Functionalized TiO2 Surfaces Facilitate Selective Receptor

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C: Surfaces, Interfaces, Porous Materials, and Catalysis 2

Functionalized TiO Surfaces Facilitate Selective Receptor-Recognition and Modulate Biological Function of Bone Morphogenetic Protein-2 Menghao Wang, Qun Wang, Kefeng Wang, and Xiong Lu J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.8b09492 • Publication Date (Web): 03 Dec 2018 Downloaded from http://pubs.acs.org on December 9, 2018

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The Journal of Physical Chemistry

Functionalized TiO2 Surfaces Facilitate Selective Receptor-recognition and Modulate Biological Function of Bone Morphogenetic Protein-2 Menghao Wang1, Qun Wang12, Kefeng Wang3*, and Xiong Lu1*

1 Key

Lab of Advanced Technologies of Materials, Ministry of Education,

School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu, Sichuan, 610031, China

2 College

of Life Science and Biotechnology, MianYang Teachers’ College, Mianyang 621006, Sichuan, China

3 National

Engineering Research Center for Biomaterials,

Genome Research Center for Biomaterials Sichuan University, Chengdu, Sichuan, 610064, China *Corresponding

author. Tel.: +86-28-87634023 Fax: +86-28-87601371 E-mail: [email protected] E-mail: [email protected]

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ACS Paragon Plus Environment

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Abstract Titanium dioxide (TiO2) is a promising biomedical material because it can be modified with various functional groups. However, the mechanism of the interaction between functionalized TiO2 and biomolecules, especially how the modified TiO2 regulates subsequent protein functions, still needs further investigation. In this study, we studied the interaction of bone morphogenetic protein-2 (BMP-2) with hydroxyl- and phosphite-grafted TiO2 surfaces. A set of force field parameters was developed for hydroxyl and phosphite groups on TiO2 surfaces, and the adsorption energy between the surface of the functionalized TiO2 and BMP-2 was calculated. Different coverages of functional groups were applied to the surface to investigate the influence of the functional group density. Grafting phosphite groups on the surface of TiO2 can significantly increase the adsorption energy of the protein and change the orientation of BMP-2 so that the wrist epitope of the BMP-2 molecules is pointing upward. This configuration specifically binds to the BMP receptor Type-I on the cell membrane and activates the SMAD1/5/8 signaling pathway for the purpose of enhancing the expression of bone growth and regeneration-related protein. This study shows that it is possible to regulate the function of a protein by deliberately modifying the material surface, which can guide the design of new materials through function-oriented surface modification.

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1.

Introduction

Titanium dioxide (TiO2) has attracted the attention of many researchers as a material with significant potential in many fields. Scientists and surgeons have developed a considerable number of biomedical devices based on TiO2. These devices have been mainly used for the repair/regeneration of bones1, cartilage2, teeth3 and other hard tissue4. This material is also used in the imaging5 and detection for specific biomolecules6. All these applications require us to understand the properties of TiO2 and its interactions with biomolecules. To acquire further understanding of the use of TiO2 in biomaterials, especially to clarify their mechanism of interaction with biomolecules, computer simulation methods have been adopted by many researchers. For instance, the adsorption kinetics between anatase and a titanium-binding polypeptide were investigated using molecular dynamics by Polimeni et al.7. They found that the adsorption of polypeptide/material occurred between polar groups on the side chain of the peptides and the water layer adsorbed on the TiO2 surface. They also indicated that the adsorption did not affect the configuration of the polypeptide. Brandt et al.8 employed molecular dynamics to study the adsorption between the rutile (100) surface of TiO2 and two biomolecules: amino acid side chain analogs and titanium-binding peptides (RKLPDA), and the importance of the water layer on the TiO2 surface during the adsorption process has also been emphasized. Our previous work indicated that a TiO2-graphene nanocomposite can synergistically enhance the adsorption of glycine9.

Researchers have introduced a variety of functional groups on TiO2 to improve its performance. The hydroxyl group is the most common functional group for the modification of this material. 3



It can act not only as a functional group, but can also serve as an attachment site for other groups such as carboxyl groups and phosphates, which give the TiO2 more potential for biomedical applications. Researchers have developed a variety of hydroxylation methods that can change the state of the TiO2 surface from completely hydroxylated to completely reduced10. Computational investigations have also been performed to study the influence of the functional groups. Zheng et al.11 used molecular dynamics to calculate the adsorption of a ProlineHydroxyproline-Glycine tripeptide on a hydroxylated rutile (110) surface in an aqueous environment containing Ca2+ ions. They demonstrated that the adsorption of the tripeptide on the surface of TiO2 aided by calcium ions was more stable than direct adsorption, and the final binding energy was also higher. Tilocca et al.12 used first-principle calculations and a CarParrinello molecular dynamics method to study the adsorption mechanism and reactivity of methanol on the surface of pure and hydroxylated anatase (101). Their research showed that molecular adsorption was more likely to occur on the surface of pure TiO2. On the hydroxylated surface, oxygen deficiency causes the energy balance be inclined to molecular separation, which maintains the dynamic balance between adsorption and separation. Ab initio methods have also been employed to study the influence of hydroxyl groups on the adsorption of biomolecules. Guo et al.13 studied the adsorption characteristics of aspartic acid on the surface of nitrogen-doped and calcium-doped TiO2. Their results indicated that the presence of water significantly affects the adsorption, and the adsorption of aspartic acid on TiO2 was attributed mainly to the amino group and carbonyl group.

The phosphate group is an important component in bone tissue and has been commonly used 4


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for the modification of TiO2. González et al.14 prepared phosphate-functionalized porous TiO2 on a silicon substrate and confirmed that this material improved the efficiency of sewage treatment. In the field of biomaterials, surface phosphorylation can introduce phosphate groups into the biological microenvironment and facilitate the time collaboration of gene expression, regulate mineralization and specific signaling pathways15. Other researchers have also discovered that inorganic phosphates are a nutrient signal that activates the protein kinase-A signaling pathway16, which has been considered by many studies to play an important role in bone differentiation. In addition, there are some experimental studies that illustrate the role of inorganic phosphates in osteogenesis from another perspective. They believe that phosphate groups can regulate multiple signaling pathways, including NF-E2 related factor-2 (Nrf2)17. This signaling pathway not only protects cells from toxic environments, but also promotes load-induced osteogenesis and fracture repair18. Although there are numerous studies of phosphate-functionalized TiO2 biomaterials, the biological properties of phosphatefunctionalized materials still need further clarification, especially the mechanism of the effect of phosphate on the protein conformation and regulation of cellular signaling pathways.

Bone morphogenetic protein-2 (BMP-2) is an important member of the transforming growth factor-β family. This cytokine has multiple effects on the development and homeostasis of bone and cartilage, and plays an important role in the osteogenesis process19. The use of BMP-2 in medical implants can improve the biocompatibility of the implants20. BMP-2 has alpha helix and two antiparallel beta sheets on each monomer, which constitute nine discrete beta-sheet chains. Furthermore, the pre-helix in BMP-2, which contains a unique small β-chain (β5α), is 5



the structural basis for hydrogen bonding with the β2, β5, β6 and β9 chains (see Fig. 1a). It can also stabilize the four-stranded antiparallel β-fold fragment. These unique structures of BMP2 enable its dimer to attain a conformation in which the two knuckle sites on the bulge (which constitutes the binding interface of the BMP receptor protein II) and two wrist sites on the cave (which constitutes the binding interface of the type I BMP receptor), which facilitates specific interaction with BMP receptors (see Fig. 1b).

Since BMP-2 is an important protein, researchers have performed many of experimental and computational research on BMP-2. For example, Zhang et al.21 studied the drug loading capacity of TiO2 nanotubes for several drugs including BMP-2, and they found that nanotubes loaded with BMP-2 have a good promoting effect on the adhesion and growth of mesenchymal stem cells. Ma et al.22 grafted the knuckle epitope of BMP-2 on the surface of TiO2 nanotubes through polydopamine and their results demonstrated that such surface treatment can enhance the osteogenic effect in animal experiments. Molecular dynamics methods have also been applied to study the properties of BMP-2. Utesch et al.23 studied the adsorption of BMP-2 on the surface of TiO2 and graphite. BMP-2 models with six different configurations were built and the results showed that the hydration layer on the surface of TiO2 prevented stable adsorption between BMP-2 and the surfaces. During the adsorption process, neither the graphite nor the TiO2 surface induced protein inactivation at a nanosecond timescale. Oliveira et al.24 studied the conformational changes of BMP-2 in an aqueous environment using a CHARMM27 field. They believe that the BMP-2 configuration in a water environment has a higher energy than the configuration in vacuum, and the unfolding tendency of BMP-2 in water 6


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is compensated by the electrostatic interaction between the protein and the water molecules. Kausar and Nayeem25 employed molecular dynamics methods to study the influence of temperature and hydration on the configuration of BMP-2.

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Fig. 1. Schematic diagram of the BMP-2 structure. a) The BMP-2 monomer; the circles indicate the position at which the acidic amino acid residue is located. b) BMP-2 dimer; the circles indicate the wrist (upper) and knuckle (lower) epitopes of BMP-2 dimer. Although considerable research on the interaction of BMP-2 and TiO2 has been performed, there are still some aspects that require further investigation. As mentioned in the previous section, various surface modification method has been performed to improve the biological performance of TiO2. However, the current computational investigations have mainly focused on pure TiO2. There are only a few studies about the surfaces with the grafted groups. The main reason for this phenomenon is the lack of force field parameters for the functionalization groups. In this study, we investigated the interaction between hydroxylated and phosphorylated TiO2 surfaces and BMP-2 by using theoretical calculations to study. We developed a new set of force field parameters for hydroxylated and phosphorylated TiO2 surfaces and used it to calculate the effect of these two functional groups on BMP-2 adsorption. The subsequent biological function of BMP-2 after its adsorption on TiO2 was discussed.

2.

Computational Details

2.1 BMP-2: The original structure of BMP-2 was acquired from RCSB protein data bank and determined by X-ray diffraction (the RCSB Code: 3BMP) with a resolution of 2.7 Å. We took the A chain from this dimer as our target molecule. After solvating the protein, we added 0.15 mol/L of Na+ and Cl- ions to the water box. To convert the protein from a crystalline state to its configuration in solution, we gradually heated the system to 310 K with a temperature difference of 10 K per step followed by 100 picoseconds of pre8


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equilibration. The relative positions of the surface and the protein are shown in Fig. S1-S4 in the Supplementary Material.

2.2 Surfaces: TiO2 has three different crystal forms, namely anatase, rutile and brookite, of which rutile and anatase are relatively stable at normal temperature. For this reason, the first two tend to receive more attention.

Pure TiO2 surface: In this study, we selected two crystal faces of rutile R (110) and anatase A (101) (see Fig. 2a, 2b) as the pure surface. The top view of these two surfaces are shown in Fig. 2c and Fig. 2d. The pure surfaces not only act as an interaction surface, but also is the base material for modification. In these two different crystalline forms, we constructed 10 models with four kinds of modification combinations. The ratio of the graft groups was also under consideration. Three kinds of modification groups were employed: hydroxyl groups, phosphite groups and phosphite/hydroxyl groups. According to previous studies, the functional groups were grafted on the 5-fold titanium atoms. We also constructed models with different ratios of the functional groups.

Hydroxyl-modified TiO2 surfaces: Two sets of hydroxyl-modified TiO2 surface models were constructed. The surface coverage of the first set was 4:1, in which one of every four 5-fold Ti atoms was grafted with hydroxyl groups. According to the different crystalline forms, these two models were referred to as ROH1 and AOH1 (see Fig. 3a, b). The second set of models had a greater degree of hydroxylation, and the surface coverage of 5-fold Ti and hydroxyl 9



groups was increased to 4:2; these two models were identified as ROH2 and AOH2, respectively (Fig. 3c, d). Phosphite-modified TiO2 surfaces: Two sets of models were constructed. In the first set, the surface coverage of 5-fold Ti with phosphite groups was set to 1:16, and named ROP2 and AOP2 depending on the crystalline form (see Fig. 4a, b). The surface coverage in the second set of models was increased to 2:16, referred to as ROP2 and AOP2 depending on the crystalline forms (see Fig. 4c, d).

Hydroxyl and Phosphite co-modified TiO2 surfaces: On this set of surfaces, only phosphite groups were regarded as variable. All the 5-fold Ti atoms were modified with hydroxyl groups. Then, phosphite groups were introduced to substitute for hydroxyl groups. The surface coverage of phosphite groups was the same as that of the previous model, and was named as RHP1/AHP1 and RHP2/AHP2 depending on the crystalline form (shown in Fig.5).

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Fig. 2. Structure of pure TiO2 unit cells. Side view of a) rutile (110) surface and b) anatase (101) surface; top view of c) rutile (110) surface and anatase (101) surface. The size of the cell and location of the 5-fold Ti atoms is indicated with arrows in two directions

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Fig. 3. Structure of hydroxyl-modified TiO2 surface unit cells. White stands for hydrogen, red stands for oxygen, and gray stands for 5-fold titanium. The structure underneath the 5-fold titanium atoms is shown as a line. R and A stand for rutile and anatase, respectively. The number in the tail stands for the modification ratio. One indicates that there is one hydroxyl group grafted on every four 5-fold titanium atoms and two means there are two hydroxyl groups grafted on every four 5-fold titanium atoms.

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Fig. 4. The structure of the phosphite-modified TiO2 surface unit cells. R and A stand for rutile and anatase, respectively. The last digits stand for the modification ratio: one means there is one phosphite group grafted on every sixteen 5-fold titanium atoms and two means there are two phosphite groups grafted on every sixteen 5-fold titanium atoms.

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Fig. 5. Hydroxyl and Phosphite co-modified TiO2 surfaces. R and A stand for rutile and anatase, respectively. The last digits stand for the modification ratio: one means there is one phosphite group grafted on every sixteen 5-fold titanium atoms and two means there are two phosphite groups grafted on every sixteen 5-fold titanium atoms. All other 5-fold Ti are grafted with hydroxyl groups.

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2.3 Calculation setup Molecular dynamics calculations were performed using NAMD 2.10 code26. Periodic boundary conditions were applied to all systems and a TIP3P water model was employed. A cutoff distance of 12 Å was applied to the real-space electrostatic interactions and van der Waals interactions. Long-range electrostatic interactions were described by the Particle-Mesh-Ewald method. In the calculation, we set the time step to 1 femtosecond and applied the rigid bond model to hydrogen atoms through a SHAKE algorithm. The behavior of TIP3P water molecules and proteins in the calculation is described by the CHARMM36 force field27. TiO2 was simulated by the parameters we fitted. The theoretical basis of the fitting was the firstprinciples calculation result by Matsui et al.28. The force field parameters of the functional group were developed through the Force Field Toolkit (ffTK) plugin in the VMD package. The atomic charges of hydroxyl and phosphate were defined according to a similar structure in the force field of CHARMM36. The calculated force field parameters and their verification are described in the supplementary materials section. The total adsorption energy (𝐸𝑡𝑜𝑡𝑎𝑙) in each model was calculated, which is the sum of electrostatic adsorption energy (𝐸𝑒𝑙𝑒𝑐) and the Van der Waals adsorption energy (𝐸𝑉𝐷𝑊): 𝐸𝑡𝑜𝑡𝑎𝑙 = 𝐸𝑒𝑙𝑒𝑐 + 𝐸𝑉𝐷𝑊 The calculated adsorption energy is listed in Table 1

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Results

Table 1 Adsorption energy in each model (Kcal/mol) Surface

Electrostatic

Van der Waals

energy

energy

R(110)

-204.04

3.55×10-3

-204.04

A(101)*

-357.32

-0.93

-358.26

ROH1

-450.20

-10.20

-460.40

ROH2*

-515.62

8.62

-502.58

AOH1

-605.87

6.75

-599.12

AOH2

-451.66

46.75

-404.92

ROP1

-2087.89

-8.75

-2096.65

ROP2

-5695.29

29.44

-5665.84

AOP1

-2189.05

8.04

-2181.01

AOP2

-3484.80

-3.22

-3488.02

AHP1

-2687.54

19.88

-2667.66

AHP2

-3984.64

72.95

-3911.12

RHP1

-3436.72

90.06

-3346.79

RHP2

-5314.29

14.76

-5299.53

*

mean value of maximum energy

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Total energy

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3.1 Pure TiO2 surfaces The adsorption energy consists of two parts, in which the electrostatic force accounts for the vast majority of the adsorption energy, whereas the van der Waals force represents the nonpolar effect in the system. There is a slight difference in the adsorption energy of BMP-2 on the surface of two pure TiO2 surfaces according to their adsorption energies. In the R (110) system, the electrostatic adsorption energy of BMP-2 was slightly smaller than that in the A (101) system; therefore, the anatase system had a higher total adsorption energy. This result is consistent with Utesch’s research

23,

in which BMP-2 could not form a stable adsorption

configuration on a pure TiO2 surface.

The adsorption mechanism also revealed the instability of the protein-surface interaction. In the R (110) system, BMP-2 moves randomly on the surfaces. During the simulation, only two amino acid residues at the starting end (ARG9, LYS11) showed a tendency to approach the surface; however, this trend did not result in a stable adsorption structure (Fig. 6). The same phenomenon also occurred in the A (101) system. The difference is that the rotation of BMP2 on the A (101) surface was slower than that on the R (110) surface. Meanwhile, the change of the adsorption site of BMP-2 on the A (101) surface was relatively small, which can be explained by the higher surface energy of anatase29.

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Fig. 6. The BMP-2 configuration changes continuously with time on pure TiO2 surfaces. a), b) and c) stand for the same system (rutile (110) surface) in different simulation time.

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3.2 Hydroxyl-modified TiO2 surfaces The adsorption energy of the hydroxylated surface was slightly higher than that of the pure surface. This phenomenon was identical in both the rutile and anatase systems and was consistent with the Car-Parrinello calculations reported by Köppen et al.30. Electrostatic interactions still played a dominant role in the total adsorption energies (see Fig. S6). This was attributed to the polarity of the rutile and anatase surface. The adsorption energies of these four models did not stabilize, indicating that the adsorption process between the surfaces of the BMP-2 was dynamic and no immobilization occurred.

In the hydroxyl modified TiO2 system, as the water molecules in the medium was replaced with hydroxyl groups, the adsorption configurations of BMP-2 were like that in the pure TiO2 systems. Waters formed two dense layers on these surfaces and competed for adsorption sites with BMP-2. Stable adsorption of BMP-2 also did not occur in the AOH1and AOH2 system (see Fig. 7 and Fig. 8). However, the presence of hydroxyl groups resulted in the adsorption of a small amount of polar amino acid residues and longer adsorption time. As shown in Fig. 7a, c, in the ROH1 system, ASP9 and ARG11 were two active amino acids on the head of the sequence, which only adsorbed on the surface for a nanosecond.

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Fig. 7. BMP-2 configuration in the AOH system. Top view: a) AOH1, b) AOH2; side view: c) AOH1, d) AOH2

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Fig. 8. BMP-2 configuration in the ROH system. Top view: a) ROH1, b) ROH2; side view: c) ROH1, d) ROH2

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3.3 Phosphite-modified TiO2 surface The adsorption energy between BMP-2 and a phosphite-modified TiO2 surface was significantly higher than that of the previous two systems. Although the van der Waals forces between them did not show much difference, the phosphite-modified surfaces provide greater electrostatic interaction than the pure surfaces and hydroxylated surfaces. The interaction between phosphite and ASP/GLU made a considerable contribution to the total adsorption energy. Another important phenomenon was that the adsorption energy in the ROP2 system was much larger than that in the ROP1 system; similar results were also found in AOP1/2, indicating that the larger grafting ratio also had a higher adsorption energy.

Phosphite groups have an important influence on the orientation of the protein. A stable interaction occurs between the weakly basic hydrogen phosphate and the weakly acidic amino acid residue (see Fig. 9 and Fig. 10). This interaction changed the orientation of the protein, resulting in a significant change in the adsorption configuration of BMP-2 in this system. The wrist epitope in the protein pointed to the top of the system. The adsorption between specific amino acid residues and the phosphite-modified TiO2 surface can be seen in Fig. 11. In short, the presence of hydrogen phosphate prevented the formation of a dense hydration layer, decreased the inference of water molecules and allowed residues to bind directly and stably to the surface. Thus, a stable adsorption mode of BMP-2 was achieved and maintained.

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Fig. 9. The morphology of the BMP-2 in the AOP system. Top view: a) AOP1, b) AOP2; side view: c) AOP1, d) AOP2

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Fig. 10. The morphology of the BMP-2 in the ROP system. Top view: a) ROP1, b) ROP2; side view: c) ROP1, d) ROP2

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Fig. 11 The influence of phosphite groups on BMP-2 adsorption. a) The distribution of acidic amino acid residues on BMP-2. ASPs are shown as red stick. GLUs are shown as blue sticks. b) The adsorption was mainly contributed by the interaction between GLU/ASP and phosphite groups.

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3.4 Hydroxyl/phosphite co-modified TiO2 surfaces Phosphite groups grafted on the surface of TiO2 are often accompanied by hydroxyl groups. First, the grafting process tends to occur in solution, which means the surface was fully hydrated31. Second, the hydroxyl groups are often grafted onto the surface as further modification sites for the phosphite groups. Therefore, hydroxyl/phosphite co-modified TiO2 is more commonly used in experimental approaches. In our calculations, the adsorption between the hydroxyl/phosphite-modified surface and BMP-2 resulted in higher stability. The wrist-pointing-up phenomenon also appeared in these systems, which was similar to a phosphite-modified surface. Furthermore, the presence of hydroxyl groups on the surface filled the gap between the phosphite and the surface. On phosphite-modified surfaces, proteins are formed only on the “finger” of the phosphite groups; whereas on the hydroxyl/phosphite comodified surfaces, the hydroxyl group filled the gap between the surface of the TiO2 and the phosphate and also provided a new binding site. Therefore, a more stable adsorption configuration was obtained (see Fig. 12).

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Fig. 12. The effects of hydroxyl/phosphite co-modified TiO2 surfaces on BMP-2 adsorption. The hydroxyl groups filled the gap between the phosphite groups.

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4

Discussion

4.1 Surface functionalization affects the BMP signaling pathway Specific surface functionalization, such as phosphite modification, has the potential to modulate the MP signaling pathway and regulate stem cell differentiation. The bioactive BMP2 structure in the human body is a dimer. It consists of two BMP-2 monomers with the same epitope on the same sides. Similar with other members of the TGF-β family32, the wrist epitopes on the BMP-2 dimer recognizes the BMP receptor type-I. The recognition mechanism on the wrist epitopes occurs when the hydrophobic residues on the BMP receptor interact with the “pocket” formed by BMP-2 dimers. During the binding process, the structure called the pre-α helix, which is beside the wrist epitope, undergoes a conformational change to accommodate and bind to the structure of the BMP receptor protein. The BMP receptor protein I is a transmembrane protein that, when bound to BMP-2, activates the intracellular SMAD1/5/8 signaling pathway (see Fig. 13). The knuckle epitope, on the other side, mainly interacts with BMP receptor protein type-II and inhibin (see Fig. 1). These transcription factors are oligomerized with SMAD4, which are translocated into the nucleus and regulate the transcription of the response gene along with transcriptional co-activation/co-repression factors. This signaling pathway is the most primary and direct signaling pathway for communication between the extracellular matrix and the nucleus. This signaling pathway can be activated by BMP-2 as well as other members of the transforming growth factor-β family.

Our calculations show that the most important residues for BMP-2 adsorption on the phosphite surfaces are aspartic acid and glutamic acid. These two amino acids are mostly distributed at, 28


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or near, the knuckle epitope of BMP-2, making the wrist epitope of this material-protein complex point upward. Because the adsorption on phosphite groups interferes the orientation of BMP-2, the pocket formed by the wrist epitope of the BMP-2 dimer also points upward (see Fig. 13). This structure specifically recognizes the BMP receptor protein I, and thereby modulates the biological functions through surface modification of the biomaterial.

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Fig. 13 Schematic diagram of the SMAD signaling pathway under the influence of a phosphitemodified TiO2 surface.

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4.2 Surface functionalization affects the water layer Surface functionalization can affect the formation of water layers on TiO2, thereby affecting the interaction mechanism between proteins and materials. Because TiO2 is a polar inorganic material, its surface tends to form a few dense water layers. The adsorption between the protein and pure/hydroxylated TiO2 surfaces is indirect, which means that water intercalates the BMP2/TiO2 system. This also implies a strong competitive adsorption relationship between BMP-2 and water molecules. This conclusion has also been confirmed by other researchers, who employed a reactive force field33. Their study reported the adsorption of multiple layers of water on the surface of pure TiO2. Furthermore, they also pointed out the differences between these water layers. The water layer closest to the surface reacts with the surface, whereas the subsequent hydration layer only undergoes molecular adsorption, and its chemical structure does not change. In addition, Koparde et al. studied the adsorption of water on TiO2 nanoparticles and reached similar conclusions34. In their study, the two hydration layers closest to the surface were clearly observed, whereas the water molecules outside the two layers did not form a particularly significant layered structure.

In our results, the density distribution of oxygen atoms in water indicated that the surface of the pure TiO2 and the surface of the hydroxyl-modified TiO2 had two regions in which water molecules were concentrated. The water molecules gathered in these two regions on both surfaces. On the pure rutile surface (see Fig. 14a), the hydrogen atoms in the water point to the under-coordinated oxygen atoms on the surface and several layers of ordered water molecules covered the surface. These water layers form a competitive adsorption relationship with 31



proteins, which is an important cause of unstable adsorption between the protein and the surface. The layered structure of the water became sparser as the distance from the surface increased. The water molecules closest to the surface were most closely adsorbed, and the protein occasionally discharged water molecules in the outer water layer and made direct contact the first water layer. A water layer was also formed on the surface of the hydroxyl-modified TiO2 surface (Fig. 14b), and the structure was similar to that on the pure TiO2 surfaces.

However, on the surface of the phosphite-modified and phosphite/hydroxyl co-modified surfaces, there was only one region in which water molecules concentrated. As shown in Fig. 14c and d, there was no significant difference in the distribution of the water molecules in the original hydration zone to those in other locations, which indicated that the phosphite groups on the surface changed the characteristics of the water layer, especially when the surface was not fully covered. The water layer on the surface of phosphite-modified TiO2 lost the consistency of orientation, and it was easier to attract amino acids for adsorption. The water layer also lost its dense structure and could no longer hinder the interaction between the proteins and the materials. Comparing the water layer on TiO2 surfaces with different functional groups, it is concluded that the functional group on the TiO2 material can change the surface microenvironment and alter its biological function.

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Fig. 14 Density profile (left) and atomic distribution (right) of water oxygen. a) Pure surface: 2 density peaks (left); 2 significant water layers (right). b) Hydroxyl-modified surface: 2 density peaks (left); 2 distinctive water layer (right). c) Phosphite-modified surface: 1 density peak (left); 1 water layer (right) and d) hydroxyl/phosphite co-modified surface, 1 density peak (left); one insignificant water layer (right).

33



5

Conclusion

Titanium dioxide has a wide range of applications in the field of bone regeneration. However, previous studies often focused on structural variation of small biomolecules when they interact with material surfaces. and the connections between structure/ properties of biomaterials and biological function of proteins are rarely discussed.

In this study, by choosing the right modification group and target protein, we investigated the effects of surface modification on the biological function of BMP-2 and found a direct link between surface structures of TiO2 and biological functions of proteins. Our results indicated that the presence of phosphite groups can influence the functions of BMP-2. Different functional group densities also affect the behavior of proteins on the surfaces of TiO2. The specific conclusions are as follows: 

The crystalline forms of TiO2 have a slight influence on the adsorption of BMP-2; the adsorption of BMP-2 on anatase is higher than that on rutile. However, BMP-2 cannot be stably adsorbed on both surfaces. The main reason is that water molecules form a competitive adsorption relationship with proteins on the surface, which hinders the formation of stable adsorption structures between the proteins and the surfaces.



Hydroxylation does not have much effect on the adsorption mode between TiO2 and BMP2, but the adsorption energy slightly increases because the surface hydroxyl groups provide nanostructures to the material and can adsorb proteins more efficiently.



Phosphorylation of the TiO2 surface significantly enhances the adsorption of BMP-2. Phosphite groups provide a site for adsorption and eliminate the effects of the water layer 34


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on the TiO2 surface. They adsorb acidic aspartic acid and glutamic acid residues mainly through the P-O-H structure. The phosphite-modified TiO2 surface adsorbs the protein’s knuckle epitope, which contains the majority of the ASPs and GLUs in BMP-2. This phenomenon makes the wrist epitope point upward, activating the SMAD1/5/8 signaling pathways and promoting osteoblast and bone fracture repair. 

The co-modification of the TiO2 surface with phosphite and hydroxyl groups improved the stability of the BMP-2 adsorption. This was mainly because the hydroxyl groups filled the gap between the hydrogen phosphate and the surface.

Our calculational approach proves the feasibility of biological function regulation via material modification, and provides a useful reference for upcoming rapid development of new biological materials, especially those who using physical/chemical methods for regulating biological functions of proteins. For instance, to modulate the functions of proteins with unique structures like BMP-2, we can modify the surface of biomaterial with basic functional groups. This is because there are only two kinds of acidic amino acids in BMP-2 molecules, i.e. GLU and ASP, which grants them uniqueness that can be manipulate specifically. Meanwhile, the interaction between acidic amino acids and polar surfaces is strong, which makes the interaction between amino acids and biomaterials stronger. These characteristic acidic amino acid residues have potential as a target interaction site for future biomaterial design. Through the combination of various material factors and unique molecular structure of target protein, we can design better biomaterials for selective controlling specific biological functions of proteins. 35



Supporting information. Including the initial structures of material-BMP-2 complex, adsorption energy profiles, the development and validation of force field parameters.

Acknowledgements This work was financially supported by the National Key Research and Development Program of China (2016YFB0700800), NSFC (81671824), and Fundamental Research Funds for the Central Universities (2682018QY02, 2682018ZT30).

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Functionalized TiO2 Surfaces Facilitate Selective Receptor

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