Multiscale reconstruction of a synthetic biomimetic micro-niche for enhancing and monitoring the differentiation of stem cells

Accepted Manuscript Multiscale reconstruction of a synthetic biomimetic micro-niche for enhancing and monitoring the differentiation of stem cells Rui Li, Jinming Li, Jianbin Xu, Dexter Siu Hong Wong, Liming Bian PII:

S0142-9612(18)30328-4

DOI:

10.1016/j.biomaterials.2018.05.001

Reference:

JBMT 18643

To appear in:

Biomaterials

Received Date: 6 February 2018 Accepted Date: 1 May 2018

Please cite this article as: Li R, Li J, Xu J, Hong Wong DS, Bian L, Multiscale reconstruction of a synthetic biomimetic micro-niche for enhancing and monitoring the differentiation of stem cells, Biomaterials (2018), doi: 10.1016/j.biomaterials.2018.05.001. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Multiscale reconstruction of a synthetic biomimetic micro-niche for enhancing and monitoring the differentiation of stem cells

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Rui Li a,1, Jinming Li b,1, Jianbin Xu c, Dexter Siu Hong Wong a, and Liming Bian a,d,e,f*

a

*

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Department of Biomedical Engineering, The Chinese University of Hong Kong, Shatin, New Territories 999077, Hong Kong, P. R. China b MOE Key Laboratory of Laser Life Science and Institute of Laser Life Science, Collage of Biophotonics, South China Normal University, Guangzhou, Guangdong, 510631, P. R. China c Biomedical Research Center, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang, 310016, P. R. China d Shenzhen Research Institute, The Chinese University of Hong Kong, Shatin, New Territories 999077, Hong Kong, P. R. China e China Orthopedic Regenerative Medicine Group (CORMed), Hangzhou, P.R. China f Centre for Novel Biomaterials, The Chinese University of Hong Kong, Shatin, New Territories 999077, Hong Kong, P. R. China

Equal contributed to this work.

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Corresponding author. Department of Biomedical Engineering, The Chinese University of Hong Kong, Shatin, New Territories 999077, Hong Kong, P. R. China E-mail: [email protected] (Liming Bian)

ACCEPTED MANUSCRIPT

Abstract Stem cells reside in a three-dimensional (3D) niche microenvironment, which provides specific cues, including cell-matrix interactions and soluble factors, that are essential to the

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differentiation of stem cells in vivo. Herein we demonstrate a general approach to the synthetic reconstruction of 3D biomimetic niche environment of stem cells by the multiscale combination of macroscopic porous hydrogels and a nanoscale upconversion nanoparticles

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(UCNP)-based nanocomplex. The porous biopolymeric hydrogels emulate the spongy bone

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microstructure and provide 3D environment conducive to the differentiation of seeded stem cells. The UCNP-based nanocomplex (Pur-UCNP-peptide-FITC), which is stably encapsulated in the porous hydrogels, emulates the repertoire of inductive factors in bone matrix by maintaining localized long-term delivery of inductive small molecules. The

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nanocomplex also generates biomarker-specific reporting emissions that correlate with the extent and stage of differentiation of the stem cells in synthetic niche, thereby allowing

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long-term tracking of stem cell fate in a non-contact, non-destructive, and potentially high-throughput manner in living cultures. To the best of our knowledge, this is first

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demonstration of synthetic niche reconstruction. The modular nature of this synthetic niche platform allows various parameters to be easily tuned to accommodate a variety of fundamental studies of dynamic cellular events under controlled settings.

Keyword: synthetic biomimetic niche, real-time monitoring, stem cell, nanocomplex, lineage commitment

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1. Introduction Human mesenchymal stem cells (hMSCs) are promising resources for repairing diseased tissues, including bones. The differentiation of stem cells into the osteogenic lineage requires

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the precisely coordinated delivery of differentiation-inducing molecules to their intracellular or membrane-attached target receptors.[1-4] For instance, purmorphamine effectively induces 6]

thereby

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osteogenesis by targeting the transmembrane receptor smoothened (SMO),[5,

triggering Indian Hedgehog signaling and upregulating the expression of RUNX2,[7] a key

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transcription factor responsible for osteogenesis.[8] However, many inductive small molecules, including purmorphamine, are hydrophobic, which hinders their efficient and sustained delivery to cells in an aqueous physiological environment. Therefore, an effective

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nanoparticle-based drug-delivery platform is required for the delivery of hydrophobic inductive agents without the use the cytotoxic organic solvents. Many research groups have developed various nanomaterial-based delivery vehicles for effective drug delivery, including

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copolymer nanoparticles,[9-12] quantum dots,[13-15] iron oxide nanoparticles[16], silica

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particles,[17-20] graphene nanoparticles,[21, 22] and upconversion nanoparticles (UCNPs).[23, 24] In recent years, UCNPs have emerged as a promising nanostructure for drug delivery and deep tissue imaging because their advantages include strong emissions,[25] photostability, and good

biocompatibility.[26-29]

We

have

previously

developed

multifunctional

quantum-dot-based and UCNP-based carriers to deliver hydrophobic small molecular drugs[30] and small interfering RNAs (siRNAs) to boost the differentiations of hMSCs while tracking their locations in vivo. [31, 32]

ACCEPTED MANUSCRIPT Detecting the biomarkers of stem cell differentiation in three-dimensional (3D) culture is critical to understanding the differentiation process and mechanism. Traditional methods, such as western blotting and reverse transcription (RT)–PCR involve the sacrifice of large

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numbers of cells and only provide a snapshot at limited time points along the dynamic and protracted process of stem cell differentiation.[33] The osteogenic differentiation of hMSCs typically requires up to 4 weeks in vitro.[34-36] Therefore, the ability to monitor the extent of

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differentiation in living hMSCs over a long period is critical to clarifying the osteogenesis

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process in detail. Few studies have reported nanoprobes for detecting the biomarkers of stem cell differentiation in living cultures, although we have previously developed a nanoprobe to detect osteogenic microRNA.[37] Matrix metallopeptidase 13 (MMP13) is an important biomarker of osteogenesis that can be detected extracellularly.[38-40] Tuckermann et al. showed

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that osteoblasts involved in endochondral and intramembranous ossification express elevated MMP13 levels,[41] and Johansson et al. reported that MMP13 is expressed during human fetal

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bone development.[42] We believe that UCNPs modified with an MMP13-sensitive reporting system can be used to track the osteogenesis of hMSCs in living cultures.

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Whereas nanoparticles can be used to monitor cellular activity on the molecular scale, bulk scaffold biomaterials are also essential to provide a 3D environment to support the adhesion and differentiation of hMSCs and the retention of the extracellular matrix secreted by the differentiated cells.[43-50] We have developed porous hyaluronic acid (HA) hydrogels as effective 3D scaffolds to support the osteogenesis of hMSCs and bone regeneration in animal bone defects.[51] The rationale is that HA stimulates CD44 and CD168,[52] both of which facilitate osteogenesis.[53,

54]

However, few studies have combined the unique strength of

ACCEPTED MANUSCRIPT nanoparticles with hydrogels to simultaneously induce osteogenesis and track the degree of differentiation of hMSCs in a 3D environment.[20, 55-57] In the present study, we developed a multiscale biomaterial platform by integrating

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drug-eluting nanoparticles in the 3D matrix of porous hydrogels to promote and track the osteogenesis of hMSCs in a 3D culture. We used mesoporous silica-coated UCNPs to mediate the sustained delivery of an osteogenic small molecule, purmorphamine. The UCNPs were

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labeled with a fluorescent tag (fluorescein isothiocyanate, FITC) via an MMP13-sensitive

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peptide linker. This allowed MMP13 detection, a marker of osteogenesis, by examining the Förster resonance energy transfer (FRET)-induced change in the emission spectrum.[58] Specifically, the upregulated MMP13 expression in hMSCs differentiating into the mature osteogenic phenotype leads to the cleavage of the MMP-sensitive peptide linker and the

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release of FITC, thereby altering the fluorescence emission profile. The UCNP@mSiO2– peptide–FITC nanocomplex generated was loaded with purmorphamine and incorporated

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within the matrices of porous HA hydrogels, which provided a 3D environment that mimicked the structure of trabecular bone.[59] After the hMSCs were seeded in the porous

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hydrogels and subjected to osteogenic induction for 14 days, the incorporated nanocomplex effectively increased the expression of the osteogenic marker genes relative to their expression in the control group, in which the culture medium was directly supplemented with the same amount of purmorphamine. The significant change in the fluorescence emission intensity at 525 nm in the hydrogels containing the nanocomplex also indicated the successful osteogenic differentiation of the hMSCs. We believe that our study demonstrates an effective multiscale platform on which to promote and track stem cell differentiation in a biomimicking

ACCEPTED MANUSCRIPT 3D environment. This multiscale platform may also facilitate fundamental studies of stem cell differentiation in 3D.[60, 61]

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2. Materials and Methods 2.1 Synthesis of UCNPs and UCNP@mSiO2

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NaYF4:Yb/Tm (30/0.5 mol%) upconverted nanoparticles (UCNPs) and a silica coating containing amine were synthesized with a previously reported protocol.[26] In brief, RECl3

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(0.4 M, RE = Y, Yb, and Tm; Sigma, St. Louis, MO, USA) dissolved in methanol was added to a 100 mL flask containing 6 mL of oleic acid and 14 mL of 1-octadecene. The solution was heated to 160 °C where it was maintained for 30 min to fully dissolve the components. The solution was cooled naturally to room temperature with stirring at 500 rpm. NH4F (3.2 mmol;

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Sigma) and NaOH (2 mmol; Sigma) dissolved in 10 mL of methanol were slowly added dropwise, and the solution was stirred for 30 min at 100 °C. After the complete evaporation of

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the methanol under reduced pressure, we increased the temperature to 300 °C under nitrogen

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protection for 1.5 h, and then cooled the solution naturally to room temperature. After the addition of acetone, the resultant UCNPs were precipitated and collected by centrifugation (6000 × g, 10 min), washed three times with methanol and ethanol, and finally resuspended in 20 mL of cyclohexane. To add the amorphous silica coating, 4 mg of UCNPs was suspended in 16 mL of cyclohexane, to which was added 2 mL of Triton X-100 (Sigma), 2 mL of 1-hexanol, 680 µL of deionized (DI) water, and the mixture was stirred at 600 rpm for 30 min. Then,

10

µL

of

tetraethyl

orthosilicate

(TEOS;

Sigma)

and

2

µL

of

ACCEPTED MANUSCRIPT (3-aminopropyl)triethoxysilane (APTES; Sigma) were added to the mixture. After stirring for 6 h, 200 µL of NH4OH (30.0% NH3 basis; Sigma) was added, and the solution stirred for another 48 h to produce a 20 nm thick silica layer. UCNP@SiO2–NH3 was isolated with

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centrifugation and washed thoroughly three times with ethanol. To coat the construct with mesoporous silica by NaOH etching, 20 mg of UCNP@SiO2–NH3 nanoparticles were suspended in 10 mL of deionized water, and ultrasonicated for 15 min. We then adjusted the

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pH of the solution to 10 with NaOH, and heated the suspension to 60 °C for 1 h.

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UCNP@mSiO2 nanoparticles were isolated by centrifugation at 6000 × g for 15 min, washing thoroughly with ethanol and methanol, and suspension in 10 mL of ethanol. The as-prepared UCNP@mSiO2 particles were characterized with transmission electron microscopy (TEM). The fluorescence emission spectra of UCNPs and UCNP@mSiO2 were measured with an

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imaging system with a laser excitation light source at 980 nm (Fluorescence Spectrophotometer F-7000, Hitachi, Tokyo, Japan).

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2.2 Conjugating MMP13-sensitive peptides to UCNP@mSiO2

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UCNP@mSiO2 (10 mg) were dissolved in 500 µL of dimethyl formamide (DMF), together with 1 µmol maleimide–tetra(ethylene glycol)–N-hydroxysuccinimide (MAL–[PEG]4–NHS; Sigma) and 1 µL of N,N-diisopropylethylamine (DIPEA; Sigma), and stirred overnight at 800 rpm. On the following day, the MAL–(PEG)4–UCNP@SiO2 nanoparticles were isolated by centrifugation and washed three times with ethanol. The MAL–(PEG)4–UCNP@SiO2 was then dissolved and stirred vigorously overnight in FITC-tagged MMP13-sensitive peptide solution (0.001 mmol in DMF) with 1 µL of N,N-Diisopropylethylamine (DIPEA) to form the

ACCEPTED MANUSCRIPT UCNP@mSiO2–peptide–FITC nanocomplex. The final product was isolated by centrifugation, washed three times with ethanol, lyophilized to powder, and stored at 4 °C.

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2.3 Loading purmorphamine into the UCNP@mSiO2–peptide–FITC nanocomplex

To prepare the purmorphamine-loaded UCNP@mSiO2–peptide–FITC nanocomplex, 2 µM purmorphamine and 10 mg of UCNP@mSiO2–peptide–FITC nanocomplex were mixed

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together in 3 mL of phosphate-buffered saline (PBS) and stirred overnight at 700 rpm.

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Purmorphamine and UCNP@SiO2–peptide–FITC nanocomplex were combined in excessive molar ratios.

2.4 Release profile of purmorphamine from the UCNP@mSiO2–peptide–FITC

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nanocomplex

An aliquot (5 mg) of the purmorphamine-loaded UCNP@mSiO2–peptide–FITC nanocomplex

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(Pur–UCNP–peptide–FITC nanocomplex) suspended in 1 mL of PBS was incubated at 37 °C for up to 7 days. We collected supernatant samples on day 1, day 3, and day 7 after

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centrifuging the solution at 5000 × g for 6 min. The amount of purmorphamine released was determined with UV–Vis spectrophotometry. The cumulative release profiles of purmorphamine from the nanocomplex and from the mesoporous UCNP@SiO2 particles were calculated by dividing the cumulative amount released by the original amount of purmorphamine loaded into the nanocomplex. The release profiles for the nanocomplex and mesoporous UCNP@SiO2 were expressed as two release curves.

ACCEPTED MANUSCRIPT 2.5 Detection of MMP13 in PBS with the nanocomplex

To detect the MMP13 enzyme in a PBS solution, MMP13 was prepared in MMP buffer (SensoLytePlus MMP-13 Assay Kit, AnaSpec, Inc, Fremont, California, USA) for 30 min at

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37 °C. The UCNP@SiO2–peptide–FITC nanocomplex was then mixed with MMP13 solution (1 mg mL-1 nanocomplex in 10 nM MMP13 solution) and incubated at 37 °C for 1 h. After

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MMP13 digestion, the nanocomplex was centrifuged and resuspended in 1 mL of PBS. The samples were then collected to quantify the photoluminescence emitted, with a fluorescence

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microplate reader. The emission spectrum of the nanocomplex was recorded at 300–600 nm to check the changes in the conjugated FITC and UCNPs. To detect MMP13 with near-infrared (NIR)-mediated FRET, an NIR laser generator (980 nm) was placed in the

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fluorescence microplate reader as the excitation light source and the UV lamp of the microplate reader was temporarily shut down. Therefore, the upconverted blue light (480 nm) emitted from the UCNPs was the only light source to excite FITC. The enzyme specificity of

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the reaction was tested with bovine serum albumin (BSA), cathepsin B, MMP3, and MMP7

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with a similar NIR excitation process.

2.6 Synthesis of methacrylated HA and porous hydrogel fabrication

Methacrylated HA (MeHA) macromolecules were synthesized from sodium hyaluronate powder (MW ~74 kDa; Lifecore, Chaska, MI, USA), as previously reported.[62] Briefly, 100 mL of 1% (w/v) sodium hyaluronate solution was reacted for 24 h with 2 mL of methacrylic anhydride at pH 9.5, adjusted with 2 M NaOH solution. After complete dialysis and

ACCEPTED MANUSCRIPT lyophilization, 100% methacrylation was confirmed with 1H nuclear magnetic resonance (NMR). An RGD peptide (GCGYGRGDSPG) (GenScript, Nanjing, Jiangsu, China) with a cysteine amino acid at the C-terminal was conjugated to the MeHA backbone with a Michael

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addition reaction between the methacrylate groups and the thiol groups in basic phosphate buffer (pH 8.0) containing 10 µM tris(2-carboxyethyl)phosphine at 37 °C. The molar ratio of methacrylate to peptide thiol was 100:3. The nanocomplex-containing RGD-functionalized

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porous MeHA hydrogels were fabricated by curing 1.5 mg of the UCNP@mSiO2–peptide–

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FITC nanocomplex or UCNP@mSiO2 nanoparticles and 50 µL of peptide-conjugated MeHA solution (3% w/v, 100% methacrylation) for 2 h, with dithiothreitol as the crosslinker, in round polyvinyl chloride molds fully packed with a poly(methyl methacrylate) (PMMA) microsphere porogen (Ø200 µm). The constructs generated were immersed in acetone and

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shaken at 90 rpm to dissolve the porogen, sterilized with 75% ethanol for 1 day, and rinsed three times with sterile PBS. In the directly purmorphamine-supplemented MeHA group and

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blank MeHA group, we only used the RGD-functionalized porous MeHA solution (3% w/v,

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100% methacrylation) to fabricate the porous hydrogels, with no embedded nanocomplex.

2.7 Scanning electron microscopy (SEM) imaging of the surface morphology of nanocomplex-containing porous hydrogels and porous MeHA hydrogels

SEM was used to evaluate the surface morphology of the Pur–UCNP–peptide–FITC– nanocomplex-containing porous MeHA hydrogels and the porous MeHA hydrogel with no embedded nanocomplex. All 3% (w/v) MeHA hydrogel samples were snap frozen in liquid nitrogen, lyophilized, and fixed to silicon wafers, which were then subjected to platinum

ACCEPTED MANUSCRIPT sputtering before imaging. The size of the embedded UCNP@mSiO2–peptide–FITC nanocomplex was measured with the ImageJ software (NIH, Bethesda, Maryland, USA) in 30

2.8 Cell culture and osteogenic differentiation study

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different particles from three different images, and is shown as the mean ± standard error.

Passage-4 human hMSCs (Lonza, Walkersville, Maryland, USA) were expanded in basal

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growth medium (α-minimal essential medium [MEM] supplemented with 16.7% [v/v] fetal

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bovine serum [FBS], 100U mL-1 penicillin/streptomycin [P/S], 2mM L-glutamine). Growth medium (50 µL) containing 106 hMSCs (2 × 107 cells mL-1) was injected into one semidry porous MeHA–RGD hydrogel, which was incubated in 37 °C for 3.5 h to allow cell attachment to the hydrogel. We then added 1 mL of osteogenic medium (α- MEM, 16.67%

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FBS, 1% P/S, 2mM L-glutamine, 10 mM β-glycerophosphate disodium, 50 mg mL-1 L-ascorbic acid 2-phosphate, 100 nM dexamethasone) to all the hydrogels, and renewed the

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medium every 2 days. Purmorphamine solution (2 mM, 1 µL) was added to the control group, which was treated directly with purmorphamine only at the beginning of osteogenic culture.

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Samples were collected on day 7 and day 14 to evaluate the degree of osteogenesis with traditional quantitative PCR (qPCR) and western blotting. Cell viability was determined with an Alamar Blue assay (Invitrogen, Carlsbad, California, USA) after 7 days in osteogenic culture. Live/dead staining was performed by adding 3 µM calcein AM and 3 µM propidium iodide (Thermo Fisher Scientific, Waltham, Massachusetts, USA) to the hydrogels. After incubation for 30 min at 37 °C, the hydrogels were washed three times with PBS and fluorescent images were taken with confocal microscopy (Nikon C2, Tokyo, Japan).

ACCEPTED MANUSCRIPT 2.9 Gene expression analysis with RT–qPCR

All samples were homogenized in 1 mL of Trizol Reagent (Invitrogen), and the total RNA was extracted according to the manufacturer’s protocol. The RNA concentration was

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measured with a NanoDrop™ One spectrophotometer (NanoDrop Technologies, Waltham, Massachusetts, USA). The total RNA (1 µg) was reverse transcribed into cDNA with the

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RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific). We performed qPCR with the Applied Biosystems 7300 Real-Time PCR System with TaqMan primers and probes

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(Applied Biosciences, Waltham, Massachusetts, USA) specific for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and other osteogenic genes, including those encoding RUNX2, alkaline phosphatase (ALP), and collagen type I . The sequences of the TaqMan

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primers and probes used are listed in Table S1 in the Supplemental Information. The relative osteogenic gene expression was normalized to that of GAPDH and the relative expression

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levels were calculated with the 2−∆∆Ct method.[63]

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2.10 Western blotting

Total proteins were extracted with RIPA Lysis and Extraction Buffer (Thermo Fisher Scientific) and Halt Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific) on ice for at least 30 min, according to manufacturer’s protocol. The supernatant was then collected with centrifugation at 12,000 × g at 4 °C, and the protein concentration was determined with the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). All the samples were then separated electrophoretically and spread well through a 10% Bis–Tris polyacrylamide gel,

ACCEPTED MANUSCRIPT before they are transferred to 0.45 µm polyvinylidene difluoride membranes (Millipore, Burlington, Massachusetts, USA). After the membranes were washed three times with PBS containing Tween 20, the protein-loaded membranes were blocked with 5% fat-free milk

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powder solution for at least 2 h at room temperature, and then incubated overnight at 4 °C with various primary antibodies (anti-GAPDH, diluted 1:3000; #sc-32251, Santa Cruz Biotechnology, Santa Cruz, CA, USA; anti-MMP13, diluted 1:500; #sc-30073, Santa Cruz

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Biotechnology; and anti-β-catenin, diluted 1:1000; #sc-59737, Santa Cruz Biotechnology). A

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horseradish peroxidase (HPR)-coupled secondary antibody (anti-mouse IgG antibody, diluted 1:2000; Santa Cruz Biotechnology) was then applied to the membranes and incubated for 90 min at room temperature. The fluorescent signals were visualized with a chemiluminescent ECL substrate WBKLS0500 (Millipore) with the ChemiDoc™ Touch Imaging System

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(Bio-Rad, Hercules, California, USA). A densitometric analysis was conducted with the open source software ImageJ (NIH). The protein levels were normalized to that of GAPDH.

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2.11 Histological assessment in vitro

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Hydrogel samples were fixed overnight in 4% paraformaldehyde at 4 °C, dehydrated in a graded series of ethanol, crystalized in a graded series of xylene, and embedded in paraffin. The histological sections (7 µm) were stained for collagen type I with the Vectastain ABC Kit and the DAB Substrate Kit (Vector Laboratories, Burlingame, California, USA). In brief, we applied 0.5 g L-1 hyaluronidase to the rehydrated samples to predigest them at 37 °C for 30 min. The samples were then transferred to 4 °C, incubated with 0.5 N acetic acid for 4 h to induce swelling, and incubated overnight with a primary antibody directed against collagen

ACCEPTED MANUSCRIPT type I at 4 °C (anti-collagen type I, diluted 1:200; sc-59772; Santa Cruz Biotechnology). Calcification of phosphates and calcium ions was identified with von Kossa staining and Alizaran Red S staining, respectively. Briefly, von Kossa stain was applied to the rehydrated

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sections, as previously reported.[51] To stain with Alizarin Red S, 1 mL of 0.5% (w/v) Alizaran Red S solution (Sigma) was applied to each hydrogel sample, which was incubated

were taken with bright-field microscopy (Nikon).

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for 5 min at room temperature before washing, dehydration, clearing, and sealing. Images

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2.12 Cell differentiation detected as the change in NIR-mediated fluorescence in the porous MeHA hydrogels

The hydrogels were washed three times with PBS to remove the nonembedded nanocomplex,

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and then exposed to NIR irradiation in a fluorescence plate reader. The fluorescence emission spectra were collected at 300–600 nm to record the initial fluorescence spectra of the

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nanocomplex-embedding and UCNP@mSiO2-embedding porous MeHA hydrogels. After hMSC seeding, the hydrogels were cultured in osteogenic medium for 14 days to induce

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osteogenesis. After 14 days, the hydrogels were placed in a fluorospectrophotometer to measure the emission spectra after osteogenic inducement. Under 980 nm excitation, NIR-mediated blue light was emitted from the nanocomplex to further excite the FITC by FRET. The fluorescent signals were collected at 300–600 nm because the wavelengths for UCNP emission are 350–480 nm and that for FITC emission is 525 nm.

2.13 Statistical analysis

ACCEPTED MANUSCRIPT All data are presented as means ± standard deviations. Statistica (Statsoft, Tulsa, OK, USA) was used to perform the statistical analyses using two-way analysis of variance (ANOVA) and the Tukey’s honest significant difference (HSD) post hoc test of the means (n = 3), using

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the period of culture and the experimental group as independent variables.

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3. Results and discussion

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3.1 Successful fabrication of UCNP@mSiO2–peptide–FITC nanocomplex

We first synthesized the UCNPs and mesoporous-silica-coated UCNPs as previously reported. [16]

Briefly, the UCNPs generated were treated with TEOS and APTES to form silica-coated

UCNPs (UCNP@SiO2) carrying reactive surface amino groups. We then etched the

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UCNP@SiO2 with NaOH to form the mesoporous-silica-coated UCNPs (UCNP@mSiO2, Scheme 1). TEM images showed that the average sizes of UCNPs, UCNP@SiO2, and

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UCNP@mSiO2 were approximately 17 ± 2.7 nm, 35 ± 3.5 nm, and 35 ± 1.8 nm, respectively

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(Figure 1A and S1). We fabricated the MMP13-sensitive nanocomplex by conjugating the FITC-tagged MMP13 substrate peptide (FITC-PLGVRGKGGC to the surface of UCNP@mSiO2. The size of the UCNP@mSiO2–peptide–FITC nanocomplex was 37.73 ± 4.71 nm, when determined with TEM (Figure S1). Dynamic light scattering showed that the average size of UCNP@mSiO2–peptide–FITC was 52.73 nm in PBS (Figure S2). The UV– Vis absorption spectrum of the nanocomplex showed an absorption band at around 480 nm, and the fluorescence emission spectrum of the nanocomplex showed emission bands at

ACCEPTED MANUSCRIPT around 350 nm (UV), 480 nm (blue), and 525 nm (green) (Figure 1B, 1C, and 1D). The emission spectrum of the UCNP@mSiO2–peptide–FITC nanocomplex at 525 nm confirmed the successful conjugation of peptide–FITC to UCNP@mSiO2. The robust emission of

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upconverted blue light (480 nm) from the UCNPs excited the conjugated FITC tag via FRET, giving rise to FITC emission at 525 nm. The osteogenic differentiation of hMSCs led to a cascade of events, including upregulated MMP13 expression, cleavage of the MMP13

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substrate linker, release of the FITC tag, and diminished FITC emission at 525 nm. The

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UCNPs and UCNP@mSiO2 nanoparticles showed similar emission intensities at 480 nm at the same concentration, indicating that the mesoporous silica coating had minimal effect on the upconversion property of the UNCPs (Figure S3). The silica coating provides multiple reactive sites for functional modifications and was previously reported to enhance the

3.2

Sustained

release

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biocompatibility of UCNP-based nanoprobes.[64]

of

purmorphamine

from

the

purmorphamine-loaded

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UCNP@mSiO2–peptide–FITC nanocomplex

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The mesoporous silica layer was designed to carry hydrophobic drugs within the surface channels of the UCNP@mSiO2–peptide–FITC nanocomplex, from which purmorphamine is continuously released into the surrounding environment. The UV–Vis absorption spectrum of the purmorphamine-loaded UCNP@mSiO2–peptide–FITC

nanocomplex (Pur–UCNP–

peptide–FITC) showed the absorption peak characteristic of purmorphamine at 286 nm, confirming the successful loading of purmorphamine into the mesoporous silica coating of the nanocomplex (Figure 2A).

ACCEPTED MANUSCRIPT We then analyzed the release kinetics of purmorphamine from the UCNP@mSiO2–peptide– FITC nanocomplex at 37 °C by measuring the absorption spectrum of the supernatant of the nanocomplex suspension at multiple time points. The prolonged release of purmorphamine

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from the Pur–UCNP–peptide–FITC nanocomplex in PBS (pH 7.4) was observed, with a cumulative release of 81.2 ± 5.3% of the total loaded amount of purmorphamine after incubation for 7 days (Figure 2B). Similarly, the sustained release of the cargo drug

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(purmorphamine) from the purmorphamine-loaded UCNP@mSiO2 nanoparticles was observed,

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with a faster release rate and a cumulative release of 95.4 ± 1.2% of the loaded purmorphamine after incubation for 7 days at 37 °C (Figure 2B). These findings suggest that purmorphamine, loaded in either the Pur–UCNP–peptide–FITC nanocomplex or the UCNP@mSiO2 nanoparticles, underwent sustained release for over 7 days at 37 °C in PBS. This should allow

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the use of the Pur–UCNP–peptide–FITC nanocomplex to effectively induce the osteogenesis of stem cells. The observed difference in the rates of release (Figure 2B) from the nanocomplex

3.3

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the nanocomplex.

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and nanoparticles is attributable to the peptide conjugated to the mesoporous silica surface of

MMP13

digestion

specifically

reduced

the

emission

intensity

of

the

UCNP−peptide−FITC nanocomplex

Under NIR exposure, the FITC-tagged MMP13 substrate peptide allowed MMP13 to be detected as the loss of FRET, which was induced by MMP13 cleavage. The released FITC tag diffused away from the surface of the UCNP–peptide–FITC nanocomplex, reducing the emission intensity at 525 nm (FITC emission wavelength) and increasing the emission

ACCEPTED MANUSCRIPT intensity at 480 nm from the UCNPs (FITC excitation wavelength) under NIR laser irradiation. Upon treatment with MMP13 for 1 h at 37 °C, the PBS suspension of the UCNP@mSiO2–peptide–FITC nanocomplex showed clearly reduced emission at 525 nm and

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an increase in emission at 480 nm (Figure 2C). These results are consistent with the reduced excitation and emission of FITC when the MMP13-sensitive peptide is cleaved. To check the specificity of MMP13 detection, the UCNP@mSiO2–peptide–FITC nanocomplex was

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incubated with several protein and enzyme solutions, including BSA, cathepsin B, MMP3,

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and MMP7 (all 10 nM) at 37 °C for 1 h, and the fluorescence emission was analyzed. Treatment with 10 nM BSA, cathepsin B, MMP3, or MMP7 only slightly reduced the intensity of the FITC emission from the nanocomplex at 525 nm (Figure 2D). Only treatment with MMP13 significantly reduced the emission intensity at 525 nm, on average by 78.50%.

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These results indicate that the UCNP@mSiO2–peptide–FITC nanocomplex has high specificity for the catalytic activity of MMP13 in PBS.

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3.4 Porous MeHA hydrogel encapsulating the Pur–UCNP–peptide–FITC nanocomplex

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promotes the osteogenesis of hMSCs by emulating a pro-osteogenic niche

The purmorphamine-loaded UCNP@mSiO2–peptide–FITC (Pur–UCNP–peptide-–FITC) nanocomplex was encapsulated in porous nanocomposite MeHA hydrogels to emulate the pro-osteogenic niche in the spongy bone.[51, 65, 66] The dense crosslinking network of the hydrogels trapped the nanocomplex within the hydrogel, with no significant loss of the encapsulated nanocomplex after washing, sterilization, or long-term cell culture for 14 days, detected as the constant intensity of the NIR-excited emission (Figure 3A and 3B). SEM

ACCEPTED MANUSCRIPT images showed that the surface roughness was greater in the nanocomplex-containing porous hydrogels than in the nanocomplex-free porous hydrogels, which may be attributable to the presence of the nanocomplex (Figure 3C and 3D). After microspheric PMMA porogen was

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dissolved in acetone and the hydrogels were sterilized with 75% ethanol, most of the nanocomplex was still contained within the hydrogels. Under the same excitation condition, the emission intensities of the nanocomplex-containing hydrogels at 480 nm before and after

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382.20 ± 9.005 a.u., respectively (Figure 3E).

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rigorous washing with acetone and ethanol were 443.05 ± 9.786 arbitrary units (a.u.) and

We seeded hMSCs in the porous MeHA hydrogels containing the Pur–UCNP–peptide–FITC nanocomplex to allow the cells to adhere to the walls of the interconnected pores within the

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hydrogels before they were cultured in osteogenic medium for another 14 days.[51] After 7 days, the hMSCs seeded in the hydrogels were highly viable, as shown with calcein/PI (live/dead) staining (Thermo Fisher Scientific) (Figure 3F). As well as hydrogels containing

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the Pur–UCNP–peptide–FITC nanocomplex (PM+NP group), we also included the following

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control groups: hydrogel encapsulating empty (no loaded drug) UCNP–peptide–FITC nanocomplex (NP group); hydrogel encapsulating empty UCNP–peptide–FITC nanocomplex with direct purmorphamine supplementation into osteogenic medium (PM group); and porous hydrogels without nanoparticles (Blank group). The amount of purmorphamine used to supplement the medium in the PM group was the same as that loaded into the nanocomplex in the PM+N group. After 7 days in osteogenic culture, an Alamar Blue assay showed that the majority of cells were viable, with 99.16%, 93.52%, 89.12%, and 90.48% cell viability in the

ACCEPTED MANUSCRIPT NP group, NP+PM group, PM group, and Blank group, respectively (Figure S5). This indicates that the embedded Pur–UCNP–peptide–FITC nanocomplex and the released purmorphamine were not cytotoxic, which is consistent with the previously reported cytocompatibility of

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UCNP@mSiO2 nanoparticles. The osteogenic differentiation of hMSCs was investigated with RT–qPCR analyses of the major osteogenic markers (ALP, RUNX2, and collagen type I). The RT–qPCR data showed

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that the direct supplementation of medium with purmorphamine (PM) and the sustained

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release of purmorphamine from the Pur–UCNP–peptide–FITC nanocomplex (NP+PM) both promoted the expression of osteogenic marker genes after 7 days of culture compared with the control groups without purmorphamine supplementation (NP and Blank) (Figure 4). Specifically, the controlled delivery of purmorphamine from the nanocomplex (PM+NP)

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induced the highest osteogenic gene expression among all the groups, with an average increase of 58.40% in ALP expression, 40.28% in RUNX2 expression, and 67.73% in

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collagen type I expression relative to the Blank control group. The direct supplementation of the osteogenic medium with purmorphamine (PM) only increased ALP expression by 36.12%

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compared with the “Blank” control group, and no significant changes in osteogenic gene expression were observed in the NP group relative to the Blank control group (Figure 4A,4B and 4C).

After 14 days in osteogenic culture, the hydrogel encapsulating the Pur–UCNP–peptide–FITC nanocomplex (PM+NP group) showed a 181.21% increase in ALP expression compared with that in the porous MeHA hydrogel (Blank group), and also significantly higher expression of all osteogenic marker genes compared with their expression levels on day 7. Specifically, the

ACCEPTED MANUSCRIPT average expression of the osteogenic genes encoding ALP, RUNX2, and collagen type I increased by 74.35%, 26.18%, and 78.22%, respectively (Figure S6). Collectively, these data confirm that our Pur-–UCNP–peptide–FITC nanocomplex successfully sustained the

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localized delivery of purmorphamine in the porous hydrogels during 14 days of osteogenic culture. This sustained localized delivery of an osteogenic agent in a 3D cellular microenvironment, mediated by the Pur–UCNP–peptide–FITC nanocomplex, promoted the

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osteogenesis of hMSCs significantly better than the direct supplementation of the culture

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medium with purmorphamine (PM group), in which situation the osteogenic agent was diluted throughout the entire volume of the culture medium rather than concentrated in the cellular microenvironment.

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Consistent with the results for osteogenic gene expression, histological staining of the osteogenic matrix was significantly more intense in the hydrogels encapsulating the Pur– UCNP–peptide–FITC nanocomplex (NP+PM group) than in the empty porous MeHA

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hydrogels (Blank group) (Figure 5). After 14 days in osteogenic culture, more type I collagen

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and mineralization had accumulated in the NP+PM hydrogels than in any of the control groups, in response to the prolonged and localized release of purmorphamine from the nanocomplex encapsulated in the porous hydrogels. Direct supplementation of the medium with purmorphamine (with an amount identical to that loaded in the nanocomplex) also increased osteogenic matrix deposition relative to that in the non-purmorphamine supplemented groups (NP and Blank groups) (Figure 5). Collectively, the osteogenic matrix staining results suggest that the sustained and localized delivery of purmorphamine by the

ACCEPTED MANUSCRIPT Pur–UCNP–peptide–FITC nanocomplex encapsulated in the porous hydrogel enhanced the formation of an osteogenic matrix by the differentiated hMSCs.

3.5 Biomimetic pro-osteogenic niche established by porous hydrogels encapsulating the

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Pur–UCNP–peptide–FITC nanocomplex promoted the biosynthesis of MMP13 and β-catenin by hMSCs

receptor

SMO,

upregulating

RUNX2

expression

and

enhancing

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transmembrane

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Purmorphamine has been reported to activate hedgehog signaling by targeting the

osteogenesis.[5-7] Together with RUNX2 expression, canonical WNT signaling is widely reported to be activated during osteogenesis.[67, 68] RUNX2 expression and the activation of WNT signaling both contribute to the downstream upregulated expression of MMP13 during

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osteogenesis.[69-71]

A western blotting analysis indicated that the expression of MMP13 and β-catenin was

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significantly higher in the biomimetic pro-osteogenic niche established by the porous

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hydrogel encapsulating the Pur–UCNP–peptide–FITC nanocomplex (NP+PM group) than in the control groups throughout the 14 days of osteogenic culture (Figure 6A). A quantitative analysis revealed that β-catenin and MMP13 biosynthesis were upregulated in the NP+PM group by 605% and 163%, respectively, compared with their synthesis in the NP and Blank groups. In contrast, direct supplementation of the medium with purmorphamine (PM group) caused no significant changes in β-catenin and MMP-13 expression compared with their levels in the NP or Blank group (Figure 6B).

ACCEPTED MANUSCRIPT These findings suggest that the localized and sustained release of purmorphamine mediated by the Pur–UCNP–peptide–FITC nanocomplex in the biomimetic pro-osteogenic niche promoted osteogenesis by activating the canonical WNT–β-catenin signaling pathway, thus

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upregulating MMP13 biosynthesis. This finding confirms the efficacy of using MMP13 expression as the indicator for tracking the extent of osteogenesis in hMSCs.

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differentiation in the biomimetic pro-osteogenic niche

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3.6 UCNP@mSiO2–peptide–FITC nanocomplex allows long-term tracking of hMSC

We then evaluated the efficacy of the UCNP@mSiO2–peptide–FITC nanocomplex in tracking the degree of differentiation in living hMSCs by examining the change in the NIR-induced FRET signal between the UCNP and the conjugated FITC tag during osteogenic culture. We

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measured the NIR-induced emission spectra of identical hydrogels encapsulating either Pur– UCNP–peptide–FITC (“NP+PM”) or the non-drug-loaded nanocomplex (“NP”) on day 0 and

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day 14 of osteogenic culture. The emission profiles of both the NP+PM and NP hydrogels were similar on day 0, shortly after cell seeding, with intense emission at 525 nm due to the

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FRET effect between the FITC tag and UCNPs (Figure S7). After 14 days in osteogenic culture, the fluorescence emission intensity of the NP+PM hydrogels was significantly reduced at 525 nm and increased at 480 nm compared with that on day 0, indicating a substantial reduction in the FRET signal due to the considerable cleavage of the MMP13-sensitive linker and the release of the FITC tag (Figure 7A and 7B). In contrast, the emission intensity of the NP hydrogels decreased less at 525 nm but was higher at 480 nm than for the NP+PM hydrogels, indicating less MMP13 activity and the release of less FITC

ACCEPTED MANUSCRIPT (Figure 7A and 7B). Quantification of the changes in the 525nm fluorescence emission intensities showed reductions of 76% and 55% in the NP+PM and NP hydrogels, respectively, compared with the signals on day 0 (Figure 7C). Notably, the FRET emission at 525 nm in

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the NP+PM hydrogels remained detectable on day 14, indicating the possibility that the degree of differentiation of stem cells can be tracked in the long term. When the FRET signal was lost after the MMP13-mediated release of the FITC tag, the emission intensity at 480 nm

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(from the UCNPs) increased after 14 days in culture. Quantification of the change in the

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emission intensity at 480 nm showed increases of 68.96% and 34.50% in the NP+PM and NP hydrogels, respectively, compared with the intensity on day 0 (Figure 7A).

These differential changes in the emission profiles of the different groups of hydrogels

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correlated well with the extent of osteogenesis of the seeded hMSCs, determined with RT– qPCR and histological staining. In the porous hydrogels encapsulating the Pur-–UCNP– peptide–FITC nanocomplex (NP+PM group), the significant reduction in emission at 525 nm

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and the increase at 480 nm indicated that the expression of MMP13 was upregulated by the

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seeded hMSCs (Figure 7B), consistent with the significantly upregulated expression of the genes encoding RUNX2, ALP, and collagen type I and the abundant accumulation of the osteogenic matrix, detected with histological staining (Figure 5). In contrast, the smaller changes in the FRET signals in the control groups (NP, PM, and Blank groups) were consistent with the lower expression of osteogenic marker genes and less accumulation of osteogenic matrix. These data show that the extent of osteogenic differentiation of seeded

ACCEPTED MANUSCRIPT hMSCs can be determined by examining the emission profile of the synthetic niche under NIR irradiation.

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4. Conclusions In conclusion, we reconstructed the 3D synthetic biomimetic niche environment of stem cells with a multiscale combination of macroscopic porous hydrogels and a nanoscale UCNP-based

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nanocomplex. On the one hand, the porous biopolymeric hydrogels emulated the spongy bone

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microstructure and provided a 3D environment conducive to the attachment, proliferation, and biosynthesis of seeded hMSCs. On the other hand, the multifunctional UCNP-based nanocomplex, which was encapsulated in the porous hydrogels, emulated the repertoire of inductive agents in the bone matrix by sustaining the localized and long-term delivery of an

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inductive small molecule to the synthetic niche microenvironment. The nanocomplex also generated reporting emissions that correlated well with the extent and stage of differentiation

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of the stem cells resident in the synthetic niche, thereby allowing the long-term tracking of stem cell fate in a living culture. Our findings show that this multiscale synthetic niche not

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only enhances the differentiation of the seeded stem cells, but also allows the stage of differentiation of the living stem cells to be tracked in the long term with a noncontact, nondestructive, and potentially high-throughput method. To the best of our knowledge, our study is the first to demonstrate the multiscale combination of functional biomaterials to construct a controlled stem cell niche microenvironment to enhance and track stem cell differentiation. The modular nature of this synthetic niche platform allows the various parameters to be easily tuned, including the microstructure morphology, scaffold stiffness,

ACCEPTED MANUSCRIPT inductive molecule, and the peptide linker, to accommodate a wide array of fundamental

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studies of dynamic cellular events in the long term.

Acknowledgments

Project 31570979 is supported by the National Natural Science Foundation of China. The

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work described in this paper is supported by a General Research Fund grant from the Research Grants Council of Hong Kong (Project No. 14202215 and 14220716). This research

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is also supported by project BME-p3-15 of the Shun Hing Institute of Advanced Engineering, The Chinese University of Hong Kong, The Chinese University of Hong Kong. This work is supported by the Health and Medical Research Fund, the Food and Health Bureau, the Government of the Hong Kong Special Administrative Region (reference no.: 04152836). This research is supported by the Chow Yuk Ho Technology Centre for Innovative Medicine,

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The Chinese University of Hong Kong. Rui Li and Jinming Li contributed equally to this work. The authors thank Qian Feng and Rui Li for proofreading the manuscript. The authors thank Dr. Kongchang Wei, Dr. Heng Chen, Kunyu Zhang, Xiaoyu Chen, Pengchao Zhao, and

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Weiping Li for valuable discussions.

Appendix A. Supporting Information and Declaration Supporting Information is available from the Elsevier online website or from the author. The authors declare no conflict of interests.

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Mesoporous silica

NHS-PEG-MAL

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FITC tagged MMP-13 sensitive peptide

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Purmorphamine (hydrophobic)

UCNP NH 2

UCNP@mSiO2

UCNP@mSiO2-peptide-FITC

Synthetic micro-niche with reporting function reconstructed by multiscale biomaterials 980nm

Purmorphamine promotes Runx2 and MMP13 expression.

Porous hydrogels

980nm

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Osteogenic culture

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FITC

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Day 14, Osteogenically committed hMSCs, MMP-13 expression is high↑

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Spongy bone niche microenvironment

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Scheme 1. Synthesis of the UCNP–peptide–FITC nanocomplex and its loading with purmorphamine to form the Pur–UCNP–peptide–FITC nanocomplex. The Pur–UCNP– peptide–FITC nanocomplex emulated the repertoire of inductive agents in the bone matrix by sustaining the localized and long-term delivery of inductive small molecules into the synthetic niche microenvironment. Purmorphamine activates hedgehog signaling by targeting the transmembrane receptor smoothened (SMO), leading to the upregulation of RUNX2 expression and enhanced osteogenesis. RUNX2 expression contributes to the upregulated MMP13 expression during osteogenesis.

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A

B

300

Blue Emission (480nm)

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FITC Emission (525nm)

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Figure 1. Characterization of the UCNP@mSiO2–peptide–FITC nanocomplex. A) TEM image of UCNP@mSiO2. B) Photoluminescence (PL) spectrum of the UCNP@mSiO2 nanoparticles. C) UV–Vis absorption spectra of the UCNP@mSiO2 (black) and UCNP@mSiO2–peptide–FITC nanocomplexes showed obvious absorption at 480 nm (red). D) PL spectrum of the UCNP@mSiO2–peptide–FITC nanocomplex showed strong emission at 525 nm due to the FITC–peptide conjugation.

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Figure 2. Loading/release of purmorphamine into/from UCNP@mSiO2–peptide–FITC, and MMP13 detection with NIR-mediated FRET in UCNP@mSiO2–peptide–FITC. A) UV–Vis spectra of purmorphamine and purmorphamine-loaded UCNP–peptide–FITC nanocomplex. B) Cumulative drug release profile of UCNP@SiO2 nanocarrier. C) Photoluminescence intensity of UCNP@mSiO2–peptide–FITC before and after the addition of MMP13. D) Specific detection of MMP13 by UCNP@mSiO2–peptide–FITC in PBS. UCNP@mSiO2–peptide– FITC, 1 mg mL-1; MMP13, 10 nM; incubation for 1 h at 37 °C.

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C

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pore

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MeHA hydrogel embedding MeHA hydrogel embedding Pur-UCNP-peptide-FITC Pur-UCNP-peptide-FITC

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Non-embedded MeHA hydrogel

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Figure 3. Fabrication process and the characterization of methacrylated HA hydrogel containing the UCNP–peptide–FITC nanocomplex. A) Scheme shows the fabrication of the nanocomplex-containing porous MeHA hydrogel, hMSC seeding, osteogenic culture, and the experiments used to examine the degree of osteogenesis. B) Image of the porous MeHA hydrogel with embedded UCNP–peptide–FITC nanocomplex. (10×image and general picture inset [d=5 mm, h=2.2 mm]. Scale bar, 50um) C) and D) SEM images showing the morphology of the nanocomplex-containing porous MeHA hydrogels (C) and the porous MeHA hydrogel with no embedded nanocomplex (D) at a magnification of 500×. Scale bar, 100 µm. E) Under the same 980 nm excitation conditions, the intensities of the emission at 480 nm in the nanocomplex-containing hydrogels were 443.05 ± 9.786 a.u. and 382.20 ± 9.005 a.u. before and after washing with acetone and sterilization with 75% ethanol, respectively. F) Live/dead staining showed hMSCs attached to the interconnected pores of the MeHA hydrogels. Calcein staining showed the morphology and strong viability of the cells. Scale bar, 200 µm.

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RGD RGD

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PM: Hydrogels encapsulating Blank: Hydrogels containing no empty nanocomplex with nanocomplex without direct supplementation of purmorphamine supplementation. purmorphamine in media

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Figure 4. Osteogenic gene expression after 7 days in differentiation culture. Porous hydrogels encapsulating the Pur–UCNP–peptide–FITC nanocomplex (NP+PM) induced higher gene expression than was observed in the control groups. A) ALP expression in all groups on day 7. B) RUNX2 expression in all groups on day 7. C) Collagen I expression in all groups on day 7. Data are reported as mean ± SD (n = 9). *p < 0.05, **p < 0.01.

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Figure 5. Pur–UCNP–peptide–FITC nanocomplex enhanced the deposition of the osteogenic matrix in hMSC-seeded porous hydrogels after 14 days in osteogenic culture, as shown with von Kossa staining, Alizarin Red S staining, and immunohistochemical staining (IHC) for type I collagen. Scale bar, 100 µm.

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GAPDH

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Figure 6. A. MMP13 and β-catenin protein expression in hydrogels encapsulating the Pur– UCNP–peptide–FITC nanocomplex and other control hydrogels after 14 days in osteogenic culture. Data are means ± SD of three experiments. **p < 0.01, ***p < 0.001. B. Expression of MMP13 and β-catenin was significantly higher in the NP+PM group than in the control hydrogels (NP, PM, and Blank groups). Intense expression of β-catenin indicated the robust activation of canonical WNT signaling in the NP+PM group.

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Blue Emission (480nm) increases↑

Day 0 - NP+PM Day 14- NP+PM Day 14- NP

FITC Emission (525nm) decreases↓

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Figure 7. Changes in NIR-excited FRET in the multiscale synthetic niche correlated with the extent of hMSC osteogenesis. A. Emission profiles of hydrogels encapsulating Pur–UCNP– peptide–FITC nanocomplex (NP+PM) and hydrogels encapsulating non-drug-loaded UCNP– peptide–FITC nanocomplex (NP) on days 0 and 14 of osteogenic culture under NIR irradiation. B. In the presence of low MMP13 activity (early culture time point or lack of osteogenic differentiation), the conjugated FITC tag was excited by the blue emission (480 nm) from the UCNPs under NIR excitation, yielding robust emission at 525 nm. With elevated MMP13 activity (late culture time point and substantial osteogenic differentiation of hMSCs), the release of the FITC tag by linker cleavage reduced the FRET effect, i.e., increased emission at 480 nm and reduced emission at 525 nm. C, D. Quantification of emission intensity at 525 nm (C) and 480 nm (D) of hydrogels encapsulating non-drug-loaded UCNP–peptide–FITC nanocomplex (NP) or Pur–UCNP–peptide–FITC nanocomplex (NP+PM) on days 0 and 14 of osteogenic culture under NIR irradiation. E. Quantitation of the 525/480 nm fluorescence intensity ratio for the hydrogels encapsulating non-drug-loaded UCNP–peptide–FITC nanocomplex (NP) or Pur–UCNP–peptide–FITC nanocomplex (NP+PM) on days 0 and 14 of osteogenic culture under NIR irradiation.

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Supporting Information

Multiscale reconstruction of a synthetic biomimetic microniche for enhancing and

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monitoring the differentiation of stem cells

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Rui Li1, Jinming Li1, Jianbin Xu, Dexter Siu Hong Wong, and Liming Bian*

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Figure S1. Characterization of the UCNP@mSiO2–peptide–FITC nanocomplex. TEM images showed that, the size of the UCNP(A), UCNP@mSiO2 (B), and UCNP@mSiO2-peptide-FITC

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nanocomplex (C) were measured to be 17 ± 2.7nm, 35 ± 3.5 nm, and 35 ± 1.8 nm, respectively (scale bar=50nm).

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Figure S2. The dynamic light scattering (DLS) analysis showed that the average diameter of

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the UCNP@mSiO2–peptide–FITC nanocomplex was 52.73nm in PBS solution.

Figure S3. Photoluminescence (PL) spectrum of the UCNPs and UCNP@mSiO2 nanoparticles showed similar emission at 525 nm. The mesoporous silica coating didn’t hinder the 525nm fluorescence emission of the original UCNPs obviously.

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Figure S4. 1H nuclear magnetic resonance (1H NMR [400 MHz, D2O, δ]) results showed that

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the modification rates of the methacrylated hyaluronic acid (MeHA) polymer was 104%.

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Figure S5. Alamar Blue assay after 7 days of the osteogenic culture showed that the majority of the cells in “NP”, “NP+PM”, “PM”, and “Blank” groups were viable, with 99.16%, 93.52%, 89.12% and 90.48% cell viability in “NP” group, “NP+PM” group, “PM” group and

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“Blank” group, respectively.

Figure S6. After 14 days in osteogenic culture, the hydrogel encapsulating the Pur–UCNP– peptide–FITC nanocomplex (PM+NP group) showed a 181.21% increase in ALP expression compared with that in the porous MeHA hydrogel (Blank group), and also significantly

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and collagen type I increased by 74.35%, 26.18%, and 78.22%, respectively.

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Figure S7. Photoluminescence intensity of the hydrogels from “NP+PM” and “NP” groups

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were highly similar at day 0 of the osteogenic culture.

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Table S1. The sequence of the primers and probes used for real-time PCR is listed. The primer and probe sequences of the Runx2 are proprietary (Applied Biosystem) and not disclosed. Gene GAPDH ALP

Collagen I

Forward primer

Reverse primer

Probe

AGGGCTGCTTTTAACTCTGGTAAA

GAATTTGCCATGGGTGGAAT

CCTCAACTACATGGTTTAC

CGGAACTCCTGACCCTTGAC

TGTTCAGCTCGTACTGCATGTC

TCGAAGAGACCCAATAGGT

AGGACAAGAGGCATGTCTGGTT

GGACATCAGGCGCAGGAA

TTCCAGTTCGAGTATGGC