Modularity-based mathematical modeling of ligand inter-nanocluster connectivity for unraveling reversible stem cell regulation

Ethics declaration

All mouse experiments were performed after obtaining approval from the Institutional Animal Care and Use Committee of Korea University (KOREA-2021-0006). The mice were housed in a standardized environment with a 12 h light/12 h dark cycle at 18–25 °C under 50 ± 5% relative humidity in a semi-specific pathogen-free (SPF) environment at Laboratory Animal Research Center of Korea University College of Medicine. The animals were monitored daily for any clinical symptoms (e.g., re-epithelialization, weight loss, inflammation, infection, bleeding). After each experiment, mice were euthanized via CO₂ asphyxiation in a sealed chamber, with CO₂ gas concentration gradually increased at a rate of 10–30% per minute and maintained for at least 5 min. After respiration ceased, the animals were checked for heartbeat cessation to confirm complete euthanasia.

Tuning only the anisotropy of silica-enveloped nano-blocker precursors irrespective of the projected area

For the meticulous tuning of the anisotropy degree (i.e., low, moderate, and high) of remotely manipulable nanorods (nano-blockers), precursor nanorods in akaganeite phase (β-ferric oxyhydroxide) prior to the formation of silica envelop were first synthesized in different dimensions. Their nanorod structures were achieved through the hydrolysis process of iron(III) chloride (FeCl₃), resulting in the preferential growth of the akaganeite phase (monoclinic structure) in the [010] direction. The growth rate of the akaganeite nanorods is directly proportional to the FeCl₃ concentration, in which their higher concentration results in the nanorods with higher anisotropies. Hence, the akaganeite nanorods with varied anisotropies were synthesized through a sequential procedure. First, controlled amounts (2.5 g, 5.0 g, or 7.5 g) of FeCl₃ · 6H₂O were dissolved in deionized (DI) water and left at 85 °C overnight, resulting in the akaganeite nanorods with low, moderate, and high anisotropies, respectively. They were then isolated by washing with centrifugation using ethanol and then suspended in 10 mL of DI water. Finally, the precursor akaganeite nanorods in various anisotropies prior to the formation of silica envelop were stabilized in 90 mL of DI water containing 2 wt% polyvinylpyrrolidone (PVP, molecular weight of 10 kDa) overnight, washed with centrifugation using DI water, and finally suspended in 12 mL of DI water.

For the adjustment of their projected area to be similar and their structural preservation during the phase transformation from akaganeite to magnetite phase (via reduction), a silica envelope was formed on the surfaces of each precursor akageneite nanorods of different anisotropies. The thickness of silica envelops on each akaganeite nanorod differed depending on the desired anisotropy. To this end, 5 mL of DI water containing 1.25 mL of ammonium hydroxide was added to 1 mL of stabilized akaganeite nanorods in 25 mL of ethanol, followed by stirring for 20 min. The number of times TEOS was added to the mixture was controlled depending on the desired anisotropies (low, moderate, and high) of the silica-enveloped nanorods. For the low or moderate anisotropy of the nanorod structure of akageneite enveloped by silica (nano-blocker precursors) (“Low aniso. nano-blocker precursor” and “Moder. aniso. nano-blocker precursor”), 12.5 μL of TEOS was added eight or four times with 15-min interval between each addition to the akaganeite nanorods with low or moderate anisotropies, respectively. For high-anisotropy nano-blocker precursors (“High aniso. nano-blocker precursor”), 10 μL of TEOS was added once to the akaganeite nanorods with high anisotropy. In each case, the mixture was then stirred at room temperature for 2 h, washed with centrifugation using ethanol, and suspended in 3 mL of DI water.

In situ phase transformation for converting anisotropic nano-blocker precursors to anisotropic nano-blockers

The annealing-mediated reduction was applied to mediate in situ phase transformation of the akaganeite phase in the anisotropic nano-blocker precursors into the magnetite phase to produce the nanorod structure of magnetite enveloped by silica envelope (nano-blocker) that is remotely and reversibly manipulable. To this end, 1.5 mL of nano-blocker precursors in various anisotropies were each mixed with 10 mL of triethylene glycol (TEG) and heated at 340 °C for 2 h under a nitrogen atmosphere. After the treatment via in situ phase transformation, the resulting anisotropic nano-blockers were washed with centrifugation using ethanol and suspended in 6 mL of ethanol. Each “Low aniso. nano-blocker precursor”, “Moder. aniso. nano-blocker precursor”, and “High aniso. nano-blocker precursor” transformed into “Low aniso. nano-blocker”, “Moder. aniso. nano-blocker”, and “High aniso. nano-blocker”, respectively.

For the verification of in situ phase transformation of converting the akageneite phase (monoclinic structure) in anisotropic nano-blocker precursors into the magnetite phase (inverse spinel structure) during the reduction, in situ, TEM, HR-TEM, and selected area electron diffraction (SAD) imaging were performed by using a Titan^TM 80-300. FFT analysis was also performed on the images (aligned along the zone axis) acquired from in situ HR-TEM imaging for the crystal structure analysis. The analyses of the phase transformation under anneal conditions at 340 °C for 2 h were performed using representative moderate-anisotropy nano-blocker precursors at an accelerating voltage of 200 kV with a heating rate of 0.45 °C/s. For the in situ SAD pattern, 60 frames were captured over 130 min and the resulting in situ movie was produced at 780 times faster than in real-time speed. The distinctive rings of diffraction for the (310) plane corresponding to the akaganeite phase and the (220) plane corresponding to the magnetite phase were colored in turquoise and red, respectively.

In situ TEM analysis

For the examination of homogeneous shapes and sizes of the anisotropic nano-blocker precursors, anisotropic nano-blockers, and GNPs, TEM imaging was conducted with Talos G2 apparatus from Thermo Fisher⁷⁵. Analysis of the obtained TEM images using ImageJ software allowed the computation of anisotropic nanorod length, width, anisotropy [calculated by dividing the nanorod length (major axis) by its width (minor axis)], and projected area.

In situ SAD analysis

For crystalline atomic structure characterizations, anisotropic nano-blocker precursors and anisotropic nano-blockers were characterized for the crystal structure verification of the akaganeite in the nano-blocker precursors and magnetite in the nano-blocker via SAD analysis using a Titan 80-300 with a camera length of 60 mm. The results were presented in the SAD pattern that showed multiple rings of diffraction corresponding to various planes, such as (200), (103), (211), (310), and (411) for the akaganeite phase and (220), (311), (400), (511), and (440) for the magnetite phase.

In situ HR-TEM analysis

The anisotropic nano-blocker precursors, anisotropic nano-blockers, and GNPs were characterized for the atomic structure visualizations of the akaganeite in the nano-blocker precursors, magnetite in the nano-blockers, and gold in GNPs via HR-TEM analysis using a Titan 80-300 apparatus at an accelerating voltage of 300 kV. The average d-spacing between the successive lattice planes (approximately 5.2–5.4 Å for akaganeite, 3.1 Å for magnetite, and 2.4 Å for gold) was measured and identified based on available data for the respective crystalline structures.

In situ FFT analysis

The images (aligned along the zone axis) acquired from HR-TEM imaging were exploited for the crystal structure analysis of the akaganeite phase in the nano-blocker precursors and the magnetite phase in the nano-blockers via FFT characterization. The results showed that periodic bright spots corresponded to the (200) plane of the akaganeite in the nano-blocker precursors and the (220) plane of the magnetite in the nano-blockers.

Linear profiles of elemental EDS mapping

For the structural and elemental analyses of anisotropic nano-blocker precursors and anisotropic nano-blockers, HAADF-STEM and EDS mapping were performed using the Talos G2 apparatus. HAADF-STEM imaging was carried out for the nanostructure examination of anisotropic nano-blocker precursors and anisotropic nano-blockers. The specific imaging conditions include a 200-kV acceleration voltage, a collection semi-angle of 38–200 mrad, a convergence semi-angle of 11.8 mrad, a pixel dwell time of 3 μs, a 1024 × 1024-pixel area, an electron probe size of 0.2 nm, an emission current of 185 μA, and a probe current of 185 pA.

EDS mapping and analyses (elemental spectra and line profile) of anisotropic nano-blocker precursors and anisotropic nano-blockers were conducted for the elemental composition (Fe, O, and Si elements) examination of the nanorods. The specific imaging was carried out at 200 kV and 2.13 nA with a speed of 15 min/image. The EDS mapping showed that Fe was present solely in the core, O was evenly distributed, and Si was solely present in the silica envelop of nano-blocker precursors and nano-blockers. The EDS line profiles, taken from the midsection of nano-blocker precursors and nano-blockers, confirmed individually optimized silica envelops for each varying anisotropy.

HR-STEM

The anisotropic nano-blockers and GNPs were subjected to HR-STEM characterization using Titan 80-300 apparatus for the atomic structure examinations of crystalline magnetite in the nano-blockers and gold in the GNPs. The imaging was performed at 300 kV with a Cs-corrected TM 80-300 probe at a magnification of 10 million times under conditions including 47.5–200-mrad collection semi-angle, 12-mrad convergence semi-angle, 8-μs pixel dwell time, 2048 × 2048 pixel area, 0.08-Å electron probe size, 197-μA emission current, and 62.5-pA probe current. The lattice parameters of the (100) plane of the magnetite (8.4 Å) and (111) planar spacing of the gold (2.4 Å) were calculated to confirm their respective crystalline structures.

XRD analysis

For the crystalline plane examination of the akaganeite in the nano-blocker precursors and magnetite in the nano-blockers, XRD analysis was performed using a D/MAX-2500V/PC apparatus from Rigaku with Cu Kα radiation. The typical diffraction peaks such as (103), (211), (310), (411), and (512) for the akaganeite phase and (220), (311), (400), (511), and (440) for the magnetite phase were identified for the respective crystalline planes.

Magnetic property analysis

For the examination of non-magnetic anisotropic nano-blocker precursor properties and magnetically reversible anisotropic nano-blocker properties, VSM measurement was conducted using an EV9-380 apparatus (Microsense). The measured magnetic moments were assessed as hysteresis loops under a magnetic field and presented after normalization to the respective dry weight of the material.

Amine-functionalization of anisotropic nano-blockers

For the versatile application of anisotropic nano-blockers, their silica envelops were functionalized with amine groups by mixing 10 mL of each nano-blocker in various anisotropies with 60 mL of ethanol. The suspension was mixed with 1 mL of (3-aminopropyl) triethoxysilane (APTES) and stirred at room temperature for 16 h. The resulting anisotropic nano-blockers with amine-functionalized silica envelopes were washed with centrifugation using ethanol and then suspended in 40 mL of DI water.

Polymer linker-coupling of anisotropic nano-blockers

For the reversible tuning of the number of inter-cluster edges (referred to as “# inter-cluster edges”), nano-blockers in various anisotropies (“Low aniso. nano-blocker”, “Moder. aniso. nano-blocker”, and “High aniso. nano-blocker”) were coated with polymer linkers. The procedure involved adding 0.5 mg of maleimide-poly(ethylene glycol)-N-hydroxy-succinimide (Mal-PEG-NHS, molecular weight of 10 kDa from Polysciences), 20 μL of phosphine hydrochloride (TCEP), and 2 μL of N, N-diisopropylethylamine (DIPEA) to 1 mL of amine-functionalized anisotropic nano-blockers, then vortexing the mixture for 16 h in the dark at 25 °C. This procedure produced polymer linker-coupled amine-functionalized anisotropic nano-blockers ready for grafting to the materials with the interconnected ligand nodes (“Low aniso.”, “Moder. aniso.”, and “High aniso.”).

Zeta potential analysis

For the verification of the changes in the surface charge before and after coupling polymer linkers to the amine-functionalized anisotropic nano-blockers, zeta potential analysis was conducted using a Zetasizer Nano ZS90 apparatus from Malvern Panalytical.

FTIR analysis

For the verification of specific chemical bonds formed after the amine-functionalization and polymer linker-coupling of the anisotropic nano-blockers, FTIR analysis was conducted using a Nicolet iS10 apparatus from Thermo Fisher Scientific. Beforehand, samples in the suspension state were dried, embedded in KBr pellets, and measured for the FTIR. The absorption peaks were identified based on their matching with the chemical bonds of the anisotropic nano-blockers exhibiting Si-O and Fe-O bonds. The absorption peaks after the change in the chemical bond with identified at the peaks of O=C−NH and C−O bonds signifying the successful polymer linker-coupling.

Synthesis of the GNPs of distinctly different sizes

For the obvious discrimination of smaller GNPs acting as ligand nodes of the interconnected ligands on the material surface and larger GNPs utilized for tagging integrin β1 of the recruited stem cell, GNPs of distinctly different sizes were separately prepared. For their synthesis of smaller- and larger-sized GNPs, 20 mL of 1-mM hydrogen tetrachloroaurate (III) trihydrate (HAuCl₄·3H₂O) was first shaken at 100 °C for 30 min. Followingly, 1.6 mL (for smaller GNPs in the diameter of 20 nm) or 2.5 mL (for larger GNPs in the diameter of 40 nm) of 38.8-mM trisodium citrate (Na₃C₆H₅O₇) was added and stirred for 15 min. The GNPs were each collected when the color of the solution changed from yellow to burgundy red, signifying the completion of the reaction.

DLS analysis

For the confirmation of homogeneous GNPs in smaller (in the diameter of 20 nm) and larger (in the diameter of 40 nm) sizes used in this study, a DLS examination was conducted with a Zetasizer Nano ZS90 apparatus from Malvern Panalytical.

Development of materials displaying a reversibly tunable number of inter-cluster edges within interconnected ligand networks

To develop materials displaying a reversibly tunable number of inter-cluster edges within interconnected ligand networks, the nano-blockers of each anisotropy were grafted via polymer linkers over the interconnected ligand nodes (liganded smaller GNPs) presented on the material surface. To decouple other parameters, the nano-blockers of each anisotropy were designed to exhibit similar projected areas, which were grafted to the material surface at similar density, enabling the variation of only anisotropy in the nano-blockers to present a tunable number of inter-cluster edges of interconnected ligands. To this end, glass coverslips (22 × 22 mm², cell culture grade) used as the material were sterilized by washing with a 1:1 hydrochloric-methanol solution for 45 min, followed by rinsing with DI water three times. Afterward, the coverslips were treated with sulfuric acid (H₂SO₄) for 70 min to activate their surface with hydroxyl (−OH) groups, followed by washing with DI water and methanol. The hydroxyl-activated surface was then thiolated for 70 min in a solution of (3-mercaptopropyl)trimethoxysilane (MPTMS) and ethanol (1:19) in the dark, followed by rinsing with ethanol and DI water and drying in an oven at 100 °C for 70 min.

To fabricate the interconnected ligand network, the thiolated surfaces of the materials were first incubated with 300 μL of smaller GNPs (in the diameter of 20 nm) at 25 °C for 16 h in the dark for their grafting through gold-thiol bonding. Successive treatment with a solution of 0.2 nM of thiolated RGD tripeptide ligand (CDD RGD from Gl Biochem) and 10 mM of tris(2-carboxyethyl)phosphine (TCEP) for 12 h in the dark followed by DI water washing resulted in the material-grafted liganded GNPs via the gold-thiol bond, thereby turning them into completely interconnected ligand nodes. In this procedure, TCEP was used to prevent any non-specific grafting of the thiolated ligand to the thiolated surface through disulfide bonds.

The material surfaces exhibiting completely interconnected ligands were then incubated with 300 μL of polymer linker-coupled nano-blockers of each anisotropy for 16 h in the dark, followed by rinsing with DI water. The polymer linker-coupled anisotropic nano-blockers were grafted to the material surface not covered with the interconnected ligand nodes through maleimide-thiol bonding. The residual surfaces not covered with either the ligand nodes or anisotropic nano-blockers were treated with 2 mL of DI water containing 0.75 mg of methoxy-PEG-maleimide (MeO-PEG-Mal, molecular weight of 750 Da from Sigma–Aldrich) at 25 °C for 2 h in the dark to prevent non-specific cell adhesion to the non-liganded surfaces. The material surfaces were then washed with DI water.

To linearize the anisotropic nano-blockers [“Low aniso. (Lin.)”, “Moder. aniso. (Lin.)”, and “High aniso. (Lin.)”], the materials were subjected to three rounds of magnetic annealing for 15 min each during their incubation period with the polymer linker-coupled anisotropic nano-blockers. The uniform magnetic field produced by a 13.5-A electric current applied to the electromagnets at both ends of the materials linearized the anisotropic nano-blockers in the direction of the magnetic field. The magnetic annealing system was optimized so that the nano-blockers could be linearized without being attracted to the magnets. In our study, the anisotropic nano-blockers were assumed to be randomly oriented unless the linearization was stated.

Systematic analysis of tunable # ligand inter-cluster edges

The SEM imaging was conducted with a Quanta 250 FEG SEM (FEI) to examine the arrangement of the liganded GNPs (ligand nodes) and anisotropic nano-blockers on the material surface displaying reversibly tunable average number of inter-cluster edges within the interconnected ligand networks. Beforehand, the materials were dried under a vacuum and coated with platinum before SEM imaging. Analysis of the obtained SEM images using ImageJ software revealed a highly homogeneous arrangement of the ligand nodes with their similar values of inter-distance and surface density of the anisotropic nano-blockers, as well as different values of the average number of inter-cluster edges and the average inter-angle between the nano-blockers (in absolute value). The highly consistent regular distribution of the ligand nodes on the material surfaces allowed cells to sense them as interconnected ligands that could be analyzed to compute the average number of inter-cluster edges. Moreover, the identical surface density of anisotropic nano-blockers in each group suggests the difference in the average number of inter-cluster edges in each group to be mainly attributed to the anisotropy difference of nano-blockers.

Analysis of the interconnected ligands using the average number of inter-cluster edges

To quantitatively analyze the interconnected ligand clusters, the notion of the average number of inter-cluster edges based on graph theory was utilized^76,77. In our system, the liganded GNPs that each acted as ligand nodes were homogeneously arranged with equal inter-distances (approximately 200 nm). To facilitate graph theory-based analysis, the edges between ligand nodes were established using Delaunay triangulation, which ensures geometrically consistent connections between neighboring ligand nodes by maximizing the minimum angle of each triangle to achieve tessellation close to equilateral triangles^78,79. Each liganded GNP node in the network structure in the SEM images was mapped as coordinates using ImageJ software, then connected by edges by applying Delaunay triangulation in Python. Following the construction of the ligand network, the edges that are disconnected by the presence of anisotropic nano-blockers were removed, resulting in the modeling of the ligand network involving anisotropic nano-blockers. Subsequently, these network models were partitioned into clusters by maximizing the modularity using the Louvain algorithm in Python. Finally, the average number of inter-cluster edges (referred to as “# inter-cluster edges”) was calculated by dividing the number of interconnected edges between clusters by the number of connected cluster pairs. The “Low aniso.”, “Moderate aniso.”, and “High aniso.” groups present different # inter-cluster edges of highly organized ligand nodes in the presence of anisotropic nano-blockers where the obstruction of connected edges between ligand clusters is stimulated with escalating nano-blocker anisotropy that reduces # inter-cluster edges. Remote control of irreversible linearization (“Lin.”) of high-anisotropy nano-blockers yields their presentation in an ordered manner, resulting in more locally unobstructed ligand node connections and thus partly enhancing # inter-cluster edges as compared with the non-linearized group. Remote control of elevating anisotropic nano-blockers (“E.”) reconnects the interconnected ligand nodes allowing cell infiltration under the elevated nano-blockers, which are disconnected in the presence of non-elevated nano-blockers (“NE.”), resulting in a significant escalation of # inter-cluster edges.

Optimization of the polymer linker density to reversibly control # ligand inter-cluster edges

To confirm the optimized polymer linker density used to graft the anisotropic nano-blockers to the material surface that allows cells to sense the ligand nodes to be interconnected across the elevated anisotropic nano-blockers and thus infiltrate through the nano-gap under them, Ellman’s assay was performed. To this end, each 20 μL of “High aniso. nano-blocker” (approximately 654 k nanorods per 1 μL) coupled with either a low (150 μg) or high (6000 μg) amount of polymer linker was first reacted four times independently with 3.6 μg of thiolated L-cysteine via thiol-ene bond at room temperature. Successive applications of Ellman’s assay enabled a comparative analysis of the density of polymer linkers coupled on each nano-blocker by computing the concentration of L-cysteine reacted with the polymer linkers coupled to the nano-blockers, in which a high number of reacted L-cysteine signified high density of polymer linkers coupled on the nano-blockers. For such calculation, the amount of unreacted L-cysteine determined by the amount of residual Ellman’s reagent in the supernatant was subtracted from the total amount of added L-cysteine. SEM images of the IGNP tagging of recruited integrin in stem cells confirmed that the low density of polymer linkers used to graft the nano-blockers to the materials (“High aniso.”) was optimized for cells to infiltrate under the elevated nano-blockers, while the high density of polymer linkers blocked the cell infiltration albeit under the elevated state.

In situ imaging of the cyclic remote control of the anisotropic nano-blocker

For the examination of cyclic remote control of anisotropic nano-blockers on the materials enabling reversibly tunable # inter-cluster edges of interconnected ligands, AFM was performed with an XE-100 System from Asylum Research at room temperature in air mode using an SSS-SEIHR-20 AFM cantilever (spring constant: 5–37 N/m, resonance frequency: 96–175 kHz) from Nanosensors. The remote modulation of the anisotropic nano-blockers situated over the interconnected ligand nodes (liganded smaller GNPs in the diameter of 20 nm) that reversibly controls their # inter-cluster edges was examined via peak height change. The application of an upward magnetic field pulls the anisotropic nano-blockers away from the ligand nodes situated under them, thereby escalating # ligand inter-cluster edges by reconnecting the ligand nodes that were originally inaccessible to cells. In the absence of the magnetic field, anisotropic nano-blockers obstruct the interconnection of ligands reducing # ligand inter-cluster edges.

The identical area of the “High aniso.” material displaying nano-blockers in high-anisotropy over smaller GNPs was cyclically imaged underemployment (“E.”) and non-employment (“NE.”) a piece of permanent magnet (295 mT) above the material. The differences in linear height profile and the computed peak height of the high-anisotropy nano-blockers between the elevated [“High aniso. (E.)”] and non-elevated [“High aniso. (NE.)”] states were analyzed by using the Igor Pro 6.12 A and ImageJ software to verify the cyclic tuning of # inter-cluster edges of interconnected ligands in situ.

Stem cell regulation by tuning # ligand inter-cluster edges only

The materials displaying reversibly tunable # inter-cluster edges of interconnected ligand networks were first sterilized for 3 h via UV light irradiation. They were employed to culture human mesenchymal stem cells (hMSCs, passage 5) provided and authenticated by Lonza (catalog number: PT-2501) to probe the effect of tuning # ligand inter-cluster edges on integrin recruitment, focal adhesion, mechanotransduction, and following differentiation. Owing to the relatively large size of stem cells on the hundreds of micrometer scale, they were employed as a model cell type whose responses to materials can be systematically analyzed via the average # ligand inter-cluster edges that can be generalized for microscale ligand modeling by calculating the number of connected edges between ligand clusters for each ligand cluster pair in SEM images.

In specific, to probe the effect of # ligand inter-cluster edges on stem cell adhesion, stem cells (100k cells per cm²) were seeded only at the initial stage of culture (without additions at the later stage) onto the materials under the conditions of 37 °C and 5% CO₂ using growth medium prepared by supplementing 10% fetal bovine serum (FBS), 4 mM L-glutamine, and 50 U/mL of penicillin and streptomycin into high glucose Dulbecco’s modified Eagle medium (DMEM). To this end, each material displays nano-blockers of different anisotropy (“Low aniso.”, “Moder. aniso.”, and “High aniso.”) and linearized nano-blockers [“High aniso. (Lin.)”] were used to assess the adherent stem cells after 48 h of culturing. To verify the # RGD ligand inter-cluster edges-specific regulation of stem cells, the following control groups were employed: “Non-decorated material”, “GNP-decorated material”, “Liganded GNP-decorated material”, “Low aniso. free of ligand”, “Moder. aniso. free of ligand”, and “High aniso. free of ligand”. To compare the effectiveness of stem cell regulation by ligand ordering, the following control groups were employed: both non-linearized and linearized nano-blockers of different anisotropy including “Low aniso.”, “Low aniso. (Lin.)”, “Moder. aniso.”, and “Moder. aniso. (Lin.)” groups.

Regulation of stem cell behaviors via cyclic tuning of # ligand inter-cluster edges

Cyclic tuning of # ligand inter-cluster edges was achieved by controlling the elevation of anisotropic nano-blockers was analyzed for the adherent cells after 72 h of culturing on each material displaying anisotropic nano-blockers with the employment “High aniso. (E.)” or the non-employment “High aniso. (NE.)” of a piece of permanent magnet (295 mT) above the materials that were switched or maintained after every 24 h up to 72 h for the “High aniso.” group (“NE.-NE.-NE.”, “NE.-E.-NE.”, “E.-NE.-E.”, and “E.-E.-E.”). To compare the effectiveness of stem cell adhesion by manipulating the elevation of the nano-blockers, the following control groups were employed: both low-anisotropy and moderate-anisotropy nano-blockers including “Low aniso. (E.)”, “Low aniso. (NE.)”, “Moder. aniso. (E.)”, and “Moder. aniso. (NE.)” groups.

Cycling tuning of # ligand inter-cluster edges was also applied to evaluate its effect on the mechanosensing and differentiation of stem cells by culturing them in osteogenic differentiation medium, which was prepared by supplementing 100 nM of dexamethasone, 50 µM of ascorbic acid-2-phosphate, and 10 mM of glycerophosphate into the growth medium, using all the other experimental conditions and groups used in the adhesion experiments.

Regulation of mechanotransduction-mediated stem cell differentiation via tuning # ligand inter-cluster edges was probed either at 48 h (mechanotransduction) or 72 h (differentiation) after culturing in a growth medium or osteogenic differentiation medium, respectively. Each medium used in both conditions was supplemented with one of the following cell adhesion-related inhibitors: ML9 (myosin II-inhibitor, 0.1 µM), Swinholide A (actin polymerization-inhibitor, 0.1 µM), or Y27632 [ROCK-inhibitor, 50 µM] or none for the control group.

Fluorescent immunostaining and quantification analysis

For the investigation of the stem cell-regulatory effect of tuning # ligand inter-cluster edges, fluorescent immunostaining was carried out. Stem cells after culture were fixed in 4% paraformaldehyde (PFA) at room temperature for 10 min, followed by washing with PBS three times. The fixed cells were then blocked using a blocking buffer (3% bovine serum albumin (BSA) and 0.1% Triton X in PBS) at 37 °C for 40 min. The blocked cells were then treated with a blocking buffer containing primary antibodies [Paxilin (Santa Cruz, sc-365379), integrin β1 (Santa Cruz, sc-374429), YAP (Santa Cruz, sc-101199), RUNX2 (Abcam, ab192256), and HuNu (Sigma–Aldrich, MAB1281)] and then incubated at 4 °C for 16 h, followed by washing with 0.5% Tween 20 in PBS four times. The cells were serially incubated in a blocking buffer containing fluorescent secondary antibodies [Alexa Fluor 488 (Thermo Fisher, A11001 and A21206), and Alexa Fluor 546 (Thermo Fisher, A11030)] and phalloidin (Thermo Fisher, A12379) at room temperature for 45 min in the dark, washed four times with 0.5% Tween 20 in PBS, and then immersed in DAPI (Thermo Fisher, P36931) antifade solution.

For the analysis of fluorescently immunostained stem cells, imaging was performed with an LSM700 confocal microscope and Lattcie SIM 5 (NFEC-2024-11-301127) both from Carl Zeiss using the same laser exposure conditions for all groups. Using the confocal images, ImageJ software was utilized to compute the density of adherent stem cells (calculating the number of DAPI-positive nuclei per unit area), the number of focal adhesions per cell (calculating the number of paxillin-positive clusters larger than 1 µm² in each cell), the area per cell (actin-positive area), and the aspect ratio (the longest length divided by the shortest length) of adhered cells⁸⁰. The fluorescence intensities of integrin β1 and the nuclear/cytoplasmic ratios of YAP and RUNX2 fluorescence intensities were determined by analyzing integrin β1-stained images or YAP- or RUNX2-stained images along with the co-staining of phalloidin and DAPI.

For the quantitative analysis of stem cell differentiation, western blotting analysis was conducted after 72 h of cell culturing under the cyclic tuning of # ligand inter-cluster edges in the “High aniso.” group. The proteins of interest in the adherent cells, RUNX2 (Santa Cruz, sc-101145, 60 kDa) and ALP (Abcam, ab67228, 75 kDa), were extracted via centrifugation using PRO-PREPTM solution (iNtRON Biotechnology) and a protease-inhibitor. After mixing with loading dye and denaturing, protein quantification was performed using a BCA Protein Assay Kit from Thermo Scientific. Denatured proteins were separated on 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) gels at 110 V for 1 h and then electroblotted onto polyvinylidene difluoride (PVDF) membranes at 120 V for 1.5 h. The membranes were serially blocked with TBST blocking buffer (5% skimmed milk) for 1 h, incubated with primary antibodies (RUNX2, ALP, and GAPDH) for 16 h, washed with TBST buffer, and then incubated with secondary antibodies conjugated with anti-horse radish peroxidase. Protein expression was visualized and computed using a Linear Image Quant LAS 4000 mini chemiluminescent imaging system after normalization to GAPDH expression. The levels of RUNX2 and ALP protein expressions were determined relative to GAPDH (Santa Cruz, sc-365062, 37 kDa) as an internal control using a chemiluminescent imaging system.

IGNP tagging of recruited integrin

For the examination of stem cell integrin β1 recruitment to the interconnected ligand nodes depending on their tunable # inter-cluster edges, IGNP tagging with analysis was performed. For their obvious discrimination in SEM imaging from smaller GNPs (in the diameter of 20 nm) that act as the ligand nodes, homogeneous larger GNPs (in the diameter of 40 nm) were used for IGNP tagging. To allow larger GNPs for tagging stem cell integrins, they were first treated with a buffer (1% BSA, and 0.1% Tween 20 added to 0.1 M of 1,4 piperazine bis (2-ethanosulfonic acid) (PIPES) buffer at pH 7.4) containing secondary antibodies [goat anti-mouse (H+L) IgG (Abcam, ab6708)] under mild shaking at 37 °C for 16 h.

The adherent stem cells on the materials of different groups were washed with PIPES buffer, fixed in 4% PFA for 10 min, and washed with PBS. The fixed cells were then permeabilized with 0.5% Triton X-100, blocked with 1% BSA for 1 h, treated with integrin β1 primary antibodies at 37 °C for 1 h, and washed six times with 1% BSA. Consequent treatment of the integrin β1 primary antibody-treated cells with secondary antibody-conjugated IGNPs for 16 h allowed the IGNPs to bind to integrin, thereby tagging it in the stem cells. The cells were then washed with PIPES buffer and fixed with 2.5% glutaraldehyde for 7 min. To enhance the contrast for their clear visualization in SEM images, the cells were further treated with 1% osmium tetroxide, washed with PIPES buffer, filtered with DI water, and dried at 37 °C for 1 h.

For the analysis of cellular integrins recruited to the interconnected ligand nodes depending on # inter-cluster edges, the resulting IGNP-tagged stem cells were subjected to SEM imaging using FEI TENEO VS and EDS mapping (for Fe element in nano-blocker core) using an EDAX Octane Elect Super EDS System. Representative analysis of the former enabled the precise visualization of the recruited stem cell integrins to the ligand nodes on the materials, while the latter confirmed the presence and arrangement of the anisotropic nano-blockers that alter # inter-cluster edges of interconnected ligand nodes. The images of cells infiltrating under the elevated high-anisotropy nano-blockers [“High aniso. (E.)”] confirmed that cells sense the ligand nodes to be interconnected across the elevated anisotropic nano-blockers (low polymer linker density), which escalates the overall # ligand inter-cluster edges accessible by cells. For their clear identification in the acquired images, stem cells and IGNPs were each colored green and white, respectively. The average number of IGNPs-tagged integrin of stem cells at the cell boundary was calculated per unit area (μm²).

In vivo stem cell regulation via time-resolved tuning of # ligand inter-cluster edges

40 male BALB/c nude mice (8-week-old) were used in the investigation of the effect of modulating # ligand inter-cluster edges on the focal adhesion, mechanosensing, and following differentiation of stem cells in vivo. Each mouse was first anesthetized via intraperitoneal injection of 60 µL of mixture (alfaxan and rompun in 3:1 ratio) after which an incision was made on their backs, the prepared material implanted in a subcutaneous pocket, and then sutured. The materials (12 × 12-mm² glass) displaying reversibly tunable # inter-cluster edges of interconnected ligands were implanted into the subcutaneous pockets of mice. For such implantation, tweezers were used to lift the subcutaneous tissues and carefully implant the materials so that the surface displaying reversibly tunable # inter-cluster edges of interconnected ligands would face upward. Stem cells (300 k hMSCs per the implant) were then injected onto the implanted material surface and the mice were kept anesthetized for 6 h to immobilize them to ensure stable adherence of the stem cells to the materials without any breakage and leakage. A piece of permanent magnet (295 mT) was coupled to the backs of the mice using a bandage to elevate high-anisotropy nano-blockers [“High aniso. (E.)”] in vivo. The coupling and uncoupling were either switched or maintained after 3 h (“NE.-NE.”, “NE.-E.”, “E.-NE.”, and “E.-E.”). After implantation for 6 h, the mice were sacrificed and the implants were retrieved by carefully opening up the suture with operating scissors and picking up the materials using tweezers, followed by washing with PBS three times for the fluorescent immunostaining analysis of confirming adherent stem cells [human-specific nuclei antigen (HuNu)], focal adhesion, mechanosensing, and their following differentiation. The implants were also retrieved after 24 h for SEM imaging to validate in vivo preservation of the anisotropic nano-blockers and the liganded GNPs on the material surfaces after remote tuning of # inter-cluster edges of the interconnected ligands.

Local and systemic toxicity evaluation of the materials displaying reversibly tunable # ligand inter-cluster edges

For the assessment of the local and systemic toxicity, histological analysis was conducted on subcutaneous tissue collected near the implanted site and the major organs (liver, heart, spleen, and kidney) of the mice before and 7 d after implantation. The collected tissues and organs were first fixed in 4% PFA for 2 d and then washed three times with DI water. Before being embedded in paraffin at 62 °C for 16 h, the samples were sequentially dehydrated with increasing concentrations of ethanol (50%, 75%, 90%, 95%, and 100%) for 1 h each. After embedding, a HistoCore Multicut microtome from Leica RM2125 RTS Biosystem was used to cut the samples into 6 μm-thick sections for mounting onto glass slides. The sections were deparaffinized and rehydrated, then stained with hematoxylin and eosin (H&E) for the visualization of cellular structures within the extracellular matrix in tissues and organs. Nikon Eclipse Ts2 optical microscopy was used to examine the stained samples. The results showed that the materials displaying reversibly tunable # ligand inter-cluster edges inflicted inappreciable local and systemic toxicity.

Statistics and reproducibility

The results reported in this study were validated by independently repeating all experiments at least three times to ensure the production of consistent datasets. The computation of the data was performed using GraphPad Prism software (version 8.0.2). One-way analysis of variance (ANOVA) along with the Tukey–Kramer post hoc test is used to compare multiple groups and determine statistical significance. The level of significance is indicated by asterisks next to the p-values (*p < 0.05, **p < 0.01, and ***p < 0.001). The number of replicates in each experiment is represented by the “n” value.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

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• Source: https://www.nature.com/articles/s41467-024-54557-8