Bionanocomposites for tissue regeneration

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ECM – Cells interactions

Cells interact with the ECM via transmembrane proteins. In animal cells, the principal family of such proteins is the integrin one. Integrins are heterodimer glycoproteins (subunits α and β) that act as a linker between the ECM and the actin cytoskeleton of mammalian cells. When they create weak bonds with macromolecules of the ECM, it activates intracellular signaling pathways that communicate to the cell the characteristics of the ECM. Many different kinds of integrins exist (at least 22 in mammals), which interact specifically with collagen, laminin or fibronectin domains. The affinity is relatively low between integrins and their ligand, which enable to have dynamic bindings.  Three amino acid sequences have been found to play a key role in ECM-cell interactions:
(i) the RGD motif is a tripeptide sequence (arginine (R), glycine (G) and aspartic acid (D)) that was first discovered on fibronectin, but is also present on laminin (Figure I-3). When integrins specifically bind to RGD on fibronectin, this triggers cytoskeleton reorganization and formation of focal adhesion. Focal adhesions are large structures clustering integrins, but also many other proteins that anchor the cells to the ECM. The formation of focal adhesions concentrates actin stress fibers, which helps maintaining cells shape and adhesion to the matrix. This triggers adhesion-dependent signal transduction inside the cells, allowing either cell adhesion, spreading or motility.21–24 RGD is also present in some laminins and collagens but it is less accessible because of the molecules conformation
(ii) the PHSRN sequence (proline (P), histidine (H), serine (S), arginine (R) and asparagine (N)) is localized on fibronectin near RGDS (Figure I-3-A). Although it is itself not biologically active, it enhances the cell-adhesive activity of RGD synergistically. RGD is the primary recognition site for α5β1 integrins, in fibronectin III10 repeat and PHSRN is the synergy site for α5β1, in fibronectin III9 repeat. The presence of both peptides, in native conformation and spacing, allows α5β1-mediated adhesion, thus instructing cells to adhere, spread, differentiate, migrate, and, in the case of osteogenic cells, mineralize more efficiently than RGD alone.
(iii) In laminins and collagens other amino acid motifs are known to serve as alternative selective binding modules. For example, on laminin, another important peptide sequence IKVAV (isoleucine (I)-lysine (K)-valine (V)-alanine (A)-valine (V)) is located on the C-terminal end of laminin. This sequence promotes cell adhesion, neurite outgrowth, collagenase IV activity, angiogenesis, plasminogen activation, cell growth, tumor growth and differentiation of progenitor cells.
ECMs being the key element in which cells live and work so as to preserve healthy and functional tissues and organs, it is particularly important to understand how their bio-chemical, structural and physical features, including stiffness, fiber orientation, and ligand presentation trigger specific cellular behaviors. Such an understanding should provide fruitful guidelines for the design of biomimetic scaffolds for tissue engineering.

Biomaterials to mimic the ECM

Tissues can be damaged by diseases, injuries or traumas and necessitate treatments to help their repair, replacement or regeneration. The autograft, transplant of an organ from one site to the other within the same patient, is currently the gold standard. However, it presents several disadvantages such as having to suffer another injury for the patient, with possible complications and pain on the site of the organ removal and the possible mismatch of function between the damaged and donor organs. The alternative is the allograft, i.e. the transplantation of an organ from another donor. However, this possibility also has a lot of constraints, such as difficulties of accessing available compatible tissues, risks of rejection by the patient’s immune system and the possibility of introducing infection or disease from the donor to the patient. Additionally, immunosuppressive treatments required to decrease organ rejection compromise the immune system, leading to weakening of the patient.
The field of tissue engineering is an interesting alternative to those treatments. How to regenerate damaged tissues with a minimum of surgical work? Indeed, the body has intrinsic self-healing abilities. However, extent of repair varies amongst different tissues, the severity of injuries or diseases and the age and state of health of the patient.34 That is where biomaterials come in, to restore or improve tissue integrity.

Biomaterials specifications

The definition of biomaterials is not easy to establish because of the diversity of applications and processes. According to the International Union of Pure and Applied Chemistry (IUPAC), it is the material exploited in contact with living tissues, organisms, or microorganisms.35 It can be, in particular, a matrix providing cells with structural scaffolding, chemical signaling and ideal mechanical properties to regenerate a tissue. Since antiquity, humans have been taking materials (glass, metals or polymers) to replace body parts that have been damaged by disease or injury. Bioengineering approaches link biological tools and engineering principles. They have the advantages over grafts of having low immunogenicity while avoiding the creation of a second injury. The material provides a direct framework for tissue regeneration with minimum surgery work. This scaffold needs to simulate the environment required for cell growth and consequently has to fit specific requirements.
First of all, the material must be biocompatible. It is a complex notion, the organism must accept it and in parallel the material should be functional and beneficial for the organism. In particular, the body’s immune reaction should be minimal, without severe inflammatory response that could lead to rejection of the scaffold.
The scaffold should provide mechanical and structural properties similar to the initial tissue. Native ECMs have a fibrillar architecture in 3D. Using a hierarchical structuration is important to feature the properties at all scales from the nanometer to millimeter level.37 The influence of the mechanical properties on cell differentiation is evidenced by mesenchymal stem cells: they differentiate into different cell types, such as neurons, myoblasts and osteoblasts on increasing stiffer substrates.38 The mechanical properties can be modulated by different approaches depending on the nature of the materials and applications such as using cross-linkers,39 controlling crystallinity during processing,40 and using inorganic reinforcing fillers…41 Additionally, to facilitate its surgical implementation, it should be solid enough to be manipulated without hampering its integrity. An ideal scaffold also needs to combine these interesting mechanical properties with porosity to allow vascularization, supply in nutrients, and cell colonization. The size and connectivity of the pores should also be modulated depending on the target tissue.
Because cells need to adhere to the biomaterial to differentiate, the cell-material interface is of primary importance. The surface chemistry of materials is one of the key parameters. Many studies have been conducted by directly using ECM proteins such as collagen, fibronectin or laminin as materials or as coatings.43 Another option is to incorporate only the bioactive ligand such as RGDS or IKVAV by chemical binding or physical adsorption.
Another challenge in engineering an interesting scaffold is to address the display of biochemical signals, in particular when it comes to control their spatial distribution. Indeed, more than the chemical nature of a biological component, its clustering is a key signal to rule biological activity and trigger cell behaviors such as adhesion, migration, proliferation, and differentiation.52–57 A typical example is the formation of focal adhesions during cell adhesion that is triggered only after the formation of an effective integrin cluster.
A combination of optimal size, architecture, and surface properties may lead to biomaterials that allow the formation of a new ECM in the body and create a favorable environment for tissue regeneration to occur. Finally, the biomaterials should be able to degrade by itself to be replaced by this new ECM. The products of this biodegradation should not be toxic and be cleared from the body without any damage.

Tissue-specific structural properties

To successfully engineer a biomaterial for tissue regeneration, a crucial parameter to take into account is to mimic the specific structure of the native tissue. The biochemical nature of the ECM is the first step, but the architecture of the scaffold is at least as important. Biomaterials can be synthesized from synthetic or biological materials. Biological materials have intrinsically ideal properties to interact with surrounding native tissues. Many of them have been used as materials for neural tissue engineering, including fibronectin, silk fibroin, chitosan and collagen.59–61 The molecules of collagen are present in all tissues, but in different density and with specific structures. Table I-1 presents the properties of engineered biomaterials that are required when mimicking different types of tissues .
Alternatively, synthetic polymers are synthesized to mimic the structural characteristics and properties of biological macromolecules. They have tailorable mechanical properties, good biocompatibility, and easy processability. Moreover, it is usually easier to reach a good reproducibility and obtain higher yields and purity than with extracted biomolecules.
They can be covalently linked polymers, such as Poly(Glycolic Acid), Polylactic Acid, Poly(ethylene Glycol).62–64 However, they lack the biological properties of their natural counterparts. Innovative attempts to design biomimetic molecules that can assemble in an ECM-like manner have been described. For example, a covalent network by sol–gel polymerization of a silylated peptide bearing a sequence derived from the consensus collagen sequence [Pro-Hyp-Gly] was reported.65 Other collagen-mimicking peptides have been produced and form a supramolecular self-assembly.66–68 Supramolecular polymers are dynamic and self-assemble hierarchically, similar to the native proteins. The monomeric building blocks are interacting with each other by multiple noncovalent intermolecular interactions such as hydrogen bonding, metal–ligand coordination, π – π stacking, and hydrophobic interactions. Numerous peptides, not always with collagen-similar sequences, have been synthesized in order to engineer a scaffold for tissue engineering, such as peptide amphiphiles.

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Synthesis of inorganic nanoparticles

Nanoparticles can be synthesized by top-down or bottom-up strategies. In the top-down approach, large objects are converted by mechanical forces or irradiation into smaller ones (e.g. by fragmentation, etching or lithography).134,135 However, the disadvantage of the top-down approach to synthesize nanoparticles is the large distribution and poor morphological control in case of mechanical procedure, or time and energy consuming process for lithography techniques. The bottom-up strategy is the opposite approach. From small objects (i.e. atoms, ions or molecules) larger structures are built, by keeping a control over the size of the resulting nanoscale object. The synthesis can occur in gas or liquid, and is performed in two steps: (1) a nucleation phase, were the first object form a small and stable assembly; and (2) a growth phase, by adding precursors at the surface of the nuclei or by associating nuclei together. The synthesis stops when there is no longer precursor available or the surface reactivity is no longer high enough for the reaction. This process is ruled by thermodynamic law. The key parameters to control the size and dispersity of the nanoparticles are the concentration, the surface reactivity, the temperature and stirring.
As an example, we shortly introduce the use of the sol-gel process to synthesize metal oxide nanomaterials. This inorganic polymerization from a metal ion Mz+ or an alcoolate M-(O-R)n can occur in aqueous solution or in organic solvents and includes in both cases two steps (1) hydroxylation and (2) condensation.
(1) hydroxylation of the precursor to create M-OH bonds or M-O- bonds.
(2) condensation process leading to the departure of a water molecule: M-OH + M-OH M-O-M +H2O M-OH + M-O-R M-O-M + R-OH.
Condensed species are linked by oxygen atoms. Reaction parameters (pH, temperature…) need to be adjusted to get nanoparticle via this sol-gel process. One typical synthesis method using this sol-gel route is the Stöber process.137 From tetraethylorthosilicate Si(OC2H5)4 (TEOS), silica nanoparticles with tunable size can be obtained in ethanol solution with basic catalysis.

Biofunctionalization of inorganic nanoparticles

Nanoparticles have a wide range of applications in the biomedical field, such as targeted drug delivery, hyperthermia, bioimaging and biosensors.139,140 Some of these applications need a biofunctionalization of the surface of the nanoparticles. The conjugation of biomolecules at the surface to create hybrid particles allowed to target a receptor at the surface of cells, to improve their colloidal stability or to interact with objects of interest and display information to the cell for example. The large surface-to-volume ratio of nanoparticles enable a large number of molecules to be grafted at the surface. The molecules can be covalently linked at the surface of the particles (peptide linkage, click chemistry…) or be stabilized at the surface by electrostatic interactions. Silica nanoparticle and other hydroxylated surfaces can be functionalized with silicon-based functional reagents. Commercially available alkoxysilanes can be grafted via Si–O–Si bonds to the surface in a condensation reaction with the surface silanol groups. Many kinds of groups can be displayed at the surface of the particles such as amines, polyethylene glycol or thiols. They can be further used for the conjugation of biomolecules.
Because of the diversity of the chemical nature, the numerous possibilities of functionalization and their intrinsic physical properties, inorganic nanoparticles are ideal building blocks to be incorporated within nanocomposites to enhance their functionality. However, those approaches can be extended to organic particles, especially that interesting progresses have been realized in the last decades.145

Bionanocomposite engineering

Bionanocomposites can by synthesized following different procedures. The first one is a mechanical mixing between the two phases in solution (Figure I-5-A).125 The gelation of the resulting material is induced only after mixing the nanoparticles with the biopolymer. The great advantage of this method is that the nanoparticles are already synthesized at the required size with the appropriate functionalization. Additionally, the conditions of nanoparticle synthesis are regularly not ideal to preserve the native state of the biopolymer (e.g. aggressive pH, solvents, temperature…). The main shortcoming of this method is to reach a good dispersion of the nanoparticles in the matrix in solution, with an increasing difficulty when the polymer is viscous, for example at high concentration or because of the interactions with nanoparticles.146 Inhomogeneity in the nanocomposite is an issue for its characterization and application. The inorganic compounds need to be stable in the biopolymer mixing medium and under the gelation conditions, such as change in the pH (collagen), temperature (gelatin) or addition of crosslinking agents (alginate).
Another approach consists in using the biopolymer as a template to synthesize the inorganic nanoparticles within the matrix (Figure I-5-B).125,147 The particle precursors (typically ions) are dispersed in the polymer, usually before gelation. By modifying the external conditions, they are converted into inorganic colloids, with limited issues in their dispersion.
Similarly, bifunctional ions can be inserted within the biopolymer matrix and that can act both as inorganic nanoparticle precursors and network cross-linkers (Figure I-5-C).125 In this case, ions have a key role in hydrogel formation and become afterward precursors for the templated synthesis of inorganic particles.
In the above detailed strategies to elaborate bionanocomposites, the bio/inorganic interface between the biomacromolecule and the particle surface has to be subtly defined and investigated to fit the required molecular and supramolecular features of the composite. Indeed, the presence of inorganic colloids may disturbe the formation of the hydrogel. This will depend on the particle/polymer ratio, the surface chemistry of the particles and the synthesis conditions.

Table of contents :

1. The extracellular matrix
1.1. Structure and function
1.2. Collagens
1.3. Glycoproteins
1.4. ECM – Cells interactions
2. Biomaterials to mimic the ECM
2.1. Biomaterials specifications
2.2. Tissue-specific structural properties
2.3. A concrete example: peripheral nerve regeneration
3. Bionanocomposites for tissue regeneration
3.1. What is a bionanocomposite?
3.2. Nanoparticles
3.2.1. Definition and properties
3.2.2. Synthesis of inorganic nanoparticles
3.2.3. Biofunctionalization of inorganic nanoparticles
3.3. From nanoparticle to bionanocomposite
3.3.1. Bionanocomposite engineering
3.3.2. Application as a biomaterial
3.4. Silica nanoparticle – collagen bionanocomposites
3.4.1. Why combining silica and collagen?
3.4.2. Collagen- Silica for tissue engineering
4. References 

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