Osteopromoting Reservoir of Stem Cells: Bioactive Mesoporous

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Osteopromoting reservoir of stem cells: Bioactive mesoporous nanocarrier / collagen gel through slow-releasing FGF18 and the activated BMP signaling Chinmaya Mahapatra, Rajendra Kumar Singh, Jung-Ju Kim, Kapil Dev Patel, Roman A Perez, Jun-Hyeog Jang, and Hae-Won Kim ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b09769 • Publication Date (Web): 20 Sep 2016 Downloaded from http://pubs.acs.org on September 21, 2016

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ACS Applied Materials & Interfaces

Osteopromoting Reservoir of Stem Cells: Bioactive Mesoporous Nanocarrier / Collagen Gel through Slow-Releasing FGF18 and the Activated BMP Signaling Chinmaya Mahapatra1,2, Rajendra K Singh1,2, Jung-Ju Kim1,2, Kapil D Patel1,2 , Roman A Perez1,2, Jun-Hyeog Jang3, Hae -Won Kim1,2,4* 1

Institute of Tissue Regeneration Engineering (ITREN), Dankook University, Cheonan 330-714, South

Korea 2

Department of Nanobiomedical Science & BK21 PLUS NBM Global Research Center for Regenerative

Medicine, Dankook University, Cheonan 330-714, South Korea 3

Department of Biochemistry, Inha University School of Medicine, Incheon 22212, Republic of Korea

4

Department of Biomaterials Science, School of Dentistry, Dankook University, Cheonan 330-714, South

Korea

--------------------*

Corresponding author: Tel: +82 41 550 3081; Fax: +82 41 550 3085; E-mail: [email protected]

For: ACS Applied Materials & Interfaces

ACS Paragon Plus Environment


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Abstract

Providing an osteogenic stimulatory environment is a key strategy to construct stem cell-based boneequivalent tissues. Here we design a stem cell delivering gel matrix made of collagen (Col) with bioactive glass nanocarriers (BGn) that incorporate osteogenic signaling molecule, fibroblast growth factor 18 (FGF18); a reservoir considered to cultivate and promote osteogenesis of mesenchymal stem cells (MSCs). The presence of BGn in the gel was shown to enhance the osteogenic differentiation of MSCs, possibly due to the therapeutic role of ions released. The mesoporous nature of BGn was effective in loading FGF18 at large quantity, and the FGF18 release from the BGn-Col gel matrix was highly sustainable with almost a zero-order kinetics; over 8 weeks of a complete release confirmed by the green fluorescence protein signal change. The released FGF18 was effective in accelerating osteogenesis (alkaline phosphatase activity and bone related gene expressions) and bone matrix formation (osteopontin, bone sialoprotein, and osteocalcin production) of MSCs. This was attributed to the bone morphogenetic protein (BMP) signaling pathway, where the FGF18 release stimulated the endogenous secretion of BMP2 and the downstream signal Smad1/5/8. Taken together, the FGF18-BGn/Col gel is considered an excellent osteopromoting depot to support and signal MSCs for bone tissue engineering.

Keywords: Nanocomposite gel; bioactive glass nanocarrier; growth factor delivery; mesenchymal stem cells; BMP signaling; bone tissue engineering


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1. Introduction Stem cell-based tissue engineering holds great promise for the regeneration of injured and dysfunctional tissues, including bone. Biomatrices allow the anchorage of stem cells, and their growth, migration, and lineage differentiation.

1,2

Among the biomatrices,

3-5

hydrogels can encapsulate cells, providing 3D

environment that largely mimic the native tissue extracellular matrix (ECM), and can deliver them directly 6-10

to the damaged tissue space with minimal invasiveness.

Optimized hydrogels demand properties tuned to target tissues, in terms of mechanical stiffness, native ECM composition, and signaling molecules, to drive stem cells encapsulated to differentiate toward lineage cells.

8-10

For bone, rather stiffer matrix (with stiffness of approximately 30-50 kPa and even

higher) is preferred.

11-13

Moreover, collagen type I, as a major component of bone ECM, is widely used to

provide nanofibrous network in the gel matrix.

14,15

Also, for the cellular maturation and calcification,

signaling molecules like bone morphogenetic protein and calcium ions are promiscuously introduced.

16-19

Nanocomposite hydrogels made of organic and inorganic phase have been developed as promising matrices for hard tissues. Recently, bioactive glass nanospheres (BGn) have been introduced into biopolymer hydrogel networks, significantly improving the physical and chemical stability, mechanical properties and the bone bioactivity and mineralization of cells.

20-22

These properties are considered

favorable for the culture and driving stem cells toward lineage cells for hard tissue regeneration. Some of the most intriguing aspects of BGn incorporated as the nanocomponent include i) the release of ions to exert therapeutic effects, and ii) the mesoporous nature that can load signaling molecules to stimulate stem cell functions.

23-25

With this in mind, here we explore the Col/BGn hydrogel system for the culture of mesenchymal stem cells (MSCs) and their differentiation into an osteogenic lineage. For this, an osteopromoting growth factor (fibroblast growth factor 18; FGF18) is introduced within the tailored mesopore structure of BGn, and a sustainable release hydrogel system is enabled to spatiotemporally trigger the MSC behaviors. Thus a combinatory role of BGn, i.e., the delivery of extrinsic growth factor (FGF18) and intrinsic ions (Si and Ca), is envisaged to synergize in stimulating MSCs in the hydrogel depot. We characterize the nanocomposite hydrogels briefly, examine the effects of BGn on the cell survivability and differentiation capacity, and then detail the delivery of FGF18 and the impact on the osteogenic promoting events, including bone matrix formation and the underlying signaling of endogenously produced bone morphogenetic protein (BMP).



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2. Experimental details 2.1. Large pore BGn and nanocomposite gel As the nanocomponent for the gel, BGn with a large mesopore size was prepared according to the 24

procedures described previously with slight modification. One gram of cetrimonium bromide (CTAB) was dissolved in an emulsion made of 150 mL DW, 2 mL aqueous ammonia, 40 mL ethyl ether, 20 mL ethanol, and 0.1125 g calcium nitrate (Ca(NO3)2.4H2O). After vigorously stirring at room temperature for 30 min, 600 µl tetraethyl orthosilicate (TEOS) was added to the mixture. The molar ratio of Ca:Si is 15:85. The resulting mixture was vigorously stirred at 30°C (a temperature determined to increase the pore size) for 4 h. A white precipitate was obtained, filtered, washed with pure water, dried in air at 60°C for 24 h, and calcined at 550°C for 5 h to prepare BGn. The BGn/Col nanocomposite gel was prepared as follows: BGn were added to collagen solution at varying ratios (1:1, 2:1 and 4:1 by weight), and the mixture was homogenized, after this, 0.5 ml 10xDMEM and 0.1 M NaOH were added optimally to neutralize the gel o

21

and to allow gelation at 37 C. The gelation time was recorded using a tube inversion test.

The micro- and nano-structure of the composite gel was observed by scanning electron microscopy (SEM; JSM-6500F, JEOL, Japan) and field emission SEM (FE-SEM; S-4300, JEOL, Japan). The thermal behavior of the gels was characterized by differential scanning calorimetry (DSC: Q-20, TA, USA). Samples were heated from 25 to 100ºC at a ramping rate of 5ºC in air, and the endothermic peak and Tg value were obtained. The mechanical properties of the gels were measured by dynamic mechanical analysis (DMA; MetraVib, DMA25N) using a parallel plate configuration. Each gel was molded into a cylinder (6 mm diameter x 4 mm height) and the mechanical testing was performed using dynamic frequency sweep (0.1 to 10 Hz) at 37ºC and with strain amplitude of 5%. The force was ramped from 0.001 to 0.2 N and the maximum allowed strain was set at 10%. The storage modulus (E′) and loss modulus (E″) of the samples were measured. 2.2. Preparation of recombinant FGF18 As an osteogenic stimulating molecule, FGF18 in a recombinant form was used. For the FGF18 production, the E. coli cells were grown overnight in a LB-Amp medium at 37°C. When the culture reached an A600 value of 0.5, induction was initiated with 0.02% (w/v) L-arabinose. After 3 h, bacteria were pelleted by centrifugation, lysed, and sonicated. Crude protein from cell extract was purified by binding His-tag (located at the N-terminal end of FGF18) to the nickel-nitrilotriacetic acid resin column. The purity of the recombinant protein was examined under denaturing condition by coomassie blue staining of 12% SDS-PAGE gel. Upon induction with L-arabinose, E. coli TOP10 produced a protein of molecular weight (MW) ca. 25 kDa (as estimated by SDS-PAGE), which was the size expected for a fusion protein consisting of FGF18 and the amino-terminal His6 tag. The expression of recombinant FGF18 was confirmed by western blotting using monoclonal anti-polyhistidine antibody, as detailed elsewhere. 2.3. Loading and release of FGF18


26

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The loading of proteins onto BGn was first investigated using a model protein cytochrome C (cyt C) (Sigma–Aldrich, Cat No. 12222). One milligram of BGn was dispersed in various concentrations of cyt C solutions made in PBS, and incubated at 37ºC for 6 h. After centrifugation, the absorbance of supernatant was measured using a UV–Vis spectrophotometer (Libra S22, Biochrom, UK), and the loading amount was deduced from the value. For the cyt C release test, 10 mg of sample was incubated in 5 ml of PBS at 37ºC and the amount of cyt C released was studied for up to 21 days. The medium was refreshed at each run of the measurement and the cumulative values were recorded. After this, FGF18 release study was carried out. To track the release profile, FGF18 was conjugated with green florescence protein (FGF18-GFP), which allows the fluorescence-based retention assay.

27,28

After

loading 40 µg/ml FGF18 onto 1 mg BGn (a condition to ensure a complete loading), the FGF-BGn was centrifuged, and then redispered in PBS and incubated at 37ºC. At each time point, the supernatant of FGF18 released was analyzed by the fluorescent assay. The fluorescent intensity was quantified by using a florescence plate reader. The release of FGF18 from the BGn/Col gel was then examined after preparing the FGF18-BGn/Col nanocomposite gel. The gel sample was placed in each well of 24-well plates, and incubated in 1 ml of PBS at 37ºC, with the medium renewed every day. The fluorescent intensity of each medium was recorded for up to 21 days. Moreover, the fluorescence intensity of gel images was also recorded for a longer period of incubation (up to 4 weeks). 2.4. Rat mesenchymal stem cell culture Mesenchymal stem cells (MSCs) derived from rat bone marrow were harvested from the femora and tibiae of adult rats (180–200 g). The harvested product was centrifuged and the supernatant was collected and suspended within a culture flask containing a normal growth medium, which consists of αminimal essential medium (α-MEM) supplemented with 10% fetal bovine serum (FBS) and 100 U/ml penicillin and 100 mg/ml streptomycin in a humidified atmosphere of 5% CO2 in air at 37°C. After incubation for 1 day, the medium was refreshed and cultured until the cells reached near confluence. The cells were sub-cultured by trypsinization and maintained in the normal culture condition. Cells at 2–3 passages were used for further tests. 2.5. Cell encapsulation in gel 4

Each hydrogel encapsulating 3 x 10 cells was prepared in each well of 24-well plates (a dimension of 15 mm diameter x 6 mm height). After 1 h of incubation, 2.5 mL additional medium was added to each well and cultured in a humidified incubator at 37ºC with 5% CO2. Culture medium was changed every three days until harvest. For the osteogenesis of cells, an osteogenic medium was used; normal growth medium (described above) plus 10 mM beta-glycerophosphate, 50 µg/ml ascorbic acid and 10 nM dexamethasone. To harvest cells from the gel, type I collagenase was used. In brief, the medium was



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removed and the hydrogel was washed (3x) with Dulbecco's phosphate-buffered saline (DPBS) and subjected to an enzymatic treatment with 2 mg/ml collagenase (type I) solution diluted in α-MEM, and prefiltered with 0.45 µm syringe filter (Millipore, USA) for 20 min at 37ºC with 5% CO2 followed by centrifugation at 1500 rmp for 5 min. The cell pellets collected were used for the further cellular assays (viability, mRNA level, and protein production). 2.6. Cell proliferation and viability Samples were analyzed at 1, 3, 7, 14 and 21 days of culture in a growth medium. At each period, the cellgel constructs were washed (3x) with PBS, followed by a replacement with 700 µl medium containing 70 o

µl MTS reagent. The treated sample was incubated at 37 C for 3 h, and 100 µl of the formazan color formed was transferred to a 96 well plate and analyzed at an absorbance of 490 nm (biorad iMark) in triplicate. The cell survivability was examined by Live/Dead assay (Invitrogen Corporation, Carlsbad, CA). For the cell images, samples were fixed with 4% PFA for 10 min, permeabilized with 0.1% Triton X-100 for 5 min, blocked with 1% BSA, and then incubated with Alexa fluro 546 (Invitrogen) phalloidin stain. The nuclei were stained with DAPI followed by mounting with anti-fade VICTASHEILD (H-1000). The images were captured using a confocal laser scanning microscope (M700, ZEISS, Germany). Three replicate samples were used for each condition (n = 3). 2.7. DNA quantification The cell growth was also quantified by using the double-stranded DNA detection kit (Quanti-iT Picogreen, Invitrogen). For this, the cultured gels were washed and then dissolved with type I collagenase (2 mg/ml) (Sigma) to collect cell pellet, as described above. The DNA quantity was measured by means of fluorescence intensity according to the manufacturer's instructions. Three replicate samples were used for each condition (n = 3). 2.8. Alkaline phosphatase (ALP) activity Samples cultured for up to 21 days in an osteogenic medium were used for measuring ALP activity. The cell pellets were gathered from the gels, and added with cell lysis buffer (0.1% triton X-100, 1 M tris-HCl, 5 M NaCl, and 0.5 M ethylenediaminetetraacetic acid). The enzymatic product, p-nitrophenol (pNP), formed via alkaline phosphatase reaction in the presence of MgCl2, was quantified by the absorbance at 405 nm using a spectrophotometer. The ALP activity level was normalized to dsDNA. Three replicate samples were used for each condition (n = 3). 2.9. Gene expression analysis After culture, cells were gathered from the gels. RNA samples (1 µg) isolated using RNA isolation kit (RNeasy mini kit 74104, Qiagen, CA) were reverse transcribed to cDNA in 40 µl using Quantitect RT kit o

(Qiagen) according to manufacturer’s protocol. The reaction was allowed to proceed at 95 C for 5 min. A similar reaction mixture without the reverse transcriptase enzyme was prepared and used as a template to demonstrate the absence of contaminating genomic DNA. One microliter of cDNA was subjected to


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qPCR amplification in spectrofluorometric thermal cycler (Rotor-Gene 3000, Corbett Research, Korea). The primer sequences of genes (Bioneer, Korea) used in this study are summarized in Table 1. PCR o

o

o

reactions were carried out at 95 C for 30 s, 58 C for 30 s, and 75 C 60 s for 40 cycles, and performed in triplicate. The CT value of target genes was normalized to endogenous GAPDH transcripts to obtain -∆∆Ct

finally 2

value expressed in fold expression. Table 1. Primary sequences of genes used for RT-PCR.

Primer

Forward primer

Reverse primer

GAPDH

5’-CTGGAAGATGGTGATGG-3’

5’-GATTTGGTCGTATTGGGCG-3’

Col I

5’-GAGAGGTGAACAAGGTCCCG-3’

5’-AAACCTCTCTCGCCTCTTGC-3’

OCN

5’-AGACTCCGGCGCTACCTCAACAAT-3’

5’-CAGCTGTGCCGTCCATACT-3’

OPN

5’-GCTGAAGCCTGACCCATCTC-3’

5’-TGCTTGGAAGAGTTTCTTGCTTAA-3’

BSP

5’-ACAGCTGACGCGGGAAAGTTG-3’

5’-ACCTGCTCATTTTCATCCACTTC-3’

ALP

5’-ACTGGTACTCGGACAATGAG-3’

5’-ATCGATGTCCTTGATGTTGT-3’

BMP2

5’-CGTCAAGCCAAACACAAACAGC-3’

5’-GAGCCACAATCCAGTCATTCCAC-3’

BMPR1A

5’-GATTCACCAAAAGCCCAG-3’

5’-CCCATCCATACTTCTCCATA-3’

BMPR1B

5’-CTCTGGGAGATTGCAAGGAG-3’

5’-TCATAAGCTTCCCCATTTGC-3’

BMPR2

5’-GCT TCGCAGAATCAAGAACG-3’

5’-GTGGACTGAGTGGTGTTGTG-3’

Smad1

5’-TTGTTTAGAAATGAATGGGTT-3’

5’-ACAGTTAGAGGAATTAACCAGCTG-3’

Smad5

5’-CAGATGGGCTCTCCGCTGAAC-3’

5’-TCGTTTACAATACTTTTGAAA-3’

Smad8

5’-ACTTCCGGCCAG TTTGCTAC-3’

5’-TGGGGATCTTGCAGACAGTG-3’

2.10. Immunocytochemistry The production of bone matrix proteins (OPN, BSP, and OCN) was revealed by co-fluorescence immunostaining with mouse anti-OPN antibody (1:100, Santa Cruz Ins), anti-BSP antibody (1:50, Hybridoma bank, IOWA), and rabbit anti-OCN antibody (1:100, Santa Cruz Ins). After 14 and 21 days of culture, the cell-gel constructs were fixed with 4% PFA for 5 min. The samples were then combined with OCT for the cryosectioning to prepare ~30 µm thickness sections. Sectioned samples were air-dried at room temperature, and the OCT compound was removed by washing with PBS solution (3x). Samples were permeabilized with 0.1% Triton X-100 for 10 min, and then blocked with 1% BSA, after which they º

were probed with primary antibodies incubated overnight at 4 C, washed with PBS (3x), and incubated with secondary antibodies labeled Rhode for anti-mouse primary antibody and FITC labeled secondary antibody for anti-rabbit primary antibody. The nuclei were stained with DAPI, mounted with VECTOSHEILD for anti-fate, and then the images were captured using a confocal microscope (M700, ZEISS, Germany). 2.11. Western blot analysis



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Western blot analysis was carried out to quantify the production of bone matrix proteins. The extracts of the harvested cells were prepared with lysis buffer (Pre-Prot, iNTRON, South Korea), after which protein samples were resolved on 8% sodium dodecyl sulphated/polyacrylamide gels, transferred to nitrocellulose (NC) membranes, blocked with 5 % fat free milk in Tris-buffered saline with 0.5 % Tween-20 for 1 h at room temperature, and then incubated with primary antibodies: rabbit anti-OCN, -BSP, SMAD1/5/8 antibody and mouse anti-OPN, -BMP2 antibody (Santa Cruz In, Dilution 1:200) overnight at o

4 C. The blots were then incubated with horseradish peroxidase-conjugated anti rabbit or mouse secondary IgG for 1 h at room temperature, and immunoreactive bands were detected using ECL detection reagent (Pierce, Rockford, IL). The band imaging and intensity was analyzed by ImageQuant LAS4000 mini (GE Healthcare, CA) when normalized to endogenous GAPDH or β-actin expression. The experiments were performed in triplicate. 2.12. Statistics Data were presented as the mean ± one standard deviation. Statistical comparisons were made using the Student’s t-test. P < 0.05 was considered to be statistically significant.

3. Results and Discussion 3.1. Nanocomposite gel organized at the nanoscale with high physico-chemical stability As the functional nanocomponent for the stem cell culture depot, bioactive and mesoporous nanospheres (BGn) were introduced within collagen gel; in particular, the mesopores were enlarged to deliver osteogenic signaling protein. The TEM images of BGn revealed uniform sized nanospheres with highly mesoporous structure (Fig. 1a). The properties of BGn summarized also showed high mesoporosity, 2

3

including high surface area (554 m /g) and mesopore volume (0.67 cm /g), and large pore size (10.3 nm). The ζ-potential of BGn was negative (-17.6 mV). These characteristics of BGn are effective in loading target growth factor, FGF18, which has a molecular dimension of 25 kDa, a ligand affinity-dissociation constant (KD) of 38 nM and a net positive charge at physiological pH.

26,29,30


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Fig. 1. Characteristics of BGn and BGn/Col gel. (a) TEM images of BGn at different magnifications and the characteristics summarized. (b,c) SEM images of freeze-dried gels at different magnifications; (b) Col and (c) BGn/Col.

The Col gel, when freeze-dried, showed fibrous networks and nanofibrillar structure at high magnification, 31,32

a feature typical of collagen-based scaffolds (Fig. 1b).

The BGn/Col gel matrix also showed a highly

fibrous scaffold structure, and a closer examination revealed a number of nanospheres intercalated in the collagen nanofibrils (Fig. 1c). The physico-chemical properties of the gels are summarized in Table 2. The gelation time of the BGn/Col was recorded to be shorter than that of pure Col gel (73 s vs. 126 s). o

The thermal behavior of the nanocomposite gel showed a higher glass transition temperature (Tg; 67 C o

vs. 47 C), implying the BGn addition increased the thermal stability of the gel. The mechanical properties of gels were then characterized by a dynamic mechanical analyzer. The nanocomposite gel showed enhanced storage modulus (E’) than Col gel (153 kPa vs. 89 kPa), indicating an increase in stiffness. The shortened gelation time, and the enhanced thermal and mechanical stability were due to the nanoparticle-collagen fibril interactions.

33,34

In fact, the non-covalent and weak charge-charge interactions 2+

between the amino acids in collagen and the hydroxyl groups and ions (e.g., Ca ) in silica-based bioactive glasses have also been implicated.

21,35

Even though the collagen gels have shown excellent

36

cellular compatibility, the poor physical and mechanical stability, and the accompanied early degradation and gel shrinkage limit potential applications for the cell culture matrix in hard tissues.


34,37

Thus methods


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to tackle this issue have been developed, including physical gel compression, blending with other natural polymers, and calcium phosphate addition,

38-40

and the current approach of using BGn is considered an

alternative promising way of improving those properties; even more, the capacity of BGn to release ions and deliver signaling molecules – a therapeutic role to be played in stimulating stem cells within the gel matrix – is also envisaged. Table 2. Physico-chemical properties of gels. Col gel

BGn/Col gel

Gelation Time (Sec)

126

73

DSC Tg (Degree)

47.1

67.3

E′ (kPa)

89.2 ± 5.9

153.9 ± 1.5

E″ (kPa)

6.53 ± 0.58

14.1 ± 3.8

tanδ

0.07 ± 0.01

0.09 ± 0.03

3.2. BGn/Col gel as a 3D depot for rMSC cultivation and osteogenic commitment The BGn/Col gel could encapsulate cells during the gelation. Rat MSCs (30,000) were encapsulated within 100 µl of each gel, and the survivability during culture for up to 7 days was recorded by a live/dead stain assay (Fig. 2a,b). Cells at day 1 to 7 were highly survivable within Col gel (with 80-90% of live cell fractions). Within BGn/Col gel, the cells appeared to show low survivability initially at day 1 (~60% survivability), which however, recovered quickly with prolonged cultures (~70% and 80% survivability at day 3 and 7, respectively). The initially low cell viability was due to the possible pH rise associated with 2+

the cationic (mostly Ca ) release from BGn, and this has often been observed in the culture with bioactive glasses.

41-43

This phenomenon is considered to be spatiotemporal as the cells regain the

viability shortly after a few days, when the cells become more stable supported by substantially enhanced anchorages to a surrounding matrix and neutralized medium conditions. The cytoskeletal morphology of rMSCs, as observed by confocal microscopy, revealed an active extension of α-actins progressively with culture time in both gels (Fig. 2c). The MTT assay was further conducted to ascertain the long-term cellular viability within the gels up to 21 days (Fig. 2d). The cell viability in a growth medium gradually increased with time for both gels. Although the cells in Col-BGn gel showed somewhat lower cell viability at the very initial period (within 3 days, similar to the finding and discussion in live/dead cell assay), they regained the proliferative capacity quickly, showing the growth pattern similar to that in Col gel, implying that the gel matrix provided a favorable cell proliferation environment. The possibility of the rMSCs encapsulated in the gel to undergo osteogenic commitment under an osteogenic medium was briefly examined by the ALP activity, an early osteogenic marker (Fig. 2e). The


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ALP activity increased with time from 7 to 14 days in both gels, a phenomenon widely observed in MSCs, i.e., time-dependent ALP increase in an osteogenic medium. Noteworthy was the significantly higher ALP level recorded at day 14 in the nanocomposite gel than in the Col gel, suggesting the BGn were influential on the osteogenesis of rMSCs. The ions (Ca and Si) possibly released from the BGn may stimulate the osteogenic commitment of rMSCs, as this has also been implicated in the culture of osteoblasts and 44,45

MSCs with other bioactive glasses and related composites.

Taken from the results, the BGn-Col

nanocomposite gel is thought to provide rMSCs appropriate 3D matrix conditions to multiply and ionic cues to develop into bone forming cells.

Fig. 2. Effects of BGn on rMSC behaviors in Col gel. (a,b) Cell survivability in gel; live/dead cell stained image and the quantification at day 1, 3 and 7. (c,d) Cell proliferation in gel; cell morphology at



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day 3 and 7 observed by F-actin staining with alexafluro 546 and the proliferation kinetics up to 21 days recorded by MTS method. (e) ALP activity of cells showing an osteogenic commitment. The ALP level was normalized to dsDNA content at day 7 and 14. Scale bar = 100 µm. Statistical significance considered at **P < 0.01 for n = 3.

3.3. BGn/Col gel as an osteogenic reservoir of slowly-releasing growth factor FGF18 In order to confer more osteopromoting environment to MSCs, FGF18 was loaded onto BGn prior to a gelation with collagen. The FGF18 to be released from BGn is considered to recapitulate the microenvironment of MSCs, signaling osteogenic responses in the vicinity (as illustrated in Fig. 3a). FGF18, as one of the growth factors in FGF family, is involved in the osteochondral bone development through the regulation of chondrogenesis and osteogenesis.

46,47

Furthermore, the presence of FGF18 is

known to up-regulate the production of bone morphogenetic protein 2 (BMP2), a key molecule in the osteogenic maturation and bone formation.

48-50

To establish the loading and release conditions, cyt C

protein was pre-tested as the model molecule. Varying concentrations of cyt C were first loaded onto 1 mg of BGn in 5 ml PBS for 6 h. Results showed a high loading capacity of ~10% (0.1 mg cyt C maximally loaded per 1 mg BGn), which was due to the large sized pores (10.3 nm) and highly mesoporous nature of nanoparticles. The release study of cyt C from BGn performed in PBS showed a gradual release for up 2

to 10 days (Fig. 3b). The release pattern fitted well (R value = 0.98) to the Ritger–Peppas empirical equation, and the kinetic exponent n value was 0.29, implying the release mechanism was anomalous diffusion-controlled, a phenomenon widely observed in other mesoporous nanoparticles.

51,52

Based on this, we incorporated FGF18 within BGn at the loading amount of 40 µg FGF18 per 1 mg BGn, which is considered to load the FGF18 almost completely and the level considered to have a therapeutic efficacy (Fig. 3c). The release of FGF18 from BGn was also recorded by the fluorescent intensity of GFP which was tagged to FGF18. The release profile was similar to that observed in cyt C, i.e., FGF18 released gradually up to 10 days without showing an initial burst release. Next, the FGF18 release from the BGn-Col gel matrix was recorded. The FGF18 released was quantified by the GFP fluorescence intensity (Fig 3d). The FGF18 release from the gel matrix was very slow, profiling an almost linear pattern 2

with time for up to 4 weeks; a linear regression revealed a linearity of 0.384 with R of 0.96. This near zero-order kinetic pattern of molecules from the biomaterials has been recognized to be optimal as the therapeutic molecules can be released at a constant rate for a long-term period. The fluorescence retention image of gels was also captured during the incubation for long-term periods of up to 7 weeks (Fig 3e). The fluorescence level decreased very slowly, suggesting a high retention of FGF18 in the nanocomposite gel.


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Fig. 3. FGF18 delivery system through BGn/Col gel. (a) Illustration of therapeutic nanocomposite gel depot, where FGF18 delivered through BGn/Col signals and stimulates MSCs. (b) Cyt C release profile from BGn used as a model protein. (c) FGF18 release profile from BGn. (d) FGF18 release profile from BGn/Col gel, measured up to 21 days. GFP-tagged FGF18 used for the fluorescence intensity measurement during incubation. (e) BGn(FGF18)/Col gel mages showing the fluorescence retained over a long-term incubation period (4 weeks), and the intensity quantified.



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Collectively, while the FGF18 was released from the BGn pore channels through a diffusion-controlled mechanism, up to ~10 days, with a slow-down in release rate with time, the overall release from the BGn/Col gel through the matrix was highly prolonged over weeks to months and profiled almost zeroorder kinetics (at least up to 21 days, a measured period); the results suggest the BGn nanocarrier and collagen matrix played a combined role in sustaining the FGF18 release. It is considered that the FGF18 molecules released from BGn might bind stably to collagen fibrils, as collagen amino acid sequences have also been shown to enable chemical interactions with many growth factors, including transforming growth factor beta 1 (TGF-β1) , fibroblast growth factor 2 (FGF2), and vascular endothelial growth factor 9, 53,54

(VEGF),

which yet remained as further investigation.

3.4. FGF18-releasing BGn/Col gel in the osteogenesis promotion of MSCs After confirming the nanocomposite gel could release FGF18 slowly and constantly we next examined the biological effects on rMSCs encapsulated in the gel. First, we embedded FGF18-BGn locally in one part of the gel to check if any chemo-attractant (otherwise chemo-repulsive) influence on the surrounding MSCs (as depicted in Fig. 4a). As shown in the CLSM image, the rMSCs appeared to be attractant toward the embedded FGF18-BGn, showing a cellular migration and a gradient cell density along the FGF18-direction. This demonstrates the release of FGF18 to the surrounding gel matrix where MSCs are present and the possible triggering of cells through FGF18. In fact, FGF18 has also been reported to enhance the chemotactic cellular migration.

55,56

Next, the FGF18-BGn was homogeneously distributed in the gel and the MSC proliferation was examined. At 3 and 7 days, the cell morphology was observed by confocal microscopy (Fig. 4b). Cells extended cytoskeletal processes actively in both gels (containing FGF18 or not). The dsDNA content was quantified for up to 14 days. Cells cultured in FGF18-free gel grew continuously with time until 14 days. On the other hand, the FGF18-releasing gel stimulated the cell proliferation only by 7 days, after which the cellular growth appeared to cease and even dropped (Fig. 4c). The on-going increase in rMSC proliferation in a normal growth medium has been well documented, however, a drop in proliferative potential at a prolonged period often signs a switch to differentiation, where osteogenic molecules are supplemented or other osteopromoting parameters are engaged.

57

Therefore, the rMSCs cultivated in the FGF18-releasing

gel are thought to undergo substantial differentiation at this culture period (14 days); based on this the FGF18 released is considered to accelerate osteogenesis of rMSCs and can shorten the differentiation time, which assists in designing further differentiation tests.


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Fig. 4. Effects of FGF18 release on the rMSC migration and proliferation. (a) Design to examine the FGF18-attractant effect on cell migration; FGF18-BGn was embedded locally in one part of the gel and confocal microscopy of cell image showing migratory-stimulatory effect. (b,c) The FGF18 released from BGn distributed homogenously in gel stimulated early phase of cell proliferation; confocal cytoskeletal extension image at day 3 and 7 (b), and dsDNA quantification up to 14 days (c). (Statistical significance considered at *P < 0.05 and **P < 0.01, n = 3).

To further verify the osteopromoting role of FGF18 within the gel, the ALP activity of cells was assessed at early time points (up to 7 days). Cells cultured in the BGn/Col gel free of FGF18 showed a small change in ALP activity with time, whereas those in FGF18-releasing gel exhibited significantly stimulated ALP level at day 7 (Fig. 5a). The expression of a series of osteogenic genes including Col I, ALP, BSP, OPN and OCN, was then time-sequence analyzed. While Col I and ALP are considered relatively initial markers, BSP, OPC and OCN are mid-to-late markers of osteogenesis of rMSCs.

58

There was little

change in Col I expression with time in both gels with and w/o FGF18. The ALP gene was expressed at significantly higher levels (2~4 fold increase) in FGF18-releasing gel. Of note was the expression of BSP, OPN and OCN genes in the FGF18-releasing gel particularly at 21 days; 10 to 20 times higher than that in FGF18-free gel. The gene expression results confirmed the FGF18 release accelerated the rMSCs toward osteogenesis through the culture period, with time-sequenced up-regulation of related osteogenic markers.



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Fig. 5. Osteogenesis of rMSCs promoted by the FGF release in the BGn/Col gel. (a) ALP activity at day 7 and 14, normalized to total dsDNA. (b) mRNA levels of bone specific makers expressed in MSCs, as measured by RT-PCR at day 7, 14, and 21. The genes representative of early-, mid- and late-stage osteogenesis including Col I, ALP, BSP, OPN, and OCN were analyzed. Each gene level normalized to endogenous GAPDH. Statistical significance considered at *P < 0.05 and **P < 0.01 for n = 3.

The production of proteins was further analyzed. Western blotting of key osteogenic proteins involved in mid-to-late stage (BSP, OPN and OCN) showed clear band expressions of all proteins. The BSP expression was clear at day 14 although the signal was attenuated at day 21. Above all, the OPN band was the strongest among the proteins at both 14 and 21 days, and the OCN band was also clear at both periods (Fig. 6a). When the band intensities were quantified, the FGF18-releasing group produced those osteogenic proteins to significantly higher levels, and particularly the production of OPN was the most contrasted between the gels with and without FGF18. The immunochemical staining of cells qualitatively supports the protein production (Fig. 6b). The significantly higher secretion of osteogenic proteins indicates MSCs have already committed to produce a mature gel matrix that mimics the native bone ECM. In particular, OPN and OCN have been recognized to get heavily involved in cellular calcium deposition and mineral nodule formation, a status considered a final in vitro fate of cells those committed bone


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formation and this is also envisaged in our system based on the series of gene and protein expressions, which though needs clarification in further studies.

Fig. 6. Osteogenic protein expressions in rMSCs stimulated by the FGF18 delivery. (a) Western blot analysis and (b) immunocytochemical staining at day 14 and 21. Essential osteogenic markers at mid-tolate stage differentiation, OPN, BSP and OCN, were analyzed. Western band intensities were quantified by densitometer and normalized to GAPDH (n = 3). Scale bar = 100 µm. Statistical significance considered at *P < 0.05 and **P < 0.01 for n = 3.

3.5. FGF18-activated BMP2 production and the signaling events Those osteogenic events of rMSCs stimulated by the FGF18 release were considered to involve BMP2, as FGF18 has recently been shown to active the endogenous production of BMP2 through Smad signaling pathways.

49,50

We thus analyzed the gene expression of FGF18-activated BMP2 and the related

molecules in the Smad signaling process, including BMP2-receptors (R1 and R2) and Smad1/5/8. First,



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the expression of BMP2 gene was shown to increase with time in the FGF18-releasing gel, and the gene level was significantly higher than in the gel without FGF18 (Fig. 7a). The gene expressions of other molecules in the BMP signaling process was also analyzed. BMP receptor 1 (BMPR1 both A and B subunits) was significantly up-regulated in the FGF-releasing gel as early as day 3; however, BMPR2 was not significantly altered by the FGF2. Among the Smads analyzed, Smad 1 and 8 were highly upregulated but Smad 5 was not in the FGF-releasing gel (Fig. 7b). The western blot analysis of representative proteins (BMP2 and phosphorylated form of Smad1/5/8) further clarified the significantly activated protein molecules in the FGF18-releasing gel, particularly at day 14 (Fig. 7c). The FGF18 has recently proven to activate BMP receptor, especially receptor 1 (R1), which is primarily related with the osteogenic stimulation of cells and is directly linked with Smad1/5/8 signaling process where Runx2 is a key transcription factor.

59,60

Fig. 7. FGF18-activated BMP signaling in rMSCs. The expression of associated molecules including BMP2, BMP receptors, and Smad1/5/8 was analyzed. (a) Gene expression of BMP2. Band intensities were quantified by densitometer and normalized to GAPDH (n = 3). (b) Expression of internal signaling molecules, BMP receptors (BMP1A, BMP1B, BMPII) and Smad genes (SMAD1, SMAD5, and SMAD8). (c) Western blot analysis of BMP2 and phosphorylated Smad1/5/8. Western band intensities were quantified by densitometer and normalized to α-actin (n = 3).


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The signaling events that stimulate rMSCs encapsulated in the FGF18-releasing BGn-containing nanocomposite gel matrix toward an osteogenic differentiation are schematically illustrated in Fig. 8. The released FGF18, not only is signaled through FGFR, but also activates BMP2 receptor (mainly R1) in rMSCs, and the intracellular signal processes through Smad1/5/8 and the stimulated Runx2 transcriptional factor in turn up-regulate the secretion of BMP2 as well as osteogenic molecules (BSP, OPN and OCN). The endogenously produced BMP2 contributes paracrine stimulation of osteogenesis while the FGF18 slowly releases to cycle the signaling process. The produced bone matrix proteins further accelerate cellular maturation at prolonged culture periods, where the Ca and Si ions released from BGn can also support the mineralization, mimicking bone equivalent tissues. Therefore, the currently designed rMSCs-encapsulating gel depot may be i) developed through long-term cultures ex vivo into bone-mimic constructs, or ii) implanted in vivo to provide osteopromoting signaling environments for rMSCs to actively engage in regenerating bone defects. Here we used FGF18 as one of the potent BMP2 signaling molecules, instead of directly using BMP2. For sure, the biological roles and effects of FGF18 and BMP2 are different. Not just the effect on osteogenesis and bone formation, but other important roles of FGF18 have also been found in the biological interactions with cells, including the growth of stem cells, chemo-attractive cell migration, and the repair and regeneration of soft tissues.

55,56,61,62

In fact, the use of BMP2, even with its potent

osteoinductive effects on bone formation, has often resulted in abnormal bone formation in spine canal, osteoclast activation and tissue inflammation in clinics, which considered a drawback for the extensive use of BMP2.

63-65

Thus, the use of FGF18 is hoped to relieve some of this aspect of BMP2 while

reinforced with other biological responses such as the proliferation and migration of stem cells, which is yet to be clarified thus needs further investigations. As demonstrated, here we observed the potential of current designed gels through a series of in vitro events, however, the findings need confirmation under in vivo conditions where the possible difference in therapeutic range of growth factors released and the complicated cellular interactions with native tissue environments should also be considered, which warrants further study.



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Fig. 8. Schematic illustration of the osteogenic promoting events in the current system. The FGF18 delivery through BGn/Col gel triggered the encapsulated rMSCs through BMP receptor and the Smad signaling molecules, and the endogenously produced BMP2 in turn stimulated a series of osteogenic processes in rMSCs, including the expression of osteogenic markers and maturation. The Ca and Si ionic releases from BGn also play additional roles.


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4. Conclusions An osteopromoting gel matrix made of BGn/Col nanocomposite with FGF18 was designed to cultivate rMSCs and to drive their potential osteogenesis. The bioactive nanocomponent BGn was effective in stimulating osteogenic differentiation of rMSCs, thanks to the intrinsic properties like ionic release. More potentially, the FGF18 loaded within BGn was delivered sustainably to the surrounding rMSCs, and accelerated osteogenesis as early as 1-2 weeks, with significant expressions of osteogenic markers at the gene and protein level. This event was ascribed to the FGF18-activated BMP2 production through Smad 1/5/8 signaling pathway, where the endogenous BMP could in turn stimulate the secretion of osteogenic bone matrix proteins. The currently designed BGn-FGF18/Col gel can be a potential therapeutic depot to culture rMSCs and their accelerated osteogenesis and maturation for bone tissue engineering.

Acknowledgement The work was supported by Global Research Laboratory Program (2015-032163) and Priority Research Centers Program (2009-0093829), National Research Foundation, Republic of Korea.



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Legend of Figures & Tables

Fig. 1. Characteristics of BGn and BGn/Col gel. (a) TEM images of BGn at different magnifications and the characteristics summarized. (b,c) SEM images of freeze-dried gel samples at different magnifications; (b) Col and (c) BGn/Col. Fig. 2. Effects of BGn on rMSC behaviors in Col gel. (a,b) Cell survivability in gel; live/dead cell stained image and the quantification at day 1, 3 and 7. (c,d) Cell proliferation in gel; cell morphology at day 3 and 7 observed by F-actin staining with alexafluro 546 and the proliferation kinetics up to 21 days recorded by MTS method. (e) ALP activity of cells showing an osteogenic commitment. The ALP level was normalized to dsDNA content at day 7 and 14. Scale bar = 100 µm. Statistical significance considered at **P < 0.01 for n = 3. Fig. 3. FGF18 delivery system through BGn/Col gel. (a) Illustration of therapeutic nanocomposite gel depot, where FGF18 delivered through BGn/Col signals and stimulates MSCs. (b) Cyt C release profile from BGn used as a model protein. (c) FGF18 release profile from BGn. (d) FGF18 release profile from BGn/Col gel, measured up to 21 days. GFP-tagged FGF18 used for the fluorescence intensity measurement during incubation. (e) BGn(FGF18)/Col gel mages showing the fluorescence retained over a long-term incubation period (7 weeks), and the intensity quantified. Fig. 4. Effects of FGF18 release on the rMSC migration and proliferation. (a) Design to examine the FGF18-attractant effect on cell migration; FGF18-BGn was embedded locally in one part of the gel and confocal microscopy of cell image showing migratory-stimulatory effect. (b,c) The FGF18 released from BGn distributed homogenously in gel stimulated early phase of cell proliferation; confocal cytoskeletal extension image at day 3 and 7 (b), and dsDNA quantification up to 14 days (c). (Statistical significance considered at *P < 0.05 and **P < 0.01, n = 3). Fig. 5. Osteogenesis of rMSCs promoted by the FGF release in the BGn/Col gel. (a) ALP activity at day 7 and 14, normalized to total dsDNA. (b) mRNA levels of bone specific makers expressed in rMSCs, as measured by RT-PCR at day 7, 14, and 21. The genes representative of early-, mid- and late-stage osteogenesis including Col I, ALP, BSP, OPN, and OCN were analyzed. Each gene level normalized to endogenous GAPDH. Statistical significance considered at *P < 0.05 and **P < 0.01 for n = 3. Fig. 6. Osteogenic protein expressions in rMSCs stimulated by the FGF18 delivery. (a) Western blot analysis and (b) immunocytochemical staining at day 14 and 21. Essential osteogenic markers at mid-tolate stage differentiation, OPN, BSP and OCN, were analyzed. Western band intensities were quantified by densitometer and normalized to GAPDH (n = 3). Scale bar = 100 µm. Statistical significance considered at *P < 0.05 and **P < 0.01 for n = 3.


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Fig. 7. FGF18-activated BMP signaling in rMSCs. The expression of associated molecules including BMP2, BMP receptors, and Smad1/5/8 was analyzed. (a) Gene expression of BMP2. Band intensities were quantified by densitometer and normalized to GAPDH (n = 3). (b) Expression of internal signaling molecules, BMP receptors (BMP1A, BMP1B, BMPII) and Smad genes (SMAD1, SMAD5, and SMAD8). (c) Western blot analysis of BMP2 and phosphorylated Smad1/5/8. Western band intensities were quantified by densitometer and normalized to α-actin (n = 3). Fig. 8. Schematic illustration of the osteogenic promoting events in the current system. The FGF18 delivery through BGn/Col gel triggered the encapsulated rMSCs through BMP receptor and the Smad signaling molecules, and the endogenously produced BMP2 in turn stimulated a series of osteogenic processes in rMSCs, including the expression of osteogenic markers and maturation. The Ca and Si ionic releases from BGn also play additional roles. Table 1. Primary sequences of genes used for RT-PCR. Table 2. Physico-chemical properties of gels.



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Osteopromoting Reservoir of Stem Cells: Bioactive Mesoporous

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