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- Atif, Abdul Raouf, 1996-, et al.
(författare)
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A microfluidics-based method for culturing osteoblasts on biomimetic hydroxyapatite
- 2021
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Ingår i: Acta Biomaterialia. - : Elsevier. - 1742-7061 .- 1878-7568. ; 127, s. 327-337
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Tidskriftsartikel (refereegranskat)abstract
- The reliability of conventional cell culture studies to evaluate biomaterials is often questioned, as in vitro outcomes may contradict results obtained through in vivo assays. Microfluidics technology has the potential to reproduce complex physiological conditions by allowing for fine control of microscale features such as cell confinement and flow rate. Having a continuous flow during cell culture is especially advantageous for bioactive biomaterials such as calcium-deficient hydroxyapatite (HA), which may otherwise alter medium composition and jeopardize cell viability, potentially producing false negative results in vitro. In this work, HA was integrated into a microfluidics-based platform (HA-on-chip) and the effect of varied flow rates (2, 8 and 14 µl/min, corresponding to 0.002, 0.008 and 0.014 dyn/cm2, respectively) was evaluated. A HA sample placed in a well plate (HA-static) was included as a control. While substantial calcium depletion and phosphate release occurred in static conditions, the concentration of ions in HA-on-chip samples remained similar to those of fresh medium, particularly at higher flow rates. Pre-osteoblast-like cells (MC3T3-E1) exhibited a significantly higher degree of proliferation on HA-on-chip (8 μl/min flow rate) as compared to HA-static. However, cell differentiation, analysed by alkaline phosphatase (ALP) activity, showed low values in both conditions. This study indicates that cells respond differently when cultured on HA under flow compared to static conditions, which indicates the need for more physiologically relevant methods to increase the predictive value of in vitro studies used to evaluate biomaterials.
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| 2. |
- Atif, Abdul Raouf, 1996-, et al.
(författare)
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Influence of flow in the adhesion and proliferation of cells on hydroxyapatite integrated in a microscale culture
- 2021
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- INTRODUCTION: Synthetic biomaterials, such as calcium phosphate cements (CPCs), are a promising alternative to autologous bone to enhance bone regeneration. Calcium-deficient hydroxyapatite (CDHA), the end-product of apatite cements, matches the inorganic phase of the bone and exhibits excellent biocompatibility in vivo [1]. However, in vitro, CDHA uptakes calcium ions (Ca2+) from cell culture medium [2], causing detrimental effects on cell activity and function [3]. The aim of this work was to integrate CDHA into a microfluidic chip that provides continued culture medium supply, and to evaluate cell adhesion and proliferation as compared to standard well plates.METHODS:CDHA was integrated in a polydimethylsiloxane (PDMS)-glass microfluidic chip (CDHA-on-chip). PDMS was cured in a 3D-printed mould at 60°C for 2h. α-tricalcium phosphate was mixed with 2.5% w/v Na2HPO4(aq) (liquid-to-powder of 0.65 ml/g) and the CPC was cast within a PDMS pocket. The CPC was immersed in an aqueous solution at 37°C for 10 days to ensure full transformation to CDHA. Through plasma treatment, a glass slide was bonded to the PDMS holding the CDHA, thus forming a 0.5mm channel above the CDHA. CDHA samples were pre-incubated for 24h in minimum essential media (MEM) supplemented with 10% FBS and 1% penicillin-streptomycin (sMEM). Pre-osteoblasts (MC3T3-E1) were seeded at 50,000 cells/cm2 and after a cell adhesion period of 2h, flow was applied for 72h through the chip at different rates: 2, 8 and 14 μl/min. A static (0 μl/min) chip condition was included, where sMEM was manually replaced every 24h. CDHA discs (⌀=6mm, h=2mm) placed in a 96-well plate were used as a standard static control (200 μl sMEM replaced every 24h). At 6h and 72h, the cells were stained with a calcein, propidium iodide and Hoechst triple-stain to assess their adhesion and proliferation, respectively. In a separate experiment, sMEM was flown through the chips for 24h at the aforementioned flow rates, and Ca2+ concentration was quantified via inductively coupled plasma-optical emission spectroscopy (ICP-OES). As control, sMEM in contact with CDHA discs for 24h was evaluated.RESULTS:A larger number of cells adhered on the CDHA-on-chip under flow as opposed to both static CDHA-on-chip and CDHA disc in a well plate. Differences in cell adhesion between the flow conditions were negligible. Cell proliferation at 72h was significantly increased under flow compared to CDHA disc samples (Fig.1A). Static CDHA-on-chip showed almost no viable cells. 2 and 8 μl/min flow conditions showed the greatest cell counts, followed by the 14 μl/min flow condition. At higher flow rates, Ca2+ concentrations were closer to in fresh medium (Fig.1B)DISCUSSION & CONCLUSIONS:The static CDHA-on-chip and disc samples displayed a low degree of cell adhesion and proliferation, which seemed to indicate that ionic exchange led to detrimental cell behaviour. Cells displayed the greatest degree of adhesion and proliferation at a flow rate of 2 and 8 μl/min, probably due to more optimal Ca2+ concentrations. At 14 μl/min, the degree of cell adhesion and proliferation decreased, which could be ascribed to adverse effects of shear stress.
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| 3. |
- Atif, Abdul Raouf, 1996-, et al.
(författare)
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Modelling adsorption of proteins and cells on biomimetic hydroxyapatite
- 2021
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Calcium-deficient hydroxyapatite (CDHA), a biomaterial similar to the inorganic bone matrix, can be used in non-load bearing areas to promote bone regeneration. Upon implantation, CDHA is exposed to blood, leading to serum protein deposition on the surface and enabling cell attachment via membrane-bound receptors. In cell culture studies, biomaterials are often pre-incubated in serum supplemented medium to mimic this process. In this work, to study the extent the protein layer assists in cell adhesion, a Langmuir isotherm-based model for protein and cell adhesion kinetics was used.
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