Co–culture systems of osteoblasts and osteoclasts: Simulating in vitro bone remodeling in regenerative approaches

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Co–culture systems of osteoblasts and osteoclasts: Simulating in vitro bone remodeling in regenerative approaches

Bone is an extremely dynamic tissue, undergoing continuous remodeling for its whole lifetime, but its regeneration or augmentation due to bone loss or defects are not always easy to obtain. Bone tissue engineering (BTE) is a promising approach, and its success often relies on a “smart”scaffold, as a sup- port to host and guide bone formation through bone cell precursors. Bone homeostasis is maintained by osteoblasts (OBs) and osteoclasts (OCs) within the basic multicellular unit, in a consecutive cycle of resorption and formation. Therefore, a functional scaffold should allow the best possible OB/OC cooper- ation for bone remodeling, as happens within the bone extracellular matrix in the body. In the present work OB/OC co-culture models, with and without scaffolds, are reviewed. These experimental systems are intended for different targets, including bone remodeling simulation, drug testing and the assessment of biomaterials and 3D scaffolds for BTE. As a consequence, several parameters, such as cell type, cell ratio, culture medium and inducers, culture times and setpoints, assay methods, etc. vary greatly. This review identifies and systematically reports the in vitro methods explored up to now, which, as they al- low cellular communication, more closely resemble bone remodeling and/or the regeneration process in the framework of BTE

Extrusion 3D printing of a multiphase collagen-based material: An optimized strategy to obtain biomimetic scaffolds with high shape fidelity

Extrusion printing represents one of the leading additive manufacturing techniques for tissue engineering purposes due to the possibility of achieving accurate control of the final shape and porosity of the scaffold. Despite many polymeric materials having already been optimized for this application, the processing of biopolymer-based systems still presents several limitations mainly ascribed to their poor rheological properties. Moreover, the introduction of inorganic components into the biomaterial formulation may introduce further difficulties related to system homogeneity, finally compromising its extrudability. In this context, the present study aimed at developing a new multi-phase biomaterial ink able to mimic the native composition of bone extracellular matrix, combining type-I-collagen with nano-hydroxyapatite and mesoporous bioactive glass nanoparticles. Starting from a comprehensive rheological assessment, computational-fluid-dynamics-based models were exploited to describe the material flow regime and define the optimal printing process planning. During printing, a gelatin-based bath was exploited to support the deposition of the material, while the gelation of collagen and its further chemical crosslinking with genipin enabled the stabilization of the printed structure, characterized by high shape fidelity. The developed strategy enables the extrusion printing of complex multi-phase systems and the design of high-precision biomimetic scaffolds with great potential for bone tissue engineering

Protocol of Co-Culture of Human Osteoblasts and Osteoclasts to Test Biomaterials for Bone Tissue Engineering

open5no: New biomaterials and scaffolds for bone tissue engineering (BTE) applications require to be tested in a bone microenvironment reliable model. On this assumption, the in vitro laboratory protocols with bone cells represent worthy experimental systems improving our knowledge about bone homeostasis, reducing the costs of experimentation. To this day, several models of the bone microenvironment are reported in the literature, but few delineate a protocol for testing new biomaterials using bone cells. Herein we propose a clear protocol to set up an indirect co-culture system of human-derived osteoblasts and osteoclast precursors, providing well-defined criteria such as the cell seeding density, cell:cell ratio, the culture medium, and the proofs of differentiation. The material to be tested may be easily introduced in the system and the cell response analyzed. The physical separation of osteoblasts and osteoclasts allows distinguishing the effects of the material onto the two cell types and to evaluate the correlation between material and cell behavior, cell morphology, and adhesion. The whole protocol requires about 4 to 6 weeks with an intermediate level of expertise. The system is an in vitro model of the bone remodeling system useful in testing innovative materials for bone regeneration, and potentially exploitable in different application fields. The use of human primary cells represents a close replica of the bone cell cooperation in vivo and may be employed as a feasible system to test materials and scaffolds for bone substitution and regeneration.This project has received funding from the European Research Council (ERC) under the European Unions Horizon 2020 research and innovation program (grant agreement No 681798-BOOST). Available online: http://www.ercprojectboost.eopenBorciani, Giorgia; Montalbano, Giorgia; Baldini, Nicola; Vitale-Brovarone, Chiara; Ciapetti, GabrielaBorciani, Giorgia; Montalbano, Giorgia; Baldini, Nicola; Vitale-Brovarone, Chiara; Ciapetti, Gabriel

Synthesis and incorporation of rod-like nano-hydroxyapatite into type I collagen matrix: A hybrid formulation for 3D printing of bone scaffolds

Abstract Over the recent years, nanometric hydroxyapatite (HA) has gained interest as constituent of hybrid systems for bone scaffold fabrication, due to its biomimicry and biocompatibility. In this study, rod-like nano-HA particles were introduced in a type I collagen matrix to create a composite mimicking the bone composition. HA nano-rods (40−60 nm × 20 nm) were synthesised by hydrothermal method involving the use of an ammonium-based dispersing agent (Darvan 821-A) and fully characterised. The homogeneous dispersion of HA nanoparticles throughout the final hybrid formulation was achieved through their suspension in a collagen solution in presence of Darvan 821-A. The resulting homogeneous collagen/nano-HA suspension proved to be suitable for extrusion printing applications, showing shear thinning and sol-gel transition upon simil-physiological conditions. Furthermore, mesh-like structures were printed in a gelatine-supporting bath by means of a commercial bioprinter further demonstrating the potential of the designed hybrid system for the fabrication of 3D bone-like scaffolds

Type I Collagen and Strontium-Containing Mesoporous Glass Particles as Hybrid Material for 3D Printing of Bone-Like Materials

Bone tissue engineering offers an alternative promising solution to treat a large number of bone injuries with special focus on pathological conditions, such as osteoporosis. In this scenario, the bone tissue regeneration may be promoted using bioactive and biomimetic materials able to direct cell response, while the desired scaffold architecture can be tailored by means of 3D printing technologies. In this context, our study aimed to develop a hybrid bioactive material suitable for 3D printing of scaffolds mimicking the natural composition and structure of healthy bone. Type I collagen and strontium-containing mesoporous bioactive glasses were combined to obtain suspensions able to perform a sol-gel transition under physiological conditions. Field emission scanning electron microscopy (FESEM) analyses confirmed the formation of fibrous nanostructures homogeneously embedding inorganic particles, whereas bioactivity studies demonstrated the large calcium phosphate deposition. The high-water content promoted the strontium ion release from the embedded glass particles, potentially enhancing the osteogenic behaviour of the composite. Furthermore, the suspension printability was assessed by means of rheological studies and preliminary extrusion tests, showing shear thinning and fast material recovery upon deposition. In conclusion, the reported results suggest that promising hybrid systems suitable for 3D printing of bioactive scaffolds for bone tissue engineering have been developed

Analysis of multiple protein detection methods in human osteoporotic bone extracellular matrix: From literature to practice

The punctual analysis of bone Extracellular Matrix (ECM) proteins represents a pivotal point for medical research in bone diseases like osteoporosis. Studies in this field, historically done to appreciate bone biology, were mainly conducted on animal samples and, up to today, only a few studies on protein detection in human bone are present. The challenges in bone ECM protein extraction and quantitation protocols are related to both the separation of proteins from the mineral content (i.e. hydroxyapatite) and the difficulty of avoiding protein denaturation during the extraction processes. The aim of the present work was to define appropriate protocol(s) for bone ECM protein extraction that could be applied to investigate both normal and pathological conditions. We compared and optimised some of the most used protocols present in the literature, modifying the protein precipitation method, the buffer used for resuspension and/or the volume of reagent used. Bradford and BCA assays and Western Blotting were used to evaluate the variations in the total protein recovery and the amount of selected proteins (Type I Collagen, TGF-β, IGF-1, Decorin, Osteopontin, Bone Sialoprotein-2 and Osteocalcin). Collectively, we were capable to draw-up two single-extract protocols with optimal recovery and ideal protein content, that can be used for a detailed analysis of ECM proteins in pathological bone samples. Time-consuming multi-extract procedures, optimised in their precipitation methods, are however crucial for a precise detection of specific proteins, like osteocalcin. As the matter of fact, also the demineralization processes, commonly suggested and performed in several protocols, could hinder an accurate protein detection, thus inherently affecting the study of a pathological bone ECM. This study represents a starting point for the definition of appropriate strategies in the study of bone extracellular matrix proteins involved in the onset and maintenance of bone diseases, as well as a tool for the development of customized scaffolds capable to modulate a proper feedback loop in bone remodelling, altered in case of diseases like osteoporosis

Analysis of multiple protein detection methods in human osteoporotic bone extracellular matrix: From literature to practice

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