Self-assembling biomaterials: liquid crystal phases of cholesteryl oligo(L-lactic acid) and their interactions with cells

Abstract

We report here on the synthesis and characterization of a series of self-assembling biomaterials with molecular features designed to interact with cells and scaffolds for tissue regeneration. The molecules of these materials contain cholesteryl moieties, which have universal affinity for cell membranes, and short chains of lactic acid, a common component of biodegradable tissue engineering matrices. The materials were synthesized in good yields with low polydispersities in the range of 1.05-1.15, and their characterization was carried out by small-angle x-ray diffraction, transmission electron microscopy, electron diffraction, differential scanning calorimetry, and atomic force microscopy. These molecular materials form layered structures that can be described as smectic phases and can also order into single-crystal stacks with an orthorhombic unit cell. Their layer spacings range from 58 to 99 A, corresponding to bilayers of oligomers with an average of 10 and 37 lactic acid residues, respectively. The self-organized layered structures were found to promote improved fibroblast adhesion and spreading, although the specific mechanism for this observed response remains unknown. The ability of self-assembling materials to present ordered and periodic bulk structures to cells could be a useful strategy in tissue engineering.

Figure 1

Chemical structure of C-LA_n̄.

Figure 2

Characteristic birefringent texture under crossed polars for C-LA at 25°C.

Figure 3

DSC scan of C-LA revealing a glass transition at 32°C, a thermotropic liquid crystalline transition (smectic to smectic phase) at 57°C, and isotropization at 88°C.

Figure 4

SAXS scans of C-LA_n̄ (n̄ = 10, 24, 37), reveal (001) and (002) reflections indicating lamellar organization. (Inset) A molecular graphics model showing the proposed packing of C-LA molecules into a highly interdigitated bilayer with spacing of 58 Å. [Reproduced with permission from ref. (Copyright 2002, Am. Chem. Soc.).]

Figure 5

(a) TEM image and electron diffraction pattern of single crystal C-LA. (b) TEM image of a film of C-LA_8̄ revealing multilayer domains formed by spiral dislocations. (Inset) Electron diffraction pattern (rings correspond to evaporated Au), indicating that the material is not a single crystal but a smectic E_h liquid crystalline phase. (c) TEM image of a precipitate C-LA_8̄ showing a multilayered lozenge-shaped single crystal formed by spiral dislocations. (Inset) Electron diffraction pattern revealing an orthorhombic unit cell.

Figure 6

AFM image showing the surface topography of C-LA films. The image is 106 × 106 microns.

Figure 7

(a) 3T6 fibroblasts on a PLA surface 72 hr after cell seeding. The fibroblasts aggregate and do not spread on these surfaces. (b) 3T6 fibroblasts on C-LA 72 hr after seeding. The cells spread preferentially along the boundaries of focal conic domains.

Figure 8

Surface area of adhered cells on various biomaterials. Error bars are ± a 95% confidence level. All sample sets at each timepoint were compared by using ANOVA to the PLA control. *, P < 0.05; †, P < 0.01.^‡

Figure 9

3T3 fibroblasts after 3 days of exposure to (a) a PLA surface and (b) a PLA surface modified with C-LA. A higher population of cells is found on the surface in (b) modified with self-assembling multilayers.

Figure 10

Chemical structure of dehydroergosterol.

Figure 11

3T3 fibroblasts after 6 hr of exposure to a surface of DHE-(l-lactic acid)_n. Image was taken under epifluorescence illumination by using a 4′,6-diamidino-2-phenylindole-type filter set. The fluorescent material is visible throughout the cell cytoplasm and membrane.