Calcium phosphate/polyelectrolyte multilayer coatings for sequential delivery of multiple growth factors

摘要

Introduction: Many of the most promising strategies for regenerative tissue-engineering aim to replicate natural cellular microenvironments using biomimetic, resorbable materials capable of growth factor delivery. Combinations of growth factors synergistically enhance tissue regeneration, but require sequential, rather than co-delivery delivery for maximum efficacy. Polyelectrolyte multilayer (PEM) coatings retain growth factor activity, however, staggered, sequential delivery from these coatings requires barrier layers to prevent interlayer diffusion of multiple factors that results in co-delivery. Here we demonstrate that the incorporation of a biomimetic calcium phosphate (bCaP) layer into PEM can prevent interlayer diffusion of biomolecuies and enable staggered delivery. This modified PEM system is one of few aqueous, low temperature systems available for sequential, multifactor delivery and has potential to significantly impact the regenerative tissue-engineering field. Methods: bCaP coatings were prepared on sandblasted, 22 mm, treated tissue culture plastic disks, (NUNC, Rochester, NY) using a simulated body fluid method. The PEM coatings (8-30 bilayers) were applied on top of the bCaP layer by automated alternate 10 min dipping into 1 mg/ml poly L glutamic acid (-) or poly-L lysine (+) solutions (Sigma, St. Louis, MO) with saline rinses between. Coatings were characterized with x-ray diffraction and scanning electron microscopy. To demonstrate sequential delivery a combination of a proliferative factor, recombinant human fibroblast growth factor-2 (FGF-2) (150 ng/disk) (R & D Systems, Minneapolis, MN), and a cytotoxic factor antimycin A (AntiA) (213 μg/disk)(Sigma, St. Louis, MO) was used. In vitro cellular activity on the prepared coatings was assessed with MC3T3-E1 mouse calvariai osteoprogenitor cells (ATCC, Manassas, VA) seeded at 10 or 40 × 103 cells/cm2. Proliferative and cytotoxic effects were quantified using the LIVE/DEAD® assay (Invitrogen Life Technologies, Grand Island, NY). Statistical significances were determined by unpaired t-tests and one-way ANOVA with Tukey post-tests. Results: Uniform nanocrystalline coatings of bCaP (Fig. 1 A) and bCaP plus 30 bilayers of PEM (CaP-PEM30) (Fig. 1B) on the disks were observed via SEM. MC3T3-E1 s cultured directly on adsorbed AntiA with no bCaP or PEM coating (AntiA) resulted in 90% cell death on day 1 (Fig. 2A). MC3T3-E1s cultured on AntiA embedded under 8 bilayers of PEM (AntiA-PEM8) resulted in 47% cell death on day 1 due to diffusion of AntiA through PEM8. MC3T3-E1s cultured on AntiA embedded under bCaP and 8 bilayers of PEM (AntiA-CaP-PEM8) resulted in 15% cell death, significantly better than PEM only (p＜0.001). These data demonstrate that the addition of bCaP to PEM coatings inhibits interlayer diffusion. Active FGF-2 was delivered from 30 bilayers of PEM above CaP (CaP-PEM30-FGF2) as demonstrated by a significant increase in MC3T3-E1 proliferation on day 1 as compared to cells cultured on the FGF-2-free control, (Fig. 2B). No growth factor release was detected by ELISA without cells present. Sequential access of cells to FGF-2 followed by AntiA was also demonstrated (Fig. 3A). On day 1, no differences were observed between CaP-PEM30-FGF2 and AntiA-CaP-PEM30-FGF2 indicating access to AntiA was successfully blocked by bCaP. On day 5 however, cell-mediated degradation of PEM30 and bCaP resulted in a significant decrease in LIVE staining between groups (Fig. 3A) indicating AntiA delivery. Conclusions: Addition of a bCaP barrier layer to a PEM delivery system prevents unwanted interlayer diffusion of embedded factors and enables cell-mediated sequential delivery of multiple factors. This technology can be utilized in multiple research applications where a sequential delivery profile activated by cell degradation of matrix is desired.