Biomineralized hydrogel with enhanced toughness by chemical

Subscriber access provided by UNIV OF NEW ENGLAND ARMIDALE

Tissue Engineering and Regenerative Medicine

Biomineralized hydrogel with enhanced toughness by chemical bonding of alkaline phosphatase and vinylphosphonic acid in collagen framework LU Chen, Ke Yang, Huan Zhao, Amin Liu, Wanying Tu, Chengheng Wu, Suping Chen, Zhenzhen Guo, Hongrong Luo, Jing Sun, and Hongsong Fan ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/ acsbiomaterials.8b01197 • Publication Date (Web): 15 Feb 2019 Downloaded from http://pubs.acs.org on February 15, 2019

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 40 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 60

ACS Biomaterials Science & Engineering

Biomineralized hydrogel with enhanced toughness by chemical bonding of alkaline phosphatase and vinylphosphonic acid in collagen framework Lu Chen, † Ke Yang, † Huan Zhao, † Amin Liu, † Wanying Tu, † Chengheng Wu, † Suping Chen, † Zhenzhen Guo, § Hongrong Luo, † Jing Sun, † Hongsong Fan * † †National Engineering Research Center for Biomaterials, Sichuan University, Chengdu 610064, Sichuan, China §Department of Gastroenterology, Hospital of the University of Electronic Science and Technology of China, Sichuan Provincial People's Hospital, Sichuan, Chengdu 610072, China *Corresponding author: Prof. Hongsong Fan E-mail address: [email protected] Tel.: +86-28-85410703 Fax: +86-28-85410246

1

ACS Paragon Plus Environment

ACS Biomaterials Science & Engineering 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 60

ABSTRACT

Alkaline phosphatase (ALP) and phosphoproteins participated collagen mineralization is one of the most important physiological process of bone formation. Simulating the natural mineralization process with the involvement of ALP and phosphoproteins is a powerful tool for the preparation of bone repair scaffolds. Searching for compatible approaches to chemically bond ALP with collagen molecules, and introducing phosphoprotein-like molecules into collagen network is the challenge of the day. Here, we synthesized alkaline phosphatase methacrylamide (ALP-MA) by amidation reaction to enable ALP to be grafted uniformly into the photo-crosslinking collagen gel, achieving the homogeneous enzymatic mineralization. Furthermore, vinylphosphonic acid (VAP), a phosphoprotein-like molecule containing phosphonate groups, was successfully introduced on collagen molecular through photo-crosslinking, playing the role of phosphoprotein for inducing mineralized CaP clusters deposited on collagen backbone. Hence, hydrogel termed as CAV was synthesized by photo-chemical reaction among collagen methacrylamide (Col-MA), ALP-MA and VAP (active mineral bonding site) for in situ mineralization. We found that the binding between collagen network and CaP clusters would lead to generation of mechanical enhanced mineralized collagen hydrogel. Encapsulated bone marrow stromal cells (BMSCs) exhibited well growth and proliferation in the in situ enzymatic mineralization process. Besides, the simulated mineralization system is highly favorable for micro-patterned structure construction and 3D-bioprinting. In brief, we designed a novel approach to effectively simulate the physiological mineralization process of bone formation. The 2


Page 2 of 40

Page 3 of 40 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 60


approach incorporated with great photo-crosslinking performance and enzymatic mineralization activity was benefited for in situ cell encapsulation and excellent cellcompatible 3D printing, hold a great promise of bone tissue engineering research.

KEYWORDS: enzymatic mineralization, collagen hydrogel, alkaline phosphatase, biomimetic biomaterials, bone repair scaffold

1. INTRODUCTION Bone is a hierarchically structured composite material with mineralized collagen fibril as the basic building block, where the collagen is the scaffold for nucleation and growth of apatite.1-3 As a typical orthophosphoric-monoester phosphohydrolase, alkaline phosphatase (ALP) acts as the extracellular matrix macromolecules and participates in the nucleation, growth and structure of the apatite mineral phases.4-6 By enzymolysis of organic phosphate esters to generate inorganic phosphate, ALP is conducive to producing a pro-mineral extracellular environment and stimulating mineralization.4-6 ALP also binds directly to type I collagen to form a scaffold for the propagation of matrix mineralization.5 In addition, phosphoproteins are critical in mineralization processes of bone, cartilage, dentin and other vertebrate mesenchymal tissue,7-10 due to their in vivo collagen-binding capacity, and phosphorylation generating anionic charged phosphonate groups distributed around the collagen, which is responsible for the nucleation of apatite and further adhesion to collagen.8, 11-13 Inspired by the process of extracellular matrix mineralization, the design of biomimetic bone repair materials for bone tissue regeneration by simulating the development of mineralized collagen 3



fibril become popular. In the past decades, mineralized collagen hydrogels with calcium phosphate (CaP) deposition offered a promising option for bone defects repair, either as freeze dried scaffolds or injectable hydrogel.14-20 Specifically, the injectable collagen hydrogel with CaP mineralization along with micro/nano-scaled collagen fibril and uniformly distribution inside the hydrogel are especially expectable. To this end, it will be constructive by simulating the natural mineralization process with ALP and phosphoproteins. There are some reports that apply ALP to achieve enzymatic inducing mineralization of collagen.21-23 However, most of them blend ALP and collagen directly, resulting in rapid release of ALP from the collagen hydrogel and occurrence of mineralization beyond the collagen matrix as well as gel mineralization in a low degree.21-22 Therefore, by means of covalent cross-linking ALP with collagen, such as applying aldehyde or EDC/NHS to crosslink ALP with collagen might inhibit the occurrence of mineralization beyond the collagen matrix and promote the deposition of CaP on the collagen fibers.23 However, the common way of covalent cross-linking is cytotoxic, which is incompatible for in situ cellular encapsulation. Basing on this meaning, it’s urgent to develop a simpler and more biocompatible way to prepare enzymatic mineralized hydrogel that can both simulate native physiological mineralization process and realize in situ cell encapsulation. Meanwhile, so important as phosphoproteins is in tissue mineralization process,8 it’s reasonable to introduce some phosphoproteins or monomers containing phosphonate groups to induce interaction between collagen and Ca2+ ions and binding of collagen to CaP clusters, 4


Page 4 of 40

Page 5 of 40 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 60


which is potential for uniform and high mineralization. In this study, we developed a facile and biocompatible way to prepare mineralized collagen hydrogel with both ALP and phosphoprotein-like molecules participating. Collagen and ALP were decorated with methyl acryl groups through amidation reaction between methacrylic anhydride and protein side-chain amino. Collagen can be photocrosslinked quickly with ALP methacrylamide (ALP-MA) to achieve uniform grafting of ALP for inducing homogeneous mineralization. At the same time, vinylphosphonic acid (VAP), one kind of phosphoprotein-like small molecules with phosphonate groups, can bind with Ca2+ due to the similar capacity as phosphoprotein.24 Also, it can be photo-grafted on collagen molecular by ene-ene reaction for simulation of phosphoprotein to promote CaP nucleation. Then the CaP cluster formed by the mineralization can be combined with the phosphate on collagen network to obtain enhanced mechanical properties. Thus, the creation of enzymatic mineralized collagen hydrogels with prospected properties could be accomplished (scheme 1.). Afterwards, we investigated the effect of ALP-MA and VAP on the mineralization of photocrosslinked collagen-based hydrogels, as well as the application of CAV hydrogel system on bone tissue engineering.

5



Scheme 1. Design of photo-crosslinked Col-MA/ALP-MA/VAP (CAV) hydrogel and the biomimetic mineralization. 2. MATERIALS AND METHOLDS 2.1 Materials Type I collagen (Col) was prepared from calf skin through pepsin treatment and salt precipitation according to our previous accumulating research and experience and the triple helical structure of type I collagen was confirmed by Circular dichroism (CD) analysis(Fig. S1).25-26 . Alkaline phosphatase (ALP), glycerol phosphate calcium salt hydrate (CaGP), methacrylic anhydride (MA), vinylphosphonic acid (VAP), Irgracure 2959 (I2959), propidium iodide (PI), fluorescein diacetate (FDA) and 4’,6-diamidino2-phenylindole dihydrochloride (DAPI) were purchased from Sigma-Aldrich (USA). BCIP/NBT Alkaline Phosphatase Color Development Kit (P3206) and Alkaline Phosphatase Assay Kit (P0321) were obtained from Beyotime Biotechnology (China). Other chemical reagents, if not specified, were purchased from Chengdu Kelong

6


Page 6 of 40

Page 7 of 40 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 60


Reagent Co., Ltd (China). 2.2 Synthesis of Col-MA and ALP-MA Col-MA and ALP-MA were prepared by amidation reaction between collagen/ALP and MA respectively.27-28 In brief, 100mg Col and ALP were dissolved in 50ml 0.2M acetic acid (HAc) and 0.1 M natrium aceticum (NaAc) respectively, and then gently stirred till completely dissolved at 4 oC. Then the solution was adjusted to pH 8.0 by adding NaOH. MA (20µl or 10µl respectively)was added to the solution for 4 hours, after that, collagen or ALP reaction solution was dialyzed against 0.2M HAc or 0.1 M NaAc respectively for 3 ～ 5 days at 4 oC with frequent changes, and then lyophilized and stored at -20 oC. 2.3 Preparation of photo-crosslinked hydrogel Firstly, lyophilized Col-MA was dissolved in 0.5M HAc, lyophilized ALP-MA was dissolved in ultrapure water, and Irgracure 2959(I2959) was dissolved in ultrapure water. The prepolymer solutions of different component gels were configured as follows: (a) Col-MA (CM) network. Col-MA solution and I2959 were added, gently mixed, then the solution was cooled on ice and adjusted to pH 7.4 by NaOH with the final concentration of 7 mg/mL Col-MA, 0.05% (w/v) I2959, respectively. (b) Col-MA/ALP-MA (CA) or Col-MA/ALP (CAn) network. Col-MA solution, ALP-MA (or ALP) solution and I2959 were added, gently mixed, and the solution was cooled on ice and adjusted to pH 7.4 by adding NaOH with the final concentration of 7 mg/mL Col-MA, 5 mg/mL ALP-MA 7



Page 8 of 40

(or ALP), 0.05% (w/v) I2959, respectively. (c) Col-MA/ALP-MA/VAP (CAV) network. Col-MA solution, ALP-MA solution, I2959 and VAP liquid were added, gently mixed, and the solution was cooled on ice and adjusted to pH 7.4 by adding NaOH with the final concentration of 7 mg/mL Col-MA, 5 mg/mL ALP-MA, 0.05% w/v I2959, 1% w/v VAP (CAV1) or 5% w/v VAP (CAV5), respectively. The obtained pre-polymer solution was poured onto the non-adhesive surface, and placed one cover glass with spacer on that surface, then

put it under UV light

(Omnicures S1500, 320～500nm, 8 W cm-2) for 30 seconds to form the hydrogel. Then, the formed gel was carefully stripped and immersed in ultrapure water and stored in 4℃. For cylindrical hydrogels, the resulting pre-polymer solution was poured into the molds with typical size of Ø8mm×2mm, then irradiated with UV light (320～500nm, 8 W cm-2) for 30 seconds. 2.4 Mineralization 1% CaGP solution was added into a bottle with ALP-loaded hydrogels (Ø8mm×2mm) and incubated at room temperature for varied period of time. After mineralization, the gel was rinsed thoroughly with deionized water and kept in deionized water for testing. 2.5 Analytical methods 2.5.1 The Alkaline Phosphatase Activity Assay The activity of ALP enzyme was detected by commercially Alkaline Phosphatase Assay Kit before and after methacrylamide. The principle of ALP enzyme activity 8


Page 9 of 40 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 60


detection was based on the conversion of colorless p-nitrophenyl phosphate (p-NPP) to colored p-nitrophenol (p-NP). Briefly,10μl of 2 ～ 20μg/ml ALP or ALP-MA was placed in 96 well plates containing 40 μl buffer solution and 50 μl chromogenic substrates (containing p-NPP) co-incubation for 30 min, then 100μl stop solution was added. The absorbance was measured at 405 nm. A calibration curve of p-NP with concentrations ranging from 0 to 200μg mg /mL served as a reference. The ALP or ALP-MA activity was calculated by the following equation 29: 𝐶𝑝 × 𝑉𝑎𝑙𝑖𝑞𝑢𝑜𝑡

Activity = 𝐶𝑎 × 𝑉𝑟𝑒𝑎𝑐𝑡𝑖𝑜𝑛 × 𝑡

(1𝐷𝐸𝐴 𝑢𝑛𝑖𝑡 𝑚𝑔 ―1 = 1𝜇𝑚𝑜𝑙 ∙ 𝑚𝑖𝑛 ―1 ∙ 𝑚𝑔 ―1)

（1）

where Cp (μmol/L) is the concentration of p-nitrophenol converted by ALP, Ca (mg/L) is the concentration of ALP, Valiquot is the volume of the aliquot read (100μl), Vreaction is the volume of the reaction (10μl), t is the reaction times. 2.5.2 Release of active ALP and ALP-MA from Hydrogels CA (or CAn) hydrogels loaded with ALP-MA (or ALP) (Ø8mm×2mm) were incubated in deionized water at 37 oC ALP released from the hydrogels was measured at 0.5, 1, 2, 4, 6, 24, 48 hours after the incubation by Alkaline Phosphatase Assay Kit (n = 6). The release curve of ALP was calculated by the release amount at different time. For visible observation, the heterogeneous distribution of ALP in the gel after one day incubation in water was characterized by BCIP/NBT Alkaline Phosphatase Color Development Kit. 2.5.3 Nuclear Magnetic Resonance (NMR) analysis 9



The modification of methacrylate groups onto Col and ALP molecules were characterized by NMR spectrometer (AV II-600 MHz, Bruker) analysis using Dimethyl sulfoxide-d6 (DMSO-d6) and deuteroxide (D2O) respectively. 2.5.4 Characterizations of Mineralized Collagen-based Hydrogels After mineralized for one day the samples were dried and characterized by scanning electron microscope (SEM) and energy dispersion spectrum (EDS) analysis (FE-SEM, Hitachi S-4800), Fourier transform infrared (FTIR) spectrophotometer (Nicolet 6700, Thermo Fisher Scientific Inc.), x-ray diffractometer (XRD, X'Pert Pro, PHILIPS), and thermal gravimetric analysis (TGA; Netzsch STA 49 C). 2.5.5 Mechanical properties The compression modulus of the hydrogel samples (Ø8mm×2mm) were recorded by dynamic mechanical analysis (DMA, TA-Q800). The amplitude was adjusted to 20μm and the prestress was 5mN. The stress–strain curves of tensile test with typical dimensions of 8×15mm2 (width× length) and a thickness of 1.0±0.1mm were characterized by DMA in force control mode at a force ramp of 0.5 N min-1 with 0.05 N preload force until the sample fractured. The samples were fixed between two clamps with 8mm interval and kept wet with water during measurement. Young’s modulus of the hydrogel was measured by the slope of the stress-strain curve about 5% elongation. Fracture elongation was calculated as percent elongation at break. The corresponding curve integral area (AR) was calculated 10


Page 10 of 40

Page 11 of 40 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 60


as follow: 𝜀

（2）

AR = ∫0𝜎(𝜀)𝑑𝜀 Where ε is the strain (as a percentage), σ is the stress (in kPa).

2.6 The biocompatibility evaluation of CAV hydrogel and the mineralization process 2.6.1 Isolation and culture of BMSCs Bone marrow stromal cells (BMSCs) were isolated from neonatal rabbits according to a previously published method.28 The experiment was approved by the Institutional Animal Care and Use Committee of Sichuan University. 2.6.2 3D cell encapsulation For encapsulation, BMSCs were suspended in neutralized CM or CAV1 prepolymer solution at a density of 5×106 cells per mL. The cell–sol composites were irradiated under UV light (320～500nm, 8 W cm-2) for 30 seconds to form hydrogels. Both the cell-encapsulated CM and CAV1 gels were cultured in a 5% CO2 incubator at 37℃. Then, the culture medium was changed for the first time after 2 hours and the second time after 12 hours; then, CAV1 hydrogels were cultured in culture medium containing 1%CaGP for 0h, 4h, 8h, 1d respectively (denoted as CAV-0h, CAV-4h, CAV-8h, CAV1d, respectively), after that, mineralization medium was replaced with normal medium and changed every 2 days. 2.6.3 Cell viability and morphology. 11



The cell-encapsulated gels were harvested at 1d, 3 d and 6 d for FDA/PI staining, and then observed with confocal laser scanning microscopy (CLSM, Leica-TCSSP5). For actin cytoskeleton staining, the hydrogels were fixed in 4% paraformaldehyde solution for 10～15 min and then incubated in 5μg/mL Alexa Fluor-594 phalloidin for 40 min. The hydrogels were then rinsed in PBS and incubated with 5μg/mL DAPI to stain the nuclei for 5 min, after which the hydrogels were washed with PBS for imaging. 2.7 Fabrication of micro-patterned hydrogels and three-dimensional (3D) printing To prepare micro-patterned hydrogels, CAV1 prepolymer solution (Section 2.4) was transferred between two slides with a height of 300mm distance holder. And then, a photo-mask was placed on the top slide and irradiated under UV light (320～500nm, 8 W cm-2) for 30 seconds. To prepare 3D printing hydrogels, CAV1 prepolymer solution (Section 2.4) was printed using a 23 gauge needle (450µm inner diameter) to form 5-layer porous scaffolds with a length of 15 mm, width of 12 mm and a height of 2 mm and then irradiated under UV light (320～500nm, 8 W cm-2) for 30 seconds. 3D printing cell-encapsulated gels were prepared by printing the cell-hydrogel composite (Section 2.7.2) as described before, and then cultured in a 5% CO2 incubator at 37℃. 2.8 Statistical analysis Statistical significance was measured by two-way ANOVA-test, the level of significance was set at P < 0.05 (*). 12


Page 12 of 40

Page 13 of 40 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 60


3、RESULTS AND DISCUSSION 3.1 Characterization of Col-MA and ALP-MA and the release behavior in collagenbased gel

Figure 1. NMR spectra of Col and ALP before and after methacrylamide. (a) 1H-NMR spectra of Col and Col-MA. (b) 1H-NMR spectra of ALP and ALP-MA. Under alkaline conditions, photo-active Col-MA or ALP-MA was obtained by the reaction of lysine on the collagen or ALP with methacrylic anhydride. As shown in Fig. 1a, the appearance of methacrylamide proton peaks27 (methylene at 5.63 and 5.28 ppm, methyl at 1.84 ppm) in the spectrum of Col-MA, and the ration of methyl and methylene peaks for 3:2 (Fig. S2a) confirmed the incorporation of methylpropylene photo-active moieties. In Fig. 1b, the present of methylene at 6.22 and 5.76 ppm, methyl peak at 1.85 ppm, and the ration of methyl and methylene peaks for 3:2 (Figure S2b) proved the incorporation of methacrylamide photo-active moieties in ALP-MA.

13



Figure 2. Activity of ALP-MA and the release behavior in collagen gel. (a) Activity of ALP and ALP-MA. (b) Release of ALP from ALP-loaded Collagen hydrogels. Release of ALP from CA (CA5 with 5mg/mL ALP-MA or CA10 with 10mg/mL ALP-MA) was significantly lower than CAn (CAn1 with 1mg/mL ALP or CAn5 with 5mg/mL ALP) from photo-crosslinked collagen hydrogel. (c) The heterogeneous distribution of ALP in collagen-based hydrogel. (d) The asymmetric mineralization by the release of ALP from collagen-based hydrogel. The enzyme activity before and after methacrylamide modification is showed in Fig. 2a, with the value of 26.6±1.6 DEA units/mg and 10.3±1.1DEA units/mg for ALP and ALP-MA respectively, indicating that the activity of the modified enzyme was decreased to 40%. Release profiles of ALP from both ALP-loaded hydrogels are 14


Page 14 of 40

Page 15 of 40 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 60


showed in Fig. 2b. The cumulative release of ALP from CAn sample was about 10% of the total amount within 2 hours and increased to 18% after 6 hours and 25% after 1days， resulting the heterogeneous distribution of ALP in the CAn as shown in Fig. 2c and corresponding asymmetric mineralization in CAn hydrogel (Fig. 2d). In contrast, ALP release from ALP-MA loaded CA gels showed the low value of 1% with little increase from 1 day to 2days in incubation. This lower release from CA should be attributed to the covalent crosslinking between ALP-MA and Col-MA. Though the molecular weight of ALP has been reported to be 140,000～160,000 g mol1,30

physical packaged ALP with collagen may result in the leaching of the enzyme

molecules from collagen hydrogels into the mineralization solution and the precipitation of CaP happened in the surrounding solution,21, 31 thus it may lead to the low efficiency and inhomogeneous mineralization inside the hydrogel networks (Fig.2d). Both Col and ALP are macromolecules with many functional reactive groups, 32-34

and the e-amino groups of lysine in the collagen or ALP molecules are likely to

bind with MA by amidation reaction.27-28, 34 Due to the photo-active moieties in both Col-MA and ALP-MA, ALP-MA can be grafted uniformly into the photo-crosslinking collagen gel, achieving the following enzymatic mineralization inside the hydrogels. Meanwhile, as the lysine and arginine groups are high related with the enzyme catalytic activity,32, 35 the MA modification can certainly decrease the enzyme activity. Since the predominant active site of amidation reaction is the ε-amino of lysine that lies on collagen or ALP side termination27-28, 34, ALP-MA retained about 40% activities of ALP by the retention of arginine and lysine that is not involved in amidation reaction and the 15



ALP-MA/Col-MA hydrogel still kept a high mineralization activity in mineralization solution. Therefore, through methacrylamide modification, ALP-MA could be grafted uniformly into photo-crosslinking collagen hydrogel, thus resulting in a biomimetic homogeneous enzymatic mineralization. 3.2 Effect of VAP on gelation properties of ALP-MA/Col-MA composite gels

Figure 3. Effect of VAP on gelation properties of ALP-MA/Col-MA composite gels. (a) Storage modulus of hydrogels with different polymer composition. (b) Loss modulus of hydrogels with different polymer composition. (c) The composition of different gels. (d) FTIR spectra of freeze-dried hydrogels with different polymer composition.

16


Page 16 of 40

Page 17 of 40 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 60


In order to simulate the role of phosphoproteins, vinyl-phosphonic acid (VPA), a phosphoprotein-like molecule containing C-PO43 groups, which can serve as calcium ions binding sites during mineralization,8, 24 was copolymerized into ALP-MA/Col-MA hydrogel. Four prepolymer solutions with different components were prepared, as shown in Fig. 3c. Dynamic mechanical analysis of the storage modulus and loss modulus of the photo-crosslinked collagen-based hydrogels are showed in Fig. 3a and 3b. As shown in Fig.3a, compared with CM, the storage modulus of the CA composite hydrogels increased significantly, however, the storage modulus of the CAV hydrogels (CAV1, CAV5) significantly decreased and exhibited a downward trend with increased VAP concentration. For all the 4 samples, loss modulus (Fig. 3b) was significantly lower than storage modulus and shared a similar trend with storage modulus. The phenomenon indicates that the gel elasticity was higher than viscosity, and gelation performance was good. FTIR spectra in Fig. 3d showed that in all hydrogels, the amide characteristic bands36-37 in collagen at ～1642 cm-1, ～1553 cm-1 and ～1238 cm-1 were well presented. Compared with CM, the amide I peak of CA shifted from 1642.7 to 1647.7 by combination of ALP-MA and CM, which should be resulted by the photocrosslink and uniform grafting of ALP-MA in the gel. The peak at 1077cm-1 is assigned to the asymmetric stretching vibration of the P-O-H group in VAP 38and a part of the “fingerprint” region in collagen39. The phosphate group peak 1077cm-1 normalized to the amide peak （ 1642 cm-1 ）（ denoted as A1077/A1642 ） increased as VAP content increasing, which indicated the presence of VAP and the binding of VAP to Col-MA by photo-crosslinking. Since VPA reacting with Col-MA consumed Col-MA’s c=c 17



double bonds24, cross-linking of collagen would be dropped off, that’s why mechanical properties of CAV hydrogels significant declined. However, the weak CAV hydrogel can be stiffed upon mineralization. 3.3 Mineralization of Collagen-based Hydrogels. The effect of VAP on mineralization in CAV hydrogels were characterized by SEM, EDS, XRD, TGA and FTIR observation.

Figure 4. SEM micrographs and EDS test results of CaP deposited in mineralized collagen hydrogels. After 1 day mineralization, SEM images in Fig. 4a showed that, in all cases, the mineralization occurred inside the hydrogels, but CaP was more evenly deposited along the gel network in CAV1 and CAV5 compared with that in CA. EDS analysis showed that Ca:P ratio of the obtained mineralized hydrogels was similar between 1.48-1.52 (Fig. 4b). EDS mapping in Fig. S3 showed that the Ca and P elements were uniformly 18


Page 18 of 40

Page 19 of 40 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 60


distributed in mineralized collagen hydrogel, indicating uniform mineralization of the gel.

Figure 5. TGA (a), XRD (b), FTIR (c) and corresponding analysis results (d) of freezedried CA, CAV1, CAV5 hydrogels after 1 day of mineralization were characterized. (e) Changes of the appearance of the gel during mineralization. TGA measurement in Fig. 5a showed that all three samples were with similar curve and with little difference in CaP content (Fig. 5d). XRD spectra of mineralized hydrogels in Fig.5b showed that all the samples had similar spectra with two broad characteristic diffractions corresponding to the amorphous calcium phosphate.40 FTIR spectra of gels 19



before and after 1 day are showed in Fig. 5c. After mineralization, amide peak (~ 1647, ~ 1553, ~ 1238 cm-1) declined, while the peaks around ~ 1077 cm-1 and ~ 600 cm-1 intensified due to introduction of phosphate salt, and the phosphate group peak 1077cm1

normalized to the amide peak (1642 cm-1) (denoted as A1077/A1642）increased as VAP

content increasing (Fig.5d), indicating that CaP deposition was more along with Col network in CAV hydrogels. One important difference between the mineralized samples of CA and CAV should be noted, as shown in Fig. 5e, the transparency of CAV is higher than CA. The results showed that either with VAP or not, the composite of ALPMA could induce an effective mineralization and the precipitation of amorphous CaP with little variation of CaP content, but the present of VAP changed the transparency of the mineralized hydrogel, which might be due to the different binding between the polymer chains and CaP clusters. It is known that the mineralization of CaP is highly affected by the variations of the surrounding functional moieties.15, 41 The adhesion of polymer matrix to CaP could be realized through the electrostatic interaction between negatively charged phosphonate groups and the positively charged region of the CaP surface.15 Based on this principle, in CAV1 and CAV5, C-PO43- of VAP can act as nucleation sites to promote the binding between CaP ion clusters and Col matrix molecules. Therefore, there are more CaP deposition along the gel network (Fig. 4a) and the transparency of the mineralized gel increased with the increase of VAP content (Fig. 5e); on the contrary , in CA without VAP as CaP binding sites, only the charged functional groups that exist on the collagen surface as the nucleation sites for CaP deposits,42 that’s why a large number of CaP 20


Page 20 of 40

Page 21 of 40 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 60


generated by ALP were randomly distributed in the gel network (Fig. 4a). Combined the results of TGA (Fig. 5a) and FTIR (Fig. 5c), the inconsistent of the ration of A1077 / A1647 among three gels revealed that the effect of CaP deposits on C=O stretching vibration was different, which should be attributed to the different binding modes of CaP deposits with gel network molecules in CA and CAV. In CAV1 and CAV5, CaP deposited more along the gel network, leading to the reduction of C=O stretching vibration, and with the increase of VAP content, this effect showed an upward trend. 3.4 Mechanical properties of mineralized collagen-based hydrogels. The above results confirmed that the combination of VAP in ALP-MA/Col-MA hydrogel resulted in binding between mineralized CaP and gel network, which would affect the mechanical properties of the mineralized gel.

21



Figure 6. Mechanical properties of mineralized collagen-based hydrogels. (a) Storage modulus of collagen-based hydrogels with different mineralization time. (b) Stressstrain curves of collagen-based hydrogels with different mineralization time and the corresponding Young’s modulus (c), fracture elongation (d) and curve integral area (e) calculated from Stress-strain curves. (f) Storage modulus of CAV1 hydrogels with different mineralization solution concentration at day 1 mineralization. The compression modulus of CA, CAV1 and CAV5 gels at different time of mineralization in 1%CaGP mineralization solution are showed in Fig. 6a. The storage modulus increased dramatically and reached 1 ～ 2 MPa within 1 day in CAV1 and CAV5 groups. Moreover, the storage modulus of CAV1 and CAV5 was significantly 22


Page 22 of 40

Page 23 of 40 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 60


higher than CA without VAP as CaP binding sites at any time. Stress–strain curves (Fig. 6b, Fig. S4) and the corresponding Yong’s modulus (Fig. 6c) of CA, CAV1 and CAV5 hydrogels with different mineralization time showed similar trend with the compression strength that the initial strength of gels was decreased with the increase of VAP content, while the mechanical strength of mineralized gels was gradually increased with VAP content increasing and CAV5 held the highest mechanical strength after one day mineralization. Otherwise, the fracture elongation (Fig. 6d) and curve integral area (Fig.6e) increased initially and decreased afterward with the extension of mineralization time, and both that of CAV1 and CAV5 were greater than CA at corresponding mineralization times. The results demonstrated that excessive CaP deposition in hydrogels might result in an increase in gel brittleness and a decrease in toughness. Besides the effect of VAP, the storage modulus of CAV1 in different concentrations of mineralization solution in Fig. 6f showed that the storage modulus of the mineralized hydrogel increased significantly with the increase of mineralization solution concentration, which should be attributed to the increase of deposited CaP minerals with mineralization concentrations. Because the mechanical properties of the collagen-based composites varied with mineralization time and mineralization solution concentration, it’s easily to get a collagen-mineralized hydrogel with tunable mechanical behavior. Especially, a mineralized CAV hydrogel with enhanced strength and toughness (no fracture of the bend test, Fig. S5) could be obtained with incorporation of VAP, and its storage modulus, Young’s modulus, fracture elongation rate and stress-strain curve integral 23



area were significantly higher than CA without VAP as CaP binding sites. Considering that there was no significant difference in the amount of CaP deposition (Fig. 5a, 5d; Fig. S6a), water content (Fig. S6b) and degree of swelling (Fig. S6c) among CA, CAV1 and CAV5, the significant difference in mechanical properties between CA and CAV should be ascribed to the binding between CaP and collagen networks, instead of the content of CaP deposits or the change of water-swollen state. The strong electrostatic interaction between the negatively charged phosphonate groups (in VAP) and the positively charged region of the deposited CaP surface15 would lead to good adhesion of the CaP to the CAV network (Fig. 4a). This strong interaction between CaP and polymer chains also acted as a supplement for crosslinking,43-44 resulting in higher strength and toughness including the storage modulus, Young’s modulus, fracture elongation rate and curve integral area of stress-strain curve of the CAV group. More VAP would induce more binding of polymer matrix to CaP minerals, thus the mechanical strength of CAV mineralized hydrogel would increase correspondingly. The above results showed that the introduction of ALP-MA and VAP into collagen gel was conducive to the preparation of collagen gel with excellent mineralization performance. Indeed, the uniform mineralized CaP/collagen-based gel was easily obtained by uniformly grafting ALP-MA in the collagen skeleton; moreover, attractive binding between CaP and collagen matrix was improved by grafted VAP in the collagen framework and thus enhanced the mechanical properties of the gel. Therefore, through the introduction of ALP-MA and VAP, the uniform mineralized hydrogels can be obtained, and the mineralization degree and mechanical properties of 24


Page 24 of 40

Page 25 of 40 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 60


the gels can be regulated by the mineralization time and the mineralization concentration. 3.5 In situ encapsulated cell viability and potential application of CAV hydrogel

25



Figure 7 FDA (green)/PI (red) (a) and Phalloidin (red)/DAPI (blue) (b) staining of BMSCs encapsulated within CM hydrogels and CAV1 hydrogels (with 0h, 4h, 8h, 1d mineralization of CAV1-0h, CAV1-4h, CAV1-8h, CAV1-1d, respectively ) after 3 and 6 days culture. The biocompatibility of the photo-crosslinked CAV hydrogel and the effect of inmineralization process on the cellular activity were further investigated. Here, CM hydrogel with good biocompatibility was selected as control, BMSCs were encapsulated within CAV1 and CM hydrogels in situ. FDA/PI staining in Fig. 7a and Fig. S7a showed that the cells in CM and CAV1-0h (without mineralization) hydrogel maintained high activity and spread well after 6 days culture. The morphological changes could be precisely observed by phalloidin/DAPI staining (Fig. 7b) The BMSCs in CM and CAV1-0h hydrogel showed clear cytoskeletons and a spindle or polygon shape after 6 days culture. With cells encapsulated and varied mineralization in 1% CaGP mineralization solution for 4 hours (CAV1-4h), 8hours (CAV1-8h) and 1day (CAV1-1d), the inside cells showed different growth and proliferation correspondingly. In CAV1-4h, the cell spread and proliferated well; In CAV1-8h and CAV1-1d, the cell proliferation and spreading showed a slight downward trend with the extension of mineralization time, but the cells still kept a well survivability and begun to spread out, which was consistent with the MTT tests (Fig. S7b). Meanwhile, cells spread well on mineralized CAV1 hydrogels surface (Fig. S7c). This result suggests that our material with ALP-MA and VAP incorporated is biocompatible for in situ cell encapsulation.

26


Page 26 of 40

Page 27 of 40 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 60


Figure 8. Potential application of CAV1 hydrogel. (a) Photo-patterned CAV1 microgels with different shapes. For the circle, cross, square, and ring microgels, fluorescent microspheres were encapsulated and observed by inverted fluorescence microscopy. (b) Schematic illustration of the design and preparation of the biofunctional bioink for 3D printing. (c) Images of bioprinted acellular 3D constructs (15 (L) × 12 (W) × 2.0 (H) mm3). (d) Representative confocal microscopy images of FDA/PI staining of BMSCs embedded within the cell-laden 3D porous constructs 3 days postprinting.

27



Due to the facile photo-crosslink and in situ cell encapsulation within the collagenbased hydrogel network, as well as the effective enzymatically mineralization with great biocompatibility, this material system provides a gentle and simple way for biofabrication of complex tissue structure. Microgels with different explicit shapes, such as circle, cross, square and ring were fabricated respectively by photo-mask based stereolithography (Fig.8a), and hollow tubular hydrogel was successfully prepared by injection molding (Fig. S7d), indicating the feasibility of preparation of complex tissue units by photo-initiated technology. The applying of biofunctional bioink for 3D printing are showed in Fig. 8b. The porous scaffold and ring shape structure prepared by 3D printing are showed in Fig. 8c and Fig. S7e, respectively. As shown in Fig. 8b and 8d, BMSCs encapsulated in 3D printed porous CVA1 hydrogel maintained high viability and activity, and exhibited a typical spindle shapes at day 3, suggesting that the CAV network was potential for 3D printing fabrication. The ability to encapsulate cells and subsequently support survive is important for engineered tissue construct. Natural bone forming process is a stiffening process with progressing mineral deposition. Meanwhile, bone cellular behavior is regulated via multiple micro-environment including mechanical stiffness.45-46 In our system, collagen network incorporated with ALP and VAP effectively simulate the physiological mineralization process, and with the extension of mineralization time, the mechanical strength increased progressively, which is similar with the stiffening process of bone forming. Combined with good in situ cell encapsulation and biocompatibility, photocrosslink performance, enzymatic mineralization activity, as well as excellent cell28


Page 28 of 40

Page 29 of 40 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 60


compatible printing performance, the printed 3D porous hydrogel construct can be enzymatically mineralized in the mineralization solution to get stiffing mechanical strength mimicking the physiological bone forming process, thus showing a good application prospect in bone tissue engineering.

4、Conclusion In this study, a facile and biocompatible method was developed to prepare mineralized collagen hydrogel with involvement of both ALP and phosphoprotein-like molecules. On the one hand, ALP decorated with methyl acryl groups through amidation reaction was covalently grafted uniformly onto photo-crosslinking collagen gel, achieving homogeneous enzymatic mineralization. On the other hand, VAP, a phosphoproteinlike molecules containing C-PO43- groups, was bond with collagen molecules and simulated the role of phosphoprotein to induce CaP deposition along collagen matrix propagation, resulting in the mineralized CaP clusters binding with collagen molecular and the formation of uniform mineralized hydrogels. The binding between collagen network and CaP minerals leaded to enhanced mechanical properties of the mineralized hydrogel. Thus, in this system, the crosslinking ALP and VAP in collagen network effectively simulate the physiological mineralization process and the stiffening process of bone development. The approach incorporated with great photo-crosslinking performance and enzymatic mineralization activity was benefited for in situ cell encapsulation and excellent cell-compatible 3D printing, hold a great promise of bone tissue engineering research. 29



ASSOCIATED CONTENT Supporting Information The following files are available free of charge.

Stress-strain curve of mineralized collagen-based hydrogels ， change of swelling degree, water content and CaP deposition during the mineralization process ， cell viability and proliferation in mineralized CAV1 hydrogel, hollow tubular CAV1 hydrogel , ring construct prepared by 3D printing（PDF） AUTHOR INFORMATION Corresponding Author ∗ E-mail: [email protected]; Fax: +86 28 85410246; Tel: +86 28 85410703 Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (Contract Grant Nos. 51473098, 51603030 and 51673128). 30


Page 30 of 40

Page 31 of 40 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 60


REFERENCES (1) Olszta, M. J.; Cheng, X.; Jee, S. S.; Kumar, R.; Kim, Y.-Y.; Kaufman, M. J.; Douglas, E. P.; Gower, L. B. Bone structure and formation: A new perspective. Materials Science & Engineering R 2007, 58 (3-5), 77-116. DOI: 10.1016/j.mser.2007.05.001. (2) Liu, Y.; Luo, D.; Wang, T. Hierarchical Structures of Bone and Bioinspired Bone Tissue Engineering. Small 2016, 12 (34), 4611-32. DOI: 10.1002/smll.201600626. (3) Palmer, L. C.; Newcomb, C. J.; Kaltz, S. R.; Spoerke, E. D.; Stupp, S. I. Biomimetic Systems for Hydroxyapatite Mineralization Inspired By Bone and Enamel. Chemical reviews 2008, 108 (11), 4754-4783. DOI: 10.1021/cr8004422. (4) Golub, E. E. Biomineralization and matrix vesicles in biology and pathology. Seminars in immunopathology 2011, 33 (5), 409-17. DOI: 10.1007/s00281-0100230-z. (5)Haarhaus, M.; Brandenburg, V.; Kalantar-Zadeh, K.; Stenvinkel, P.; Magnusson, P. Alkaline phosphatase: a novel treatment target for cardiovascular disease in CKD. Nature Reviews Nephrology 2017, 13 (7), 429-442. DOI: 10.1038/nrneph.2017.60. (6) Golub, E. E.; Boesze-Battaglia, K. The role of alkaline phosphatase in mineralization. Current Opinion in Orthopaedics 2007, 18, 444-448. DOI: 10.1097/BCO.0b013e3282630851. (7) Dahl, T.; Sabsay, B.; Veis, A. Type I collagen-phosphophoryn interactions: specificity of the monomer-monomer binding. Journal of structural biology 1998, 31



123 (2), 162-168. DOI: 10.1006/jsbi.1998.4025. (8) George, A.; Veis, A. Phosphorylated Proteins and Control over Apatite Nucleation, Crystal Growth, and Inhibition. Chem. Rev 2008, 108 (11), 64704693. DOI: 10.1021/cr0782729. Epub 2008 Oct 3. (9) Deshpande, A. S.; Fang, P. A.; Zhang, X.; Jayaraman, T.; Sfeir, C.; Beniash, E. Primary structure and phosphorylation of dentin matrix protein 1 (DMP1) and dentin phosphophoryn (DPP) uniquely determine their role in biomineralization. Biomacromolecules 2011, 12 (8), 2933-45. DOI: 10.1021/bm2005214. (10) Mertz, E. L.; Leikin, S. Interactions of inorganic phosphate and sulfate anions with collagen. Biochemistry 2004, 43, 14901-14912. DOI: 10.1021/bi048788b. (11) Boskey, A. L. Osteopontin and Related Phosphorylated Sialoproteins: Effects on Mineralizationa. Annals of the New York Academy of Sciences 1995, 760 (1), 249256. DOI: 10.1111/j.1749-6632.1995.tb44635.x. (12) Hunter, G. K.; Goldberg, H. A. Nucleation of hydroxyapatite by bone sialoprotein. Proceedings of the National Academy of Sciences 1993, 90 (18), 8562-8565. DOI: 10.1073/pnas.90.18.8562. (13) Hunter, G. K.; Kyle, C. L.; Goldberg, H. A. Modulation of crystal formation by bone phosphoproteins: structural specificity of the osteopontin-mediated inhibition of hydroxyapatite formation. Biochemical Journal 1994, 300 (3), 723728. DOI: 10.1042/bj3020175. (14) Grayson, W. L.; Fröhlich, M.; Yeager, K.; Bhumiratana, S.; Chan, M. E.; Cannizzaro, C.; Wan, L. Q.; Liu, X. S.; Guo, X. E.; Vunjak-Novakovic, G. 32


Page 32 of 40

Page 33 of 40 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 60


Engineering anatomically shaped human bone grafts. Proc Natl Acad Sci U S A 2010, 107(8):3299-3304. DOI: 10.1073/pnas.0905439106. (15) Gkioni, K.; Leeuwenburgh, S. C.; Douglas, T. E.; Mikos, A. G.; Jansen, J. A. Mineralization of hydrogels for bone regeneration. Tissue Eng Part B Rev 2010, 16 (6), 577-585. DOI: 10.1089/ten.teb.2010.0462. (16) Kuttappan, S.; Mathew, D.; Nair, M. B. Biomimetic composite scaffolds containing bioceramics and collagen/gelatin for bone tissue engineering-A mini review. International journal of biological macromolecules 2016, 93 (Pt B), 13901401. DOI: 10.1016/j.ijbiomac.2016.06.043. (17) Yang, H. S.; La, W. G.; Bhang, S. H.; Lee, T. J.; Lee, M.; Kim, B. S. Apatitecoated collagen scaffold for bone morphogenetic protein-2 delivery. Tissue Eng Part A 2011, 17 (17-18), 2153-2164. DOI: 10.1089/ten.TEA.2010.0702. (18) Marelli, B.; Ghezzi, C. E.; Barralet, J. E.; Boccaccini, A. R.; Nazhat, S. N. ThreeDimensional Mineralization of Dense Nanofibrillar Collagen-Bioglass Hybrid Scaffolds. Biomacromolecules 2010, 11, 1470-1479. DOI: 10.1021/bm1001087. (19) Wang, S.; Yang, Y.; Zhao, Z.; Wang, X. M.; Mikos, A. G.; Qiu, Z.; Song, T.; Sun, X.; Zhao, L.; Zhang, C.; Cui, F. Z. Mineralized Collagen-Based Composite Bone Materials for Cranial Bone Regeneration in Developing Sheep. ACS biomaterials science & engineering 2017, 3 (6), 1092-1099. DOI: 10.1021/acsbiomaterials.7b00159. (20) Hung, B. P.; Naved, B. A.; Nyberg, E. L.; Dias, M.; Holmes, C. A.; Elisseeff, J. H.; Dorafshar, A. H.; Grayson, W. L. Three-Dimensional Printing of Bone 33



Extracellular Matrix for Craniofacial Regeneration. ACS biomaterials science & engineering 2016, 2 (10), 1806-1816. DOI: 10.1021/acsbiomaterials.6b00101. (21) Douglas, T. E.; Messersmith, P. B.; Chasan, S.; Mikos, A. G.; de Mulder, E. L.; Dickson, G.; Schaubroeck, D.; Balcaen, L.; Vanhaecke, F.; Dubruel, P.; Jansen, J. A.; Leeuwenburgh, S. C. Enzymatic mineralization of hydrogels for bone tissue engineering by incorporation of alkaline phosphatase. Macromolecular bioscience 2012, 12 (8), 1077-1089. DOI: 10.1002/mabi.201100501. (22) Yamauchi, K.; Goda, T.; Takeuchi, N.; Einaga, H.; Tanabe, T. Preparation of collagen/calcium phosphate multilayer sheet using enzymatic mineralization. Biomaterials 2004, 25 (24), 5481-5489. DOI: 10.1016/j.biomaterials.2003.12.057. (23) Doi, Y.; Horiguchi, T.; Moriwaki, Y.; Kitago, H.; Kajimoto, T.; Iwayama, Y. Formation of apatite-collagen complexes. Journal of Biomedical Materials Research Part B Applied Biomaterials 1996, 31 (1), 43-49. DOI: 10.1002/(SICI)1097-4636(199605)31:1;3.0.CO;2-Q. (24) Zhang, X.; Xu, B.; Gao, F.; Zheng, P.; Liu, W. Repair of volumetric bone defects with a high strength BMP-loaded-mineralized hydrogel tubular scaffold. Journal of Materials Chemistry B 2017, 5 (28), 5588-5596. DOI: 10.1039/c7tb01279a. (25) Bo Jiang, Zhihong Wu, Huichuan Zhao, Fangyuan Tang, Jian Lu, Qingrong Wei, Xingdong Zhang. Electron beam irradiation modification of collagen membrane. Biomaterials 2006, 27,15-23. DOI:10.1016/j.biomaterials.2005.05.091. (26) Zheng, L., Sun, J., Chen, X., Wang, G., Jiang, B., Fan, H. & Zhang, X. In Vivo Cartilage Engineering with Collagen Hydrogel and Allogenous Chondrocytes 34


Page 34 of 40

Page 35 of 40 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 60


After Diffusion Chamber Implantation in Immunocompetent Host. Tissue Engineering Part A 2009, 15(8), 2145-2153. DOI: 10.1089/ten.tea.2008.0268. (27) Brinkman, W. T.; Nagapudi, K.; Thomas, B. S.; Chaikof, E. L. Photo-crosslinking of type I collagen gels in the presence of smooth muscle cells: mechanical properties, cell viability, and function. Biomacromolecules 2003, 4 (4), 890-895. DOI: 10.1021/bm0257412. (28) Yang, K.; Sun, J.; Wei, D.; Yuan, L.; Yang, J.; Guo, L.; Fan, H.; Zhang, X. Photo-crosslinked mono-component type II collagen hydrogel as a matrix to induce chondrogenic differentiation of bone marrow mesenchymal stem cells. Journal of Materials Chemistry B 2017, 5 (44), 8707-8718. DOI: 10.1039/c7tb02348k. (29) Taylor, R. H.; Fournier, S. M.; Simons, B. L.; Kaplan, H.; Hefford, M. A. Covalent protein immobilization on glass surfaces: Application to alkaline phosphatase. Journal of Biotechnology 2005, 118 (3), 265-269. DOI: 10.1016/j.jbiotec.2005.05.007. (30) NEUMANN, H.; LUSTIG, A. The activation of alkaline phosphatase by effector molecules: A combined kinetic and hydrodynamic study. European journal of biochemistry 1980, 109 (2), 475-480. DOI: 10.1111/j.1432-1033.1980.tb04818.x. (31) Rauner, N.; Meuris, M.; Zoric, M.; Tiller, J. C. Enzymatic mineralization generates ultrastiff and tough hydrogels with tunable mechanics. Nature 2017, 543 (7645), 407-410. DOI: 10.1038/nature21392. (32) Chappelet-Tordo, D.; Fosset, M.; Iwatsubo, M.; Gaché, C.; Lazdunski, M. 35



Intestinal alkaline phosphatase. Catalytic properties and half of the sites reactivity. Biochemistry 1974, 13 (9), 1788-1795. DOI: 10.1021/bi00706a002. (33) Fosset, M.; Chappelet-Tordo, D.; Lazdunski, M. ntestinal Alkaline Phosphatase. Physical Properties and Quaternary Structure. Biochemistry 1974, 13 (9), 17831788. DOI: 10.1021/bi00706a001. (34) Tronci, G.; Russell, S. J.; Wood, D. J. Photo-active collagen systems with controlled triple helix architecture. Journal of Materials Chemistry B 2013, 1 (30), 3705-3715. DOI: 10.1039/C3TB20720J. (35) Chen, H. T.; Xie, L. P.; Yu, Z. Y.; Xu, G. R.; Zhang, R. Q. Chemical modification studies on alkaline phosphatase from pearl oyster (Pinctada fucata): a substrate reaction course analysis and involvement of essential arginine and lysine residues at the active site. The international journal of biochemistry & cell biology 2005, 37 (7), 1446-1457. DOI: 10.1016/j.biocel.2005.02.002. (36) Noreen, R.; Chien, C. C.; Chen, H.-H.; Bobroff, V.; Moenner, M.; &, S. J.; Hwu, Y.; Petibois, C. FTIR spectro-imaging of collagen scaffold formation during glioma tumor development. Anal Bioanal Chem 2013, 405, 8729-8736. DOI: 10.1007/s00216-013-7337-8. (37) Eklouh-Molinier, C.; Happillon, T.; Bouland, N.; Fichel, C.; Diébold, M.-D.; Angiboust, J. F.; Manfait, M.; Brassart-Pasco, S.; Piot, O. Investigating the relationship between changes in collagen fiber orientation during skin aging and collagen/water interactions by polarized-FTIR microimaging. Analyst 2015, 140, 6260-6268. DOI: 10.1039/c5an00278h. 36


Page 36 of 40

Page 37 of 40 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 60


(38) Seuring, J.; Agarwal, S. Non-Ionic Homo-and Copolymers with H-Donor and HAcceptor Units with an UCST in Water. Macromolecular Chemistry and Physics 2010, 211 (19), 2109-2117. DOI: 10.1002/macp.201000147. (39) Cao, H.; Xu, S. Y. Purification and characterization of type II collagen from chick sternal cartilage. Food chemistry 2008, 108 (2), 439-445. DOI: 10.1016/j.foodchem.2007.09.022. Epub 2007 Sep 18. (40) Combes, C.; Rey, C. Amorphous calcium phosphates: synthesis, properties and uses in biomaterials. Acta Biomaterialia 2010, 6 (9), 3362-3378. DOI: 10.1016/j.actbio.2010.02.017. (41) Song, R. Q.; Cölfen, H. Additive controlled crystallization. CrystEngComm 2011, 13 (5), 1249-1276. DOI: 10.1039/c0ce00419g. (42) Rhee, S. H.; Lee, J. D. Nucleation of Hydroxyapatite Crystal through Chemical Interaction with Collagen. J. Am. Ceram. Soc 2000, 83 (11), 2890-2892. DOI: 10.1111/j.1151-2916.2000.tb01656.x. (43) Xu, B.; Zheng, P.; Gao, F.; Wang, W.; Zhang, H.; Zhang, X.; Feng, X.; Liu, W. A mineralized high strength and tough hydrogel for skull bone regeneration. Advanced Functional Materials 2017, 27 (4), 1604327. DOI: 10.1002/adfm.201604327. (44) Han, Y.; Bai, T.; Liu, Y.; Zhai, X.; Liu, W. Zinc ion uniquely induced triple shape memory effect of dipole-dipole reinforced ultra-high strength hydrogels. Macromolecular rapid communications 2012, 33 (3), 225-231. DOI: 10.1002/marc.201100683. Epub 2011 Dec 20. 37



(45) Wang, N.; Tytell, J. D.; Ingber, D. E. Mechanotransduction at a distance: mechanically coupling the extracellular matrix with the nucleus. Nature reviews Molecular cell biology 2009, 10 (1), 75. DOI: 10.1038/nrm2594. (46) Ingber, D. E. Cellular mechanotransduction: putting all the pieces together again. FASEB J 2006, 20 (7), 811-827. DOI: 10.1096/fj.05-5424rev.

For Table of Contents Use Only

38


Page 38 of 40

Page 39 of 40 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 60


Biomineralized hydrogel with enhanced toughness by chemical bonding of alkaline phosphatase and vinylphosphonic acid in collagen framework Lu Chen, † Ke Yang, † Huan Zhao, † Amin Liu, † Wanying Tu, † Chengheng Wu, † Suping Chen, † Zhenzhen Guo, § Hongrong Luo, † Jing Sun, † Hongsong Fan * †

Alkaline phosphatase (ALP) and phosphoprotein-like molecule of vinylphosphonic acid (VAP) were photo-crosslinked into collagen network to simulate the physiological mineralization process. With improved uniform CaP deposition and mineralized clusters bonding, as well as gentle biomimetic process, mineralized CAV hydrogel with optimized strength and compatibility for in situ cell encapsulation was obtained, and the approach was appreciated as bioactive ink for 3D printing and relative 39



biofabrication.

40


Page 40 of 40

Biomineralized hydrogel with enhanced toughness by chemical

Recommend Documents