Response of Primary Human Bone Marrow Mesenchymal Stromal Cells and Dermal Keratinocytes to Thermal Printer Materials In Vitro

Abstract

Advancement in thermal three-dimensional printing techniques has greatly increased the possible applications of various materials in medical applications and tissue engineering. Yet, potential toxic effects on primary human cells have been rarely investigated. Therefore, we compared four materials commonly used in thermal printing for bioengineering, namely thermally printed acrylonitrile butadiene styrene, MED610, polycarbonate, and polylactic acid, and investigated their effects on primary human adult skin epidermal keratinocytes and bone marrow mesenchymal stromal cells (BM-MSCs) in vitro. We investigated indirect effects on both cell types caused by potential liberation of soluble substances from the materials, and also analyzed BM-MSCs in direct contact with the materials. We found that even in culture without direct contact with the materials, the culture with MED610 (and to a lesser extent acrylonitrile butadiene styrene) significantly affected keratinocytes, reducing cell numbers and proliferation marker Ki67 expression, and increasing glucose consumption, lactate secretion, and expression of differentiation-associated genes. BM-MSCs had decreased metabolic activity, and exhibited increased cell death in direct culture on the materials. MED610 and acrylonitrile butadiene styrene induced the strongest expression of genes associated to differentiation and estrogen receptor activation. In conclusion, we found strong cell-type-specific effects of the materials, suggesting that materials for applications in regenerative medicine should be carefully selected not only based on their mechanical properties but also based on their cell-type-specific biological effects.

Keywords: Biocompatibility; Bone marrow mesenchymal stromal cell; Keratinocyte; MED610; Thermal printer material.

Fig. 1

Schematic layout of experimental setup. Cultures of cells (brown) were performed in wells (dark grey) of cell culture plates that contained detachable polyester inserts (blue) with 0.4-μm pore size. The size of the pores permits unrestricted exchange of medium (pink) but not cells. The setup allowed for investigation of effects on cells caused by direct contact with materials (green) and liberation of substances from materials, as well as for investigation of effects on cells caused by potential liberation of substances from materials only. For bone marrow mesenchymal stromal cells (I) negative controls included wells containing neither cells nor materials (I a), and positive controls included cells seeded in inserts without materials (I b). Additional negative controls included wells containing materials only (without cells) (I c). Cells (I) were seeded on inserts with materials placed at the bottom of the dish (I d) or seeded directly on materials placed in inserts (I e). Keratinocytes (II) were cultured on collagen-1-coated bottoms of cell culture dishes. Negative controls included wells containing neither cells nor materials (II a), and positive controls included cells seeded on bottoms of dishes without materials in inserts (II b). Additional controls included wells containing only materials in inserts (without cells) (II c). Keratinocytes were seeded on bottoms of cell culture dishes and materials were placed in inserts for investigation of effects on cells caused by potential liberation of substances from materials (II d). Proportions are not to scale

Fig. 2

Numbers of viable keratinocytes. Numbers of cells were analyzed by MTT assay after 48 h in culture on collagen-1-coated dishes, with inserts having discs of acrylonitrile butadiene styrene M30i (ABS), PolyJet photopolymer MED610 (MED), polycarbonate-ISO (PC), and polylactic acid (PLA), respectively, immersed into cell culture medium. Controls were performed with keratinocytes cultured with empty inserts (no material) immersed into cell culture medium. Data are given as mean ± standard deviation from three biological repeats. Asterisk indicates statistically significant difference relative to control obtained using Student’s t test (*p < 0.05)

Fig. 3

Numbers of viable bone marrow mesenchymal stromal cells. Numbers of cells were analyzed by MTT assay after 48 h in culture with or on various materials. In indirect culture, cells were cultured on inserts, having discs of acrylonitrile butadiene styrene M30i (ABS), PolyJet photopolymer MED610 (MED), polycarbonate-ISO (PC), and polylactic acid (PLA), respectively, in cell culture plate bottom well immersed into medium; in direct culture, cells were cultured on discs of either ABS, MED, PC, or PLA, with disks placed in inserts immersed into culture medium. Controls were performed with cells cultured on inserts without any discs. Data are given as mean ± standard deviation from five biological repeats. Asterisk indicates statistically significant difference relative to control obtained using Student’s t test (*p < 0.05)

Fig. 4

Cell imaging of bone marrow mesenchymal stromal cells. Distribution of cells and cytoskeleton protein expression after 48 h in culture on various materials was imaged using fluorescence confocal microscopy. Cells were cultured on discs of acrylonitrile butadiene styrene M30i (ABS), PolyJet photopolymer MED610 (MED), polycarbonate-ISO (PC), and polylactic acid (PLA), respectively. After fixation, cell nuclei were stained with DAPI (blue, upper panel) and cytoskeleton-specific F-actin was stained with AF568-phalloidin (red, lower panel). Scale bar = 200 μm

Fig. 5

Material surface topography analyses. Surface topographies of discs of acrylonitrile butadiene styrene M30i (ABS), PolyJet photopolymer MED610 (MED), polycarbonate-ISO (PC), and polylactic acid (PLA) were investigated using laser scanning microscopy. Scale bar = 200 μm

Fig. 6

Cell viability of keratinocytes. Culture media of keratinocytes were analyzed for LDH activity after 48 h in culture on collagen-1-coated dishes, with inserts having discs of acrylonitrile butadiene styrene M30i (ABS), PolyJet photopolymer MED610 (MED), polycarbonate-ISO (PC), and polylactic acid (PLA), respectively, immersed into cell culture medium. Controls were performed with keratinocytes cultured with empty inserts immersed into cell culture medium. Data are given as mean ± standard deviation from three biological repeats

Fig. 7

Cell viability of bone marrow mesenchymal stromal cells. Cell culture media were analyzed for LDH activity after 48 h in culture. In indirect culture, cells were cultured on inserts, having discs of acrylonitrile butadiene styrene M30i (ABS), PolyJet photopolymer MED610 (MED), polycarbonate-ISO (PC), and polylactic acid (PLA), respectively, in cell culture plate bottom well immersed into medium; in direct culture, cells were cultured on discs of either ABS, MED, PC, or PLA, with disks placed in inserts immersed into culture medium. Controls were performed with cells cultured on inserts without any discs. Data are given as mean ± standard deviation from five biological repeats. Asterisk indicates statistically significant difference relative to control obtained using Student’s t test (*p < 0.05)

Fig. 8

Metabolic activity of keratinocytes. a Glucose consumption and b lactate production per cell were measured in cell culture media after 48 h in culture with inserts having discs of acrylonitrile butadiene styrene M30i (ABS), PolyJet photopolymer MED610 (MED), polycarbonate-ISO (PC), and polylactic acid (PLA), respectively, immersed into cell culture medium. Controls were performed with keratinocytes cultured with empty inserts immersed into cell culture medium. Data are given as mean ± standard deviation from three biological repeats. Asterisk indicates statistically significant difference relative to controls obtained using Student’s t test (*p < 0.05)

Fig. 9

Metabolic activity of bone marrow mesenchymal stromal cells. a Glucose consumption and b lactate production per cell were measured in cell culture media after 48 h in culture. In indirect culture, cells were cultured on inserts, having discs of acrylonitrile butadiene styrene M30i (ABS), PolyJet photopolymer MED610 (MED), polycarbonate-ISO (PC), and polylactic acid (PLA), respectively, in cell culture plate bottom well immersed into medium; in direct culture, cells were cultured on discs of either ABS, MED, PC, or PLA, with disks placed in inserts immersed into culture medium. Controls were performed with cells cultured on inserts without any discs. Data are given as mean ± standard deviation from five biological repeats. Asterisk indicates statistically significant difference relative to controls obtained using Student’s t test (*p < 0.05)