战场严重四肢伤分级救治系列产品的研究
|
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-2
ELISA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。
Objective
To preliminarily study therapeutic products applied during rescue by stages forcombat severer extremity trauma.
Materials and Methods
Preliminary Study on Hemostatic Mechanism and Modification ofChitosan-based First Aid Hemostat
Firstly, 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 Shunt
Firstly, 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 Bone
We 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.
Prothrombin
Firstly, 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.
Results
Preliminary Study on Hemostatic Mechanism and Modification ofChitosan-based First Aid Hemostat
FTIR 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 Shunt
Arterial 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 Bone
Angiogenesis 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.
Conclusion
Preliminary Study on Hemostatic Mechanism and Modification ofChitosan-based First Aid Hemostat
Using 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 Shunt
Compared 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 Bone
Freeze-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.
Prothrombin
Prothrombins (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.
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-2
ELISA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。
Objective
To preliminarily study therapeutic products applied during rescue by stages forcombat severer extremity trauma.
Materials and Methods
Preliminary Study on Hemostatic Mechanism and Modification ofChitosan-based First Aid Hemostat
Firstly, 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 Shunt
Firstly, 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 Bone
We 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.
Prothrombin
Firstly, 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.
Results
Preliminary Study on Hemostatic Mechanism and Modification ofChitosan-based First Aid Hemostat
FTIR 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 Shunt
Arterial 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 Bone
Angiogenesis 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.
Conclusion
Preliminary Study on Hemostatic Mechanism and Modification ofChitosan-based First Aid Hemostat
Using 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 Shunt
Compared 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 Bone
Freeze-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.
Prothrombin
Prothrombins (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.
引文
[1] Stannard A, Brown K, Benson C, et al. Outcome after vascular trauma in a deployedmilitary trauma system. Br J Surg.2011,98(2):228-234.
[2] HUGHES CW. The primary repair of wounds of major arteries; an analysis ofexperience in Korea in1953. Ann Surg.1955,141(3):297-303.
[3] Rich NM, Baugh JH, Hughes CW. Acute arterial injuries in Vietnam:1,000cases. JTrauma.1970,10(5):359-369.
[4] Rich NM, Hughes CW. Vietnam vascular registry: a preliminary report. Surgery.1969,65(1):218-226.
[5] Wedmore I, McManus JG, Pusateri AE, et al. A special report on the chitosan-basedhemostatic dressing: experience in current combat operations. J Trauma.2006,60(3):655-658.
[6] D'Alleyrand JC, Dutton RP, Pollak AN. Extrapolation of battlefield resuscitative care tothe civilian setting. J Surg Orthop Adv.2010,19(1):62-69.
[7] Pusateri AE, Holcomb JB, Kheirabadi BS, et al. Making sense of the preclinicalliterature on advanced hemostatic products. J Trauma.2006,60(3):674-682.
[8] Holcomb J, MacPhee M, Hetz S, et al. Efficacy of a dry fibrin sealant dressing forhemorrhage control after ballistic injury. Arch Surg.1998,133(1):32-35.
[9] Larson MJ, Bowersox JC, Lim RJ, et al. Efficacy of a fibrin hemostatic bandage incontrolling hemorrhage from experimental arterial injuries. Arch Surg.1995,130(4):420-422.
[10] Jackson MR, Friedman SA, Carter AJ, et al. Hemostatic efficacy of a fibrinsealant-based topical agent in a femoral artery injury model: a randomized, blinded,placebo-controlled study. J Vasc Surg.1997,26(2):274-280.
[11] Holcomb J, MacPhee M, Hetz S, et al. Efficacy of a dry fibrin sealant dressing forhemorrhage control after ballistic injury. Arch Surg.1998,133(1):32-35.
[12] Holcomb JB, Pusateri AE, Harris RA, et al. Effect of dry fibrin sealant dressings versusgauze packing on blood loss in grade V liver injuries in resuscitated swine. J Trauma.1999,46(1):49-57.
[13] Pusateri AE, Modrow HE, Harris RA, et al. Advanced hemostatic dressing developmentprogram: animal model selection criteria and results of a study of nine hemostatic dressings ina model of severe large venous hemorrhage and hepatic injury in Swine. J Trauma.2003,55(3):518-526.
[14] Holcomb JB, Pusateri AE, Harris RA, et al. Dry fibrin sealant dressings reduce bloodloss, resuscitation volume, and improve survival in hypothermic coagulopathic swine withgrade V liver injuries. J Trauma.1999,47(2):233-240,240-242.
[15] Pusateri AE, Holcomb JB, Harris RA, et al. Effect of fibrin bandage fibrinogenconcentration on blood loss after grade V liver injury in swine. Mil Med.2001,166(3):217-222.
[16] Pusateri AE, Kheirabadi BS, Delgado AV, et al. Structural design of the dry fibrinsealant dressing and its impact on the hemostatic efficacy of the product. J Biomed Mater ResB Appl Biomater.2004,70(1):114-121.
[17] Morey AF, Anema JG, Harris R, et al. Treatment of grade4renal stab wounds withabsorbable fibrin adhesive bandage in a porcine model. J Urol.2001,165(3):955-958.
[18] Sondeen JL, Pusateri AE, Coppes VG, et al. Comparison of10different hemostaticdressings in an aortic injury. J Trauma.2003,54(2):280-285.
[19] Kheirabadi BS, Acheson EM, Deguzman R, et al. Hemostatic efficacy of two advanceddressings in an aortic hemorrhage model in Swine. J Trauma.2005,59(1):25-34,34-35.
[20] Acheson EM, Kheirabadi BS, Deguzman R, et al. Comparison of hemorrhage controlagents applied to lethal extremity arterial hemorrhages in swine. J Trauma.2005,59(4):865-874,874-875.
[21] Chan MW, Schwaitzberg SD, Demcheva M, et al. Comparison of poly-N-acetylglucosamine (P-GlcNAc) with absorbable collagen (Actifoam), and fibrin sealant (Bolheal)for achieving hemostasis in a swine model of splenic hemorrhage. J Trauma.2000,48(3):454-457,457-458.
[22] Thatte HS, Zagarins S, Khuri SF, et al. Mechanisms of poly-N-acetyl glucosaminepolymer-mediated hemostasis: platelet interactions. J Trauma.2004,57(1Suppl): S13-S21.
[23] Thatte HS, Zagarins SE, Amiji M, et al. Poly-N-acetyl glucosamine-mediated red bloodcell interactions. J Trauma.2004,57(1Suppl): S7-S12.
[24] Favuzza J, Hechtman HB. Hemostasis in the absence of clotting factors. J Trauma.2004,57(1Suppl): S42-S44.
[25] Valeri CR, Srey R, Tilahun D, et al. In vitro effects of poly-N-acetyl glucosamine onthe activation of platelets in platelet-rich plasma with and without red blood cells. J Trauma.2004,57(1Suppl): S22-S25, S25.
[26] Cole DJ, Connolly RJ, Chan MW, et al. A pilot study evaluating the efficacy of a fullyacetylated poly-N-acetyl glucosamine membrane formulation as a topical hemostatic agent.Surgery.1999,126(3):510-517.
[27] Alam HB, Uy GB, Miller D, et al. Comparative analysis of hemostatic agents in a swinemodel of lethal groin injury. J Trauma.2003,54(6):1077-1082.
[28] King DR, Cohn SM, Proctor KG. Modified rapid deployment hemostat bandageterminates bleeding in coagulopathic patients with severe visceral injuries. J Trauma.2004,57(4):756-759.
[29] Connolly RJ. Application of the poly-N-acetyl glucosamine-derived rapid deploymenthemostat trauma dressing in severe/lethal Swine hemorrhage trauma models. J Trauma.2004,57(1Suppl): S26-S28.
[30] Vournakis JN, Demcheva M, Whitson AB, et al. The RDH bandage: hemostasis andsurvival in a lethal aortotomy hemorrhage model. J Surg Res.2003,113(1):1-5.
[31] Jewelewicz DD, Cohn SM, Crookes BA, et al. Modified rapid deployment hemostatbandage reduces blood loss and mortality in coagulopathic pigs with severe liver injury. JTrauma.2003,55(2):275-280,280-281.
[32] Alam HB, Burris D, DaCorta JA, et al. Hemorrhage control in the battlefield: role ofnew hemostatic agents. Mil Med.2005,170(1):63-69.
[33] Wedmore I, McManus JG, Pusateri AE, et al. A special report on the chitosan-basedhemostatic dressing: experience in current combat operations. J Trauma.2006,60(3):655-658.
[34] Wright JK, Kalns J, Wolf EA, et al. Thermal injury resulting from application of agranular mineral hemostatic agent. J Trauma.2004,57(2):224-230.
[35] Ward KR, Tiba MH, Holbert WH, et al. Comparison of a new hemostatic agent tocurrent combat hemostatic agents in a Swine model of lethal extremity arterial hemorrhage. JTrauma.2007,63(2):276-283,283-284.
[36] Kheirabadi BS, Mace JE, Terrazas IB, et al. Safety evaluation of new hemostatic agents,smectite granules, and kaolin-coated gauze in a vascular injury wound model in swine. JTrauma.2010,68(2):269-278.
[37] Pusateri AE, McCarthy SJ, Gregory KW, et al. Effect of a chitosan-based hemostaticdressing on blood loss and survival in a model of severe venous hemorrhage and hepaticinjury in swine. J Trauma.2003,54(1):177-182.
[38] Alam HB, Chen Z, Jaskille A, et al. Application of a zeolite hemostatic agent achieves100%survival in a lethal model of complex groin injury in Swine. J Trauma.2004,56(5):974-983.
[39]范杰,李云龙,李辉.一种基于浙江缙云天然沸石紧急救生止血剂的制备方法,20102010-12-23.
[40]吕小星,陈绍宗,李学拥,等.新型战伤急救止血剂体内抗菌活性的实验研究.现代生物医学进展.2011,11(8):1467-1468.
[41]吴伟萍,彭承宏,韩宝三,等.羧甲基壳聚糖在大鼠创伤出血模型的止血效果的观察.外科理论与实践.2010,15(3):240-244.
[42] Gegel B, Burgert J, Cooley B, et al. The effects of BleedArrest, Celox, and TraumaDexon hemorrhage control in a porcine model. J Surg Res.2010,164(1): e125-e129.
[43] Silverman HG, Roberto FF. Understanding marine mussel adhesion. Mar Biotechnol(NY).2007,9(6):661-681.
[44] Ivankovic H, Gallego FG, Tkalcec E, et al. Preparation of highly porous hydroxyapatitefrom cuttlefish bone. J Mater Sci Mater Med.2009,20(5):1039-1046.
[45] Lu G, Hong W, Tong L, et al. Drying enhanced adhesion of polythiophene nanotubulearrays on smooth surfaces. ACS Nano.2008,2(11):2342-2348.
[46] HUGHES CW. Arterial repair during the Korean war. Ann Surg.1958,147(4):555-561.
[47] Eger M, Golcman L, Goldstein A, et al. The use of a temporary shunt in themanagement of arterial vascular injuries. Surg Gynecol Obstet.1971,132(1):67-70.
[48] Kragh JJ, Walters TJ, Baer DG, et al. Survival with emergency tourniquet use to stopbleeding in major limb trauma. Ann Surg.2009,249(1):1-7.
[49] Woodward EB, Clouse WD, Eliason JL, et al. Penetrating femoropopliteal injury duringmodern warfare: experience of the Balad Vascular Registry. J Vasc Surg.2008,47(6):1259-1264,1264-1265.
[50] Clouse WD, Rasmussen TE, Peck MA, et al. In-theater management of vascular injury:2years of the Balad Vascular Registry. J Am Coll Surg.2007,204(4):625-632.
[51] Sohn VY, Arthurs ZM, Herbert GS, et al. Demographics, treatment, and early outcomesin penetrating vascular combat trauma. Arch Surg.2008,143(8):783-787.
[52] Peck MA, Clouse WD, Cox MW, et al. The complete management of extremityvascular injury in a local population: a wartime report from the332nd Expeditionary MedicalGroup/Air Force Theater Hospital, Balad Air Base, Iraq. J Vasc Surg.2007,45(6):1197-1204,1204-1205.
[53] Clouse WD, Rasmussen TE, Perlstein J, et al. Upper extremity vascular injury: acurrent in-theater wartime report from Operation Iraqi Freedom. Ann Vasc Surg.2006,20(4):429-434.
[54] Fox CJ, Patel B, Clouse WD. Update on wartime vascular injury. Perspect Vasc SurgEndovasc Ther.2011,23(1):13-25.
[55] Vertrees A, Fox CJ, Quan RW, et al. The use of prosthetic grafts in complex militaryvascular trauma: a limb salvage strategy for patients with severely limited autologous conduit.J Trauma.2009,66(4):980-983.
[56] Granchi T, Schmittling Z, Vasquez J, et al. Prolonged use of intraluminal arterial shuntswithout systemic anticoagulation. Am J Surg.2000,180(6):493-496,496-497.
[57] Borut LT, Acosta CJ, Tadlock LC, et al. The use of temporary vascular shunts inmilitary extremity wounds: a preliminary outcome analysis with2-year follow-up. J Trauma.2010,69(1):174-178.
[58] Rasmussen TE, Clouse WD, Jenkins DH, et al. The use of temporary vascular shunts asa damage control adjunct in the management of wartime vascular injury. J Trauma.2006,61(1):8-12,12-15.
[59]汪忠镐,谷涌泉,张建.下肢动脉损伤.北京:人民卫生出版社,2010.
[60] Bolder SB, Verdonschot N, Schreurs BW. Technical factors affecting cup stability inbone impaction grafting. Proc Inst Mech Eng H.2007,221(1):81-86.
[61] Buma P, Arts JJ, Gardeniers JW, et al. No effect of bone morphogenetic protein-7(OP-1) on the incorporation of impacted bone grafts in a realistic acetabular model. J BiomedMater Res B Appl Biomater.2008,84(1):231-239.
[62] Karrholm J, Hourigan P, Timperley J, et al. Mixing bone graft with OP-1does notimprove cup or stem fixation in revision surgery of the hip:5-year follow-up of10acetabularand11femoral study cases and40control cases. Acta Orthop.2006,77(1):39-48.
[63] Deakin DE, Bannister GC. Graft incorporation after acetabular and femoral impactiongrafting with washed irradiated allograft and autologous marrow. J Arthroplasty.2007,22(1):89-94.
[64] Xu ZJ, He RX. Selection of allografts for impaction bone grafting for bone defectreconstruction on the acetabular side. Chin Med J (Engl).2010,123(21):3143-3147.
[65] Mahadevan D, Challand C, Keenan J. Revision total hip replacement: predictors ofblood loss, transfusion requirements, and length of hospitalisation. J Orthop Traumatol.2010,11(3):159-165.
[66] Dunlop DG, Brewster NT, Madabhushi SP, et al. Techniques to improve the shearstrength of impacted bone graft: the effect of particle size and washing of the graft. J BoneJoint Surg Am.2003,85-A(4):639-646.
[67] Arts JJ, Verdonschot N, Buma P, et al. Larger bone graft size and washing of bonegrafts prior to impaction enhances the initial stability of cemented cups: experiments using asynthetic acetabular model. Acta Orthop.2006,77(2):227-233.
[68] Phillips A, Pankaj P, May F, et al. Constitutive models for impacted morsellisedcortico-cancellous bone. Biomaterials.2006,27(9):2162-2170.
[69] Voor MJ, White JE, Grieshaber JE, et al. Impacted morselized cancellous bone:mechanical effects of defatting and augmentation with fine hydroxyapatite particles. JBiomech.2004,37(8):1233-1239.
[70] Bolder SB, Schreurs BW, Verdonschot N, et al. Particle size of bone graft and methodof impaction affect initial stability of cemented cups: human cadaveric and synthetic pelvicspecimen studies. Acta Orthop Scand.2003,74(6):652-657.
[71] Bavadekar A, Cornu O, Godts B, et al. Stiffness and compactness of morselized graftsduring impaction: an in vitro study with human femoral heads. Acta Orthop Scand.2001,72(5):470-476.
[72] Lu H, Pei G, Zhao P, et al. Cyclosporine-impregnated allograft bone sterilized withlow-temperature plasma. J Tissue Eng Regen Med.2010,4(8):638-651.
[73] Cornu O, Bavadekar A, Godts B, et al. Impaction bone grafting with freeze-driedirradiated bone. Part II. Changes in stiffness and compactness of morselized grafts:experiments in cadavers. Acta Orthop Scand.2003,74(5):553-558.
[74]杨颖,罗晖.血小板内皮细胞粘附分子-1(PECAM-1)的研究与疾病.川北医学院学报.6(23):627-631.
[75] Feyen JH, van der Wilt G, Moonen P, et al. Stimulation of arachidonic acid metabolismin primary cultures of osteoblast-like cells by hormones and drugs. Prostaglandins.1984,28(6):769-781.
[76] Lerner UH, Ransjo M, Ljunggren O. Bradykinin stimulates production of prostaglandinE2and prostacyclin in murine osteoblasts. Bone Miner.1989,5(2):139-154.
[77] Abraham LA, MacKie EJ. Modulation of osteoblast-like cell behavior by activation ofprotease-activated receptor-1. J Bone Miner Res.1999,14(8):1320-1329.
[78] Pagel CN, de Niese MR, Abraham LA, et al. Inhibition of osteoblast apoptosis bythrombin. Bone.2003,33(4):733-743.
[79] Mohle R, Green D, Moore MA, et al. Constitutive production and thrombin-inducedrelease of vascular endothelial growth factor by human megakaryocytes and platelets. ProcNatl Acad Sci U S A.1997,94(2):663-668.
[80]张永刚,卢世璧.骨折修复与细胞因子调节.中华外科杂志.1997,35(5):316-318.
[81]王鸿利.止血与凝血机制研究进展.继续医学教育.2006,20(26):13-19.
[82] Karlstrom E, Norgard M, Hultenby K, et al. Localization and expression of prothrombinin rodent osteoclasts and long bones. Calcif Tissue Int.2011,88(3):179-188.
[83] Pagel CN, Sivagurunathan S, Loh LH, et al. Functional responses of bone cells tothrombin. Biol Chem.2006,387(8):1037-1041.
[84] Jenkins AL, Bootman MD, Taylor CW, et al. Characterization of the receptorresponsible for thrombin-induced intracellular calcium responses in osteoblast-like cells. JBiol Chem.1993,268(28):21432-21437.
[85] Abraham LA, Jenkins AL, Stone SR, et al. Expression of the thrombin receptor indeveloping bone and associated tissues. J Bone Miner Res.1998,13(5):818-827.
[86] Frost A, Jonsson KB, Ridefelt P, et al. Thrombin, but not bradykinin, stimulatesproliferation in isolated human osteoblasts, via a mechanism not dependent on endogenousprostaglandin formation. Acta Orthop Scand.1999,70(5):497-503.
[87] Song SJ, Pagel CN, Campbell TM, et al. The role of protease-activated receptor-1inbone healing. Am J Pathol.2005,166(3):857-868.
[88] Ljunggren O, Johansson H, Ljunghall S, et al. Thrombin increases cytoplasmic Ca2+and stimulates formation of prostaglandin E2in the osteoblastic cell line MC3T3-El. BoneMiner.1991,12(2):81-90.
[89] Kozawa O, Tokuda H, Kaida T, et al. Thrombin regulates interleukin-6synthesisthrough phosphatidylcholine hydrolysis by phospholipase D in osteoblasts. Arch BiochemBiophys.1997,345(1):10-15.
[90] Pagel CN, Song SJ, Loh LH, et al. Thrombin-stimulated growth factor and cytokineexpression in osteoblasts is mediated by protease-activated receptor-1and prostanoids. Bone.2009,44(5):813-821.
[91] Hanratty BM, Ryaby JT, Pan XH, et al. Thrombin related peptide TP508promotedfracture repair in a mouse high energy fracture model. J Orthop Surg Res.2009,4(1.
[92] Wang Y, Wan C, Szoke G, et al. Local injection of thrombin-related peptide (TP508) inPPF/PLGA microparticles-enhanced bone formation during distraction osteogenesis. J OrthopRes.2008,26(4):539-546.
[93] Amir LR, Li G, Schoenmaker T, et al. Effect of thrombin peptide508(TP508) on bonehealing during distraction osteogenesis in rabbit tibia. Cell Tissue Res.2007,330(1):35-44.
[94] Li X, Wang H, Touma E, et al. TP508accelerates fracture repair by promoting cellgrowth over cell death. Biochem Biophys Res Commun.2007,364(1):187-193.
[95] Ryaby JT, Sheller MR, Levine BP, et al. Thrombin peptide TP508stimulates cellularevents leading to angiogenesis, revascularization, and repair of dermal and musculoskeletaltissues. J Bone Joint Surg Am.2006,88Suppl3(132-139.
[96] Li G, Ryaby JT, Carney DH, et al. Bone formation is enhanced by thrombin-relatedpeptide TP508during distraction osteogenesis. J Orthop Res.2005,23(1):196-202.
[97] Sheller MR, Crowther RS, Kinney JH, et al. Repair of rabbit segmental defects with thethrombin peptide, TP508. J Orthop Res.2004,22(5):1094-1099.
[98] Wang H, Li X, Tomin E, et al. Thrombin peptide (TP508) promotes fracture repair byup-regulating inflammatory mediators, early growth factors, and increasing angiogenesis. JOrthop Res.2005,23(3):671-679.
[99] Axelrad TW, Kakar S, Einhorn TA. New technologies for the enhancement of skeletalrepair. Injury.2007,38Suppl1(S49-S62.
[100] Mann KG. Prothrombin. Methods Enzymol.1976,45(123-156.
[101] BARNHART MI. Cellular site for prothrombin synthesis. Am J Physiol.1960,199(360-366.
[102] Jamison CS, Degen SJ. Prenatal and postnatal expression of mRNA coding for ratprothrombin. Biochim Biophys Acta.1991,1088(2):208-216.
[103] Norris LA. Blood coagulation. Best Pract Res Clin Obstet Gynaecol.2003,17(3):369-383.
[104] Zoubine MN, Ma JY, Smirnova IV, et al. A molecular mechanism for synapseelimination: novel inhibition of locally generated thrombin delays synapse loss in neonatalmouse muscle. Dev Biol.1996,179(2):447-457.
[105] Glazner GW, Yadav K, Fitzgerald S, et al. Cholinergic stimulation increases thrombinactivity and gene expression in cultured mouse muscle. Brain Res Dev Brain Res.1997,99(2):148-154.
[106] Flynn PD, Byrne CD, Baglin TP, et al. Thrombin generation by apoptotic vascularsmooth muscle cells. Blood.1997,89(12):4378-4384.
[107] Festoff BW, Smirnova IV, Ma J, et al. Thrombin, its receptor and protease nexin I, itspotent serpin, in the nervous system. Semin Thromb Hemost.1996,22(3):267-271.
[108] Suzuki K, Tanaka T, Miyazawa K, et al. Gene expression of prothrombin in human andrat kidneys: basic and clinical approach. J Am Soc Nephrol.1999,10Suppl14(S408-S411.
[109] Trudel G, Uhthoff HK, Laneuville O. Prothrombin gene expression in articular cartilagewith a putative role in cartilage degeneration secondary to joint immobility. J Rheumatol.2005,32(8):1547-1555.
[110] Osterud B, Lindahl U, Seljelid R. Macrophages produce blood coagulation factors. FebsLett.1980,120(1):41-43.
[111] Shim K, Zhu H, Westfield LA, et al. A recombinant murine meizothrombin precursor,prothrombin R157A/R268A, inhibits thrombosis in a model of acute carotid artery injury.Blood.2004,104(2):415-419.
[112] Norris LA. Blood coagulation. Best Pract Res Clin Obstet Gynaecol.2003,17(3):369-383.
[113] Cook SD, Wolfe MW, Salkeld SL, et al. Effect of recombinant human osteogenicprotein-1on healing of segmental defects in non-human primates. J Bone Joint Surg Am.1995,77(5):734-750.
[114]赖翼,吴民泸,代娟,等.两种凝血酶原提取方法的比较.成都医学院学报.2009,01):
[115] Tabata II. The importance of drug delivery systems in tissue engineering. Pharm SciTechnolo Today.2000,3(3):80-89.
[116]陈英杰,许向阳,周建平.乳酸-羟基乙酸共聚物微球的研究进展.中国药科大学学报.2007,38(2):186-189.
[117] Liu J, Meisner D, Kwong E, et al. A novel trans-lymphatic drug delivery system:implantable gelatin sponge impregnated with PLGA-paclitaxel microspheres. Biomaterials.2007,28(21):3236-3244.
[118] Jaklenec A, Wan E, Murray ME, et al. Novel scaffolds fabricated from protein-loadedmicrospheres for tissue engineering. Biomaterials.2008,29(2):185-192.
[119] Kelly JF, Ritenour AE, McLaughlin DF, et al. Injury severity and causes of death fromOperation Iraqi Freedom and Operation Enduring Freedom:2003-2004versus2006. J Trauma.2008,64(2Suppl): S21-S26, S26-S27.
[120] DeBAKEY ME, SIMEONE FA. Battle injuries of the arteries in World War II; ananalysis of2,471cases. Trans South Surg Assoc.1946,57(65-110.
[121] HUGHES CW. Acute vascular trauma in Korean War casualties; an analysis of180cases. Surg Gynecol Obstet.1954,99(1):91-100.
[122] HUGHES CW. The primary repair of wounds of major arteries; an analysis ofexperience in Korea in1953. Ann Surg.1955,141(3):297-303.
[123] Rich NM, Baugh JH, Hughes CW. Popliteal artery injuries in Vietnam. Am J Surg.1969,118(4):531-534.
[124] JAHNKE EJ, HUGHES CW, HOWARD JM. The rationale of arterial repair on thebattlefield. Am J Surg.1954,87(3):396-401.
[125] Gawande A. Casualties of war--military care for the wounded from Iraq andAfghanistan. N Engl J Med.2004,351(24):2471-2475.
[126] Starnes BW, Beekley AC, Sebesta JA, et al. Extremity vascular injuries on thebattlefield: tips for surgeons deploying to war. J Trauma.2006,60(2):432-442.
[127] Fox CJ, Gillespie DL, O'Donnell SD, et al. Contemporary management of wartimevascular trauma. J Vasc Surg.2005,41(4):638-644.
[128] Holcomb J, Caruso J, McMullin N, et al. Causes of death in US Special OperationsForces in the global war on terrorism:2001-2004. US Army Med Dep J.2007,24-37.
[129] Wolf SE, Kauvar DS, Wade CE, et al. Comparison between civilian burns and combatburns from Operation Iraqi Freedom and Operation Enduring Freedom. Ann Surg.2006,243(6):786-792,792-795.
[130] JAHNKE EJ, HOWARD JM. Primary repair of major arterial injuries; report offifty-eight casualties. AMA Arch Surg.1953,66(5):646-649.
[131] JAHNKE EJ, SEELEY SF. Acute vascular injuries in the Korean War. Ann Surg.1953,138(2):158-177.
[132] Rich NM, Rhee P. An historical tour of vascular injury management: from its inceptionto the new millennium. Surg Clin North Am.2001,81(6):1199-1215.
[133] Beekley AC, Watts DM. Combat trauma experience with the United States Army102ndForward Surgical Team in Afghanistan. Am J Surg.2004,187(5):652-654.
[134]陈景云,等.抗美援朝卫生工作经验总结——战伤外科部分:抗美援朝卫生工作经验总结委员会,1953.
[135] Clouse WD, Rasmussen TE, Peck MA, et al. In-theater management of vascular injury:2years of the Balad Vascular Registry. J Am Coll Surg.2007,204(4):625-632.
[136] Owens BD, Kragh JJ, Wenke JC, et al. Combat wounds in operation Iraqi Freedom andoperation Enduring Freedom. J Trauma.2008,64(2):295-299.
[137] Ramasamy A, Harrisson SE, Clasper JC, et al. Injuries from roadside improvisedexplosive devices. J Trauma.2008,65(4):910-914.
[138] Brown KV, Ramasamy A, Tai N, et al. Complications of extremity vascular injuries inconflict. J Trauma.2009,66(4Suppl): S145-S149.
[139] Spencer FC. Historical vignette: the introduction of arterial repair into the US MarineCorps, US Naval Hospital, in July-August1952. J Trauma.2006,60(4):906-909.
[140] SPENCER FC, GREWE RV. The management of arterial injuries in battle casualties.Ann Surg.1955,141(3):304-313.
[141] Welling DR, Burris DG, Hutton JE, et al. A balanced approach to tourniquet use:lessons learned and relearned. J Am Coll Surg.2006,203(1):106-115.
[142] Walters TJ, Wenke JC, Kauvar DS, et al. Effectiveness of self-applied tourniquets inhuman volunteers. Prehosp Emerg Care.2005,9(4):416-422.
[143] Walters TJ, Mabry RL. Issues related to the use of tourniquets on the battlefield. MilMed.2005,170(9):770-775.
[144] Wenke JC, Walters TJ, Greydanus DJ, et al. Physiological evaluation of the U.S. Armyone-handed tourniquet. Mil Med.2005,170(9):776-781.
[145] Rasmussen TE, Clouse WD, Jenkins DH, et al. Echelons of care and the management ofwartime vascular injury: a report from the332nd EMDG/Air Force Theater Hospital, BaladAir Base, Iraq. Perspect Vasc Surg Endovasc Ther.2006,18(2):91-99.
[146] Beekley AC, Starnes BW, Sebesta JA. Lessons learned from modern military surgery.Surg Clin North Am.2007,87(1):157-184.
[147] Sebesta J. Special lessons learned from Iraq. Surg Clin North Am.2006,86(3):711-726.
[148] Beekley AC, Starnes BW, Sebesta JA. Lessons learned from modern military surgery.Surg Clin North Am.2007,87(1):157-184.
[149] Kragh JJ. Use of tourniquets and their effects on limb function in the modern combatenvironment. Foot Ankle Clin.2010,15(1):23-40.
[150] Porter JM, Ivatury RR, Nassoura ZE. Extending the horizons of "damage control" inunstable trauma patients beyond the abdomen and gastrointestinal tract. J Trauma.1997,42(3):559-561.
[151] Aucar JA, Hirshberg A. Damage control for vascular injuries. Surg Clin North Am.1997,77(4):853-862.
[152] Brohi K, Singh J, Heron M, et al. Acute traumatic coagulopathy. J Trauma.2003,54(6):1127-1130.
[153] MacLeod JB, Lynn M, McKenney MG, et al. Early coagulopathy predicts mortality intrauma. J Trauma.2003,55(1):39-44.
[154] Holcomb JB, Jenkins D, Rhee P, et al. Damage control resuscitation: directly addressingthe early coagulopathy of trauma. J Trauma.2007,62(2):307-310.
[155] Alam HB, Rhee P. New developments in fluid resuscitation. Surg Clin North Am.2007,87(1):55-72.
[156] Borgman MA, Spinella PC, Perkins JG, et al. The ratio of blood products transfusedaffects mortality in patients receiving massive transfusions at a combat support hospital. JTrauma.2007,63(4):805-813.
[157] Gonzalez EA, Moore FA, Holcomb JB, et al. Fresh frozen plasma should be givenearlier to patients requiring massive transfusion. J Trauma.2007,62(1):112-119.
[158] Hess JR, Holcomb JB, Hoyt DB. Damage control resuscitation: the need for specificblood products to treat the coagulopathy of trauma. Transfusion.2006,46(5):685-686.
[159] Kauvar DS, Holcomb JB, Norris GC, et al. Fresh whole blood transfusion: acontroversial military practice. J Trauma.2006,61(1):181-184.
[160] Perkins JG, Schreiber MA, Wade CE, et al. Early versus late recombinant factor VIIa incombat trauma patients requiring massive transfusion. J Trauma.2007,62(5):1095-1099,1099-1101.
[161] Fox CJ, Gillespie DL, Cox ED, et al. Damage control resuscitation for vascular surgeryin a combat support hospital. J Trauma.2008,65(1):1-9.
[162] Borgman MA, Spinella PC, Perkins JG, et al. The ratio of blood products transfusedaffects mortality in patients receiving massive transfusions at a combat support hospital. JTrauma.2007,63(4):805-813.
[163] Fox CJ, Starnes BW. Vascular surgery on the modern battlefield. Surg Clin North Am.2007,87(5):1193-1211.
[164] Alam HB, Rhee P. New developments in fluid resuscitation. Surg Clin North Am.2007,87(1):55-72.
[165] Engstrom M, Schott U, Nordstrom CH, et al. Increased lactate levels impair thecoagulation system--a potential contributing factor to progressive hemorrhage after traumaticbrain injury. J Neurosurg Anesthesiol.2006,18(3):200-204.
[166] Lou HC. Correction of increased plasma pyruvate and plasma lactate levels using largedoses of thiamine in patients with Kearns-Sayre syndrome. Arch Neurol.1981,38(7):469.
[167] Nahas GG, Sutin KM, Fermon C, et al. Guidelines for the treatment of acidaemia withTHAM. Drugs.1998,55(2):191-224.
[168] Fox CJ, Perkins JG, Kragh JJ, et al. Popliteal artery repair in massively transfusedmilitary trauma casualties: a pursuit to save life and limb. J Trauma.2010,69Suppl1(S123-S134.
[169] Dawson DL, Putnam AT, Light JT, et al. Temporary arterial shunts to maintain limbperfusion after arterial injury: an animal study. J Trauma.1999,47(1):64-71.
[170] Borut LT, Acosta CJ, Tadlock LC, et al. The use of temporary vascular shunts inmilitary extremity wounds: a preliminary outcome analysis with2-year follow-up. J Trauma.2010,69(1):174-178.
[171] Starnes BW, Arthurs ZM. Endovascular management of vascular trauma. Perspect VascSurg Endovasc Ther.2006,18(2):114-129.
[172] White R, Krajcer Z, Johnson M, et al. Results of a multicenter trial for the treatment oftraumatic vascular injury with a covered stent. J Trauma.2006,60(6):1189-1195,1195-1196.
[173] Lin PH, Bush RL, Zhou W, et al. Endovascular treatment of traumatic thoracic aorticinjury--should this be the new standard of treatment? J Vasc Surg.2006,43SupplA(22A-29A.
[174] Feliciano DV, Mattox KL, Graham JM, et al. Five-year experience with PTFE grafts invascular wounds. J Trauma.1985,25(1):71-82.
[175] Lin PH, Bush RL, Weiss VJ, et al. Subclavian artery disruption resulting fromendovascular intervention: treatment options. J Vasc Surg.2000,32(3):607-611.
[176] Fox CJ, Gillespie DL, Weber MA, et al. Delayed evaluation of combat-relatedpenetrating neck trauma. J Vasc Surg.2006,44(1):86-93.
[177] Holcomb JB, Salinas J, McManus JM, et al. Manual vital signs reliably predict need forlife-saving interventions in trauma patients. J Trauma.2005,59(4):821-828,828-829.
[178] Davis JW, Shackford SR, Mackersie RC, et al. Base deficit as a guide to volumeresuscitation. J Trauma.1988,28(10):1464-1467.
[179] Eastridge BJ, Owsley J, Sebesta J, et al. Admission physiology criteria after injury onthe battlefield predict medical resource utilization and patient mortality. J Trauma.2006,61(4):820-823.
[180] Sebesta J. Special lessons learned from Iraq. Surg Clin North Am.2006,86(3):711-726.
[181] Holcomb JB. Methods for improved hemorrhage control. Crit Care.2004,8Suppl2(S57-S60.
[182] Holcomb JB. Use of recombinant activated factor VII to treat the acquired coagulopathyof trauma. J Trauma.2005,58(6):1298-1303.
[183] Mohr AM, Holcomb JB, Dutton RP, et al. Recombinant activated factor VIIa andhemostasis in critical care: a focus on trauma. Crit Care.2005,9Suppl5(S37-S42.
[184] Holcomb JB, Hoots K, Moore FA. Treatment of an acquired coagulopathy withrecombinant activated factor VII in a damage-control patient. Mil Med.2005,170(4):287-290.
[185] Klemcke HG, Delgado A, Holcomb JB, et al. Effect of recombinant FVIIa inhypothermic, coagulopathic pigs with liver injuries. J Trauma.2005,59(1):155-161,161.
[186] McMullin NR, Kauvar DS, Currier HM, et al. The clinical and laboratory response torecombinant factor VIIA in trauma and surgical patients with acquired coagulopathy. CurrSurg.2006,63(4):246-251.
[187] Schreiber MA, Holcomb JB, Rojkjaer R. Preclinical trauma studies of recombinantfactor VIIa. Crit Care.2005,9Suppl5(S25-S28.
[188] White CE, Schrank AE, Baskin TW, et al. Effects of recombinant activated factor VII intraumatic nonsurgical intracranial hemorrhage. Curr Surg.2006,63(5):310-317.
[189] Johansen K, Daines M, Howey T, et al. Objective criteria accurately predict amputationfollowing lower extremity trauma. J Trauma.1990,30(5):568-572,572-573.
[190] Bechara C, Huynh TT, Lin PH. Management of lower extremity arterial injuries. JCardiovasc Surg (Torino).2007,48(5):567-579.
[191] Walters TJ, Holcomb JB, Cancio LC, et al. Emergency tourniquets. J Am Coll Surg.2007,204(1):185-186,186-187.
[192] Brigham RA, Eddleman WL, Clagett GP, et al. Isolated venous injury produced bypenetrating trauma to the lower extremity. J Trauma.1983,23(3):255-257.
[193] Rich NM. Management of venous trauma. Surg Clin North Am.1988,68(4):809-821.
[194] Dardik H, Wengerter K, Qin F, et al. Comparative decades of experience withglutaraldehyde-tanned human umbilical cord vein graft for lower limb revascularization: ananalysis of1275cases. J Vasc Surg.2002,35(1):64-71.
[195] Gui L, Muto A, Chan SA, et al. Development of decellularized human umbilical arteriesas small-diameter vascular grafts. Tissue Eng Part A.2009,15(9):2665-2676.
[196] Zeljko B, Lovrc Z, Amc E, et al. War injuries of the extremities: twelve-year follow-updata. Mil Med.2006,171(1):55-57.
[197] Rasmussen TE, Clouse WD, Jenkins DH, et al. The use of temporary vascular shunts asa damage control adjunct in the management of wartime vascular injury. J Trauma.2006,61(1):8-12,12-15.
[198] Clouse WD, Rasmussen TE, Perlstein J, et al. Upper extremity vascular injury: a currentin-theater wartime report from Operation Iraqi Freedom. Ann Vasc Surg.2006,20(4):429-434.
[199] Taller J, Kamdar JP, Greene JA, et al. Temporary vascular shunts as initial treatment ofproximal extremity vascular injuries during combat operations: the new standard of care atEchelon II facilities? J Trauma.2008,65(3):595-603.
[2] HUGHES CW. The primary repair of wounds of major arteries; an analysis ofexperience in Korea in1953. Ann Surg.1955,141(3):297-303.
[3] Rich NM, Baugh JH, Hughes CW. Acute arterial injuries in Vietnam:1,000cases. JTrauma.1970,10(5):359-369.
[4] Rich NM, Hughes CW. Vietnam vascular registry: a preliminary report. Surgery.1969,65(1):218-226.
[5] Wedmore I, McManus JG, Pusateri AE, et al. A special report on the chitosan-basedhemostatic dressing: experience in current combat operations. J Trauma.2006,60(3):655-658.
[6] D'Alleyrand JC, Dutton RP, Pollak AN. Extrapolation of battlefield resuscitative care tothe civilian setting. J Surg Orthop Adv.2010,19(1):62-69.
[7] Pusateri AE, Holcomb JB, Kheirabadi BS, et al. Making sense of the preclinicalliterature on advanced hemostatic products. J Trauma.2006,60(3):674-682.
[8] Holcomb J, MacPhee M, Hetz S, et al. Efficacy of a dry fibrin sealant dressing forhemorrhage control after ballistic injury. Arch Surg.1998,133(1):32-35.
[9] Larson MJ, Bowersox JC, Lim RJ, et al. Efficacy of a fibrin hemostatic bandage incontrolling hemorrhage from experimental arterial injuries. Arch Surg.1995,130(4):420-422.
[10] Jackson MR, Friedman SA, Carter AJ, et al. Hemostatic efficacy of a fibrinsealant-based topical agent in a femoral artery injury model: a randomized, blinded,placebo-controlled study. J Vasc Surg.1997,26(2):274-280.
[11] Holcomb J, MacPhee M, Hetz S, et al. Efficacy of a dry fibrin sealant dressing forhemorrhage control after ballistic injury. Arch Surg.1998,133(1):32-35.
[12] Holcomb JB, Pusateri AE, Harris RA, et al. Effect of dry fibrin sealant dressings versusgauze packing on blood loss in grade V liver injuries in resuscitated swine. J Trauma.1999,46(1):49-57.
[13] Pusateri AE, Modrow HE, Harris RA, et al. Advanced hemostatic dressing developmentprogram: animal model selection criteria and results of a study of nine hemostatic dressings ina model of severe large venous hemorrhage and hepatic injury in Swine. J Trauma.2003,55(3):518-526.
[14] Holcomb JB, Pusateri AE, Harris RA, et al. Dry fibrin sealant dressings reduce bloodloss, resuscitation volume, and improve survival in hypothermic coagulopathic swine withgrade V liver injuries. J Trauma.1999,47(2):233-240,240-242.
[15] Pusateri AE, Holcomb JB, Harris RA, et al. Effect of fibrin bandage fibrinogenconcentration on blood loss after grade V liver injury in swine. Mil Med.2001,166(3):217-222.
[16] Pusateri AE, Kheirabadi BS, Delgado AV, et al. Structural design of the dry fibrinsealant dressing and its impact on the hemostatic efficacy of the product. J Biomed Mater ResB Appl Biomater.2004,70(1):114-121.
[17] Morey AF, Anema JG, Harris R, et al. Treatment of grade4renal stab wounds withabsorbable fibrin adhesive bandage in a porcine model. J Urol.2001,165(3):955-958.
[18] Sondeen JL, Pusateri AE, Coppes VG, et al. Comparison of10different hemostaticdressings in an aortic injury. J Trauma.2003,54(2):280-285.
[19] Kheirabadi BS, Acheson EM, Deguzman R, et al. Hemostatic efficacy of two advanceddressings in an aortic hemorrhage model in Swine. J Trauma.2005,59(1):25-34,34-35.
[20] Acheson EM, Kheirabadi BS, Deguzman R, et al. Comparison of hemorrhage controlagents applied to lethal extremity arterial hemorrhages in swine. J Trauma.2005,59(4):865-874,874-875.
[21] Chan MW, Schwaitzberg SD, Demcheva M, et al. Comparison of poly-N-acetylglucosamine (P-GlcNAc) with absorbable collagen (Actifoam), and fibrin sealant (Bolheal)for achieving hemostasis in a swine model of splenic hemorrhage. J Trauma.2000,48(3):454-457,457-458.
[22] Thatte HS, Zagarins S, Khuri SF, et al. Mechanisms of poly-N-acetyl glucosaminepolymer-mediated hemostasis: platelet interactions. J Trauma.2004,57(1Suppl): S13-S21.
[23] Thatte HS, Zagarins SE, Amiji M, et al. Poly-N-acetyl glucosamine-mediated red bloodcell interactions. J Trauma.2004,57(1Suppl): S7-S12.
[24] Favuzza J, Hechtman HB. Hemostasis in the absence of clotting factors. J Trauma.2004,57(1Suppl): S42-S44.
[25] Valeri CR, Srey R, Tilahun D, et al. In vitro effects of poly-N-acetyl glucosamine onthe activation of platelets in platelet-rich plasma with and without red blood cells. J Trauma.2004,57(1Suppl): S22-S25, S25.
[26] Cole DJ, Connolly RJ, Chan MW, et al. A pilot study evaluating the efficacy of a fullyacetylated poly-N-acetyl glucosamine membrane formulation as a topical hemostatic agent.Surgery.1999,126(3):510-517.
[27] Alam HB, Uy GB, Miller D, et al. Comparative analysis of hemostatic agents in a swinemodel of lethal groin injury. J Trauma.2003,54(6):1077-1082.
[28] King DR, Cohn SM, Proctor KG. Modified rapid deployment hemostat bandageterminates bleeding in coagulopathic patients with severe visceral injuries. J Trauma.2004,57(4):756-759.
[29] Connolly RJ. Application of the poly-N-acetyl glucosamine-derived rapid deploymenthemostat trauma dressing in severe/lethal Swine hemorrhage trauma models. J Trauma.2004,57(1Suppl): S26-S28.
[30] Vournakis JN, Demcheva M, Whitson AB, et al. The RDH bandage: hemostasis andsurvival in a lethal aortotomy hemorrhage model. J Surg Res.2003,113(1):1-5.
[31] Jewelewicz DD, Cohn SM, Crookes BA, et al. Modified rapid deployment hemostatbandage reduces blood loss and mortality in coagulopathic pigs with severe liver injury. JTrauma.2003,55(2):275-280,280-281.
[32] Alam HB, Burris D, DaCorta JA, et al. Hemorrhage control in the battlefield: role ofnew hemostatic agents. Mil Med.2005,170(1):63-69.
[33] Wedmore I, McManus JG, Pusateri AE, et al. A special report on the chitosan-basedhemostatic dressing: experience in current combat operations. J Trauma.2006,60(3):655-658.
[34] Wright JK, Kalns J, Wolf EA, et al. Thermal injury resulting from application of agranular mineral hemostatic agent. J Trauma.2004,57(2):224-230.
[35] Ward KR, Tiba MH, Holbert WH, et al. Comparison of a new hemostatic agent tocurrent combat hemostatic agents in a Swine model of lethal extremity arterial hemorrhage. JTrauma.2007,63(2):276-283,283-284.
[36] Kheirabadi BS, Mace JE, Terrazas IB, et al. Safety evaluation of new hemostatic agents,smectite granules, and kaolin-coated gauze in a vascular injury wound model in swine. JTrauma.2010,68(2):269-278.
[37] Pusateri AE, McCarthy SJ, Gregory KW, et al. Effect of a chitosan-based hemostaticdressing on blood loss and survival in a model of severe venous hemorrhage and hepaticinjury in swine. J Trauma.2003,54(1):177-182.
[38] Alam HB, Chen Z, Jaskille A, et al. Application of a zeolite hemostatic agent achieves100%survival in a lethal model of complex groin injury in Swine. J Trauma.2004,56(5):974-983.
[39]范杰,李云龙,李辉.一种基于浙江缙云天然沸石紧急救生止血剂的制备方法,20102010-12-23.
[40]吕小星,陈绍宗,李学拥,等.新型战伤急救止血剂体内抗菌活性的实验研究.现代生物医学进展.2011,11(8):1467-1468.
[41]吴伟萍,彭承宏,韩宝三,等.羧甲基壳聚糖在大鼠创伤出血模型的止血效果的观察.外科理论与实践.2010,15(3):240-244.
[42] Gegel B, Burgert J, Cooley B, et al. The effects of BleedArrest, Celox, and TraumaDexon hemorrhage control in a porcine model. J Surg Res.2010,164(1): e125-e129.
[43] Silverman HG, Roberto FF. Understanding marine mussel adhesion. Mar Biotechnol(NY).2007,9(6):661-681.
[44] Ivankovic H, Gallego FG, Tkalcec E, et al. Preparation of highly porous hydroxyapatitefrom cuttlefish bone. J Mater Sci Mater Med.2009,20(5):1039-1046.
[45] Lu G, Hong W, Tong L, et al. Drying enhanced adhesion of polythiophene nanotubulearrays on smooth surfaces. ACS Nano.2008,2(11):2342-2348.
[46] HUGHES CW. Arterial repair during the Korean war. Ann Surg.1958,147(4):555-561.
[47] Eger M, Golcman L, Goldstein A, et al. The use of a temporary shunt in themanagement of arterial vascular injuries. Surg Gynecol Obstet.1971,132(1):67-70.
[48] Kragh JJ, Walters TJ, Baer DG, et al. Survival with emergency tourniquet use to stopbleeding in major limb trauma. Ann Surg.2009,249(1):1-7.
[49] Woodward EB, Clouse WD, Eliason JL, et al. Penetrating femoropopliteal injury duringmodern warfare: experience of the Balad Vascular Registry. J Vasc Surg.2008,47(6):1259-1264,1264-1265.
[50] Clouse WD, Rasmussen TE, Peck MA, et al. In-theater management of vascular injury:2years of the Balad Vascular Registry. J Am Coll Surg.2007,204(4):625-632.
[51] Sohn VY, Arthurs ZM, Herbert GS, et al. Demographics, treatment, and early outcomesin penetrating vascular combat trauma. Arch Surg.2008,143(8):783-787.
[52] Peck MA, Clouse WD, Cox MW, et al. The complete management of extremityvascular injury in a local population: a wartime report from the332nd Expeditionary MedicalGroup/Air Force Theater Hospital, Balad Air Base, Iraq. J Vasc Surg.2007,45(6):1197-1204,1204-1205.
[53] Clouse WD, Rasmussen TE, Perlstein J, et al. Upper extremity vascular injury: acurrent in-theater wartime report from Operation Iraqi Freedom. Ann Vasc Surg.2006,20(4):429-434.
[54] Fox CJ, Patel B, Clouse WD. Update on wartime vascular injury. Perspect Vasc SurgEndovasc Ther.2011,23(1):13-25.
[55] Vertrees A, Fox CJ, Quan RW, et al. The use of prosthetic grafts in complex militaryvascular trauma: a limb salvage strategy for patients with severely limited autologous conduit.J Trauma.2009,66(4):980-983.
[56] Granchi T, Schmittling Z, Vasquez J, et al. Prolonged use of intraluminal arterial shuntswithout systemic anticoagulation. Am J Surg.2000,180(6):493-496,496-497.
[57] Borut LT, Acosta CJ, Tadlock LC, et al. The use of temporary vascular shunts inmilitary extremity wounds: a preliminary outcome analysis with2-year follow-up. J Trauma.2010,69(1):174-178.
[58] Rasmussen TE, Clouse WD, Jenkins DH, et al. The use of temporary vascular shunts asa damage control adjunct in the management of wartime vascular injury. J Trauma.2006,61(1):8-12,12-15.
[59]汪忠镐,谷涌泉,张建.下肢动脉损伤.北京:人民卫生出版社,2010.
[60] Bolder SB, Verdonschot N, Schreurs BW. Technical factors affecting cup stability inbone impaction grafting. Proc Inst Mech Eng H.2007,221(1):81-86.
[61] Buma P, Arts JJ, Gardeniers JW, et al. No effect of bone morphogenetic protein-7(OP-1) on the incorporation of impacted bone grafts in a realistic acetabular model. J BiomedMater Res B Appl Biomater.2008,84(1):231-239.
[62] Karrholm J, Hourigan P, Timperley J, et al. Mixing bone graft with OP-1does notimprove cup or stem fixation in revision surgery of the hip:5-year follow-up of10acetabularand11femoral study cases and40control cases. Acta Orthop.2006,77(1):39-48.
[63] Deakin DE, Bannister GC. Graft incorporation after acetabular and femoral impactiongrafting with washed irradiated allograft and autologous marrow. J Arthroplasty.2007,22(1):89-94.
[64] Xu ZJ, He RX. Selection of allografts for impaction bone grafting for bone defectreconstruction on the acetabular side. Chin Med J (Engl).2010,123(21):3143-3147.
[65] Mahadevan D, Challand C, Keenan J. Revision total hip replacement: predictors ofblood loss, transfusion requirements, and length of hospitalisation. J Orthop Traumatol.2010,11(3):159-165.
[66] Dunlop DG, Brewster NT, Madabhushi SP, et al. Techniques to improve the shearstrength of impacted bone graft: the effect of particle size and washing of the graft. J BoneJoint Surg Am.2003,85-A(4):639-646.
[67] Arts JJ, Verdonschot N, Buma P, et al. Larger bone graft size and washing of bonegrafts prior to impaction enhances the initial stability of cemented cups: experiments using asynthetic acetabular model. Acta Orthop.2006,77(2):227-233.
[68] Phillips A, Pankaj P, May F, et al. Constitutive models for impacted morsellisedcortico-cancellous bone. Biomaterials.2006,27(9):2162-2170.
[69] Voor MJ, White JE, Grieshaber JE, et al. Impacted morselized cancellous bone:mechanical effects of defatting and augmentation with fine hydroxyapatite particles. JBiomech.2004,37(8):1233-1239.
[70] Bolder SB, Schreurs BW, Verdonschot N, et al. Particle size of bone graft and methodof impaction affect initial stability of cemented cups: human cadaveric and synthetic pelvicspecimen studies. Acta Orthop Scand.2003,74(6):652-657.
[71] Bavadekar A, Cornu O, Godts B, et al. Stiffness and compactness of morselized graftsduring impaction: an in vitro study with human femoral heads. Acta Orthop Scand.2001,72(5):470-476.
[72] Lu H, Pei G, Zhao P, et al. Cyclosporine-impregnated allograft bone sterilized withlow-temperature plasma. J Tissue Eng Regen Med.2010,4(8):638-651.
[73] Cornu O, Bavadekar A, Godts B, et al. Impaction bone grafting with freeze-driedirradiated bone. Part II. Changes in stiffness and compactness of morselized grafts:experiments in cadavers. Acta Orthop Scand.2003,74(5):553-558.
[74]杨颖,罗晖.血小板内皮细胞粘附分子-1(PECAM-1)的研究与疾病.川北医学院学报.6(23):627-631.
[75] Feyen JH, van der Wilt G, Moonen P, et al. Stimulation of arachidonic acid metabolismin primary cultures of osteoblast-like cells by hormones and drugs. Prostaglandins.1984,28(6):769-781.
[76] Lerner UH, Ransjo M, Ljunggren O. Bradykinin stimulates production of prostaglandinE2and prostacyclin in murine osteoblasts. Bone Miner.1989,5(2):139-154.
[77] Abraham LA, MacKie EJ. Modulation of osteoblast-like cell behavior by activation ofprotease-activated receptor-1. J Bone Miner Res.1999,14(8):1320-1329.
[78] Pagel CN, de Niese MR, Abraham LA, et al. Inhibition of osteoblast apoptosis bythrombin. Bone.2003,33(4):733-743.
[79] Mohle R, Green D, Moore MA, et al. Constitutive production and thrombin-inducedrelease of vascular endothelial growth factor by human megakaryocytes and platelets. ProcNatl Acad Sci U S A.1997,94(2):663-668.
[80]张永刚,卢世璧.骨折修复与细胞因子调节.中华外科杂志.1997,35(5):316-318.
[81]王鸿利.止血与凝血机制研究进展.继续医学教育.2006,20(26):13-19.
[82] Karlstrom E, Norgard M, Hultenby K, et al. Localization and expression of prothrombinin rodent osteoclasts and long bones. Calcif Tissue Int.2011,88(3):179-188.
[83] Pagel CN, Sivagurunathan S, Loh LH, et al. Functional responses of bone cells tothrombin. Biol Chem.2006,387(8):1037-1041.
[84] Jenkins AL, Bootman MD, Taylor CW, et al. Characterization of the receptorresponsible for thrombin-induced intracellular calcium responses in osteoblast-like cells. JBiol Chem.1993,268(28):21432-21437.
[85] Abraham LA, Jenkins AL, Stone SR, et al. Expression of the thrombin receptor indeveloping bone and associated tissues. J Bone Miner Res.1998,13(5):818-827.
[86] Frost A, Jonsson KB, Ridefelt P, et al. Thrombin, but not bradykinin, stimulatesproliferation in isolated human osteoblasts, via a mechanism not dependent on endogenousprostaglandin formation. Acta Orthop Scand.1999,70(5):497-503.
[87] Song SJ, Pagel CN, Campbell TM, et al. The role of protease-activated receptor-1inbone healing. Am J Pathol.2005,166(3):857-868.
[88] Ljunggren O, Johansson H, Ljunghall S, et al. Thrombin increases cytoplasmic Ca2+and stimulates formation of prostaglandin E2in the osteoblastic cell line MC3T3-El. BoneMiner.1991,12(2):81-90.
[89] Kozawa O, Tokuda H, Kaida T, et al. Thrombin regulates interleukin-6synthesisthrough phosphatidylcholine hydrolysis by phospholipase D in osteoblasts. Arch BiochemBiophys.1997,345(1):10-15.
[90] Pagel CN, Song SJ, Loh LH, et al. Thrombin-stimulated growth factor and cytokineexpression in osteoblasts is mediated by protease-activated receptor-1and prostanoids. Bone.2009,44(5):813-821.
[91] Hanratty BM, Ryaby JT, Pan XH, et al. Thrombin related peptide TP508promotedfracture repair in a mouse high energy fracture model. J Orthop Surg Res.2009,4(1.
[92] Wang Y, Wan C, Szoke G, et al. Local injection of thrombin-related peptide (TP508) inPPF/PLGA microparticles-enhanced bone formation during distraction osteogenesis. J OrthopRes.2008,26(4):539-546.
[93] Amir LR, Li G, Schoenmaker T, et al. Effect of thrombin peptide508(TP508) on bonehealing during distraction osteogenesis in rabbit tibia. Cell Tissue Res.2007,330(1):35-44.
[94] Li X, Wang H, Touma E, et al. TP508accelerates fracture repair by promoting cellgrowth over cell death. Biochem Biophys Res Commun.2007,364(1):187-193.
[95] Ryaby JT, Sheller MR, Levine BP, et al. Thrombin peptide TP508stimulates cellularevents leading to angiogenesis, revascularization, and repair of dermal and musculoskeletaltissues. J Bone Joint Surg Am.2006,88Suppl3(132-139.
[96] Li G, Ryaby JT, Carney DH, et al. Bone formation is enhanced by thrombin-relatedpeptide TP508during distraction osteogenesis. J Orthop Res.2005,23(1):196-202.
[97] Sheller MR, Crowther RS, Kinney JH, et al. Repair of rabbit segmental defects with thethrombin peptide, TP508. J Orthop Res.2004,22(5):1094-1099.
[98] Wang H, Li X, Tomin E, et al. Thrombin peptide (TP508) promotes fracture repair byup-regulating inflammatory mediators, early growth factors, and increasing angiogenesis. JOrthop Res.2005,23(3):671-679.
[99] Axelrad TW, Kakar S, Einhorn TA. New technologies for the enhancement of skeletalrepair. Injury.2007,38Suppl1(S49-S62.
[100] Mann KG. Prothrombin. Methods Enzymol.1976,45(123-156.
[101] BARNHART MI. Cellular site for prothrombin synthesis. Am J Physiol.1960,199(360-366.
[102] Jamison CS, Degen SJ. Prenatal and postnatal expression of mRNA coding for ratprothrombin. Biochim Biophys Acta.1991,1088(2):208-216.
[103] Norris LA. Blood coagulation. Best Pract Res Clin Obstet Gynaecol.2003,17(3):369-383.
[104] Zoubine MN, Ma JY, Smirnova IV, et al. A molecular mechanism for synapseelimination: novel inhibition of locally generated thrombin delays synapse loss in neonatalmouse muscle. Dev Biol.1996,179(2):447-457.
[105] Glazner GW, Yadav K, Fitzgerald S, et al. Cholinergic stimulation increases thrombinactivity and gene expression in cultured mouse muscle. Brain Res Dev Brain Res.1997,99(2):148-154.
[106] Flynn PD, Byrne CD, Baglin TP, et al. Thrombin generation by apoptotic vascularsmooth muscle cells. Blood.1997,89(12):4378-4384.
[107] Festoff BW, Smirnova IV, Ma J, et al. Thrombin, its receptor and protease nexin I, itspotent serpin, in the nervous system. Semin Thromb Hemost.1996,22(3):267-271.
[108] Suzuki K, Tanaka T, Miyazawa K, et al. Gene expression of prothrombin in human andrat kidneys: basic and clinical approach. J Am Soc Nephrol.1999,10Suppl14(S408-S411.
[109] Trudel G, Uhthoff HK, Laneuville O. Prothrombin gene expression in articular cartilagewith a putative role in cartilage degeneration secondary to joint immobility. J Rheumatol.2005,32(8):1547-1555.
[110] Osterud B, Lindahl U, Seljelid R. Macrophages produce blood coagulation factors. FebsLett.1980,120(1):41-43.
[111] Shim K, Zhu H, Westfield LA, et al. A recombinant murine meizothrombin precursor,prothrombin R157A/R268A, inhibits thrombosis in a model of acute carotid artery injury.Blood.2004,104(2):415-419.
[112] Norris LA. Blood coagulation. Best Pract Res Clin Obstet Gynaecol.2003,17(3):369-383.
[113] Cook SD, Wolfe MW, Salkeld SL, et al. Effect of recombinant human osteogenicprotein-1on healing of segmental defects in non-human primates. J Bone Joint Surg Am.1995,77(5):734-750.
[114]赖翼,吴民泸,代娟,等.两种凝血酶原提取方法的比较.成都医学院学报.2009,01):
[115] Tabata II. The importance of drug delivery systems in tissue engineering. Pharm SciTechnolo Today.2000,3(3):80-89.
[116]陈英杰,许向阳,周建平.乳酸-羟基乙酸共聚物微球的研究进展.中国药科大学学报.2007,38(2):186-189.
[117] Liu J, Meisner D, Kwong E, et al. A novel trans-lymphatic drug delivery system:implantable gelatin sponge impregnated with PLGA-paclitaxel microspheres. Biomaterials.2007,28(21):3236-3244.
[118] Jaklenec A, Wan E, Murray ME, et al. Novel scaffolds fabricated from protein-loadedmicrospheres for tissue engineering. Biomaterials.2008,29(2):185-192.
[119] Kelly JF, Ritenour AE, McLaughlin DF, et al. Injury severity and causes of death fromOperation Iraqi Freedom and Operation Enduring Freedom:2003-2004versus2006. J Trauma.2008,64(2Suppl): S21-S26, S26-S27.
[120] DeBAKEY ME, SIMEONE FA. Battle injuries of the arteries in World War II; ananalysis of2,471cases. Trans South Surg Assoc.1946,57(65-110.
[121] HUGHES CW. Acute vascular trauma in Korean War casualties; an analysis of180cases. Surg Gynecol Obstet.1954,99(1):91-100.
[122] HUGHES CW. The primary repair of wounds of major arteries; an analysis ofexperience in Korea in1953. Ann Surg.1955,141(3):297-303.
[123] Rich NM, Baugh JH, Hughes CW. Popliteal artery injuries in Vietnam. Am J Surg.1969,118(4):531-534.
[124] JAHNKE EJ, HUGHES CW, HOWARD JM. The rationale of arterial repair on thebattlefield. Am J Surg.1954,87(3):396-401.
[125] Gawande A. Casualties of war--military care for the wounded from Iraq andAfghanistan. N Engl J Med.2004,351(24):2471-2475.
[126] Starnes BW, Beekley AC, Sebesta JA, et al. Extremity vascular injuries on thebattlefield: tips for surgeons deploying to war. J Trauma.2006,60(2):432-442.
[127] Fox CJ, Gillespie DL, O'Donnell SD, et al. Contemporary management of wartimevascular trauma. J Vasc Surg.2005,41(4):638-644.
[128] Holcomb J, Caruso J, McMullin N, et al. Causes of death in US Special OperationsForces in the global war on terrorism:2001-2004. US Army Med Dep J.2007,24-37.
[129] Wolf SE, Kauvar DS, Wade CE, et al. Comparison between civilian burns and combatburns from Operation Iraqi Freedom and Operation Enduring Freedom. Ann Surg.2006,243(6):786-792,792-795.
[130] JAHNKE EJ, HOWARD JM. Primary repair of major arterial injuries; report offifty-eight casualties. AMA Arch Surg.1953,66(5):646-649.
[131] JAHNKE EJ, SEELEY SF. Acute vascular injuries in the Korean War. Ann Surg.1953,138(2):158-177.
[132] Rich NM, Rhee P. An historical tour of vascular injury management: from its inceptionto the new millennium. Surg Clin North Am.2001,81(6):1199-1215.
[133] Beekley AC, Watts DM. Combat trauma experience with the United States Army102ndForward Surgical Team in Afghanistan. Am J Surg.2004,187(5):652-654.
[134]陈景云,等.抗美援朝卫生工作经验总结——战伤外科部分:抗美援朝卫生工作经验总结委员会,1953.
[135] Clouse WD, Rasmussen TE, Peck MA, et al. In-theater management of vascular injury:2years of the Balad Vascular Registry. J Am Coll Surg.2007,204(4):625-632.
[136] Owens BD, Kragh JJ, Wenke JC, et al. Combat wounds in operation Iraqi Freedom andoperation Enduring Freedom. J Trauma.2008,64(2):295-299.
[137] Ramasamy A, Harrisson SE, Clasper JC, et al. Injuries from roadside improvisedexplosive devices. J Trauma.2008,65(4):910-914.
[138] Brown KV, Ramasamy A, Tai N, et al. Complications of extremity vascular injuries inconflict. J Trauma.2009,66(4Suppl): S145-S149.
[139] Spencer FC. Historical vignette: the introduction of arterial repair into the US MarineCorps, US Naval Hospital, in July-August1952. J Trauma.2006,60(4):906-909.
[140] SPENCER FC, GREWE RV. The management of arterial injuries in battle casualties.Ann Surg.1955,141(3):304-313.
[141] Welling DR, Burris DG, Hutton JE, et al. A balanced approach to tourniquet use:lessons learned and relearned. J Am Coll Surg.2006,203(1):106-115.
[142] Walters TJ, Wenke JC, Kauvar DS, et al. Effectiveness of self-applied tourniquets inhuman volunteers. Prehosp Emerg Care.2005,9(4):416-422.
[143] Walters TJ, Mabry RL. Issues related to the use of tourniquets on the battlefield. MilMed.2005,170(9):770-775.
[144] Wenke JC, Walters TJ, Greydanus DJ, et al. Physiological evaluation of the U.S. Armyone-handed tourniquet. Mil Med.2005,170(9):776-781.
[145] Rasmussen TE, Clouse WD, Jenkins DH, et al. Echelons of care and the management ofwartime vascular injury: a report from the332nd EMDG/Air Force Theater Hospital, BaladAir Base, Iraq. Perspect Vasc Surg Endovasc Ther.2006,18(2):91-99.
[146] Beekley AC, Starnes BW, Sebesta JA. Lessons learned from modern military surgery.Surg Clin North Am.2007,87(1):157-184.
[147] Sebesta J. Special lessons learned from Iraq. Surg Clin North Am.2006,86(3):711-726.
[148] Beekley AC, Starnes BW, Sebesta JA. Lessons learned from modern military surgery.Surg Clin North Am.2007,87(1):157-184.
[149] Kragh JJ. Use of tourniquets and their effects on limb function in the modern combatenvironment. Foot Ankle Clin.2010,15(1):23-40.
[150] Porter JM, Ivatury RR, Nassoura ZE. Extending the horizons of "damage control" inunstable trauma patients beyond the abdomen and gastrointestinal tract. J Trauma.1997,42(3):559-561.
[151] Aucar JA, Hirshberg A. Damage control for vascular injuries. Surg Clin North Am.1997,77(4):853-862.
[152] Brohi K, Singh J, Heron M, et al. Acute traumatic coagulopathy. J Trauma.2003,54(6):1127-1130.
[153] MacLeod JB, Lynn M, McKenney MG, et al. Early coagulopathy predicts mortality intrauma. J Trauma.2003,55(1):39-44.
[154] Holcomb JB, Jenkins D, Rhee P, et al. Damage control resuscitation: directly addressingthe early coagulopathy of trauma. J Trauma.2007,62(2):307-310.
[155] Alam HB, Rhee P. New developments in fluid resuscitation. Surg Clin North Am.2007,87(1):55-72.
[156] Borgman MA, Spinella PC, Perkins JG, et al. The ratio of blood products transfusedaffects mortality in patients receiving massive transfusions at a combat support hospital. JTrauma.2007,63(4):805-813.
[157] Gonzalez EA, Moore FA, Holcomb JB, et al. Fresh frozen plasma should be givenearlier to patients requiring massive transfusion. J Trauma.2007,62(1):112-119.
[158] Hess JR, Holcomb JB, Hoyt DB. Damage control resuscitation: the need for specificblood products to treat the coagulopathy of trauma. Transfusion.2006,46(5):685-686.
[159] Kauvar DS, Holcomb JB, Norris GC, et al. Fresh whole blood transfusion: acontroversial military practice. J Trauma.2006,61(1):181-184.
[160] Perkins JG, Schreiber MA, Wade CE, et al. Early versus late recombinant factor VIIa incombat trauma patients requiring massive transfusion. J Trauma.2007,62(5):1095-1099,1099-1101.
[161] Fox CJ, Gillespie DL, Cox ED, et al. Damage control resuscitation for vascular surgeryin a combat support hospital. J Trauma.2008,65(1):1-9.
[162] Borgman MA, Spinella PC, Perkins JG, et al. The ratio of blood products transfusedaffects mortality in patients receiving massive transfusions at a combat support hospital. JTrauma.2007,63(4):805-813.
[163] Fox CJ, Starnes BW. Vascular surgery on the modern battlefield. Surg Clin North Am.2007,87(5):1193-1211.
[164] Alam HB, Rhee P. New developments in fluid resuscitation. Surg Clin North Am.2007,87(1):55-72.
[165] Engstrom M, Schott U, Nordstrom CH, et al. Increased lactate levels impair thecoagulation system--a potential contributing factor to progressive hemorrhage after traumaticbrain injury. J Neurosurg Anesthesiol.2006,18(3):200-204.
[166] Lou HC. Correction of increased plasma pyruvate and plasma lactate levels using largedoses of thiamine in patients with Kearns-Sayre syndrome. Arch Neurol.1981,38(7):469.
[167] Nahas GG, Sutin KM, Fermon C, et al. Guidelines for the treatment of acidaemia withTHAM. Drugs.1998,55(2):191-224.
[168] Fox CJ, Perkins JG, Kragh JJ, et al. Popliteal artery repair in massively transfusedmilitary trauma casualties: a pursuit to save life and limb. J Trauma.2010,69Suppl1(S123-S134.
[169] Dawson DL, Putnam AT, Light JT, et al. Temporary arterial shunts to maintain limbperfusion after arterial injury: an animal study. J Trauma.1999,47(1):64-71.
[170] Borut LT, Acosta CJ, Tadlock LC, et al. The use of temporary vascular shunts inmilitary extremity wounds: a preliminary outcome analysis with2-year follow-up. J Trauma.2010,69(1):174-178.
[171] Starnes BW, Arthurs ZM. Endovascular management of vascular trauma. Perspect VascSurg Endovasc Ther.2006,18(2):114-129.
[172] White R, Krajcer Z, Johnson M, et al. Results of a multicenter trial for the treatment oftraumatic vascular injury with a covered stent. J Trauma.2006,60(6):1189-1195,1195-1196.
[173] Lin PH, Bush RL, Zhou W, et al. Endovascular treatment of traumatic thoracic aorticinjury--should this be the new standard of treatment? J Vasc Surg.2006,43SupplA(22A-29A.
[174] Feliciano DV, Mattox KL, Graham JM, et al. Five-year experience with PTFE grafts invascular wounds. J Trauma.1985,25(1):71-82.
[175] Lin PH, Bush RL, Weiss VJ, et al. Subclavian artery disruption resulting fromendovascular intervention: treatment options. J Vasc Surg.2000,32(3):607-611.
[176] Fox CJ, Gillespie DL, Weber MA, et al. Delayed evaluation of combat-relatedpenetrating neck trauma. J Vasc Surg.2006,44(1):86-93.
[177] Holcomb JB, Salinas J, McManus JM, et al. Manual vital signs reliably predict need forlife-saving interventions in trauma patients. J Trauma.2005,59(4):821-828,828-829.
[178] Davis JW, Shackford SR, Mackersie RC, et al. Base deficit as a guide to volumeresuscitation. J Trauma.1988,28(10):1464-1467.
[179] Eastridge BJ, Owsley J, Sebesta J, et al. Admission physiology criteria after injury onthe battlefield predict medical resource utilization and patient mortality. J Trauma.2006,61(4):820-823.
[180] Sebesta J. Special lessons learned from Iraq. Surg Clin North Am.2006,86(3):711-726.
[181] Holcomb JB. Methods for improved hemorrhage control. Crit Care.2004,8Suppl2(S57-S60.
[182] Holcomb JB. Use of recombinant activated factor VII to treat the acquired coagulopathyof trauma. J Trauma.2005,58(6):1298-1303.
[183] Mohr AM, Holcomb JB, Dutton RP, et al. Recombinant activated factor VIIa andhemostasis in critical care: a focus on trauma. Crit Care.2005,9Suppl5(S37-S42.
[184] Holcomb JB, Hoots K, Moore FA. Treatment of an acquired coagulopathy withrecombinant activated factor VII in a damage-control patient. Mil Med.2005,170(4):287-290.
[185] Klemcke HG, Delgado A, Holcomb JB, et al. Effect of recombinant FVIIa inhypothermic, coagulopathic pigs with liver injuries. J Trauma.2005,59(1):155-161,161.
[186] McMullin NR, Kauvar DS, Currier HM, et al. The clinical and laboratory response torecombinant factor VIIA in trauma and surgical patients with acquired coagulopathy. CurrSurg.2006,63(4):246-251.
[187] Schreiber MA, Holcomb JB, Rojkjaer R. Preclinical trauma studies of recombinantfactor VIIa. Crit Care.2005,9Suppl5(S25-S28.
[188] White CE, Schrank AE, Baskin TW, et al. Effects of recombinant activated factor VII intraumatic nonsurgical intracranial hemorrhage. Curr Surg.2006,63(5):310-317.
[189] Johansen K, Daines M, Howey T, et al. Objective criteria accurately predict amputationfollowing lower extremity trauma. J Trauma.1990,30(5):568-572,572-573.
[190] Bechara C, Huynh TT, Lin PH. Management of lower extremity arterial injuries. JCardiovasc Surg (Torino).2007,48(5):567-579.
[191] Walters TJ, Holcomb JB, Cancio LC, et al. Emergency tourniquets. J Am Coll Surg.2007,204(1):185-186,186-187.
[192] Brigham RA, Eddleman WL, Clagett GP, et al. Isolated venous injury produced bypenetrating trauma to the lower extremity. J Trauma.1983,23(3):255-257.
[193] Rich NM. Management of venous trauma. Surg Clin North Am.1988,68(4):809-821.
[194] Dardik H, Wengerter K, Qin F, et al. Comparative decades of experience withglutaraldehyde-tanned human umbilical cord vein graft for lower limb revascularization: ananalysis of1275cases. J Vasc Surg.2002,35(1):64-71.
[195] Gui L, Muto A, Chan SA, et al. Development of decellularized human umbilical arteriesas small-diameter vascular grafts. Tissue Eng Part A.2009,15(9):2665-2676.
[196] Zeljko B, Lovrc Z, Amc E, et al. War injuries of the extremities: twelve-year follow-updata. Mil Med.2006,171(1):55-57.
[197] Rasmussen TE, Clouse WD, Jenkins DH, et al. The use of temporary vascular shunts asa damage control adjunct in the management of wartime vascular injury. J Trauma.2006,61(1):8-12,12-15.
[198] Clouse WD, Rasmussen TE, Perlstein J, et al. Upper extremity vascular injury: a currentin-theater wartime report from Operation Iraqi Freedom. Ann Vasc Surg.2006,20(4):429-434.
[199] Taller J, Kamdar JP, Greene JA, et al. Temporary vascular shunts as initial treatment ofproximal extremity vascular injuries during combat operations: the new standard of care atEchelon II facilities? J Trauma.2008,65(3):595-603.