Study On The Freeze-drying Theory Of Porcine Aortic Vascular And Its Application

Vascular graft is an effective method for the treatment of some seriouscardiovascular disease except the medical treatment. In the choice of the graft material,natural biological tissue can overcome the disadvantages of artificial materials intechnical and functional aspects. While how to process and preserve these biologicalmaterial has always been a concerning problem. In this subject, vacuum freeze-dryingtechnology is used in processing porcine aortic blood vessels, it has the uniqueadvantages in terms of physical and mechanical properties, as well as the structurekeeping after vascular freeze-dried. At the same time, surgeries for allotransplantationsof freeze-dried vascular and decellularized vascular after freeze-dried have been tried, inorder to study and vascular study and evaluate the feasibility of preparing the vesselscaffold by freeze-drying technology and expect to provide high-qualified and reliablevascular graft for the clinical treatment of the related diseases.Obvious shrinkage and wall thinning had been found on the wall of the porcinvasculars after they had been freeze-dried which showed that the internal structure ofthe vessel must have been occurred collapse and destruction, therefore, this subjectfirstly aimed to optimize the freeze-drying method. Through adjustments of theexperimental scheme for many times, assisted by surface observation, Micro-CTscanning of internal structure, porosity calculated and three-dimensional composition,the structural changes in degree had been verifid, so the optimized measures of thefreeze-drying the vessels were found, simultaneously interpreting and analyzing reasonsfor vessels shrunken, and pointed out that the judgment on the end of primary dryinghad a great influence on the quality of the dried samples. Vasculars freeze-dried byoptimized method, collapse and shrinkage had been eliminated, the porosity of thefreeze-dried samples was about34.42%, dehydration rate was70.91%approximately,and the rehydration capacity was about2.37, which were superior to usual way whosefreeze-dried porosity was29.26%, dehydration rate was70.37%and rehydrationcapacity was2.28. Experimental result also included the water content of porcin aortic,(72.01±0.50)%measured by vacuum drying.On the other hand of theoretical study was simulation to the temperature changesof quick cooling of the vessel, using FLUENT software. According to the recorded actual datas, simulated results above zero were found closer to the actual temperaturechanges, while there existed deviation to subzero when the vessel was freezing, butwhole trend was consistent with actual process. The interaction of the convective heattransfer and radiative heat transfer with surrounding environment caused the minimumtemperature of the blood vessels (-40~-44℃) at cooling course can not reach the shelftemperature (-70℃), so this study estimated the outside of mutual heat exchange wasapproximately1273.5J from start of cooling until to the minimum temperature. Inprimary and secondary drying, actual latent sublimation heat of the water in vascularand changes about quality were calculated every half an hour, comparing with the latentsublimation heat of pure water in ice phaseion at the same temperature. Combined withthe changed quality value, illustrations were taken that how the dried interface moved,and the path water escaping, various difficulty degrees in each stage and their reasons,finally, mechanism of freeze-drying were further researched.Freeze-dried vessels were rehydrated, the whole process was scanned by Micro-CTalong with the cross-sectional wall-thickness changes. Shape changing from therehydrated0.5h to1h can be seen reached to maximum, and during rehydration, theporosity gradually decreased until it closed to the fresh vessel, and rehydration capacityand the change of rehydration rate were also calculated. Results showed that therehydration from0.5h to1h was the phase of rapid moisture absorption, and all of thedatas had the obvious changes. From1h to3h, moisture absorptions were taken onlinear growth. After3h, vascular moisture and rehydration rate were dynamicadjustments and changes, absorbent achieving saturated basically. Detail mechanismanalysises were carried on, explaining the reason for the various changes, and finallypointed out, porcine aortic vasculars can be applied on transplanted when they weresoaked in saline for2～2.5h under normal temperature.Mechanical properties as circumferential tesile, axial tensile and puncture weretested on rehydrated vasculars for exploring whether the vascular transplanted into thebody can withstand the pressure of the blood in all directions. Macroscopic mechanicaltest results were combined with the microscopic histological observations which thefresh and the rehydrated vessels were sliced and stained. Results showed that: duringthe circumferential tensile, within4mm around of the stretched length was the processof elastic deformation, while this deformation of the axial tensile appeared within2～ 2.5mm around. These are related to the structure characteristics of the fibers in vessel.Specific trend on curves of the rehydrated circumferential tensile, axial tensile andpuncture process were closer to these curves of fresh vessels, expressing that structualcomponents and their arrangement ways of the freeze-dried vessels were not changedgreatly. Stress-strain rate relations presented two phases in circumferential tensile whichcan be noted as elastic modulus from small to large, and the axial tensile showing thefour stages of the elastic modulus firstly small then increased, and suddenly decreasedthen increased, while these changes of the rehydrated vessel were more obvious. This iscaused by the emergence of inter-fiber pores and the elastic fibers longitudinally aligned.In the aspect of stretched length, elongation rate of the circumferential tensile of thefresh and freeze-dried vessels were177%and311%respectively, these values of theaxial tensile were459%and300%respectively, and the maximum circumferentialtensile stress of the rehydrated vessel reduced18.6%by the fresh and rehydrated axialtensile stress, puncture force increased27.0%and9.88%respectively. Reasons for themacroscopic mechanical changes were explained, with the results of microscopicobservation.Experimental studies above were maily for providing the theoretical basises to gethigh-quality transplanted vasculars. In the actual clinical application, elimination of theimmune rejection after implantation in hosts was another important factor in migration,as well as in this subjuct.Decellularized scaffold made of extracellular matrix (ECM) was received withacellular treatment of the freeze-dried vessel. By means of the porosity caused byfreeze-drying, acellular reagents only needed lower concentrations and shorteracellulared time. When the cells of freeze-dried vessel were removed completely withthe experimental concentrations and acellulared time, the middle of wall in the freshvessel still had residual cells. That means the fresh vessels required higherconcentrations of reagents and the longer time, which would inevitably lead to thedestruction of the organizational structure and various fibers, in addition, acellularingthe freeze-dried vessels would have a greater advantage than the fresh in themaintenance of the integrity of the structures, mechanical properties.In order to test the acellular degree of vessel, beside of histological observation,Transmission Electron Microscopy (TEM) and Scanning Electron Microscopy (SEM) were used to observe the ultrastructure in depth. No cells are seen, and fibers werearranged orderly, whole state was relatively good. Results of antigen expression contentfor MHC-Ⅰ and MHC-Ⅱ results revealed the freeze-dried vasculars afterdecellularized was significantly lower (P <0.01) in MHC-Ⅰ and MHC-Ⅱ antigenexpressions, as well, removal of DNA molecule by electrophoresis test was quiteobvious.Surgeries transplanted the decellularized freeze-dried vasculars and freeze-dried(not decellularized) vasculars respectively for comparison. After3months histologicalobservation displayed that it existed visible inflammatory response and microthrombi insome part which woud seriously affect the long-term patency rate and the host life,however, in freeze-dried acellulared vessel, these phenomena were not discovereddistinctively. In these vessels, there were the intima regenerated and host cells infiltratedin intima, tunica media and extima. Except for the not fully soakage in some part of thetunica media, the rest of the structures all found the new cells. Dispending onangiocardiography, higher patency degree was taken on the freeze-dried acellularedvessel, in addition, diameter stenosis and hemangioma were almost not found whilethere were some signs of narrow diameter and embolization in the transplantedfreeze-dried vessel which illuminated the decellularized freeze-dried vessel’sallotransplantation had a good state in many aspects.All the theoretical studies and application tests showed that, the decellularizedfreeze-dried vascular stent used for clinical transplantation is feasible.