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 support 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 cooperation 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 allow cellular communication, more closely resemble bone remodeling and/or the regeneration process in the framework of BTE. STATEMENT OF SIGNIFICANCE: Bone is a dynamic tissue under continuous remodeling, but spontaneous healing may fail in the case of excessive bone loss which often requires valid alternatives to conventional treatments to restore bone integrity, like bone tissue engineering (BTE). Pre-clinical evaluation of scaffolds for BTE requires in vitro testing where co-cultures combining innovative materials with osteoblasts (OBs) and osteoclasts (OCs) closely mimic the in vivo repair process. This review considers the direct and indirect OB/OC co-cultures relevant to BTE, from the early mouse-cell models to the recent bone regenerative systems. The co-culture modeling of bone microenvironment provides reliable information on bone cell cross-talk. Starting from improved knowledge on bone remodeling, bone disease mechanisms may be understood and new BTE solutions are designed.

Bone-tissue regeneration induced by biomimetic bioactive materials is the most promising approach alternative to the clinical ones used to treat bone loss caused by trauma or diseases such as osteoporosis. The goal is to design nanostructured bioactive constructs able to reproduce the physiological environment: By mimicking the natural features of bone tissue, the cell behavior during the regeneration process may be addressed. At present, 3D-printing technologies are the only techniques able to design complex structures avoiding constraints of final shape and porosity. However, this type of biofabrication requires complex optimization of biomaterial formulations in terms of specific rheological and mechanical properties while preserving high biocompatibility. In this work, we combined nano-sized mesoporous bioactive glasses enriched with strontium ions with type I collagen, to formulate a bioactive ink for 3D-printing technologies. Moreover, to avoid the premature release of strontium ions within the crosslinking medium and to significantly increase the material mechanical and thermal stability, we applied an optimized chemical treatment using ethanol-dissolved genipin solutions. The high biocompatibility of the hybrid system was confirmed by using MG-63 and Saos-2 osteoblast-like cell lines, further highlighting the great potential of the innovative nanocomposite for the design of bone-like scaffolds.

Gelatin is a natural biopolymer extensively used for tissue engineering applications due to its similarities to the native extracellular matrix. However, the rheological properties of gelatin formulations are not ideal for extrusion-based bioprinting. In this work, we present an approach to improve gelatin bioprinting performances by using pectin as a rheology modifier of gelatin and (3-glycidyloxypropyl)trimethoxysilane (GPTMS) as a gelatin–pectin crosslinking agent. The preparation of gelatin–pectin formulations is initially optimized to obtain homogenous gelatin–pectin gels. Since the use of GPTMS requires a drying step to induce the completion of the crosslinking reaction, microporous gelatin–pectin–GPTMS sponges are produced through freeze-drying, and the intrinsic properties of gelatin–pectin–GPTMS networks (e.g., porosity, pore size, degree of swelling, compressive modulus, and cell adhesion) are investigated. Subsequently, rheological investigations together with bioprinting assessments demonstrate the key role of pectin in increasing the viscosity and the yield stress of low viscous gelatin solutions. Water stable, three-dimensional, and self-supporting gelatin–pectin–GPTMS scaffolds with interconnected micro- and macroporosity are successfully obtained by combining extrusion-based bioprinting and freeze-drying. The proposed biofabrication approach does not require any additional temperature controller to further modulate the rheological properties of gelatin solutions and it could furthermore be extended to improve the bioprintability of other biopolymers.

Decellularized bone matrix is receiving much attention as biological scaffolds and implantable biomaterials for bone tissue regeneration. Here, we evaluated the efficacy of a cell-free demineralized bone matrix on mesenchymal stem cells (MSCs) survival and differentiation in vitro. The seeding of human umbilical cord-derived MSCs (hUC-SCs) on decellularized bone matrices up to 14 days was exploited, assessing their capability of scaffold colonization and evaluating gene expression of bone markers. Light and Scanning Electron Microscopies were used. The obtained cell-free decalcified structures showed elastic moduli attributable to both topology and biochemical composition. Morphological observation evidenced an almost complete colonization of the scaffolds after 14 days of culture. Moreover, in hUC-SCs cultured on decalcified scaffolds, without the addition of any osteoinductive media, there was an upregulation of Collagen Type I (COL1) and osteonectin (ON) gene expression, especially on day 14. Modifications in the expression of genes engaged in stemness were also detected. In conclusion, the proposed decellularized bone matrix can induce the in vitro hUC-SCs differentiation and has the potential to be tested for in in vivo tissue regeneration.

Graded lattice scaffolds based on rhombic dodecahedral (RD) elementary unit cell geometry were manufactured in 316L stainless steel (SS) by laser powder bed fusion (LPBF). Two different strategies based on varying strut thickness layer-by-layer in the building direction were adopted to obtain the graded scaffolds: a) decreasing strut size from core to edge to produce the dense-in (DI) structure and b) increasing strut size in the same direction to produce the dense-out (DO) structure. Both graded structures (DI and DO) were constructed with specular symmetry with respect to the central horizontal axis. Structural, mechanical, and biological characterizations were carried out to evaluate feasibility of designing appropriate biomechanical performances of graded scaffolds in the perspective of bone tissue regeneration. Results showed that mechanical behavior is governed by graded geometry, while printing parameters influence structural properties of the material such as density, textures, and crystallographic phases. The predominant failure mechanism in graded structures initiates in correspondence of thinner struts, due to high stress concentrations on strut junctions. Biological tests evidenced better proliferation of cells in the DO graded scaffold, which in turn exhibits mechanical properties close to cortical bone. The combined control of grading strategy, printing parameters and elementary unit cell geometry can enable implementing scaffolds with improved biomechanical performances for bone tissue regeneration.