Biomaterials,
which release active compounds after implantation, are an essential
tool for targeted regenerative medicine. In this study, thin multilayer
films loaded with lipid/DNA complexes (lipoplexes) were designed as
surface coatings for in situ transfection applicable in tissue engineering
and regenerative medicine. The film production and embedding of lipoplexes
were based on the layer-by-layer (LbL) deposition technique. Hyaluronic
acid (HA) and chitosan (CHI) were used as the polyelectrolyte components.
The embedded plasmid DNA was complexed using a new designed cationic
lipid formulation, namely, OH4/DOPE 1/1, the advantageous characteristics
of which have been proven already. Three different methods were tested
regarding its efficiency of lipid and DNA deposition. Therefore, several
surface specific analytics were used to characterize the LbL formation,
the lipid DNA embedding, and the surface characteristics of the multilayer
films, such as fluorescence microscopy, surface plasmon resonance
spectroscopy, ellipsometry, zeta potential measurements, atomic force
microscopy, and scanning electron microscopy. Interaction studies
were conducted for optimized lipoplex-loaded polyelectrolyte multilayers
(PEMs) that showed an efficient attachment of C2C12 cells on the surface.
Furthermore, no acute toxic effects were found in cell culture studies,
demonstrating biocompatibility. Cell culture experiments with C2C12
cells, a cell line which is hard to transfect, demonstrated efficient
transfection of the reporter gene encoding for green fluorescent protein.
In vivo experiments using the chicken embryo chorion allantois membrane
animal replacement model showed efficient gene-transferring rates
in living complex tissues, although the DNA-loaded films were stored
over 6 days under wet and dried conditions. Based on these findings,
it can be concluded that OH4/DOPE 1/1 lipoplex-loaded PEMs composed
of HA and CHI can be an efficient tool for in situ transfection in
regenerative medicine.
DiTT4 lipoplexes have exhibited excellent transfection efficiency in a complex tissue together with a biocompatibility profile that makes it a prospective vehicle for gene delivery.
A gene-activated surface coating is presented as a strategy to design smart biomaterials for bone tissue engineering. The thin-film coating is based on polyelectrolyte multilayers composed of collagen I and chondroitin sulfate, two main biopolymers of the bone extracellular matrix, which are fabricated by layer-by-layer assembly. For further functionalization, DNA/lipid-nanoparticles (lipoplexes) are incorporated into the multilayers. The polyelectrolyte multilayer fabrication and lipoplex deposition are analyzed by surface sensitive analytical methods that demonstrate successful thin-film formation, fibrillar structuring of collagen, and homogenous embedding of lipoplexes. Culture of mesenchymal stem cells on the lipoplex functionalized multilayer results in excellent attachment and growth of them, and also, their ability to take up cargo like fluorescence-labelled DNA from lipoplexes. The functionalization of the multilayer with lipoplexes encapsulating DNA encoding for transient expression of bone morphogenetic protein 2 induces osteogenic differentiation of mesenchymal stem cells, which is shown by mRNA quantification for osteogenic genes and histochemical staining. In summary, the novel gene-functionalized and extracellular matrix mimicking multilayer composed of collagen I, chondroitin sulfate, and lipoplexes, represents a smart surface functionalization that holds great promise for tissue engineering constructs and implant coatings to promote regeneration of bone and other tissues.
Surface coatings prepared by layer‐by‐layer technique permit loading of growth factors (GFs) and their spatially controlled release. Here, native chondroitin sulfate (nCS), oxidized CS (oCS100), or mixture of both (oCS50) are combined with collagen I (Col I) to fabricate polyelectrolyte multilayers (PEMs) that exhibit structural, mechanical, and biochemical cues like the natural extracellular‐matrix. The use of oCS enables intrinsic cross‐linking of PEM that offers higher stability, stiffness, and better control of bone morphogenetic protein‐2 (BMP‐2) release compared to nCS. oCS100 PEMs have enhanced stiffness, promote Col I fibrillization, and present BMP‐2 in a matrix‐bound manner. oCS50 PEMs show intermediate effects on osteogenesis, soft surface, high water content but also moderately slow BMP‐2 release profile. C2C12 myoblasts used for osteogenesis studies show that oCS PEMs are more stable and superior to nCS PEMs in supporting cell adhesion and spreading as well as in presenting BMP‐2 to the cells. oCS PEMs are triggering more osteogenesis as proved by the quantitative real‐time polymerase chain reaction, immune and histochemical staining. These findings show that intrinsic cross‐linking in oCS/Col I multilayers provides a successful tool to control GFs delivery and subsequent cell differentiation which opens new opportunities in regenerative therapies of bone and other tissues.
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