用于评估骨替代材料中骨生长与新生血管形成的兔颅骨骨增量模型

摘要

本文介绍了一种在兔子中进行的外科手术方案，旨在评估骨替代材料在骨再生能力方面的性能。通过将聚醚醚酮（PEEK）圆柱体固定在兔颅骨上，可在活体或安乐死动物中评估材料所诱导的成骨传导、成骨诱导、成骨生成以及血管生成作用。

摘要

兔颅骨模型的基本原理是在颅骨皮质部分的上方垂直生长新骨组织。该模型可用于评估骨替代材料在口腔和颅面骨再生方面对骨生长和新生血管形成的支持作用。动物麻醉并进行通气（经气管插管）后，将四个由聚醚醚酮（PEEK）制成的圆柱体分别固定于颅骨上，位于矢状缝和冠状缝两侧。在每个圆柱体所限定的骨区域内钻五个髓内孔，以允许骨髓细胞流入。将待测材料样本置入圆柱体内，随后封闭圆柱体。最后缝合手术部位，使动物苏醒。可通过显微CT对活体动物进行骨生长的评估。动物安乐死后，可利用显微CT、免疫组织化学和免疫荧光技术进一步评估骨生长及新生血管形成情况。由于材料评估需要最大程度的标准化与校准，颅骨模型显得尤为理想。该模型操作简便，使用定义明确的圆柱体有助于实现校准与标准化，并可同时评估四个样本。此外，可对活体动物进行连续断层扫描，从而有望显著减少需安乐死的动物数量。

引言

颅骨骨增量模型于20世纪90年代建立，旨在优化口腔及颅面外科领域内的引导性骨再生（GBR）概念。该模型的基本原理是在颅骨的皮质骨部分上方垂直生长新的骨组织。为此，将一个反应器（例如钛制穹顶、圆柱或笼状结构）固定于颅骨上，以保护由移植物（如水凝胶、骨替代材料等）介导的骨再生过程。借助该模型，研究人员已对钛或陶瓷笼^1,2,3,4,5,6、GBR膜^7,8,9,10、成骨因子^{11,12,13,14,15,16,17}、新型骨替代材料^{12,16,17,18,19,20,21,22,23,24,25,26,27,28,29}，以及骨再生过程中新生血管化的机制³⁰进行了评估。

从转化医学的角度来看，颅骨模型代表了一种单壁缺损，可与颌骨的IV类缺损相比较³¹。该模型的目标是在皮质骨区域上方生成新骨，且无内源性骨壁提供的侧向支撑。因此，该模型极为严格，能够评估在骨皮质部分实现垂直骨传导的真实潜力。尽管本文所述模型主要用于评估骨替代材料的骨传导性，但也可用于评估成骨作用和/或骨诱导作用，以及血管生成能力^{1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30}。

出于伦理、实际和经济方面的考虑，兔颅骨模型被开发出来，因为兔的骨代谢和骨结构与人类具有较高的相关性³²。上述引用的30篇文献中，80%采用了兔颅骨模型^{1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,17,22,23,26,27,28,29,30,33}，从而证明了该动物模型的重要性。2008年，Busenlechner 研究组将颅骨模型转移至猪，以实现同时比较八种骨替代材料²⁰（相比之下，兔模型仅能比较两种骨替代材料）。另一方面，我们的研究组则将兔颅骨模型应用于绵羊。简而言之，将钛制穹顶置于绵羊颅骨上，用于表征一种新型3D打印骨替代材料的骨引导性能。这些研究使我们得以开发并掌握颅骨模型及其分析方法^16,21。

最近引用的三项研究^16,20,21，以及另外多项研究^{12,17,18,19,22,23,24,26,27,28,29}，共同证实了颅骨模型作为筛选和表征模型的巨大潜力。然而，尽管所获得的结果相当令人满意，这些研究也指出了某些局限性：（1）使用了钛制穹顶，阻碍了X射线的穿透，从而无法进行活体微计算机断层扫描（micro-CT）检测。由于组织学处理前无法移除钛制穹顶，研究人员被迫将样本包埋于聚甲基丙烯酸甲酯树脂（PMMA）中，导致后续分析在很大程度上仅限于表面形貌观察。（2）高昂的经济成本，尤其是动物本身的费用，以及与动物运输、饲养和手术相关的成本。（3）在大型动物实验中获得伦理审批存在困难。

Polo 等人最近的一项研究²⁶在兔模型上进行了大幅改进。用可闭合的圆柱体替代了钛制穹顶，该圆柱体可填充固定体积的材料。将四个此类圆柱体置于兔颅骨上。实验结束后，可移除圆柱体，使活检样本不含金属，从而在样本处理方面提供了更大的灵活性。兔颅骨模型因此成为一种具有吸引力的模型，可用于同步测试，且成本较低、动物操作简便、样本处理便捷。利用这些最新进展，我们进一步改进了该模型，使用聚醚醚酮（PEEK）替代钛来制造圆柱体，从而允许X射线穿透，并可在活体动物上应用显微断层扫描技术。

本文将介绍麻醉和手术操作流程，并展示使用该方案可能获得的结果示例，即通过（免疫）组织学、组织形态计量学、活体及离体显微断层成像技术来评估骨再生机制，并定量分析骨替代材料所支持的新骨形成。

方案

In line with Swiss legal requirements, the protocol was approved by an academic committee and supervised by the cantonal and federal veterinary agencies (authorizations n° GE/165/16 and GE/100/18).

1. Specific devices and animals

1. Cylinders
   1. Machine cylinders with lateral stabilizing tabs out of PEEK to have inner diameter of 5 mm, outer diameter of 8 mm and a height of 5 mm (Figure 1).
   2. Machine PEEK caps with a design allowing to clip precisely onto the top of the cylinder (thickness 1 mm).
   3. Sterilize PEEK cylinders and caps by autoclaving before surgery.
2. Screws
   1. Use self-drilling micro screws (made of commercial pure titanium (grade 5)) to fix the cylinders (1.2 mm in diameter, 4 mm in length). Sterilize by autoclaving before surgery.
3. Animals
   1. Purchase three-month-old New Zealand white rabbits (male or female), weighing ~2.5 kg each.
      NOTE: We obtained rabbits by breeding at the University of Geneva.

2. Surgery

1. Surgical tray
   1. Keep scalpels, scissors, two forceps, periosteal elevator, syringes (1, 2, 5, 50 mL), surgical motor, round surgical burs (0.8 mm diameter), needles, sterile saline, four cylinders, eight screws, and screwdriver ready.
2. Preclinical treatment
   1. Acclimate the animals one week prior to surgery.
   2. Provide a prophylactic antibiotic daily (5–10 mg/kg by mouth (PO)) starting 2 h before surgery up to 3 days after surgery.
3. Anesthesia and intubation
   1. Sedate the animals by intramuscular (IM) injection of ketamine (25 mg/kg, 50 mg/mL, 0.5 mL/kg) + xylazin (3 mg/kg, 20 mg/mL, 0.15 mL/kg). Wait ~20 min for the animals to sleep
      deeply (complete muscular atony).
      NOTE: This premedication will allow a simple, fast and painless intubation process. Deep analgesia and anesthesia is induced as described in step 2.3.8.
   2. Place an intravenous (IV) cannula into the marginal vein from the ear and keep it closed until intubation is completed.
      NOTE: This IV line will serve to perfuse fentanyl and propofol for deep analgesia and anesthesia, respectively (see step 2.3.8).
   3. Maintain anesthesia by supplying 5% sevoflurane in pure oxygen until intubation is performed.
      NOTE: This step is necessary only if the animal shows signs of awakening (eye movements, muscular contractions).
   4. Anesthetize the trachea locally by spraying 10% lidocaine. Place the rabbit in prone position and maintain its head in vertical extension.
   5. Slide the first endotracheal tube of small diameter (2.5 mm) into the rabbit trachea until airflow can be heard in the tube. This will open the larynx and facilitate the insertion of the definitive tube.
   6. Insert a guide (intubation catheter) in the tube to fix the position of the tube into the trachea. Remove the small diameter tube and slide the definitive endotracheal tube (4.9 mm) on the guide.
   7. Remove the guide and inflate the balloon at the end of the endotracheal tube to seal and block the device into the trachea. The tube will stay in place but it may be secured by using a lace tied around the forehead.  Immediately ventilate (7 mL/kg, frequency of 40/min) the animal with 3% sevoflurane in pure oxygen.
   8. Continuously perfuse (ear vein) fentanyl (0.01 mg/mL, 2–4 mL/h) to induce analgesia, 2–4 mg/kg of (2%) propofol (20 mg/mL, 4–8 mL/h) to induce anesthesia, and 4 mL/kg/h of Ringer's acetate to maintain iso-volumetric conditions.
   9. Place a rectal temperature probe. Also monitor heart function, temperature and oxygen saturation during the entire process.
   10. Control the depth of anesthesia by monitoring autonomous breathing; if the animal shows signs of autonomous breathing, dispense a small bolus of propofol and fentanyl.
4. Site preparation
   1. Place the rabbit on a heated pad (39 °C) covered by a mattress pad (to avoid burns) on the surgery table. Shave the scalp.
   2. Apply a lubricating gel on the eyes to avoid irritation and dryness. Disinfect the site by scrubbing the skin with povidone iodine (10%). Then drape the rabbit with a sterile surgical drape and cut out an access area for the skull.
   3. Disinfect the surgical site with povidone iodine (10%) for a second time. Apply a lubricating gel on the eyes to avoid irritation and dryness.
   4. Prepare a draped table (sterile drape) on which to place the complete surgical tray.
5. Surgical site opening
   1. Anesthetize locally with a subcutaneous (SC) injection of lidocaine 2% (1 mL) on the skull.
   2. Incise through the skin (with a scalpel) along the calvarial sagittal line, from the orbits to the external occipital protuberance (~4 cm in length). Ensure that the periosteum is incised.
   3. Gently elevate the periosteum (with a periosteal elevator) on both side of the incision. Rinse the site with sterile saline.
6. Cylinder placement
   1. Locate the median and coronal sutures on the skull (Figure 2A,B). Note that these anatomical lines form a cross. The cylinders will be placed in each of the quadrants defined by the cross, ensuring that the edge of the cylinder is not over the suture (Figure 2C).
   2. Place the first cylinder on the left upper quadrant (left frontal bone), and try to lay the device flat. Fix in the position with strong hand pressure and screw a micro-screw, until resistance is felt. Ensure that the screw head is flush with the surface of the cylinder tab.
   3. Repeat the same procedure on the other tab to fix the cylinder tightly onto the skull. Ensure that the cylinder is hermetically fixed to the bone.
   4. Repeat the procedure on the right upper quarter (right frontal bone), left lower quarter (left parietal bone) and right lower quarter (right parietal bone).
7. Bone drilling of 5 intramedullary holes within the area circumscribed by the cylinders (Figure 1)
   1. Drill an intramedullary hole under saline irrigation (0.8 mm in diameter, ~1 mm in depth) with a round bur on the bone, in the center of the area circumscribed by the cylinder. Ensure that bleeding appears.
   2. Drill two more intramedullary holes along the axis passing through the two tab screws, at the inner edges of the cylinder. Along the perpendicular axe, drill two more intramedullary holes at the inner edges of the cylinder. Ensure that bleeding appears.
   3. Repeat the operation within the three other cylinders.
8. Filling cylinders with material samples and capping (Figure 3)
   1. Prepare the desired bone substitute material according to manufacturer instructions or material specifications.
   2. Fill the first cylinder to the brim with the material sample and close the cylinder by fitting the cap. Repeat the process in the 3 other cylinders.
9. Surgical site closure
   1. Close the skin above the cylinders with an intermittent non-resorbable suture.
   2. Apply a sprayable dressing onto the wound.

3. Post-surgical treatment

1. Stop analgesia and anesthesia (propofol and fentanyl perfusion arrest) supply and check the recovery of autonomous breathing.
2. Stop the ventilation once the animal has recovered autonomous breathing. Maintain the animal under pure oxygen before complete awakening.
3. Inject buprenorphine hydrochloride SC (0.02 mg/kg, 0.03 mg/mL, 0.67 mL/kg) and repeat the injection every 6 h for 3 days as post-surgical analgesia.
4. Transfer the animal into its usual housing with water and complete feeding.
5. Remove the sutures after about 10 days of wound healing.

结果

本文所述模型专用于评估骨替代材料的骨传导性。此外，也可评估经（预）细胞化或负载生物活性分子的骨替代材料的成骨作用和/或骨诱导作用，以及血管生成^{1,2,3,4,5,6,7,8,9,10,11,}

讨论

本文所述模型操作简便，只要遵循所有步骤并使用合适的设备，即可轻松建立。由于本方案属于外科手术方法，所有步骤均至关重要，必须严格遵守。操作人员必须接受过动物实验培训，尤其是兔类动物的饲养管理和麻醉技术。如有需要，应毫不犹豫地寻求专业麻醉师和兽医的帮助。必须强调在缝线拆除前后对动物进行每日视觉观察。尽管颅骨部位皮肤较厚、丰富且松弛，但圆柱体固定后会产生较大张力。若过早拆除缝线，伤口可能重新裂开，需要再缝合一周并重新进行抗生素治疗。若伤口裂开部位超出切口线范围，外科医生需评估是否仍可进行缝合。如无法缝合，则需考虑样本废弃。

除了方案部分所述的关键步骤外，以下细节可能有助于正确实施本方案。从技术角度来看，由于所使用的兔子年龄较小且体型较小，因此必须采用方案中描述的两步插管法。 第二根（也是最终的）导管过大，不能用于首次插管，否则存在明显的“错误路径”风险，可能导致严重损害甚至死亡。
根据所测试的材料，可考虑从已放置的耳缘静脉留置管中采集少量新鲜血液。这种方法可用于在植入前用天然细胞和生长因子预处理骨替代材料。新鲜凝固的血液也可作为理想的假手术对照样本。

材料

本文使用的材料清单

姓名	公司	目录编号	评论
药物
恩诺沙星 Baytril 10%	Bayer		抗生素
芬太尼	Bischel		用于镇痛
Ketalar 50mg/ml	Pfizer		用于麻醉的氯胺酮
Lidohex	Bichsel		眼部润滑凝胶
Opsite	Smith and Nephew	66004978	可喷洒敷料
聚维酮碘 10%，Betadine	Mundipharma		抗感染剂
丙泊酚 2%	Braun	3538710	用于麻醉
Rapidocain 2%	sintetica		局部麻醉
林格氏醋酸盐	Fresenius Kabi		容量补充
Rompun 2%	Bayer		用于麻醉的赛拉嗪
七氟烷 5%	Abbvie		用于麻醉
无菌生理盐水	Sintetica
Temgesic	Reckitt Benckiser		盐酸丁丙诺啡，用于镇痛
硫喷妥钠 Inresa	Ospediala		用于麻醉
Xylocaine 10% 喷雾	Astra Zeneca		用于插管
名称	公司	目录编号	备注
设备
Fresenius Vial pilot C	Imexmed		输液泵
加热垫	Harvard Apparatus
Suction dominant 50	Medela
Suction tubing Optimus	Promedical	80342.2
外科电机	Schick dental	Qube	用于钻制髓内孔
通气设备	Maquet Servo1
名称	公司	目录编号	备注
材料
圆柱体及盖帽	Boutyplast	定制	成分：PEEK（聚醚醚酮）
手动自持式套管	GlobalD	ACT1K
自持式套管的移动手柄	GlobalD	MTM
自攻螺钉	GlobalD	VA1.2KL4	十字驱动螺钉，由钛合金5级（ISO 5832-3）制成
名称	公司	目录编号	备注
手术托盘
Shiley 气管插管，直径 2.5 mm	Covidien	86233	用于插管
Shiley 气管插管，直径 4.9 mm	Covidien	107-35G	用于插管
Ethicon prolene 4-0	Ethicon	8581H	不可吸收缝线
镊子	Marcel Blanc	BD027R	145 mm
插管导管	Cook medical		插管引导
持针器	Marcel Blanc	BM008R
BD Microlance3 针头	Becton Dickinson	300300/304622	26G；18G
骨膜剥离器	HU-Friedy	P9X
圆形外科钻头	Patterson	78000	直径 0.8 mm，用于钻制髓内孔
手术刀	Swann-Morton		编号°10 和编号°15
剪刀	Marcel Blanc	00657	180 mm
Omnifix 注射器	Braun	4616057V	5 ml、10 ml 和 50 ml
Venflon G22	Braun	42690985-01	耳部静脉通路用 Vasofix 安全型留置针