Nanostructuration of 3D surfaces: Clusterization on silica nanoparticles

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Biomaterials for tissue regeneration

The extracellular matrix

Structure and function

The extracellular matrix (ECM) is the noncellular component present within all tissues and organs. The ECM is composed mostly of water and macromolecules organized in a 3D network (Figure I-1). These macromolecules, mostly proteins and polysaccharides, provide a scaffold into which cells are embedded and also regulate many cellular processes including growth, migration, differentiation, survival, homeostasis, and morphogenesis.1–3
The composition and topology of the ECM is tissue-dependent. During tissue development, cells (e.g. epithelial and endothelial cells, fibroblasts, adipocytes) synthesize the constitutive elements of the ECM, that influence its organization and buffering (e.g. water retention) properties. The ECM further evolved via biophysical and biochemical modifications induced by cells and the environment. The direct consequence is the variation in the tensile and compressive strength, elasticity and protection abilities. ECM features are indeed varying remarkably from one tissue to another: the same characteristics are not required for lungs versus skin or bones.1,2,4
ECM components are interacting with cells via biochemical and biomechanical cues (Figure I-1-A). They present chemical signals directly on the macromolecular network or through specialized proteins such as binding growth factors (GF), interacting with cell-surface receptors and triggering signal transduction. This process directly modifies the regulation of the gene transduction and subsequently cell behavior.1,2,4
The ECM has a highly heterogeneous and dynamic organization. Components are constantly synthesized, degraded and reshaped by cells. The remodeling is particularly high during development, wound repair or after a disease. It is initiated by a complex interplay between cells, via ECM receptors, and action of enzymes such as matrix metalloproteases involving mechanical modulations.1,2,4
The three major components of the ECM are proteoglycans (PGs), collagens, and glycoproteins. PGs are the combination between a protein and a glycosaminoglycan (GAG, e.g. long unbranched polysaccharides). They fill the extracellular interstitial space within the tissue, creating a hydrated gel. The diversity of structures translates a multitude of functions: protection, lubrication, biomechanical resistance to pressure, GF reservoir…1,2,5–7 Collagen and glycoproteins are fibrillary proteins of particular interest in this PhD work.


Collagens are the most abundant proteins in the human body (ca. 30% of its protein mass). At this day, 28 collagen types have been described in the literature, with various sequences of polypeptide chains. All collagen molecules consist of three polypeptide chains, called α-chains, and contain at least one domain composed of repeating Gly-X-Y sequences in each of the constituent chains. X and Y positions are frequently occupied by the two amino acids proline and 4-hydroxyproline. Because of the high content of proline, 4-hydroxyproline and glycine, α-chains exhibit a tendency to spontaneously form left-handed helices without any formation of intrachain hydrogen bonds. Those three left-handed helices coiled into a common axis to form a right-handed triple helix. This superstructure is allowed because of the presence of glycine, the smallest amino acid, on every third position that enables the close packing. The triple helix is stabilized by interchain hydrogen bonds, mostly due to 4-hydroxyproline amino acids.1,2,5,6,9–11
Some collagens can self-assemble into fibrils, such as the types I, II, III, V and XI. They are mostly present in tissues that need to resist shear, tensile force or pressure. Consequently, they are a structural key element of connective tissues. Other collagens can form a non-fibrillar network (IV, VII, X) principally found in the basal membrane. The most abundant type of collagen is the fibrillar collagen I, present throughout the body, except in cartilage. Moreover, it is the principal collagen in the dermis, fasciae, and tendons and it is massively present in mature scar tissues.
Fibrillar collagen is responsible not only for structural integrity of the ECM but also contributes to cell adhesion, chemotaxis, migration and direct tissue development.12
Fibrillar collagen molecules are mostly synthesized by fibroblasts directly in the stroma or from adjacent tissues. The α-chains synthesized in the endoplasmic reticulum self-assemble inside the cell, goes through post translational modifications and are secreted by exocytosis (Figure I-2-A). This initial triple helix is the procollagen bearing propeptides at the N- and C-terminal ends of their polypeptide chains (Figure I-2-B). As monomer, the triple helix is approximately 300 nm in length and 1.5 nm in diameter. Metalloprotease enzymes cleave the terminal procollagen peptides, which triggers the self-assembly of the triple helix into fibrils that further self-assemble into collagen fibers (Figure I-2-C). The self-assembly of triple helices produce “overlap” and “gap” areas creating a typical pattern of fibrillar collagen identified by electron microscopy (Figure I-2-D-E). This structure presents a characteristic 67 nm axial periodicity, independently of the fibrils diameters.
Type I collagen molecules are soluble as triple helix in acidic conditions. Their fibrillation can be induced in vitro without any enzyme by modification of physico-chemical parameters such as temperature or pH.13–17 In our group, a usual technique to trigger collagen fibrillation is to raise the pH, by ammonia vapor or neutralization in aqueous medium.


Fibronectin and laminin are two key glycoproteins. Fibronectin is the major glycoprotein of connective tissues, while laminin is present is basement membranes.
Fibronectin is composed of two subunits covalently connected by disulfide bonds at their C-termini (Figure I-3-A). Each subunit is a repetition of three modules, Type I, II and III differing by their amino-acid sequence.1,2,5,20 Fibronectin is also synthesized by cells, mostly fibroblasts, via a soluble state: a soluble protein dimer. This precursor self-assembles in solution into an insoluble complex of fibers. Fibronectin is critical for cell attachment and migration.
Laminins constitute a family of about 20 glycoproteins assembled into a cross-linked web.2,5,6 They are heterodimers formed by three chains (α, β and γ) linked together by disulfide bonds (Figure I-3-B). They are synthesized by a wide variety of cells, depending on the  considered tissue (e.g. endothelial cells, skeletal and cardiac muscle cells). Many laminins self-assemble into networks within the ECM. Laminins have a major role in embryonic development and organogenesis. They trigger cell adhesion, migration, and differentiation. They are notably well-known to be crucial for axonal regeneration, Schwann cell differentiation and myelination in the injured peripheral nervous system.
Collagens, fibronectin and laminins interact with each other, and other molecules such as elastin, to create an insoluble supramolecular network. Some domains are directly interacting with other molecules of the ECM, as indicated in Figure I-3, but they also have the ability to interact with cells via transmembrane proteins.

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.25
(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.26,27
(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.28,29
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.30–33
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.36
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.30,37,42
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.44–52
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.30,58

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.44,69–74
Altogether a wide variety of molecules, being natural or synthetic, are available or can be designed for the development of tissue engineering scaffolds. However, each targeted tissue has its own intrinsic properties, including its specific pathways of regeneration. Therefore, there is no universal scaffold and, even for the same targeted tissue or organs, requirements can depend on the origin and extent of the damages as well as on the patient age or health state. Thus for each situation, it is first important to precisely define the objectives of the therapeutic device and to set up adapted technical specifications. In the following section, we will focus on peripheral nerve regeneration.

A concrete example: peripheral nerve regeneration

Our nervous system is mainly composed of two parts: the central nervous system (CNS, brain and spinal cord) and peripheral nerve systems (PNS, nerves). PN tissues are able to regenerate by themselves when the gaps after injuries are smaller than 6 mm. For larger gaps, a graft is usually required, but with the drawbacks detailed above. In addition, in the case of PN, there is the possibility of generating a painful neuroma (i.e. tumor of nervous tissue) on the injured zone or on the site of the organ removal.28-30
PN are composed of highly ordered and aligned bundles of axons. Consequently, to help the regeneration, bioengineered devices should be longitudinally oriented to provide the physical support to bridge gap and contact guidance for neurite regrowth, while maintaining the biological functionality.101 The ECM of PNS will be detailed in chapter IV.
PN scaffold materials have been synthesized from many synthetic or biological materials.102 For example, Neurotube®, made of poly(glycolic acid) (PGA) has been approved by governmental regulation authorities and is currently commercialized. This device allows to get comparable result to autografts in the treatment of lesions with a maximum length of 3 cm and a small diameter, but do not allow a complete functional recovery, especially for more extensive injuries.103 Interesting results have also been obtained with polyaminodoamines (PAA) in vivo on animal models but for a maximum of 5 mm gap injury on PN. This material shows interesting elasticity properties and helps to improve axon size and density.104 With a rigid material from poly(lactic-co-glycolic acid )(PLGA),105,106, axons with large diameters were obtained leading to thick myelin sheaths. PN regeneration has also been nicely evidenced in vitro with aligned polymer fiber-based constructs, such as poly(acrylonitrile-co-methylacrylate)107 or polycaprolactone (PCL).108 With the latter, confocal microscopy images show that the direction of neurite out-growth from the cell body on 5 μm PCL fibers corresponds to the direction of fiber alignment (see arrow, Figure I-4-A1-2).108 On the contrary, as expected, neurite out-growth was observed in random directions on flat substrates (Figure I-4-A3). However, synthetic materials can induce inflammatory reactions and be rejected by the body.109

Table of contents :

I. Biomaterials for tissue regeneration
1. The extracellular matrix
2. Biomaterials to mimic the ECM
3. Bionanocomposites for tissue regeneration
4. References
II. Influence of the bio-chemical signal clusterization on cell adhesion in a Peptide Amphiphile – SiNP composite
1. Introduction to Peptide Amphiphiles
2. Chemical Design
3. Preparation and characterization of the composite
4. Biological activity
5. Conclusion
6. Experimental methods
7. References
III. Nanostructuration of 3D surfaces: Clusterization on silica nanoparticles
1. Introduction to Patchy Particles
2. Synthesis of self-assembling silanes bearing an aromatic moiety
3. Self-assembly of the synthesized alkoxysilanes
4. Transfer of An self-assemblies to the SiNP surface to create Patchy SiNPs
5. Bifunctional patchy particles
6. Conclusion and perspectives
7. Experimental section
8. References
IV. Biomaterials engineering for neural type cells differentiation
1. Peripheral nerve regeneration
2. Engineering a bionanocomposite
3. Bionanocomposites of the study
4. PC12 differentiation on collagen-SiNP threads
5. Discussion
6. Conclusion and perspectives
7. Experimental methods
8. References
V. Self-supported collagen-based matrices by electrospinning
1. Electrospinning
2. A self-supported membrane
3. Investigating the native structure of collagen by circular dichroism (CD)
4. Characterizing the collagen structure within the membrane
5. Cell adhesion on the different matrices
6. Conclusion and perspectives
7. Experimental methods
8. References
I. Patchy Particles by grafting Py precursor
II. Conjugation of di-mercaptosuccinic- acid modified Fe2O3 particles
III. Synchrotron-radiation Oriented Linear Dichroism of collagen
List of abbreviations


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