Bioinspired Silk Fibroin-Based Composite Grafts as Bone Tunnel Fillers for Anterior Cruciate Ligament Reconstruction.

Anterior cruciate ligament (ACL) replacement is still a big challenge in orthopedics due to the need to develop bioinspired implants that can mimic the complexity of bone-ligament interface. In this study, we propose biomimetic composite tubular grafts (CTGs) made of horseradish peroxidase (HRP)-cross-linked silk fibroin (SF) hydrogels containing ZnSr-doped ß-tricalcium phosphate (ZnSr-ß-TCP) particles, as promising bone tunnel fillers to be used in ACL grafts (ACLGs) implantation. For comparative purposes, plain HRP-cross-linked SF hydrogels (PTGs) were fabricated. Sonication and freeze-drying methodologies capable of inducing crystalline ß-sheet conformation were carried out to produce both the CTGs and PTGs. A homogeneous microstructure was achieved from microporous to nanoporous scales. The mechanical properties were dependent on the inorganic powder's incorporation, with a superior tensile modulus observed on the CTGs (12.05 ± 1.03 MPa) as compared to the PTGs (5.30 ± 0.93 MPa). The CTGs presented adequate swelling properties to fill the space in the bone structure after bone tunnel enlargement and provide a stable degradation profile under low concentration of protease XIV. The in vitro studies revealed that SaOs-2 cells adhered, proliferated and remained viable when cultured into the CTGs. In addition, the bioactive CTGs supported the osteogenic activity of cells in terms of alkaline phosphatase (ALP) production, activity, and relative gene expression of osteogenic-related markers. Therefore, this study is the first evidence that the developed CTGs hold adequate structural, chemical, and biological properties to be used as bone tunnel fillers capable of connecting to the ACL tissue while stimulating bone tissue regeneration for a faster osteointegration.

Horseradish Peroxidase-Crosslinked Calcium-Containing Silk Fibroin Hydrogels as Artificial Matrices for Bone Cancer Research.

Hydrogels, being capable of mimicking the extracellular matrix composition of tissues, are greatly used as artificial matrices in tissue engineering applications. In this study, the generation of horseradish peroxidase (HRP)-crosslinked silk fibroin (SF) hydrogels, using calcium peroxide as oxidizer is reported. The proposed fast forming calcium-containing SF hydrogels spontaneously undergo SF conformational changes from random coil to ß-sheet during time, exhibiting ionic, and pH stimuli responsiveness. In vitro response shows calcium-containing SF hydrogels' encapsulation properties and their ability to promote SaOs-2 tumor cells death after 10 days of culturing, upon complete ß-sheet conformation transition. Calcium-containing SF hydrogels' angiogenic potential investigated in an in ovo chick chorioallantoic membrane (CAM) assay, show a high number of converging blood vessels as compared to the negative control, although no endothelial cells infiltration is observed. The in vivo response evaluated in subcutaneous implantation in CD1 and nude NCD1 mice shows that calcium-containing SF hydrogels are stable up to 6 weeks after implantation. However, an increased number of dead cells are also present in the surrounding tissue. The results suggest the potential of calcium-containing SF hydrogels to be used as novel in situ therapeutics for bone cancer treatment applications, particularly to osteosarcoma.

Antibacterial Nanostructured Ti Coatings by Magnetron Sputtering: From Laboratory Scales to Industrial Reactors.

Based on an already tested laboratory procedure, a new magnetron sputtering methodology to simultaneously coat two-sides of large area implants (up to ~15 cm2) with Ti nanocolumns in industrial reactors has been developed. By analyzing the required growth conditions in a laboratory setup, a new geometry and methodology have been proposed and tested in a semi-industrial scale reactor. A bone plate (DePuy Synthes) and a pseudo-rectangular bone plate extracted from a patient were coated following the new methodology, obtaining that their osteoblast proliferation efficiency and antibacterial functionality were equivalent to the coatings grown in the laboratory reactor on small areas. In particular, two kinds of experiments were performed: Analysis of bacterial adhesion and biofilm formation, and osteoblasts-bacteria competitive in vitro growth scenarios. In all these cases, the coatings show an opposite behavior toward osteoblast and bacterial proliferation, demonstrating that the proposed methodology represents a valid approach for industrial production and practical application of nanostructured titanium coatings.

Lactoferrin-Hydroxyapatite Containing Spongy-Like Hydrogels for Bone Tissue Engineering.

The development of bioactive and cell-responsive materials has fastened the field of bone tissue engineering. Gellan gum (GG) spongy-like hydrogels present high attractive properties for the tissue engineering field, especially due to their wide microarchitecture and tunable mechanical properties, as well as their ability to entrap the responsive cells. Lactoferrin (Lf) and Hydroxyapatite (HAp) are bioactive factors that are known to potentiate faster bone regeneration. Thus, we developed an advanced three-dimensional (3D) biomaterial by integrating these bioactive factors within GG spongy-like hydrogels. Lf-HAp spongy-like hydrogels were characterized in terms of microstructure, water uptake, degradation, and concomitant release of Lf along the time. Human adipose-derived stem cells (hASCs) were seeded and the capacity of these materials to support hASCs in culture for 21 days was assessed. Lf addition within GG spongy-like hydrogels did not change the main features of GG spongy-like hydrogels in terms of porosity, pore size, degradation, and water uptake commitment. Nevertheless, HAp addition promoted an increase of the pore wall thickness (from ~13 to 28 µm) and a decrease on porosity (from ~87% to 64%) and mean pore size (from ~12 to 20 µm), as well as on the degradability and water retention capabilities. A sustained release of Lf was observed for all the formulations up to 30 days. Cell viability assays showed that hASCs were viable during the culture period regarding cell-laden spongy-like hydrogels. Altogether, we demonstrate that GG spongy-like hydrogels containing HAp and Lf in high concentrations gathered favorable 3D bone-like microenvironment with an increased hASCs viability with the presented results.

Scaffolding Strategies for Tissue Engineering and Regenerative Medicine Applications.

During the past two decades, tissue engineering and the regenerative medicine field have invested in the regeneration and reconstruction of pathologically altered tissues, such as cartilage, bone, skin, heart valves, nerves and tendons, and many others. The 3D structured scaffolds and hydrogels alone or combined with bioactive molecules or genes and cells are able to guide the development of functional engineered tissues, and provide mechanical support during in vivo implantation. Naturally derived and synthetic polymers, bioresorbable inorganic materials, and respective hybrids, and decellularized tissue have been considered as scaffolding biomaterials, owing to their boosted structural, mechanical, and biological properties. A diversity of biomaterials, current treatment strategies, and emergent technologies used for 3D scaffolds and hydrogel processing, and the tissue-specific considerations for scaffolding for Tissue engineering (TE) purposes are herein highlighted and discussed in depth. The newest procedures focusing on the 3D behavior and multi-cellular interactions of native tissues for further use for in vitro model processing are also outlined. Completed and ongoing preclinical research trials for TE applications using scaffolds and hydrogels, challenges, and future prospects of research in the regenerative medicine field are also presented.

Enzymatically Cross-Linked Silk Fibroin-Based Hierarchical Scaffolds for Osteochondral Regeneration.

Osteochondral (OC) regeneration faces several limitations in orthopedic surgery, owing to the complexity of the OC tissue that simultaneously entails the restoration of articular cartilage and subchondral bone diseases. In this study, novel biofunctional hierarchical scaffolds composed of a horseradish peroxidase (HRP)-cross-linked silk fibroin (SF) cartilage-like layer (HRP-SF layer) fully integrated into a HRP-SF/ZnSr-doped ß-tricalcium phosphate (ß-TCP) subchondral bone-like layer (HRP-SF/dTCP layer) were proposed as a promising strategy for OC tissue regeneration. For comparative purposes, a similar bilayered structure produced with no ion incorporation (HRP-SF/TCP layer) was used. A homogeneous porosity distribution was achieved throughout the scaffolds, as shown by micro-computed tomography analysis. The ion-doped bilayered scaffolds presented a wet compressive modulus (226.56 ± 60.34 kPa) and dynamic mechanical properties (ranging from 403.56 ± 111.62 to 593.56 ± 206.90 kPa) superior to that of the control bilayered scaffolds (189.18 ± 90.80 kPa and ranging from 262.72 ± 59.92 to 347.68 ± 93.37 kPa, respectively). Apatite crystal formation, after immersion in simulated body fluid (SBF), was observed in the subchondral bone-like layers for the scaffolds incorporating TCP powders. Human osteoblasts (hOBs) and human articular chondrocytes (hACs) were co-cultured onto the bilayered structures and monocultured in the respective cartilage and subchondral bone half of the partitioned scaffolds. Both cell types showed good adhesion and proliferation in the scaffold compartments, as well as adequate integration of the interface regions. Osteoblasts produced a mineralized extracellular matrix (ECM) in the subchondral bone-like layers, and chondrocytes showed GAG deposition. The gene expression profile was different in the distinct zones of the bilayered constructs, and the intermediate regions showed pre-hypertrophic chondrocyte gene expression, especially on the BdTCP constructs. Immunofluorescence analysis supported these observations. This study showed that the proposed bilayered scaffolds allowed a specific stimulation of the chondrogenic and osteogenic cells in the co-culture system together with the formation of an osteochondral-like tissue interface. Hence, the structural adaptability, suitable mechanical properties, and biological performance of the hierarchical scaffolds make these constructs a desired strategy for OC defect regeneration.

In Vitro Mimetic Models for the Bone-Cartilage Interface Regeneration.

In embryonic development, pure cartilage structures are in the basis of bone-cartilage interfaces. Despite this fact, the mature bone and cartilage structures can vary greatly in composition and function. Nevertheless, they collaborate in the osteochondral region to create a smooth transition zone that supports the movements and forces resulting from the daily activities. In this sense, all the hierarchical organization is involved in the maintenance and reestablishment of the equilibrium in case of damage. Therefore, this interface has attracted a great deal of interest in order to understand the mechanisms of regeneration or disease progression in osteoarthritis. With that purpose, in vitro tissue models (either static or dynamic) have been studied. Static in vitro tissue models include monocultures, co-cultures, 3D cultures, and ex vivo cultures, mostly cultivated in flat surfaces, while dynamic models involve the use of bioreactors and microfluidic systems. The latter have emerged as alternatives to study the cellular interactions in a more authentic manner over some disadvantages of the static models. The current alternatives of in vitro mimetic models for bone-cartilage interface regeneration are overviewed and discussed herein.

Rapidly responsive silk fibroin hydrogels as an artificial matrix for the programmed tumor cells death.

Timely and spatially-regulated injectable hydrogels, able to suppress growing tumors in response to conformational transitions of proteins, are of great interest in cancer research and treatment. Herein, we report rapidly responsive silk fibroin (SF) hydrogels formed by a horseradish peroxidase (HRP) crosslinking reaction at physiological conditions, and demonstrate their use as an artificial biomimetic three-dimensional (3D) matrix. The proposed SF hydrogels presented a viscoelastic nature of injectable hydrogels and spontaneous conformational changes from random coil to ß-sheet conformation under physiological conditions. A human neuronal glioblastoma (U251) cell line was used for screening cell encapsulation and in vitro evaluation within the SF hydrogels. The transparent random coil SF hydrogels promoted cell viability and proliferation up to 10 days of culturing, while the crystalline SF hydrogels converted into ß-sheet structure induced the formation of TUNEL-positive apoptotic cells. Therefore, this work provides a powerful tool for the investigation of the microenvironment on the programed tumor cells death, by using rapidly responsive SF hydrogels as 3D in vitro tumor models.

Bioceramics for Osteochondral Tissue Engineering and Regeneration.

Considerable advances in tissue engineering and regeneration have been accomplished over the last decade. Bioceramics have been developed to repair, reconstruct, and substitute diseased parts of the body and to promote tissue healing as an alternative to metallic implants. Applications embrace hip, knee, and ligament repair and replacement, maxillofacial reconstruction and augmentation, spinal fusion, bone filler, and repair of periodontal diseases. Bioceramics are well-known for their superior wear resistance, high stiffness, resistance to oxidation, and low coefficient of friction. These specially designed biomaterials are grouped in natural bioceramics (e.g., coral-derived apatites), and synthetic bioceramics, namely bioinert ceramics (e.g., alumina and zirconia), bioactive glasses and glass ceramics, and bioresorbable calcium phosphates-based materials. Physicochemical, mechanical, and biological properties, as well as bioceramics applications in diverse fields of tissue engineering are presented herein. Ongoing clinical trials using bioceramics in osteochondral tissue are also considered. Based on the stringent requirements for clinical applications, prospects for the development of advanced functional bioceramics for tissue engineering are highlighted for the future.

Silk Fibroin-Based Hydrogels and Scaffolds for Osteochondral Repair and Regeneration.

Osteochondral lesions treatment and regeneration demands biomimetic strategies aiming physicochemical and biological properties of both bone and cartilage tissues, with long-term clinical outcomes. Hydrogels and scaffolds appeared as assertive approaches to guide the development and structure of the new osteochondral engineered tissue. Moreover, these structures alone or in combination with cells and bioactive molecules bring the mechanical support after in vitro and in vivo implantation. Moreover, multilayered structures designed with continuous interfaces furnish appropriate features of the cartilage and subchondral regions, namely microstructure, composition, and mechanical properties. Owing the potential as scaffolding materials, natural and synthetic polymers, bioceramics, and composites have been employed. Particularly, significance is attributed to the natural-based biopolymer silk fibroin from the Bombyx mori silkworm, considering its unique mechanical and biological properties. The significant studies on silk fibroin-based structures, namely hydrogels and scaffolds, towards bone, cartilage, and osteochondral tissue repair and regeneration are overviewed herein. The developed biomimetic strategies, processing methodologies, and final properties of the structures are summarized and discussed in depth.

Clinical Trials and Management of Osteochondral Lesions.

Osteochondral lesions are frequent and important causes of pain and disability. These lesions are induced by traumatic injuries or by diseases that affect both the cartilage surface and the subchondral bone. Due to the limited cartilage ability to regenerate and self-repair, these lesions tend to gradually worsen and progress towards osteoarthritis. The clinical, social, and economic impact of the osteochondral lesions is impressive and although therapeutic alternatives are under discussion, a consensus is not yet been achieved. Over the previous decade, new strategies based on innovative tissue engineering approaches have been developed with promising results. However, in order those products reach the market and help the actual patient in an effective manner, there is still a lot of work to be done. The current state of the implications, clinical aspects, and available treatments for this pathology, as well as the ongoing preclinical and clinical trials are presented in this chapter.

Commercial Products for Osteochondral Tissue Repair and Regeneration.

The osteochondral tissue represents a complex structure composed of four interconnected structures, namely hyaline cartilage, a thin layer of calcified cartilage, subchondral bone, and cancellous bone. Due to the several difficulties associated with its repair and regeneration, researchers have developed several studies aiming to restore the native tissue, some of which had led to tissue-engineered commercial products. In this sense, this chapter discusses the good manufacturing practices, regulatory medical conditions and challenges on clinical translations that should be fulfilled regarding the safety and efficacy of the new commercialized products. Furthermore, we review the current osteochondral products that are currently being marketed and applied in the clinical setting, emphasizing the advantages and difficulties of each one.

Natural-based nanocomposites for bone tissue engineering and regenerative medicine: a review.

Tissue engineering and regenerative medicine has been providing exciting technologies for the development of functional substitutes aimed to repair and regenerate damaged tissues and organs. Inspired by the hierarchical nature of bone, nanostructured biomaterials are gaining a singular attention for tissue engineering, owing their ability to promote cell adhesion and proliferation, and hence new bone growth, compared with conventional microsized materials. Of particular interest are nanocomposites involving biopolymeric matrices and bioactive nanosized fillers. Biodegradability, high mechanical strength, and osteointegration and formation of ligamentous tissue are properties required for such materials. Biopolymers are advantageous due to their similarities with extracellular matrices, specific degradation rates, and good biological performance. By its turn, calcium phosphates possess favorable osteoconductivity, resorbability, and biocompatibility. Herein, an overview on the available natural polymer/calcium phosphate nanocomposite materials, their design, and properties is presented. Scaffolds, hydrogels, and fibers as biomimetic strategies for tissue engineering, and processing methodologies are described. The specific biological properties of the nanocomposites, as well as their interaction with cells, including the use of bioactive molecules, are highlighted. Nanocomposites in vivo studies using animal models are also reviewed and discussed.