Inkjet-based biopatterning of bone morphogenetic protein-2 to spatially control calvarial bone formation

Abstract

The purpose of this study was to demonstrate spatial control of osteoblast differentiation in vitro and bone formation in vivo using inkjet bioprinting technology and to create three-dimensional persistent bio-ink patterns of bone morphogenetic protein-2 (BMP-2) and its modifiers immobilized within microporous scaffolds. Semicircular patterns of BMP-2 were printed within circular DermaMatrix human allograft scaffold constructs. The contralateral halves of the constructs were unprinted or printed with BMP-2 modifiers, including the BMP-2 inhibitor, noggin. Printed bio-ink pattern retention was validated using fluorescent or (125)I-labeled bio-inks. Mouse C2C12 progenitor cells cultured on patterned constructs differentiated in a dose-dependent fashion toward an osteoblastic fate in register to BMP-2 patterns. The fidelity of spatial restriction of osteoblastic differentiation at the boundary between neighboring BMP-2 and noggin patterns improved in comparison with patterns without noggin. Acellular DermaMatrix constructs similarly patterned with BMP-2 and noggin were then implanted into a mouse calvarial defect model. Patterns of bone formation in vivo were comparable with patterned responses of osteoblastic differentiation in vitro. These results demonstrate that three-dimensional biopatterning of a growth factor and growth factor modifier within a construct can direct cell differentiation in vitro and tissue formation in vivo in register to printed patterns.

FIG. 1.

Custom inkjet printing system (A) used to create three-dimensional printed constructs within DermaMatrix. (B, C) Growth factor bioink droplets averaged 14 pl (D). Color images available online at www.liebertonline.com/ten.

FIG. 2.

DermaMatrix as a three-dimensional printing substrate. (A) Tiled scanning electron micrograph of 5 mm DermaMatrix disk. (B) Scanning electron micrograph of DermaMatrix cross section. Right side represents epidermal layer. (C) Fluorescent image of Cy5-bone morphogenetic protein-2 (BMP-2) printed in a gradient upon DermaMatrix. (D) Fluorescent image of Cy5-BMP-2 printed on half of 5 mm DermaMatrix disk.

FIG. 3.

Binding and retention of printed BMP-2 and noggin on DermaMatrix.

FIG. 4.

BMP-2 printed on DermaMatrix stimulates osteogenic cell differentiation in C2C12 cells. All DermaMatrix pieces had right and left semicircular halves printed independently as labeled. Alkaline phosphatase (ALP) staining (blue) shows increased ALP expression with increased doses of BMP-2 (A–C). Comparison of different protein combinations showed that noggin was more capable of inhibiting undesired ALP staining (on the left side of the implants) compared with unprinted or transforming growth factor-β1 (TGF-β1)-printed DermaMatrix (D–F). Control stimulatory growth factor (GDF-5) showed increased ALP activity on the left side of the DermaMatrix (side not printed with BMP-2) (G). C2C12 cells seeded randomly onto DermaMatrix show an ALP staining dose response to printed BMP-2 (H). Higher concentrations of BMP-2 are achieved by increasing the number of bio-ink overprints (OPs). Images are representative of at least three separate experiments. Color images available online at www.liebertonline.com/ten.

FIG. 5.

Applying BMP-2-printed DermaMatrix constructs in a critical-sized mouse defect model to spatially control bone formation. A) Photograph (left), radiograph (center), and 3D CT (right) of implanted skull 2 weeks postoperative. B) 3D CT reconstructions of one animal 2 weeks, 4 weeks, and 8 weeks postoperative. Color images available online at www.liebertonline.com/ten.

FIG. 6.

The effect of various applied patterns on bone fill in register to applied pattern. Treatment groups were defects with unfilled (Defect Control, n = 20), paired nonprinted (NP) DermaMatrix disks (NP Control, n = 10), NP control (n = 20) adjacent to BMP-2 (100 μg/mL BMP-2, 12 OPs, n = 20) pairs, and noggin (100 μg/mL BMP-2, 12 OPs, n = 18) adjacent to BMP-2 (n = 18). Animals were sacrificed after 4 weeks and evaluated by radiography. Data are presented as mean percentage of pattern fill ± standard error of the mean.

FIG. 7.

Hematoxylin and eosin (H&E) histological staining for representative unprinted DermaMatrix (A) and DermaMatrix printed half with 100 μg/mL BMP-2, 12 OPs (B) and half left unprinted (C). Large black arrows denote calvarial defect perimeter. IC, invading undefined cells; WB, woven bone (magnification, × 50).

FIG. 8.

Higher magnification of H&E histological staining for representative DermaMatrix printed disks at 4 weeks (A) and 8 weeks (B) using 100 μg/mL BMP-2, 12 OPs. IC, invading undefined cells; WB, woven bone; BM, bone marrow space; BV, blood vessel; C, endochondral bone; LB, lamellar bone (magnification, × 200).