Living biointerfaces based on non-pathogenic bacteria to direct cell differentiation

Abstract

Genetically modified Lactococcus lactis, non-pathogenic bacteria expressing the FNIII7-10 fibronectin fragment as a protein membrane have been used to create a living biointerface between synthetic materials and mammalian cells. This FNIII7-10 fragment comprises the RGD and PHSRN sequences of fibronectin to bind α5β1 integrins and triggers signalling for cell adhesion, spreading and differentiation. We used L. lactis strain to colonize material surfaces and produce stable biofilms presenting the FNIII7-10 fragment readily available to cells. Biofilm density is easily tunable and remains stable for several days. Murine C2C12 myoblasts seeded over mature biofilms undergo bipolar alignment and form differentiated myotubes, a process triggered by the FNIII7-10 fragment. This biointerface based on living bacteria can be further modified to express any desired biochemical signal, establishing a new paradigm in biomaterial surface functionalisation for biomedical applications.

Regulation of Stem Cell Fate by Nanomaterial Substrates

“…The non-pathogenic bacterium Lactococcus lactis has been genetically modified to express the RGD containing fibronectin fragment FNIII [7][8][9][10] 148 and thus affect the adhesion and morphology of C2C12 cells. 149 While the bacteria-modified surface was not shown to be directly cell-responsive, one can envision that the bacteria film would be responsive to changes in the biological environment and may be able to respond to changes in its surroundings that are brought about by the mammalian cells culture on the surface.…”

Toward Light‐Regulated Living Biomaterials

“…Attached bacteria induced with IPTG were able to express the proteins on their surface, as indicated by the red fluorescence in the microscopy images (Figure 2b). Initially, we observed rapid bacterial growth on the surface, resulting in the formation of multilayers within 6 h. Retardation of bacterial metabolism was achieved by addition of tetracycline antibiotic 12. Bacterial growth and protein expression were followed over the span of a day in the presence of tetracycline at concentrations spanning 1–10 µg mL −1 (Figure S1a, Supporting Information).…”

Functional living biointerfaces to direct cell-material interaction

“…Despite its low EPS production, L. lactis adheres to and colonizes a broad range of surfaces such as synthetic polymers, glass, metals and live tissues. These biofilms remain stable for weeks and support cell adhesion, growth and differentiation [211], as will be further discussed. pT1NX has been the chosen expression vector in this work.…”

“…Several cell lines, namely fibronectin-null fibroblasts (Fn − /Fn − ), NIH3T3 and C2C12 adhere, spread and develop focal adhesion complexes on the engineered L. lactis biofilm, behaving in a similar way as when cultured directly on a FN-coated surface. [216] This strain produces stable biofilms that support cellular growth, adhesion and differentiation [211] . The formation of the biofilm starts when some individual cells adhere to the surface, a process governed by weak non-specific forces -Van der Waals and polar Lewis acid-base interactions [43,303].…”

“…cremoris MG1363 is a strain with a low production of exopolysaccharides, so biofilms usually show a monolayered morphology, lacking the horizontal stratified structure found in other strains. [211,304,305] Hence, the formation of L. lactis biofilms seems limited to a monolayer [306] of strongly attached bacteria, in almost any abiotic surface such as polymers, glass or metallic surfaces. Usually, the adhesion of the bacterium to the substrate is mediated by the bacterial fimbriae, also called adhesion pili [307].…”