Find best premium and Free Joomla templates at GetJoomlaTemplatesFree.com

Development of Artificial Blood Vessel Using Bacterial Cellulose

Kwon Mun Hyok¹ , Han Gyong Ae²

¹ Department of Pediatric Orthopaedics, Pyongyang Medical University Hospital, DPR Korea

² Department of Biochemistry, KIM IL SUNG University, DPR Korea

 

 

Correspondence:

Kwon Mun Hyok

Paediatric Orthopaedic Department, Pyongyang Medical University Hospital

Ryon Hua Dong, Central District, Pyongyang City, DPR Korea

E-mail : This email address is being protected from spambots. You need JavaScript enabled to view it.

 

Abstract

Background: Development of new artificial blood vessel is very important issue in vascular surgery because vascular injuries are growing higher and especially, most of vascular substitutes are not available in the surgery related to small diameter vessel less than 6mm. The aim of this study was to review basic characteristics for application of bacterial cellulose into artificial blood vessel.

Materials and Methods: We used Acetobacter xylinum 10 for production of bacterial cellulose and manufactured several devices to gain tubular bacterial cellulose and investigated its effectiveness mechanically and pathologically.

Results: Suitable carrier was 4µm-thick polyethylene for high cellulose production and suitable culture period was18 days. Young’s modulus (GPa) of tubular Bacterial Cellulose gained in new culture device was 13.6 GPa and strain (%) was five times higher than cellophan. Tubular Bacterial Cellulose (BC) had a quite low porosity (0.00107 ± 0.00011 ml/mm²· min·120mmHg) and thickness of capsule around the graft was peak (42.2±0.84 µm) in the 60 days after implantation and significantly thinner than PTFE throughout investigated period and change of inflammatory cell numbers had same trend with that of capsule.

Conclusion: Our results suggest that tubular bacterial cellulose has suitable mechanical properties and good biocompatibility for application of artificial blood vessel.

Key words: bacterial cellulose, artificial blood vessel, biocompatibility

  

Introduction

Cellulose is most abundant polymer in the world and mainly, produced by plants. In 1886, one researcher had first found that microorganisms produce a thick gelatinous material at air/culture medium interface in the presence of carbohydrates[1,3]. Many studies on this material were performed since this discovery and it was shown that this material was cellulose which had same composition and structure with cellulose produced by plants, but different high-order structures. This cellulose can be produced by using microorganisms, especially Acetic acid bacteria a cellulose produced by microorganisms will be referred to as “bacterial cellulose (BC)” hereinafter and used as industrial, medical and chemical material.

For example, in the medical field, the hollow bacterial cellulose can be used as substitute for internal hollow organ such as ureter, trachea, digestive tract, lymphatic vessel or blood vessel and many studies are performing to reveal its practical effectiveness.

The bacterial cellulose has network structure in which fine ribbon-shaped fibers composed of highly crystalline and uniaxially oriented cellulose are complicatedly entangled with one another[3]. On the other hand, this network structure contains a large quantity of water in the interior voids and has a gel-like appearance. Interestingly, water contained in the voids is present as free water and can be easily squeezed out[4]. Regardless of this, bacterial cellulose has a high strength even in wet state. It is because bacterial cellulose is composed of very fine ribbon-shaped fibers smaller than 100nm in width and diameter[1,2]. On the other hand , since the bacterial cellulose has no plasticity and the contained liquid components is not restrained , it is difficult to directly mold gelatinous bacterial cellulose into desirable shape or size while utilizing original structure and characteristics.

For example, hollow product such as pipe, cannot be obtained from bacterial cellulose obtained by conventional technique and therefore, application of bacterial cellulose in medical fields is remarkably restricted with regard to its shape, size and processibility.

So we performed this study to estimate whether tubular BC obtained by several devices we made can be used as artificial blood vessel.

Table 1. Porosity of tubular cellulose obtained by new culture device.

Experimental groups Porosity(ml/mm² * min * 120mmHg)
Bacterial cellulose-0.010mm 0.00125±0.00013
Bacterial cellulose-0.025mm 0.00107±0.00011
428-polyester artificial blood vessel 1650±0.55
bacterial cellulose 1 bacterial cellulose 2
Figure1. Straight-and Y-shape culture devices for tubular BC production. Figure 2. Amount of cellulose production according to culture period in culture device.

Methods & Methods

We used Acetobacter xylinum 10 isolated by biochemistry department, KIM IL SUNG University. We first tested many membranes in the regard to O2 permeability for high cellulose production. We compared with cellulose productability of various membranes on the air/culture medium surface and then selected suitable membrane for manufacture of culture device. Secondly, we manufactured several culture devices having straight-and Y-shape that resembles blood vessels located in human body using selected membrane. After that, we measured Young’s modulus, strain and porosity of tubular BC obtained by selected culture device (estimation of mechanical properties). Thirdly, we implanted tubular BC into muscle of rabbits weighting 1.8~2.4kg regardless of its gender and pathologically investigated the thickness of capsule and changes of inflammatory cell numbers around implanted BC and ePTFE (expended polytetrafluoroethylene) was selected as control and statistically, all data were shown as ± SEM and other supplementary data were not presented here in detail.

Results and discussion

From data on cellulose production, we found out that 4µm-thick polyethylene membrane was suitable in view of cellulose productability, so we made culture devices using this membrane for production of tubular bacterial cellulose. We also gained straight- and Y-shaped tubular BC with different diameters depending on diameter and shape of the carrier in the static culture using this devices(Fig.1). And then we investigated the amount of cellulose production according to culture period in interior surface of this device after filling with culture medium containing Acetobacter xylinum10 (Fig.2). Figure 2 showed that amount of cellulose in this device was no longer increased after 18 days of culture. So we determined that suitable culture period for cellulose production was 18 days. In the view of cellulose productability , we re-estimated usefulness of this device.

Table 2. Thickness of connective tissue’s capsule after implantation of grafts into muscle of rabbits (µm)

Groups
(number)
Period(d) 30 Period(d) 60 Period(d) 90 Period(d) 180
BC(n=12) 10.1±0.61* 42.2±0.84* 38.6±0.89* 28.2±0.55*
ePTFE(n=12) 14.8±0.89 54.4±0.69 47.9±0.72 32.1±0.61

 

This device was useful, but had limitation, that is, the amount of cellulose production was reduced in case that the carrier became less than 6~8mm in diameter, since broth volume per surface area of carrier becomes smaller. Thickness of tubular BC produced by this device was so thin and fragile. So we developed new culture device in order to increase cellulose production and therefore obtain thick tubular BC. Because A. xylinum 10 has no ability of gas formation, we focused only on a reduction of broth to keep original tubular shape of carrier.

The cross-sectional view of new device was shown in Figure 3.

bacterial cellulose 3 bacterial cellulose 4
 Figure 3. Cross-sectional view of new culture device.  Figure 4. Young’s modulus and strain of tubular cellulose obtained by new culture device.

Using this device we could actively control the broth volume per surface area through changes of glass diameter(R) when tubular BC is formed in this device and gain the thick tubular BC enough to resist external force. Then we measured mechanical properties of tubular BC by using new culture device in order to determine whether this cellulose tube is appropriate or not for vascular substitute.  As shown in Figure 4, Young’s modulus (13.6GPa) of tubular BC gained by this culture device was smaller than that of plate BC , but strain was five times higher than cellophan.

Tubular BC had enough strength and strain to resist external force in wet state, but didn’t keep its original shape and size when it was expressed in room temperature. That is, this tubular BC was rapidly changed into very thin dry sheet because this gelatinous BC couldn’t restrain water in the voids. So we immersed this gelatinous BC into glycerin solution after finishing its culture and investigated its shape change in the same conditions. This BC also took same routine, but had the nearly original shape when it was re-immersed into water. This fact showed we should have more research to improve a quality of tubular BC as a substitute for blood vessel. Based on these data, we measured its porosity because porosity is important element for vascular substitute. Tubular BC had a quite low porosity and this data showed no need for preclotting when this material was applied as a substitute for blood vessel.

Bacterial cellulose had enough strength and strain and porosity suitable for artificial blood vessel. After determining its mechanical properties, we investigated biocompatibility of tubular BC after implantation of tubular cellulose into muscle of rabbits. We selected expended polytetrafluoroethylene(ePTFE) as a control, which is a original material of Gore-tex artificial blood vessel. Thickness of capsule around the graft was peak (42.2±0.84 µm) in the 60 days after implantation and significantly thinner than ePTFE throughout investigated period. This result showed that this material had less irritation and foreign reaction than control (ePTFE) which is known as good biocompatibility with living tissue because thickness of capsule around graft depends mainly upon local inflammatory reactions. In addition to capsule thickness, we also pathologically investigated the change of inflammatory cell numbers in the tissues around graft (fig. 5). This result agreed with the above data referred to capsule’s thickness, that is, this means tubular BC had less inflammation reactions than control (ePTFE). From these data, we recognized tubular BC obtained by our method had good mechanical properties and biocompatibility suitable for artificial blood vessel.

bacterial cellulose 5

Figure 5. Change of numbers of infiltrated inflammatory cells and fibrocytes in tissues around grafts according to investigated period.

Summary

We developed new culture device using 4 µm-thick polyethylene membrane appropriate for manufacture of tubular BC and estimated mechanical properties of tubular BC and then tested its biocompatibility with a living tissue. All results showed that tubular BC we made was suitable for the artificial blood vessel and had good biocompatibility. Bacterial cellulose has these advantages, but still has limitation. In medical field, hollow fiber is available,but we didn’t make it into hollow cellulose and therefore we should have more researches to make tubular BC into hollow fibers. In the future we also should investigate in situ changes of tubular BC and its blood compatibility when it is really used as a substitute for blood vessel in experimental animals (blood compatibility) and estimate its usefulness in human.

 

 

REFERENCES

1. Han Gyong Ae. A study on massive production and some applied developments of bacterial cellulose. A thesis of biological doctor 2003; 110~129

2. Kim Jong Pok. Manufacturing of ultrafiltration membrane by bacterial cellulose. KIM IL SUNG university articles(natural science) 2002; 48(4):111~113

3. Aika S. et al..Screening of bacterial cellulose-producing strains suitable for sucrose as a carbon source. Biosci. Biotech. Biochem. 1997; 61(4) : 725~738

4. Gehr K. D. et al..Non woven fabric-like product using a bacterial cellulose binder and method for its preparation. PCT WO89/01047