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Purification and biocompatibility of fermented hyaluronic acid for its applications to biomaterials.

Choi S, Choi W, Kim S, Lee SY, Noh I, Kim CW - Biomater Res (2014)

Bottom Line: While low molecular weight impurities and insoluble impurities were successfully removed using a ultrafiltration membrane with 50 KDa molecular weight cut, endotoxins, high molecular weight proteins and nucleic acids were removed from the broth by employing adsorbents such as alumina and activated carbons.Alumina showed the best results for the removal of endotoxins, all of the activated carbons were very effective in the removal of high molecular weight proteins and nucleic acids.We obtained high molecular weight HA with excellent biocompatibility as judged by both measurement of cell proliferation and viability, indicating high possibility of its applications to biomaterials.

View Article: PubMed Central - PubMed

Affiliation: School of Life Science and Biotechnology, Korea University, Seoul, 136-701 Republic of Korea ; Department of Bioplant, Hanmi Pharm. Co, Pyeongtaek, 451-805 Korea.

ABSTRACT

Background: Hyaluronic acid (HA) is of importance due to its diverse applications in pharmaceuticals and medical devices such as dermal filler, adhesion barriers, carrier for cells and bioactive molecules as well as scaffold biomaterials for tissue engineering. Evaluations of purification and biocompatibility of HA are required for its applications to biomaterials.

Results: After synthesizing HA by fermentation of streptococcus zooepidemicus for 25 hr, extensively purification of the fermented broth was performed to remove impurities using a filtration process for insoluble components and cells, and diverse adsorbents for soluble impurities. Its in vitro biocompatibility has been evaluated by measurement of cell counting and assay of cell live and dead. 60% yield of white HA powder was obtained, having 15-17 dL/g intrinsic viscosity with a molecular weight of approximately 1,000 kDa. While low molecular weight impurities and insoluble impurities were successfully removed using a ultrafiltration membrane with 50 KDa molecular weight cut, endotoxins, high molecular weight proteins and nucleic acids were removed from the broth by employing adsorbents such as alumina and activated carbons. Alumina showed the best results for the removal of endotoxins, all of the activated carbons were very effective in the removal of high molecular weight proteins and nucleic acids. The purified HA solution showed excellent cell compatibility with no cell damages as observed by both measurement of cell proliferation and observation of cell viability.

Conclusions: We obtained high molecular weight HA with excellent biocompatibility as judged by both measurement of cell proliferation and viability, indicating high possibility of its applications to biomaterials.

No MeSH data available.


Related in: MedlinePlus

Particle numbers and sizes under the different conditions described in Table4.
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Fig4: Particle numbers and sizes under the different conditions described in Table4.

Mentions:  After HA purification, the used adsorbents needed to be removed by filtration to obtain the pure HA products. Even though all the activated carbons were removed from the processed broth, Norit C Gran, which is a granular activated carbon, blocked the filter during filtration when a 1% broth sample was used. To better understand the reason for this result, particles sizes and their distributions were measured for samples containing 2% Norit C Gran and 0.3% hyaluronic acid after stirring for 5 hr (Table 4 and Figure 4). When Norit C Gran without the addition of HA and stirring was used, the broth was easily filtered through a 0.45 μm filter (PR sterile 40, Begerow Inc., Germany) and only 108 microparticles were observed with a diameter of 2 μm (83%). When we added HA (3%) without Norit C Gran adsorbents, the particles seemed to have become entangled, and the numbers of particles with higher sizes increased from 108 to 1,196. When we added the granular activated carbons to the HA solution, the number of particles with higher sizes increased by a factor of two from 1,196 to 2,642. The particle sizes were, however, still mostly less than 5 μm, indicating that the broth obtained was still filterable in this experiment. Significant increases in particle sizes and numbers were observed when we stirred fermented broth containing granular activated carbons for 5 and 13 hrs. 65,151 particles were generated by stirring for 5 hr and their numbers increased to 84,010 after 13 hr of stirring. Furthermore, particles with sizes greater than 5 μm also increased from 12% to 49% under these conditions. Based on these results, Norit C Gran and 5 hr of stirring clearly had an effect on the particle sizes and number and 87,713 particles with smaller sizes were generated and 24% of these were larger than 5 μm in size. These results indicated that under stirring conditions, granular activated carbons broke down. Overall, we did not observe significant effects of pH, BET, methylene blue adsorption on the removal of high molecular weight proteins, nucleic acid and endotixins. However, the types of activated carbons and species of adsorbents significantly affected their removals of those impurities. HA had an effect on their breakdowns and entanglement with HA resulted in an increase in particle size, which lead to blockage of the membrane’s pores.Table 4


Purification and biocompatibility of fermented hyaluronic acid for its applications to biomaterials.

Choi S, Choi W, Kim S, Lee SY, Noh I, Kim CW - Biomater Res (2014)

Particle numbers and sizes under the different conditions described in Table4.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4549129&req=5

Fig4: Particle numbers and sizes under the different conditions described in Table4.
Mentions:  After HA purification, the used adsorbents needed to be removed by filtration to obtain the pure HA products. Even though all the activated carbons were removed from the processed broth, Norit C Gran, which is a granular activated carbon, blocked the filter during filtration when a 1% broth sample was used. To better understand the reason for this result, particles sizes and their distributions were measured for samples containing 2% Norit C Gran and 0.3% hyaluronic acid after stirring for 5 hr (Table 4 and Figure 4). When Norit C Gran without the addition of HA and stirring was used, the broth was easily filtered through a 0.45 μm filter (PR sterile 40, Begerow Inc., Germany) and only 108 microparticles were observed with a diameter of 2 μm (83%). When we added HA (3%) without Norit C Gran adsorbents, the particles seemed to have become entangled, and the numbers of particles with higher sizes increased from 108 to 1,196. When we added the granular activated carbons to the HA solution, the number of particles with higher sizes increased by a factor of two from 1,196 to 2,642. The particle sizes were, however, still mostly less than 5 μm, indicating that the broth obtained was still filterable in this experiment. Significant increases in particle sizes and numbers were observed when we stirred fermented broth containing granular activated carbons for 5 and 13 hrs. 65,151 particles were generated by stirring for 5 hr and their numbers increased to 84,010 after 13 hr of stirring. Furthermore, particles with sizes greater than 5 μm also increased from 12% to 49% under these conditions. Based on these results, Norit C Gran and 5 hr of stirring clearly had an effect on the particle sizes and number and 87,713 particles with smaller sizes were generated and 24% of these were larger than 5 μm in size. These results indicated that under stirring conditions, granular activated carbons broke down. Overall, we did not observe significant effects of pH, BET, methylene blue adsorption on the removal of high molecular weight proteins, nucleic acid and endotixins. However, the types of activated carbons and species of adsorbents significantly affected their removals of those impurities. HA had an effect on their breakdowns and entanglement with HA resulted in an increase in particle size, which lead to blockage of the membrane’s pores.Table 4

Bottom Line: While low molecular weight impurities and insoluble impurities were successfully removed using a ultrafiltration membrane with 50 KDa molecular weight cut, endotoxins, high molecular weight proteins and nucleic acids were removed from the broth by employing adsorbents such as alumina and activated carbons.Alumina showed the best results for the removal of endotoxins, all of the activated carbons were very effective in the removal of high molecular weight proteins and nucleic acids.We obtained high molecular weight HA with excellent biocompatibility as judged by both measurement of cell proliferation and viability, indicating high possibility of its applications to biomaterials.

View Article: PubMed Central - PubMed

Affiliation: School of Life Science and Biotechnology, Korea University, Seoul, 136-701 Republic of Korea ; Department of Bioplant, Hanmi Pharm. Co, Pyeongtaek, 451-805 Korea.

ABSTRACT

Background: Hyaluronic acid (HA) is of importance due to its diverse applications in pharmaceuticals and medical devices such as dermal filler, adhesion barriers, carrier for cells and bioactive molecules as well as scaffold biomaterials for tissue engineering. Evaluations of purification and biocompatibility of HA are required for its applications to biomaterials.

Results: After synthesizing HA by fermentation of streptococcus zooepidemicus for 25 hr, extensively purification of the fermented broth was performed to remove impurities using a filtration process for insoluble components and cells, and diverse adsorbents for soluble impurities. Its in vitro biocompatibility has been evaluated by measurement of cell counting and assay of cell live and dead. 60% yield of white HA powder was obtained, having 15-17 dL/g intrinsic viscosity with a molecular weight of approximately 1,000 kDa. While low molecular weight impurities and insoluble impurities were successfully removed using a ultrafiltration membrane with 50 KDa molecular weight cut, endotoxins, high molecular weight proteins and nucleic acids were removed from the broth by employing adsorbents such as alumina and activated carbons. Alumina showed the best results for the removal of endotoxins, all of the activated carbons were very effective in the removal of high molecular weight proteins and nucleic acids. The purified HA solution showed excellent cell compatibility with no cell damages as observed by both measurement of cell proliferation and observation of cell viability.

Conclusions: We obtained high molecular weight HA with excellent biocompatibility as judged by both measurement of cell proliferation and viability, indicating high possibility of its applications to biomaterials.

No MeSH data available.


Related in: MedlinePlus