Laminin functionalized biomimetic apatite to regulate the adhesion and proliferation behaviors of neural stem cells

Abstract

Background: Functionalizing biomaterial substrates with biological signals shows promise in regulating neural stem cell (NSC) behaviors through mimicking cellular microenvironment. However, diverse methods for immobilizing biological molecules yields promising results but with many problems. Biomimetic apatite is an excellent carrier due to its non-toxicity, good biocompatibility, biodegradability, and favorable affinity to plenty of molecules. Therefore, it may provide a promising alternative in regulating NSC behaviors.

Methods: Biomimetic apatite immobilized with the extracellular protein - laminin (LN) was prepared through coprecipitation process in modified Dulbecco's phosphate-buffered saline (DPBS) containing LN. The amount of coprecipitated LN and their release kinetics were examined. The adhesion and proliferation behaviors of NSC on biomimetic apatite immobilized with LN were investigated.

Results: The coprecipitation approach provided well retention of LN within biomimetic apatite up to 28 days, and supported the adhesion and proliferation of NSCs without cytotoxicity. For long-term cultivation, NSCs formed neurosphere-like aggregates on non-functionalized biomimetic apatite. A monolayer of proliferated NSCs on biomimetic apatite with coprecipitated LN was observed and even more stable than the positive control of LN coated tissue-culture treated polystyrene (TCP).

Conclusion: The simple and reproducible method of coprecipitation suggests that biomimetic apatite is an ideal carrier to functionalize materials with biological molecules for neural-related applications.

Keywords: adhesion; biomimetic apatite; coprecipitation; laminin; neural stem cell; proliferation.

Figure 1

Schematic illustration of coprecipitating LN within biomimetic apatite. Abbreviations: LN, laminin; mDPBS, modified Dulbecco’s phosphate-buffered saline.

Figure 2

SEM images of surfaces morphologies of (A) biomimetic apatite, (B) apatite/LN5, (C) apatite/LN10, (D) apatite/LN20, and (E) apatite/LN40. Scale bar is 20 µm in length. Abbreviations: LN, laminin; SEM, scanning electron microscope.

Figure 3

XPS analysis of representative samples. Notes: (A) XPS peaks of biomimetic apatite and apatite with coprecipitated LN (apatite/LN5, apatite/LN10, apatite/LN20, and apatite/LN40). Magnified graph shows nitrogen (N) peaks in the biomimetic apatite and apatite/LN in the bottom right panel. (B) Quantification of atomic compositions on the surfaces of representative samples. Abbreviations: LN, laminin; XPS, X-ray photoelectron spectroscopy.

Figure 4

(A) In vitro release of apatite/FITC-LN20 in PBS over 28-day time period. Values are represented as mean±SD (n=3). (B) Side depth profiles obtained by stacking each series of confocal images through the thickness of apatite/FITC-LN20 after immersing in PBS solution for 28 days and control sample. Abbreviations: FITC-LN, fluorescein isothiocyanate-conjugated LN; LN, laminin.

Figure 5

NSCs adhesion on surfaces of biomimetic apatite and apatite with coprecipitated LN (apatite/LN5, apatite/LN10, apatite/LN20, and apatite/LN40) samples in growth medium after 12-hour culture. Notes: (A) Attached cells were assayed by CCK8. Values are represented as mean±SD (n=3). Statistical significance relative to the group of biomimetic apatite: *P<0.05; **P<0.01. (B) SEM images of NSCs attached on biomimetic apatite and apatite/LN20. The red circle pointed the processes from the cell body. Scale bar is 5 µm in length. Abbreviations: CCK8, Cell Counting Kit-8; Ctr+, positive control; Ctr−, negative control; LN, laminin; NSC, neural stem cell; SEM, scanning electron microscopy.

Figure 6

NSCs proliferation on surfaces of biomimetic apatite and apatite/LN20 samples in growth medium for 3, 5, 7 and 10 days. Notes: Cells were assayed by CCK8. Values are represented as mean±SD (n=3). Statistical significance relative to the group of biomimetic apatite: *P<0.05; **P<0.01. Abbreviations: CCK8, Cell Counting Kit-8; Ctr+, positive control; Ctr−, negative control; LN, laminin; NSC, neural stem cell.

Figure 7

NSCs were cultured in indirect contact with biomimetic apatite and apatite/LN20 for 48-hour incubation to evaluate the cytotoxicity. Notes: (A) Cell viability was quantitatively analyzed by CCK8. Values are represented as mean±SD (n=3). Statistical significance relative to the group of negative control: **P<0.01. (B) Cytotoxicity was qualitatively observed by the live/dead viability/cytotoxicity kit (green color: live cells, red color: dead cells). Scale bar is 200 µm in length. Abbreviations: CCK8, Cell Counting Kit-8; Ctr+, positive control; Ctr−, negative control; LN, laminin; NSC, neural stem cell.

Figure 8

Immunofluorescence images of nestin (a NSC marker, green) and DAPI (blue) staining of NSCs proliferation on surfaces of biomimetic apatite and apatite/LN20 samples in growth medium for 7 and 10 days. Scale bar is 200 µm in length. Abbreviations: Ctr+, positive control; Ctr−, negative control; LN, laminin; NSC, neural stem cell.