Résumé

L’ingénierie tissulaire est une alternative prometteuse aux autogreffes, aux allogreffes ou aux biomatériaux pour le traitement des lésions osseuses de tailles critiques. Généralement les produits d’ingénierie tissulaire associent un échafaudage et des cellules et sont implantés ou injectés au sein de la lésion. Les cellules doivent être intégrés au sein de l’échafaudage biocompatible et approprié afin d’offrir un environnement favorable à leur survie et leur différenciation en cellules osseuses. Au cours de nos travaux, nous avons développé un hydrogel composite par l’association d’un hydrogel de collagène de type 1, une protéine de la matrice extracellulaire largement utilisée dans plusieurs applications thérapeutiques, avec un hydrogel synthétique généré à partir d’une molécule amphiphile. Cet hydrogel composite a présenté des propriétés mécaniques et biologiques améliorés par rapport à celles présentées par chacun des hydrogels utilisés individuellement. L’incorporation de l’hydrogel synthétique participe à limitation de l’effet de compaction de l’hydrogel de collagène et a augmenté le module élastique de l’hydrogel. L’hydrogel composite a permis l’adhésion, la survie et la prolifération cellulaire in vitro et in vivo. De plus, il a favorisé la différenciation des hASCs en l’absence de facteurs ostéogéniques. In vivo, les cellules ensemencées au sein de l’hydrogel composite et injectées par voie sous-cutanée chez la souris ont produit un tissu lamellaire et se sont différenciées en ostéoblastes. Cette étude présente ce nouvel hydrogel composite comme une échafaudage prometteur pour une application en ingénierie tissulaire osseuse.

Mots-clés

Os, Ingénierie tissulaire, Hydrogels, Ostéogénique, Biomatériaux, Cellules souches, Échafaudages

Abstract

Tissue engineering is a promising alternative to autografts, allografts or biomaterials to address the treatment of severe and large bone lesions. Classically, tissue engineering products associate a scaffold and cells, and are implanted or injected into the lesion. These cells must be embedded in an appropriate biocompatible scaffold, which offers a favourable environment for their survival and differentiation. Here, we designed a composite hydrogel composed of collagen 1, an extracellular matrix protein widely used in several therapeutic applications, which we associated with a physical hydrogel generated from a synthetic small amphiphilic molecule. This composite showed improved mechanical and biological characteristics as compared with gels obtained from each separate compound. Incorporation of the physical hydrogel prevented shrinkage of collagen and cell diffusion out of the gel, and yielded a gel with a higher elastic modulus than those of gels obtained with each component alone. The composite hydrogel allowed cell adhesion and proliferation in vitro, and long-term cell survival in vivo. Moreover, it promoted the differentiation of hASCs in the absence of any osteogenic factors. In vivo, cells embedded in the composite gel and injected subcutaneously in immuno-deficient mice produced lamellar osteoid tissue and differentiated into osteoblasts. This study points this new composite hydrogel as a promising scaffold for bone tissue engineering applications.

Keywords

Introduction

Bone injuries and defects are serious health problems, especially those caused by complex fractures or bone defects arising from malformation, osteoporosis and tumors. Severe bone lesions cause hundreds of millions of surgical procedures each year around the world. Lesions which are too large to self-heal require the use of autografts, allografts or grafting of exogenous biomaterials which ensure mechanical stability and provide the appropriate environment for efficient healing[136,137]. However, autographs are often associated with tissue morbidity and present limitations in terms of the obtention of sufficient tissue, and immunogenic rejection and risk of infection are still an unsolved question in the case of allografts[138]. In the case of the use of biomaterials and bone substitutes, limitations have been reported related to poor vascularization leading to poor integration and insufficient bone repair. As such, in some cases, the biomaterial alone is insufficient and exogenous cells must be associated to the biomaterial, as to further support bone regeneration. Moreover, solid biomaterials such as ceramics do not easily fit the size and shape of the defect.

Mesenchymal stromal cells are of considerable interest for many therapeutic applications including repair of damaged tissues, restoration of infarcted heart areas, or anti-cancer therapy[370,371]. These cells exert two types of effects: they can directly take part as building block in tissue regeneration, or they can stimulate a number of host responses which ultimately favor tissue repair or, in the case of cancer, inhibit tumor growth. The latter effect is due to the secretion by these cells of a large number of cytokines and growth factors which can have immuno-modulatory, angiogenic and chemotactic effects[371–375]. In the case where MSCs must be grafted locally, as to exert their therapeutic effect, their efficiency largely dependents on their capacity to persist over a sufficient period within the site of implantation or injection. Additionally, many experimental studies in rodents have shown that MSCs have a short life span after, their engraftment, with commonly less than 10% of the grafted cells still present and alive as early as 10 days post grafting[283,284,376,377].

As such, the design of a biocompatible scaffold that allows MSCs survival, hinders their

diffusion and drives their differentiation towards the desired cell type is therefore an

important challenge to improve the efficiency, and eventually broaden the field of

application of these cells. In this context, injectable hydrogels for application in tissue

engineering, in the field of orthopedic sciences, produces many benefits[155].

Therapeutic applications of injectable hydrogels include the filling of large bone

defects[136]as they can be formed in situ and fit the defect shape and geometry[155].

They should be able to create an environment where exogenous cells can generate

functional osteoblasts, when the large lesion hinders the efficient recruitment of

endogenous host progenitor cells. It must also favor vascularization of the implant, as to

ensure cell survival and allow further recruitment of host cells such as osteoclasts. In

recent orthopedic research reports, hydrogels have often been used to maintain the actors

of bone regeneration (i.e. cells, growth factors, hydroxyapatite particles) together, within

the bone lesion[378]. For applications in bone tissue engineering, scaffolds should be

fully biocompatible, while eliciting a moderate inflammatory reaction by the host tissue,

and thus preventing fibrosis, to avoid damages or encapsulation of the graft[158]. It

should also be easy to handle, allowing the incorporation of cells and eventually growth

factors, without the need for cross-linking agents or ultraviolet light that could damage

cells or proteins[149]. Degradable scaffolds are often desired, aiming to the replacement

of the exogenous material by a newly synthetized ECM. Finally, and ideally, scaffold

should be injectable to allow minimal and less invasive surgery procedures[155].

As an alternative to polymers, a new generation of physically cross-linked hydrogels has emerged. They are obtained by the self-assembly of building block molecules, without the need for a chemical cross-linker. Gel structure is maintained by weak interactions, which may depend on temperature or ionic strength, and confer to the gel the possibility of a reversible gel-sol transition, that may be useful for drug delivery purposes. We previously described the development of new types of hydrogels, obtained from the self-assembly of small amphiphilic nucleotide-based molecules, glycol-nucleo-lipids[120,379,380], that feature minimal cytotoxicity and that can be implanted or injected into animal tissues[123,381]. In particular, gels based on glyco-nucleo-lipids containing a fluorinated carbon chain (GNF) have been shown to be biocompatible, eliciting a moderate inflammatory reaction and reduced fibrosis[123]. Their half-life was evaluated at circa 30 days, both in vitro and in vivo, and we have also shown that these gels supported the survival of adipose tissue-derived mesenchymal stromal cells (hASCs) aggregates. However, cell adhesion to these gels was limited, obviating an efficient and homogeneous colonization by the seeded cells. Several studies have shown the benefit of designing hybrid hydrogels, in order to combine the properties of each component[137,219]. In this context, the use of collagen gels does have a number of benefits: i) collagen is one of the main components of the ECM of native vascular tissues; ii) it is abundantly available and can be easily purified from living organisms; iii) it is non-immunogenic and biocompatible; iv) collagen gels can be directly and uniformly seeded

remodeled by smooth muscle cells (SMCs) and fibroblasts (FBs)[382]. Indeed, type I collagen is widely used for tissue engineering and tissue regeneration applications[107,383] as it offers a non-toxic, cell-adhesive and tissue-biocompatible environment. In addition, studies were shown that integrin-mediated adhesion to type I collagen enhances osteogenic differentiation of mesenchymal stem cells[384]. However, collagen shows weak mechanical properties and can be rapidly degraded by endogenous proteases. It is possible to stabilize it with crosslinking agents, or by the use of enzymes[165], but it is difficult to control its stiffness.

The aim of this work was to combine GNF and type I collagen to obtain blend hydrogels using a thermo-gelation process. This composite hydrogel was used as scaffold for cell embedding. After in vitro characterization, this composite hydrogel was tested in a subcutaneous implantation model to assess its osteo-inductive properties. This scaffold provided a suitable environment for the cells, conferring them long-term viability and driving their differentiation towards osteoblasts.

4.2. Materials and Methods