Nanocomposite bioinks for 3D bioprinting

Tremendous advances in tissue engineering and regenerative medicine have revealed the potential of fabricating biomaterials to solve the dilemma of bone and articular defects by promoting osteochondral and cartilage regeneration. Three-dimensional (3D) bioprinting is an innovative fabrication technology to precisely distribute the cell-laden bioink for the construction of artificial tissues, demonstrating great prospect in bone and joint construction areas. With well controllable printability, biocompatibility, biodegradability, and mechanical properties, hydrogels have been emerging as an attractive 3D bioprinting material, which provides a favorable biomimetic microenvironment for cell adhesion, orientation, migration, proliferation, and differentiation. Stem cell-based therapy has been known as a promising approach in regenerative medicine; however, limitations arise from the uncontrollable proliferation, migration, and differentiation of the stem cells and fortunately could be improved after stem cells were encapsulated in the hydrogel. In this review, our focus was centered on the characterization and application of stem cell-laden hydrogel-based 3D bioprinting for bone and cartilage tissue engineering. We not only highlighted the effect of various kinds of hydrogels, stem cells, inorganic particles, and growth factors on chondrogenesis and osteogenesis but also outlined the relationship between biophysical properties like biocompatibility, biodegradability, osteoinductivity, and the regeneration of bone and cartilage. This study was invented to discuss the challenge we have been encountering, the recent progress we have achieved, and the future perspective we have proposed for in this field.

Biomaterial-based 3D bioprinting strategy for orthopedic tissue engineering.

212Three-dimensional (3D) printing is a modern, computer-aided, design-based technology that allows the layer-by-layer deposition of 3D structures. Bioprinting, a 3D printing technology, has attracted increasing attention because of its capacity to produce scaffolds for living cells with extreme precision. Along with the rapid development of 3D bioprinting technology, the innovation of bio-inks, which is recognized as the most challenging aspect of this technology, has demonstrated tremendous promise for tissue engineering and regenerative medicine. Cellulose is the most abundant polymer in nature. Various forms of cellulose, nanocellulose, and cellulose derivatives, including cellulose ethers and cellulose esters, are common bioprintable materials used to develop bio-inks in recent years, owing to their biocompatibility, biodegradability, low cost, and printability. Although various cellulose-based bio-inks have been investigated, the potential applications of nanocellulose and cellulose derivative-based bio-inks have not been fully explored. This review focuses on the physicochemical properties of nanocellulose and cellulose derivatives as well as the recent advances in bio-ink design for 3D bioprinting of bone and cartilage. In addition, the current advantages and disadvantages of these bio-inks and their prospects in 3D printing-based tissue engineering are comprehensively discussed. We hope to offer helpful information for the logical design of innovative cellulose-based materials for use in this sector in the future.

Recently, tissue engineering and regenerative medicine, especially bone tissue engineering and regenerative medicine have developed rapidly. Bone tissue engineering is a complicated and dynamic bone remodeling process that starts with recruitment and migration of osteoblasts and then regulates their proliferation, differentiation, and mineralization. Scaffold is typically biodegradable material that plays a crucial role as one of the three elements of bone tissue engineering and regenerative medicine. Bone scaffold provides the mechanical support during reformation and repair of damaged or diseased bone. For more than a decade, a large amount of research work has been carried out on the designing of scaffolds, processing techniques, performance assessment, and medical application. Significant progress has been made toward scaffold materials for structural support for desired osteogenesis and angiogenesis abilities. To date, depending on the advanced technologies, the bioresorbable scaffolds possess controlled porosity and high mechanical properties are possible used as bone scaffold. Natural bone has delicate architectural structure that spans nanoscale to macroscopic dimensions, which determine the unique mechanical properties of bone. The hierarchical structure of natural bone can be defined as a nanocomposite consisting of inorganic nanocrystalline hydroxyapatite (HA), organic components (mostly are collagens) and water. The outside proteins assemble together to form an extracellular matrix (ECM) with nanostructured which influences the adhesion, proliferation, differentiation, and mineralization of several cell types, such as bone lining cell, mesenchymal stem cells, osteoblasts, osteocytes, and osteoclasts. To better mimic the nanostructure in natural ECM, many research groups over the past decade developed nano-based scaffolds (nanotubes, nanofibers, nanoparticles, and hydrogel) to resemble the ECM for promising candidates in replacing defective tissues. The high biomimetic properties and excellent mechanical features of nano-based scaffolds plays a vital role in stimulating cell adhesion and proliferation, as well as directing bone tissue formation through mimetic the nanometer dimension of natural bone tissue. Besides, mechanical properties are key factors for bone regeneration and bone local function. Nowadays, a large number of research groups have tried to manipulate the mechanical properties of scaffolds through designing the different nanosturctures, including nanofiber, nanoparticle, polymer matrices to mimic the nanocomposite structure of natural bone. These materials have highly stiffness, strength, and toughness properties that are promising in bone tissue engineering. Nanobiomaterials, especially inorganic nanobiomaterials, have excellent mechanical properties and biocompatibility, and are ideal for the preparation of scaffold materials for bone tissue engineering and regenerative medicine, showing a broad application prospect. In this article, we systematically review the application of inorganic nanomaterials, including hydroxyapatite, silicon-based nanomaterials, carbon-containing nanomaterials and several metal nanomaterials in bone tissue engineering and regenerative medicine. Although nanotechnology represents a major frontier with potential to significantly advance the field of bone tissue engineering, the application in the clinical regenerative engineering has limited. Currently, there are several limitations in regenerative strategies, including low efficient cellular proliferation, differentiation and mineralization, insufficient mechanical strength of scaffolds, and limited production of growth factors necessary for efficient osteogenesis. Therefore, there are still some issues on the nano-based scaffold should be cleared: (1) The interaction of nano-based scaffold and bioactive molecules, growth factors, and genetic material, (2) the biocomptibility, cytotoxicity, and biodegradation of nano-based scaffold, and (3) nano-based scaffold mechanical stability, cellular survival, and molecular mechanism of inducing bone formation. As the development of nanotechnology, ultimate translation to the clinical application may allow for improved therapeutic outcomes in patients with large bone deficits and osteodegenerative diseases.

In the era of personalised medicine, novel therapeutic approaches raise increasing hopes to address currently unmet medical needs by developing patient-customised treatments. Three-dimensional (3D) bioprinting is rapidly evolving and has the potential to obtain personalised tissue constructs and overcome some limitations of standard tissue engineering approaches. Bioprinting could support a wide range of biomedical applications, such as drug testing, tissue repair or organ transplantation. There is a growing interest for 3D bioprinting in the orthopaedic field, with remarkable scientific and technical advances. However, the full exploitation of 3D bioprinting in medical applications still requires efforts to anticipate the upcoming challenges in translating bioprinted products from bench to bedside. In this review we summarised current trends, advances and challenges in the application of 3D bioprinting for bone and cartilage tissue engineering. Moreover, we provided a detailed analysis of the applicable regulations through the 3D bioprinting process and an overview of available standards covering bioprinting and additive manufacturing.

Methacrylated gelatin (GelMA) has been widely used as a tissue-engineered scaffold material, but only low-concentration GelMA hydrogels were found to be promising cell-laden bioinks with excellent cell viability. In this work, we reported a strategy for precise deposition of 5% (w/v) cell-laden GelMA bioinks into controlled microarchitectures with high cell viability using extrusion-based three-dimensional (3D) bioprinting. By adding gelatin into GelMA bioinks, a two-step cross-linking combining the rapid and reversible thermo-cross-linking of gelatin with irreversible photo-cross-linking of GelMA was achieved. The GelMA/gelatin bioinks showed significant advantages in processability because the tunable rheology and the rapid thermo-cross-linking of bioinks improved the shape fidelity after bioprinting. Here, the rheology, mechanical properties, and swelling of GelMA/gelatin bioinks with different concentration ratios were carefully characterized to obtain the optimized bioprinting setup. We successfully printed the 5% (w/v) GelMA with 8% (w/v) gelatin into 3D structures, which had the similar geometrical resolution as that of the structures printed by 30% (w/v) GelMA bioinks. Moreover, the cell viability of 5/8% (w/v) GelMA/gelatin bioinks was demonstrated by in vitro culture and cell printing of bone marrow stem cells (BMSCs). Larger BMSC spreading area was found on 5/8% (w/v) GelMA/gelatin scaffolds, and the BMSC viability after the printing of 5/8% (w/v) GelMA/gelatin cell-laden bioinks was more than 90%, which was very close to the viability of printing pure 5% (w/v) GelMA cell-laden bioinks. Therefore, this printing strategy of GelMA/gelatin bioinks may extensively extend the applications of GelMA hydrogels for tissue engineering, organ printing, or drug delivery.

Polysaccharide hydrogels, which can mimic the natural extracellular matrix and possess appealing physicochemical and biological characteristics, have emerged as significant bioinks for 3D bioprinting. They are highly promising for applications in tissue engineering and regenerative medicine because of their ability to enhance cell adhesion, proliferation, and differentiation in a manner akin to the natural cellular environment. This review comprehensively examines the fabrication methods, characteristics, and applications of polysaccharide hydrogel-driven 3D bioprinting, underscoring its potential in tissue engineering, drug delivery, and regenerative medicine. To contribute pertinent knowledge for future research in this field, this review critically examines key aspects, including the chemistry of carbohydrates, manufacturing techniques, formulation of bioinks, and characterization of polysaccharide-based hydrogels. Furthermore, this review explores the primary advancements and applications of 3D-printed polysaccharide hydrogels, encompassing drug delivery systems with controlled release kinetics and targeted therapy, along with tissue-engineered constructs for bone, cartilage, skin, and vascular regeneration. The use of these 3D bioprinted hydrogels in innovative research fields, including disease modeling and drug screening, is also addressed. Despite notable progress, challenges, including modulating the chemistry and properties of polysaccharides, enhancing bioink printability and mechanical properties, and achieving long-term in vivo stability, have been highlighted.

Chapter 12 - Cartilage 3D Printing

This study aimed to address the dilemma of low peripheral blood-derived mesenchymal stromal cell (PBMSC) activity and reduced phenotype in bone or cartilage tissue engineering. Rat PBMSCs (rPBMSCs) were obtained by density gradient centrifugation, and stromal cell characteristics were confirmed by flow cytometry (FCM) and multi-differentiation potential induction experiments. Cell growth curve, viability experiments, and clone formation experiments were performed by [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] (MTS) and cell counting, and the cell cycle was confirmed by cell FCM. The proliferation signal pathway and stemness-related proteins were detected by molecular methods including Western blot and real-time polymerase chain reaction. CD73, CD90, and CD105 were highly expressed, and CD14, CD19, CD34, CD45, and HLA-DR were barely expressed in rPBMSCs. rPBMSCs possessed the potential to differentiate into chondrocytes, adipocytes, and osteoblasts under their respective induction conditions. Cell growth curve and viability experiments were performed under hypoxic conditions: 19% O2, 5% O2, and 1% O2. Specifically, 5% O2 accelerated the proliferation and expression of the stemness of PBMSCs. Cycle experiments proved that hypoxia promoted the cell transition from the G1 phase to the S phase. Molecular experiments confirmed that 5% O2 hypoxia significantly elevated the expressions of hypoxia-inducible factor 1α and β-catenin and simultaneously the expressions of cycle-related genes including CyclinE/CDK2 and stemness-related genes including Nanog and SOX2. The appropriate concentration of hypoxia (i.e., 5% O2) enhanced the proliferation and stemness of rPBMSCs and increased the multidirectional differentiation potential of stromal cells. The proposed culture method could improve the viability and maintain the phenotype of rPBMSCs in cartilage or bone tissue engineering.

Chapter 12 - Impacts of nanotechnology in tissue engineering

The introduction of 3D bioprinting is expected to revolutionize the field of tissue engineering and regenerative medicine. The 3D bioprinter is able to dispense materials while moving in X, Y, and Z directions, which enables the engineering of complex structures from the bottom up. In this study, a bioink that combines the outstanding shear thinning properties of nanofibrillated cellulose (NFC) with the fast cross-linking ability of alginate was formulated for the 3D bioprinting of living soft tissue with cells. Printability was evaluated with concern to printer parameters and shape fidelity. The shear thinning behavior of the tested bioinks enabled printing of both 2D gridlike structures as well as 3D constructs. Furthermore, anatomically shaped cartilage structures, such as a human ear and sheep meniscus, were 3D printed using MRI and CT images as blueprints. Human chondrocytes bioprinted in the noncytotoxic, nanocellulose-based bioink exhibited a cell viability of 73% and 86% after 1 and 7 days of 3D culture, respectively. On the basis of these results, we can conclude that the nanocellulose-based bioink is a suitable hydrogel for 3D bioprinting with living cells. This study demonstrates the potential use of nanocellulose for 3D bioprinting of living tissues and organs.

Nanoparticle-based bioactive agent release systems for bone and cartilage tissue engineering.

Osteoarthritis (OA) can arise from various factor including trauma, overuse, as well as degeneration resulting from age or disease. The specific treatment options will vary based on the severity of the condition, and the affected joints. Some common treatments for OA include lifestyle modifications, medications, physical therapy, surgery and tissue engineering (TE). For cartilage tissue engineering (CTE), three-dimension (3D) scaffolds are made of biocompatible natural polymers, which allow for the regeneration of new cartilage tissue. An ideal scaffold should possess biological and mechanical properties that closely resemble those of the cartilage tissue, and lead to improved functional of knee. These scaffolds are specifically engineered to serve as replacements for damaged and provide support to the knee joint. 3D-bioprinted scaffolds are made of biocompatible materials natural polymers, which allow for the regeneration of new cartilage. The utilization of 3D bioprinting method has emerged as a novel approach for fabricating scaffolds with optimal properties for CTE applications. This method enables the creation of scaffolds that closely mimic the native cartilage in terms of mechanical characteristics and biological functionality. Alginate, that has the capability to fabricate a cartilage replacement customized for each individual patient. This polymer exhibits hydrophilicity, biocompatibility, and biodegradability, along with shear-thinning properties. These unique properties enable Alginate to be utilized as a bio-ink for 3D bioprinting method. Furthermore, chondrogenesis is the complex process through which cartilage is formed via a series of cellular and molecular signaling. Signaling pathway is as a fundamental mechanism in cartilage formation, enhanced by the incorporation of biomolecules and growth factors that induce the differentiation of stem cells. Accordingly, ongoing review is focusing to promote of 3D bioprinting scaffolds through the utilization of advanced biomolecules-loading of Alginate-based that has the capability to fabricate a cartilage replacement tailored specifically to each patient's unique needs and anatomical requirements.

Abstract3D bioprinting is the most popular additive manufacturing method, in which solid objects are constructed by depositing several layers in sequence for the fabrication of biostructures. Bioprinted structures, like tissues, organs, and Organ-on-chip, in 3D, are now widely used to study and understand the functions of the human body. The 3D bioprinted structures are closely similar to naturally occurring biologics systems, and studies performed on 3D biostructures can be more biologically relevant than in vitro studies conducted in 2D. 3D bioprinting has advanced over the years and is commonly used in tissue engineering and regenerative medicine. The fabrication of biomaterials like cells, tissues, and organs using 3D bioprinting is becoming an alternative and favorable tissue and organ transplantation approach. Bioprinting is a relatively novel method and holds great promise but has several challenges and limitations. To this end, this chapter summarizes the concept of 3D bioprinting, bioinks and their classification, different methods of bioprinting, and their applications in areas of health, pharmaceutical science, and biomedical engineering. The chapter also highlights the challenges associated with the clinical utilization of 3D bioprinting. 3D bioprinting and its applications in personalized medicine and tissue bioengineering are continuously growing. In the future, this technology could provide advanced tools to the researchers to develop targeted treatment and improve patient outcomes.Keywords3D bioprintingTissue engineeringRegenerative medicineBioinksPharmaceuticalsMedical devicesOrgan-on-chip

The characteristics of the umbilical cord extracellular matrix are discussed relatively of their potential use for tissue engineering. The purpose of this review is to assess the current knowledge about using of homologous biomaterials with regenerative properties to create bioengineered structures. One of the most important components of tissue engineering - matrix (scaffold), resident cells can migrate, attach to it and function. Due to their structure, matrices should be easily integrated into the patient’s tissue and provide conditions for cell growth and differentiation. The cells that populate the matrix in the bioreactor before the transplantation of this construction, or resident cells recruited into the transplanted extracellular matrix), and cell- matrix interactions are absolutely necessary components of tissue engineering. Available commercial bioengineering products made from mammalian tissues have certain advantages and significant disadvantages due to the risks of immunological reactions and transmission of infectious agents. The transplantation of products from xenogenic materials is prohibited by law in the Russian Federation. The donor material is limited, receipt of human cadaver material requires a long period of legal registration, which has a detrimental effect on the biomaterial. Therefore, finding a suitable homologous biomaterial is ongoing. Due to the peculiarities of the embryonic phenotype, extraembryonic tissues have special biological properties, one of which is the scarless healing of wounds. Low immunogenicity, optimal mechanical properties of extracellular matrix, presence of cell adhesion molecules and growth factors promoting regeneration provide anti-inflammatory, anti-fibrotic, anti- scarring properties for tissue engineering structures from umbilical cord and amniotic membrane. Umbilical cord and amnion due to the availability and non-invasiveness of obtaining from healthy young donors are an excellent source of homologous biomaterial for extracting matrices, cells and hydrogels for tissue engineering and regenerative medicine.