and in some patients, hypothermia and sedation therapy was given to relieve brain swelling and brain edema. Active treatments for nerve nutrition and improvement of microcirculation of the central nervous system were preferred. For coma patients, enteral and parenteral nutrition support and early rehabilitation were necessary to promote the recovery of neurological function.

Ozone-enriched autologous blood transfusion
In the supine, blood samples were extracted from the cubital vein. First, the blood transfusion apparatus was inserted into saline and exhausted. Then, the short tube of the apparatus was inserted into the blood transfusion bag, and full of anticoagulant agents from the blood transfusion bag via the open access. Blood samples from the vein, 100 mL, were collected via puncture followed by shutting the blood transfusion path, and the saline pathway was open for intravenous infusion. Ozone sample, 47 mg/g, was extracted and injected into the blood bag following gently shaking until the blood was fully ozonized. Then, the saline pathway was closed, and the transfusion path was open. The ozonized blood was transferred back to the patients. After blood transfusion, the saline pathway was open until the blood was completely into the body.

Complications and follow-up
Patients were subject to periodic head CT examination after surgery, and underwent routine cranioplasty at postoperative 3-6 months. After repair, complications associated with the artificial meninges were observed and recorded, and all patients were followed up for 1 year.

Intracranial pressure monitoring: At 1, 7, 14 postoperative days, only lumbar puncture was employed in intracranial pressure measurement prior to the usage of mannitol. Closed conduit was connected to the brain pressure gauge with no release of cerebrospinal fluid, and mannitol was transfused immediately after pressure measurement to prevent herniation. By intracranial pressure measurement, we could assess the effect of surgical decompression and analyze the changes in intracranial pressure.

Neuroimaging evaluation: At 1, 7, 14 days after surgery, head CT examination was done prior to the usage of mannitol. Based on OM baseline, CT scan was done at a thickness and interval of 7 mm. CT results were used to assess the midline shift level and distance of brain tissue expanded from the bone window on the same level. CT results combined with the value of intracranial pressure monitoring could be used for assessing the therapeutic efficacy of surgical decompression.
Complication evaluation: The observation indexes included (1) inflammation, (2) adhesion, and (3) subflap effusion.

Main outcome measures
Head CT examination results and complications.

Statistical analysis
Data were expressed as mean±SD and analyzed statistically using the SPSS 19.0 (IBM, USA). Paired t test was done for evaluation of intracranial pressure and midline shift level. A value of P < 0.05 was considered statistically significant.

讨论

Traumatic brain injury is often combined with extensive brain contusion and inadequate blood supply and cerebral hypoxia, thereby leading to brain swelling[16-18], and seriously affecting the prognosis of patients. Direct exposure of the dura can result in the brain tissue in direct contact with the scalp, which is prone to cause cerebrospinal fluid leakage, subcutaneous fluid, pseudo-swelling of the meninges, wound dehiscence, and increased incidence of subcutaneous fluid^[19-23]. The key to the surgery for traumatic brain injury is to recover the normal anatomical structure of the brain and mitigate the stimulation to the cerebral cortex from external factors as far as possible. Therefore, choosing a good artificial meningeal material is crucial for the successful treatment of traumatic brain injury. Ideal dura repair materials should be: (1) stable biologically inert that do not cause acute and chronic inflammation; (2) safe, non-toxic, non-carcinogenic, teratogenic (the biofilm type is required not to spread known or unknown potential infections); (3) with good histocompatibility and no rejection; (4) with good compactness, no permeability, to prevent cerebrospinal fluid leakage and protect brain tissue; (5) resilient and able to be sutured; (6) able to promote regeneration of the meninges and not to cause meningeal adhesion; (7) simple and easy to use and sterilize; (8) with a wide range of sources and inexpensive^[24]. Currently, there are four kinds of dura repair materials, including autologous materials, allograft materials, synthetic materials and xenogeneic biomaterials.

The ozone gas cannot be injected intravenously, because the gas mixture contains over 95% volume fraction of oxygen that can result in oxygen embolism^[25]. Currently, the autotransfusion therapy is the most mature and reliable method. Depending on the patient's body weight, the blood volume used is not equal. Then, the same volume of gas mixture is added, and the ozone concentration is precisely controlled that oxygen molecules cannot be easy to penetrate, thus hypoxia is aggravated to cause a vicious circle of “edema-hypoxia- edema^”[26]. Ozone can (1) promote the delivery of oxygen, glucose, ATP to the ischemic tissues so as to ease brain and whole body tissue hypoxia, reduce brain cell degeneration and necrosis, thereby to relieve cerebral edema and rapidly decrease the intracranial pressure. (2) Ozone can enhance angiogenesis, promote hematopoietic stem cell implantation, thereby increasing the formation of new blood vessels and tissue regeneration to clear hematom and necrotic tissues, accelerate the regeneration of collagen fibers and blood capillary, and speed up the repair of lesions^[27-31]. (3) Ozone can upregulate antioxidant enzymes and heme oxygenase I so as to strengthen free radical scavenging and antioxidant capacity, and reduce reperfusion injury[32-34]. (4) Ozone can trigger neurons neurotransmitter reaction to accelerate the recovery of consciousness, in order to maintain the normal activities of life functions and thereby improve the quality of life of patients[35]. Therefore, ozone has a good role in promoting the treatment and recovery of traumatic brain injury. Research has shown that ozone can improve the symptoms of brain injury, relieve brain injury-caused cerebral edema, decrease intracranial pressure, improve oxygen supply to the brain tissue, facilitate awakening of coma patients so as to restore the nervous system and reduce complications^[36-38]. Taken together, ozone treatment is safe and effective for brain injury, which is better than traditional medicines, do not increase the suffering of patients, and has fewer adverse reactions. Patients are also willing to accept ozone treatment rather than drug treatment.

In short, the collagen sponge artificial dura mater with ozone therapy is a simple, safe and effective method for treatment of traumatic brain injury, which cannot only keep the intact anatomical structure, exert sufficient decompression role, maintain brain function, but also have the same therapeutic efficacy to the dural suture. This combined therapy also creates favorable conditions for the latter skull repair, reduce complications and protect brain function, which should be popularized in neurosurgery.