Artigo Células-Tronco

Subscriber access provided by NATIONAL UNIV OF SINGAPORE
Biological and Medical Applications of Materials and Interfaces

Supramolecular Hydrogels Based on Nanoclay and Guanidine-
Rich Chitosan: Injectable and Moldable Osteoinductive Carriers
Xiao Zhang, Jiabing Fan, Chung-Sung Lee, Soyon Kim, Chen Chen, and Min Lee
ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.0c01241 • Publication Date (Web): 16 Mar 2020
Downloaded from pubs.acs.org on March 17, 2020
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,

Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.
Page 1 of 36 ACS Applied Materials & Interfaces
1
2
3
4
5
6
7
8
Supramolecular Hydrogels Based on Nanoclay and
9
10
11
12
Guanidine-Rich Chitosan: Injectable and Moldable
13
14
15
16
Osteoinductive Carriers
17
18
19
20
21 Xiao Zhang, † Jiabing Fan, † Chung-Sung Lee, † Soyon Kim, † Chen Chen† and Min Lee*，†，‡
22
23
24 † Division of Advanced Prosthodontics, University of California at Los Angeles, 10833 Le Conte
25
26 Avenue, Los Angeles, California 90095, United States
27
28
29
30
‡ Department of Bioengineering, University of California at Los Angeles, 420 Westwood Plaza,
31
32 Los Angeles, California 90095, United States
33
34
35 KEYWORDS. supramolecular hydrogels, nanoclay, chitosan, osteogenesis, demineralized bone
36
37 matrix
38
39
40
41
42
43 ABSTRACT. Supramolecular hydrogels have great potential as biomaterials for tissue engineering
44
45
46 applications or vehicles for delivering therapeutic agents. Herein, a self-healing and pro-osteogenic
47
48 hydrogel system is developed based on the self-assembly of laponite nanosheets and
49
50 guanidinylated chitosan, where laponite works as a physical crosslinker with osteoinductive
51
52
53
property to form a network structure with a cationic guanidine group on chitosan chains. The
54
55 hydrogels can be prepared with varying ratios of chitosan to laponite and display self-healing and
56
57
58
59
60 ACS Paragon Plus Environment
1
ACS Applied Materials & Interfaces Page 2 of 36
1
2
3 injectable properties due to supramolecular forces as well as osteoinductive activity due to
4
5
6 nanoclay. They enhance cell adhesion and promote osteogenic differentiation of mesenchymal
7
8 stem cells by activating the Wnt/β-catenin signaling pathway. In addition, the hydrogel is used as
9
10 a malleable carrier for demineralized bone matrix (DBM). The loading of DBM does not affect
11
12
13
the self-healing and injectable natures of hydrogels while enhancing osteogenic capacity,
14
15 indicating advanced allograft bone formulations with carriers can facilitate handling and bone
16
17 healing. This work provides the first demonstration of therapeutic supramolecular design for the
18
19
treatment of bone defects.
20
21
22
23 1. INTRODUCTION
24
25
26
27
Hydrogels, networks of hydrophilic polymer chains, have received great attention in regenerative
28
29 medicine for use as tissue engineering scaffolds and carriers of therapeutic cells or drugs.1-7
30
31 Traditional hydrogels are fabricated by various chemical reactions (e.g. Michael type addition,
32
33
condensation reactions), radical polymerization or high energy irradiation to form crosslinked
34
35
36 networks in aqueous solutions.8-11 However, crosslinking agents and chemical reactions used in
37
38 conventional gel formation may have detrimental effects on living cells and bioactive molecules.9
39
40 Moreover, hydrogel networks created by chemical crosslinking are not injectable and do not
41
42
43 recover after rupture.12-14 On the contrary, supramolecular hydrogels are driven by reversible non-
44
45 covalent bonds, including ionic interactions, hydrophobic interactions hydrogen bonding, and
46
47 possess self-healing and injectable properties due to their dynamic bonding nature. These are
48
49
50
critical features for tissue engineering applications.11, 15-18
51
52 Nanoclays are two-dimensional (2D) inorganic silicate particles and have been shown to be able
53
54 to form supramolecular gel networks from various hydrogel precursors with self-healing nature
55
56
57
58
59
2
1
2
3 and shape-memory behavior.19-22 Laponite (Na+0.7[(Mg5.5Li0.3)Si8O20(OH)4]-0.7), is a synthetic
4
5
6 disk-shaped smectite approximately 25 nm in diameter and 0.92 nm in thickness.23 The
7
8 degradation products of laponite (e.g. magnesium, lithium and orthosilicic acid) have been shown
9
10 to induce osteogenic differentiation of mesenchymal stem cells and promote bone formation by
11
12
13
regulating cell adhesion and the Wnt/β-catenin signaling pathway.23-30 The incorporation of
14
15 laponite into various polymer matrices (e.g. chitosan, PEG, PEO) not only enhanced the
16
17 mechanical properties of nanocomposite hydrogels but also stimulated osteogenic differentiation
18
19
even without supplementation of growth factors.31-34
20
21
22 In this work, we demonstrate a therapeutic supramolecular assembly of chitosan and nanoclays for
23
24 bone regeneration applications (Figure 1). Novel supramolecular hydrogel networks are
25
26 assembled from laponite nanosheets (LNSs) and guanidinylated chitosan (GC) binder via
27
28
29 formation of salt-bridge between guanidine ion pendants in the GC binder and oxyanions on the
30
31 LNS surface. These networks have several key features: (i) they are a simple and nontoxic system
32
33 suitable for biomedical applications, (ii) they have injectable and self-healing features for the
34
35
36
administration in a minimally invasive manner and the integration to the defect site without
37
38 hydrogel fragmentation, (iii) they demonstrate an intrinsic osteoinductive nature and enhanced
39
40 bone healing. We also demonstrate the potential of supramolecular hydrogels as malleable carriers
41
42
for bone graft materials such as demineralized bone matrix (DBM) to enhance osteogenic capacity.
43
44
45
46 2. MATERIALS AND METHODS
47
48
49 2.1. Chemical and Materials. Glycol chitosan, 1H-Pyrazole-1-carboxamidine hydrochloride, N,
50
51 N-Diisopropylethylamine (DIPEA), sodium polyacrylate (ASAP), rhodamine, methylene blue, 5-
52
53
54 bromo-4-chloro-3-indoxylphosphate (BCIP), Nitro Blue tetrazolium (NBT), Alizarin red S, L-
55
56 ascorbic acid, p-nitrophenol phosphate, β-glycerophosphate, dexamethasone were purchased from
57
58
59
3
1
2
3 Sigma Aldrich (St.Louis, MO). Laponite XLG was obtained from Best of Chemicals (Shirley,
4
5
6 NY). alamarBlue™, Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS),
7
8 penicillin/streptomycin (P/S), Trizol reagents, cDNA transcription kit, Calcein AM, Pico 488
9
10 Green and Ethidium homodimer (EthD-1) were purchased from Life Technologies (Grand Island,
11
12
13
NY).
14
15
16 2.2. Guanidinylation of Chitosan. The primary amino groups of glycol chitosan were
17
18 guanidinylated by 1H-Pyrazole-1-carboxamidine hydrochloride. In brief, 1.0 g glycol chitosan and
19
20
3.85 g 1H-Pyrazole-1-carboxamidine hydrochloride (5 equiv, according to the number of primary
21
22
23 amino groups) were dissolved in 20 mL deionized water. Then 4.34 mL DIPEA (5 equiv, according
24
25 to the number of primary amino groups) was added and reacted for 24 h at room temperature.
26
27 Subsequently, the raw product was dialyzed in a dialysis bag (MWCO 3500 Da) against deionized
28
29
30 water to remove free 1H-Pyrazole-1-carboxamidine hydrochloride. Dialyzed product was freeze
31
32 dried to obtain guanidinylated chitosan, which was confirmed by 1H NMR (400 MHz, D2O).
33
34
35 2.3. Preparation and Characterization of Hydrogel. Firstly, laponite was added into deionized
36
37
38
water with magnetic stirring (1000 rpm, 30 min). ASAP was added with stirring magnetically (500
39
40 rpm) for 10 min. Finally, guanidinylated chitosan was added and mixed by a vortex for 5 s.
41
42 Different amounts of laponite and chitosan were used to obtain hydrogels with various
43
44
laponite/guanidinylated-chitosan ratios. Hydrogel prepared of laponite/glycol chitosan was used
45
46
47 as a negative control. To make them easy to see, hydrogels (laponite/guanidinylated-chitosan ratio:
48
49 3 wt %/0.5 wt %) were incubated in either rhodamine B or methylene blue solutions for 30 min to
50
51 generate pink and blue hydrogels, respectively. Both pink hydrogel and blue hydrogel were cut
52
53
54 into two pieces. One piece of pink hydrogels and one piece of blue hydrogels were touched and
55
56 formed into an integrated hydrogel. Then these two healed hydrogels were incubated in phosphate
57
58
59
4
1
2
3 buffer solutions (PBS) for 3 h to detect their stability. The new hydrogels were re-cut into two
4
5
6 pieces. Two pieces of pink-blue hydrogels were re-joined together and formed a new hydrogel. In
7
8 addition, whole pink hydrogel and blue hydrogel were touched with each other for 10 min, and
9
10 transfer the new pink/blue gel into deionized water for 10 min to detect its stability. Each step was
11
12
13
photographed.
14
15
16 Storage modulus (G’) and loss modulus (G’’) of hydrogels with 3 wt % LNSs were monitored
17
18 using Discovery Hybrid Rheometers HR-3 (TA Intruments). Frequency sweep of the hydrogel was
19
20
performed from 0.1 to 10 Hz frequency at γ = 5.0%. Strain sweep of the hydrogel was performed
21
22
23 from 0.02% to 300 % strain at fxed 1.6 Hz frequency to study shear-thinning properties of
24
25 hydrogels. Continuous step strain sweep was performed with alternating shear strains of low (1%,
26
27 300 s)-high (300%, 300 s)-low (1%, 300 s) strain.
28
29
30
31 The degradation of hydrogels was carried out for 40 days. Hydrogels with 3 wt % LNSs were
32
33 incubated at 37 °C in PBS (pH 7.4) or cell culture medium (DMEM, 10% FBS, and 1% P/S),
34
35 which were replaced every 5 days. At a given time, hydrogels were lyophilized for weight
36
37
38
measurement. The present residual weight of hydrogels was calculated using the following
39
40 equation: Residual gel weight = Mt/M0*100%, where M0 and Mt refer to the weight of hydrogels
41
42 at time 0 (hydrogels did not undergo degradation) and t, respectively.
43
44
45
2.4. Cell Encapsulation in 3D Hydrogel. The mouse bone marrow stromal cell line (BMSCs, D1
46
47
48 ORL UVA [D1], D1 cell, CRL-12424) was purchased from American Type Culture Collection
49
50 (ATCC, Manassas, VA) and cultured in DMEM with 10% FBS and 1% P/S at 37 °C in a
51
52 humidified atmosphere containing 5% CO2. Cell culture media DMEM, penicillin, streptomycin,
53
54
55 and FBS were purchased from Gibco (Manassas, VA). First, 2×107 BMSCs were suspended in 50
56
57
58
59
5
1
2
3 μL guanidinylated-chitosan solution (5 wt%, PBS). Different volumes (from 250 to 450 μL) of
4
5
6 laponite (5 wt%) and PBS (from 200 to 0 μL) were added maintaining a final volume of 500 μL
7
8 and the mixture vortexed for 5 s immediately. 50 μL of each the resulting cell-encapsulated
9
10 hydrogels was cultured with 1 mL DMEM culture medium in separate wells of a 24-well plate.
11
12
13
The culture medium was changed every 2 days.
14
15
16 2.5. Cell Viability Assay. To estimate the cytotoxicity of laponite hydrogel to BMSCs, cell
17
18 viability was detected using alamarBlue™. BMSCs loaded hydrogel was cultured for 1, 3, or 7
19
20
days. Then the hydrogel was stained with 1 mM calcein AM and 2 mM EthD-1 for CLSM
21
22
23 observation. Cells were incubated with 100 μL culture medium containing 10% alamarBlue™ for
24
25 another 2 h at 37 ℃. The fluorescence intensity was detected by a microplate reader using 570 nm
26
27 excitation and 585 nm emission. The additional cytocompatibility test was performed by placing
28
29
30 the hydrogels in tissue culture plates (TCP) in which cells were cultured and exposed to the
31
32 hydrogel samples. The hydrogel and cells were separated by 70 μm mesh inserts in culture
33
34 medium. The alamarBlue™ fluorescence was measured at day 1, 4, or 7 and was normalized to
35
36
37 untreated cells grown on TCP.
38
39
40 2.6. Alkaline Phosphatase (ALP) Expression and Activity. After incubation in osteogenic
41
42 medium (DMEM medium containing 10% FBS, 50 μg/mL L-ascorbic acid, 10 mM β-
43
44
45
glycerophosphate, 1% P/S and 100 nM dexamethasone) for 3, 7 or 14 days, hydrogels were fixed
46
47 using 10% formalin solution for 20 min and immersed in ALP solutions (100 mM Tris, 50 mM
48
49 MgCl2, 100 mM NaCl, pH 8.5) for 10 min. Then hydrogels were stained with BCIP/NBT for 2 h
50
51 and imaged using an Olympus IX71 microscope (Olympus, Tokyo, Japan). For ALP activity
52
53
54 detection, hydrogels were washed with PBS and lysed using 0.1% Tween-20 buffer at 4 ℃ for 30
55
56 min. The supernatant was collected after centrifugation at 12000 rpm for 5 min and reacted with
57
58
59
6
1
2
3 phosphatase substrate for 2 h at 37 ℃. ALP concentration was measured at a wavelength of 405
4
5
6 nm using a microplate reader, and values were normalized to DNA content measured using Pico
7
8 488 Green.
9
10
11 2.7. Mineralization Assay. After incubation in osteogenic medium for 7, 14, or 21 days, hydrogels
12
13
14 were stained with 0.2% Alizarin red S solution for 5 min. Once washed with PBS for 3 times,
15
16 hydrogels were imaged with an Olympus IX71 microscope and quantified using acetic acid with
17
18 colorimetric measurement at 405 nm. The quantitative results were normalized with the weight of
19
20
21
the hydrogels.
22
23
24 2.8. Quantitative Polymerase Chain Reaction (qPCR) Analysis. At given times, total RNA was
25
26 obtained from hydrogels using Trizol reagent. Then cDNA was synthesized using a cDNA
27
28 transcription kit (Invitrogen). Amplification reactions including 1 μL of cDNA template, 1 μL of
29
30
31 primer, 8 μL of distilled deionized water and 10 μL of SYBR Green were carried out using a
32
33 LightCycler 480 PCR (Indianapolis, IN) for 55 cycles. The sequences of primers are listed in Table
34
35 S1.
36
37
38
39 3. RESULTS AND DISCUSSION
40
41
42 3.1. Preparation and Characterization of Hydrogels. First, primary amines of glycol chitosan
43
44 were guanidinylated by 1H-Pyrazole-1-carboxamidine hydrochloride and DIPEA, leading to the
45
46
47
synthesis of GC binder.35-37 1H NMR spectroscopy confirmed that the signals corresponding to the
48
49 protons next to primary amines (b, δ = 1.75 ppm) were gradually shifted downfield (b’, δ = 1.83
50
51 ppm) after guanidinylation (Figure 2A).35 After dispersed with ASAP, LNS solutions were mixed
52
53 with GC binder for the preparation of hydrogels.38-41 As shown in Figure 2B, LNS solutions could
54
55
56 form hydrogels after interaction with GC binder for 100 s. However, the mixture of LNSs and
57
58
59
7
1
2
3 glycol chitosan was not able to form hydrogels, indicating that there was a stronger supramolecular
4
5
6 interaction between LNSs and GC binder after guanidinylation. When LNS amount was increased
7
8 from 2.5 to 4.5 wt %, the gelation time was reduced from 220 to 5 s. Increased amounts of GC
9
10 binder could also decrease gelation time, indicating that the formation of hydrogels was affected
11
12
13
by supramolecular interactions including hydrogen bonding and electrostatic interaction between
14
15 LNSs and GC binder (Figure S1-S4). Meanwhile, glycol chitosan without guanidinylation was
16
17 used as a control group (Figure S5). Hydrogels consisting of LNSs and glycol chitosan at a ratio
18
19
of 4.0 wt %/0.5 wt % formed slower (90 s) than LNSs/GC hydrogels with the same ratio (10 s),
20
21
22 due to the weaker supramolecular interactions between LNSs and glycol chitosan.
23
24
25 Next, the self-healing ability of LNSs/GC hydrogels was evaluated using the macroscopic
26
27 method.42-43 After staining with rhodamine B and methylene blue, hydrogels with two different
28
29
30 colors were cut into two pieces and held together along the cut surface at 25 ℃ without any external
31
32 intervention. The boundaries between different pieces had blurred after 10 min and the two pieces
33
34 were completely merged into a single object. Furthermore, newly formed hydrogels could maintain
35
36
37 their shape and integrity in aqueous environment up to 3 h. After the second cut, the two pieces of
38
39 pink-blue hydrogels also could form an integral new hydrogel again. (Figure 2C). Consistently,
40
41 two uncut dyed hydrogels also fused into one object, and the fused hydrogels could also keep their
42
43
44
shape and integrity in aqueous environment (Figure 2D). These results manifested that our
45
46 supramolecular hydrogels exhibited excellent self-healing ability thanks to dynamic non-covalent
47
48 bonds in the hydrogel networks (Figure 2E). In addition, hydrogels with injectable properties have
49
50 the potential for minimally invasive therapies.44-46 Our newly fabricated hydrogels, using various
51
52
53 amounts of LNSs (2.5 to 4.5 wt %) exhibited an injectable feature, shown as a rapid reassembly
54
55 when extruded them into a petri dish (Figure 2F). Meanwhile, supramolecular hydrogel (3.0 wt %
56
57
58
59
8
1
2
3 LNSs, 0.5 wt % GC) could be extruded through a 23-gauge needle without clogging to form “ABC”
4
5
6 hydrogels successfully (Figure2G). Similarly, different shaped molds were used to simulate
7
8 irregular tissue defects (Figure 2H). The molds could be filled with extruded hydrogels using a
9
10 23-gauge needle to rebuild unabridged triangle-, oval-, heart-, and hexagon-shaped hydrogels,
11
12
13
indicating that these non-covalently driven hydrogels have great potential for therapies of irregular
14
15 tissue defects with minimally invasive approach due to their injectable performance.
16
17
18 To quantify the mechanical performance associated with shear-thinning and the subsequent
19
20
recovery, we measured the rheological properties of the LNSs/GC hydrogels. The hydrogel with
21
22
23 3 wt % LNSs and 0.5 wt % GC had a storage modulus (G’) of ~215 Pa and a loss modulus (G’’)
24
25 of ~183 Pa as functions of angular frequency at a fixed strain, γ = 5.0% (Figure 3A). As shown in
26
27 Figure 3B, strain sweeps of the hydrogels revealed an elastic response characters of hydrogels.
28
29
30 The G’ and G’’ values decreased fleetly at a critical shear stress level, indicating a collapse of
31
32 hydrogels. Furthermore, cyclic high and low stresses were applied to the hydrogels (Figure 3C).
33
34 At a low stress (1%) level, the value of G’ was higher than G’’, indicating that the hydrogel was
35
36
37
stable. Once the stress increased to 300%, the value of G’ was lower than G’’, demonstrating that
38
39 the hydrogel was changed into a liquid-like state. After removing the high stress, the hydrogel was
40
41 recovered immediately. The degradation of hydrogels was evaluated and displayed in Figure 3D.
42
43
The weight of hydrogels with 3 wt % LNSs and 0.5 wt % GC decreased 48.4% and 45.2% after
44
45
46 incubation for 40 days in PBS (pH 7.4) and culture medium, respectively.
47
48
49 3.2. Cytotoxicity and Osteoinduction of Hydrogels. Once the expected self-healing and
50
51 injectable features were verified successfully, both biocompatibility and osteoinductive capacity
52
53
54 were evaluated using mouse bone marrow stromal cells (BMSCs).47-50 BMSCs were cultured in
55
56 tissue culture plates and exposed to the hydrogel samples. The viability of the cell cultures exposed
57
58
59
9
1
2
3 to the hydrogels was not significantly different compared to untreated cells (Figure S6). Cell
4
5
6 proliferation in hydrogel 3D culture using alamarBlue™ assay was shown in Figure 4A. There
7
8 was no obvious difference in cell growth rate in hydrogels with different concentrations of LNSs
9
10 from 2.5 to 4.5 wt %. The fluorescence intensity of hydrogel (LNSs = 3.0 wt %) increased to ~3.4
11
12
13
times at day 3 compared with day 1. The growth rate slowed down from day 3 to 7, maybe due to
14
15 BMSCs that were directed to differentiation rather than proliferation. Moreover, we employed a
16
17 Calcein AM and EthD-1 staining approach to distinguish live (green) and dead (red) cells (Figure
18
19
S7 and Figure S8). The cells grew very well and there were no apparent dead cells after 7 days of
20
21
22 culture, which agreed well with the alamarBlue™ staining results. Therefore, our supramolecular
23
24 hydrogels display good biocompatibility and may act as a favorable substrate for cell growth and
25
26 differentiation.
27
28
29
30 To further evaluate the osteogenic efficiency of the supramolecular hydrogels for bone
31
32 regeneration, the activity and expression of ALP, an early marker of osteogenic differentiation,
33
34 was first tested in BMSCs encapsulated with hydrogels.47-50 As shown in Figure 4B, ALP activity
35
36
37
revealed a significant increase at day 3 and reached the highest value at day 14. Hydrogels
38
39 containing 4.5 wt % LNSs displayed 1.5-fold higher ALP activity than hydrogels containing 2.5
40
41 wt % LNSs. In contrast, increasing the amount of GC binder did not have a significant impact on
42
43
ALP activity (Figure S9). ALP expression was also observed after staining with BCIP/NBT using
44
45
46 a microscope directly (Figure 4C). Consistent with prior results of ALP activity, ALP expression
47
48 increased over time and was significantly elevated in hydrogels with increased LNS concentration.
49
50 Moreover, hydrogels displayed moderate osteogenic ability under basal culture medium (Figure
51
52
53 S10). To assess mineralization (late osteogenic marker) in hydrogels, Alizarin red S was
54
55 subsequently used to stain calcium (Figure 4D). Intense Alizarin red Stain was observed during
56
57
58
59
10
1
2
3 14 day culture, indicating deposition of calcium. Staining was more intense as the amount of LNSs
4
5
6 increased. The quantification of calcium deposition shown that hydrogels with 4.0 wt % and 4.5
7
8 wt % LNSs displayed the highest values at all of the time points, which was consistent with the
9
10 corresponding staining (Figure S11). The staining and quantitative data indicated the elevated
11
12
13
osteogenic capacity resulting from inherent osteoinductivity of LNSs.
14
15
16 3.3. Osteogenic Gene Expression of Hydrogels. To uncover the osteogenesis efficiency on
17
18 molecular level, qPCR analysis was used to detect the level of osteogenic gene expression
19
20
including ALP (a marker in the early stage), Runx2 (an osteoblast-specific marker) and OCN (a
21
22
23 marker in the later stage).47-50 As exhibited in Figure 5A, hydrogels with increased amounts of
24
25 LNSs had higher levels of ALP, Runx2 and OCN expression at day 14 when compared with day 1.
26
27 Of note, hydrogels with 4.5 wt % LNSs increased the level of ALP and Runx2 to 1.6- and 1.9-fold
28
29
30 respectively at day 14, and increased the level of OCN to 1.5-fold at day 21 when compared with
31
32 hydrogels with 2.5 wt % LNSs. Overall, the increase of LNSs from 2.5 to 4.5 wt % could give rise
33
34 to the considerable upregulation of osteogenic genes, consistent with the evidence for increased
35
36
37
ALP and mineral accumulation. Given the Wnt/β-catenin signaling activation by the degradation
38
39 products of lithium and orthosilicic acid from LNSs, the mRNA levels of several key proteins in
40
41 Wnt/β-catenin signaling including Gsk3 and β-catenin were also determined using qPCR assay
42
43
(Figure 5B).51-52 The level of β-catenin in hydrogels with 3.0 wt % LNSs was increased up to 1.7-
44
45
46 fold, while the levels of Gsk3, which negatively regulates β-catenin signal were decreased up to
47
48 0.42-fold at day 14. In addition, the mRNA levels of Integrin α and Integrin β were investigated
49
50 to evaluate the cell adhesion induced by magnesium (Figure 5C). The levels of Integrin α and
51
52
53 Integrin β were upregulated with the increased amounts of LNSs in hydrogels, and about 1.5-fold
54
55 and 1.6-fold increase was detected in hydrogels containing 4.5 wt % LNSs as compared with
56
57
58
59
11
1
2
3 hydrogels containing 2.5 wt % LNSs 2 at day 14. Together, these results suggested that
4
5
6 supramolecular hydrogels composed of LNSs and GC could stimulate cell osteogenic
7
8 differentiation through activating Wnt/β-catenin signaling and enhance cell adhesion mediated by
9
10 their degradation products including lithium, orthosilicic acid and magnesium.
11
12
13
14
3.4. Properties of DBM-Loaded Hydrogels. DBM has been widely used in clinical treatment of
15
16 bone defects due to its inherent osteoconductivity and osteoinductivity derived from its growth
17
18 factor and protein matrix components, such as type I collagen, bone morphogenetic protein-2
19
20
(BMP-2), and osteopontin (OPN).53-54 However, more extensive use of DBM particles is limited
21
22
23 in large defect areas due to difficulties in handling, concerns about particle migration, and
24
25 insufficient bone formation capacity.55-57 Although synthetic inert carriers such as glycerol and
26
27 hyaluronic acid have been used in clinical applications, they are rapidly dissolved in a body and
28
29
30 DBM particles may migrate out of the defect site.58-59 This may induce severe inflammation and
31
32 the desired localized effect of BMPs present in DBM may not manifest. Taking the above into
33
34 account, we explored our supramolecular hydrogels as polymeric carriers for DBM, to enhance
35
36
37
handling properties and osteogenic potential. Since LPN concentrations of 3 wt% or higher
38
39 presented desired gelation time (<100 s) to rapidly entrap cells and bioactive components, 3 wt%
40
41 LPNs were tested in the DBM-loaded hydrogels as a starting concentration. Hydrogels containing
42
43
3 wt % LNSs could be formed after the addition of 1.5 to 6.0 wt % DBM, and their gelation times
44
45
46 (100 s) were similar to that of DBM-free hydrogels containing equal LNSs (Figure S12). To
47
48 investigate their self-healing properties, hydrogels prepared using 3.0 wt % LNSs and 3.0 wt %
49
50 DBM were treated with rhodamine B and methylene blue to obtain two different colored hydrogels
51
52
53 (Figure 6A and Figure 6B). After intimate contact, pink and blue hydrogels were healed into a
54
55 single integrated hydrogel. Also, hydrogel samples cut into two pieces could merge into one piece
56
57
58
59
12
1
2
3 and the integral hydrogel was stable in deionized water. These results indicated that the loading of
4
5
6 DBM had no influence on the self-healing nature of hydrogels.
7
8
9 The injectable properties of DBM-loaded hydrogels were investigated by extruding the hydrogels
10
11 into a petri dish, molds with various shapes, or aqueous environment. As shown in Figure 6C and
12
13
14
Figure 6D, DBM-loaded hydrogels not only could be injected into deionized water without any
15
16 breakage but also could be injected using a syringe to form “DBM” characters. Also, four kinds
17
18 of molds were employed to imitate irregular tissue defect sites and were filled with DBM-loaded
19
20
hydrogels (Figure 6E). The extruded hydrogel fragments were healed into an integral object
21
22
23 holding the same shapes with the original molds. Picrosirius Red was employed to stain DBM
24
25 distribution in supramolecular hydrogels. As shown in Figure 6F, DBM could be distributed in
26
27 hydrogels uniformly, maintaining its own particle morphology. These results indicate that
28
29
30 supramolecular hydrogels well maintained their injectability after loading with DBM, and thus
31
32 could provide convenient delivery of DBM for clinical usage.
33
34
35 3.5. Osteogenic Ability of DBM-Loaded Hydrogels. To explore the cytocompatibility and
36
37
38
potential osteoinduction of DBM-loaded hydrogels, BMSCs were encapsulated and cell viability
39
40 was assessed using alamarBlue™ assay (Figure 7A). The growth rate of cells in DBM-loaded
41
42 hydrogels was similar to that of cells in DBM-free hydrogels after incubation in culture medium
43
44
for 7 days. Hydrogels containing 3.0 wt % DBM showed the fastest growth rate with a 3.5-fold
45
46
47 increase at day 3 as compared with day 1. The growth rate slowed from day 3 to 7, suggesting that
48
49 hydrogels coupled with DBM not only provided a suitable 3D microenvironment for cell growth
50
51 but also may induce osteogenic differentiation. Additionally, hematoxylin and eosin (H&E) stain
52
53
54 showed that BMSCs cultured in DBM-free scaffolds exhibited homogenous cell proliferation with
55
56 a spindle-like shape, while cells in DBM-loaded hydrogels proliferated primarily along the surface
57
58
59
13
1
2
3 of DBM (Figure 7B). For the evaluation of osteogenic capability, both ALP activity and
4
5
6 expression were investigated after incubation at days 3, 7, and 14. ALP activity per ng DNA was
7
8 10.74 μM/ng in hydrogels with 3.0 wt % DBM at day 14, while there was 8.74 μM ALP activity
9
10 per ng DNA in DBM-free hydrogels (Figure 7C). There was higher ALP expression in hydrogels
11
12
13
with 3.0 and 6.0 wt % DBM than hydrogels with 1.5 wt % DBM at day 7 (Figure 7D and Figure
14
15 S13). Alizarin red S staining showed more mineralization in supramolecular hydrogels containing
16
17 3.0 and 6.0 wt % DBM after incubation for 14 days (Figure 7D, and Figure S14). Moreover, the
18
19
levels of expression of osteogenic genes including ALP, Runx2 and OCN were were measured by
20
21
22 qPCR (Figure 7E). After treatment in osteogenic medium for 14 days, the mRNA level of ALP in
23
24 hydrogels loaded with 3.0 wt % DBM was increased 3.6-fold compared with DBM-free hydrogels,
25
26 and the level of Runx2 in hydrogels with 3.0 and 6.0 wt% DBM was increased 2.5-fold and 2.6-
27
28
29 fold, respectively. At day 21, the level of OCN increased 2.1-fold, 2.7-fold and 2.8-fold in
30
31 hydrogels with 1.5, 3.0, and 6.0 wt % DBM, respectively, when compared with DBM-free
32
33 hydrogels. These results collectively suggest that DBM could cooperatively stimulate osteogenesis
34
35
36
induced by LNSs and our supramolecular hydrogels can serve as an effective delivery system for
37
38 DBM. The data obtained from this study may not indicate the most optimal LPN+DBM
39
40 combination for osteoinduction. Further studies will be need to evaluate the dose-dependent effect
41
42
of LPNs.
43
44
45
46 4. CONCLUSIONS
47
48
49 In summary, we have extended a new strategy on the development of supramolecular hydrogels to
50
51 explore their use for inducing bone regeneration. These hydrogels could be prepared by a simple
52
53
54 mixing procedure using LNSs and guanidine-rich polysaccharides, which create a 3-D network via
55
56 salt-bridge formation by hydrogen bonding and electrostatic interaction. In particular, the created
57
58
59
14
1
2
3 hydrogels with 3 wt % LNSs displayed self-healing and injectable properties that could facilitate,
4
5
6 minimally invasive delivery to irregular defect sites and integration with surrounding tissues.
7
8 Moreover, the hydrogel showed inherent osteogenic properties by activating the Wnt/β-catenin
9
10 signaling pathway without the addition of exogenous growth factors. Lastly, the supramolecular
11
12
13
hydrogel system was effective to deliver DBM and could serve as a versatile moldable vehicle,
14
15 facilitating handling and osteogenic potential. This work demonstrates a novel therapeutic
16
17 supramolecular design that can create injectable and self-healing polymer networks with high
18
19
osteoinductivity. The supramolecular hydrogel presents a versatile platform for encapsulating
20
21
22 cells, therapeutic agents, and synthetic carriers for malleable bone graft constructs to promote
23
24 clinical bone repair.
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
15
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40 Figure 1. Schematic illustration of supramolecular hydrogels with self-healing and injectable
41
42
properties for bone regeneration with no need for growth factors, including (A) guanidine
43
44
45 modification of glycol chitosan to obtain GC binder, (B) preparation of supramolecular hydrogel
46
47 based on nanoclay and GC binder via salt-bridge formation by hydrogen bonding and electrostatic
48
49 interaction, DBM loading and stem cell osteogenic differentiation along with activation of Wnt/β-
50
51
52 catenin signaling and promotion of cell adhesion.
53
54
55
56
57
58
59
16
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
17
1
2
3 Figure 2. Physical and chemical properties of supramolecular hydrogels. (A) 1H NMR spectra of
4
5
6 the glycol chitosan (blue line) and GC binder (red) in D2O. (B) Images of LNSs with glycol
7
8 chitosan and LNSs with GC binder. (C) Self-healing processes of supramolecular hydrogels: (1)
9
10 two disk-shaped hydrogels stained with rhodamine B and methylene blue were cut into equal two
11
12
13
pieces; (2) one piece of pink hydrogel and one piece of blue hydrogel were joined together to form
14
15 two new hydrogels; (3) these two new hydrogels were immersed in PBS; (4) PBS was removed
16
17 after 3 h; (5) these two new hydrogels were re-cut into equal two pieces; (6) two pieces of pink-
18
19
blue hydrogels were joined together to form two new hydrogels; (7) these two new hydrogels were
20
21
22 immersed in PBS again. (D) Self-healing of two hydrogels after attachment directly: (1) pink
23
24 hydrogel and blue hydrogel were attached directly; (2) the new hydrogel was immersed into
25
26 deionized water for 10 min; (3) the hydrogel was transferred from deionized water and maintained
27
28
29 its shape. (E) Scheme diagram of the self-healing process. (F) Images of hydrogels injected from
30
31 23 G needle into water. (G) “ABC” gel could be formed after injection from 23 G needle. (H)
32
33 Hydrogels with different shape could be obtained after injection from 23 G needle.
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
18
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
Figure 3. Rheological and degradation properties of supramolecular hydrogels. (A) G’, G’’ and
38
39 Eta values of hydrogels with 3.0 wt % LNSs on frequency sweep at γ = 5.0%. G’ and G’’ values
40
41 of hydrogels with 3.0 wt % LNSs on strain sweep at frequency = 1.6 Hz. (B) and in continuous
42
43
step strain measurements (C). (D) Degradation kinetics of hydrogels in PBS and culture medium
44
45
46 for 40 days by measuring the weight.
47
48
49
50
51
52
53
54
55
56
57
58
59
19
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41 Figure 4. Osteoinductive efficiency of supramolecular hydrogels. (A) Fluorescence intensity of
42
43 alamarBlue towards MSCs after incubation in culture medium for 1, 3 and 7 days (n=3). (B) ALP
44
45 activity (n=3, *p < 0.05, **p < 0.001 by one-way analysis of variance, ANOVA) and (C) ALP
46
47
48
expression of cell-encapsulated hydrogels with LNSs ranging from 2.5 to 4.5 wt % treated in
49
50 osteogenic medium for 1, 3, 7 and 14 days. Scar bar repents 1 mm. (D) Mineralization of cell-
51
52 encapsulated hydrogels with LNSs ranging from 2.5 to 4.5 wt % after incubation with osteogenic
53
54
medium for 3, 7, 14 and 21 days. Scar bar repents 1 mm.
55
56
57
58
59
20
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
Figure 5. Gene expression of cell-encapsulated supramolecular hydrogels. (A) Osteogenic genes
48
49
50 ALP and Runx2 were tested at day 1, 7 and 14. OCN was evaluated at day 1, 14 and 21 (n=3, *p <
51
52 0.05, **p < 0.001 by one-way analysis of variance, ANOVA). (B) Wnt/β-catenin signaling
53
54 markers Gsk3 and β-catenin and (C) cell adhesion markers Integrin α and Integrin β were evaluated
55
56
57
58
59
21
1
2
3 after incubation for 1, 7 and 14 days (n=3, *p < 0.05, **p < 0.001by one-way analysis of variance,
4
5
6 ANOVA).
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
22
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40 Figure 6. Physical and chemical properties of DBM-loaded hydrogels. (A) Self-healing processes
41
42
43
of DBM-loaded supramolecular hydrogels: (1) two disk-shaped DBM-loaded hydrogels stained
44
45 with rhodamine B and methylene blue were cut into equal two pieces; (2) one piece of pink
46
47 hydrogel and one piece of blue hydrogel were joined together to form two new hydrogels; (3)
48
49
These two new hydrogels were immersed in deionized water for 10 min; (4) the hydrogels were
50
51
52 transferred from deionized water. (B) Self-healing of two DBM-loaded hydrogels after attachment
53
54 directly: (1) pink hydrogel and blue hydrogel were attached directly; (2) the new hydrogel was
55
56
57
58
59
23
1
2
3 immersed into deionized water for 10 min; (3) the hydrogel was transferred from deionized water
4
5
6 and maintained its shape. (C) Images of DBM-loaded hydrogels injected from a syringe without
7
8 needle into water. (D) “DBM” gel could be formed after injection from a syringe without needle.
9
10 (E) Hydrogels with different shapes could be obtained after injection from a syringe without
11
12
13
needle. (F) Images of hydrogels and DBM-loaded hydrogels after staining with Picrosirius Red.
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
24
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48 Figure 7. Osteoinductive efficiency of DBM-loaded hydrogels. (A) Fluorescence intensity of
49
50
51 alamarBlue™ towards cell-encapsulated DBM-loaded hydrogels after incubation in culture
52
53 medium for 1, 3 and 7 days (n=3). (B) Histological images for H&E staining of cell-encapsulated
54
55 DBM-loaded hydrogels after incubation in osteogenic medium for 21 days. Scar bar repents 100
56
57
58
59
25
1
2
3 μm. (C) ALP activity of cell-encapsulated DBM-loaded hydrogels treated in osteogenic medium
4
5
6 for 3, 7 and 14 days (n=3, *p < 0.05, **p < 0.001 by one-way analysis of variance, ANOVA). (D)
7
8 ALP expression DBM-loaded hydrogels in osteogenic medium for 7 days and mineralization of
9
10 cell-encapsulated DBM-loaded hydrogels after incubation with osteogenic medium for 14 days.
11
12
13
Scar bar repents 1 mm. (E) mRNA levels of ALP and Runx2 at day 14 and OCN at day 21 (n=3,
14
15 *p < 0.05, **p < 0.001by one-way analysis of variance, ANOVA).
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
26
1
2
3 ASSOCIATED CONTENT
4
5
6
7 Supporting Information. Additional data, Figures S1-S14 and Table S1. This material is
8
9 available free of charge via the Internet at http://pubs.acs.org.
10
11
12 AUTHOR INFORMATION
13
14
15 Corresponding Author
16
17
18 *E-mail: leemin@ucla.edu. Phone: +1-310-825-6674. Fax: +1-310-825-6345.
19
20
21 ORCID
22
23
Xiao Zhang: 0000-0002-0242-1939
24
25
26 Jiabing Fan: 0000-0002-7485-8275
27
28 Chung-Sung Lee: 0000-0001-5813-6056
29
30 Soyon Kim: 0000-0003-1030-8482
31
32
33 Min Lee: 0000-0003-2813-2091
34
35
Notes
36
37
38 The authors declare no competing financial interest.
39
40
41 ACKNOWLEDGMENT
42
43
44 This work was supported by grants from the National Institutes of Health (R01 DE027332), the
45
46 Department of Defense (W81XWH-18-1-0337), and the MTF Biologics.
47
48
49 REFERENCES
50
51
52
1. Moroni, L.; Burdick, J. A.; Highley, C.; Lee, S. J.; Morimoto, Y.; Takeuchi, S.; Yoo, J. J.
53
54 Biofabrication strategies for 3D in vitro models and regenerative medicine. Nat. Rev. Mater. 2018,
55
56 3, 21-37.
57
58
59
27
1
2
3 2. Balakrishnan, B.; Banerjee, R. Biopolymer-Based Hydrogels for Cartilage Tissue
4
5
6 Engineering. Chem. Rev. 2011, 111, 4453-4474.
7
8
9 3. Hoffman, A. S. Hydrogels for biomedical applications. Adv. Drug. Deliver. Rev. 2012, 64,
10
11 18-23.
12
13
14
15
4. Lee, K. Y.; Mooney, D. J. Hydrogels for Tissue Engineering. Chem. Rev. 2001, 101, 1869-
16
17 1880.
18
19
20 5. Peppas, N. A.; Hilt, J. Z.; Khademhosseini, A.; Langer, R. Hydrogels in Biology and
21
22
Medicine: From Molecular Principles to Bionanotechnology. Adv. Mater. 2006, 18, 1345-1360.
23
24
25
26 6. Slaughter, B. V.; Khurshid, S. S.; Fisher, O. Z.; Khademhosseini, A.; Peppas, N. A.
27
28 Hydrogels in Regenerative Medicine. Adv. Mater. 2009, 21, 3307-3329.
29
30
31 7. Liu, M.; Zeng, X.; Ma, C.; Yi, H.; Ali, Z.; Mou, X.; Li, S.; Deng, Y.; He, N. Injectable
32
33
34 hydrogels for cartilage and bone tissue engineering. Bone Res. 2017, 5, 17014.
35
36
37 8. Yu, L.; Ding, J. Injectable hydrogels as unique biomedical materials. Chem. Soc. Rev. 2008,
38
39 37, 1473-1481.
40
41
42
43
9. Hennink, W. E.; van Nostrum, C. F. Novel crosslinking methods to design hydrogels. Adv.
44
45 Drug. Deliver. Rev. 2012, 64, 223-236.
46
47
48 10. Du, X.; Zhou, J.; Shi, J.; Xu, B. Supramolecular Hydrogelators and Hydrogels: From Soft
49
50
Matter to Molecular Biomaterials. Chem. Rev. 2015, 115, 13165-13307.
51
52
53
54 11. Appel, E. A.; del Barrio, J.; Loh, X. J.; Scherman, O. A. Supramolecular polymeric
55
56 hydrogels. Chem. Soc. Rev. 2012, 41, 6195-6214.
57
58
59
28
1
2
3 12. Chen, H.; Yang, F.; Chen, Q.; Zheng, J. A Novel Design of Multi-Mechanoresponsive and
4
5
6 Mechanically Strong Hydrogels. Adv. Mater. 2017, 29, 1606900.
7
8
9 13. Costa, S. A.; Simon, J. R.; Amiram, M.; Tang, L.; Zauscher, S.; Brustad, E. M.; Isaacs, F.
10
11 J.; Chilkoti, A. Photo-Crosslinkable Unnatural Amino Acids Enable Facile Synthesis of
12
13
14
Thermoresponsive Nano- to Microgels of Intrinsically Disordepink Polypeptides. Adv. Mater.
15
16 2018, 30, 1704878.
17
18
19 14. Ouyang, L.; Highley, C. B.; Sun, W.; Burdick, J. A. A Generalizable Strategy for the 3D
20
21
Bioprinting of Hydrogels from Nonviscous Photo-crosslinkable Inks. Adv. Mater. 2017, 29,
22
23
24 1604983.
25
26
27 15. Dou, X.-Q.; Feng, C.-L. Amino Acids and Peptide-Based Supramolecular Hydrogels for
28
29 Three-Dimensional Cell Culture. Adv. Mater. 2017, 29, 1604062.
30
31
32
33 16. Miyamae, K.; Nakahata, M.; Takashima, Y.; Harada, A. Self-Healing, Expansion–
34
35 Contraction, and Shape-Memory Properties of a Preorganized Supramolecular Hydrogel through
36
37 Host–Guest Interactions. Angew. Chem. Int. Edit. 2015, 54, 8984-8987.
38
39
40
41
17. Voorhaar, L.; Hoogenboom, R. Supramolecular polymer networks: hydrogels and bulk
42
43 materials. Chem. Soc. Rev. 2016, 45, 4013-4031.
44
45
46 18. Hu, X.; Vatankhah-Varnoosfaderani, M.; Zhou, J.; Li, Q.; Sheiko, S. S. Weak Hydrogen
47
48
Bonding Enables Hard, Strong, Tough, and Elastic Hydrogels. Adv. Mater. 2015, 27, 6899-6905.
49
50
51
52 19. Dawson, J. I.; Kanczler, J. M.; Yang, X. B.; Attard, G. S.; Oreffo, R. O. C. Clay Gels For
53
54 the Delivery of Regenerative Microenvironments. Adv. Mater. 2011, 23, 3304-3308.
55
56
57
58
59
29
1
2
3 20. Dawson, J. I.; Oreffo, R. O. C. Clay: New Opportunities for Tissue Regeneration and
4
5
6 Biomaterial Design. Adv. Mater. 2013, 25, 4069-4086.
7
8
9 21. Han, L.; Lu, X.; Liu, K.; Wang, K.; Fang, L.; Weng, L.-T.; Zhang, H.; Tang, Y.; Ren, F.;
10
11 Zhao, C.; Sun, G.; Liang, R.; Li, Z. Mussel-Inspired Adhesive and Tough Hydrogel Based on
12
13
14
Nanoclay Confined Dopamine Polymerization. ACS Nano 2017, 11, 2561-2574.
15
16
17 22. Laurenti, M.; Al Subaie, A.; Abdallah, M.-N.; Cortes, A. R. G.; Ackerman, J. L.; Vali, H.;
18
19 Basu, K.; Zhang, Y. L.; Murshed, M.; Strandman, S.; Zhu, J.; Makhoul, N.; Barralet, J. E.; Tamimi,
20
21
F. Two-Dimensional Magnesium Phosphate Nanosheets Form Highly Thixotropic Gels That Up-
22
23
24 Regulate Bone Formation. Nano. Lett. 2016, 16, 4779-4787.
25
26
27 23. Tomás, H.; Alves, C. S.; Rodrigues, J. Laponite®: A key nanoplatform for biomedical
28
29 applications? Nanomed.-Nanotechnol. 2018, 14, 2407-2420.
30
31
32
33 24. Hedgepeth, C. M.; Conrad, L. J.; Zhang, J.; Huang, H.-C.; Lee, V. M. Y.; Klein, P. S.
34
35 Activation of the Wnt Signaling Pathway: A Molecular Mechanism for Lithium Action. Dev. Biol.
36
37 1997, 185, 82-91.
38
39
40
41
25. Han, P.; Wu, C.; Xiao, Y. The effect of silicate ions on proliferation, osteogenic
42
43 differentiation and cell signalling pathways (WNT and SHH) of bone marrow stromal cells.
44
45 Biomater. Sci. 2013, 1, 379-392.
46
47
48
26. Luthringer, B. J. C.; Willumeit-Römer, R. Effects of magnesium degradation products on
49
50
51 mesenchymal stem cell fate and osteoblastogenesis. Gene 2016, 575, 9-20.
52
53
54
55
56
57
58
59
30
1
2
3 27. Zhang, F.; Phiel, C. J.; Spece, L.; Gurvich, N.; Klein, P. S. Inhibitory Phosphorylation of
4
5
6 Glycogen Synthase Kinase-3 (GSK-3) in Response to Lithium: EVIDENCE FOR
7
8 AUTOREGULATION OF GSK-3. J. Biol. Chem. 2003, 278, 33067-33077.
9
10
11 28. Clément-Lacroix, P.; Ai, M.; Morvan, F.; Roman-Roman, S.; Vayssière, B.; Belleville, C.;
12
13
14
Estrera, K.; Warman, M. L.; Baron, R.; Rawadi, G. Lrp5-independent activation of Wnt signaling
15
16 by lithium chloride increases bone formation and bone mass in mice. Proc. Natl. Acad. Sci. USA
17
18 2005, 102, 17406-17411.
19
20
21
29. Yoshizawa, S.; Brown, A.; Barchowsky, A.; Sfeir, C. Magnesium ion stimulation of bone
22
23
24 marrow stromal cells enhances osteogenic activity, simulating the effect of magnesium alloy
25
26 degradation. Acta Biomater. 2014, 10, 2834-2842.
27
28
29 30. Zhang, J.; Ma, X.; Lin, D.; Shi, H.; Yuan, Y.; Tang, W.; Zhou, H.; Guo, H.; Qian, J.; Liu,
30
31
32 C. Magnesium modification of a calcium phosphate cement alters bone marrow stromal cell
33
34 behavior via an integrin-mediated mechanism. Biomaterials 2015, 53, 251-264.
35
36
37 31. Xavier, J. R.; Thakur, T.; Desai, P.; Jaiswal, M. K.; Sears, N.; Cosgriff-Hernandez, E.;
38
39
40
Kaunas, R.; Gaharwar, A. K. Bioactive Nanoengineepink Hydrogels for Bone Tissue Engineering:
41
42 A Growth-Factor-Free Approach. ACS Nano 2015, 9, 3109-3118.
43
44
45 32. Gaharwar, A. K.; Mihaila, S. M.; Swami, A.; Patel, A.; Sant, S.; Reis, R. L.; Marques, A.
46
47
P.; Gomes, M. E.; Khademhosseini, A. Bioactive Silicate Nanoplatelets for Osteogenic
48
49
50 Differentiation of Human Mesenchymal Stem Cells. Adv. Mater. 2013, 25, 3329-3336.
51
52
53 33. Gaharwar, A. K.; Kishore, V.; Rivera, C.; Bullock, W.; Wu, C.-J.; Akkus, O.; Schmidt, G.
54
55 Physically Crosslinked Nanocomposites from Silicate-Crosslinked PEO: Mechanical Properties
56
57
58
59
31
1
2
3 and Osteogenic Differentiation of Human Mesenchymal Stem Cells. Macromol. Biosci. 2012, 12,
4
5
6 779-793.
7
8
9 34. Yao, C.; Liu, Z.; Yang, C.; Wang, W.; Ju, X.-J.; Xie, R.; Chu, L.-Y. Smart Hydrogels with
10
11 Inhomogeneous Structures Assembled Using Nanoclay-Cross-Linked Hydrogel Subunits as
12
13
14
Building Blocks. ACS Appl. Mater. & Inter. 2016, 8, 21721-21730.
15
16
17 35. Choi, J.-y.; Ryu, K.; Lee, G. J.; Kim, K.; Kim, T.-i. Agmatine-Containing Bioreducible
18
19 Polymer for Gene Delivery Systems and Its Dual Degradation Behavior. Biomacromolecules 2015,
20
21
16, 2715-2725.
22
23
24
25 36. Kim, T.-i.; Lee, M.; Kim, S. W. A guanidinylated biopinkucible polymer with high nuclear
26
27 localization ability for gene delivery systems. Biomaterials 2010, 31, 1798-1804.
28
29
30 37. Wender, P. A.; Rothbard, J. B.; Jessop, T. C.; Kreider, E. L.; Wylie, B. L. Oligocarbamate
31
32
33 Molecular Transporters: Design, Synthesis, and Biological Evaluation of a New Class of
34
35 Transporters for Drug Delivery. J. Am. Chem. Soc. 2002, 124, 13382-13383.
36
37
38 38. Lee, J. H.; Kim, C.; Jung, J. H. Control of the rheological properties of clay nanosheet
39
40
41
hydrogels with a guanidinium-attached calix[4]arene binder. Chem. Commun. 2015, 51, 15184-
42
43 15187.
44
45
46 39. Wang, Q.; Mynar, J. L.; Yoshida, M.; Lee, E.; Lee, M.; Okuro, K.; Kinbara, K.; Aida, T.
47
48
High-water-content mouldable hydrogels by mixing clay and a dendritic molecular binder. Nature
49
50
51 2010, 463, 339-343.
52
53
54
55
56
57
58
59
32
1
2
3 40. Tamesue, S.; Ohtani, M.; Yamada, K.; Ishida, Y.; Spruell, J. M.; Lynd, N. A.; Hawker, C.
4
5
6 J.; Aida, T. Linear versus Dendritic Molecular Binders for Hydrogel Network Formation with Clay
7
8 Nanosheets: Studies with ABA Triblock Copolyethers Carrying Guanidinium Ion Pendants. J. Am.
9
10 Chem. Soc. 2013, 135, 15650-15655.
11
12
13
14
41. Li, Z.; Zhang, Y.-M.; Wang, H.-Y.; Li, H.; Liu, Y. Mechanical Behaviors of Highly
15
16 Swollen Supramolecular Hydrogels Mediated by Pseudorotaxanes. Macromolecules 2017, 50,
17
18 1141-1146.
19
20
21
42. Yang, X.; Liu, G.; Peng, L.; Guo, J.; Tao, L.; Yuan, J.; Chang, C.; Wei, Y.; Zhang, L.
22
23
24 Highly Efficient Self-Healable and Dual Responsive Cellulose-Based Hydrogels for Controlled
25
26 Release and 3D Cell Culture. Adv. Funct. Mater. 2017, 27, 1703174.
27
28
29 43. Wei, Z.; Yang, J. H.; Liu, Z. Q.; Xu, F.; Zhou, J. X.; Zrínyi, M.; Osada, Y.; Chen, Y. M.
30
31
32 Novel Biocompatible Polysaccharide-Based Self-Healing Hydrogel. Adv. Funct. Mater. 2015, 25,
33
34 1352-1359.
35
36
37 44. Basu, S.; Pacelli, S.; Feng, Y.; Lu, Q.; Wang, J.; Paul, A. Harnessing the Noncovalent
38
39
40
Interactions of DNA Backbone with 2D Silicate Nanodisks To Fabricate Injectable Therapeutic
41
42 Hydrogels. ACS Nano 2018, 12, 9866-9880.
43
44
45 45. Tseng, T.-C.; Tao, L.; Hsieh, F.-Y.; Wei, Y.; Chiu, I.-M.; Hsu, S.-h. An Injectable, Self-
46
47
Healing Hydrogel to Repair the Central Nervous System. Adv. Mater. 2015, 27, 3518-3524.
48
49
50
51 46. Rodell, C. B.; MacArthur Jr., J. W.; Dorsey, S. M.; Wade, R. J.; Wang, L. L.; Woo, Y. J.;
52
53 Burdick, J. A. Shear-Thinning Supramolecular Hydrogels with Secondary Autonomous Covalent
54
55 Crosslinking to Modulate Viscoelastic Properties In Vivo. Adv. Funct. Mater. 2015, 25, 636-644.
56
57
58
59
33
1
2
3 47. Kim, S.; Cui, Z.-K.; Koo, B.; Zheng, J.; Aghaloo, T.; Lee, M. Chitosan–Lysozyme
4
5
6 Conjugates for Enzyme-Triggered Hydrogel Degradation in Tissue Engineering Applications.
7
8 ACS Appl. Mater. & Inter. 2018, 10, 41138-41145.
9
10
11 48. Cui, Z.-K.; Kim, S.; Baljon, J. J.; Doroudgar, M.; Lafleur, M.; Wu, B. M.; Aghaloo, T.;
12
13
14
Lee, M. Design and Characterization of a Therapeutic Non-phospholipid Liposomal Nanocarrier
15
16 with Osteoinductive Characteristics To Promote Bone Formation. ACS Nano 2017, 11, 8055-8063.
17
18
19 49. Cui, Z.-K.; Kim, S.; Baljon, J. J.; Wu, B. M.; Aghaloo, T.; Lee, M. Microporous
20
21
methacrylated glycol chitosan-montmorillonite nanocomposite hydrogel for bone tissue
22
23
24 engineering. Nat. Commun. 2019, 10, 3523.
25
26
27 50. Cui, Z.-K.; Sun, J. A.; Baljon, J. J.; Fan, J.; Kim, S.; Wu, B. M.; Aghaloo, T.; Lee, M.
28
29 Simultaneous delivery of hydrophobic small molecules and siRNA using Sterosomes to direct
30
31
32 mesenchymal stem cell differentiation for bone repair. Acta Biomater. 2017, 58, 214-224.
33
34
35 51. Kahn, M. Can we safely target the WNT pathway? Nat. Rev. Drug. Discov. 2014, 13, 513-
36
37 532.
38
39
40
41
52. Cohen, P.; Goedert, M. GSK3 inhibitors: development and therapeutic potential. Nat. Rev.
42
43 Drug. Discov. 2004, 3, 479-487.
44
45
46 53. Holt, D. J.; Grainger, D. W. Demineralized bone matrix as a vehicle for delivering
47
48
endogenous and exogenous therapeutics in bone repair. Adv. Drug. Deliver. Rev. 2012, 64, 1123-
49
50
51 1128.
52
53
54
55
56
57
58
59
34
1
2
3 54. Song, J. J.; Ott, H. C. Organ engineering based on decellularized matrix scaffolds. Trends
4
5
6 Mol Med 2011, 17, 424-432.
7
8
9 55. Shi, J.; Sun, J.; Zhang, W.; Liang, H.; Shi, Q.; Li, X.; Chen, Y.; Zhuang, Y.; Dai, J.
10
11 Demineralized Bone Matrix Scaffolds Modified by CBD-SDF-1α Promote Bone Regeneration via
12
13
14
Recruiting Endogenous Stem Cells. ACS Appl. Mater. & Inter. 2016, 8, 27511-27522.
15
16
17 56. Hu, Q.; Liu, M.; Chen, G.; Xu, Z.; Lv, Y. Demineralized Bone Scaffolds with Tunable
18
19 Matrix Stiffness for Efficient Bone Integration. ACS Appl. Mater. & Inter. 2018, 10, 27669-27680.
20
21
22
57. Gupta, V.; Lyne, D. V.; Laflin, A. D.; Zabel, T. A.; Barragan, M.; Bunch, J. T.; Pacicca,
23
24
25 D. M.; Detamore, M. S. Microsphere-Based Osteochondral Scaffolds Carrying Opposing
26
27 Gradients Of Decellularized Cartilage And Demineralized Bone Matrix. ACS Biomaterials
28
29 Science & Engineering 2017, 3, 1955-1963.
30
31
32
33 58. Zheng, Y.; Huang, K.; You, X.; Huang, B.; Wu, J.; Gu, Z. Hybrid hydrogels with high
34
35 strength and biocompatibility for bone regeneration. Int J Biol Macromol 2017, 104, 1143-1149.
36
37
38 59. Man, Z.; Hu, X.; Liu, Z.; Huang, H.; Meng, Q.; Zhang, X.; Dai, L.; Zhang, J.; Fu, X.; Duan,
39
40
41
X.; Zhou, C.; Ao, Y. Transplantation of allogenic chondrocytes with chitosan hydrogel-
42
43 demineralized bone matrix hybrid scaffold to repair rabbit cartilage injury. Biomaterials 2016, 108,
44
45 157-167.
46
47
48
49
50
51
52
53
54
55
56
57
58
59
35
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17 TOC
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
36

Artigo Células-Tronco

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Artigo Células-Tronco

Uploaded by

Copyright:

Available Formats

Subscriber access provided by NATIONAL UNIV OF SINGAPORE

Biological and Medical Applications of Materials and Interfaces

is published by the American Chemical Society. 1155 Sixteenth Street N.W.,

You might also like