Bioactivation Routes of Gelatin-Based Scaffolds to Enhance at Nanoscale Level Bone Tissue Regeneration

The present work is focused on the development of gelatin-based scaffolds crosslinked through carbodiimide reaction and their bioactivation by two different methods: (i) surface modification by inorganic signals represented by hydroxyapatite nanoparticles precipitated on scaffold through biomimetic treatment; (ii) analog of BMP-2 peptide decoration. The results showed the effects of polymer concentration and crosslinking time on the physico-chemical, morphological, and mechanical properties of scaffolds. Furthermore, a comparative study of biological response for both bioactivated structures allowed to evaluate the influence of inorganic and organic cues on cellular behavior in terms of adhesion, proliferation and early osteogenic marker expression. The bioactivation by inorganic cues induced positive cellular response compared to neat scaffolds in terms of increased cell proliferation and early osteogenic differentiation of human mesenchymal stem cell (hMSC), as evidenced by the Alkaline phosphatase (ALP) expression. Similarly BMP-2 peptide decorated scaffolds showed higher values of ALP than biomineralized ones at longer time. The overall results demonstrated that the presence of bioactive signals (either inorganic or organic) at nanoscale level allowed an osteoinductive effect on hMSC in a basal medium, making the modified gelatin scaffolds a promising candidate for bone tissue regeneration

Viscoelastic Properties of Rapid Prototyped Magnetic Nanocomposite Scaffolds for Osteochondral Tissue Regeneration

Poly(ϵ-caprolactone) and poly(ethylene glycol) based magnetic nanocomposite scaffolds were fabricated using fused deposition modeling and stereolithography approaches, and a hybrid scaffold was obtained by combining these additive manufacturing technologies. Viscoelastic properties in compression were investigated at 37 °C, spanning a range frequency of four decades. Results suggest that poly(ϵ-caprolactone) and poly(ethylene glycol) based scaffolds adequately reproduce viscoelastic properties of subchondral bone and articular cartilage tissues, respectively. By combining fused deposition modeling and stereolithography it is possible to manufacture a hybrid scaffold suitable for osteochondral tissue regeneration. Poly(ϵ-caprolactone) and poly(ethylene glycol) based magnetic nanocomposite scaffolds were fabricated using fused deposition modeling and stereolithography approaches, and a hybrid scaffold was obtained by combining these additive manufacturing technologies. Viscoelastic properties in compression were investigated at 37 °C, spanning a range frequency of four decades. Results suggest that poly(ϵ-caprolactone) and poly(ethylene glycol) based scaffolds adequately reproduce viscoelastic properties of subchondral bone and articular cartilage tissues, respectively. By combining fused deposition modeling and stereolithography it is possible to manufacture a hybrid scaffold suitable for osteochondral tissue regeneration

Advanced application of collagen-based biomaterials in tissue repair and restoration

AbstractIn tissue engineering, bioactive materials play an important role, providing structural support, cell regulation and establishing a suitable microenvironment to promote tissue regeneration. As the main component of extracellular matrix, collagen is an important natural bioactive material and it has been widely used in scientific research and clinical applications. Collagen is available from a wide range of animal origin, it can be produced by synthesis or through recombinant protein production systems. The use of pure collagen has inherent disadvantages in terms of physico-chemical properties. For this reason, a processed collagen in different ways can better match the specific requirements as biomaterial for tissue repair. Here, collagen may be used in bone/cartilage regeneration, skin regeneration, cardiovascular repair and other fields, by following different processing methods, including cross-linked collagen, complex, structured collagen, mineralized collagen, carrier and other forms, promoting the development of tissue engineering. This review summarizes a wide range of applications of collagen-based biomaterials and their recent progress in several tissue regeneration fields. Furthermore, the application prospect of bioactive materials based on collagen was outlooked, aiming at inspiring more new progress and advancements in tissue engineering research. Graphical Abstrac

From 3D Hierarchical Scaffolds for Tissue Engineering to Advanced Hydrogel-Based and Complex Devices for in situ Cell or Drug Release☆

Abstract In the past few years, researchers have focused on the development of three-dimensional (3D) advanced scaffolds and multifunctional hydrogel-based materials. As reported in literature, 3D polymer-based composite scaffolds for tissue engineering have been manufactured through conventional and advanced manufacturing techniques, and different injectable materials and hydrogel-based systems have been proposed and studied. The aim of the current research was to define an approach in the development of multifunctional tools spanning from 3D hierarchical scaffolds for soft tissue engineering to advanced hydrogel-based devices for in situ cell or drug release. The mechanical/rheological behaviour as well as the structural/functional features of the designed devices were discussed and analyzed

Biofunctionalization of 3D printed collagen with bevacizumab-loaded microparticles targeting pathological angiogenesis

Pathological angiogenesis is a crucial attribute of several chronic diseases such as cancer, age-related macular degeneration, and osteoarthritis (OA). In the case of OA, pathological angiogenesis mediated by the vascular endothelial growth factor (VEGF), among other factors, contributes to cartilage degeneration and to implants rejection. In line with this, the use of the anti-VEGF bevacizumab (BVZ) has been shown to prevent OA progression and support cartilage regeneration. The aim of this work was to functionalize a medical grade collagen with poly (lactic-co-glycolic acid) (PLGA) microparticles containing BVZ via three-dimensional (3D) printing to target pathological angiogenesis. First, the effect of several formulation parameters on the encapsulation and release of BVZ from PLGA microparticles was studied. Then, the anti-angiogenic activity of released BVZ was tested in a 3D cell model. The 3D printability of the microparticle-loaded collagen ink was tested by evaluating the shape fidelity of 3D printed structures. Results showed that the release and the encapsulation efficiency of BVZ could be tuned as a function of several formulation parameters. In addition, the released BVZ was observed to reduce vascularization by human umbilical vein endothelial cells. Finally, the collagen ink with embedded BVZ microparticles was successfully printed, leading to shape-stable meniscus-, nose- and auricle-like structures. Taken altogether, we defined the conditions for the successful combination of BVZ-loaded microparticles with the 3D printing of a medical grade collagen to target pathological angiogenesisThis project has received funding from the European Union's Horizon 2020 research and innovation program under grant agreement No 814444 (MEFISTO). The authors thank mAbxience-GH Genhelix for the kind donation of Bevacizumab (Avastin®) and Geistlich Pharma AG for providing the medical grade collagen. AA acknowledges funding from “la Caixa” Foundation (ID 100010434) with a fellowship code LCF/BQ/PR22/11920003. RL acknowledges funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (grant agreement No. 949806, VOLUME-BIO). RL and JM acknowledge funding from the Dutch Artritis Foundation (LLP-12 and LLP-22)S

Biofunctionalization of 3D printed collagen with bevacizumab-loaded microparticles targeting pathological angiogenesis

Pathological angiogenesis is a crucial attribute of several chronic diseases such as cancer, age-related macular degeneration, and osteoarthritis (OA). In the case of OA, pathological angiogenesis mediated by the vascular endothelial growth factor (VEGF), among other factors, contributes to cartilage degeneration and to implants rejection. In line with this, the use of the anti-VEGF bevacizumab (BVZ) has been shown to prevent OA progression and support cartilage regeneration. The aim of this work was to functionalize a medical grade collagen with poly (lactic-co-glycolic acid) (PLGA) microparticles containing BVZ via three-dimensional (3D) printing to target pathological angiogenesis. First, the effect of several formulation parameters on the encapsulation and release of BVZ from PLGA microparticles was studied. Then, the anti-angiogenic activity of released BVZ was tested in a 3D cell model. The 3D printability of the microparticle-loaded collagen ink was tested by evaluating the shape fidelity of 3D printed structures. Results showed that the release and the encapsulation efficiency of BVZ could be tuned as a function of several formulation parameters. In addition, the released BVZ was observed to reduce vascularization by human umbilical vein endothelial cells. Finally, the collagen ink with embedded BVZ microparticles was successfully printed, leading to shape-stable meniscus-, nose- and auricle-like structures. Taken altogether, we defined the conditions for the successful combination of BVZ-loaded microparticles with the 3D printing of a medical grade collagen to target pathological angiogenesis

A NOVEL ROUTE TOWARDS THE DESIGN OF 3D MORPHOLOGICALLY CONTROLLED MAGNETIC SCAFFOLDS FOR ADVANCED BONE TISSUE ENGINEERING

Tissue engineering is an interdisciplinary field that has the goal of creating new tissues and organs. Ideal bone scaffold, which is the key element,should possess important chemical, biochemical and biophysical properties, whilst the biomechanical environment introduces another level of complexity. Scaffold needs to be able to withstand external forces, and it is known that bone regeneration, modeling and remodeling is mediated by mechanical stimuli known as mechanotransduction. Mechanical stimuli transferred by scaffolds to cells rely exclusively on intrinsic scaffold properties, such as material stiffness and architecture. Consequently, the introduction of rapid prototyping technologies in the biomedical field has allowed to obtain scaffolds characterized by a precise control of its internal architecture, including precise pore size, pore geometry, spatial distribution of pores and interconnectivity, which may be considered as critical features to the their in vivo, biological and mechanical performances. In order to obtain a complete histomorphologically and biologically mature tissue, as bone, the restoration of the mechanical resistance to physiological stresses should be also followed by angiogenesis, which is a crucial aspect in the development of regenerative medicine approaches that require rapid vascularization of tissue-engineered structures. The main driving idea of this work is creating a conceptually new type of bioactive scaffold able to be manipulated in situ by means of magnetic forces in order to repair large bone and osteochondral defects. As first step, the design of 3D fiber deposited poly(ε-caprolactone)/iron oxide nanocomposite scaffolds has been described. The effect of iron oxide nanoparticle inclusion on morphological, mechanical, magnetic and biological performances has been assessed. Successively, in order to avoid the dangerous problem of leaving any non bioresorbable magnetic inclusion (for example, magnetite) inside the repaired tissue, poly(ε-caprolactone)/iron-doped hydroxyapatite substrates were designed and characterized using different polymer-to-particle weight ratios. The effect of iron-doped hydroxyapatite nanoparticle inclusion on morphological, mechanical, magnetic and biological performances has been assessed. This has allowed to choose the optimal polymer-to-particle weight ratio. In particular, a nanoparticle amount of 20% by weight embedded into the polymeric matrix has shown the best compromise between all the above reported features and then, 3D morphologically controlled nanocomposite magnetic scaffolds have been manufactured. The effect of a sinusoidal magnetic stimulation on adhesion and proliferation of cells seeded on 3D scaffolds has been studied. Future works will be focused on the effect of a variable magnetic field on cell differentiation. This work may represent a first approach towards the design of morphologically controlled and fully biodegradable nanocomposite magnetic scaffolds, which should be able to improve cell recruitment and cell loading efficiency. Furthermore, preliminary histological analyses have highlighted very interesting results

Development of a highly concentrated collagen ink for the creation of a 3D printed meniscus

The most prevalent extracellular matrix (ECM) protein in the meniscus is collagen, which controls cell activity and aids in preserving the biological and structural integrity of the ECM. To create stable and high-precision 3D printed collagen scaffolds, ink formulations must possess good printability and cytocompatibility. This study aims to overlap the limitation in the 3D printing of pure collagen, and to develop a highly concentrated collagen ink for meniscus fabrication. The extrusion test revealed that 12.5 % collagen ink had the best combination of high collagen concentration and printability. The ink was specifically designed to have load-bearing capacity upon printing and characterized with respect to rheological and extrusion properties. Following printing of structures with different infill, a series of post-processing steps, including salt stabilization, pH shifting, washing, freeze-drying, crosslinking and sterilization were performed, and optimised to maintain the stability of the engineered construct. Mechanical testing highlighted a storage modulus of 70 kPa for the lower porous structure while swelling properties showed swelling ratio between 9 and 11 after 15 min of soaking. Moreover, human avascular and vascular meniscus cells cultured on the scaffolds deposited a meniscus-like matrix containing collagen I, II and glycosaminoglycans after 28 days of culture. Finally, as proof-of-concept, human size 3D printed meniscus scaffold were created

Reverse engineering of mandible and prosthetic framework: Effect of titanium implants in conjunction with titanium milled full arch bridge prostheses on the biomechanics of the mandible

This study aimed at investigating the effects of titanium implants and different configurations of full-arch prostheses on the biomechanics of edentulous mandibles. Reverse engineered, composite, anisotropic, edentulous mandibles made of a poly(methylmethacrylate) core and a glass fibre reinforced outer shell were rapid prototyped and instrumented with strain gauges. Brånemark implants RP platforms in conjunction with titanium Procera one-piece or two-piece bridges were used to simulate oral rehabilitations. A lateral load through the gonion regions was used to test the biomechanical effects of the rehabilitations. In addition, strains due to misfit of the one-piece titanium bridge were compared to those produced by one-piece cast gold bridges. Milled titanium bridges had a better fit than cast gold bridges. The stress distribution in mandibular bone rehabilitated with a one-piece bridge was more perturbed than that observed with a two-piece bridge. In particular the former induced a stress concentration and stress shielding in the molar and symphysis regions, while for the latter design these stresses were strongly reduced. In conclusion, prosthetic frameworks changed the biomechanics of the mandible as a result of both their design and manufacturing technology