Spatial patterning of BMP-2 and BMP-7 on biopolymeric films and the guidance of muscle cell fate

Abstract

In the cellular microenvironment, growth factor gradients are crucial in dictating cell fate. Towards developing materials that capture the native microenvironment we engineered biomimetic films that present gradients of matrix-bound bone morphogenetic proteins (BMP-2 and BMP-7). To this end layer-by-layer films composed of poly(L-lysine) and hyaluronan were combined in a simple microfluidic device enabling spatially controlled growth factor diffusion along the film. Linear long-range gradients of both BMPs induced the trans-differentiation of C2C12 myoblasts towards the osteogenic lineage in a dose dependent manner with a different signature for each BMP. The osteogenic marker alkaline phosphatase (ALP) increased in a linear manner for BMP-7 and non-linearly for BMP-2. Moreover, an increased expression of the myogenic marker troponin T was observed with decreasing matrix-bound BMP concentration, providing a substrate that it is both osteo- and myo-inductive. Lastly, dual parallel matrix-bound gradients of BMP-2 and -7 revealed a complete saturation of the ALP signal. This suggested an additive or synergistic effect of the two BMPs. This simple technology allows for determining quickly and efficiently the optimal concentration of matrix-bound growth factors, as well as for investigating the presentation of multiple growth factors in their solid-phase and in a spatially controlled manner.

Keywords: Bone morphogenetic proteins; Gradient; Layer-by-layer; Microfluidic; Myogenesis; Osteoinduction.

FIGURE 1

ALP expression of C2C12 myoblasts in response to soluble BMP-2 and BMP-7 added at increasing concentrations. ALP was measured after 3 days of culture using a colorimetric assay. Dose response curves yields inflections points of 117 ng/mL and 858 ng/mL respectively for BMP-2 and BMP-7. Error bars indicate standard deviation of five samples.

FIGURE 2

(A) Adsorbed amounts of BMPs on (PLL/HA) films as a function of initial loading concentration of BMP-2 and BMP-7 in solution. BMP-7 data were fitted with a ligand binding curve (K_d=56 μg/mL) whereas BMP-2 data were fitted with a linear regression (B) Relative ALP activity of C2C12 myoblasts on BMP-loaded (PLL/HA) films as a function of the adsorbed amount of BMP measured using a colorimetric assay. The highest BMP concentration for each condition was set at 100%. Error bars indicate standard error of three different experiments. BMP-7 data were fitted with a sigmoidal curve (inflection point at 0.8 μg/cm²) whereas BMP-2 data were fitted with a ligand binding curve (K_d=0.07 μg/cm²).

FIGURE 3

Fluorescence images of matrix-bound gradients of fluorescein-labeled BMP-7 (A) and rhodamine-labeled BMP-2 (B) over one microchannel (scale bar: 1 mm). (C) Profiles of matrix-bound BMP gradients as a function of length of the channels. Adsorbed amounts were calculated from calibration curves obtained from a fluorescent plate reader and fluorescent microscopy (SI Figure 1). Linear fits yields slopes of 0.58 μg/cm³ and 1.24 μg/cm³ for BMP-2 and BMP-7 respectively. Error bars indicate standard deviation between three different channels.

FIGURE 4

(A) Luminescence imaging of C2C12 myoblasts transfected with a BMP-2 responsive element on a matrix-bound BMP-2 gradient, indicating dose-response activation of the SMAD pathway. Boxed region indicates approximate localization of the matrix-bound BMP-2 gradient. (B) Corresponding intensity profile as a function of the distance along the gradient.

FIGURE 5

Differentiation of C2C12 myoblasts on BMP-2 gradients. (A) Overview image and representative images of ALP staining confirm osteogenic differentiation, while immuno-fluorescent imaging reveals a decrease of troponin T positive cells (undergoing myogenic differentiation) with increasing BMP-2 concentration. Top images: ALP (purple); bottom images ALP (black), troponin T (green), actin (gray), nucleus (blue). (B) Quantification of ALP expression by measuring the intensities of the cyan and magenta channels in CMYK images, and amount of troponin T positive cells per field of view.

FIGURE 6

Differentiation of C2C12 myoblasts on BMP-7 gradients. (A) Overview image and representative images of ALP staining confirms osteogenic differentiation, while immuno-fluorescent imaging reveals a decrease of troponin T positive cells with increasing BMP-7 concentration. Top images: ALP (purple); bottom images ALP (black), troponin T (green), actin (gray), nucleus (blue). (B) Quantification of ALP expression by measuring the intensities of the cyan & magenta channels in CMYK images, and amount of troponin T positive cells per field of view.

FIGURE 7

(A) ALP expression of C2C12 myoblasts on (PLL/HA) films loaded with BMP-7 (gray bar), BMP-2 (white bars) or both (black bars). BMP-7 loading concentration was fixed to 10 μg/mL (corresponding to 0.38 μg/cm²) and BMP-2 loading concentration to 3 or 6 μg/mL (corresponding to 0.19 and 0.38 μg/cm²). On the right hand side is plotted ALP signal when BMP-7 is incorporated from a solution at 50 μg/mL (corresponding to 1.76 μg/cm²) and BMP-2 from a solution at 25 μg/mL (corresponding to 1.16 μg/cm²). Additive effect on ALP is observed when both BMPs are delivered from the same film. (B) Profiles of matrix-bound dual parallel BMP-2 and -7 gradients as a function of length of the channels. (C) Profiles of dual opposite BMP-2 and BMP-7 gradients. Adsorbed amounts were calculated as indicated in Figure 3. Error bars indicate standard deviation between three different channels.

FIGURE 8

Differentiation of C2C12 myoblasts on dual parallel BMP-2 and -7 gradients. (A) Overview image and representative images of ALP staining confirms osteogenic differentiation. no troponin T positive cells were found. (B) Quantification of ALP expression by measuring the intensities of the cyan & magenta channels in CMYK images.

SCHEME 1

Schematic representation of the different steps of matrix-bound gradient formation using microfluidics on a glass slide coated with (PLL/HA) films prepared via layer-by-layer assembly.