Development and biological evaluation of Ti6Al7Nb scaffold implants coated with gentamycin-saturated bacterial cellulose biomaterial

Abstract

Herein we present an innovative method of coating the surface of Titanium-Aluminium-Niobium bone scaffold implants with bacterial cellulose (BC) polymer saturated with antibiotic. Customized Ti6Al7Nb scaffolds manufactured using Selective Laser Melting were immersed in a suspension of Komagataeibacter xylinus bacteria which displays an ability to produce a 3-dimensional structure of bio-cellulose polymer. The process of complete implant coating with BC took on average 7 days. Subsequently, the BC matrix was cleansed by means of alkaline lysis and saturated with gentamycin. Scanning electron microscopy revealed that BC adheres and penetrates into the implant scaffold structure. The viability and development of the cellular layer on BC micro-structure were visualized by means of confocal microscopy. The BC-coated implants displayed a significantly lower cytotoxicity against osteoblast and fibroblast cell cultures in vitro in comparison to non-coated implants. It was also noted that gentamycin released from BC-coated implants inhibited the growth of Staphylococcus aureus cultures in vitro, confirming the suitability of such implant modification for preventing hostile microbial colonization. As demonstrated using digital microscopy, the procedure used for implant coating and BC chemical cleansing did not flaw the biomaterial structure. The results presented herein are of high translational value with regard to future use of customized, BC-coated and antibiotic-saturated implants designed for use in orthopedic applications to speed up recovery and to reduce the risk of musculoskeletal infections.

Fig 1. Stages of preparation of BC-coated Ti6Al7Nb scaffold.

(A)—native Ti6Al7Nb scaffold; (B)—implant coated with unpurified BC; (C)—BC-coated implant after removal of media leftover and bacteria; (D)—BC-coated Ti6Al7Nb scaffold after partial drying.

Fig 2. Visualization of cellulose covering (A) and ingrowing (B) the pores of Ti6Al7Nb implant.

The area marked by a red circle in (A) is visualized with higher magnification in (B). The cellulose seen in (A) was intentionally mechanically disrupted (as seen in the middle of the picture) to uncover the underlying implant’s struts. Magn. 51x and 318, respectively. Zeiss EVO MA SEM Microscope. Please also refer to S3 Fig to see more Ti6Al7Nb implants with partially removed cellulose.

Fig 3. Cytotoxicity of BC-coated Ti6Al7Nb scaffolds vs. non-coated Ti6Al7Nb native scaffolds for fibroblasts and osteoblasts after 24 and 48 h of incubation.

BCS—BC-coated Ti6Al7Nb scaffolds; NS—non-coated Ti6Al7Nb native scaffolds; Asterisks mark statistically significant differences (M-W test, p<0.5) between particular columns.

Fig 4. Fibroblasts colonizing bacterial cellulose membrane.

Cellulose is visualized using laser reflection (shown in red) (A), while fibroblasts are fluorescently labeled (shown in green) (B). Merged channels are presented in (C) (view from the top), (D) and (E) (side views). The imaging was performed on a Leica SP8 confocal microscope.

Fig 5. No negative impact of the procedures applied on implant structure.

Structure of the implants coated with cellulose and subjected to a temperature of 80°C (after cellulose removal) under (A) 105x and (B) 2060x magnification, respectively. Zeiss EVO MA SEM Microscope.