Engineered Phage Matrix Stiffness-Modulating Osteogenic

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Engineered Phage Matrix Stiffness-Modulating Osteogenic Differentiation Hee-Sook Lee, Jeong-In Kang, Woo-Jae Chung, Do Hoon Lee, Byung Yang Lee, Seung-Wuk Lee, and So Young Yoo ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b17871 • Publication Date (Web): 18 Jan 2018 Downloaded from http://pubs.acs.org on January 19, 2018

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ACS Applied Materials & Interfaces

Engineered Phage Matrix Stiffness-Modulating Osteogenic Differentiation Hee-Sook Lee,1,2,† Jeong-In Kang,3,4,5† Woo-Jae Chung,6 Do Hoon Lee,7 Byung Yang Lee,7 Seung-Wuk Lee,1* and So Young Yoo,3,4,* 1

Bioengineering, University of California, Berkeley, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA; 2Ministry of Food and Drug Safety, Center for Test and Analysis, Busan 48562, Republic of Korea; 3BIO-IT Foundry Technology Institute, Pusan National University, Busan 46241, Republic of Korea; 4Research Institute for Convergence of Biomedical Science and Technology, Pusan National University Yangsan Hospital, Yangsan 50612, Republic of Korea; 5Control and Instrumentation Engineering, Korea Maritime and Ocean University, Busan 49112, Republic of Korea; 6Genetic Engineering Sungkyunkwan University, Suwon 16419, Republic of Korea; 7Mechanical Engineering, Korea University, Seoul 02842, Republic of Korea KEYWORDS: phage, matrix, stiffness, osteogenic, differentiation

ABSTRACT: Herein, we demonstrate an engineered phage mediated matrix for osteogenic differentiation with controlled stiffness by crosslinking the engineered phage displaying RGD and HPQ with various concentrations of streptavidin or polymer, PDDA. Osteogenic gene expressions showed that they were specifically increased when MC3T3 cells were cultured on the stiffer phage matrix than softer one. Our phage matrices can be easily functionalized using chemical/genetic engineering and used as a stem cell tissue matrix stiffness platform for modulating differential cell expansion and differentiation.

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1. Introduction The control of cell fate is a major interest of regenerative medicine area. For this reason, diverse materials to mimic the clues that the cells receive (including chemical and topographical cues) in their in vivo cellular environments (called “niches”) are being designed. The delivery of the form of short peptides of extracellular matrix (ECM) components on biomaterials has been proven to be successful in directing stem cell lineage.1-3 In addition, growth factors have been delivered in order to provide clues for determining stem cell differentiation via biomaterials.4 ECM-mimicking nanofibrous M13 phages after chemical or genetic modifications with cellsignaling peptides have also been considered as novel tissue engineering materials5-6 and were used to direct the desired cellular functions.1, 3, 7-9 In regulating cellular growth processes and their fate, it is well known that specific biochemical cues in tissue ECMs play a critical role. However, the role of physical/mechanical cues, such as topology and/or stiffness, has not been well studied. In terms of guiding the fate of resident stem cells, nanotopographical clues mimicking the ECM in vivo may regulate stem cell differentiation. Recently, it has been proposed that the matrix stiffness regulates stem cell differentiation and the fate of the cells by interacting with the ECM.10-11 It has been shown that the stiffness of the ECM regulates the short- and long-term cell functions, such as cell spreading and cell phenotypic changes in the substrates.12 Some studies have described the roles of mechanical cues provided by both substrate such as topography and stiffness and extrinsic microenvironment such as fluid flow, compression, hydrostatic pressure, and tension in the differentiation of MSCs as well as cellular behaviors.13-16 Phage nanofibers, such as natural biomaterial building blocks comprising refined structures, can also be utilized for studying the nanotopographical cues or stiffness effects of ECMs on cell


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differentiation through sophisticated and precise self-organization processes. We have recently established a bacteriophage-based biomimetic process,17 called “self-templating assembly,” to realize and exploit the scalable synthesis of desired nanostructures with biological functions. In addition to the bio- or chemical cues provided by the displayed functional peptides on the phage,1 the topological or mechanical cues can be provided by the biomimetic self-templating supramolecular structure of M13 phages.4 Its biomimetic, helical, monodisperse, and nanofibrous shape and its ability to display multiple functional motifs are altogether particularly advantageous for achieving the desired chemical and topographical cues. Herein, we demonstrate an engineered phage-mediated matrix with controlled stiffness for various applications and application advantages over conventional tissue engineering materials by exploiting the effects of its physical and mechanical structural features on osteogenic differentiation (Fig. 1).

2. Results and Discussion 2.1. Engineered phage-based matrix stiffness can be modulated using streptavidin or PDDA. We assembled the phage into large area spread films by pulling the substrates vertically from phage suspensions at finely controlled speeds (Fig. 1, left),17 resulting from the deposition of locally accumulated phage particles on the substrate at the meniscus via the fastest evaporation near the air-liquid-solid-contact line. Therefore, the topological and mechanical cues are modulated by phage nanofiber concentrations and the pulling speed (Fig. 1 (right) and Table S1).17


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The M13 phage is a filamentous bacterial virus mainly covered by 2700 copies of pVIII, a major coat protein comprising approximately 98% of the viral surface, on which dense and uniform peptide display with 2.7 nm spaced apart along the axis is readily available for proteinbinding interactions.18 At each end of the phage, there are minor coat proteins (pIII and pVII), of which longer amino acid sequences (compared to pVIII) can be inserted without compromising the structure or function of the phage.19 Herein, we constructed M13 phages to express integrinbinding peptides (Arg-Gly-Asp; RGD) on their pVIII major coat protein and biotin-like peptides (His-Pro-Gln; HPQ) on pIII and pVII minor coat proteins (Table 1 and Fig. 2 (top)). A detailed description of the methods regarding their construction is provided in the supporting information. The integrin-binding peptide RGD motif has been widely used for tissue engineering.20-24 Integrins are major cellular receptors binding to ECM proteins in which RGD is commonly found. We used linear RGD on the pVIII protein to prevent interference with the phage structure or propagation as well as to get better interaction with corresponding cells.25 Our previous research has reported that higher density peptides display results in better interaction between the expressed peptide on the phage body and cells.

This led to cellular responses that are

considerably better than that for the phage tail.4, 8, 23 The HPQ peptide, previously identified by through phage display, specifically binds to streptavidin.26-29 The HPQ motifs have biotin-like specificity to streptavidin.30-32 We genetically modified both pIII and pVII of phage coat proteins with circular HPQ motifs. The circular HPQ peptide produces a higher affinity for streptavidin than the linear HPQ peptide.31, 33 Integrin-binding RGD peptides were then inserted on the major coat proteins of the phage that expressed HPQ on their pIII and pVII proteins (R8H3H7, termed YSY184). Here, the HPQ motif allows binding to streptavidin while the RGD motif facilitates cell adhesion and interaction


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onto the phage matrices. HPQs on the head (pVII) and tail (pIII) of the resulting phage will easily grab streptavidin, thereby leading to the phage-to-phage crosslinking in the self-assembled phage matrix structures. Furthermore, the negatively charged phage will electrostatically interact with a positively charged PDDA polymer (Fig. 2, bottom). Therefore, streptavidin or PDDA addition to the phage matrix will provide differential matrix stiffness (for obtaining appropriate topological or mechanical cues), whereas the RGDs on the body (pVIII) of the resulting phage matrix will efficiently interact with cells (by providing cell-favoring chemical cues).

2.2. Various phage matrix stiffness ranges was produced by the combination of phage with different concentration of streptavidin. To mimic tissue stiffness in the human body which ranges from Young's Modulus E – several kPa of the adipose tissue34 to E – GPa, bone,35 The phage matrix stiffness can be controlled by crosslinking the engineered phage with different concentrations and using compositions with streptavidin or polymer mixtures. The resulting stiffness ranged between 1–3,000 kPa. We used 4 mg/mL of the phage solution and 2% PDDA or 4 mg/mL of streptavidin. The gold-coated silicon substrates were vertically pulled from the phage solution (4 mg/mL) and then sequentially from PDDA (2%) or different concentrations of streptavidin solutions (0.04 mg/mL, 0.4 mg/mL, or 4 mg/mL) using a modified computer-controlled syringe pump. The surface stiffness significantly decreased and increased when PDDA and streptavidin were added to the phage substrate, respectively. As expected, different concentrations of streptavidin gave different ranges of stiffness values for the phage matrix produced using YSY184 (Fig. 3). The surface Young’s moduli (E) measured using an atomic force microscope (AFM) demonstrated that the values followed the


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following relationship: YSY184 + PDDA (3.6 ± 1.1 kPa) < YSY184 (57.2 ± 10.4 kPa) < 1:0.01 ratio of YSY184 + streptavidin (351.7 ± 42.6 kPa) < 1:0.1 ratio of YSY184 + streptavidin (2,562±241.5 kPa) < 1:1 ratio of YSY184 + streptavidin (4,330 ± 496.8 kPa) (Fig. 3). However, RGD8 (R8) with streptavidin (E ~ 97.9 kPa; 1:1 ratio) did not exhibit a similar stiffness to YSY184 with streptavidin (E ~ 4,330 kPa) in which the specific streptavidin binding was present with YSY184. On another note, the stiffness of simple streptavidin mixed with R8 (E ~ 97.9 kPa) was higher than that of phage-only matrix (E ~ 57.2 kPa) possibly because a non- specific streptavidin mixed with the R8 phage may also result in changes in the topology or stiffness.

2.3. Surface pattern characterizations of the phage-PDDA, phage, phage-streptavidin show that the stiffness may be produced by interfibrillar incorporation of streptavidin into the phage matrix. Next, we examined whether the surface pattern or thickness of the tissues is associated with their stiffness. A patterned tissue of RGD and HPQ displaying our engineered phage layers (YSY184) can serve as a platform to provide sufficient surface area in order to enhance the interaction of the phage matrix tissue with the integrin receptors on cellular surfaces. The engineered phage pattern can be re-pulled from PDDA or streptavidin solution to modulate the stiffness of the final phage pattern. The AFM images of phage–PDDA, phage, and phage– streptavidin show no significant differences in phage patterning (Fig. 4, top) and phage thickness (Fig. 4, bottom left). The slightly reduced thickness of the phage pattern re-pulled in PDDA or streptavidin compared to the phage-only pattern may be due to the interfibrillar incorporation of PDDA or streptavidin (Fig. 2, bottom). On the other hand, the surface stiffness significantly decreased and increased for the phage matrices re-pulled in PDDA and streptavidin, respectively,


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compared with the phage-only matrix with corresponding stiffness values of ~3.6 kPa for phagePDDA, ~57 kPa for phage, and ~4,330 kPa for phage-streptavidin (Fig. 4, bottom right).

2.4. Morphology of preosteoblasts on the phage tissue matrices with different stiffness ranges shows that the aspect ratio of the cells increased with an increase in the phage tissue surface stiffness. In our previous study, we found that the peptide-specific osteogenic stem cell responses to the functional peptide displayed on the phage in the early stage of osteogenic differentiation (biochemical cues provided by the engineered phage) 1. However, its peptide-specific responses of osteogenic differentiation can be generally limited by up to ~5 days. The most pronounced expression of the osteogenic protein marker was found in the cells on the DGEA phage, compared to those on the RGD and RGE controls at an early stage; however, a little effect of peptides itself displayed on the phages was examined at later stages of the osteogenic differentiation. It maybe because the cells secrete more ECM proteins with prolonged culture periods (after 5 days), thereby diluting the effects of the peptide ligands themselves displayed on the engineered phage. To exclude any other effects, we next investigated whether phage substrate stiffness affects osteogenic differentiation at an early stage (within three-day cultures without osteogenic differentiation induction). We generated differential stiffness using our phage matrix with varied ratios of streptavidin and cultured MC3T3 preosteoblast cells on the phage matrices. We were able to generate 30–60 kPa (phage-only), 80–100 kPa (phage:streptavidin = 1:0.0001), 300–400 kPa (phage to streptavidin = 1:0.01), and approximately 4000 kPa (phage to streptavidin = 1:1). This matrix has an RGD peptide on the phage body with variable mechanical stiffness via


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streptavidin incorporation. In other words, different phage matrices with the same biochemical cues but different mechanical cues can be prepared. The effect of the stiffness of our phage matrix on osteogenic stem cells was also examined (Fig. 5). Aspect ratio (AR), the ratio of length of cells’ major axis to the length of the minor axis, is an important measurement of cell shape. The AR was calculated by dividing the long axis by the short axis of each cell using ImageJ software (National Institutes of Health, Bethesda, MD). The long axis is the longest length of the cells and the short axis is the length across cellular nucleus, perpendicular to the long axis. The cell morphologies of 24-h cultures on each phage matrix with different stiffness ranges show that the AR of the cells increased with an increase in the surface stiffness (p < 0.0001; Bartlett’s test for equal variance), whereas similar cell populations (nuclei numbers) were found for all surface stiffness values (p > 0.05; analysis of variance (ANOVA)). The morphological changes caused by the phage stiffness are apparent between the phage-only matrix and the 1:1 phage–streptavidin matrix (AR; Fig. 5 (bottom left)). Interestingly, the ARs were not affected by the different functional peptides displayed by phage drop-cast films (DGEA, DGDA, EGEA, RGD, RGE, and wild; Fig. 1S), while the cellular area on DGEA phage was the highest and was correlated with the osteogenic differentiation marker in our previous studies.1 From these results, we postulate that specific interactions between cells and the peptides on the phage body lead to wider cellular spreading and increase surface-to-surface interactions. However, the surface stiffness causes cellular elongation to differentiate and constitute cell-tocell connections comprising desired organized tissues. Although we were unable to define a specific function from a specific morphology, it is believed that cellular interactions with certain cues provided by the surfaces result in different cellular responses.


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2.5. Phage matrix stiffness is associated with osteogenic differentiation. Studies on the matrix stiffness and the relation to osteogenic differentiation demonstrated that osteogenic lineage induction is enhanced with increased stiffness.36-38 It is widely considered that a stiffer matrix promotes osteogenesis while soft one is good for adiogenesis. Cell-cell interactions and substrate modulus also regulate osteogenic differentiation of mesenchymal stem cells (MSCs).39-40 To verify whether osteogenic differentiation can be controlled by the stiffness of our constructed phage matrix with different mechanical cues, MC3T3 cells were cultured on top of different phage matrices (phage–PDDA, phage, and phage–streptavidin) for 24 h without osteogenic differentiation induction. We characterized the osteogenic responses to each phage matrix stiffness by quantifying the osteogenic marker mRNA expression: alkaline phosphatase (ALP), collagen type I (COL I), osteopontin (OP), osteocalcin (OCN), and dentin matrix acidic phosphoprotein I (Dmp I). We used β-actin (BAT) as an endogenous protein control and found that osteogenic gene expressions were upregulated on the stiffer phage matrix compared to the soft phage matrix (phage–streptavidin > phage > phage–PDDA; p < 0.05; ANOVA; Fig. 6). It is likely that the stiffness provided by the phage-only matrix is sufficient to induce osteogenic differentiation in which all osteogenic markers, including COL, a general marker for osteogenic differentiation, expressed comparably in the phage-only matrix. However, the soft matrix of phage–PDDA, having a stiffness of less than 10 kPa, exhibited an effect, particularly in the DMP expression, that can be ignored.

2.6. Our engineered phage matrix stiffness can provide an important chemical, physical and mechanical cues together to modulate stem cell differentiation.


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Stem cells feel and respond to the stiffness of ECM via mechanotransduction pathway.41-42 The relationship between matrix stiffness and stem cell phenotype is also receiving more interest. AR of MSCs increases with substrate stiffness and human mesenchymal stem cells (hMSCs) on the stiffer substrate shows higher osteogenesis.43 Some papers on the cell shape effect on differentiation described that monotonic increase of osteogenic differentiation with AR, which may apply when AR is less than 8.44-46 In this study, we successfully demonstrated different stiffness ranges of matrix by using engineered phage nanofibers (Fig. 3) and confirm that our phage matrix stiffness controls the morphology of MC3T3 as well as their osteogenic differentiation (Fig. 5 &6). We then investigated the effect of our phage matrix stiffness when MC3T3 cells are cultured with osteogenic differentiation media. Interestingly, the stiffness effect was significant only in the cells on soft phage matrix (phage+PDDA, p0.05; 20 ~ 3,000 kPa) in terms of AR and osteogenic differentiation of MC3T3 cells at 18h culture on top of different phage matrices (Fig. 7A). But expression levels of osteognic markers on the phage matrices are highly elevated with osteogenic induction, compared to without osteogenic induction. ASCs showed similar results (Fig. 7B). To see the effect of presented phage surface proteins, we included wild type phage matrix. Interestingly, cells responded differently even with same stiffness (p 0.05; ANOVA). The Young’s moduli increased significantly in the order of phage–PDDA, phage, and phage–streptavidin patterns (bottom right; **p < 0.0001; ANOVA).


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Figure 5. Morphology of preosteoblasts on top of the phage tissue matrices with different stiffness ranges. The different stiffness ranges of the engineered phage tissue matrices can be generated by mixing with a varied ratio of streptavidin. The increased ratio of streptavidin to phages shows increased stiffness (top). MC3T3 cells cultured on the phage tissue matrices with different stiffness values show that the aspect ratio (AR) of cells increase with an increase in the stiffness (bottom left; *p < 0.0001; Bartlett’s test for equal variance). However, a similar cell population was found for all phage matrices, regardless of the stiffness (bottom right; **ns, p > 0.05; ANOVA).


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Figure 6. Osteogenic differentiation of MC3T3 cells associated with phage matrix stiffness. The quantification of osteogenic differentiation marker protein expression in preosteoblasts stimulated by three different phage matrices: phage–PDDA (3-8 kPa), phage-only (20-40 kPa), and phage–streptavidin (> 86 kPa) using quantitative polymerase chain reaction (qPCR). Alkaline phosphatase (ALP), collagen type I (COL I), osteopontin (OP), osteocalcin (OCN), and dentin matrix acidic phosphoprotein I (Dmp I) were quantified and normalized by β-actin. Data are shown as mean values ± standard error of mean (SEM) of three independent samples (p < 0.05; ANOVA).


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Figure 7. AR changes of stem cells responding to engineered phage matrix stiffness in their osteogenic differentiation. (A&B) Morphology of preosteoblasts (MC3T3, A) and human adipose-derived stem cells (ASCs, B) on top of different phage matrixs (left) and quantitative osteogenic differentiation marker expression of the cells on the different stiffness ranged phage matrix (right) in osteogenic differentiation media. With osteogenic induction media, there was no significant difference in AR (left) and osteogenic marker expression (right) of MC3T3s and ASCs on the different stiffness ranged phage matrix (phage only, phage+strep-1:0.0001, phage+strep-1:0.01 and phage+strep-1:1, Most of them have about 2.5 of ARs at 18h culture; **ns p>0.05, ANOVA) whereas ARs of them on phage matrix with low stiffness (3-6kPa, phage+PDDA in A & B) or phage matrix without RGD motif (20-40kPa, WT phage matrix in B) are around 1.5 (*p0.05, ANOVA) whereas that of ASCs on phage matrix with low stiffens (3-6kPa, phage+PDDA) are kept as around 1.5 (*p phage+strep1:001 > phage+strep 1:0.0001 > phage only > phage+PDDA, Scale bar = 20 µm).


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TABLES. Table 1. Genetically engineered phages used herein a) Name

pVII

pVIII

pIII

WT

MEQV

AEGDDP

SHSAET

R8

MEQV

ADSGRGDTEDP

SHSAET

YSY184

MECLHPQTCV

AGGRGDSDDYDP

SHSACHPQGPLCGGGSAET

a)

Peptide inserts are shown in italics and bold. Functional sequences are underlined.


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Supporting Information. The following files are available free of charge. Table S1, phage concentrations and the areal phage density in the self-templating assembled phage films. Table S2-S5, primers used and PCR conditions for cloning. Figure S1, morphology of MC3T3 on different phages. AUTHOR INFORMATION Corresponding Authors [email protected] (S.W. Lee); [email protected] (S.Y. Yoo) Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. †These authors (H.S. Lee and J.I. Kang) contributed equally. ACKNOWLEDGMENT This research was supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI) funded by the Ministry of Health & Welfare, Republic of Korea (HI16C1067) and by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF2016R1D1A1B03935221).


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(47) Lee, D. Y.; Lee, H.; Kim, Y.; Yoo, S. Y.; Chung, W. J.; Kim, G. Phage as Versatile Nanoink for Printing 3-D Cell-laden Scaffolds. Acta biomaterialia 2016, 29, 112-124, DOI: 10.1016/j.actbio.2015.10.004.


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TOC We demonstrate an engineered phage matrix for osteogenic differentiation with controlled stiffness by crosslinking the engineered phage displaying RGD and HPQ with streptavidin or PDDA. Our phage matrices, which can be easily functionalized using chemical/genetic engineering, can be used as a convenient tissue matrix platform for controlling stem cell expansion and differentiation Keyword: phage, matrix, stiffness, differentiation H.S. Lee, J.I. Kang, W.J. Chung, D.H. Lee, B.Y. Lee, S.W. Lee,* S.Y. Yoo* Engineered Phage Matrix Stiffness-Modulating Osteogenic Differentiation


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Engineered Phage Matrix Stiffness-Modulating Osteogenic

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