Scaffolds for Chondrogenic Cells Cultivation Prepared from Bacterial Cellulose with Relaxed Fibers Structure Induced Genetically

Abstract

Development of three-dimensional scaffolds mimicking in vivo cells’ environment is an ongoing challenge for tissue engineering. Bacterial nano-cellulose (BNC) is a well-known biocompatible material with enormous water-holding capacity. However, a tight spatial organization of cellulose fibers limits cell ingrowth and restricts practical use of BNC-based scaffolds. The aim of this study was to address this issue avoiding any chemical treatment of natural nanomaterial. Genetic modifications of Komagataeibacter hansenii ATCC 23769 strain along with structural and mechanical properties characterization of obtained BNC membranes were conducted. Furthermore, the membranes were evaluated as scaffolds in in vitro assays to verify cells viability and glycosaminoglycan synthesis by chondrogenic ATDC5 cells line as well as RBL-2H3 mast cells degranulation. K. hansenii mutants with increased cell lengths and motility were shown to produce BNC membranes with increased pore sizes. Novel, BNC membranes with relaxed fiber structure revealed superior properties as scaffolds when compared to membranes produced by a wild-type strain. Obtained results confirm that a genetic modification of productive bacterial strain is a plausible way of adjustment of bacterial cellulose properties for tissue engineering applications without the employment of any chemical modifications.

Structural changes of bacterial nanocellulose pellicles induced by genetic modification of Komagataeibacter hansenii ATCC 23769

“…The resulting constructs were transformed using the heat shock method into E. coli TOP10F’ cells (Froger and Hall 2008 ). Then, K. hansenii ATCC 23769 was transformed with control pTI99, pTI99-motA, pTI99-motB by electroporation, in accordance with the method used in previous studies (Jacek et al 2018 ). The cells were incubated at 30 °C for 3 h, diluted, and transferred to SH/agar plates containing ampicillin (200 μg/mL) for screening of recombinants (Fig.…”

“…Because of these properties, BNC has a wide range of potential biomedical applications, including wound dressings, medical implants, drug delivery, vascular grafts, and scaffolds for tissue engineering (Svensson et al 2005 ; de Oliveira Barud et al 2016 ; Picheth et al 2017 ). In recent years, many studies have been carried to develop BNC scaffolds for biomedical use (Wu et al 2016 ; Cielecka et al 2018 ; Jacek et al 2018 ). Nevertheless, the main problem for tissue engineering is the limited porosity of the BNC that makes the ingrowth of the eukaryotic cells is impossible.…”

Towards control of cellulose biosynthesis by Komagataeibacter using systems-level and strain engineering strategies: current progress and perspectives

“…b A loose BNC network produced by K. hansenii ATCC 23769 mutants overexpressing the motility-related genes motA and motB (WT-wild-type strain, Control-strain transformed with an empty vector; motAB+-mutant with motA and motB overexpression). Reprinted from Jacek et al (2018) authors expressed curdlan synthase (crdS) gene from the most efficient producer of this biopolymer, Agrobacterium sp. ATCC 31749.…”

“…Induction of gene disruption through a homologous recombination was commonly performed with the use of non-replicating plasmid disruption cassette carriers, such as pUC19, pT7Blue, pET14 or pHSG399 (Table 4 ). At present, one can expect intensification of genetic engineering of different Komagataeibacter strains due to the progress in the application of three new vector backbones for endogenous and heterologous gene expression, namely pTI99, pTSa and pSEVA331Bb (Fang et al 2015 ; Florea et al 2016 ; Jacek et al 2018 , 2019b ; Gwon et al 2019 ). Moreover, the performance of various promoters, ribosome binding sites (RBS) and terminators has been characterised in the first two studies applying synthetic biology tools in Komagataeibacter hosts (Florea et al 2016 ; Teh et al 2019 ).…”

Molecular aspects of bacterial nanocellulose biosynthesis

“…Although genetic modifications of the Komagataeibacter strains were aimed mainly at increasing the productivity, the group from Lodz University of Technology recently obtained two disruption mutants producing stiffer membranes with a more‐packed structure and three overexpression mutants producing membranes with a porous structure (Jacek et al . ) demonstrated that by manipulating genes responsible for motility and cell divisions it is possible to obtain mutants producing BNC membranes with desirable properties. These studies initiate a new promising trend in obtaining BNC membranes with changed structure.…”