【作者】 陆海波；

【导师】 卢世璧； 郭全义； 彭江； 汪爱媛；

【作者基本信息】 中国人民解放军军医进修学院 ， 骨外科学， 2012， 博士

【副题名】新型壳聚糖急救止血剂、锁定暂时性血管分流装置、自体血凝块预混的冻干异体骨、促进冻干异体骨改建的凝血酶原

【摘要】 一、目的：本研究立足于我军战时严重四肢伤分级救治的实际需要，着力研究一系列有望应用于四肢伤救治的产品和技术，具体包括新型壳聚糖急救止血剂、锁定暂时性血管分流装置、自体血凝块预混的冻干骨和凝血酶原。通过本研究，试图为上述产品的后续研发提供理论基础和实验依据。具体而言，本研究分为以下三个方面：着眼于四肢伤的战（现）场急救阶段，我们针对现有壳聚糖急救止血剂（chitosan-based first aid hemostat, CFAH）水下粘附力差，止血效果欠佳的不足，开展了壳聚糖急救止血剂化学修饰的初步研究，并对止血剂的止血及粘附机理进行初步探索，旨在为进一步提升壳聚糖急救止血剂水浸条件下的止血效果而提供理论基础和实验依据。着眼于四肢伤的紧急救治阶段，我们针对美军目前正在使用的暂时性血管分流（temporary vascular shunt, TVS）的分流管固定费时、不牢靠，脱出率高的不足，设计了独具螺纹和配套锁定螺母的分流装置——锁定暂时性血管分流（locked temporary vascular shunt, LTVS）装置，旨在进一步提高TVS技术的救治效率和安全性。着眼于四肢伤的早期治疗阶段，我们针对大段骨缺损修复时骨不连、骨延迟愈合等难题，研究了新型异体骨填充材料——自体血凝块预混的冻干辐照异体骨（autologous coagula impregnated freeze-dried irradiated allograft bone,ACIFIAB）。同时，针对现有组织工程缓释技术存在的局部因子的高浓度与因子最佳效应浓度不一致的矛盾，提出了“原料修复策略”（material repair strategy,MRS），并研究了有望促进冻干异体骨改建的新的促成骨因子——凝血酶原（prothrombin, PT），旨在进一步改善骨移植材料的血液供应和促进成骨。二、方法：在CFAH止血机制及改性的初步探索性研究方面，我们首先尝试通过Schiff碱合成和多肽合成的方法对壳聚糖急救止血剂进行化学改性，分别合成了经3,4-二羟基苯甲醛修饰的壳聚糖（DHBH modified chitosan, DMCTS）和经左旋多巴修饰的壳聚糖（DOPAmodified chitosan, DOPAMCTS），通过傅立叶红外光谱分析、凝血试验以及扫描电镜等手段对上述两种改性的壳聚糖和几种包括甲壳素、壳聚糖、Celox止血剂、乌鱼骨等在内的止血剂进行理化表征，并通过止血及粘附效果的对比分析，进一步从物理及化学两个层面对止血剂的止血及粘附机制进行深入探讨。在LTVS装置的研究方面，我们设计了独具螺纹和配套锁定螺母的分流装置。然后通过体外血管爆破压试验评价其吻合强度；通过犬股动脉、髂总动脉损伤模型评价其吻合效率；通过组织学方法评价吻合部位血管壁组织的卡压损伤情况。在ACIFAB的研究方面，我们首先通过体外限制性压缩实验来比较和评价不同异体骨材料的力学性能，随后通过异位植骨实验及其后的HE染色、血小板内皮细胞粘附分子1（platelet endothelial cell adhesion molecule-1,PECAM-1）免疫组化染色、Masson染色以及针对VEGFα的qRT-PCR等方法来评价自体血凝块对异体骨血管化和改建的影响，并用Image-Pro Plus图像分析软件对经免疫组化染色显示的双侧植入物内血管面积及数目进行定量分析。在PT的研究方面，我们首先通过ELISA、CCK8、RT-PCR及qRT-PCR等方法体外评价凝血酶原对成骨细胞增殖及分泌BMP-2的影响。继而，通过兔双侧桡骨缺损模型和术后X线评分及缺损面积定量分析的方法评价自体凝血酶原对大段骨缺损的修复效果。随后，我们通过免疫组化的方法观察新鲜骨组织内经骨库制备技术处理后凝血酶原的变化。最后，我们制备了以壳聚糖-PLGA微球为缓释载体的复合人凝血酶原冻干辐照异体骨（humanprothrombin impregnated freeze-dried irradiated allograft bone, HPIFIAB），并通过扫描电镜观察了其表面形貌，并通过ELISA测试了其于体外对PT的缓释性能。三、结果：1．CFAH的止血机制及改性初探傅立叶红外光谱图提示，Celox止血剂与国产水溶性壳聚糖均含有代表N-H键的伸缩振动峰；DMCTS样品的光谱图上出现了我们预期的C=N双键及苯环特征峰；在DOPAMCTS分子中，DOPA的邻苯二酚和氨基均链接到了壳聚糖的侧链上。凝血试验的结果提示，甲壳素不具备促凝作用；经修饰为DMCTS后，壳聚糖的促凝作用消失；而壳聚糖、Celox止血剂以及DOPAMCTS均具有明显的促凝作用，各组的凝血时间具有显著性差异（p=0.000）。另，未经研磨的乌鱼骨块，具备优异的促凝血性能，而经过反复研磨后的乌鱼骨粉其促凝作用消失，两组的凝血时间具有显著性差异（p=0.000）。扫描电镜结果提示，完整的乌鱼骨块表面高度整齐划一，而经研磨的乌鱼骨粉末其原有的表面形貌被破坏，与壳聚糖颗粒一样，表面则极不规则。经近乎相等的水流冲刷作用下，国产壳聚糖与乌鱼骨颗粒在横纹肌组织表面的存留情况明显不同。其中，粘附于横纹肌表面的壳聚糖颗粒在水流冲刷的作用下全部消失；乌鱼骨颗粒在水流冲刷的作用下仍然与组织牢固粘附；乌鱼骨与壳聚糖的等比例混合颗粒仅有极少数存留；先撒乌鱼骨，后撒壳聚糖的组织表面仍有较多颗粒存留。2．LTVS装置的研究体外爆破压测定的结果提示，经锁定螺母固定的血管残端其爆破压的实测值比经普通缝线结扎固定者高114.29%（p=0.000）。LTVS的吻合时间可比TVS的吻合时间缩短60.37%（p=0.000）。虽然经过丝线或塑料螺纹持续1周的卡压，吻合口处的血管壁组织（特别是血管肌层）仍然能够维持其结构的连续性。3．ACIFAB的研究异位植骨实验的大体标本提示，术后8周时，在同一只大鼠体内，实验侧植入物表面已经有大量的新生血管长入，而对照侧植入物表面未见明显的血管长入。HE染色提示，到了术后第4周和第8周，实验侧的新生血管密度明显高于对照侧。免疫组化染色提示，在术后1周，双侧均未见明显的管腔样组织被染成PECAM-1阳性，但在血凝块植入侧，可见大量的团块状物质被PECAM-1阳性识别；在术后4周和8周，在同一只宿主体内，实验侧的PECAM-1阳性染色区域明显较对照侧多，且大部分集中于管腔状结构周围。定量分析结果提示，在术后1周、4周及8周，实验侧植入物内的血管面积较对照侧相比均增大，且差异具有统计学意义（p=0.035）。Masson染色提示，在术后第1周，实验侧与对照侧的异体骨颗粒均呈散在分布，无明显基质生成，而且，实验侧视野内可见大小不一的血凝块颗粒；在术后第4周，实验侧的异体骨颗粒已被新生的基质包围并连成片状，其间分布着大量的蚯蚓状血管组织，而对照侧异体骨周围的基质连接并不完全，基本上仍呈各自分散的状态，其间并未见明显的血管状组织；在术后第8周，实验侧的基质进一步成熟，密度增加，大量的异体骨颗粒呈现溶解吸收，其间也可看到大量的蚯蚓状血管组织，而对照侧异体骨周围的基质融合也进一步增强，但程度不及实验侧，且未见大量的血管组织及明显的异体骨溶解。qRT-PCR的结果提示，在术后1周，实验侧植入物标本中VEGFα的表达明显高于对照组（p <0.05）。而到了术后第4周和第8周，双侧植入物标本中VEGFα的表达无显著性差异。体外限制性压缩实验的结果提示，新鲜冷冻骨的刚度（stiffness, S）和弹性模量（elastic modulus, EM）略高于复水后的冻干骨（p <0.05）。且经过新鲜血凝块或壳聚糖溶液预混后，冻干骨的S及EM进一步降低（p <0.05）。经过渐进式打压，各组异体骨块的压缩率（ratio of compression, RC）明显升高(p=0.000)，而各组骨块的压缩变化率（delta ratio of compression,ΔRC）明显降低(p=0.000)。相关分析的结果提示，ΔRC与打压次数(Impacts, I)呈负相关（Pearson相关系数为-0.711，p=0.000），与S呈负相关（Pearson相关系数为-0.229，p=0.000），与骨块高度（Height, H）呈正相关（Pearson相关系数为0.482，p=0.000），与EM无明显相关性（Pearson相关系数为-0.053，p=0.231）。以ΔRC为因变量的回归方程为,Δ R_i=0.0590.0004327585881334×I_i+0.0004563645521868×H_i该方程的R~2值为0.516，p=0.000。其中I_i代表打压次数；Hi代表在第i次打压前沿打压方向测量的骨块高度值。4．PT的研究4．1．PT促成骨细胞增殖CCK8结果提示，当体外无血清培养条件下分别以0μg/ml、0.1μg/ml、1μg/ml、10μg/ml、25μg/ml的浓度刺激大鼠成骨细胞时， PT对细胞的增殖表现出了不同程度的促进作用，且相比之下，1μg/ml的促进作用最明显；而相同浓度梯度的凝血酶（thrombin, TH）则对细胞的增殖表现出了不尽相同的作用：当TH的刺激浓度为0.1μg/ml时，无明显的促进或抑制作用，当TH的刺激浓度为1μg/ml时，表现出了一定程度的促进作用，但当TH的浓度高于10μg/ml时，则对成骨细胞的增殖呈现出抑制作用，且浓度越高，抑制作用越明显。PT+TH的促增殖作用在1μg/ml时达到了顶峰，而当刺激浓度继续升高时，则促进作用逐渐减弱，但并不呈现出明显的抑制作用。CCK8结果还提示，在含10%胎牛血清培养条件下，PT对大鼠成骨细胞的增殖能够起到一定的促进作用。特别是较低浓度的PT (0.014μg/ml)，能够达到最佳的促进效果。4．2．PT促成骨细胞表达及分泌BMP-2ELISA的结果提示，当以0.5μg/ml为起始浓度刺激大鼠成骨细胞时，TH促成骨细胞分泌BMP-2的高峰出现在刺激后的12h，随后开始下降；而PT的促分泌效果在24h的观察周期内始终呈上升趋势；TH+PT的促分泌趋势与TH相当，最高峰也出现在12h，且峰值较TH更高；空白对照组BMP-2的高峰出现得最早，且24h的浓度最低。ELISA的结果还提示，当TH分别以0、0.1、1、10、25μg/ml的浓度梯度刺激成骨细胞时，BMP-2的分泌高峰出现在10μg/ml；而PT刺激组的分泌高峰出现在1μg/ml；TH+PT刺激组的分泌高峰则出现在10μg/ml。经RT-PCR证实，1ug/ml的PT会促进成骨细胞对BMP-2的表达增强。qRT-PCR的结果也证实，经0.1mg/ml的PT刺激后，成骨细胞对BMP-2的表达明显增强(p=0.000)。4．3．自体PT促进骨修复兔双侧桡骨缺损修复术后的X线评分结果提示，术后1w时，自体PT修复侧（实验侧）与对照侧的评分值均为0。随着观察时间的延长，双侧的骨缺损都有渐进性的修复，表现在X线评分则为分值逐渐增加。而且，自术后4w始，实验侧的分值明显高于对照侧分值（p=0.021）。图像分析的结果提示，在术后1w、4w、8w及16w，实验侧缺损面积均小于对照侧，以术后8w最明显，且差异具有显著性意义（p=0.045）。4．4．骨库制备技术对骨组织中PT的破坏经大鼠凝血酶原免疫组化染色证实，新鲜皮质骨中存在有大量的PT，且大部分与骨细胞一起位于骨陷窝内。而对比发现，经脱脂、脱蛋白、冻干及辐照等处理后，皮质骨内的骨细胞基本上被彻底清除干净，遗留下许多空泡状的陷窝；而与此同时，原本存在于新鲜异体骨中的PT也被清除干净。4．5．HPIFIAB扫描电镜的结果提示，在冻干异体骨的髓腔面，可清晰看到髓腔壁的骨小梁结构，而在皮质面，则能清晰看到皮质骨表面的滋养孔。在经壳聚糖包被冻干异体骨的髓腔面及皮质面，上述骨组织表面的正常结构被均匀的壳聚糖膜覆盖。经冻干并烘烤后的人PT-PLGA微球，其外形呈表面多孔的圆饼状，直径约为100μm，厚度约为40μm。HPIFIAB髓腔面及皮质面，分布着大小不一的微球，微球直径约为2～10μm。与单纯凝血酶原微球的多孔状表面不同，负载于冻干异体骨的微球其表面尚包裹一层光滑的壳聚糖膜。通过ELISA测定出微球的包封率为74.93%，而且在体外测出了经单纯壳聚糖以及壳聚糖-PLGA微球缓释的PT浓度曲线。从曲线上各个时间点两组的PT浓度值可见，以单纯壳聚糖为缓释载体，其溶液中释放出来的PT浓度较以壳聚糖-PLGA微球为高，且各时间点的浓度均值及标准差起伏较大。相比较而言，经壳聚糖-PLGA微球缓释后，各时间点溶液中PT的浓度均较低，且起伏波动不大，各时间点的浓度标准差也较小。从整体观察周期来看，以单纯壳聚糖为缓释载体的溶液在缓释第一天内出现了“突释”现象，PT的浓度在第一天达到了峰值，而在随后的观察周期内，虽有起伏，但PT浓度整体呈下降趋势；而以壳聚糖-PLGA微球为缓释载体的溶液则看不到对PT的“突释”，在长达30天的观察周期内，PT浓度始终维持平稳，没有出现明显的突释现象。四、结论：1．CFAH的止血机制及改性初探1．1．Schiff碱合成方案与多肽合成方案均能成功地将邻苯二酚结构桥接至壳聚糖，使壳聚糖在化学层面具备了像贝壳一样发生水下粘附的物质基础。1．2．与Schiff碱合成方案相比，多肽合成方案能够在化学修饰壳聚糖的同时保留壳聚糖的止血作用，因而更适合于本项目的后续研究。1．3．游离氨基对于壳聚糖止血剂的止血性能而言，发挥着“不可或缺”的关键作用。1．4．止血剂微观表面形貌的高度一致性，是其具备粘附特性的必备条件之一。2． LTVS装置的研究2．1．与美军现有的TVS相比，LTVS的吻合时间可以缩短60.37%。2．2．与美军现有的TVS相比，LTVS的吻合强度至少可以提高114.29%。3． ACIFIAB的研究3．1．冻干辐照异体骨比新鲜冷冻异体骨软，更容易被打压。3．2．就骨块的瞬时形变率而言，冻干辐照异体骨与新鲜冷冻异体骨不分伯仲，有望为髋臼假体提供相似的初期稳定性。3．3．由于新鲜血凝块尚能促进冻干辐照异体骨的再血管化与骨改建，我们推荐在髋臼翻修时，将冻干异体骨和自体新鲜血凝块预混后一并植入。4． PT的研究4．1．PT可以促进成骨细胞的增殖及对BMP-2的分泌。4．2．与TH不同，高浓度的PT对成骨细胞的增殖没有明显的抑制作用。4．3．局部补充自体PT可以有效促进大段骨缺损的修复。4．4．现有的骨库技术对骨组织中的PT破坏严重。4．5．借助于缓释技术，PT有望成为一种新型的促成骨因子应用于临床。总之，对壳聚糖急救止血剂止血机制与改性的初步研究，从物理化学双重层面揭示了止血剂的止血和粘附机制，有望在此基础之上进一步研发兼具止血和粘附性能的优秀止血剂；锁定暂时性血管分流装置，能够缩短吻合时间，增强吻合强度，并进一步提高分流管的抗凝特性，有望进一步提高暂时性血管分流技术的救治效率和安全性；自体血凝块预混的冻干异体骨和凝血酶原，能够进一步改善骨移植材料的血液供应和促进成骨，有望应用于大段骨缺损的治疗。因此，有必要对上述产品和技术开展后续研发，从而进一步提升我军对战时严重四肢伤的整体救治水平。更多还原

【Abstract】 ObjectiveTo preliminarily study therapeutic products applied during rescue by stages forcombat severer extremity trauma.Materials and MethodsPreliminary Study on Hemostatic Mechanism and Modification ofChitosan-based First Aid HemostatFirstly, We modified chitosan with3,4-dihydroxybenzene by Schiff’ baseformation and polypeptide synthesis and respectively, developed DHBH modifiedchitosan (DMCTS) and DOPA modified chitosan (DOPAMCTS). Secondly, weverified the feasibility of these two modifying methods by FT-IR. Thirdly, weevaluated hemostatic effects of chitin, chitosan, CeloxTMHemostat, intact andground cuttlefish bones, DMCTS and DOPAMCTS by coagulation test andcompared adhesive property to muscular tissue of intact cuttlefish bones andchitosan. Fourthly, we observed the surface topography of chitosan, intact andground cuttlefish bones by scanned electronic microscopy.Locked Temporary Vascular ShuntFirstly, we updated present TVS to LOCKED TVS (LTVS) in the following twoaspects. One was to make the outer surface of TVS tube threaded. The other wasto alter stabilization method from manual suture ties to locking buckles with threaded inner surface. Secondly, we compared the anastomosis stability of TVSand LTVS by measuring arterial blasting pressure in vitro. Thirdly, we comparedanastomosis time of TVS and LTVS in canis bilateral femoral artery defect model.Fourthly, using histological methods, we examined the continuity of vessel wallscrushed for7days by threads (LTVS) and suture ties (TVS).Autologous Coagula Impregnated Freeze-dried Allograft BoneWe developed a novel impacted material, i.e. freeze-dried irradiated allograft bone(FIAB) with fresh autologous coagula, to enhance the donor-host incorporationafter impaction in hip revision, and experimentally investigated its effects onangiogenesis in a rat ectopic bone allograft implantation model, and evaluated itsdeformable property using a confined-impaction mechanical test.ProthrombinFirstly, we developed the Material Repair Strategy and using ELISA, CCK8,RT-PCR and qRT-PCR, we compared the influence of prothrombin and thrombinon BMP-2secreation and proliferation of rat osteoblasts. Secondly, usingpostoperative X-ray scoring and Image-Pro Plus software, we evaluated repairingeffect of autologous prothrombin on segmental defect in rabbit bilateral radiusdefect model. Thirdly, using immunohistochemistric stain for rat prothrombin, wecompared distribution of prothrombin within fresh femeral bone and defatted,freeze-dried and irradiated femeral bone. Fourthly, we prepared bone allograftloaded with prothrombin encapsulated by chitosan-PLGA microsphere, and usingELISA, we evaluated controlled releasing property of this novel material.ResultsPreliminary Study on Hemostatic Mechanism and Modification ofChitosan-based First Aid HemostatFTIR results revealed the similarity of chemical structures of chitosan andCelox, especially with N-H bending vibration of primary amines, and withinDMCTS and DOPAMCTS, the incorporation of3,4-dihydroxybenzene fromDMCTS and DOPA into the backbone of chitosan. The coagulation time for chitosan, Celox and DOPAMCTS was significantlyshorter than that of chitin and DMCTS (p=0.000). The coagulation time for intactcuttlefish bones was significantly shorter than that of ground cuttlefish bones (p=0.000). Blood drops touching chitosan, Celox, DOPAMCTS and intactcuttlefish bones resulted in a significant surface-tension phenomenon.SEM revealed extremely regular alignment of surface topography of intactcuttlefish bones and tangled surface topography of ground cuttlefish bones andchitosan particles.Under water scouring, intact cuttlefish bones tightly adhered to muscular tissuewhile ground cuttlefish bones and chitosan particles could not adhered to musculartissue as tightly as intact cuttlefish bones did.Locked Temporary Vascular ShuntArterial blasting pressure test revealed that, the arterial blasting pressure of LTVSgroup was significantly higher than that of TVS group (0.045±0.008MPa versus0.021±0.012MPa, p=0.000). The anastomosis time of LTVS was significantlyshorter than that of TVS (138.89±18.22s versus350.48±52.20s, p=0.000).Although being crushed by threads (LTVS) and suture ties (TVS) for7days, thestratum vasculare at the site of anastomosis still maintained its continuity.Autologous Coagula Impregnated Freeze-dried Allograft BoneAngiogenesis within FIAB on the coagula implanting (study) side was moreintense than that on the physiological saline implanting (control) side atpostoperative weeks4and8. The area of vasculars within the implant on the studyside was significantly larger than that on the control group (p=0.035) Atpostoperative week1, the relative expression of rat vascular endothelial growthfactor α on the study side was significantly higher than that on the control side (p<0.05). Delta ratio of compression, an index of transient deformable property ofbone grafts, was determined by bone height and impact frequency, but not bystiffness or elastic modulus (R2=0.514, p=0.000), although FABs were foundstiffer than FIABs.Prothrombin CCK8test revealed that, when rat osteoblasts were cultured in FBS freee DMEMmedia, prothrombin (PT) promoted proliferation of rat osteoblasts when PTconcentration reached different levels (0μg/ml,0.1μg/ml,1μg/ml,10μg/ml and25μg/ml) and the promotion effect reached summit when PT concentration was1μg/ml. The effect of thrombin (TH) on osteoblast proliferation was differentaccording to different TH concentrations. When TH concentration reaching0.1μg/ml, no significant promotion or inhibition effect on osteoblast proliferation.When TH concentration was reaching1μg/ml, TH could promote osteoblastproliferation. As TH concentration was higher than10μg/ml, TH began to inhibitproliferation of rat osteoblsts. And the higher TH concentration was, the moreseverer the inhibition effect reached. The promotion effect of PT+TH on osteoblastproliferation reached summit when concentration of PT+TH reaching1μg/ml. Asconcentration of PT+TH kept rising, the promotion effect weakened gradually butno obvious inhibition effect appeared.Results of CCK8test also revealed that, when rat osteoblasts were cultured inDMEM media containing10%FBS, PT could promote osteobast proliferation,especially when PT concentration reaching0.014μg/ml.Results of ELISA suggested that, the promotion effect of0.5μg/ml TH on ratosteoblast secreating BMP-2reached summit at12h after PT stimulation, thengradually decreased till24h. The BMP-2concentration in the PT stimulation groupgradually increased from0to24h after stimulation. The BMP-2concentration inthe TH+PT stimulation group was similar to that of TH stimulation group and thesummit of BMP-2concentration occurred at12h and was higher than that of theTH stimulation group. The summit of BMP-2concentration in the blank group(without TH or PT stimulation) was firstly seen and was the lowest at24h.Results of ELISA also suggested that, when rat osteoblasts were stimulated bydifferent concentrations of TH, PT and PT+TH (0μg/ml,0.1μg/ml,1μg/ml,10μg/ml and25μg/ml) respectively, BMP-2concentration reached summit whenthe concentration of TH was10μg/ml, or the concentration of PT was1μg/ml, orthe concentration of TH+PT was10μg/ml. Results of RT-PCR verified that,1ug/ml of PT enhanced expression of BMP-2byosteoblasts. qRT-PCR also suggested expression of BMP-2by osteoblasts could besignificantly enhanced by0.1mg/ml of PT (p=0.000).After bilaterial radial defects (11mm of length) of10rabbits were repaired bycalcium alginate sponge plus autologuous PT (study side, Left) or calcium alginatesponge alone (control side, Right), values of bilaterial X-ray scoring graduallyincreased indicating bilaterial defects being gradually repaired. From postoperative4,8weeks to16weeks, X-ray scoring values of the study side were significanthigher than those on the control side (p=0.021), and defect area on the study sidewas significantly smaller than that on the control side (p=0.045).Immunohistochemistric staining for rat PT revealed that, there were plenty of PTswithin fresh cortical bones, and most of them, along with osteocytes, locatedwithin bone lacuna. However, after fresh cortical bones being prepared bydefatting, deproteining, freeze-drying and gamma irradiation, most ofimmunogenic components within fresh cortical bones, i. e., not only osteocytes,but also PTs, were removed.SEM revealed bone trabecula on the medullary canal surface and nutrient foramenon the cortical surface, of freeze-dried irradiated allograft bone (FIAB). AfterFIAB being impregnated into chitosan solution and freezed-dried again, bonetrabecula and nutrient foramen on the surfaces were covered by homogeneouschitosan membrane. After being freeze-dried and toasted, humanprothrombin-PLGA microspheres (hPPMs) were looked like chords with poroussurface, approximate100μm of diameter, and40μm of thickness. On themedullary canal and cortical surface of human prothrombin impregnated FIABs(HPIFIABs), there were plenty of hPPMs with2to10μm of diameters covered byhomogeneous chitosan membrane.Results of ELISA revealed controlled-releasing characteristics of HPIFIABs. Theentrapment rate (ER) of PTs was74.93%. Compared with chitosan carrier (CC),chitosan-microsphere carrier (CMC) induced slower releasing of PTs with lessfluctuation. Burst releasing of PTs could be seen within the first24hrs in the CC group, and overall, the concentration of PTs in this group gradually decreasedduring the period of30days. In contrast, no obvious burst releasing could be seenin the CMC group and the PT concentration in the CMC group kept steady all thetime of30days.ConclusionPreliminary Study on Hemostatic Mechanism and Modification ofChitosan-based First Aid HemostatUsing Schiff’ base formation and polypeptide synthesis, we can successfullymodify chitosan with3,4-dihydroxybenzene. This modification might providechemical basis to chitosan-based hemostats in favor of adhesion propertyunderneath water.Compared with Schiff’ base formation, polypeptide synthesis is more suitable forfurther modification of chitosan-based hemostats because polypeptide synthesisdoes not disturb hemostatic potency of chitosan-based hemostats.Free aminos are indispensible for chitosan-based hemostat to stop bleeding.Extremely regular alignment of surface topography is one of prerequisites forhemostats to possess adhesive property.Locked Temporary Vascular ShuntCompared with TVS currently used by U.S. troops, LTVS can significantlyshorten anastomosis time (60.4%, p=0.000).Compared with TVS currently used by U.S. troops, LTVS can significantlyenhance anastomosis stability (114.29%, p=0.000).Autologous Coagula Impregnated Freeze-dried Irradiated Allograft BoneFreeze-dried irradiated allograft bones (FIABs) are softer and more easily to beimpacted than fresh-frozen allograft bones (FABs).FIABs are equivalent to FABs in transient deformative potency and therefore canprovide so enough primary stability to acetabular prothesises as fresh frozen bonescan. Because fresh cougular can also promote revascularization and remodeling ofFIABs, FIABs with fresh autologous coagula might be recommended for hiprevision impaction.ProthrombinProthrombins (PTs) can promote proliferation and BMP-2secreation ofosteoblasts.Unlike to thrombin (TH), PTs with high concentration have no obvious inhibitioneffect on osteoblast proliferation.Topical supplement of PTs can effectively promote repairing of segmental bonedefects.Currently applied bone bank preparation methods can destroy PTs within freshbones.Controlled releasing strategies might be helpful in promoting PT to be a novelosteogenic factor applied in clinic.In summary, these therapeutic products could be further investigated to promoteclinical results during rescue by stages for combat severer extremity trauma.更多还原