An Elastin-derived composite matrix for enhanced vascularized and innervated bone tissue reconstruction: from material development to preclinical evaluation.

Despite progress in bone tissue engineering, reconstruction of large bone defects remains an important clinical challenge. Here, we developed a biomaterial designed to recruit bone cells, endothelial cells, and neuronal fibers within the same matrix, enabling bone tissue regeneration. The bioactive matrix is based on modified elastin-like polypeptides (ELPs) grafted with laminin-derived adhesion peptides IKVAV and YIGSR, and the SNA15 peptide for retention of hydroxyapatite (HA) particles. The composite matrix shows suitable porosity, interconnectivity, biocompatibility for endothelial cells, and the ability to support neurites outgrowth by sensory neurons. Subcutaneous implantation led to the formation of osteoid tissue, characterized by the presence of bone cells, vascular networks, and neuronal structures, while minimizing inflammation. Using a rat femoral condyle defect model, we performed longitudinal micro-CT analysis, which demonstrates a significant increase in the volume of mineralized tissue when using the ELP-based matrix compared to empty defects and a commercially available control (Collapat). Furthermore, visible blood vessel networks and nerve fibers are observed within the lesions after a period of two weeks. By incorporating multiple key components that support cell growth, mineralization, and tissue integration, this ELP-based composite matrix provides a holistic and versatile solution to enhance bone tissue regeneration. This article is protected by copyright. All rights reserved.

Bone Regeneration in Small and Large Segmental Bone Defect Models after Radiotherapy Using Injectable Polymer-Based Biodegradable Materials Containing Strontium-Doped Hydroxyapatite Particles.

The reconstruction of bones following tumor excision and radiotherapy remains a challenge. Our previous study, performed using polysaccharide-based microbeads that contain hydroxyapatite, found that these have osteoconductivity and osteoinductive properties. New formulations of composite microbeads containing HA particles doped with strontium (Sr) at 8 or 50% were developed to improve their biological performance and were evaluated in ectopic sites. In the current research, we characterized the materials by phase-contrast microscopy, laser dynamic scattering particle size-measurements and phosphorus content, before their implantation into two different preclinical bone defect models in rats: the femoral condyle and the segmental bone. Eight weeks after the implantation in the femoral condyle, the histology and immunohistochemistry analyses showed that Sr-doped matrices at both 8% and 50% stimulate bone formation and vascularization. A more complex preclinical model of the irradiation procedure was then developed in rats within a critical-size bone segmental defect. In the non-irradiated sites, no significant differences between the non-doped and Sr-doped microbeads were observed in the bone regeneration. Interestingly, the Sr-doped microbeads at the 8% level of substitution outperformed the vascularization process by increasing new vessel formation in the irradiated sites. These results showed that the inclusion of strontium in the matrix-stimulated vascularization in a critical-size model of bone tissue regeneration after irradiation.

3D anatomical and perfusion MRI for longitudinal evaluation of biomaterials for bone regeneration of femoral bone defect in rats.

Magnetic Resonance Imaging (MRI) appears as a good surrogate to Computed Tomography (CT) scan as it does not involve radiation. In this context, a 3D anatomical and perfusion MR imaging protocol was developed to follow the evolution of bone regeneration and the neo-vascularization in femoral bone defects in rats. For this, three different biomaterials based on Pullulan-Dextran and containing either Fucoidan or HydroxyApatite or both were implanted. In vivo MRI, ex vivo micro-CT and histology were performed 1, 3 and 5 weeks after implantation. The high spatially resolved (156 × 182 × 195 µm) anatomical images showed a high contrast from the defects filled with biomaterials that decreased over time due to bone formation. The 3D Dynamic Contrast Enhanced (DCE) imaging with high temporal resolution (1 image/19 s) enabled to detect a modification in the Area-Under-The-Gadolinium-Curve over the weeks post implantation. The high sensitivity of MRI enabled to distinguish which biomaterial was the least efficient for bone regeneration, which was confirmed by micro-CT images and by a lower vessel density observed by histology. In conclusion, the methodology developed here highlights the efficiency of longitudinal MRI for tissue engineering as a routine small animal exam.

An easy-to-use and versatile method for building cell-laden microfibres.

Fibre-shaped materials are useful for creating different functional three-dimensional (3D) structures that could mimic complex tissues. Several methods (e.g. extrusion, laminar flow or electrospinning) have been proposed for building hydrogel microfibres, with distinctive cell types and with different degrees of complexity. However, these methods require numerous protocol adaptations in order to achieve fibre fabricating and lack the ability to control microfibre alignment. Here, we present a simple method for the production of microfibers, based on a core shell approach, composed of calcium alginate and type I collagen. The process presented here allows the removal of the calcium alginate shell, after only 24 hours of culture, leading to stable and reproducible fibre shaped cellular constructs. With time of culture cells show to distribute preferentially to the surface of the fibre and display a uniform cellular orientation. Moreover, when cultured inside the fibres, murine bone marrow mesenchymal stem cells show the capacity to differentiate towards the osteoblastic lineage, under non-osteoinductive culture conditions. This work establishes a novel method for cellular fibre fabrication that due to its inherent simplicity can be easily upscaled and applied to other cell types.

The effect of cyclo-DfKRG peptide immobilization on titanium on the adhesion and differentiation of human osteoprogenitor cells.

This study takes place in the field of development of a bioactive surface of titanium alloys. In this paper, titanium was functionalized with cyclo-DfKRG peptide by coating or grafting using different anchors (thiol or phosphonate) as spacers between the surface and the peptide. Cell adhesion, and differentiation of human osteoprogenitor (HOP) cells arising from human bone marrow were investigated. Our results seem to demonstrate that cyclo-DfKRG peptide coating with a phosphonate anchor and grafting procedure contributes to higher cell adhesion and a strong ALP and Cbfa1 mRNA expression, after 10 days of cell seeding. At the contrary, this peptide coated with a thiol anchor stimulates differentiation of HOP within 3 days of culture.

Human primary endothelial cells stimulate human osteoprogenitor cell differentiation.

Bone development and remodeling depend on complex interactions between bone-forming osteoblasts and other cells present within the bone microenvironment, particularly endothelial cells that may be pivotal members of a complex interactive communication network in bone. While cell cooperation was previously established between Human OsteoProgenitor cells (HOP) and Human Umbilical Vein Endothelial Cells (HUVEC) the aim of our study was to investigate if this interaction is specific to Human Endothelial cell types (ECs) from different sources. Osteoblastic cell differentiation analysis performed using different co-culture models with direct contact revealed that Alkaline Phosphatase (Al-P) activity was only increased by the direct contact of HOP with human primary vascular endothelial cell types including endothelial precursor cells (EPCs) isolated from blood cord, endothelial cells from Human Saphen Vein (HSV) while a transformed cell line, the Human Bone Marrow Endothelial Cell Line (HBMEC) did not modify osteoblastic differentiation of HOP. Because connexin 43, a specific gap junction protein, seemed to be involved in HUVEC/HOP cell cooperation, expression by RT-PCR and immunocytochemistry of this gap junctional protein was investigated in EPCs, HSV and HBMEC. Both endothelial cells are positive to this protein and the disruption of gap junction communication using 18alpha-glycyrrhetinic acid treatment decreased the positive effect of these endothelial co-cultures on HOP differentiation as was previously demonstrated for HUVEC and HOP co-cultures. These data seem to indicate that this cross talk between HOP and ECs, through gap junction communication constitutes an additional concept in cell differentiation control.