Study On Therapeutic Products Applied During Rescue By Stages For Combat Severe Extremity Trauma

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