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Biological systems have evolved an extremely diverse range of structures and materials which possess unique and valuable properties of significant interest to engineering and other areas of science (Brown 2005). There are numerous examples in the past, extending well into the last century, where scientists and engineers have taken their lead from nature. Early examples of wing-inspire flying machines are clear examples and even Gustav Eiffel designed the curves which support the weight of the Eiffel tower on structure of bone. Indeed even today there a large range of technologies which utilise biomimicry: new solar cells, robotics, artificial intelligence, anti-drag surface coatings and the development of aerospace materials (Ball 2001). In addition to these applications there has also been a move towards both studying and applying the concept of biomimicry on the molecular scale. This represents huge potential in the biomedical and the biomaterial fields and is currently a widely researched topic.
Recently there have been considerable advances in the development of biomaterial which are able to elicit specific cellular responses by way of the utilisation of the natural processes of bimolecular recognition. These biomimetic materials are engineered by approaches which involve combining biological macromolecules involved in triggering cell responses, with either synthetic or natural substrates. The macromolecules in question may be integrated intact or it might simply be recognition or structural motifs. The combined materials can be described as biofunctional and/or bioactive (Bronzio et al 2006) and could potentially be used to target a wide range of biological processes.
One application for biomimetic materials is in the development of novel bioactive substrates for use in tissue engineering and regenerative medicine. For this application the utilisation of a wide range of biological macromolecules are possible including extracellular matrix (ECM) proteins, growth factors and even modified natural proteins. It is worth mentioning that the extracellular matrices (ECM's) play large roles in tissue morphogenesis, homeostasis, and repair, and these natural scaffolds provide many characteristics worthy of mimicking to control molecular cell function, tissue structure, and regeneration (Bronzio et al 2006). Other related applications of biomimetic materials are as surface modifiers for implantable materials and also as materials to facilitate drug delivery.
The key factor in all these applications is the understanding of the mechanisms which living systems utilise on a molecular level. It is this understanding that is vital if success of these materials is to be achieved on a macroscale. Hierarchical organisation, self-regulation, adaptability, mutifunctionality, and self-repair are all important mechanism which are used in biological systems and it is these properties which are to be ideally mimicked to achieve new generations of materials and treatments.
The aim of this review to critically evaluate the current research which is being carried out in the development of biomimetic materials in areas of tissue engineering and hard tissue mineralisation and approaches that are being adopted. A further objective is to explore the future applications of biomimetic materials.
Knowing What to Mimic
When considering the potential uses for biomimetic materials for biomedical applications it is important to consider the range of natural structures and macromolecules which are to be mimicked. As mentioned previously the proteins of the Extracellular Matrix (ECM) have been the most commonly studied.
RGB (arginine-glycineaspartate sequence) sequences are peptide sequences which often used to enhance the biomimetic properties of materials. Their significance is that adhesion between cells and the ECM is medicated by cell surface receptors called integrins which bind to ligands with exposed RGB sequences. In addition to adhesion these receptors also stimulate intracellular signalling and gene expression involved in cell growth, migration, and survival which makes them vital targets in biomimetic systems.
Major ECM proteins (collagen, fibronectin, vitronectin, laminin and fibrin) which support physiological cell adhesion are all known to contain short linear, integrin-binding ligand sequences, related to the common found RGD sequences.
The primary application for biomimetic materials in the field of tissue engineering is in the manufacture of scaffold materials. There materials can be natural or synthetic and can be described as biomimetic if they mimic one or more of the characteristics of the natural ECM (Ma 2008). Below are described some of the strategies of which are applied to these materials with some examples but this does not constitute an exhaustive list of the materials or approaches.
Biodegradability is a desirable property for a scaffold material to have, ideally with the material degrading at a similarly compatible rate to the formation of new tissue to allow it to serve its purpose as a template (Ma 2005).
For this reason linear aliphatic aliphatic polyesters such as poly(lactic acid) (PLA), poly(glycolic acid) (PGA), and their copolymers poly(lactic acid-co-glycolic acid) (PLGA) are used in scaffold fabrication due to their of biodegradability in addition to their well accepted biocompatibility (Ma & Langer 1995). Some useful materials do not have the required biodegradability though do possess other desirable properties. An example of this is poly(ethylene glycol) (PEG) which is a biocompatible material that has the similar mechanical properties to some soft tissues such as cartilage. An approach which was taken to overcome this deficiency was to synthesize a copolymer of PEG with PLA, PGA or PLGA (Han & Hubbell 1997).
Another approach which is taken to impart biodegradability in an more biomimetic way, is to synthesize a PEG-based polymeric biomaterial designed to exhibit degradation by specific enzymes, e.g., matrix metalloproteases (MMPs). This approach is similar to and mimics the enzymatic biodegradability of collagen and other natural ECM components (West & Hubbell 1999).
Composite and Nano-composite materials
While many scaffold materials are usually used in their pure form there are occasions when combining two (or more) materials may produce a composite with a combination of desirable properties which could not be achieve by any one material alone. This has significance with respect to the concept of biomimicry because some natural tissues are also found to be composites. An example of this is bone, whose matrix has an organic/inorganic composite form consisting of collagen and apatites, respectively.
An example of this approach is in the creation of scaffolds for bone tissue engineering. There have been a variety of approaches involved. Most of the approaches involve the use of hydroxyapatite (HA) or calcium phosphate and the inorganic component along with a conventional degradable scaffold material such as PLGA or PLLA used as the organic component. Calcium phosphate and HA are similar to the inorganic component of natural bone and are known to provide good osteoconductivity. With the significant amount of research that has been conducted it can be summarized that HA in composite scaffolds significantly increase protein adsorption capacity, suppresses apoptotic cell death, and provides a more favourable microenvironment for bone tissue regeneration (Ma 2008).
The surfaces of scaffold materials are extremely important in tissue engineering as they interact with the biointerface of living tissue and therefore can affect cellular responses and ultimately influence tissue regeneration. An ideal, and effective, scaffold should be able to mimic the ECM and interact with the surrounds cells in a positive way. Desirable interactions include enhanced cell adhesion, growth, migration, and differentiated function (Ma 2008). Though of variety of synthetic scaffold materials have been used which are biodegradable, they usually lack biological recognition. Through the use of composite materials as discussed previously these problems can be overcome by improving cellular interactions.
In general bulk or surface modifications are applied to scaffold materials with the aim of eliciting positive cellular interactions. Bulk modification is usually achieved by copolymerisation or functional group attachment to the polymer chain prior to the fabrication of the scaffold. An example of this is work done by Robert Langer's group (Cook et al 1997), where they produced a poly(L-lactic acid-co-L-lysine) and chemically attached a RGD peptide to the lysine residue of the copolymer in order to enhance cell adhesion. However, it should be said that a disadvantage of bulk modification is that as a consequence of the process there the processing and the mechanical properties of the material are also affected.
Surface modification differs from bulk modification in that it can be carried out after a porous scaffold has been fabricated. Consequently it does not usually affect the scaffold structure and or the mechanical properties significantly
Surface modification strategies have largely concentrated adding to the surface of scaffolds a variety of biomimetic peptides (eg the RDG sequences), ECM proteins and protein fragments from a variety of proteins including collagen, laminin, fibronectin, vitronectin etc (Brown & Phillips 2007). A wide variety of these cell-binding peptide ligands have been applied to broad range of cell types under the premise that high binding specificity might resent an advantage (Shin et al 2003).
RGD sequences are the most common though others are also sometimes used. Another sequence type are YIGSR peptides have also been used, particularly in epithelial/endothelial cell types and related applications (eg vascular or skin).
It should be mentioned that a defining advantage which protein-based scaffolds are known to have over synthetic polymers is that they possess superior cell attachment and migration properties (Brown and Phillips 2007). This is also illustrated by the significant amount of research literature which is exists describing methods to alter the surface chemistry of the most common synthetic polymers (eg polylactites, polyglycolic acid, polycapriolactones ) as described above.
Bioactive molecule delivery
For tissue regeneration to occur or even in the restoration of normal cell function in implanted tissues cell signalling plays a vital role in both these and other processes. Often exogenous signalling molecules are required because the quantity and/or type of endogenous signalling molecule are not sufficient to repair tissue defects. In these circumstances it becomes important for these molecules to be delivered as part of a tissue engineering-based treatment. In addition to the requirement of signalling molecules it is important to consider that many of these molecules may have short half-lives. For these reasons it is an essential factor in tissue engineering that delivery systems are present for the successful application of biological factors.
A variety of techniques have been developed which facilitate the delivery of bioactive molecules. Biological facts have been added to polymer solutions or emulsions in the fabrication of scaffolds (Kim et al 2003) and there have also been coating techniques developed. While these methods achieve slow release kind, which is desirable, control over their release kinetics is limited.
Another method involves the trapping of PLGA microspheres in a porous scaffold by using a gas foaming process (Hile et al 2000). Here it was found that there was limited control on the pore size and shape and that the release kinetics of the imbedded microsphere were also uncontrollable.
Hard Tissue Mineralisation
An important development in the application of biomimicry is in the study of hydroxyapatite mineralisation. In this area there have been a large number of approaches which have been researched with the aim of producing hydroxyapatitie which mimics the naturally occurring form in structure, alignment, composition and the formation method.
The methods described below ware all approaches which focus on the formation hydroxyapatitie (HA) in synthetic systems which are designed with biomimicry in mind.
Protein based approaches
Collagen is on a vital structural protein found naturally. The biointerface between HA and collagen is an area where alot of research is conducted. The hierarchical structure of collagen ie structures which contain collagen fibrils, offer great opportunities to produce scaffolds which mimic autologous bone grafts (Landis et al 2006).
Due to the significance of collagen the in vivo and in vitro mineralisation of self-assembled collagen fibrils does appear to be an area of significant research. Easy studies which aimed to mimic the composition and structure of bone focused on using reconstituted type I collagen with SBF. An example of this was work done by Glimcher et al (1984). That noted that HA was nucleated in the hole zones of rabbit collagen.
Recently work done to replicate the hierarchical self-assembly of mineralized collagen into composites of nanofibrils was made by Zhang et al (2003). What they observed was that the HA crystal's grew on the surface of triple helical fibrils such that their c-axes were oriented along the long axis of the fibrils, as in natural bone. The structure of the composite and its hierarchical arrangement was observed by TEM.
Gelatin is a polymer which can be produced by the partial hydrolysis of collagen, and has also been considered in mineralisation studies. A study was done where gelatine films were used to mimic collagen and poly(acrylic acid) was used to mimic natural acidic macromolecules. After time in SBF solution, the film appeared to mineralise with spherical aggregates and later these crystals grew with preferential orientation of their c-axes along the long the organic molecules, which was taken to indicate the potential of gelatin substrates in place of collagen in HA mineralization (Bigi et al 2000).
With collagen have an important role in the natural formation of bone it dies appear to be an obvious scaffold to use in the study of synthetic mineralisation of HA. In summary, studied show that collagen is an important structural facilitator to direct HA mineralisation by protein or polymers with bone-mimicking organisation (Palmer et al 2008).
Other natural biopolymers have been used in the research of biomimetic remineralisation. There are a large number studied some of which are chitin, chitosan, ChS, starch and the polyhydroxyalkanoate polyesters. While these materials generally do posess some desirable properties such as biocompatibility and biodegradability their successful application in a biomimetic capacity is limited.
That said, a recent study three-dimensional HA/chitosan-gelatin networks of greater than 90% porosity were prepared to examine the proliferation and functions of neonatal rat calvaria osteoblast (Zhao et al 2002). With this scaffold, the cells were found to attach, proliferate, and produce extracellular matrix. Significant biomineralization was observed after 3 weeks in culture.
While the success of these polymers is limited at the moment, with further work, these could progress to being significant methods for mineralisation.
There are a number of synthetic systems, primarily polymeric and supramolecular, that can mimic the fibrous texture of collagen and can induce mineralization. There structures may not have the complete complex structure of collagen and have been described as "one-dimensional (1D) structures" (Palmer et al 2008).
The versatility of synthetic scaffolds is attractive as they can act as a scaffold and, in addition, can also act as a drug delivery device to induce bioactivity. In addition to the more established properties some are also able to entrap solvent molecules to form viscoelastic hydrogels that can lead to better localization and tuneable drug release profiles (Palmer et al 2008).
It important note that that research indicates that that carboxylic acids and phosphate groups appear in many macromolecules responsible for HA mineralisation. Consequently one of the most commonly used polymers are the poly(a-hydroxy acids). Common examples include poly-L-lactic acid (PLLA), poly(lactic-co-glycolic acid) (PLGA), polyglycolic acid
Aside the poly(a-hydroxy acids), a study was done using cross-linked polymethacrylamide and polymethacrylate hydrogels (functionalized with mineral-binding ligands) for the templated formation of HA (Song et al 2005).This study found that carboxylate and hydroxyl groups were found to give good adhesion between the organic and the inorganic materials. It was suggested by the researchers that the mineral-nucleating potential of hydroxyl groups point to a potential role for hydroxylated collagen proteins in bone mineralization. This study does highlight the potential usefulness of hydroxyl groups in biomimetic mineralization.
The above mentioned polymers are very useful in the study of HA mineralization, though by no means is it an exhaustive list, other common ones studied are PHB, PHEMA and PCL. (Palmer et al 2008).
Organoapatites (OA) are macromolecules which contain large surface area structures with nanoscale crystallites that mature slowly into HA based on analysis of Ca/P ratio. The use of these molecules was specifically utilised to mimic apatite formation in natural tissues. They were targeted for use as artificial materials to trigger bone regeneration at defect sites or interfaces with implants. The main biomimetic feature was the use of organic macromolecules to nucleate apatite crystals. Organoapatites have been shown in studies to also be an area of interest in the coating of biocompatible metals.
These systems offer the possibility to become basic models for mineralization with biomimetic features since they mimic the architecture of fibrous matrices and also have potentially higher order parameters relative to polymers.
One of the significant supremolecular systems which has showed some promise and is attracting interest to is the use of self-assembling peptide amphiphiles(PA). These molecules are designed with the aim of creating nanostructures that not incorporate bioactive epitomes in addition to chemically targeting the natural mineralisation processes. Structurally the PA's consist of a hydrophobic tail linked to an electrostatically charged peptide sequence (see figure 1). The sequence of amino acids actually affects the properties as it has beem observed that when the peptide sequence includes amino acids with a strong ß-sheet propensity, high aspect ratio cylindrical nanofibers are observed that could potentially mimic the architecture of natural collagen fibres.
Self-assembly of the PA molecules is controlled by hydrophobicity of the alkyl tail and by the hydrogen bonding between adjacent peptides. Studies have shown that creating PA's results in the created entangled network of nanofibers, which are observed macroscopically as a self-supporting gel. (Palmer et al 2008)
Studies were done with self-assebled PA's where bone biomimetic mineralisation was was observed. Interestingly another study was able to establish that the crystallographic c-axis of the HA was preferentially aligned with the long axis of the PA fibers, as in mammalian bone and dentin (Traub et al 1989).
PA have beeen studied using a variety of other methos including a 3D PA scaffold, PA nanofibres which disply RGD sequence , and in accociated with methods using them with metal coatings (NiTi and Ti-6Al-4V).
In addition to the methods listed above other compound which are being researched are , dendrimers, star polymers, polymer microgels, and Poly(amino acids).
In summary it could be said here that the biomimetic lesson here is that largely inorganic materials could be synthesized in an easily degraded but complex organic scaffold that may help create a hierarchical structure.
Figure 1: Chemical structure of the PA, consisting of a hydrophobic alkyl tail; four cysteine residues
that when oxidized may form disulfide bonds to polymerize the self-assembled structure; a
flexible linker region of three glycine residues to provide the hydrophilic head group flexibility
from the more rigid cross-linked region; a single phosphorylated serine residue that was
designed to interact strongly with calcium ions and help direct mineralization of HA; and the
cell adhesion ligand RGD. (b) Molecular model of the PA showing the overall conical shape
of the molecule going from the narrow hydrophobic tail to the bulkier peptide region.
(c)Schematic showing the self-assembly of PA molecules into a cylindrical micelle. (Palmer et al 2008).
Given the narrow scope of this paper it is difficult to fully assess the state and progress in development of biomimetic materials and techniques in tissue engineering and hard tissue mineralisation. While many approaches do adopt complex means to mimic natural biological process other do not and yet are also referred to as "biomimetic". An example of this is the utilisation of non-physiological biomolecules such as gelatine, chitin, or starch. It should be stated that any claims these approaches have towards biomimicry in mammalian systems are extremely tenuous. This is not to say they are not biocompatible or even effective as biomaterials, but only to emphasize that there is no mimicry of the natural system occurring.
With respect to the biomimetic approaches I have reviewed on tissue engineering it is clear that there is large scope here with respect to further development of better techniques and materials. Outside the scope of the current studies it likely that research will progress to further generations of scaffold materials which mimic the surrounding tissues with the inclusion of multiple biomimetic signalling in addition to possessing controllable molecular release functionality. This level of very high integration and control is currently out of reach of current research.
The biomimetic approaches concerning bone mineralisation have not at present yielded mineralisation which truly mimics the natural process. While there are some very promising result and a wealth of potential avenues to study, no one technique has risen to a convincing level of success to be compared with biological mineralisation. Work is likely to progress and as the techniques for tissue engineering improve so will the scope of improved mineralisation. That said the use of PA;s, and particularly their self assembling nature, does hold promise for future developments.
The work that has been done above is undoubtedly complex however the next level of complexity will likely be on larger scale which incorporate different types of building blocks containing different types of supramolecular assemblies.
In tissue enginnering the use of bioactive molecules with biomaterials has enabled the design of biomimetic scaffold which are able to elicit speci¬c cellular responses and direct new tissue formation. With these advances the line between tissue engineering and regenerative biology becomes blurred. Surface and bulk modi¬cation of materials with these biomimetic peptides can allow for the modulation of cellular functions such as adhesion, proliferation and migration allowing for greater control than ever. Several challenges, however, still remain including the design and research of adhesion molecules in co-ordination with speci¬c cell types as needed for guided tissue regeneration and the development of materials exhibiting the mechanical properties of living tissues.
A large amount of progress has been made over the last few decades in understanding the process of HA biomineralization in mammalian tissues. There is now undoubtedly some knowledge concerning the role certain proteins play in producing a controlled deposition of the mineral crystals as opposed to simple unstructured precipitation. The understanding is far from complete, particularly in bone. In could be said that an ideal artificial material would mimic bone mineralization would be able cue cells to regenerate these tissues.