Quantitative evaluation of osteoblast proliferation and differentiation on a biomaterial in a microfluidic device

• IntroductionCalcium phosphate cements (CPCs) are able to transform into calcium deficient hydroxyapatite (CDHA), whose crystal size and chemistry closely matches that of the inorganic phase of bone [1]. CDHA readily uptakes calcium ions, and releases phosphate, when immersed in synthetic solutions that mimic physiological fluids [1]. While CPCs are able to enhance bone regeneration in defect sites located in non-load bearing areas, the ionic imbalance that arises from dissolution may also have detrimental effects on cell behavior and function. The purpose of this study was to culture cells on CDHA embedded in a microfluidic chip, under flow, to sustain optimal ionic concentrations, and subsequently evaluate cell proliferation and differentiation.   MethodsCPC was cast into a polydimethylsiloxane (PDMS) pocket (h = 0.8 mm) and then set in a 0.9 % NaCl(aq) solution at 37°C for 10 days leading to conversion into CDHA. The CDHA embedded in PDMS were dried and bonded to glass via oxygen plasma treatment, resulting in chips with a 0.5 mm deep channel above the CDHA. In parallel, CDHA discs (⌀ = 6 mm) were set in Teflon molds for the same period of time. The CDHA chips and discs were sterilized with ethanol and pre-incubated with cell culture media overnight. MC3T3-E1 pre-osteoblasts (50,000 cells/cm2) were seeded on the CDHA, and allowed to adhere for 2 h, before initiating a flow of 8 µl/min. Cell proliferation (indirectly measured as the cytosolic lactate dehydrogenase (LDH) enzyme of cells previously adhered to the material) and cell differentiation (alkaline phosphatase activity normalized by total amount of protein) were quantified on day 1, 5 and 10. On day 10, cells were stained with Calcein, Propidium iodide (live/dead assay) and Hoechst (nucleus), and were imaged via fluorescence microscopy.   ResultsThe fabrication of the CDHA-on-chip was successful (Fig 1A). There was a faster increase of osteoblast growth on the CDHA-on-chip (under flow) than on discs (static conditions). Specifically, between day 5 and 10, cell number on-chip increased a two-fold as compared to the insignificant change on discs (Fig 1B). Cells on-chip were observed confluent at day 10 (Fig 1C) and seemed to differentiate over time (not shown).ConclusionThe integrated hydroxyapatite platform is a potential alternative for standard in vitro analysis using well plates. Application of flow ameliorates media ionic imbalance, while also providing fresh nutrients and removing waste.

Subject headings

TEKNIK OCH TEKNOLOGIER  -- Materialteknik (hsv//swe)

ENGINEERING AND TECHNOLOGY  -- Materials Engineering (hsv//eng)

Keyword

Organs-on-chip

Hydroxyapatite

Teknisk fysik med inriktning mot mikrosystemteknik

Engineering Science with specialization in Microsystems Technology

Publication and Content Type

vet (subject category)

kon (subject category)