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Nanoclay Diffusion Gels www.advhealthmat.de

Self-Assembling Nanoclay Diffusion Gels for Bioactive

Osteogenic Microenvironments
Pujiang Shi, Yang-Hee Kim, Mohamed Mousa, Roxanna Ramnarine Sanchez,
Richard O. C. Oreffo, and Jonathan I. Dawson*

a conducive microenvironment or “niche”

Laponite nanoparticles have attracted attention in the tissue engineering able to host, stimulate, and direct stem
field for their protein interactions, gel-forming properties, and, more recently, cell driven tissue formation. For this func-
osteogenic bioactivity. Despite growing interest in the osteogenic properties tion, the ability to present, in space and
over time, the biological, chemical, and
of Laponite, the application of Laponite colloidal gels to host the osteogenic
mechanical stimuli directing stem cell
differentiation of responsive stem cell populations remains unexplored. Here, behavior is a vital design constraint.[2]
the potential to harness the gel-forming properties of Laponite to generate Second, the scaffold serves as a sur-
injectable bioactive microenvironments for osteogenesis is demonstrated. A gical implant material that facilitates the
diffusion/dialysis gelation method allows the rapid formation of stable trans- delivery of stem cells or biological mol-
parent gels from injectable, thixotropic Laponite suspensions in physiological ecules to the site of damage and provides
a template for new tissue formation. For
fluids. Upon contact with buffered saline or blood serum, nanoporous gel net- this function, parameters such as han-
works exhibiting, respectively, fivefold and tenfold increases in gel stiffness dling, mode of delivery, bulk mechan-
are formed due to the reorganization of nanoparticle interactions. Laponite ical properties, and degradation and/or
diffusion gels are explored as osteogenic microenvironments for skeletal diffusion profiles come to the fore. The
stem cell containing populations. Laponite films support cell adhesion, optimization and marrying of these com-
plex design parameters remains a critical
proliferation, and differentiation of human bone marrow stromal cells in 2D.
unmet challenge.[3]
Laponite gel encapsulation significantly enhances osteogenic protein expres- Recent studies investigating clay nano-
sion compared with 3D pellet culture controls. In both 2D and 3D conditions, particles for stem cell-based regenera-
cell associated mineralization is strongly enhanced. This study demonstrates tive strategies suggest their potential to
that Laponite diffusion gels offer considerable potential as biologically active provide new opportunities for advanced
and clinically relevant bone tissue engineering scaffolds. functional scaffold design.[4] The well-
established utility of clay nanoparticles
to interact with biological molecules for
controlled or localized delivery,[5] and
1. Introduction their ability to interact with polymers to enhance mechanical
properties as seen in the development of polymer–clay nano-
In order to realize the potential of tissue engineering and composites,[6] are both of core relevance to the scaffold design
regenerative medicine, the development of advanced functional parameters outlined above. More recently various groups have
materials for stem cell therapy remains a strategic research pri- explored cellular interactions with clay nanoparticles either
ority.[1] The biomaterial scaffold serves a critical role in bridging alone or in combination with polymeric matrices.[7–10] These
the gap between the biological context of stem cell driven tissue pioneering studies have indicated new and unforeseen utility
formation and the clinical context of adult health care.[1] The for certain clays as bioactive additives able to enhance cellular
scaffold thus fulfills a dual function. It serves, first, to sustain functions including adhesion, proliferation, and differentiation,
most notably for osteogenesis.[4]
Dr. P. Shi, Dr. Y.-H. Kim, M. Mousa, R. R. Sanchez, Prof. R. O. C. Oreffo, As well as their utility as fillers or cross-linkers, certain
Dr. J. I. Dawson clays form aqueous colloidal gels in the absence of a polymeric
Bone and Joint Research Group
Centre for Human Development
phase. Of particular note in this regard is the synthetic hec-
Stem Cells and Regeneration torite clay, Laponite, a magnesium silicate (Na0.7+[(Si8Mg5.5Li0.3)
Institute of Developmental Sciences O20(OH)4]−0.7) composed of rigid disk shaped crystals of 1 nm
University of Southampton thickness and ≈30 nm diameter possessing a permanent nega-
Southampton SO16 6YD, UK tive surface charge and a weak, pH dependent rim charge. A
E-mail: jid@soton.ac.uk
substantial body of literature has been generated on Laponite as
The ORCID identification number(s) for the author(s) of this article
can be found under https://doi.org/10.1002/adhm.201800331.
a model system in soft matter physics due to the rich array of
physical states Laponite displays depending on solid concentra-
DOI: 10.1002/adhm.201800331 tion, ionic strength, and sample age.[11,12] We have previously

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explored the ability to harness Laponite colloidal gels for drug-
delivery and demonstrated the ability to localize and sustain
the activity of bioactive molecules in space and over time for
improved safety and efficacy.[13] For example, Laponite gels
were able to localize the osteoinductive molecule bone mor-
phogenetic protein 2 to achieve ectopic bone induction at the
lowest dose recorded in the literature to date.[14]
Despite intriguing data suggesting the inherent osteogenic
bioactivity of clays, no studies have yet successfully applied
clay colloidal gels as host environments for the osteogenic dif-
ferentiation of responsive stem cell populations. Here we apply
a diffusion reaction method to induce physiologically respon-
sive gelation and demonstrate the ability to harness both the
bioactivity of Laponite and its colloidal properties to develop an
injectable and osteogenic cell delivery system.
2. Results and Discussion
Given current interest in the osteogenic properties of Laponite
nanoparticles, we set out to explore the potential of Laponite col-
loidal gels to generate injectable osteogenic microenvironments
for skeletal stem cells. To this end, we have applied a dialysis/
diffusion-based approach to gelling predispersed Laponite
(XLG) where an irreversible gel is formed from a thixotropic
Laponite suspension via diffusion of ions and/or proteins from
an outer reservoir. This approach permits the direct formation
of stable gels in biologically relevant fluid environments.
2.1. Laponite Gelation in Physiological Fluids
Laponite dispersions (optimized to 2.8 wt%, to permit extru-
sion through a 25G needle) displayed instantaneous gelation
upon contact with physiological saline (phosphate buffered
saline, PBS) or blood serum (blood plasma without the pro-
teins involved in clot formation; Figure 1a,b). Importantly,
and in contrast to standard mixing approaches which result
in flocculation and phase separation, the diffusion method of
gelation preserved the low optical density (absorbance) of the
Laponite dispersions, even at very high (>1.5 m) ionic strengths
(Figure 1c; Figure S1, Supporting Information), to generate
free-floating gel phases that remained stable in cell culture
media for over 3 weeks.
Physical characterization of Laponite gels formed in saline
and blood serum was conducted using scanning electron
microscope (SEM) and rheological analysis. Laponite samples
were prepared via critical point drying and imaged using SEM
across the fracture plane after gentle crushing (Figure 1d).
Distinct morphologies were observed between Laponite in its
native state and gels formed in saline and serum, respectively
(Figure 1e). Critical-point dried Laponite suspensions pre-
sented as nanoporous networks of loosely associated string-
like aggregations of 20–50 nm thickness and 100–200 nm in
length. Laponite gelled in saline was more compact and less
porous at the 10–100 nm scale but displayed occasional larger
disconnected pores at the µm scale. In blood serum, the gel
morphology changed dramatically with the emergence of large

Figure 1.  Laponite gel formation and morphology. a) Hydrous suspensions of Laponite (28 mg mL−1) form thixotropic gels that allow passage through a
low gauge needle. Laponite suspensions stiffen into stable gels when applied as a discrete phase to physiological fluids such as saline [(a),b) phosphate
buffered saline, PBS] and blood serum [(b) fetal calf serum, FCS, scale bar = 1 mm] while preserving their low optical density c). d) Critically point dried
Laponite gels thus formed were crushed and assessed across a fracture plain [(d) scale bar = 500 µm] in their native state and following 24 h incubation
in saline or blood serum to reveal changes in microstructure between conditions [e) top panel scale bar = 2 µm, bottom panel scale bar = 500 nm].

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Figure 2.  Rheometric analysis of Laponite suspensions following exogenous addition of saline and blood serum. a) Evolution of Laponite (Cw > 2.8%)
viscoelastic properties were assessed in situ following addition of PBS or FCS. b) Addition of saline (light blue) and serum (orange) resulted in a sharp
fivefold and tenfold increase, respectively, in storage modulus compared with native state (dark blue). c) Following both saline and serum incubation
(1 h), a stress strain amplitude sweep reveals a sharp fall in G′ (solid circles) at the limit of the linear viscoelastic region and a pronounced G″ (open
circles) maximum characteristic of brittle fracturing behavior. d) The elastic stress (τ) was plotted against shear strain (γ) and yield stress (τy) and yield
strain (γy) for each treatment calculated as the point of inflection (>5%) from the linear dependence of τ on γ. e) Despite a significant (P = 0.0002) two-
fold increase in storage modulus following FCS incubation compared with PBS incubation, f) the lower yield strain of FCS incubated samples resulted
in similar yield stress values for both treatments. Plotted curves represent mean values. Error bars = SD, N = 3, **, ***, and **** indicate P < 0.01,
P < 0.001, and P < 0.0001, respectively (one way ANOVA with Tukey’s multiple comparisons test).

fibrous aggregations forming an interconnected network of
pores ranging from 10–2000 nm in diameter.
Rheological analysis of the evolution in the elastic (G′) and
viscous (G″) moduli of Laponite suspensions was undertaken
in situ following exogenous addition of saline or blood serum
(Figure 2). At 2.8 wt%, after 24 h following autoclaving, Laponite
presented as a viscoelastic solid (G′  > G″) at low applied strain
values (i.e., in the linear viscoelastic regime). Addition of saline
or serum induced a sharp fivefold or tenfold increase, respec-
tively, in both elastic and viscous moduli indicating an increase
in gel stiffness (Figure 2b). Following both saline and serum
incubation, a stress–strain amplitude sweep revealed a sharp
fall in G′ at the limit of the linear viscoelastic region and a
pronounced G″ maximum characteristic of brittle fracturing
(Figure 2c). The elastic stress (τ) was plotted against shear strain
(γ) and yield stress (τy) and yield strain (γy) for each treatment
calculated as the point of inflection (>5%) from the linear
dependence of τ on γ (Figure 2d). Despite a significant (P = 0.002)
twofold increase in storage modulus following serum incubation
compared with saline (Figure 2e), the lower yield strain of serum
incubated samples resulted in similar yield stress values for both
treatments (Figure 2f) indicating that as stiffness increased in
the presence of serum the gel became more brittle.
The phase diagram of Laponite is complex and remains the
subject of considerable interest as a model system.[11] Such com-
plexity relates to the charge anisotropy of the particles which
are subject to a range of mutual repulsive (face-to-face, edge–
edge) and attractive (face to edge) electrostatic interactions as
well as short-range van der Waals attraction. These interactions
and the resultant structure and properties of the colloid are very
sensitive to changes in solid concentration, ionic concentration,
and valence, pH, and the presence of organic compounds.[15]
At 2–3 wt% in deionized water and at its native pH = 10,
repulsive interactions arising from the negative surface charge
of Laponite disks dominate over attractive interactions resulting
in an arrested “gel-like” state more accurately defined as a
repulsive (or Wigner) glass (Figure 3).[16] Physiological solu-
tions have an ionic concentration of >150 × 10−3 m (see Table S1
in the Supporting Information for comparison of relevant
parameters between systems). This is well above the threshold
(≈20 × 10−3 m) at which Laponite suspensions characteristically
undergo flocculation driven phase separation due to compres-
sion of the electric double layer and an accumulation of attrac-
tive interactions between particles. Furthermore, at pH 7.4 in
PBS and serum, increased protonation of OH groups at the
particle edges will increase the positive rim charge (point of

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Figure 3.  Suggested mechanism for the formation of Laponite diffusion gels in physiological fluids. In deionized water at its native pH 10, mutual
face to face (F–F) repulsion between Laponite disks driven by a maximally expanded electric double layer (EDL) dominate over attractive interactions.
At Cw > 2%, crowding effects yield an arrested state termed a repulsive glass. Upon application to physiological fluids, a diffusion gradient of solutes
forms across the arrested Laponite phase causing local rotational and translational movement of particles. In saline, the diffusion of monovalent
cations results in compression of the EDL and reduced F–F repulsion. Concurrent reduction in pH increases the positive rim charge and drives the
formation of edge to face (E–F) attractive bonds. In serum, diffusion of divalent ions further reduces the EDL and increases the importance of van der
Waals F–F attraction. Diffusion of proteins results in the formation of clay–protein–clay bridges. The progressive strengthening of a bonded network
of particles across the diffusion gradient prevents subsequent rearrangement into larger aggregates and the resultant phase separation normally
observed in mixed preparations.

zero charge ≈ pH 11)[17] and strengthen edge-face attraction.
Given the dominance of attractive interactions in the system,
it is notable that the diffusion method applied here allows for
reorganization of the native glassy state into a stiff gel that
remains stable against macroscopic flocculation. Though not
our purpose to fully account for a mechanism, a diffusion gra-
dient of ions or other solutes across the interface of the arrested
Laponite phase could be envisaged. In this scenario, the electro-
static bonds formed upon initial reorientation of particles at the
gelation point are gradually strengthened as the concentration
of solutes increases, resulting in an interlocked network of par-
ticles stable against further translational movement into larger
aggregates (Figure 3).
Recent work by Bandyopadhyay and co-workers applying
cryo-SEM and rheology to characterize smectite gels report the
emergence of highly disordered open network structures, very
similar to those observed in the current study, formed by clay
aggregation under certain high salt and low pH conditions.[18–20]
For example, mixing of a low concentration of sulfuric acid to
predispersed Laponite in the absence of added salt was suffi-
cient to induce cluster formation driven by edge-face attraction
leading to volume spanning networks of aggregates.[20] In the
current study, a relatively low pH is combined with a high salt
concentration. In the case of saline, this appears to result in a
≈100 nm scale network of aggregations expected by a combi-
nation of a strong rim charge and a compressed double layer
(Figure 3). In the case of serum, additional ionic solutes such
as divalent cations would be expected to cause further reduction
in the range of the electric double layer and a corresponding
tendency toward van der Waals mediated face–face attraction.[21]
The continued presence of a strong rim charge would favor an
“overlapping coin” arrangement, which may account for the
emergence of fine elongated network branches apparent in the
SEM images.[18,21] Additionally, proteins abundant in serum
will readily adsorb to clay which is likely to further restrict
the kinetic freedom of the particles by the formation of clay–
protein–clay bridges and account for the increasing network
stiffness (Figure 3). Further studies are warranted to test these
and other possible conceptualizations.
2.2. Laponite Gels Generate Bioactive Osteogenic Cell
Environments
Having established the promising ability to generate stable
Laponite colloidal gels via simple contact with physiological
fluids, we explored their ability to host the osteogenic differ-
entiation of human skeletal stem cells. Initial studies assessed
cell viability, proliferation, and morphology of primary human
bone marrow stromal cells (HBMSCs) cultured in monolayer
upon dried substrates of varying solid concentration and prepa-
rations (Figure S2, Supporting Information). In all treatments,

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dried Laponite substrates (2.5 mg mL−1) com-

pared with the glass control surface (Figure 4;
Figure S3, Supporting Information). Upon
hydrated Laponite gels, cell spreading was
significantly reduced (P  < 0.0001), presum-
ably as a consequence of the softer substrate,
but displayed clear evidence of adhesion
through the organization of stress fibers and
the projection of numerous filopodia forming
associations with the gel surface (Figure 4).
This HBMSC morphology is consistent with
the profile of a cell-adhesive substrate of stiff-
ness in the range of 5–10 kPa observed by
Rowlands et al.[25] It is notable that human
bone marrow cell adhesion to clay colloidal
gels and film coatings is achieved without
prior addition of adhesion proteins or motifs.
Various possible mechanisms for clay
mediated cell adhesion have been suggested.
Clay adsorption of cell adhesive proteins such
as fibronectin or vitronectin from serum is
frequently cited and, indeed is, likely to play a
role.[22,26,27] Clay mediated enhancement has
also been observed in serum-free conditions;
however,[9] it is also possible that clay nano-
particles themselves may act as focal adhe-
sion sites through the provision of reactive
functional groups (e.g., >Si–OH2+) for cell
attachment.[22,26]
Osteogenic differentiation of monolayer
HBMSCs was assessed upon dried films
under osteogenic induction to further con-
firm the cytocompatibility of high concen-
Figure 4.  Cell adhesion and spreading on Laponite. a) Cytoskeletal (phalloidin) staining and
b) SEM of cells seeded upon glass versus dried and hydrated clay gel films. No difference in tration Laponite substrates (Figure 5). After
morphology is apparent between cells seeded on glass surfaces and cells seeded on surfaces 1 and 3 weeks of culture, broadly equiva-
coated with air-dried Laponite films (2.5 mg mL−1). Cell spreading was reduced on hydrated lent mRNA expression profiles (Collagen
Laponite gels, but cytoskeletal organization and focal adhesion formation were apparently con- I, Runx2, and Osteocalcin) were observed
sistent with a soft, cell-adhesive surface profile. Scale bars correspond to 500 µm (a, top), on Laponite (2.5 mg mL−1) compared with
100 µm (a, bottom), and 10 µm (b). the glass control across 5 separate patient
samples, though higher Collagen I expres-
cell viability and proliferation over 14 d on clay gels were com- sion (P  = 0.024, two-way ANOVA) was detectable at week 3
parable to the tissue culture plastic positive control up to the on Laponite due, in part, to a slight but nonsignificant (P  =
upper limit tested of 2.5 mg mL−1 though with some fluctua- 0.1665) drop in expression between week 1 and 3 on control
tion at earlier time points under certain preparation conditions. surfaces. Observed variation across the five patient samples
Previous studies involving direct dispersion of Laponite into assayed is consistent with the literature.[28] Protein expression
cell culture media observed a decrease in metabolic activity of these representative markers was assessed at the interface
at 0.1 mg mL−1.[8,22] This difference is likely due to the mode of Laponite and tissue culture plastic substrates by immunocy-
of presentation. In the high salt concentrations of cell culture tochemistry. Consistent with stable mRNA expression over the
media, even low concentration dispersions will typically gen- time course, a uniform cumulative increase of each osteogenic
erate microsized aggregations (flocs) with a tendency to accu- marker was apparent by week 3 with no obvious qualitative
mulate around cells, interfering with membrane channels, difference in staining intensity apparent between substrates
cytoskeletal organization, and cellular metabolism.[23] The gen- (Figure 5a). These results provide further evidence for the basic
eration of stable gels or films serves to minimize this inhibitory cytocompatibility of Laponite, even at relatively high concentra-
effect and, as illustrated below, allows for functional cell encap- tions, when presented as a stable gel or film.
sulation in gel phases of up to 28 mg mL−1. Gaharwar et al. applied low concentrations (up to
Laponite has been found to impart cell adhesive properties to 0.1 mg mL−1) of Laponite as a dispersed phase to cell cul-
otherwise nonadhesive polymeric hydrogels.[9,24] In the current ture media and observed a dose dependent upregulation of
study, morphological analysis of HBMSCs via cytoskeletal (phal- osteocalcin, osteopontin, and runx2 protein expression in
loidin) staining and SEM revealed equivalent cell spreading on HBMSCs including in the absence of standard osteogenic

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Figure 5.  Osteogenic differentiation on 2D Laponite films. Human bone marrow stromal cells seeded onto surfaces coated with air-dried Laponite
films (2.5 mg mL−1) displayed equivalent gene expression profiles to controls, though with higher Type I Collagen expression at week 3. Each data point
represents an individual patient sample (n = 5). a) Analysis of protein expression by immunocytochemistry across the interface of Laponite coated
and control surfaces revealed an increase in protein between week 1 and week 3 with no apparent difference between surfaces. Scale bars = 200 µm.
b) Alizarin red staining of calcium revealed strong staining only on Laponite coated surfaces in basal conditions after 3 weeks and in osteogenic condi-
tions after 1 week. Scale bars = 500 µm. c) SEM analysis also revealed strong mineral deposition on Laponite surfaces in contrast to control surfaces
under both basal and osteogenic conditions. The corresponding presence of calcium and phosphorus peaks by energy dispersive spectroscopy (EDS)
was apparent by week 1 in osteogenic conditions and by week 3 in basal conditions. Scale bars = 10 µm, curves representative of N = 3 samples. Error
bars = SD, N = 3, **, ***, and **** indicate P < 0.01, P < 0.001, and P < 0.0001, respectively (one way ANOVA with Tukey’s multiple comparisons test).

supplements (ascorbate-2-phosphate, dexamethasone, and
β-glycerophosphate).[8] The lack of such an upregulation upon
dried Laponite films again seems likely to relate to the mode of
presentation. For example, presentation as a stable film is likely
to minimize the amount of Laponite available for cytoplasmic
uptake, an event which appeared to precede differentiation
when applied as a dispersed phase. Consistent with this, the
influence of Laponite inclusion on osteogenic gene expression
in polymer composites (also limiting of cell uptake) remains
less well evidenced and difficult to identify against the back-
ground of improved cell-adhesion and growth on an otherwise
suboptimal gel or scaffold.[29] Where enhanced osteogenic gene
or protein expression has been seen over standard tissue cul-
ture controls, the effect of polymer embedded Laponite is more
modest and transient, again suggesting the necessity of cellular
uptake.[30]
Strong localized enhancement of calcified deposition was
however observed on Laponite substrates. Alizarin red S

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staining (ARS), though often presented as evidence of clay
mediated osteogenesis, should be interpreted cautiously given
that ARS will complex with free calcium exchanged upon clay
surfaces independently of calcium phosphate (CaP) deposi-
tion by cells.[31] Nevertheless, strong cell-specific staining of
calcium was apparent against this background from week 3
in basal conditions and from week 1 under osteogenic condi-
tions (Figure 5b; Figure S4, Supporting Information). Most
notably, calcium staining was strongly enhanced on Laponite
coated surfaces and remained minimal on proximal control
surfaces even after three weeks in osteogenic conditions. This
observed interface effect suggests localization of calcium ions
on Laponite surfaces and resultant depletion on control sur-
faces (Figure S4, Supporting Information). Consistent with
this, SEM and energy dispersive X-ray (EDX) analysis revealed
calcium phosphate deposition from week 1 and aggregate for-
mation (arrows) by week 3 in basal conditions on Laponite
coated surfaces. An even stronger response, again only on
Laponite coated surfaces, was observed in osteogenic condi-
tions with corresponding calcium and phosphate peaks visible
by EDX. Importantly, no calcium phosphate peaks were evident
on Laponite substrates in the absence of cells after three weeks
in osteogenic media indicating Laponite enhanced mineraliza-
tion to be cell mediated.
Strong and early enhancement of CaP mineralization
is a common feature of osteogenesis in association with
clays[8,10,32] suggesting the possibility of a direct influence
on CaP nucleation, growth, and/or deposition. Evidence for
inherent Laponite “bioactivity” in terms of its ability to initiate
biomineralization in supersaturated calcium phosphate solu-
tions has also been presented.[33] While a mechanism remains
to be defined, the affinity of silica for calcium ions together
with the heterogeneous charge structures of clay particles sug-
gests their possible function as energetically favorable envi-
ronments for cell mediated calcium phosphate deposition.[34]
It is notable in this regard that, in the current study, strongly
localized CaP deposition in cells growing on clay surfaces
was apparent despite limited evidence of Laponite mediated
enhancement of cell differentiation (Figure 5a) suggesting a
bioactive role downstream of differentiation events. Further
confirmation of specific mineralizing activity was provided
through short term cultures on Laponite dried films of two
osteosarcoma cell lines, representing stable models of early
(MG63) and late (SaOS2) stage osteoblast differentiation
(Figure S5, Supporting Information).[35] Whereas, MG63 cells
cultured on Laponite displayed no cell specific ARS staining,
strong localized staining was apparent in SAOS cells growing
on clay surfaces, again indicating cell specific enhancement of
mineralization by Laponite.
Finally, we sought to test the potential of Laponite diffu-
sion gels as 3D osteogenic microenvironments. HBMSCs were
resuspended in Laponite dispersions (28 mg mL−1) and imme-
diately added drop-wise to FCS containing cell culture media
to induce gel formation. After 24 h, cell–Laponite composites
were transferred to osteogenic media. Histological assessment
of Laponite–cell composites across 4 patients consistently
revealed strong staining for Type I Collagen and Runx 2 from
week 1 and an increase in osteocalcin between week 1 and 3
(Figure 6). Importantly, ARS staining increased over time in
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culture and colocalized strongly with osteocalcin expression
though a degree of background staining may also, in this case,
contribute to the increase over controls (Figure 6). In all cases
staining was observed to be stronger in cells at the periphery of
the gel capsule, though in certain examples, regions of strong
Collagen I, osteocalcin, and ARS staining were also present
toward the core of the gel. Overall similar staining patterns
and intensities were also observed in the scaffold free pellet-
culture control under osteogenic induction with the exception
of Runx2 which was of higher intensity in the scaffold free con-
trol due to the higher cell density. It is notable that the stained
tissue area was apparent at a considerably smaller scale in scaf-
fold free controls despite a 20-fold higher starting cell number.
Furthermore, in contrast to Laponite–cell composites which
preserved their dimensions over the course of culture, the cell
pellets reduced significantly in size between week 1 and week 3
(Figure S6, Supporting Information). Thus, across all markers,
Laponite displayed significantly higher positive staining (inte-
grated density) compared with the standard 3D culture control
(Figure 6b). SEM analysis of critically point dried Laponite gels
(Figure 7) revealed extensive reorganization of the matrix in
comparison to day 1 and unseeded samples. By week 3, exten-
sive bone-like apatite formation was apparent at both the sur-
face and center of the construct and corresponding calcium
phosphate peaks confirmed by EDX. Taken together, this study
provides strong evidence for the ability of Laponite diffusion
gels to host the osteogenic differentiation of skeletal stem cell
containing populations.
3. Conclusion
Laponite nanoparticles have attracted interest in the tissue engi-
neering field for their bioactivity, protein interactions, and gel-
forming properties. In the current study, we have demonstrated
the possibility to effectively harness these properties using a dif-
fusion gelation method to generate injectable bioactive micro-
environments for osteogenesis. Two notable observations can
be highlighted: i) the introduction of Laponite as an arrested
phase to high ionic strength, solute rich fluids, resulted in the
rapid evolution of stiff, stable, and transparent gels via a diffu-
sion–reaction process, ii) Laponite diffusion gels thus formed
could host osteogenic matrix synthesis and strongly enhance
cell-mediated matrix mineralization. This is notable since oste-
ogenesis is achieved both at the relatively high concentrations
of Laponite necessary for colloidal gel formation, and without
the structural support of a polymeric matrix, or incorpora-
tion of additional matrix molecules or adhesion motifs. These
observations suggest Laponite diffusion gels to offer consider-
able potential as biologically active and clinically relevant bone
tissue engineering scaffolds.
4. Experimental Section
Laponite Preparation: Laponite dispersions were prepared as
described previously.[29] Briefly, Laponite XLG (kindly gifted by BYK
Additives, Widnes, UK) was dispersed in type 1+ deionized water
(18.2 MΩ, pH 7) to required concentration % weight Laponite per unit
volume. The preparations were subsequently sterilized by autoclave and
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Figure 6.  Osteogenic differentiation in 3D Laponite diffusion gels. HBMSCs were suspended in Laponite suspensions (28 mg mL−1) and added to cell
culture media to induce gelation before being cultured in osteogenic conditions. More extensive staining across all markers is apparent in Laponite in
comparison to scaffold free controls by week 3. a) Images show representative parallel sections from surface and central regions. Scale bars = 100 µm.
Insets of entire constructs are included for scale. Scale bars = 400 µm. b) Stained area was quantified across multiple sections from 4 individual
patient samples by image analysis and presented as integrated density. Error bars = SD, *, ***, and **** indicate P < 0.05, P < 0.001, and P < 0.0001,
respectively, by one way ANOVA with Tukey’s multiple comparisons test.

any water evaporated during cooling replaced. Unless stated, Laponite
suspensions were used within three days of preparation. To produce clay
gel films for cell culture, unless otherwise stated, Laponite suspensions
were added to the surface of the well or glass cover slip at the required
volume before being air dried in its native state at room temperature.
All Laponite coated surfaces were sterilized for 2 h under UV prior to
cell seeding. Diffusion gels were prepared using 28 mg mL−1 Laponite
suspensions added as a discrete phase to phosphate buffered saline
(Lonza, UK) or fetal bovine serum (Sigma, UK). This was achieved
variously via careful drop-wise addition to the surface of the gelling

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Figure 7.  Matrix reorganization and mineral deposition in 3D Laponite diffusion gels. SEM β-Glycerophosphate disodium salt hydrate, and
analysis of critically point dried samples indicated matrix remodeling and mineral deposition
across the cross-section of the sample over 3 weeks under osteogenic conditions. EDS analysis were kept in basal medium as a control. Samples
confirmed strong calcium and phosphorous peaks after 3 weeks. Curves representative of N = 3
samples. Scale bars = 10 µm.
solution to form gel beads, by direct injection into the gelling solution
(as in Figure 1a), or by layering of saline or serum solutions onto the
surface of Laponite gels within a tissue culture well plate.
Scanning Electron Microscope and Energy Dispersive X-Ray Analysis:
Seeded and unseeded Laponite samples were fixed by 4% buffered
paraformaldehyde, before dehydration in 50%, 70%, 90%, and 100%
ethanol. Laponite diffusion gels prepared for structural analysis were
incubated in serum or saline for 24 h prior to harvesting for analysis.
Laponite films were air dried, while 3D samples were processed by critical biomaterial scaffolds. Recovered and washed cell pellets following
point drying. The specimens were mounted on aluminum stubs and sputter
coated in gold palladium. They were then imaged and analyzed using a FEI
Quanta 200 operated at high vacuum with secondary electron detection.
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Rheological Analysis: All rheological
measurements were conducted on an MCR 92
rheometer (Anton Paar, UK) using a 25 mm parallel
plate geometry. 1.5 mL Laponite suspensions
(28 mg mL−1) preincubated at 37 °C in a water bath
for 1 h were loaded onto the rheometer plate preset
to 37 °C with a 2.5 mm gap. Initially measurements
of storage and loss modulus over time were taken
over 15 min at a constant oscillatory strain of 0.1%
and angular frequency of 1 rad s−1. After 2 min, a
saline or serum reservoir was applied up to the level
of the lower surface of the upper plate. Following
this initial regime, an amplitude sweep covering the
range of 0.01–100% strain was applied, again at a
constant angular frequency of 1 rad s−1.
Human Bone Marrow Stromal Cell Isolation and
Culture: HBMSCs were isolated from femoral bone
marrow obtained from haematologically normal
adults undergoing total hip replacement surgery
at Southampton General Hospital with approval
from the Southampton Local Research Ethics
Committee (LREC 194/99). The HBMSCs were
isolated by repeat perfusion of the marrows and
filtration through 100 µm filter before centrifugation
at 1200 rpm for 5 min. The HBMSSCs pellets
were resuspended in minimum essential medium
alpha modification (α-MEM) with supplements of
10% fetal bovine serum (FBS) and 100 µg mL−1
penicillin/streptomycin, and culture expanded
in monolayer at 37 °C and under humidified 5%
CO2. The media was changed every 2–3 days and
cultures expanded over 2 or 3 passage before use.
HBMSC Culture on 2D Laponite Films: For
cell adhesion studies, HBMSCs were seeded
upon tissue culture glass surfaces coated with
2.8% Laponite gels or dried Laponite films (total
concentration 2.5 mg mL−1) at 3.2 × 104 cells cm2 −1
and incubated for 24 h in basal media before fixation
and phalloidin staining or processing for SEM.
Images were taken on a Nikon Eclipse Ti and image
analysis conducted on CellProfiler software.
For cell differentiation studies, HBMSCs
were seeded upon culture glass surfaces coated
with dried Laponite films (total concentration
2.5 mg mL−1) at 3.2 × 104 cells cm−2 and incubated
for 24 h in basal media being transferred to
osteogenic medium incubation (α-MEM with
supplementary of 10% FBS, 1% antibiotics,
100 × 10−6 m ascorbate-2-phosphate, 10 × 10−3 m
10 × 10−9 m dexamethasone). Some samples
were fixed and harvested for analysis after 1 and
3 weeks in osteogenic media. Each experiment
was conducted five times using separate patient
samples. Immunocytochemistry and calcium
staining were prepared as above but slides were half-coated to allow
assessment at the interface between coated and noncoated surfaces.
HBMSC Culture in 3D Conditions: To assess Laponite diffusion gels
as 3D osteogenic microenvironments, HBMSCs were encapsulated in
Laponite and cultured alongside scaffold free pellet culture controls.
Pellet cultures were selected as a comparative 3D culture system able to
support osteogenic cell differentiation and mineralized matrix formation
while avoiding certain confounding variables associated with alternative
trypsinization were resuspended in Laponite suspensions (28 mg mL−1)
at a density of 5 × 106 cells mL−1 and added drop-wise as 10 µL volumes
(containing 5 × 104 cells) into basal media. Cell pellets formed of 1 × 106
© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.advancedsciencenews.com
cells were used as a positive control. Differences in cell number were
due to 1) the necessity of a high cell number to generate pellets of a
sufficient size for histological processing and analysis and 2) challenges
associated with encapsulating higher cell numbers in Laponite due to
increasing viscosity as a result of increasing ionicity. After 24 h, samples
were transferred to osteogenic conditions and fixed and harvested for
analysis after 1 and 3 weeks culture. Each experiment was conducted
four times with separate patient samples.
Biological Assays: Total RNA of HBMSSCs from samples at week 1 and
week 3 were extracted and purified using Rneasy Plus Mini Kit (Qiagen,
Valencia, CA). RNA concentrations were measured using NanoDrop
(NanoDrop Technologies, Wilmington, DE), and complimentary DNA
synthesis was carried out using SuperScriptVILO cDNA synthesis kit
(Life Technologies, Gibco, Cambridge Bioscience). Real-time polymerase
chain reaction was done using cDNA, Power SYBR Green PCR Master
Mix (Life Technologies, Gibco, Cambridge Bioscience), and the
optimized concentration of primers. All reactions were performed on the
7500 real-time PCR system (Applied Biosystems, Forster, CA). The genes
analyzed were osteocalcin (OCN), collagen type I, osteonectin (ON),
and Runt-related transcription factor 2 (RUNX2) for ostengenesis. The
targeted genes expression levels were normalized to Glyceraldehyde
3-phosphate dehydrogenase (GAPDH), and were then calculated using
the 2−∆∆Ct formula with reference to the respective control groups. The
cells seeded and cultured on the coverslips were fixed by 4% buffered
paraformaldehyde at week 1 and week 3, and they went through alizarin
red and immunohistological staining. The 3D samples were fixed and
embedded in paraffin blocks, sections were prepared for Alizarin red S
and immunohistological staining. The slides were deparaffinized and
rehydrated, then treated by 3% H2O2 for 5 min before 1% bovine serum
albumin (BSA) incubation. The coverslips and slides were subsequently
incubated with primary antibodies for OCN, Collagen Type I, and RUNX2
(1:100) overnight before incubation with the relevant secondary antibody
and ExtrAvidin peroxidase. AEC (3-amino-9-ethylcarbazole) was used
as a chromogenic agent. Imaging was conducted using digital virtual
microscopy system (dotSlide, Olympus, Germany) and Cell Profiler
software was used to quantify the integrated staining density.
Supporting Information
Supporting Information is available from the Wiley Online Library or
from the author.
Acknowledgements
The authors would like to thank Julia Wells of the University of
Southampton Bone and Joint group for technical support and
Dr. Nicholas Evans for critical feedback on the manuscript. Funding:
This work was supported by Jonathan Dawson’s EPSRC fellowship
(grant number EP/L010259/1). PhD funding for M.M. from BYK, Altana
is also gratefully acknowledged.
Conflict of Interest
Dr. Dawson and Prof. Oreffo are co-founders and shareholders in a
University spin out company with a license to IP indirectly related to the
current manuscript.
Keywords
bioactivity, diffusion gels, Laponite, osteogenesis, skeletal stem cells
Received: March 29, 2018
Published online:
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