Medical

Collin College

DigitalCommons@Collin

Collin College Undergraduate Interdisciplinary Student Research Conference

Apr 19th, 10:00 AM - 11:15 AM

Medical

Follow this and additional works at:https://digitalcommons.collin.edu/ccuisrc

This Panel is brought to you for free and open access by DigitalCommons@Collin. It has been accepted for inclusion in Collin College Undergraduate Interdisciplinary Student Research Conference by an authorized administrator of DigitalCommons@Collin. For more information, please contact

[email protected].

Liu, Yuqing; Hatch, Nicole; Ogunbayode, Toluwani; Shelke, Siddhant; and Olszewski, Corey, "Medical" (2018).Collin College Undergraduate Interdisciplinary Student Research Conference. 9.

Investigating_​​the_​​Effects_​​of_​​Time-Mediated_​​Addition_​​of_​​Titanium_​​Dioxide_​​Nanoparticles_​​on_​​the Differentiation_​​and_​​Proliferation_​​of_​Human_{​ ​​}Dental_​​Pulp_​​Stem_​​Cells

​​Abstract

Dental_​​pulp_​​stem_​​cells_​​(DPSCs)_​​have_​​therapeutic_​​promise_​​due_​​to_​their_{​ ​​}rapid_​​proliferation_​​and multipotency_​​but_​​require_​​further_​​research_​​to_​​reach_​​their_​​full_​​potential._​​Titanium_​​dioxide nanoparticles_​​(TiO_{​2​​​}NPs)_​​possess_​​properties_​​for_​​cell_​​tracking_​​and_​imaging,_{​ ​​}but_​​their_​​harmful effects_​​on_​​cell_​​viability_​​and_​​function_​​pose_​roadblocks_{​ ​​}to_​​their_​​usage._​​This_​​study_​​investigates_​​the timing_​​of_​​TiO_​2​​NP_{​ ​​}addition_​​to_​​DPSCs,_​​a_​​commonly_​​neglected_​​variable_​​when_​​testing_​​NP_​​toxicity, and_​​its_​​effects_​​on_​​DPSC_​​proliferation_​and_{​ ​​}differentiation._​​Based_​on_{​ ​​}preliminary_​​testing,_​​DPSCs can_​​respond_​​to_​​polybutadiene_​​substrate_​​mechanics_​​after_​​a_​​4-day_​​incubation_​​period._​​Accordingly, we_​​added_​​TiO_{​2​​​}NPs_​​on_​​both_​​days_​​1_​​and_​​4_​​(NP-1_​​and_​​NP-4,_​respectively)_{​ ​​}after_​​plating_​​DPSCs_​​on hard_​​polybutadiene_​​films._​​Through_​​the_​​lens_​of_{​ ​​}mechanical_​​properties,_​this_{​ ​​}study_​​explores_​​the influence_​​of_​​time-mediated_​​TiO_{​2​​​}NP_​​addition_​​and_​​examines_​​its_​effects_{​ ​​}on_​​DPSC_​​viability, proliferation,_​​and_​​differentiation_​​before_​​and_​​after_​​recognition_​​of_​​polybutadiene-coated_​​substrate. Results_​​showed_​​that_​​NP-4_​​had_​​substantially_​​reduced_​​harm_​​to_​​DPSC_​​proliferation_​​and

differentiation_​​as_​​compared_​​to_​​NP-1,_​​suggesting_​​that_​​time-mediated_​​addition_​​can_​​prevent_​​adverse effects_​​of_​​TiO_{​2​​​}and_​​NPs_​​as_​​a_​​whole._​​These_​​results_​​can_​​be_​translated_{​ ​​}to_​​many_​​other_​​applications including_​​drug_​​delivery,_​​developmental_​biology,_{​ ​​}biosensing,_​​and_​​biological_​​imaging.

I. Introduction

(I._​​A.)_​​Background_​​Information

Stem_​​cells_​​have_​​gained_​​attention_​​from_​​scientists,_​​medical_​​professionals,_​​and_​​the_​​general public_​​for_​​their_​​possible_​​applications_​​in_​the_{​ ​​}emerging_​​fields_​of_{​ ​​}tissue_​​engineering_​​and

regenerative_​​medicine._​1​_{​​As​​undifferentiated​​cells​​with​​the​​distinct​​ability​​to​​self-renew}

indefinitely_​​and_​​differentiate_​​into_​​various_​​types_​​of_​​specialized_​​cells,_​​stem_​cells_{​ ​​}offer_​​new_​​avenues in_​​the_​​treatment_​​of_​​diseases_​​using_​​cell-based_​​therapies._​2​_{​​Research​regarding​ ​​stem​​cells​​continues}

to_​​expand_​​due_​​to_​​their_​​tremendous_​​potential_​​in_​​revolutionizing_​​medical_​​care._​3

While_​​stem_​​cells_​​can_​​originate_​​from_​​all_​​over_​​the_​​body—for_​example,_{​ ​​}skin,_​​bone_​​marrow, and_​​muscle_​​tissues—human_​​dental_​​pulp_​​stem_​​cells_​​(DPSCs)_​​specifically_​​​have_​​been_​​widely studied_​​due_​​to_​​their_​​rapid_​​proliferation_​​rates,_​​easy_​​accessibility,_​​multipotent_​​differentiation,_​​and less_​​invasive_​​harvesting_​​compared_​​to_​​stem_​​cells_​taken_{​ ​​}from_​​bone_​​marrow._​4​_{​​In​​addition,​​DPSCs}

can_​​be_​​easily_​​cryopreserved_​​and_​​revived,_​​allowing_​​for_​​more_​​flexibility_​for_{​ ​​}future_​​usage_​​in laboratories_​​and_​​therapies._​5​​​_{Moreover,​​DPSCs​​can​​differentiate​​into​​a​variety​ ​​of​​cell​​lines,}

including_​​osteoblasts,_​​odontoblasts,_​​and_​​chondrocytes,_​allowing_{​ ​​}them_​​to_​​regenerate_​​and_​​repair many_​​different_​​types_​​of_​​damaged_​​tissue._​6​_{​​Because​​of​​this,​​DPSCs​​are​​currently​​being​​tested​​to}

treat_​​a_​​variety_​​of_​​conditions_​​including_​​type_​​1_​​diabetes,_​​neurological_​​diseases,_​​immunological diseases,_​​and_​​diseases_​​of_​​the_​​bone_​​and_​cartilage._{​ ​}7​_{​​Despite​​this​​progress,​​further​​imaging​​and}

characterization_​​of_​​stem_​​cell_​​properties_​​and_​​functions_​are_{​ ​​}needed_​​for_​​DPSCs_​​to_​​reach_​​their_​​full therapeutic_​​potential._​8​_{​​Controlling​​and​​monitoring​​DPSCs​​​in​​vitro​​​for​​applications​​​in​​vivo}

This_​​goal_​​can_​​be_​​addressed_​with_{​ ​​}nanotechnology,_​​as_​​nanoparticles_​​(NPs)_​​provide

solutions_​​and_​​other_​​benefits_​​when_​​added_​​to_​​and_​inside_{​ ​​}of_​​cells._​​Among_​the_{​ ​​}most_​​popular_​​of_​​these particles_​​is_​​(rutile)_​​titanium_​​dioxide_​​(TiO_​2​),_​​a_​​well-known_​​and_​low-cost_{​ ​​}material,_​​with_​​ideal properties_​​such_​​as_​​semiconductivity._​9​ ​​_{Commercially,​​TiO​}

2​​​NPs​​are​used​ ​​in​​sunscreens,​​lotions,

toothpastes,_​​and_​​various_​​cosmetics_​​due_​​to_​​their_​​strong_​​catalytic_​​activity._​10​_{​​In​​dentistry,​​TiO​} 2​​​is

used_​​in_​​dental_​​composites_​​and_​​root_​canal_{​ ​​}surgeries_​​for_​their_{​ ​​}strong_​​antimicrobial_​​properties, biocompatibility,_​​and_​​higher_​​stiffness._​11​​​_TiO​

2​​​NPs​​thus​​possess​​a​​wide​​range​​of​​applications​​when

used_​​to_​​enhance_​or_{​ ​​}observe_​​cellular_​​development.

However,_​​despite_​​their_​substantial_{​ ​​}promise,_​​TiO_{​2​​​}NPs_​​have_​​been_​found_{​ ​​}to_​​be_​​potentially harmful_​​to_​​cells._​​Contrary_​​to_​​a_​​conventional_​​characterization_​​of_​​TiO_{​2​​​}as_​​a_​​“white_​​knight”_​​with low_​​toxicity_​​and_​​chemical_​​inertness,_​12​​​_{recent​​studies​​have​​shown​​various​adverse​ ​​effects​​as​​a}

result_​​of_​​TiO_​2​​exposure._{​ ​​}For_​​example,_​​TiO_{​2​​​​​}NPs_​​have_​​been_​​shown_​​to_​​decrease_​​cell_​​proliferation and_​​impair_​​the_​cellular_{​ ​​}functions_​​of_​​human_​​dermal_​​fibroblasts._​13​_{​Specifically​ ​​regarding​​stem​​cells,}

uptake_​​of_​​TiO_{​2​​​}NPs_​​can_​​negatively_​​affect_​​the_​​proliferation,_​​viability,_​and_{​ ​​}differentiation_​​of_​​bone marrow_​​mesenchymal_​​stem_​​cells_​​​14​_{​​and​​inhibit​​short​​term​​DPSC​proliferation​ ​​at​​concentrations​​as}

low_​​as_​​25_​​µg/mL._​15​ ​​_{More​​broadly,​​TiO​}

2​​​NPs​​have​​also​​been​​seen​​to​​cause​​oxidative​​stress,

carcinogenesis,_​​genotoxicity,_​​and_​​immune_​​disruption._​15

(I._​​B.)_​​Experimental_​​Objective_​​and_​​Rationale

This_​​study_​​seeks_​​to_​​investigate_​​the_​​risk_​​for_​​using_​​TiO_{2​ ​​​}as_​​in_​​those_​​products,_​​as_​​it_​​is_​​still_​​an open_​​question,_​​but_​​the_​​focus_​​of_​​our_​​experiment_​​is_​​the_​​modulation_​​of_​​a_​​never_​​before_​​considered variable_​​in_​​NP-cell_​​interaction,_​​which_​​we_​hypothesized_{​ ​​}could_​​potentially_​reduce_{​ ​​}or_​​change_​​the extent_​​of_​​harmful_​​effects_​​of_​​TiO_{​2​​​}in_​laboratory_{​ ​​}cellular_​​work._​​In_​doing_{​ ​​}so,_​​the_​​applications_​​of

using_​​NPs_​​and_​​TiO_{​2​​​}in_​​particular_​​can_​​be_​​recognized._​​When_​​inside_​​cells,_​TiO_{​ ​2​ ​​}NPs_​​are_​​able_​​to regulate_​​the_​​release_​​and_​​kinetics_​​of_​​critical_​​growth_​​factors_​​to_​​improve_​​​in_​​vivo_​​​conditions._​16​_​​For

DPSCs,_​​TiO_{​2​​​}NPs_​​can_​​also_​​be_​​used_​​as_​​a_​​non-invasive_​​mechanism_​​for_​​greatly_​​improving_​​cell tracking_​​​17​ ​​_{and​​imaging​}18​_{​​ ​​due​​to​​their​fluorescent​ ​​properties​​and​​ability​​to​​penetrate​​cells.​​This}

allows_​​scientists_​​to_​​examine_​​the_​​movement_​​of_​​the_​​cells_​in_{​ ​​}a_​​body_​​or_​​in_​​an_​​injected_​​gel_​​and_​​is critical_​​to_​​the_​​future_​​of_​​stem_​​cell_​​research_​and_{​ ​​}application._​​Additionally,_​​TiO_{​2​​​}NP’s_​​can_​​play_​​a role_​​in_​​developing_​​targeted_​​drug_​​delivery_​​technologies,_​​as_​​they_​​not_​​only_​​serve_​​as_​​carriers_​​and_​​can release_​​the_​​drug,_​19​_{​their​ ​​tracking​​element​​ensures​​that​​the​​correct​​areas​​are​​receiving​​the​​drug.}

TiO_{​2​​​}NPs_​​thus_​possess_{​ ​​}the_​​potential_​​for_​​a_​​wide_​​range_​​of_​​important_​​and_​​cutting-edge_​​applications when_​​used_​​to_​​enhance_​​or_​​observe_​​cellular_​​development,_​​granted_​​there_​​can_​​be_​​a_​​way_​​to_​​protect cellular_​​health_​​and_​​viability.

Understanding_​​the_​​various_​​factors_​​affecting_​​NP-cell_​​interactions_​​is_​​paramount_​​to minimizing_​​the_​​potentially_​negative_{​ ​​}effects_​​of_​​adding_​​TiO_{​2​​​}NPs._​In_{​ ​​}all_​​previous_​​investigations and_​​literature_​​regarding_​​NP-cell_​​interactions,_​​however,_​​there_​​has_​​always_​​been_​​one_​​neglected factor:_​​the_​​timing_​​of_​​the_​​addition_​​of_​​NPs_​​(they_​​had_​​always_​​been_​​added_​on_{​ ​​}the_​​first_​​day)._​​We decided_​​to_​​introduce_​​this_​​variable_​​and_​​examine_​​the_​​effects_​​of_​time-mediated_{​ ​​}NP_​​insertion,_​​which could_​​provide_​​a_​​novel_​​method_​​to_​​diminish_​​the_​harmful_{​ ​​}effects_​​of_​​TiO_{​2​​​}NPs._​​​​Previous_​​work_​​has suggested_​​that,_​​over_​​time,_​​cells_​​can_​​recognize_​the_{​ ​​}stiffness_​​of_​​their_​​substrate_​​and_​​adjust_​​their mechanical_​​properties_​​to_​​match_​​that_​​of_​​their_​​substrate,21_​ ​_{​​and​​of​​course​​all​​cells​​develop​​and}

change_​​over_​​time_​​as_​​a_​​culture_​​grows._​Furthermore,_{​ ​​}given_​​that_​​physical_​​properties_​​of_​​cells_​​have significant_​​effects_​​on_​​biological_​​function,_​22​ ​​_{especially​​stem​​cell​​maintenance​​and​​differentiation,​}23

different_​​outcomes_​​when_​​nanoparticles_​​are_​​added_​​on_​​those_​​days._​​The_​timing_{​ ​​}of_​​this_​​substrate recognition_​​determined_​​our_​​time_​​points_​​for_​​the_​​addition_​​of_​​TiO2_​NPs,_{​ ​​}which_​​preliminary_​​testing showed_​​to_​​be_​​on_​​day4,_​​and_​​we_​​chose_​​to_​use_{​ ​​}a_​​hard_​​substrate,_​​which_​​would_​​cause_​​the_​​cells_​​to adjust_​​to_​​be_​​mechanically_​​stronger_​​by_​​that_​​day.

Accordingly,_​​we_​​compared_​​two_​​experimental_​​groups_​​with_​rutile_{​ ​​}TiO_{​2​​​}NPs_​​added_​​on_​​day_​​1 of_​​culturing_​​(before_​​cells_​​responded_​​to_​​surface_​mechanics)_{​ ​​}and_​​day_​​4_​​(after_​​cells_​​responded_​​to surface_​​mechanics)_​​in_​​addition_​​to_​​a_​​control_​group_{​ ​​}without_​​NPs._​​These_​​cells_​​were_​​grown_​​to differentiate_​​into_​​osteoblasts,_​​and_​​a_​​comparison_​​of_​​the_​​three_​​groups_​​of_​​cells_​​would_​​allow_​​us_​​to determine_​​the_​​effects_​​of_​​TiO_{2​​​​}NPs_​​on_​​DPSCs_​​when_​​added_​​at_​​the_​​normal_​​time_​​and_​​a_​​later_​​time._​​We considered_​​differences_​​in_​​NP_​​uptake_​​mechanism_​​and_​​immediate_​​effect,_​​short_​​term_​​proliferation, and_​​the_​​different_​​DPSCs’_​​abilities_​​for_​​osteogenic_​​differentiation_​​after_​​a_​longer_{​ ​​}period_​​of_​​time.

II. Materials_​​and_​​Methods

(II._​​A.)_​​Cell_​​Culture_​​and_​​Fixation

The_​​first_​​step_​​was_​​to_​​create_​​an_​​environment_​for_{​ ​​}the_​​cells,_​​which_​was_{​ ​​}a_​​20_​​nanometer_​​PB film_​​(over_​​a_​​Si_​​wafer)_​​as_​​a_​​hard_​​substrate,_​​and_​​alongside_​​an_​​Alpha_​MEM_{​ ​​}growth_​​medium_​​of_​​10% FBS,_​​200μM_​​L-ascorbic_​​acid_​​2-phosphate,_​​and_​​10mM_​​b-glycerophosphate_​​is_​​an_​​environment_​​for osteogenesis._​24​_{​​Our​​goal​​was​​not​​to​​control​​when​​or​​how​​differentiation​​occurred,​​but​​to​​test​​the}

cells_​​could_​​even_​​do_​​so_​​after_​​up_​​to_​​21_​days,_{​ ​​}so_​​we_​​used_​​that_​​working_​​substrate_​​and_​​medium._​​We used_​​cut_​​wafers_​​and_​​wells_​​of_​​different_​​sizes_​for_{​ ​​}different_​​purposes,_​​but_​spin_{​ ​​}coated_​​a_​​thin,_​​hard layer_​​of_​​PB_​​over_​​each_​​of_​​them_​​and_​​cultured_​​DPSCs_​to_{​ ​​}all_​​of_​​them_​​on_​day_{​ ​​}0_​​with_​​the_​​same_​​cell solution._​​First,_​​using_​​a_​​diamond_​​cutter_​​and_​tweezers,_{​ ​​}we_​​cleaved_​​silicon_​​wafers_​​needed_​​for_​​the substrates_​​into_​​the_​​1x1_​​and_​​2x2_​​cm_​​squares,_​​with_​​the_​​small_​​samples_​​for_​​confocal_​​microscopy_​​and

SEM_​​and_​​the_​​larger_​​samples_​​for_​​AFM._​​Before_​we_{​ ​​}spin_​​casted_​​the_​cleaved_{​ ​​}wafers_​​to_​​coat_​​them, they_​​were_​​put_​​through_​​a_​​series_​​of_​washes_{​ ​​}to_​​prevent_​​possible_​​contamination_​​and_​​ensure_​​optimal conditions_​​for_​​creating_​​a_​​hard,_​​stiff_​​polybutadiene_​​(PB)_​​film._​24​​​_{​​To​​do​​so,​​we​​performed​​separate}

processes_​​to_​​remove_​​dust_​​and_​​organics_​​before_​​making_​​the_​​surface_​hydrophilic_{​ ​​}to_​​preserve_​​the cleaned_​​wafers_​​in_​​water._​​The_​​hardness_​​of_​​the_​​substrate_​​required_​​a_​​thin_​​PB_​​film,_​​with_​​thickness being_​​determined_​​by_​​the_​​concentration_​​of_​​the_​solution_{​ ​​}being_​​spin_​​casted._​​To_​​create_​​that_​​solution, we_​​measured_​​15_​​mg_​​of_​​PB_​​out_​​on_​​an_​​electronic_​​scale_​​and_​​dissolved_​​it_​​into_​​5_​​mL_​​of_​​toluene_​​to form_​​a_​​[3mg/mL]_​​PB-toluene_​​solution.

We_​​spin_​​casted_​​the_​​PB-toluene_​​solution_​​onto_​the_{​ ​​}prepared_​​silicon_​​wafers_​​to_​​create_​​the_​​20 nm_​​thick_​​hard_​​PB_​​film._​​Because_​​the_​​PB-toluene_​​solution_​​was_​hydrophobic,_{​ ​​}we_​​immersed_​​the wafers_​​in_​​H_​2O:HF_{​ ​​}=_​​30:1_​​immediately_​prior_{​ ​​}to_​​spin_​​casting_​​until_​​the_​​surface_​​became

hydrophobic._​​We_​​then_​​pipetted_​​PB-toluene_​​solution_​onto_{​ ​​}wafers_​​until_​the_{​ ​​}surface_​​was_​​fully covered._​​We_​​spin_​​casted_​​the_​​wafer_​​at_​​2500_​rpm_{​ ​​}at_​​a_​​1000_​ramp_{​ ​​}acceleration_​​for_​​30_​​seconds. Finally,_​​we_​​annealed_​​the_​​spin_​​casted_​​samples_​​in_​​a_​​high-powered_​​vacuum_​​oven_​​set_​to_{​ ​​}10_​-7​_{​​torr​​at}

150℃_​​for_​​12_​​hours_​​to_​​sterilize_​the_{​ ​​}wafer_​​and_​flatten_{​ ​​}the_​​PB_​​film.

We_​​then_​​plated_​​DPSCs_​​(cell_​​line_​​AX3)_​onto_{​ ​​}prepared_​​substrates_​​on_​​day_​​0,_​​pipetting_​​the cell_​​solution_​​onto_​​substrates_​​inside_​​wells_​along_{​ ​​}with_​​our_​aMEM_{​ ​​}medium._​​We_​​plated

approximately_​​7,500_​​and_​​15,000_​​cells_​​on_​​each_​​small_​​and_​​large_​well,_{​ ​​}respectively._​​Based_​​on previously_​​calculated_​​concentration_​​of_​​the_​​cell_​​solution_​​(by_​​counting_​​a_​sample_{​ ​​}of_​​the_​​solution with_​​a_​​hemocytometer),_​​this_​​corresponded_​​to_​​1_​​mL_​​of_​​medium_​​needed_​​for_​​small_​​1x1_​​samples_​​and 3_​​mL_​​of_​​medium_​​for_​​large_​​2x2_​​samples._​After_{​ ​​}autoclaving,_​​we_​​then_​added_{​ ​​}sterilized_​​rutile_​​TiO_​2 NPs_​​to_​​experimental_​​groups_​​NP-1_​​and_​​NP-4_​​on_​​days_​1_{​ ​​}and_​​4,_​​respectively._​​To_​​do_​​so,_​​we_​​added_​​40

mL_​​of_​​medium_​​to_​​the_​​4_​​mg_​​of_​​TiO_{​2​​​}NPs_​​to_​​create_​​a_​​0.1_​​mg/mL_​​solution._​​We_​​aspirated_​​the previous_​​medium_​​in_​​the_​​wells,_​​and_​​we_​​pipetted_​1_{​ ​​}mL_​​of_​​the_​TiO_{​ ​2​}-medium_​​solution_​​into_​​each experimental_​​well.

For_​​each_​​day_​​of_​​testing,_​​cell_​​fixation_​on_{​ ​​}every_​​sample_​​was_​​performed_​​for_​​images_​​under confocal_​​microscopy_​​and_​​scanning_​​electron_​microscopy_{​ ​​}(SEM),_​​discussed_​​later._​​To_​​prevent contamination,_​​the_​​gloves_​​and_​​fume_​​hood_​​were_​​sprayed_​​with_​​70%_​ethanol._{​ ​​}Sample_​​wells_​​were then_​​aspirated_​​until_​​only_​​the_​​cells_​​and_​​substrates_​​remained._​​1_​​mL_​​of_​​sterilized_​​PBS_​​solution_​​was then_​​pipetted_​​into_​​each_​​well_​​and_​aspirated_{​ ​​}immediately_​​afterwards._​This_{​ ​​}aspiration_​​procedure_​​was done_​​twice_​​with_​​PBS_​​and_​​then_​​placed_​​into_​10%_{​ ​​}formalin_​​for_​​15_​minutes_{​ ​​}to_​​kill_​​cells_​​while

maintaining_​​their_​​shape_​​and_​​structure,_​​completing_​​the_​​preservation_​​process._​​After_​​washing_​​with PBS_​​twice,_​​the_​​cells_​​were_​​preserved_​in_{​ ​​}a_​​4℃_​refrigerator_{​ ​​}for_​​later_​​use.

(II._​​B.)_​​Atomic_​​Force_​​Microscopy

To_​​determine_​​cell_​​mechanical_​properties_{​ ​​}in_​​relation_​​to_​​the substrate,_​​we_​​performed_​​shear_​​modulation_​​force

microscopy_​​(SMFM)_​​using_​a_{​ ​​}Digital_​​Instruments_​​Atomic Force_​​Microscope_​​(AFM)_​​set_​to_{​ ​​}contact_​​mode._​​As_​​depicted in_​​Figure_​​2,_​​a_​​tiny_​cantilever_{​ ​​}and_​​tip_​​make_​​soft_​​physical contact_​​with_​​the_​​surface_​​of_​the_{​ ​​}cells,_​​dragged_​​along_​​at_​​drive amplitudes._​​Based_​​on_​​the_​​cells’_​​resistance_​​to_​​deformation_​​and_​​corresponding_​​friction,_​​the

response_​​amplitude_​​is_​​captured_​​by_​​the_​​laser_​​and_​​photodiode,_​​thus_​measuring_{​ ​​}cell_​​stiffness._​​We plotted_​​varying_​​drive_​​and_​​subsequent_​​response_​amplitudes_{​ ​​}for_​​each_​SMFM_{​ ​​}measurement_​​for_​​a wider_​​range_​​of_​​data.

We_​​used_​​a_​​Bruker_​​Dimension_​​Icon_​​with_​​ScanAsyst_​​to_​​analyze_​the_{​ ​​}surface_​​morphology_​​of each_​​spin-coated_​​sample._​​They_​​were_​​then_​​scanned_​​in_​​peak_​​force_​tapping_{​ ​​}mode_​​and_​​analyzed using_​​Bruker’s_​​NanoScope_​​Analysis_​​software._​​The_​​data_​for_{​ ​​}this_​​software_​​would_​​later_​​be_​​used_​​to calculate_​​the_​​cells’_​​shear_​​modulus_​​values,_​​which_​​will_​​be_​​explained_​​later_​​in_​​results._​​Every_​​test_​​day, we_​​first_​​performed_​​SMFM_​​on_​​a_​​regular_​​20_​​nm_​​PB_​​film_​​before_​​testing_​to_{​ ​​}create_​​a_​​base_​​calibration for_​​later_​​SMFM_​​measurements_​​of_​​DPSC_​​cells._​​After_​​we_​​calibrated_​the_{​ ​​}substrates,_​​we_​​placed samples_​​under_​​the_​​AFM,_​​and_​​we_​​performed_​​testing_​twice_{​ ​​}for_​​3_​​chosen_​cells_{​ ​​}in_​​each_​​of_​​two samples_​​for_​​a_​​total_​​of_​​6_​​measurements_​​per_​​experimental_​​group.

(II._​​C.)_​​Scanning_​​Electron_​​Microscopy

We_​​characterized_​​cross_​​sectional_​​images_​​of_​DPSCs_{​ ​​}to_​​view_​nanoparticle_{​ ​​}uptake_​​in_​​cells via_​​focused_​​ion-beam-scanning_​​electron_​​microscopy_​​(FIB-SEM)_​​using_​​a_​​LEO/Zeiss_​​1550 emission_​​scanning_​​electron_​​microscope_​​with_​​a_​Zeiss_{​ ​​}Crossbeam_​​340_​attachment_{​ ​​}at_​​1_​​kV acceleration_​​voltage_​​and_​​5_​​mm_​​working_​distance._{​ ​​}Cells_​​were_​fixed_{​ ​​}in_​​2%_​​glutaraldehyde/2% paraformaldehyde_​​for_​​1_​​hour_​​and_​​stained_​​using_​​the_​​OTOTO_​​method_​​​26​_{​​commonly​​used​​to}

increase_​​the_​​contrast_​​of_​​SEM_​​images._​​We_​​then_​​used_​​Acetone/DI_​water_{​ ​​}mixtures_​​at_​​gradually increasing_​​concentrations_​​from_​​30%_​​to_​​100%_​to_{​ ​​}dehydrate_​​samples_​before_{​ ​​}sputter_​​coating_​​with Au_​​for_​​4_​​nm._​​After_​​samples_​​were_​​prepared,_​We_{​ ​​}deposited_​​Pd_​​onto_​cells_{​ ​​}for_​​protection_​​from_​​the focused_​​ion_​​beam._​​The_​​FIB_​​then_​​milled_​​the_​cell,_{​ ​​}exposing_​​the_​​cell_​cross_{​ ​​}section_​​and_​​allowing_​​the SEM_​​to_​​take_​​a_​series_{​ ​​}of_​​cross-sectional_​​stack_​​images.

Furthermore,_​​we_​​captured_​​the_​​elemental_​composition_{​ ​​}of_​​DPSCs_​​using_​​regular_​​SEM equipped_​​with_​​Oxford_​​energy_​​dispersive_​​X-ray_​​spectroscopy_​​(SEM/EDX)_​​on_​​day_​21_{​ ​​}in_​​order_​​to characterize_​​biomineralization,_​​or_​​the_​​cell-mediated_​process_{​ ​​}of_​​depositing_​​minerals_​​into_​​their