CHRZANOWSKA Olga ，STRUSZCZYK Marcin Henryk，KRUCINSKA Izabella

Department of Material and Commodity Sciences and Textile Metrology，Center of Advanced Technologies of Human Friendly Textiles PRO HUMANO TEX，F(xiàn)aculty of Material Technologies and Textile Design，University of Technology，Lodz 90-924，Poland

Introduction

The reconstruction of the small diameter blood vessels using the artificial prostheses (endo-or standard grafts)is still a significant problem due to the early occlusions or/and thrombosis of the grafted implants［1-3］.

The elaboration of the multilayered small diameter vascular prostheses using the melt-spinning techniques becomes more attractive.Based on Refs.［4 -5］，it is well-known the use of a combination of electrospinning using the polymer solution with melt-electrospinning or melt-spinning to manufacture the small diameter (less than 6 mm)vascular grafts.Our purpose is to produce the multilayered tubular structures using only meltelectrospinning and melt-blown techniques［6-8］.The idea of design of the semi-biodegradable vascular prosthesis was described in Ref.［6］.The biodegradable outside layer of the prosthesis made of poly (L-lactide-co-glicolide)(PLAGA)effecting on the reduction in the mass of the implant and allowing to control in-growth of tissue，is responsible for assuring the intraoperative surgical tightness of the graft.Also，the similar biodegradable inside layers supporting additionally intraoperative surgical tightness can act as carriers for endothelialisation.The middle layer of polypropylene (PP)fibrous structures is the main non-biodegradable，high strength framework of the prosthesis.

The aim of the research，as a part of the study for the elaboration of new design of the textile vascular prostheses for the microreconstruction，was to select the most optimal content of the multilayered vascular prostheses taking into account the final processing stages effect including sterilization by various sterilization agents (ethylene oxide(EO)，steam and radiation)on the main physical and mechanical properties.The isolated layers of the final multilayered structures of the final vascular graft were investigated.

1 Materials and Methods

1.1 Materials

PLAGA (85/15) was purchased from the Centre of Polymer and Carbon Materials，Polish Academy of Sciences(Poland)，whereas bormed HF840MO PP was supplied from its manufacturer Borealis (Austria).The main parameters of the raw materials are listed in Table 1.

Table 1 The main properties of the raw materials in the study

1.2 Methods

The melt-electrospinning and melt-blown were made using the HaakeMiniLab (Thermo Scientific，Germany)co-rotating twin-screws extruder.The specially made spinning head，allowing fabrication of the melt-blown and melt-electrospun structures was applied.The collector with rotating mandrel of 5 mm in diameter was also used.The two techniques of the fibrous structures fabrication:melt-electrospinning and meltblown were applied.The parameters of the process are presented in Table 2 (melt-electrospinning)and Table 3 (melt-blown technique).

Table 2 Processing parameters for the melt-electrospinning of PLAGA or PP

Table 3 Processing parameters for the melt-blown technique for PP

The melt-electrospun tubular structures of PLAGA were additionally heated in the oven at 100 ℃ for 90 s (thermal stabilization).This treatment of the melt-electrospun tubular structures of PLAGA was necessary mainly for the stabilization of received structure resulted in the increase in the mechanical properties.

Then the obtained tabular structures were packed into the medical packaging (self-seal sterilization pouches;OPM/Poland)susceptible for the radiation，steam，and EO sterilization.

The steam sterilization was carried out at the Sterilization Plant (TZMO SA/Poland)at 121 ℃according to the PN-EN ISO 17665-1∶2008 Standard.Radiation sterilization was carried out in Institute of Nuclear Chemistry and Technology (Poland)using the radiation dose of 28 kGy in validated condition according to PN-EN ISO 11137-1/2/3 Standard，whereas the EO sterilization was made in CSP Technochemia (Poland)in validated conditions according to PN-EN ISO 11135-1 ∶2009 Standard.

1.3 Analytical methods

1.3.1 Surface mass

The surface mass of the designed fibrous structures before and after sterilization processes was determined according to PNEN ISO 29073-1∶1994 Standard.

1.3.2 Thickness

The thickness of the designed fibrous structures before and after sterilization processes was determined according to PN-EN ISO 9073-2∶2002 Standard.

1.3.3 Apparent density and porosity

The apparent density and porosity of the designed fibrous structures before and after sterilization processes were determined according to ASTM D2654∶1998 Standard.

1.3.4 Mechanical properties

For determination of tubular structures tensile properties Instron 5511 testing machine was used.The tests were made according to the ISO 7198 ∶ 1998 and PN-EN 29073-3 Standards.The specimens were stretched at a steady rate of 50 mm/min.The maximum load，stress at the maximum load，elongation at the maximum load，and elastic modulus were determined in longitudinal and circumferential directions of tubular structures.

2 Results and Discussion

The macroscopic superficies of elaborated PP or PLAGA tubular，fibrous structures using the various techniques and after the sterilization by various agents:radiation，EO，and steam sterilization are shown in Fig.1.The process of the thermal stabilization that was used for the PLAGA melt-electrospinning tubular structures only，resulted in the more compacted fibrous structure and significant reduction in wall thickness (up to 0.15 mm as shown in Table 4).The process of the sterilization by EO or steam was leaded to the total destruction of the fibrous structure of the graft prototypes，whereas the application of the radiation did not make any macroscopic changes.All tested radiation agents did not influence on the macroscopic superficies of the PP originated grafts designed by the melt-electrospinning and melt-blown techniques.

Fig.1 The macroscopic superficies of elaborated PP or PLAGA tubular，fibrous structures using the various techniques and after the sterilization by various agents

The physical properties of the elaborated fibrous，tabular structures of PP or PLAGA are shown in Table 4.The thermal stabilization of the PLAGA melt-electrospun tubular structures yielded in the decrease in the porosity by approximate 15% and the increase in the apparent density by 300% as compared with initial sample.The radiation sterilization， on one hand，effectively allows sterilizing the elaborated PLAGA structures，did not significantly alter their physical properties.On the other hand，the steam sterilization of PP melt-electrospun tubular structures resulted in the decrease in the thickness of the wall of graft and it surface mass without any changes in the apparent density and porosity (Table 5).The significant increase in the surface mass was observed for the PP melt-electrospun tubular structures underwent EO or radiation sterilization without the influence for other physical parameters.

Table 4 The physical properties of PLAGA melt-electrospun tubular structures after the final processing stages and the sterilization using the various serialization agents

Table 5 The physical properties of PP melt-electrospun tubular structures after the final processing stages and the sterilization using the various serialization agents

The sterilization process using all agents:EO，steam，and radiation，did not provide the alteration in the studied physical parameters of PP melt-blown fibrous tubular structures (Table 6).

Table 6 The physical properties of PP melt-blown tubular structures after the final processing stages and the sterilization using the various serialization agents

The thermal stabilization of PLAGA melt-electrospun tubular structures yielded in the significant increase in the maximal load in both directions: longitudinal and circumferential (Tables 7 and 8).The increase in Young modulus，stress at the maximal load as well as elongation at the maximal load was also observed.The sterilization process，using the radiation as a sterilization agent，leaded to the reduction of the maximal load by approx imate 50%.Moreover，the elongation at the maximal load in both directions also decreased.The observed phenomenon is connected with the degradation effect of the radiation on the PLAGA molecules.

Table 7 The mechanical properties in longitudinal direction of PLAGA melt-electrospun tubular structures after the final processing stages and the sterilization using the various serialization agents

Table 8 The mechanical properties in circumferential direction of PLAGA melt-electrospun tubular structures after the final processing stages and the sterilization using the various serialization agents

PP melt-electrospun fibrous structures submitted to the radiation showed the similar to the initial maximal load in longitudinal direction and reduction by approx imate 50% in the parameter determined in circumferential direction (Tables 9 and 10).The elongation at the maximal load was reduced in case of the steam and radiation sterilization if measured in longitudinal direction.

Table 9 The mechanical properties in longitudinal direction of PP melt-electrospun tubular structures after the final processing stages and the sterilization using the various serialization agents

Table 10 The mechanical properties in circumferential direction of PP melt-electrospun tubular structures after the final processing stages and the sterilization using the various serialization agents

Moreover，the reduction in elongation at the maximal load in circumferential direction occurred if EO and radiation for the sterilization were used.

Application of the melt-blown for the fabrication of the vascular graft prototypes resulted in the mechanical properties that were incomparable to those received for PP meltelectrospun tubular structures， both in longitudinal and circumferential directions (Tables 11 and 12).The sterilization process (undependable of the sterilization agent used)yielded in this case in reduction in the maximal load and stress at the maximal load measured in longitudinal direction，whereas no significant changes in above-mentioned parameters was found，if they were determined in circumferential direction(except the maximal load determined after radiation sterilization showing the increase by approximate 19% as compared with initial sample).The elongation at the maximal load indicated the highest values as compared with PP meltelectrospun tubular structures.

Table 11 The mechanical properties in longitudinal direction of PP melt-blown tubular structures after the final processing stages and the sterilization using the various serialization agents

Table 12 The mechanical properties in circumferential direction of PP melt-blown tubular structures after the final processing stages and the sterilization using the various serialization agents

The research showed the effect of the various agents helpful for the modeling the proper properties of designed vascular prostheses.The elaborated multilayered structure showed significantly higher mechanical strength as compared with the natural vessels.However， there is the significantly high variance of the testing method using for the estimation of the mechanical characteristic of low diameter blood vessels (i.e.，carotid or popliteal arteries).The variance of the testing methods used makes difficult to the compare the native blood vessels and elaborated grafts mechanical behaviors.The most validated study［9］showed that the tensile strength in longitudinal or circumferential direction ranged from approximate 0.5 -2.0 MPa depending on the layer of the ceratoid arteries，whereas extensibility (elongation)did not reach 2%.In reference to the results obtained in our research，the assumption effect for the multilayered structure should be taken into the account.

In other hand， the in vivo tested electrospun polycarpolactone-collagen scaffolds［10］showed the maximal load in circumferential direction by approximate 2 MPa which is comparable to the results obtained for our research，especially if the final multilayered structure of designed grafts will be taken into the account.

3 Conclusions

Process of the thermal stabilization of the PLAGA meltelectrospun tubular，fibrous structures positively affected the final mechanical properties of the biodegradable prototypes of the vascular grafts.Additionally the resulted structure after the stabilization showed the better surgical handling behavior(described by the essential reduction in thickness as well as increase in the mechanical resistance，both in longitudinal and circumferential directions).Above phenomenon is probably related with the changes in the microstructures of the fibers resulted from the thermal alterations in microstructures of the PLAGA.The research on the impact of the thermal stabilization on physical and structural parameters of the obtained tubular structures will be continued.The choice of the optimal technique for the elaboration of the vascular prostheses is also very important step as showed in the characterization of the PP grafts made by melt-electrospinning or melt-blown technique.

The selection of the optimal sterilization agents is a most important part of the design process of the medical devices.It should be characterized by the low effect on the mechanical，physical，chemical，and biological properties.The EO and steam sterilizations had a destructive effect on the designed PLAGA melt-electrospun tubular，fibrous structures，whereas the radiation yielded the changes in mechanical and physical properties on the acceptable level，taking into account the future clinical applications.

The more complicated is the selection of the proper methods for the non-biodegradable PP fibrous tubular structures made of the two non-conventional techniques:melt-blown and melt-electrospinning.The most optimal method of the sterilization for the future-designed semi-biodegradable multilayered tubular structures is radiation sterilization，taking into account the effect of the sterilization on both PLAGA and PP tubular structures properties.

The next step of the research will be the investigation of the sterilization effect on the macro and molecular structure of the designed PP and PLAGA tubular structures to explain in more detailed way the found phenomena.

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