ARTICLE

DOI: 10.1038/s41467-018-05599-2 OPEN

A biodegradable hybrid inorganic nanoscaffold

for advanced stem cell therapy
Letao Yang 1, Sy-Tsong Dean Chueng1, Ying Li2, Misaal Patel2, Christopher Rathnam1, Gangotri Dey1,
Lu Wang1, Li Cai2 & Ki-Bum Lee1,2,3
1234567890():,;

Stem cell transplantation, as a promising treatment for central nervous system (CNS) dis-
eases, has been hampered by crucial issues such as a low cell survival rate, incomplete
differentiation, and limited neurite outgrowth in vivo. Addressing these hurdles, scientists
have designed bioscaffolds that mimic the natural tissue microenvironment to deliver phy-
sical and soluble cues. However, several significant obstacles including burst release of drugs,
insufficient cellular adhesion support, and slow scaffold degradation rate remain to be
overcome before the full potential of bioscaffold–based stem-cell therapies can be realized.
To this end, we developed a biodegradable nanoscaffold-based method for enhanced stem
cell transplantation, differentiation, and drug delivery. These findings collectively support the
therapeutic potential of our biodegradable hybrid inorganic (BHI) nanoscaffolds for advanced
stem cell transplantation and neural tissue engineering.

1 Department of Chemistry and Chemical Biology, Rutgers, The State University of New Jersey, 123 Bevier Road, Piscataway, NJ 08854, USA. 2 Department of

Biomedical Engineering, Rutgers, The State University of New Jersey, 599 Taylor Road, Piscataway, NJ 08854, USA. 3 College of Pharmacy, Kyung Hee
University, 26 Kyungheedae-ro, Dongdaemun-gu, Seoul 02447, Korea. Correspondence and requests for materials should be addressed to
K.-B.L. (email: [email protected])

NATURE COMMUNICATIONS | (2018)9:3147 | DOI: 10.1038/s41467-018-05599-2 | www.nature.com/naturecommunications 1

ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-05599-2

D
eveloping reliable therapeutic methods to treat central Results
nervous system (CNS) diseases (e.g., Alzheimer’s and Enhanced stem cell differentiation on MnO2 nanoscaffolds.
Parkinson’s diseases), degeneration in the aging brain, Recently, hybrid inorganic 2D nanomaterial-based scaffolds have
and CNS injuries (e.g., spinal cord injury (SCI) and traumatic been demonstrated to control stem cell differentiation by pro-
brain injuries) has been a major challenge due to the complex and viding controlled physical, chemical, and biological properties
dynamic cellular microenvironment during the disease that can be utilized to regulate cell-matrix interactions23,26,35,36.
progression1,2. Several current therapeutic approaches have To investigate whether our biodegradable MnO2 hybrid nanos-
aimed to restore neural signaling, reduce neuroinflammation, and caffolds have an enhanced binding affinity toward ECM proteins
prevent subsequent damage to the injured area using stem cell to promote cell adhesion, neuronal differentiation of stem cells,
transplantations3–6. Given the intrinsically limited regenerative and neurite outgrowth through the ECM-mediated integrin sig-
abilities of the CNS and the highly complex inhibitory environ- naling pathway, we first investigated the interaction between 2D
ment of the damaged tissues, stem cell transplantation has great MnO2 nanosheets and laminin proteins (Fig. 2a-b, Supplemen-
potential to regenerate a robust population of functional neural tary Fig. 1). Using a bicinchoninic acid (BCA) assay, we observed
cells such as neurons and oligodendrocytes, thereby re- significantly increased laminin adsorption on MnO2 nanosheets
establishing disrupted neural circuits in the damaged CNS (7.5-fold increase) compared to its binding toward control glass
areas4,7–10. However, several pertinent obstacles hinder advances and polymer substrates (Fig. 2c). To better understand the origin
in stem cell transplantation. First, due to the inflammatory nature of such strong binding interactions between ECM proteins and
of the injured regions, many transplanted cells perish soon after MnO2-nanosheets, we used the density functional theory (DFT)
transplantation11. Second, the extracellular matrix (ECM) of the method to calculate the binding energies between the MnO2-
damaged areas is not conducive to stem cell survival and nanosheets and a series of functional groups commonly exhibited
differentiation2,12. Therefore, to address the aforementioned in ECM proteins (Fig. 2b, d, e). The calculation results showed
issues and facilitate the progress of stem cell therapies, there is a that electrostatic and polar-π interactions are the main con-
clear need to develop an innovative approach to increase the tributors to the strong binding interactions of the biomolecules
survival rate of transplanted stem cells and to better control stem onto the MnO2-nanosheets. For example, the binding energies for
cell fate in vivo, which can lead to the recovery of the damaged methylamine and methylbenzene are about 3-fold higher than
neural functions and the repair of neuronal connections in a more that of water (Fig. 2e, Supplementary Fig. 2, Supplementary
effective manner. Table 1). Considering laminin proteins are rich in amino and
To this end, we report a biodegradable hybrid inorganic (BHI) aromatic functional groups, the DFT calculation results indicated
nanoscaffold-based method to improve the transplantation of that these interactions are critical for the strong binding of ECM
human patient-derived neural stem cells (NSCs) and to control proteins onto the MnO2-nanosheet. Given the extraordinarily
the differentiation of transplanted NSCs in a highly selective and high crystal surface of 2D MnO2 nanosheets, we speculated that
efficient way. Further, as a proof-of-concept demonstration, we the nanoscaffolds would also have strong binding interactions
combined the spatiotemporal delivery of therapeutic molecules toward small molecule drugs that contain aromatic and amine
with enhanced stem cell survival and differentiation using BHI- structures. Our DFT calculation approach was thus further uti-
nanoscaffold in a mouse model of SCI. Specifically, our developed lized to provide insight into the laminin-induced formation of 3D
three-dimensional (3D) BHI-nanoscaffolds (Fig. 1) have unique MnO2 hybrid nanoscaffolds and acted as a screening method to
benefits for advanced stem cell therapies: (i) wide-range tunable identify neurogenic or anti-inflammatory drugs that can enhance
biodegradation; (ii) upregulated ECM-protein binding affinity; survival and neuronal differentiation of NSCs in vitro and in vivo.
(iii) highly efficient drug loading with sustained drug delivery To study neuronal differentiation of stem cells using our MnO2
capability; and (iv) innovative magnetic resonance imaging hybrid nanoscaffolds, we synthesized layer-by-layer MnO2
(MRI)-based drug release monitoring (Fig. 1a-c). Hybrid bioma- nanoscaffold assembly (3D-MnO2 nanoscaffolds) using a vacuum
terial scaffolds have been demonstrated to mimic the natural filtration method that enabled us to generate highly homogeneous
microenvironment for stem cell-based tissue engineering13–22. In and reproducible 3D-MnO2 nanoscaffolds (Fig. 2f). Compared to
this regard, scientists including our group, have recently reported conventional 3D nanoscaffold-fabrication methods such as
that low-dimensional (0D, 1D, and 2D) inorganic and carbon spraying, drop-casting, and electrochemical deposition, our
nanomaterial (e.g., TiO2 nanotubes, carbon nanotubes, and gra- applied vacuum filtration method can produce large-scale,
phene)-based scaffolds, having unique biological and physio- homogeneous, free-standing, and mechanically robust 3D
chemical properties, and nanotopographies, can effectively control nanoscaffolds in a highly controllable way (Supplementary Figs. 3,
stem cell behaviors in vitro, as well as in vivo23–31. However, these 4). To perform the 3D-MnO2 nanoscaffold-based stem cell assay,
inorganic and carbon-based nanoscaffolds are intrinsically limited we chose human induced pluripotent stem cell (hiPSC)-derived
by their non-biodegradability and restricted biocompatibility, NSCs as a model system since hiPSC-derived NSCs can be
thereby delaying their wide clinical applications. On the contrary, effectively translated into clinical applications for neuro-
MnO2 nanomaterials have proven to be biodegradable in other degenerative diseases and injuries37.
bioapplications such as cancer therapies, with MRI active Mn2+ By seeding hiPSC-NSCs on laminin-coated 3D-MnO2 nanos-
ions as a degradation product32–34. Taking advantage of their caffolds, we observed a significant enhancement of neuronal
biodegradability, and incorporating their unique physiochemical differentiation (43% increase) and neurite outgrowth (11-fold
properties into stem cell-based tissue engineering, we have increase) compared to the control conditions by measuring the
developed MnO2 nanomaterials-based 3D hybrid nanoscaffolds to biomarker protein and gene expression levels (Fig. 2g-i,
better regulate stem cell adhesion, differentiation into neurons, Supplementary Figs. 5, 6). To understand the underlying
and neurite outgrowth in vitro and for enhanced stem cell mechanism of the 3D-MnO2 nanoscaffold-based enhanced
transplantation in vivo (Fig. 1d-e). Considering the difficulties of neuronal differentiation and neurite outgrowth, we investigated
generating a robust population of functional neurons and the relevant laminin-mediated focal adhesion-dependent signal-
enhancing neuronal behaviors (neurite outgrowth and axon ing pathways using a qRT-PCR (quantitative reverse
regeneration), our biodegradable MnO2 nanoscaffold can poten- transcription-polymerase chain reaction, Supplementary Table 2)
tially serve as a powerful tool for improving stem cell transplan- technique. Indeed, a substantial increase of focal adhesion kinase
tation and advancing stem cell therapy. (FAK) gene (4.7-fold) and an upregulation of a neuronal growth

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NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-05599-2 ARTICLE

a 3D-Novel hybrd nanomaterials Stem cell therapy Simulation method

ECM Neurogenic drug
proteins (N-drug)

2D MnO2 nanosheet

3D-biodegradable hybrid
Density functional theory
inorganic (BHI) nanoscaffolds
-based N-drug screening
(with drugs, ECM proteins, and stem cells)

Neurogenic drug

H
H
b (DAPT) c

H
H
H

H
H
Tunable biodegradation (MnO2)

H
H

H
H
H

H
O
H
H
H

H
O
H
Efficient drug loading and sustained release

H
N
H

O
H
FRET/MRI-based monitoring of drug release

H H

F
H
Nanomaterial-enabled advanced stem cell therapy

H
F
d SCI site
e hiPSC derived NSCs Neuronal
differetiation

Transplantation
of NSCs
Enhanced stem cell
survival and differentiation

ble
da ds
Differentiation e gra affol
d c
of stem cells Bio anos
n

Signal relaying

Fig. 1 BHI nanoscaffolds for advanced stem cell therapy. a To develop an effective method for stem cell transplantation, we synthesized a BHI nanoscaffolds
that simultaneously integrate advancements in 3D-hybrid nanomaterials and DFT calculations-based precision drug screening. Cells are labeled with green
due to their green fluorescence protein labeling. Laminin proteins are colored in blue. Drugs are represented by red-colored dots. In the simulation scheme,
blue colored atoms represent manganese and red colored atoms represent oxygen. b Compared to conventional inorganic scaffolds for stem cell
transplantation, our BHI nanoscaffold self-assembled from atomic-thin MnO2 nanosheets, ECM proteins, and therapeutic drugs has unique advantages
including: (i) Redox mediated tunable biodegradation; (ii) Efficient drug loading and sustainable release; (iii) FRET/MRI monitorable drug release; (iv)
Nanomaterials enabled advanced stem cell transplantation. c A representative SEM (Scanning Electron Microscopy) image of BHI nanoscaffolds. d–e The
unique advantages from our innovative BHI nanoscaffold effectively improved the stem cell transplantation under CNS injured microenvironments, which
typically have highly inflammatory and inhibitory microenvironments at the injury site. Blue colored and elongated cells indicate host neurons; dark-blue cells
with pink nuclei: immune cells such as macrophage; red cross: inflammatory and inhibitory microenvironments. Specifically, the significantly enhancement of
stem cell transplantation from BHI nanoscaffold is hypothesized to achieve through an improved cell adhesion and neuronal differentiation. A murine
hemisection SCI model was used to evaluate the in vivo survival and differentiation of BHI nanoscaffold-transplanted iPSC-NSCs. Scale bar: 500 nm

cone-associated GAP43 gene (36%) were observed from hiPSC- Supplementary Fig. 9). UV-Vis absorption spectrum data con-
NSC-derived neurons on 3D MnO2 nanoscaffolds, compared to firmed that 2D-MnO2 nanosheets were degraded by ascorbic acid
those cultured on a glass substrate (Fig. 2g-i, Supplementary in a dose-dependent manner. Similarly, a controllable degrada-
Figs. 6–8, Supplementary Methods). In short, these results tion rate of MnO2 nanosheets by ascorbic acid was observed using
strongly suggested that our 3D-MnO2 nanoscaffolds can improve micropatterned-MnO2 nanoscaffolds, by directly monitoring the
neuronal differentiation and neurite outgrowth, through the disappearance of the micropatterned-MnO2 nanoscaffolds and by
enhanced laminin binding and focal adhesion-related pathways. analyzing the x-ray energy dispersive spectroscopy (EDS) data
(Fig. 3c, d, Supplementary Methods). In addition, the tunability of
Controllable biodegradation of MnO2 hybrid nanoscaffolds. biodegradation rate can be effectively achieved by changing the
While low-dimensional inorganic nanomaterials have shown number of assembled layers (Supplementary Fig. 10). Further-
great potential in stem cell-based regenerative medicine, in vivo more, we investigated the redox properties of MnO2 nanoscaf-
biocompatibility and biodegradation of these nanomaterials are folds in PBS using cyclic voltammetry (CV) to study the
the most critical issues to be addressed before inorganic degradation without exogeneous bioreductants. We could detect a
nanomaterial-based stem cell applications can be fully realized. clear reduction voltage peak at −700 mV from the CV curves, at
To demonstrate the tunable biodegradation study of MnO2 which MnO2 nanoscaffolds (Fig. 3b) degrade. From these elec-
nanoscaffolds in extracellular microenvironments, we first trochemical experiments, we confirmed our hypothesis that our
investigated the degradation of 2D-MnO2 nanosheets using synthesized MnO2 nanoscaffolds could be degraded via an
aqueous solution of ascorbic acid (vitamin C, Fig. 3a, unconventional redox-mechanism. In parallel, we inserted MnO2

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ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-05599-2

a b c 100% **
Laminin
**
Atomically thin Functional

absorption
groups in H

Laminin
layered MnO2
biomolecules COO
nanosheet 50%
δ-MnO2
CONH SH

NH3+ MnO2
OH nanosheet
0
PCL Glass MnO2

d e f
Polar- π Homogeneous
12 surface
Electrostatic

–C6H5
–Binding energy
(kcal per mol)

–NH2
8

–COOH
Layered MnO2 nanoscaffold

–OH
MnO2
nanosheet 4

0 H2O
g Integrin
GAP43
Complex
Membrane h 34%, 5 μm i i 77%, 59 μm
FAK

Glass laminin

Enhanced adhesion, axonal

growth & neuronal differentiation

Laminin coated Laminin coated

glass scaffold MnO2 nanoscaffold
Nanoscaffold DAPI/Tuj DAPI/Tuj

Fig. 2 Enhanced stem cell differentiation using biodegradable MnO2 hybrid nanoscaffolds. a Representative TEM (Transmission Electron Microscopy) of
atomic-thin layered MnO2 nanosheets. Inset: HR (High Resolution) TEM image (image size: 18 Å by 14 Å). b A schematic diagram describing the
intermolecular binding between MnO2 nanosheets and selected functional groups that are commonly existent in ECM proteins and biomolecules. c A
significantly upregulated ECM protein (laminin) binding towards 2D MnO2 nanosheet, compared to control substrate [etched glass and polycaprolactone
(PCL) substrates). These upregulated laminin binding was studied by a BCA protein assay. Data are mean ± s.d. n = 3, **P < 0.01 by one-way ANOVA
(Analysis of variance) with Tukey post-hoc test. d, e, By modeling small molecule-nanosheet interactions (d), we summarized differential binding affinities
of MnO2 nanosheets towards of a library of functional groups (e). These simulation results aligned well with experimentally observed enhanced laminin
binding, as laminin structures are rich in both amino and aromatic moieties. Exemplary molecule in d: toluene. Carbon and hydrogen atoms are colored in
black and gray, respectively. f A representative SEM image showing the layered structure and homogeneous surface of MnO2 hybrid nanoscaffolds
fabricated using the vacuum filtration technique. Inset photograph: a free-standing MnO2 hybrid nanoscaffold fabricated by vacuum filtration and
manipulated by a tweezer. g A schematic diagram illustrating the proposed mechanism for the enhanced neuronal differentiation on MnO2 hybrid
nanoscaffolds. Red color indicates GAP43; Orange indicates FAK; Green indicates integrin complex. h, i Representative immunostaining data supporting
the significantly enhanced neuronal differentiation and neurite outgrowth of iPSC-NSCs on MnO2 hybrid nanoscaffolds (i) compared to control substrates
(h). Cell nuclei are in blue and TuJ1 is pseudocolored in green. Average percentages of neuron and neurite lengths were measured using NeuronJ software
(h: n = 9; i: n = 12) and labeled on the top right of each image. Scale bars: a 25 nm; f 500 nm; h, i 100 μm

nanoscaffolds in between two layers of cells, which can mimic delivery of exogenous reductants. Moreover, we could control the
in vivo transplantation conditions, to study the nanoscaffold degradation rate of MnO2 nanoscaffolds by showing a tunable
degradation profiles, as well as to investigate whether such redox- half-degradation period from a few minutes to one month. This
mediated biodegradation of MnO2 nanoscaffolds was possible in tunability of a biodegradation rate was achieved by changing the
tissue-mimicking conditions without adding any exogenous assembled layered-structures of MnO2 nanosheets and by con-
bioreductants or electrical stimuli. As a negative control experi- trolling concentrations of reductants (Fig. 3f, Supplementary
ment, graphene oxide (GO) nanoscaffolds were also inserted in Fig. 10). In short, our developed MnO2 nanoscaffolds represent
between two layers of cells. The biodegradation of both nanos- an innovative inorganic hybrid nanoscaffold system that can be
caffolds (MnO2 vs. GO) was examined daily by measuring the biodegraded in vitro. Given that CNS microenvironment contains
thickness of the dark-colored nanoscaffold layers. Consistent with highly concentrated bioreductants and different degradation
previous reports, we did not observe any noticeable degradation speeds would be needed for different applications, the con-
of GO nanoscaffolds throughout a month-long observation trollable biodegradation properties of MnO2 nanoscaffolds could
(Fig. 3e)38. In contrast, MnO2 nanoscaffolds rapidly degraded be more appealing and important in the field of neural tissue
with over 30% of the MnO2 nanoscaffolds degraded within engineering39.
one week, and a half-degradation time was around 2 weeks
(Supplementary Fig. 9). This result proved that the biodegrad- 3D MnO2 hybrid nanoscaffolds self-assembled with ECM
ability of MnO2 nanoscaffolds could be induced by cells without protein. One of the critical issues of conventional degradable

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NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-05599-2 ARTICLE

a b
40 Nanoscaffold
Assembled layered-structures
Mn2+
** ITO
of MnO2 nanosheets
20
Bioreductants

Current/μA
PBS
(e.g., vitamin C) 0 (control)

Tunable redox –20 Redox mediated

biodegradation H2O degradation
Vacuum filtrated –40
MnO2 nanoscaffolds
–1.0 –0.5 0.0 0.5 1.0

c d 30
Before bio-reductants After bio-reductants 0.6 wt%
20 Before
added added Mn

cps
degradation
10
EDS
0
30
Redox-mediated
degradation

cps
20 After
0 wt% degradation
10
Micropatterned Degraded
nanoscaffolds nanoscaffolds 0
0 2.5 5.0 7.5 10
Energy (keV)

e f
Scaffold with cell Cell layers
MnO2

Degradation

Cell induced biodegradaton

No
GO

degradation
Fast degradation of nanoscaffolds
D1 D7 D14 D21

Fig. 3 Controllable biodegradation of MnO2 hybrid nanoscaffolds. a A schematic diagram explaining an unconventional redox biodegradation mechanism of
our MnO2 hybrid nanoscaffolds. This redox biodegradation can be achieved through either bioreductants exists commonly in human body such as vitamin
C, electrically delivered reduction signals or by stem cells cultured with the nanoscaffold. Degradation products include water (colored in green) and Mn2+
(colored in blue). b Controllable redox biodegradation of MnO2 hybrid nanoscaffolds demonstrated through cyclic voltammetry. A successful degradation
of nanoscaffolds were confirmed by the disappearance of yellow color from nanoscaffold triggered by electrical stimuli. x axis indicates voltage (v).
c, d Degradation of nanoscaffold by commonly existent bioreductants (e.g., vitamin C), indicated by the decay of micropatterns from the micropatterned
nanoscaffold (c); and the disappearance of manganese elements from the substrate after degradation through EDS analysis (d). cps means count per scan.
e Time dependent biodegradation of MnO2 hybrid nanoscaffolds in cell culture without addition of any external trigger. The degradation of nanoscaffold
was examined based on the thickness of the black-colored layer sandwiched between two layers of cells. GO nanoscaffolds were used as a negative control
and no noticeable degradation was observed. Half-degradation time of MnO2 hybrid nanoscaffolds was determined to be around 2 weeks. f Controllable
fast-biodegradation of MnO2 hybrid nanoscaffolds. By controlling bioreductant (vitamin C) concentrations, a fast degradation of iPSC-NSC seeded
nanoscaffold and formation of iPSC-NSC sheet was achieved. Nanoscaffold is indicated by the dark-colored background before degradation. Size of each
image is 1 cm by 1 cm. Cells were stained with pink color for convenience of observation. Scale bar: c 100 μm

bioscaffolds is degradation-mediated disruption of cellular of laminin toward 2D-MnO2 nanosheets, where laminin can
microenvironments, which can interrupt continuous neuronal function as adhesive layers (binder) for individual MnO2
differentiation and neurite outgrowth of transplanted NSCs15,40. nanosheets. To investigate whether 3D-MnO2-laminin hybrid
To this end, biocompatible 3D bioscaffolds complexed with ECM nanoscaffolds promote the neuronal differentiation of NSCs and
proteins or peptides, such as laminin, fibronectin, and Argi- the following neuronal behaviors including neurite outgrowth, we
nylglycylaspartic acid (RGD), that enhance neuronal differentia- performed stem cell assays using three different substrates/scaf-
tion of stem cells and neurite outgrowth continuously, have folds (glass, MnO2 nanoscaffolds, and 3D-MnO2-laminin hybrid
provided a promising solution for advanced stem cell-based tissue nanoscaffolds) under the same culture conditions. After 6 days of
engineering35,41,42. As such, inspired by a recent report on the stem cell differentiation assays, we found dramatically higher cell
non-covalent preparation of hydrogels43, we developed a method counts from our 3D-MnO2-laminin hybrid nanoscaffolds com-
to generate biocompatible 3D-MnO2 hybrid nanoscaffolds com- pared to glass (740% higher) and MnO2 nanoscaffolds (270%
plexed with laminin-protein, termed, 3D-MnO2-laminin hybrid higher) controls (Fig. 4b, f-h, Supplementary Fig. 11). Further-
nanoscaffolds. Interestingly, MnO2-laminin hybrid nanoscaffold, more, through a neuronal marker, beta-III tubulin (TuJ1)
an innovative 3D-inorganic scaffold self-assembled from laminin, immunostaining, we confirmed even more significant improve-
was successfully synthesized by mixing 2D-MnO2 nanosheets ment of neuronal differentiation and neurite outgrowth from our
with laminin solutions (Fig. 4a-e, Supplementary Fig. 11). The 3D-MnO2-laminin hybrid nanoscaffolds compared to the other
self-assembly process could be achieved by the strong interactions control substrates, showing 11 times longer average neurite

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ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-05599-2

a b iPSC-NSCs
Laminin
6-day
Self-assembly
enhanced
neuronal differentiation
MnO2 nanosheets MnO2 laminin
hybrid nanoscaffold Mature neurons
c d e Cell encapsulated MnO2
laminin nanoscaffolds

MnO2 laminin
nanoscaffold
f Control g MnO2 nanoscaffold h MnO2 laminin nanoscaffold

12%, 5 μm 67%, 59 μm 81%, 85 μm

DAPI/Tuj1 DAPI/Tuj1 DAPI/Tuj1

i Region of interest j k
Time (frames at 50 Hz)

DAPI/MAP2 DAPI/Syn1

Fig. 4 3D MnO2 hybrid nanoscaffolds self-assembled with ECM proteins. a, b Schematic diagram illustrating the mechanism for the self-assembly of ECM
protein (laminin) and MnO2 nanosheets through a non-covalent crosslinking mechanism. This mechanism was utilized to synthesize MnO2 laminin hybrid
nanoscaffolds, and iPSC-NSCs cultured on MnO2-laminin hybrid nanoscaffolds successfully differentiated into mature (MAP2+) neurons after 6 days (b).
c–e Representative SEM images of MnO2 laminin hybrid nanoscaffolds (c, d), and iPSC-NSC-encapsulated hybrid nanoscaffolds (e). f–h Immunostaining
results on neuronal markers (TuJ1, labeled with green) demonstrate significant enhancements of cell adhesion and neuronal differentiation of iPSC-NSCs
differentiated on MnO2 laminin hybrid nanoscaffolds (h) compared to both control (glass, f) substrates and MnO2 hybrid nanoscaffolds (g). Average
percentage of neuron and neurite lengths were labeled on the bottom left of each image. i Time-lapse calcium imaging results of iPSC-NSC differentiated
neurons on MnO2 laminin hybrid nanoscaffolds. j, k representative immunostaining images on mature neuronal markers (i: MAP2 and k: Synapsin 1,
labeled with green) from iPSC-NSC-differentiated neurons on MnO2 laminin hybrid nanoscaffolds. Scale bars: c 10 μm; d 500 nm; e–h 100 μm; j, k 100 μm

lengths compared to laminin-coated glass and 1.4 times longer controlled delivery of soluble cues such as small organic mole-
than laminin-coated MnO2 nanoscaffold (Fig. 4f, Supplementary cules (e.g., neurogenic drugs to selectively induce stem cell neu-
Fig. 6). Meanwhile, we have also verified the neurons formed on ronal differentiation) using our 3D-hybrid inorganic
our hybrid nanoscaffolds are mature through time-dependent nanoscaffolds provides a promising solution44,46,47. Conventional
calcium imaging technique and immunostaining with mature scaffolds that typically use physical encapsulation to load drugs
neuronal markers such as Microtubule-associated protein 2 normally suffer from rapid diffusion of drugs, which leads to
(MAP2) and Synapsin (Syn1) (Fig. 4i-k, Supplementary Fig. 7). undesired damage to the transplanted cells, as well as the sur-
These results support our hypothesis that 3D-MnO2-laminin rounding tissues because of the high drug concentration initially,
hybrid nanoscaffolds can effectively and steadily promote neu- and limited neurogenic effect later on due to an insufficient
ronal differentiation of stem cells and neuronal behaviors for remaining drug concentration45–47. To this end, our developed
versatile stem cell therapies. 3D-MnO2-laminin hybrid nanoscaffolds showed improved drug-
loading capability and minimized burst-release owing to strong
Spatiotemporal controlled delivery and monitoring of drugs. drug-binding affinity to the nanoscaffolds. For a comprehensive
While some conventional biodegradable and biocompatible 3D- study of drug loading and monitoring of drug release using our
hybrid scaffolds have shown their potential to promote stem cell nanoscaffolds, we first used a fluorescent aromatic ring-
neuronal differentiation and neurite outgrowth, there is still a lot containing small molecule, Rhodamine B (RhB), as a model
of room for improvement to control stem cell differentiation and drug system. To optimize the loading and binding of drug
neuronal behaviors in a more selective and temporally controlled molecules, RhB was first loaded onto 2D-MnO2-nanosheets.
manner in vivo44–47. These requirements would be essential to Then, the RhB-loaded MnO2-nanosheets self-assembled with
achieve the full therapeutic potential of transplanted stem cells for laminin to generate 3D-MnO2-laminin hybrid nanoscaffolds
SCI treatment. Addressing this challenge, spatiotemporal (Fig. 5a). We adapted a quantitative fluorescence resonance

6 NATURE COMMUNICATIONS | (2018)9:3147 | DOI: 10.1038/s41467-018-05599-2 | www.nature.com/naturecommunications

NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-05599-2 ARTICLE

a Fluorescent molecule (RhB)

Fluorescence quenching
(as a model-drug system) Fluorescence
monitoring
Laminin
FRET OFF
Degradation Mn2+
Self-assembly Mn2+ MRI
FRET ON monitoring
BHI nanoscaffolds Mn2+
Drug loading Drug loading

b c Strong Weak
PBS Excellent
(control) drug-binding

MRI
Scaffold
RhB loaded degraded
nanoscaffolds
FRET

FRET
FRET OFF Drug
released
Controlled Vitamin C
release (testing condition)
High Low

d e
E(BE) = –18.3 kcal per mol

Notch Notch inhibition Glass

inhibitor
Glial cells (control)
(DAPT)
6.3 Å
7.8 Å 5.6 Å
5.6 A
iPSC-NSCs
DAPT-loaded
MnO2 nanosheets Neurons BHI nanoscaffolds

Fig. 5 Spatiotemporal controlled delivery of soluble cues using 3D-hybrid inorganic nanoscaffolds. a Schematic diagram of drug loading, releasing, and
monitoring on the MnO2 laminin hybrid nanoscaffolds. Drug (pink and gray circles) was first loaded onto individual nanosheets, then self-assembled with
laminin to form the drug loaded-MnO2 laminin hybrid nanoscaffolds to achieve controlled drug release. Two modalities of monitoring scaffold degradation
and drug release can be achieved by the nanoscaffold through FRET and the stoichiometrical release of T1 active Mn2+, respectively. b Fluorescence
microscopy images demonstrating excellent drug hold-up from drug-loaded hybrid nanoscaffolds, and controlled drug release by bioreductants. Release of
model drugs were monitored by the red fluorescence nearby nanoscaffold. c A MRI-based monitoring of drug release enabled by our hybrid nanoscaffold,
which is confirmed by a direct correlation between the amount of released drug (indicated by red fluorescence) and T1 MRI intensities detected from the
same nanoscaffolds. This unique drug monitoring was enabled by the strong interaction between drug and nanoscaffold, which determines the
stoichiometrical relevance between amount of drug and Mn2+ released as degradation products. 5, 2.5, 1, 0.5, 0.1 mg (left to right) of scaffold were
degraded before MRI and fluorescence measurements. Samples were incubated for 2 days before imaged. Dotted circles have diameters of 1 mm. d An
optimized binding geometry and binding energy of simulation-screened neurogenic drug (DAPT) toward nanosheets. Nitrogen atoms are colored in blue. e
Spatial control of neuronal differentiation and neurite outgrowth across the boundary between control substrates and DAPT-loaded MnO2 laminin hybrid
nanoscaffolds. DAPT-loaded MnO2 laminin hybrid nanoscaffolds enhanced neuronal differentiation of iPSC-NSCs compared to control substrate and MnO2
hybrid nanoscaffolds. Through a spatial patterning of DAPT-loaded MnO2 laminin nanoscaffolds, a spatially controlled neuronal differentiation was
successfully demonstrated. TuJ1 and nuclei staining of iPSC-NSC were indicated by green and blue, respectively. Astrocytes are colored in orange in the
scheme. Scale bar: e 100 μm

energy transfer (FRET)-based approach to monitor released or amount of drug released (Fig. 5a, c). Indeed, by inducing the
non-binding RhB molecules32. Our FRET-based method allowed nanoscaffold degradation by bioreductants, we found that the
us to assay the drug loading and release process (Fig. 5a). Based amount of released drug, measured by the fluorescence intensity
on this FRET-based drug monitoring method, we observed an of RhB was closely correlated with the intensity of MRI signal
excellent drug-binding affinity onto the 3D-MnO2-laminin (Fig. 5c). This “on/off” MRI-based monitoring of drug release has
hybrid nanoscaffolds, wherein minimal RhB release from the not yet been demonstrated in conventional scaffolds, thereby
nanoscaffolds was detected over 7 days. However, as soon as we offering a tool that can provide a much-improved investigation
introduced bioreductant (vitamin C) to the RhB-loaded 3D- on drug delivery and in vivo release48.
MnO2-laminin hybrid nanoscaffolds, the fluorescence signal of The optimized condition regarding drug loading and release,
RhB release was observed with an over 500-fold increase with a based on the fluorescent RhB molecule as a model drug, was used
sustainable delivery profile (Fig. 5b, Supplementary Figs. 12–13). to load and deliver neurogenic drugs for an enhanced neuronal
This experimental result strongly supports that our 3D-hybrid differentiation. To screen the optimal neurogenic drug, we
inorganic nanoscaffold-based drug delivery platform can control applied the DFT calculations (Fig. 2d, Supplementary Table 1)
drug release kinetics over a few weeks through degradation of and selected a neurogenic drug (DAPT: N-[N-(3,5-Difluorophe-
nanoscaffolds. Additionally, the stoichiometrically equivalent nacetyl)-L-alanyl]-S-phenylglycine t-butyl ester) based upon its
Mn2+ ion release and the MnO2 degradation (1:1 ratio) high binding energy to 2D-MnO2 nanosheet (Fig. 5d, Supple-
encouraged us to hypothesize that MRI signals from Mn2+ can be mentary Figs. 13–14). DAPT is a γ-secretase and Notch inhibitor
utilized to quantify the degradation rate of our hybrid nanos- that simultaneously promotes neuronal differentiation and
caffolds and to correlate the intensity of MRI signal with the neurite outgrowth while suppressing astrocyte differentiation49,50.

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ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-05599-2

a Hemisection SCI model

hiPSC-NSCs
Transplantion

3D-biodegradable hybrid
inorganic (BHI) nanoscaffolds
(with DAPT, laminin, and iPSC-NSC) 7 weeks post-injury
(7 WPI)

1 and 4 weeks post-injury

(1 WPI and 4 WPI) Therapeutic effect
b
Improved Enhanced Reduced
cell survival neuronal differentiation glial scar formation

Fig. 6 Pathways of enhanced stem cell transplantation into SCI sites by 3D-BHI nanoscaffolds. a, b, Schematic diagram (a) showing the enhanced
transplantation of iPSC-NSC in a murine lateral hemisection SCI model by 3D BHI nanoscaffold and the proposed mechanisms (b) for the enhanced
transplantation and potential beneficial effects on overcoming inhibitory microenvironments in the SCI sites

The calculated binding energy between DAPT and the 2D-MnO2 hemisected SCI lesion (Fig. 6a)51. To identify our transplanted
nanosheet is −18.3 kcal mol−1, an over 4-fold increase compared cells, hiPSC-NSCs were genetically labeled with green fluorescent
to the binding of solvent (water) to nanoscaffolds, indicating that protein (GFP) (i.e., hiPSC-NSC-GFP). Surgifoam inserted mice
DAPT drugs can be strongly adsorbed to the MnO2 surface. with injuries were used as a control condition. After transplan-
Indeed, by forming DAPT-loaded 3D-MnO2-laminin hybrid tation, we first evaluated nanoscaffold biodegradation in vivo by
nanoscaffolds, the spectrums from matrix-assisted laser deso- detecting the amount of degraded Mn (manganese) element in
rption/ionization (MALDI) time-of-flight (TOF) mass spectro- mouse urine samples using inductively coupled plasma mass
meter showed a high amount of DAPT loaded onto the spectrometry (ICP-MS) analysis (Supplementary Fig. 17). Con-
nanoscaffolds, while control scaffolds (glass and polymer sistent with our previous in vitro studies, we could observe rapid
substrate) did not show any noticeable peaks (Supplementary in vivo degradation of nanoscaffold. This degradation of trans-
Fig. 14). To investigate the effect of DAPT-loaded 3D-MnO2- planted nanoscaffolds was also detected by the color change
laminin hybrid nanoscaffolds on stem cell neuronal differentia- (from black to brown) in a time-dependent manner throughout
tion and neuronal behaviors, we tested hiPSC-NSC-based the first-week post-transplantation. After investigating the in vivo
neuronal differentiation assays using DAPT-loaded 3D-MnO2- biodegradability, we then studied the effects of 3D-BHI nanos-
laminin nanoscaffolds and related controlled conditions for one caffold for enhanced stem cell transplantation. We hypothesized
week. We found a strong enhancement of neuronal differentia- that our 3D-BHI nanoscaffold could robustly improve the sur-
tion from the DAPT-loaded 3D-MnO2-laminin nanoscaffold vival of hiPSC-NSCs and the differentiation of hiPSC-NSCs into
condition (a 1.4-fold enhancement of Tuj1 mRNAs and a 1.7-fold neurons in the early and intermediate stages (1–4 weeks post
enhancement of neurite outgrowth compared to 3D-MnO2 injury (WPI)) (Fig. 6, Supplementary Figs. 18–25). We also
laminin hybrid nanoscaffolds), as well as suppressed GFAP (Glial expected that the 3D-BHI nanoscaffold could reduce astroglial
fibrillary acidic protein) mRNA expression (Fig. 5e, Supplemen- scar formation in the longer term (7 WPI) (Fig. 6). To test our
tary Figs. 15, 16). Remarkably, in the boundary of DAPT-loaded hypothesis, we first performed short-term (1-week) in vivo stem
3D-MnO2-laminin hybrid nanoscaffold and glass, NSC-derived cell transplantation assay in the injured site and 3 different
neurons across the boundary, cultured under the same condition, control conditions for stem cell transplantation were included as
had a dramatic change in morphology and neurite outgrowth controls (Fig. 7, Supplementary Figs. 20–23). From our immu-
(Fig. 5e). This result provided a direct comparison between our nostaining data, we observed significantly higher populations of
3D-MnO2-laminin hybrid nanoscaffolds and conventional scaf- GFP + cells and TuJ1 + cells from our testing condition com-
folds and indicated the ability of spatiotemporal control of hiPSC- pared to the other 3 control conditions, which directly suggests an
NSC differentiation using our drug-loaded 3D-hybrid inorganic enhanced stem cell transplantation. This result is also consistent
nanoscaffolds. with our Caspase 3 immunostaining data (Fig. 7, Supplementary
Fig. 23), where fewer hiPSC-NSC-GFP cells transplanted by 3D-
BHI nanoscaffold showed apoptotic markers as compared to the
Enhanced stem cell transplantation into SCI sites. With the polymer scaffold-transplanted cells. Additionally, hiPSC-NSC-
prominent effects of the 3D-hybrid inorganic nanoscaffolds on GFP cells transplanted by 3D-BHI nanoscaffold showed more
improving the adhesion, neuronal differentiation of hiPSC-NSCs, spreading, whereas GFP + cells from two control conditions
and neurite outgrowth of differentiated neurons, we then further (Fig. 7) showed less spreading. This could be due to the pro-
tested the effects of the nanoscaffold on enhanced stem cell moted cellular adhesion and could improve neuronal
transplantation in vivo (Fig. 6). To transplant the stem cell-seeded differentiation, based on our in vitro focal adhesion studies
nanoscaffolds, we first generated a T10 thoracic hemisection (Supplementary Fig. 6). This trend of enhanced cell survival and
lesion to the spinal cord of an adult mouse, then the hiPSC-NSC increased neuronal differentiation of stem cells from 3D-BHI
seeded-nanoscaffolds (as an experimental condition) and -poly- nanoscaffold-treated condition continued until 4-WPI, as shown
caprolactone (PCL) polymer scaffolds (PCL-cell group, as a in (Supplementary Fig. 24). Furthermore, the percentage of
control condition) were rolled up and inserted into the hiPSC-NSC-GFP with more mature neuronal markers (Syn1) is

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NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-05599-2 ARTICLE

a DAPI/GFP DAPI/TuJ1 DAPI/GFP/TuJ1 DAPI/GFP DAPI/TuJ1 DAPI/GFP/TuJ1

nanoscaffold
3D-BHI
PCL + cell
control
MnO2 + cell
control
Laminin + cell
control

GFP+: cell survival GFP+ + Tuj1+: neuronal differentiation

Tuj1+: neurons
after transplantation of hiPSCs after transplantation

b c
** **
** **
Tuj1 + GFP+ cell counts

160 ** 150 ** 100

GFP+ cell counts

Tuj1+/GFP+ cells
Percentage of
80 75 50

0 0 0
Nano- PCL MnO2 laminin Nano- PCL MnO2 laminin
scaffold cell cell cell scaffold cell cell cell

Fig. 7 3D-BHI nanoscaffold enhances survival and neuronal differentiation of hiPSC-NSC-GFP. a Immunohistological staining analysis was performed on
tissue slices from 4 different animal groups transplanted with hiPSC-NSC-GFP to determine enhanced cell survival and improved neurogenesis from our
3D-BHI nanoscaffold transplanted condition. All tissue analysis was performed 1-week post transplantation and stained with DAPI (blue) and TuJ1
antibodies (red). Arrows indicate neuronal cells differentiated from hiPSC-NSC-GFP (identified by TuJ1+/GFP+ cells). b By quantifying the number of
remaining GFP+ cells, the ability of 3D-BHI nanoscaffold to retain the significant higher amount of cells after transplantation was demonstrated by
comparing to other cell transplantation groups. c Improved cell transplantation by our nanoscaffold can be further evidenced by an increased neuronal cell
population and a higher percentage of neuronal cells in GFP+ cells (area = 1 mm2). This is consistent with Supplementary Fig. 20, where co-labeling of GFP
+/TuJ1+ and GFP+/GFAP+ nearby the injured sites suggest most of the transplanted hiPSC-NSC-GFP become neuronal cells but not astroglial cells. Scale
bars: 100 μm. Error bars represent mean ± s.d.; n = 3, **P < 0.01 by one-way ANOVA with Tukey post-hoc test

also higher in 3D-BHI nanoscaffold-treated condition as com- DAPT released from nanoscaffold in the early stages12. Over-
pared to cell injection condition at this time point, indicating coming the highly inhibitory microenvironment such as inflam-
enhanced neuronal differentiation from our nanoscaffold-treated mation and astroglial scars has been considered as an effective
conditions (Supplementary Fig. 24). Equally importantly, the strategy to treat SCI11; and our results showed that 3D-BHI
improved transplantation from our 3D-BHI treated conditions nanoscaffold could be utilized for the enhanced stem cell trans-
can mitigate inhibitory microenvironmental effects in the long- plantation and for the treatment of CNS injuries. On the other
term (7-WPI) (Fig. 8). For example, immunostaining on a pro- hand, as future plans to apply our nanoscaffold for disease
liferation marker (PH3), inflammation marker (F4/80) and an treatment, it would be essential to analyze long-term cell fates of
astroglial scar marker (GFAP) revealed higher proliferation, transplanted stem cells and is critical to evaluate animal beha-
suppressed inflammation and reduced astroglial scar formation vioral recovery from 3D-BHI nanoscaffold treated conditions
from our 3D-BHI nanoscaffold, which could be largely attributed using larger animal sets52,53. Nevertheless, given its unique
to the neurotrophic factors secreted by hiPSC-NSC-GFP and properties demonstrated in vitro, and the enhanced

NATURE COMMUNICATIONS | (2018)9:3147 | DOI: 10.1038/s41467-018-05599-2 | www.nature.com/naturecommunications 9

ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-05599-2

a
7-week post-injury 7-WPI 7-WPI
(7-WPI)

nanoscaffolds
3D-BHI
Nanoscaffold Nanoscaffold

7-WPI 7-WPI
PCL-cell
group

V C
R

7-WPI 7-WPI
(control group)
Injury only

Proliferation (PH3) Glial-scar formation (GFAP) Immune cells (F4/80)

b
** ** **
**
Glial layer thickness (μm)

**
Macrophage cell counts
150
PH3+ cell counts

10 60
100 *
5 30
50

0 0 0
Nanoscaffold PCL Control Nanoscaffold PCL Control Nanoscaffold PCL Control

Fig. 8 Improved long-term effects on SCI sites from 3D-BHI enhanced stem cell transplantation. a, b Histological immunostainings images (a) and
quantifications (b) of cell proliferation markers (PH3), astroglial markers (GFAP), and immune cell markers (F4/80). These results collectively suggesting
an improved stem cell transplantation from our 3D BHI nanoscaffolds can enhance cell proliferation inside scaffolds, reduce glial-scar formation and
decrease infiltration of inflammatory cells in vivo at 7-WPI. Cell nuclei were colored in blue in a; other staining (PH3, GFAP, and F4/80) were in red. Scale
bars in a: left and middle column: 200 μm; right column: 100 μm. Error bars represent mean ± s.d.; n = 3 or 5, *P < 0.05, **P < 0.01 by one-way ANOVA
with Tukey post-hoc test

transplantation of hiPSC-NSCs in vivo compared to conventional properties of porous scaffolds and the unique physiochemical
cell transplantation methods, our developed 3D-BHI nanoscaf- properties of MnO2 nanoscaffold such as magnetic properties and
fold could represent a promising candidate for stem cell therapy degradation rate in a single platform. Recently, 2D/3D inorganic
and for advanced stem cell transplantation. and carbon nanomaterials have attracted much attention, as they
have the great potential to control stem cell neuronal differ-
entiation and neuronal behaviors. However, several pertinent
Discussion barriers including limited biodegradability and drug loading/
The development and the use of biomaterials for stem cell-based release capability hinder their broad application toward stem cell-
tissue engineering to treat CNS diseases/injuries to date have based tissue engineering. Thus, our developed biodegradable
focused on: (i) providing favorable microenvironments for hybrid inorganic nanoscaffold-based stem cell therapeutic
endogenous and exogenous cellular regeneration and (ii) serving approach would be a useful tool for improving stem cell survival
as a spatiotemporally controlled drug release platform to regulate and inducing neuronal differentiation in vivo, and thus can
pro-neuroregenerative signaling pathways. This work is based on provide insights into stem cell behaviors post-transplantation that
the development of a biodegradable hybrid inorganic nanoscaf- may lead to novel therapies for the treatment of the neurode-
fold and its utilization for the enhanced transplantation of stem generative diseases. Collectively, our developed hybrid
cells into SCI sites. Our demonstrated nanoscaffold technology nanoscaffold-based approach to control stem cell differentiation
platform can be combined with other neurogenic drugs, as well as into neurons and promote neurite outgrowth would provide an
stem cell therapeutic efforts currently in development. In the alternative to help overcome the critical barriers that limit cellular
developed hybrid nanoscaffold, we can tune the 2D/3D structural therapies for many devastating injuries and diseases.

10 NATURE COMMUNICATIONS | (2018)9:3147 | DOI: 10.1038/s41467-018-05599-2 | www.nature.com/naturecommunications

NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-05599-2 ARTICLE

Methods standard differentiation media (w/o bFGF and no exogenous compounds) for
Synthesis and characterization of MnO2 nanosheet and GO. MnO2 nanosheets different periods of time (1 day, 3 day, 7 day, 12 day, 17 day, and 22 day). The cells
were synthesized based on a previous protocol with minor modifications54. Briefly, were then fully detached using acutase for 10 min at 37 °C. Followed by washing
2.2 g Tetramethyl ammonium pentahydrate (TMAOH·5H2O, Alfa Aesar) was first with PBS and de-ionized water, 24-well plate was vacuum dried. Based on the
dissolved in 20 ml 3% wt. H2O2 (Sigma-Aldrich) by vortexing (concentration of absorption of MnO2 nanosheet at 385 nm, the degradation percentage was quan-
TMAOH is 0.6 M). In parallel, 0.594 g MnCl2·4H2O (Sigma-Aldrich) was dissolved tified by subtracting background (empty well) and normalize to the well without
in 10 ml de-ionized water (Concentration of MnCl2 is 0.3 M) through sonication. culturing the cells.
Then the TMAOH dissolved in H2O2 solution was rapidly added into MnCl2 To study the degradation of thick layered nanoscaffold, a 3-layer, cell-MnO2
solution within 10 s with fast stirring at 1200 r.p.m. (round per minute). Please note nanoscaffold-cell sandwiched structure that mimic tissue structures was formed
that gas will be generated and rapid increase of solution volume will be observed. through centrifugation at 130×g in a 15 mL Eppendorf centrifuge tubes. A similar
The solution was continued to be stirred at 600 r.p.m. overnight and centrifuged at structure of GO nanoscaffold-cell construct was formed using the same protocols
2000×g for 5 min to obtain the bulk δ-MnO2. After washing with water for 3 times as a control. The first layer contains 1 million iPSC-NSCs. The second layer is
and ethanol for 2 times through shaking and centrifuge, bulk MnO2 was dried in the composed of 1.0 mg of MnO2 nanosheet or graphene oxide. The third layer (Top
oven under ambient conditions for 12 h. After adding 100 mg into 10 ml de-ionized layer) was centrifuged down from another 1 million iPSC-NSCs. Degradation of
water, the solution was extensively sonicated in the Sonics bath sonicator for 10 h. the scaffolds was monitored by the volume change and thickness change of scaffold
Lastly, the solutions were centrifuged at 8801×g for 10 min to get rid of the on a weekly base. Based on the assumption that the scaffold has identical radius
aggregations and un-exfoliated products. The black solution was measured with and areas, the percentage of scaffold volume was normalized to the thickness that
concentration by evaporating water in the solution. MnO2 nanosheet was diluted to was measured on Day 1.
10 μg per ml for TEM (80 kv on a Philips CM12 with an AMT digital camera model To demonstrate cell-seeded nanoscaffold can fast degrade under biocompatible
XR111) and Ultra-Stem imaging. For X-ray photoluminescence spectroscopy redox conditions, an aqueous solution of 0.3 mg per ml MnO2 nanosheet was
(Thermo Scientific ESCALAB 250 Xi with a base pressure <1 × 10–9), MnO2 filtered through a cellulose membrane, then a layer of dye-labeled (food dye) cells
nanosheet solution (100 μg per ml) was drop-casted onto a silicon substrate and was formed on the MnO2 nanosheet assembled substrate using a tri-circular PDMS
dried in the vacuum. An Al-Kα monochromated X-ray source was used to obtain (Polydimethylsiloxane, Dow Corning®) chamber. After the addition of 2.0 mg per
the core level spectra, and the instrumental broadening was around 0.5 eV. The ml ascorbic acid (Sigma-Aldrich) for 5 min, most of the dark color of MnO2
hydrodynamic size and zeta potential of MnO2 nanosheets in aqueous solution were nanosheet disappeared, and a layer pink colored cell layer was formed.
measured by a ZS (Nano Zetasizer) dynamic light scattering instrument (Malvern
Instruments, Malvern, UK), with the temperature set at 25 °C and a detection angle
of 90 degrees. UV-Vis absorption spectrum of MnO2 nanosheet solution was Tunable biodegradation of MnO2 nanoscaffolds. To show the tunable biode-
measured by a Varian Cary 50 spectrophotometer using a quartz cuvette. gradability of MnO2 nanoscaffolds, we controlled the geometrical and chemical
Graphene oxide was synthesized based on our previous publications55,56. 1.0 structures of MnO2 nanoscaffold (Supplementary Fig. 10) which includes: (i)
gram of Graphite (Bay Carbon) was preoxidized in the mixture of sulfuric acid thickness [0.2 H vs. 1 H (H = 0.4 mm), shown in a and b, respectively, which is
(Sigma-Aldrich, 98%), phosphor oxide (Sigma-Aldrich) and potassium persulfate achieved by filtrating different concentrations (0.6 mg per ml and 3.0 mg per ml,
(Sigma-Aldrich) at 80 degrees for overnight. Then the pre-oxidized graphite was respectively) of MnO2 nanosheet solution while keeping solution volume (1.0 ml)
washed with water, dried and reacted with sulfuric acid and potassium and filtrating area (1075 mm2) constant]; (ii) Height to surface area ratio (from 0.4
permanganate through a 3-step process. After quenching with H2O2, a shining gold mm:1075 mm2 in Supplementary Fig. 10b to 4 mm:107.5 mm2 in Supplementary
solution appear, and the graphite oxide was purified with 10% HCl solution Fig. 10c), which was achieved by filtrating same amount of MnO2 nanosheets (1.0
(Sigma-Aldrich) and water extensively. Lastly, graphite oxide was exfoliated into ml of 3.0 mg per ml) but reduced the filtrating area by 10 times; (iii) protein
graphene oxide by tip sonication (Branson). Multi-layered graphene oxide was amount in the scaffold (MnO2 nanosheets absorbed with 1.0 mg per ml vs. with 10
centrifuged down at 16,639×g for 45 min, and the final suspension of single or few mg per ml bovine serum protein and then vacuum filtered). Degradation profile of
layered graphene oxide was obtained. different scaffolds obtained by measuring time-dependent manganese concentra-
tions in the solution using Inductively coupled plasma mass spectrometry (ICP-
MS, Fisons Instruments PlasmaQuad 2+). Each sample was measured 3 times to
Measurement of protein absorption by MnO2 nanosheet. Into the solutions of obtain error bars and standard deviation. To control scaffold degradation without
ECM protein (laminin protein from Sigma-Aldrich, stock concentration of 200 μg any additional bioreductants, different cell densities (0, 0.1, 0.5, 1, and 5 million
per ml, 0.5 ml, PBS is from Thermo Fisher), 10 μl of MnO2 nanosheet aqueous cells per well in a 24 well plate) were seeded onto MnO2 nanoscaffold, and the
solution (3 mg per ml) was added, or a piece of etched glass (first treated by complete degradation time was monitored by the full disappearance of dark color.
pirahana solution for 1 h, then oxygen plasma treated for 1 min, followed by 0.5 ml media was changed every two days. Each experiment was repeated twice.
polylysine (1 mg per ml) coating for 4 h) or a polymer scaffold (Polycaprolactone Results are summarized in Supplementary Fig. 10m.
nanofiber scaffold23, 1 mg) was inserted. The solutions turned brown immediately,
and then they were continued to incubate under 37 °C for 1 h. To remove the
MnO2 nanosheet with absorbed proteins, the solution was centrifuged for 3 times Fabrication and characterization of MnO2 hybrid nanoscaffold. To fabricate the
at 8801×g for 10 min, and precipitates were removed each time until there are no MnO2 hybrid nanoscaffolds, 10 ml of MnO2 nanosheet solution at a specific
visible precipitates anymore. The solution should be transparent at this moment. concentration was filtered through a cellulose filter paper (pore size = 20 nm)
0.1 ml supernatant solution was transferred into a 96-well plate, and BCA under the vacuum condition. Then the filter paper was taken out and cut into sizes
(bicinchoninic acid assay, Thermo Fisher, A53226) was used to quantify the per- and shapes of choice. To transfer into a transparent glass substrate, cleaned glass
centage of protein absorbed on nanosheets by subtracting the total amount of was first treated with oxygen plasma, then the MnO2 nanosheet deposited on filter
proteins remaining in the control group to the protein remained in the experi- paper was wetted with de-ionized water and pressed against the glass. A 2.0 kg per
mental groups. The assay was conducted strictly following the protocols from cm2 pressure was placed on top of the filter paper for 8–12 h, and glass attached
Thermo Fisher and absorption at 570 nm was used to quantify the protein amount with MnO2 nanosheet was detached from the weight. To remove the cellulose
for each group. These experiments were replicated for 3 times, and the values were attached with nanoscaffold, the substrate was incubated in acetone for 0.5 h and
normalized to the glass control. The amount of laminin absorbed on MnO2 then briefly washed in methanol for 1 h. The transparency of nanoscaffold can be
nanosheet was calculated by subtracting the laminin concentration after MnO2 easily tuned by using different concentrations of MnO2 nanosheet solution. The 3
nanosheet absorption from the original concentration. The percentage of laminin concentrations used in Supplementary Fig. 3 are 50 μg per ml, 100 μg per ml and
absorption was calculated by dividing the amount of laminin absorbed by the 200 μg per ml. For cellular studies, the concentration of 200 μg per ml MnO2
original laminin concentration. BCA protein assay was repeated 3 times experi- nanosheet solution was used throughout the study. Graphene oxide assembled
mentally to get error bars shown in Fig. 1. Data are mean ± s.d., n = 3, **P < 0.01 by scaffold was fabricated using an identical protocol with a graphene oxide aqueous
one-way ANOVA with Tukey post-hoc test. solution of 200 μg per ml. FESEM (Field Emission Scanning Electron Microscopy,
Zeiss with Oxford EDS) was used to characterize the nanoscaffold.
To form MnO2 laminin hybrid nanoscaffold, 400 μL of MnO2 nanosheet
In vitro biodegradation of MnO2 hybrid nanoscaffold. We first studied the aqueous solution (2 mg per ml) was quickly added to 100 μL of laminin solution
in vitro degradation of MnO2 nanosheet in physiological conditions, different PBS (1.0 mg per ml, PBS, PH = 7.4). Then the laminin conjugated MnO2 nanosheet was
solutions with varying concentrations of vitamin C (10 μg per ml, 50 μg per ml, centrifuged and re-suspended in 10 ml de-ionized water (MnO2 nanosheet
100 μg per ml, 200 μg per ml, 500 μg per ml) were prepared. Then 10 μl of 3 mg per concentration was 80 μg per ml). After vacuum filtration, the cellulose filter paper
ml MnO2 nanosheet solution was added into 3 ml of vitamin C solution, and the or PCL fiber was cut into size and shape of interest for the following cell studies.
UV-Vis spectrum of the solution was recorded every two minutes. The percentage
of nanosheet remaining was normalized to the absorption (at 385 nm) of a control
group without any vitamin C added. To study the degradation of thin layered HiPSC-NSC culture. Human iPSC-NPCs were derived from human iPSCs
nanoscaffold, we drop-casted 100 μL of MnO2 nanosheet solution (1.0 g per ml) (WT126 clone 8; and WT33 clone 1)57. iPSC-NPCs were expanded in a pro-
into the wells of 24 well plate treated with oxygen plasma. After vacuum drying for liferation media containing DMEM/F12 with Glutamax (Invitrogen), B27-
3 h, a homogeneous, yellow and transparent film formed. Then the wells were supplement (Invitrogen), N2 (Stem Cells), and 20 ng per mL FGF2 (Invitrogen).
coated with laminin (Thermo Fisher, Catlog No.: 23017015) and seeded with Tissue culture vessels were treated with Matrigel (Corning) 1:200 dilution with
human neural stem cells at a cell density of 80k per well. The cells were cultured in DMEM (Invitrogen) at 37 °C for 1 h. To initiate the neuronal differentiation

NATURE COMMUNICATIONS | (2018)9:3147 | DOI: 10.1038/s41467-018-05599-2 | www.nature.com/naturecommunications 11

ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-05599-2

process, bFGF was removed. Fresh media was exchanged every other day. iPSC- dependent calcium intensity peaks can be found in Fig. 4, which was automatically
NSCs with passage 8–11 were used in all our transplantation and in vitro studies. obtained from the Nikon ND2 software.

Dye loading on MnO2 nanoscaffold and MRI studies. To study drug loading and
Differentiation of iPSC-NSC on MnO2 nanoscaffold. The viabilities of iPSC- release on MnO2 nanoscaffold, rhodamine B was used a model drug. Briefly, 0.3 mg
NSCs and human neural progenitor cells (hNPC, Supplementary Methods) cultured rhodamine B (Alfa Aesar, Catalog Number: A13572) was added into 3.0 ml of
on MnO2 nanoscaffold were measured by presto blue cell viability assay (Thermo MnO2 nanosheet solution. After incubation at room temperature for 12 h, 5.0 ml
Fisher, Catalog No.: A13261, 10% volume ratio as compared to cell media). Into 24- PBS (PH = 7.4) was gradually added into the solution and RhB loaded MnO2
well plates, laminin (10 μg/ml) was first coated onto the glass (control), graphene nanosheet was centrifuged down at 3431 g for 5 min and extensively washed with
oxide assembled scaffold (positive control) and MnO2 nanoscaffold at a con- PBS for 6 times to remove the residual RhB solution. Then the RhB-loaded MnO2
centration of 20 μg per ml (media volume: 1.0 ml per well), for 4 h. Then the iPSC- nanosheet was re-suspended in 10 ml solution and re-assembled with laminin
NSC was seeded into each well at a cell density of 20 k in growth media (bFGF using the identical conditions for fabricating MnO2 laminin hybrid nanoscaffolds.
added, 20 ng per ml). After the cells were cultured for 48 h, cell viabilities cultured To monitor the dye hold-up, RhB-nanoscaffold was incubated with PBS for 12 h,
on different substrates were quantified using fluorescence (excitation at 570 nm and then the fluorescence of the supernatant was detected by a fluorescence spectra
emission at 590 nm) presto blue assay and normalized to the control group (glass). (Varian Cary Eclipse). The dye loading was confirmed by degrading the RhB-
For the neurite length analysis, neurites on each substrate were first automatically nanoscaffold using 1.0 mg/ml ascorbic acid PBS solution. Instant appearance of
traced and the lengths were automatically measured by NeuronJ in ImageJ software. pink color from RhB proves the loading of RhB inside nanoscaffold. RhB-
The values are all averaged from 9–12 measurements in the representative immu- nanoscaffold before and after degradation was also spotted in a glass slide in a
nostaining images58. Here is a summary of the average neurite lengths with standard close-proximity and then imaged in the fluorescent microscope. To test the cor-
deviation obtained from software: Glass control, 5.2 ± 2.3 μm; MnO2 nanoscaffold, relation between MRI signals and RhB released, different amount of RhB-
59.3 ± 19.8 μm; MnO2 laminin nanoscaffold, 84.5 ± 26.5 μm. nanoscaffold [5, 2.5, 1, 0.5, 0.1 mg (from left to right in Fig. 5c)] were degraded
For the differentiation of iPSC-NSC on glass substrates, MnO2 nanoscaffold, with ascorbic acid (1.0 mg per ml) to form a homogeneous solution. Then the same
graphene oxide assembled scaffold (GO nanoscaffold) and glass were first sterilized solution in 96-well plates was used for MRI (Aspect’s M2TM Compact High-
in the ultraviolet (UV) lamp for 5 min and then coated with laminin solution (10 Performance MRI, 1T) measurement and fluorescence measurement under Nikon
μg per ml) for 4 h. The substrates were placed in 24-well plates, and iPSC-NSCs fluorescent microscope.
were seeded into the wells at a cell density of 60 k cells per well. The cells To study the day-dependent drug (RhB) release from our MnO2-laminin hybrid
proliferated for 24 h, and the media was changed to differentiation media without nanoscaffold, PBS with 10 μg per ml vitamin C was used to incubate the RhB
bFGF. To observe the stem cell proliferation and attachment onto the substrate, the loaded nanoscaffold, and was changed regularly every day. Fluorescence images
cells were imaged in the optical microscope (Nikon Eclipse Ti-E microscope). After (Nikon fluorescent microscope) were taken at Day1, Day2, and Day7, and the
6-days’ differentiation, the cells were fixed and immunostained with nuclei intensities from 3 different experiments were used to quantify the amount of RhB
(Hoechst, Thermo Fisher, catalog number: 33346, 1:100 dilution, 0.2 mM) and released. As a control, PCL polymer was dissolved with RhB and then formed a
neuronal marker (TuJ1, Cell Signaling, catalog number: 4466, 1:500 dilution). To scaffold by drying at room temperature. Then the dye release was measured at the
quantify the neuronal markers (TuJ1) and astrocyte markers (GFAP), qRT-PCR same time points as RhB loaded nanoscaffolds. The percentage of dye release was
was conducted by using GAPDH mRNA as a control (Supplementary Table 2). all normalized to the fluorescence intensity obtained at Day1.
To load neurogenic drugs into MnO2 laminin hybrid nanoscaffold, DAPT (N-
[(3,5-Difluorophenyl)acetyl]-L-alanyl-2-phenyl]glycine-1,1-dimethylethyl ester,
Fabrication of MnO2 laminin hybrid nanoscaffold. MnO2 laminin hybrid Tocris, Catalog Number: 2634) was first dissolved in a PBS: DMF = 9:1 solution
nanoscaffold can be facilely fabricated by adding 10 μL of MnO2 nanosheet aqu- (dimethylformamide, Sigma-Aldrich) at a concentration of 0.1 mg per ml. Then 1.0
eous solution (3 mg per ml) into 100 μL laminin solution (1 mg per ml), and the ml of DAPT solution was quickly mixed with 100 μL of 3 mg per ml MnO2 nanosheet
MnO2 nanosheet will be assembled within 5 s. To fabricate larger scale MnO2 aqueous solution. After incubating for 12 h, the solution was centrifuged down and
laminin hybrid nanoscaffold, 100 μL of MnO2 nanosheet aqueous solution (3 mg washed with de-ionized water for 3 times. The successful loading of DAPT onto
per ml) was added into 500 μL laminin solution (1 mg per ml) and then vacuum MnO2 nanosheet was confirmed by MALDI-TOF (Bruker, Ultraflex) based on the Na
filtered on a cellulose paper as described above. The structure of MnO2 laminin +-DAPT peak at 455 (molecular weight to charge ratio). Briefly, 50 μL MnO
2
hybrid nanoscaffold was then analyzed in FESEM. To fabricate cell encapsulated nanosheet loaded with DAPT solution was mixed with 50 μL gold nanoparticle
MnO2 laminin-nanoscaffold, 1 million iPSC-NSCs were centrifuged down and re- solution (Ted Pella, 10 nm, 5.7 × 10 particles per ml). Then 1 μL of the mixed
12
dispersed in 25 μL laminin PBS solution. Different amount (0, 0.3, 1.5, 3, 15, and solution was drop-cast onto ITO glass and baked at 50 °C for 1 min to fully evaporate
30 μL) of MnO2 nanosheet solution (3 mg per ml) was injected into the cell laminin water. The DAPT solution was drop-cast on the same ITO glass as a reference. ITO
solution, and a iPSC-NSC encapsulated pellet was spontaneously formed after one glass was placed into the MALDI-TOF and exposed with a laser for the analysis.
hour. To investigate the interaction between MnO2 and encapsulated iPSC-NSCs, To measure the DAPT loading and releasing profile on DAPT MnO2
the medium was removed and the neurons were fixed in Formalin solution (Sigma- nanoscaffold, 3.0 mg MnO2 nanosheets were first loaded with DAPT using the
Aldrich) followed by two PBS washes. The biological samples (prepared under 15 previous protocol and then assembled into MnO2 laminin hybrid nanoscaffolds.
μL MnO2 condition) were then dehydrated to eliminate water through a series of Using UV-Vis spectroscopy, we first identified the characteristic absorption peak of
ethanol dehydration process by replacing PBS with 50% ethanol/water, 70% DAPT (20 μM in water with 1% DMF) at 264 nm. Then the drug loading amount
ethanol/water, 85% ethanol/water, 95% ethanol/water, and absolute ethanol twice was determined by the full disappearance of the 264 nm peak after incubated with
for 10 min each in succession. The biological samples were then stored in absolute nanosheets (2.0 ml PBS, 1.5 mg per ml). This corresponds to a loading efficiency of
ethanol before transferring to critical point dryer to eliminate traces of ethanol. 110 μg per 3.0 mg nanosheets and a molar ratio between DAPT and manganese
Then 20 nm of gold was sputter coated onto the surface of biological samples after atom = 1:134 (Supplementary Fig. 13). After that, to quantify the release of DAPT,
drying. FESEM was then used for micrograph acquisition. degradation and DAPT release from the nanoscaffold was initiated by incubating it
For the differentiation of iPSC-NSC on substrates, glass, MnO2 nanoscaffold and in a solution containing 10 μg per ml ascorbic acid. As DAPT form strong binding
MnO2 laminin hybrid nanoscaffold were first sterilized in the UV lamp for 5 min and complex with MnO2 nanosheets (Binding Energy = −18.43 kcal per mol), we
then coated with laminin solution (10 μg per ml) for 4 h. The substrates were placed monitored DAPT release through quantifying the amount of manganese amount at
in 24-well plates, and iPSC-NSCs were seeded into the wells at a cell density of 60,000 different time points, and estimated the drug release based on the constant molar
per well. After 6 days’ differentiation, the cells were fixed, and immunostaining on ratio between DAPT and manganese (1:134) in the MnO2-DAPT complex. The
nuclei (DAPI) and neuronal marker (TuJ1) was conducted. Stem cell assays were average daily release was quantified through dividing the total amount of
repeated 3 times to obtain statistical information unless mentioned otherwise. Student manganese released by the length of degradation. The summarized DAPT release
t-test was used for two group analysis and ANOVA with Tukey post hoc test was used profile can be found in Supplementary Fig. 13.
for multi-group (more than 3 groups) analysis. For the long-term (1 month, 30 days)
stem cell differentiation assay, identical protocol was used with twice media change
per week. Mature neuronal marker (MAP2) was used for identifying neurons DFT calculations on small molecule and MnO2 binding. DFT calculations were
differentiated from iSPC-NSCs on MnO2 nanoscaffold. carried out using the Quantum ESPRESSO software package. For the geometry
Calcium imaging of neurons differentiated from iPSC-NSCs on MnO2 laminin optimization Perdew-Burke-Ernzerhof (PBE) functional along with D2 dispersion
hybrid nanoscaffold in 12 well-plates. iPSC-NSCs were differentiated on MnO2 corrections were used59,60. The MnO2 surface and the MnO2 bound complexes
laminin hybrid nanoscaffold using an identical protocol mentioned above for were treated with DFT + U method. This is because conventional DFT functionals
6 days, then the cells were incubated with 1 ml of Fura-2 AM (Life Technologies, are unable to describe the strong correlation effect among the partially filled d
Catalog Number: F1201, 1:200 dilution, 5 μg per ml) in cell media for 1 h. states in Mn61. The Hubbard parameter ‘U’, is introduced for the Mn 3d electrons
Afterwards, cell media was changed to PBS. Under the movie mode of a to describe the on-site Coulomb interaction, as given in the well-known GGA + U
fluorescence microscope, concentrated KCl solution in PBS (50 mM, 0.1 ml) was method62. The values of U = 4 eV and J = 0 eV for MnO2 were adopted63. Spin-
added to the cells, and the movie was taken for 10 min with 60 frames per seconds. polarized calculations were performed since bulk MnO2 has an antiferromagnetic
The movies were pseudocolored, with red indicating strong calcium flux and green ground state. The electron cores were defined using ultrasoft pseudopotential for all
indicating weak calcium flux. An identical procedure was also applied for collecting the elements and were extracted from the Quantum ESPRESSO main website
calcium imaging of neurons differentiated from hNPCs. A summary of time (http://theossrv1.epfl.ch/Main/Pseudopotentials). For the k-point mesh, a γ-center

12 NATURE COMMUNICATIONS | (2018)9:3147 | DOI: 10.1038/s41467-018-05599-2 | www.nature.com/naturecommunications

NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-05599-2 ARTICLE

was used. The wave function cutoff of 60 Ry and kinetic energy cutoff of 240 Ry the sagittal sections. All tissue sections are in the center of the scaffold implants and
were used in all the cases studied. The Gaussian smearing was turned so that the then identified nearby the transplanted sites.
difference between the free energy and the total energy is less than 0.005 Ry per
atom. The energy convergence was set to 1 × 10−6 a.u. and the force convergence Data availability. The data that support the findings of this study are available
threshold for the ionic minimization was set at 1 × 10−4 a.u.
from the authors on reasonable request. New data related to this manuscript can
The binding energies on the MnO2 surface were calculated for a series of small
also be found on the following link: https://figshare.com/projects/
molecules (Supplementary Table 1) and the DAPT drug molecule. The size of the
A_Biodegradable_Hybrid_Inorganic_Nanoscaffold_for_Advanced_Stem_Cell_-
cell was taken equivalent to the size of the MnO2 surface that has 8 × 8 oxygen
Therapy/29040.
atoms at the periphery. The box size for the simulated system is 23 × 23 × 40 Å, and
periodic boundary conditions are used. This condition was chosen to mimic the 2D
MnO2 surface. We first performed geometry optimizations for the bound Received: 10 July 2017 Accepted: 17 July 2018
complexes, with the resulting energy referred to as Ecomplex. We then optimized the
structures of isolated MnO2 surface and the molecule of interest, obtaining their
energies EMnO2 and Emol, respectively. The binding energy is defined as
Eb ¼ Ecomplex  EMnO2  Emol . Negative Eb indicates binding while positive Eb
indicates repulsion to the surface. References
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14 NATURE COMMUNICATIONS | (2018)9:3147 | DOI: 10.1038/s41467-018-05599-2 | www.nature.com/naturecommunications