Introduction

In biomedical applications, there are continuous efforts to enhance methods, materials and devices. The recent development of novel biomaterials and their applications to biomedical problems have dramatically improved the treatment of many diseases and injuries.1, 2, 3 Although a various types of materials in biomedicine have been used widely, most biomaterials lack the desired functional properties to interface with biological systems and have not been engineered for optimum performance. Therefore there is an increasing demand to develop novel materials to address such problems in biomedicine arena. Biocompatible and biodegradable fusion polymers are a class of new generation of biomaterials that have demonstrated great potential for medical devices, tissue engineering scaffolds, drug delivery and biomedical-healthcare sensors.

There are numerous parameters of polymeric biomaterials that can affect the cellular behavior in a controlled manner. The underlying mechanisms for the biocompatibility of polymers at the molecular level are complex and have not been clearly demonstrated, although many theoretical and experimental efforts have been made to understand these mechanisms.4, 5 Water and proteins interactions have been recognized as fundamental for the biological response upon contact with polymers. We have proposed the ‘Intermediate Water’ concept6, 7, 8 on the basis of results on the water sorption process into polymeric biomaterials. The water exhibited clearly defined peaks for cold crystallization in the differential scanning calorimetry (DSC) chart, a strong peak at 3400 cm−1 in a time-resolved infrared spectrum and higher mobility of water in a 2H-nuclear magnetic resonance.6, 7, 8 As a result, the biocompatibility of polymers was ascribed to the predominant population of intermediate water in the hydrated polymers. Intermediate water interacts with polymer chains in a intermediate way, that is, stronger than free water but weaker than tightly bound non-freezing water. We hypothesized that intermediate water, which prevents the proteins and blood cells from directly contacting the polymer surface on the polymer surface, has an important role in the biocompatibility of polymers.

In this focus review, we describe the recent design of biocompatible and biodegradable polymeric biomaterials for various applications in medical devices. Here we present various synthetic strategies for the preparation of the biomaterials, which include characteristic properties of the biocompatibility, biodegradability and anti-microbial activity of polymer-based biomaterials in a self-organization manner. In addition, we describe the applications of polymer-based biomaterials in tissue engineering and medical devices and provide an overview of the recent experimental progress of the screening of multi-functional biocompatible polymers based on bio-interfacial water structure.

Biocompatible polymeric biomaterials

Polymeric materials for the medical devices that may come in contact with human blood should have capacity to resist protein adsorption and blood cell adhesion and thus triggering the organism’s defense systems.1 Some biocompatible polymer surfaces have been developed, and they fall into the following three categories:1, 6 (a) hydrophilic surfaces, (b) surfaces with micro-phase-separated domains, and (c) biomembrane-like surfaces, including zwitterionic groups. Physicochemical properties, including wettability, surface free energy, surface charge, stiffness, topography and the presence of specific chemical functionalities, surface bound water appears to bear an instrumental role in the biological response induced by the synthetic polymers.1, 9 New-generation polymer poly(2-methoxyethyl acrylate) (PMEA) shows excellent blood compatibility and biocompatibility and has been approved for medical use by the Food and Drug Administration.6, 7, 8 For instance, PMEA-coated circuits and tubes exhibit significantly reduced blood cell activation when used in cardiopulmonary bypass and catheters for central veins of human blood vessels. It has been maintained that PMEA’s compatibility with platelets, white and red blood cells (RBCs), complement and coagulation systems has been dictated by the presence of the intermediate water.6, 7, 8

It should be noted that the word ‘biocompatibility’ is used in general as the term evaluating properties of materials that do not cause adverse effect when the materials come into contact with living organisms, such as proteins, biological cells and tissues.5 This review primarily deals with ‘biocompatibility’ of polymer materials against various biological elements in human blood flow system.

Principle of cell attachment on polymers

Cells can attach in serum-containing medium even on polymers, such as polystyrene and polyethylene terephthalate, which do not possess any specific cell attachment ligands.10, 11 On these polymers, serum proteins (for example, fibrinogen and fibronectin) generally adsorb and change their conformation to allow the cells to attach or to function as cell attachment ligands (Figure 1a).10, 11 Protein adsorption and its conformational change are thus critical for cell attachment on polymers, and the regulation of protein adsorption leads to the control of cell attachment on polymers. We have suggested that intermediate water can influence protein adsorption on polymers.12, 13 Therefore the attachment behavior of the cells will be different between PMEA and conventional polymers such as polystyrene due to the difference of protein adsorption and its conformational change.

Figure 1
figure 1

Schematic illustration of cell attachment on polymers, (a) conventional polymers (polyethylene terephthalate (PET) and polystyrene (PSt)), (b) PTFHA, PMEA and PMEA analogous polymers. (c) PMPC and polyethylene glycol (PEG).

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Different attachment of human platelets and non-blood cells on PMEA and its analogous polymers

Cell attachment ligands are different among the cells. It has been reported that platelets require the adsorption of fibrinogen and adsorption-induced conformational change, which exposes cell attachment sites for their attachment.11 Therefore, it is necessary to prevent the adsorption and conformational change of fibrinogen on the polymers for the acquirement of blood compatibility. In contrast to platelet attachment, non-blood cells require the adsorption and the conformational change of fibronectin rather than fibrinogen for their attachment. Previously reported blood compatible polymers such as polyethylene glycol and the polymers containing 2-methacryloyloxyethyl phosphorylcholine (PMPC) have been reported to prevent the adsorption and the conformational changes of any proteins, including both fibrinogen and fibronectin, and thus any types of cells cannot attach on the substrates coated with them (Figure 1c).14, 15

PMEA and its analogous polymer, poly(tetrahydrofurfuryl acrylate) (PTHFA), have been reported as blood compatible polymers.16, 17 These polymers suppress the adsorption and conformational change of fibrinogen to prevent platelet attachment.12, 16 Recently, we have reported that PMEA and PTHFA do not suppress the conformational change of fibronectin, and the fibronectin can expose their cell attachment sites on the polymers.13 Non-blood cells can attach on PMEA and PTHFA due to such fibronectin (Figure 1b).13 PMEA and PTHFA are thus newly categorized as blood compatible polymers, which allow the attachment of non-blood cells but not platelets.13, 18

Adsorption-induced conformational change is determined by protein flexibility. The difference of conformational change between fibronectin and fibrinogen observed on PMEA and PTHFA might be due to the difference of flexibility of these proteins. Fibronectin shapes ‘beads on strings’ and shows high flexibility.19, 20 Fibronectin can change its conformation even on the polymers that prevent conformational change of adsorbed fibrinogen. Intermediate water keeps proteins away from non-freezing water, which induces conformational change of protein.12, 16 It appears that necessary amounts of intermediate water to prevent conformational change are different between fibronectin and fibrinogen due to the difference of flexibility. Therefore cell attachment can be regulated by the regulation of intermediate water content through the regulation of protein conformational change (Table 1).

Table 1 Relationship among intermediate water contents, protein conformational change, and cell attachment
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Recent advances in biology and medicine require blood-contact biomedical applications, including cell isolation from blood and endothelial cell-covered artificial blood vessels and stents. Newly categorized blood-compatible polymers, such as PMEA and PTHFA, are useful for these applications. Therefore the techniques to control intermediate water contents will strongly progress blood-contact biomedical applications through the regulation of protein adsorption and the following cell attachment.

Controls on water structure at the biointerface through precision polymer synthesis

Precision control over the polymers’ biocompatibility is a longstanding drawback in the arena of biocompatible polymeric materials, and the synthesis of well-defined polymers having precisely controlled molecular architecture is a powerful approach for the manipulation of polymer properties. This is particularly true in the development of biocompatible polymeric materials where the primary structure of polymers, for example, molecular weight, molecular weight distribution, monomer sequence distribution, stereoregularity, side-chain functionality, chain-end structures and long-chain branching, can greatly affect the biocompatibility of polymeric materials. To clarify the fundamental relationship between the biocompatible property of polymers and the chemical structure of polymeric biomaterials, we have started a study to elucidate the structure-property relationships in blood-compatible polymers by means of precision polymer synthesis.

Thus far, we have been investigating the relationship between the polymer primary structures and their blood compatibility by utilizing vinyl polymers having hydrophilic functional groups. In our previous studies, we have reported that PMEA, which has a quite simple chemical structure, exhibits superior blood compatibility;21, 22, 23 and PMEA possesses the unique hydration water structure, intermediate water in the hydrated state.24, 25, 26, 27, 28, 29 We have further investigated the blood compatibility of PMEA analogous polymers (Figure 2) having differences in the backbone structure (acrylate or methacrylate), oligo(ethylene glycol) (EG) side-chain lengths (number of units=1 to 3) and side-chain terminal groups (methyl or ethyl).30

Figure 2
figure 2

Chemical structures of poly(2-methoxyethyl acrylate) (PMEA) and PMEA analogous polymers (POEG(M)A).

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Side-chain modification

The modification with oligoEGs is a well-established methodology to tune the hydrophilicity of polymeric materials.31, 32, 33, 34, 35, 36, 37, 38, 39 Poly[oligo(ethylene glycol)(meth)acrylate]s (POEG(M)As) consist of poly(meth)acrylate backbones and oligo(ethylene glycol) side-chains, consequently, the EG functionalized poly(meth)acrylate is one of the most readily accessible hydrophilic polymers. Although POEG(M)As have simple chemical structures and numerous research studies have been conducted to date, there is still plenty of room for controlling hydrophilicity/hydrophobicity by modifying the chemical structure of side-chains. The basic way to modify the side-chain structure is by tuning the number of EG units and chain-end terminal group. Hydrophilicity of the polymer increases with the number of EG units, as the polymers have longer side-chains, the polymers become soluble in water and typically show lower critical solution temperature (LCST) in aqueous solutions.33 The number of carbon atoms in terminal alkoxy group also affects the water solubility and some of the polymers having longer alkyl terminal group show LCST below 37 °C.40 The DSC measurement revealed that intermediate water content was increased by tuning the chemical structure of polymer to be more hydrophilic (much EG units with less terminal carbons), and a decrement trend was observed in the number of adhered platelets with increasing the intermediate water content.

Zwitterionic polymers are known as the promising biocompatible materials for medical devices.40 For example, poly(2-methacryloyloxyethyl phosphorylcholine) (PPBMA: poly(phosphobetaine methacrylate), generally known as PMPC)) is a biomimetic material containing phosphorylcholine group for resisting nonspecific protein adsorption and platelet adhesion.41, 42 Recently, synthetic polymers containing zwitterionic structures similar to PPBMA, such as poly{[2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide} (poly(sulfobetaine methacrylate)),43, 44 and poly(1-carboxy-N,N-dimethyl-N-(2’-methacryloyloxyethyl)methanaminium) (poly(carboxybetaine methacrylate)),45, 46, 47 bearing sulfo- and carboxy- betaine group, respectively, are also reported as blood-compatible polymers, which show good plasma protein-fouling resistance. Most recently, poly(serine methacrylate) was reported as a new family of a zwitterionic polymer having an amino acid, L-serine, as the side-chain group.48 (Figure 3)

Figure 3
figure 3

Chemical structures of zwitterionic polymers (phospho-betaine, sulfo-betaine and calboxy-betaine), which possess the intermediate water in their hydrated state.

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Backbone modification

Changing the polymer backbone structure is also an effective approach to tune the polymer properties.49, 50 Poly[oligo(ethylene glycol) vinyl ether] is an analog of POEG(M)A. The structural difference between the two polymers is only in the side-chain linkage that the former OEG side-chains were connected to polymer backbone through ether bonds instead of ester connections (Figure 4). However, the difference engenders large differences in the molecular mobility and the hydrophilicity of the polymers. For instance, most of poly(vinyl ether)s having OEG side-chains show quite low glass transition temperature (Tg<−60 °C) and are soluble in water or exhibit an LCST in aqueous media. Some of the poly(vinyl ether)s (for example, poly(2-ethoxyethyl vinyl ether), LCST=21 °C) are insoluble at body temperature, and the human platelets adhesion test could be performed at 37 °C. Accordingly, we have analyzed the hydration water structure in POEG(M)A and their poly(vinyl ether) analogs by DSC, and the poly(vinyl ether)s showed a cold crystallization of water at around −40 °C and exhibited low platelet adhesion as well as the case of POEG(M)A.51

Figure 4
figure 4

Poly(2-alkoxyethyl acrylate)s and their poly(vinyl ether) analogs. Poly(2-ethoxyethyl vinyl ether) (R=C2H5) exhibits LCST at 21 °C.

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Modification on side-chain branch spacing

As mentioned above, the structural control over the macromolecular chemical structure is an effective approach to modify/control the hydration water structure and the blood compatibility of polymeric materials. There remains ample scope for further modification in the chemical structure of polymers, for example, tacticity, side-chain linkage and side-chain branch placement. The structural control over the side-chain placement along the polymer backbone is one of the most challenging topics in vinyl polymer synthesis. Fortunately, an effective pathway to achieve the model sequence-regulated vinyl polymers was reported, and the methodology utilizing the regio- and stereo-selective ring-opening metathesis polymerization (ROMP) of allyl-substituted cycloalkenamers52 opened a new window to precisely control the side-chain branch placement.53, 54, 55, 56, 57, 58 Based on the works, we have started a study to elucidate the structure–property relationships in biocompatible polymeric materials by means of precision polymers synthesized through regio- and stereo-selective ROMP (Figure 5).

Figure 5
figure 5

Synthesis of polymers having precisely placed side-chain branches by regio- and stereo-selective ROMP of allyl-substituted cis-cyclooctenes.

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The single substitution of a functional group at the allyl-position of cis-cyclooctene (COE) allows to achieve the regioregular polymers by means of ROMP with the second-generation Grubbs catalyst (G2).59 Thus we have synthesized COEs having hydrophilic functional groups at allyl-position,60 for example, polymerized the COEs with G2 in CHCl3. ROMP of the allyl-substituted COEs proceeded in a regio- and stereo-selective manner to afford polymers exhibiting remarkably high head-to-tail regioregularity and high trans- stereo-regularity as we previously reported. Figure 6 shows olefinic region of 1H nuclear magnetic resonance and 1H-1H correlated spectra of 3-methoxy-substituted COE. The coupling constant for the two olefinic signals is Jab=15.5 Hz, indicating that the double bond has trans- configuration. The dd and dt multiplicities for Ha and Hb, respectively, and the correlation between Ha and Hb reveal the near-perfect trans- head-to-tail regularity.

Figure 6
figure 6

Olefinic region of 1H NMR spectra and 1H-1H correlated spectra of poly(3-methoxy-1-cyclooctene).

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Polymers having precisely placed branches on every eighth backbone carbons were obtained upon hydrogenation. Water contact angle measurement confirmed the presence of hydrophilic surface for all polymers. The water structure in hydrated polymers was determined by DSC, cold crystallization of water and/or low melting of ice in hydrated polymers were observed on heating process. Cold crystallization of water is the clear evidence for the presence of intermediate water, and the content was able to be varied by changing the polymer structure. A human platelet adhesion test was employed to assess the blood compatibility of regioselective ROMP-produced polymers. The number of adhered platelets was also varied by changing the polymer structure, and we found out that the number was suppressed by introducing the longer EG side-chains. The platelets adhesion number was decreased with increasing the content of intermediate water regardless of the polymer structure. This result suggests that our hypothesis could be true that the presence of intermediate water is the key to provide the polymer materials with antithrombotic character, and the blood compatibility of polymers should be controlled by tuning the water structure at the bio-interface through precision polymer synthesis.60

Biodegradable synthetic polymers used/studied in medical applications

Some biomedical devices, especially for temporary use or disposable purpose, such as surgical suture, bone-fixation materials and drug-eluting stents comprise biodegradable synthetic polymers, including polylactides, polyglycolide, poly(É›-caprolactone), poly(trimethylene carbonate) (PTMC) and poly(p-dioxanone), as shown in Figure 7.61, 62 These polymers are degraded by hydrolysis with/without enzyme and absorbed in the body through metabolic pathway, although the duration of these existing biodegradable polymers in the body varies.63 They have drawn keen attention as alternatives to biopolymers such as peptides, nucleic acids and polysaccharides that cost high to produce and purify and potentially possess the risk of antigenicity and infection.

Figure 7
figure 7

Biodegradable polymers used/studied in medical applications.

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Such implantable medical devices need to be compatible with host cells to reduce adverse effects. As it has been confirmed that the aforementioned polymers exhibit minimal or acceptable cytotoxicity,64 most of those polymers are approved for medical application. Few reports have ever described the relationship between the biocompatibility and structural features of those polymers. In the case of PMEA, the ester and ether groups on the side-chains contribute to the hydration and generation of intermediate water.29 The hydration generally occurs through hydrogen bonding between polar moieties in the polymer and water molecules. This concept may be extended to the aforementioned polymers comprising ester or carbonate linkage and alkyl- or alkyloxy-chains of varying lengths. The detailed study for hydration and intermediate water in those polymers is now in progress by our group. Next examples also imply that the intermediate water concept should be employed to explain the observed biocompatibility.

Biodegradable antimicrobial polymers with low hemolytic property

In recent years, a PTMC analog bearing a side group of quaternary ammonium salt have demonstrated potent antimicrobial activities but showed minimal hemolytic properties (Figure 8).65, 66 In contrast, most of the cationic polymers are well known to interact with negatively charged bacterial cell membranes, subsequently inducing the membrane disruption.67 As the cationic polymers physically destroy cells, drug resistance is hard to develop differently from the use of conventional antibodies. However, this electrostatic interaction often influences mammalian cells resulting in cytotoxicity, which is a serious issue to be solved in developing antimicrobial polymers with positive charges. The first antimicrobial polycarbonate reported in 2011 shows efficient antimicrobial activity but displays no hemolytic property.65 This polymer has amphiphilic triblock nature to form nano-sized micelles by conjugating hydrophobic PTMC as peripheral blocks (Figure 8a). Accumulation of positive charges on micelle surface might contribute to differentiating bacteria and mammalian cells. In similar speculation proposed by Kuroda and colleagues, localization of charges and segregation of the hydrophobic part by micellization suppress the interaction with mammalian cell membrane with less negative charges than those of bacterial cell membrane.68, 69 Considering that the other PTMC analogs bearing different side-chains have also exhibited little cytotoxicity;70, 71, 72 however, this low hemolytic property may also be supported by the contribution of hydration involving the carbonate linkages in the main chain. In particular, as both RBCs and platelets are blood cells, the inactive behavior of the polymer to RBCs is likely to occur in a similar way that PMEA shows excellent compatibility to platelets.16 IBM propounds to call a series of these antimicrobial biodegradable polycarbonates ‘Ninja Polymer’ describing the function to work behind the scenes and eventually disappear.

Figure 8
figure 8

Synthetic biodegradable antimicrobials in different active forms. (a) spherical micelles (critical micelle concentration (CMC)=28 μg ml−1), (b) rod-like micelles (CMC=25 μg ml−1), (c) rod-like aggregate (CMC=6 μg ml−1).

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Supramolecularly bolstered antimicrobial activity and blood compatibility

Multiple activities against several types of bacteria present another challenge for the design of antimicrobial materials. The first antimicrobial polycarbonate described above shows the efficacy only against Gram-positive bacteria and their drug-resistant strains such as Bacillus subtilis and (methicillin-resistant) Staphylococcus aureus, respectively.65 Because Gram-negative bacteria and fungi are not as negatively charged as Gram-positive bacteria are,73 other artifices should be integrated into the macromolecular architecture. Lipophilicity and hydrophobicity are generally required for valid antimicrobial activity against Gram-negative bacteria such as Escherichia coli and Pseudomonas aeruginosa, owing to affinity to superficial lipopolysaccharide on the cell wall. However, increased hydrophobicity of the cationic polymers often develops hemolytic property. Fukushima et al.66 introduced a rigid hydrogen bonding motif in the middle of a center hydrophobic segment of a triblock copolymer composed of poly(L-lactide) (PLLA) and the cationic polycarbonate, forming fibrous micelles by orientation of self-assembly (Figure 8b). Interestingly, this polymer shows antimicrobial activity against a wide range of bacteria covering Gram-positive/negative bacteria and fungi but induced no hemolysis. In all cases, minimum inhibitory concentration of this polymer was higher than critical micelle concentration, supporting that the polymer serves as aggregates. It turns out that the fibrous shape is somewhat responsible for the improved antimicrobial activity. Later, Fukushima et al.74 have also reported supramolecular antifungals where the same rigid hydrogen bond motif is used directly to attach low molecular cationic primary ammonium at the both ends instead of the cationic polycarbonate (Figure 8c). The antifungal becomes active against fungi such as Candida albicans and Cryptococcus neoformans only in the form of nanofiber that indicates glass transition at 120 °C similar to molecular glass.75

In these above two cases, the hemolytic activity of the polymer also remained minimal. The cationic moiety obviously affects bacterial cell membranes, but the interaction with RBCs is mitigated even though the assembly form varies. At the latter case, especially, the interaction of molecules with cells, such as cytotoxicity, antimicrobial activity and biocompatibility, is managed by cooperation of primary structure of the peripheral functional groups to tune the chemical functions and higher-order structure forming specific shape to restrict or expand the chemical function as biological system generally adopts. The cationic moiety usually forms hydration layer, including strongly oriented water molecules that are categorized as non-freezing water, often causing adverse effects. If the intermediate water is responsible for the low hemolytic property of the cationic fibrous assemblies, the following hypothesis would be supposed: By assembling to such fibrous form, the surface hydration layer is disorganized with electrostatic repulsion of condensed cationic groups, which may trigger forming intermediate water from non-freezing water. In fact, a similar insight has been reported for generation of intermediate water by disorganization of hydration layer of non-freezing water in a copolymer of poly[N-methyl-N-(4-vinylphenethyl)ethylenediamine] with a small amount of additional poly(2-hydroxyethyl methacrylate).76

Control over the geometry of polymeric aggregates often entails non-covalent interactions, including hydrogen bond, π-π stacking, charge transfer complex and ion complex. These interactions also differentiate nano-rheology and dynamics of the aggregates. According to Stupp and colleagues, strength of the interaction at the internal domain of cationic supramolecular aggregates affects accumulation on and disaggregation of mammalian cell membrane.77 The aggregates with strong ‘internal bond strength’ interact with the cell membrane resulting in membrane disruption, while those with weak internal bond strength remain dynamic nature to release unimers upon approaching cells leading to no damage of the cells. It turns out that the selection of types and direction of bond (covalent vs non-covalent) significantly involves regulation of biocompatibility and cytotoxicity. In consequence, design of high-performance biomaterials in future should actively employ supramolecular chemistry in terms of geometry control, subsequent development of secondary function and dynamic behavior of the material besides primary chemical functions.

The research on clarification of relationship between biocompatibility of these polymer systems and water structure is ongoing.78 The intermediate water was only found in hydrated biopolymers (proteins, polysaccharides and nucleic acid; DNA and RNA) and hydrated biocompatible synthetic polymers but not in hydrated non-biocompatible synthetic polymers.79, 80, 81, 82 Therefore we propose intermediate water concept for directional design of functional polymeric biomaterials, but it is needed for the quantitative and precise description of biocompatibility driven by novel interface-sensitive approaches, such as spectroscopic (including sum-frequency generation and dielectric spectroscopy), X-ray and neutron scattering, and force curve measurements combined with computer simulations under the physiological condition.83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94

Conclusion

Surfaces made of biocompatible and biodegradable polymers profoundly influence cell behavior at all hierarchical levels. The interaction of polymers with cells is managed by cooperation of primary structure including backbone and functional groups at the side-chain to tune the chemical functions and higher-order structure forming specific shape to restrict or expand the chemical function as biological system generally adopts. Using principles of intermediate water, which is common in hydrated biopolymers and in only biocompatible synthetic polymers, the synthetic and supramolecular methodology to create novel biocompatible polymers moves toward a more high-throughput way. Such well-defined polymeric biomaterials could find application in the age of personalized medicine.