Two-Site Internally Cooperative Mechanism for Enzyme Kinetics in a Hydrogel Forming Recombinant Protein.

A recombinant protein, ATCTA, consisting of three domains, α-helix (A), thrombin cleavage site (T), and water-soluble coil (C), forms hydrogels via the self-association of its flanking α-helices into tetrameric bundles, which act as cross-links for the hydrogel network. In the presence of thrombin, the hydrogel degrades due to the thrombin cleavage sites. To better understand the proteolysis reaction in ATCTA, we performed a series of kinetic experiments on the proteins ATC, CTA, CTATC, and ATCTA. The KM and kcat of ATC and CTA were determined to be 88 ± 5 µM and 6.4 ± 0.1 s(-1) and 91 ± 9 µM and 6.1 ± 0.1 s(-1), respectively. Using these kinetic parameters, a model based on a two-site internally cooperative mechanism was developed to describe the kinetics of proteins containing two cleavage sites. This model was then validated by comparing predicted results with kinetic data from the proteolysis of ATCTA.

Combinatorial screen of the effect of surface energy on fibronectin-mediated osteoblast adhesion, spreading and proliferation.

In order to accelerate tissue-engineering research, a combinatorial approach for investigating the effect of surface energy on cell response has been developed. Surface energy is a fundamental material property that can influence cell behavior. Gradients in surface energy were created by using an automated stage to decelerate a glass slide coated with a self-assembled monolayer (SAM, n-octyldimethylchlorosilane) beneath a UV lamp such that the SAM is exposed to the UV-light in a graded fashion. UV exposure causes oxidation of the SAM such that a longer exposure correlates with increased hydrophilicity. This approach yielded substrates having a linear gradient in surface energy ranging from 23 to 62 mN/m (water contact angles ranging from 25 degrees to 95 degrees ). Using the gradient specimen approach enables all surface energies from 23 to 60 mN/m to be screened on each slide. Before cell culture, surface energy gradients were coated with fibronectin to allow a study of the effect of surface energy on fibronectin-mediated cell response. Cells were seeded on the fibronectin-coated gradients and adhesion, spreading and proliferation were assessed with automated fluorescence microscopy. Surface energy did not affect initial cell adhesion at 8h. However, the rate of proliferation was linearly dependent on surface energy and increased with increasing hydrophobicity. Cell spread area was unaffected by changes in surface energy over the majority of the gradient although cells were significantly smaller on the most hydrophilic region. These results show that fibronectin-mediated cell spreading and proliferation are dependent on surface energy and establish a new combinatorial approach for screening cell response to changes in surface energy.

Combinatorial approach to study enzyme/surface interactions.

A fast combinatorial approach to access information about the immobilization behavior and kinetics of enzymes on a variation of surfaces is presented. As a test system, Candida Antarctica Lipase B was immobilized on a self-assembled monolayer bearing a gradient of surface energy. The respective immobilization behavior was monitored by Fourier transform infrared micro-spectroscopy. In addition, the activity of the immobilized enzyme was monitored over the entire film in real time with a specially developed fluorescence activity assay embedded into a siloxane gel. It was found that the highest amount of active protein was immobilized on the hydrophilic end of the gradient surface. This effect is associated with a higher surface roughness of this area resulting in hydrophobic micro-environments in which the enzyme gets immobilized.

Combinatorial screening of cell proliferation on poly(L-lactic acid)/poly(D,L-lactic acid) blends.

We have combined automated fluorescence microscopy with a combinatorial approach for creating polymer blend gradients to yield a rapid screening method for characterizing cell proliferation on polymer blends. A gradient in polymer blend composition of poly(L-lactic acid) (PLLA) and poly(D,L-lactic acid) (PDLLA) was created in the form of a strip-shaped film and was annealed to allow PLLA to crystallize. Fourier transform infrared (FTIR) microspectroscopy was used to determine the composition in the gradients and atomic force microscopy was used to characterize surface topography. Osteoblasts were cultured on the gradients and proliferation was assessed by automated counting of cells using fluorescence microscopy. Surface roughness varied with composition, was smooth on PDLLA-rich regions and was rough on the PLLA-rich regions. Cell adhesion was similar on all regions of the gradients while proliferation was faster on the smooth, PDLLA-rich end of the gradients than on the rough, PLLA-rich end of the gradients. These results demonstrate the feasibility of a new, combinatorial approach for evaluating cell proliferation on polymer blends.

Biocompatibility of sorbitol-containing polyesters. Part I: Synthesis, surface analysis and cell response in vitro.

A series of sorbitol-containing polyesters were synthesized via a one-pot lipase-catalyzed condensation polymerization. Thin films were prepared by spin coating on silicon wafers and surfaces were analyzed by tapping mode atomic force microscopy and contact angle measurements. Surface morphologies and surface energies across the series of polyester films, including a poly(epsilon-caprolactone) (PCL) control were nearly indistinguishable. Biocompatibility of the sorbitol-containing polyester series was evaluated against a PCL control by measuring cell spreading and proliferation of a mouse fibroblast 3T3 cell line in vitro. Results confirmed that the sorbitol-containing polyester surfaces elicited cell behavior similar to the PCL control. These results establish the sorbitol-containing polyester series as a promising material for tissue engineering research and development.

High-throughput investigation of osteoblast response to polymer crystallinity: influence of nanometer-scale roughness on proliferation.

A high-throughput method for analyzing cellular response to crystallinity in a polymer material is presented. Variations in crystallinity lead to changes in surface roughness on nanometer length scales, and it is shown that cells are exquisitely sensitive to these changes. Gradients of polymer crystallinity were fabricated on films of poly(L-lactic acid) using a gradient in annealing temperature. The resultant morphologies were characterized using an atomic force microscope. Root-mean-square (rms) roughness values ranging from 0.5 to 13 nm were created on a single sample. MC3T3-E1 osteoblastic cells were cultured for 1, 3 and 5 d, and the number of cells was measured using automated fluorescence microscopy. It is shown that the rate of proliferation on the smooth regions of the films is much greater than that on the rough regions, and a monotonic variation in rate is observed as a function of roughness. The critical rms roughness, above which a statistically significant reduction in rate of proliferation occurs, was approximately 1.1 nm. Fluorescence microscopy measurements on immunostained cells indicate there is no significant change in cell area, the number or type of adhesions formed, or the degree of actin polymerization. Results from enzyme-linked immunofluorescence assays indicated that there was no detectable change in adhesion protein accessibility, suggesting the cells directly respond to substrate topography. The use of the gradient library approach yielded the functional dependence of cell proliferation on nanometer-scale roughness and gave a sensitive estimate of the critical roughness for which a decrease in proliferation is observed.