Spatio-temporal modification of collagen scaffolds mediated by triple helical propensity

Abstract

Functionalized collagen that incorporates exogenous compounds may offer new and improved biomaterials applications, especially in drug-delivery, multifunctional implants, and tissue engineering. To that end, we developed a specific and reversible collagen modification technique utilizing associative chain interactions between synthetic collagen mimetic peptide (CMP) [(ProHypGly) chi; Hyp = hydroxyproline] and type I collagen. Here we show temperature-dependent collagen binding and subsequent release of a series of CMPs with varying chain lengths indicating a triple helical propensity driven binding mechanism. The binding took place when melted, single-strand CMPs were allowed to fold while in contact with reconstituted type I collagens. The binding affinity is highly specific to collagen as labeled CMP bound to nanometer scale periodic positions on type I collagen fibers and could be used to selectively image collagens in ex vivo human liver tissue. When heated to physiological temperature, bound CMPs discharged from the collagen at a sustained rate that correlated with CMP's triple helical propensity, suggesting that sustainability is mediated by dynamic collagen-CMP interactions. We also report on the spatially defined modification of collagen film with linear and multi-arm poly(ethylene glycol)-CMP conjugates; at 37 degrees C, these PEG-CMP conjugates exhibited temporary cell repelling activity lasting up to 9 days. These results demonstrate new opportunities for targeting pathologic collagens for diagnostic or therapeutic applications and for fabricating multifunctional collagen coatings and scaffolds that can temporally and spatially control the behavior of cells associated with the collagen matrices.

Figure 1

Density of CMPs bound to collagen films (type I, bovine) after treatment with CMP solutions followed by extensive washing. CMP solutions preincubated at four different temperatures were applied to cell culture plates at room temperature coated with stable type I collagen film. The amount of CMP remaining on the collagen film was determined by fluorescence intensity of wash solutions. M marks the maximum binding condition for each CMP-X, which was used to determine initial dissociation index (IDI). Error bars represent ± SD.

Figure 2

Transmission electron micrographs of reconstituted type I collagen fiber from mouse tail tendon after incubation with gold nanoparticles displaying CysCMP-7 (a) or CysCMP-R7 (b). The white arrows indicate periodic positions of nanoparticles on collagen fibers located precisely at one interface between the dark and the white bands facing the collagen molecule’s C-terminal end.

Figure 3

^FCMP-6 binds specifically to collagens of human liver tissue. (a, b) Fluorescence confocal microscopy of human liver carcinoma stained with either ^FCMP-6 (green) (a) or ^FCMP-R10 (green) (b), and anti-CD31 mAb (red) and anticollagen I-V pAb (blue). Frozen, unfixed liver sections were thawed, washed with acetone and PBS, pH 7.4, and blocked with fetal bovine serum. A 50 µM solution of ^FCMP-6 or ^FCMP-R10 was then applied along with anti-CD31-(R)-PE and anticollagen I~V. The anticollagen antibody was visualized using antirabbit secondary antibodies conjugated to Alexa Fluor 647. As the overlay image indicates, the basement membrane and endothelial staining observed in ^FCMP-6 staining matches the pattern observed in anticollagen I~V staining (a); however, no discernible staining is observed for ^FCMP-R10, whereas the pattern observed in the anticollagen I~V antibody is very clear (b). For the blocking experiment (c), liver sections were pretreated with 100 µM solution of GlyGlyGly-(ProHypGly)₆ before staining with ^FCMP-6 and antibodies as above. ^FCMP-6’s affinity to collagens in the liver tissue is negated by the pretreatment with GlyGlyGly-(ProHypGly)₆.

Figure 4

Cumulative release profiles of ^FCMP-X from collagen films in 37 °C, PBS buffer solution (pH 7.4). Initially, 20 nmol of ^FCMP-X in PBS solution at predetermined temperature was applied to collagen film (type I bovine skin; area: 0.0616 cm²). Day 0 represents ^FCMP-X released after extensive washing with 4 °C PBS buffer. Collagen films were incubated in 37 °C buffer solution and the concentrations of released ^FCMP-X were determined by measuring the UV–vis absorbance of buffer solutions at 493 nm. Data is reported as the mean ± SD of the four samples.

Figure 5

Density profiles of ^FPEG-CMPXs and ^FMPEG-CMP8 remaining on collagen films during incubation at 37 °C, PBS buffer solution (pH 7.4). The charging and release conditions are the same as Figure 4. The quenched sample was first heated to 80 °C, cooled to 25 °C within 4 min, and immediately applied to the collagen film. Inset: optical micrographs of PEG-CMP10 treated collagen films seeded on day 0 with human breast epithelial cells (MCF-7) and incubated at 37 °C. The inset pictures correspond directly to the density profiles. Only the areas to the right side of the dotted line were treated and the whole collagen film was seeded with the cell suspension (5.6 × 10⁵ cells/mL).