Chapter 22 - Musculoskeletal tissue engineering

From its inception, tissue engineering has had three tenets: cells, biomaterial scaffolds and signaling molecules. Among the triad, cells are the center piece, because cells are the building blocks of tissues. For decades, cell therapies have focused on the procurement, manipulation and delivery of healthy cells for the treatment of diseases or trauma. Given the complexity and potential high cost of cell delivery, there is recent and surging interest to orchestrate endogenous cells for tissue regeneration. Biomaterial scaffolds are vital for many but not all, tissue-engineering applications and serve to accommodate or promote multiple cellular functions. Signaling molecules can be produced by transplanted cells or endogenous cells, or delivered specifically to regulate cell functions. This review highlights recent work in tissue engineering and cell therapies, with a focus on harnessing the capacity of endogenous cells as an alternative or adjunctive approach for tissue regeneration.

Tissue engineering, as an interdisciplinary approach, is seeking to create tissues with optimal performance for clinical applications. Various factors, including cells, biomaterials, cell or tissue culture conditions and signaling molecules such as growth factors, play a vital role in the engineering of tissues. In vivo microenvironment of cells imposes complex and specific stimuli on the cells, and has a direct effect on cellular behavior, including proliferation, differentiation and extracellular matrix (ECM) assembly. Therefore, to create appropriate tissues, the conditions of the natural environment around the cells should be well imitated. Therefore, researchers are trying to develop biomimetic scaffolds that can produce appropriate cellular responses. To achieve this, we need to know enough about biomimetic materials. Scaffolds made of biomaterials in musculoskeletal tissue engineering should also be multifunctional in order to be able to function better in mechanical properties, cell signaling and cell adhesion. Multiple combinations of different biomaterials are used to improve above-mentioned properties of various biomaterials and to better imitate the natural features of musculoskeletal tissue in the culture medium. These improvements ultimately lead to the creation of replacement structures in the musculoskeletal system, which are closer to natural tissues in terms of appearance and function. The present review article is focused on biocompatible and biomimetic materials, which are used in musculoskeletal tissue engineering, in particular, cartilage tissue engineering.

Tissue engineering is rapidly progressing toward clinical application. In the musculoskeletal field, there has been an increasing necessity for bone and cartilage replacement. Despite the promising translational potential of tissue engineering approaches, careful attention should be given to the quality of developed constructs to increase the real applicability to patients. After a general introduction to musculoskeletal tissue engineering, this narrative review aims to offer an overview of methods, starting from classical techniques, such as gene expression analysis and histology, to less common methods, such as Raman spectroscopy, microcomputed tomography, and biosensors, that can be employed to assess the quality of constructs in terms of viability, morphology, or matrix deposition. A particular emphasis is given to standards and good practices (GXP), which can be applicable in different sectors. Moreover, a classification of the methods into destructive, noninvasive, or conservative based on the possible further development of a preimplant quality monitoring system is proposed. Biosensors in musculoskeletal tissue engineering have not yet been used but have been proposed as a novel technology that can be exploited with numerous advantages, including minimal invasiveness, making them suitable for the development of preimplant quality control systems.

The field of tissue engineering continues to advance with the discovery of new biomaterials, growth factors and scaffold fabrication techniques. However, for the ultimate success of a tissue engineered construct the issue of nutrient transport to the scaffold interior needs to be addressed. Often, the requirements for adequate nutrient supply are at odds with other scaffold design parameters such as mechanical properties as well as scaffold fabrication techniques, leading to incongruities in finding optimal solutions. The goal of this review article is to provide an overview of the various engineering design factors that promote movement of nutrients, waste and other biomolecules in scaffolds for musculoskeletal tissue engineering applications. The importance of diffusion in scaffolds and how it is influenced by porosity, permeability, architecture, and nutrient mixing has been emphasized. Methods for measuring porosity and permeability have also been outlined. The different types of biomaterials used, scaffold fabrication techniques implemented and the pore sizes/porosities obtained over the past 5 years have also been addressed.

Tissue grafting is mostly used for repair and replacement of severely damaged tissues, the key challenges are compatibility, availability of the grafts, complex surgical process and post-operative complications. Hence, additive technologies such as three-dimensional (3D) bioprinting have emerged as promising alternative for tissue engineering in order to ensure safety, compatibility, and rapid healing. The aim of this chapter is to give an elaborate account of 3D printed scaffolds for bone, cartilage, cardio-vascular and nerve tissue engineering. Various components such as polycaprolactone, poly (lactic-co-glycolic acid), and β-tricalcium phosphate, bioglass 45S5, and nano-hydroxyapatite are combined with collagen and its derivatives to achieve specific pore size in the scaffolds for effective restoration of the defects of soft or hard tissues. Likewise, proanthocyanidin, oxidized hyaluronic acid, methacrylated gelatin, are used in collagen based 3D printed scaffolds for cartilage tissue engineering. Bioink with collagen as active component is also used for developing cardio-vascular implants with recellularizing properties. Collagen in combination with silk fibroin, chitosan, heparin sulphate and others are ideal for fabrication of elastic nerve guidance conduits. In view of the background, collagen-supplemented hydrogels can revolutionize future biomedical approaches for the development of complex scaffolds for tissue engineering.

Chitin, the second most abundant natural biomaterial composed of β(1→4)-linked 2-acetamido-2-deoxy-β-d-glucose units (or N-acetylglucosamine units), is well recognized as a substance providing many valuable biological properties, namely, biocompatibility, hemostasis, and antibacterial/anti-infectious property (Jayakumar et al., 2011). It is a fairly cytocompatible material that promotes the attachment and spreading of diverse cells such as normal human keratinocytes, broblasts, osteoblasts, and many more. Chitin is highly biodegradable in nature due to the activity of lysozymes present in the human body (Kurita et al., 2000; Noh et al., 2006). It is being used in the fabrication of a variety of medical devices and regenerative medical components due to its high crystallinity, biochemical signicance, and biocompatibility (Yimin et al., 2008). The excellent and unique properties of chitin offer a large and active surface area, making it suitable for different applications, and hence, these properties are being explored in the elds of engineering, technology, and medicine (Muzzarelli, 2011). The main disadvantage of chitin is its insolubility in organic solvents and acids. However, chitin is soluble in saturated CaCl2-methanol solvent system (Tamura et al., 2011). Using this solvent system, chitin solution was prepared (Tamura et al., 2011). This chitin solution can be easily regenerated into gel or nanogel by the addition of water/methanol (Sanoj Rejinold et al., 2011, 2012; Tamura et al., 2011). Chitin hydrogel is subjected to lyophilization to obtain chitin scaffolds (Madhumathi et al., 2010; Jayakumar et al., 2011;CONTENTS34.1 Introduction ........................................................................................................................ 673 34.2 Chitin Nanobers in Tissue Engineering and Wound Dressing ................................ 674 34.3 Chitin Nanocomposite Scaffolds in Tissue Engineering and Wound Healing ........ 675 34.4 Chitin Nanoparticles in Drug Delivery .......................................................................... 676 34.5 Chitin Nanogels in Drug Delivery and Imaging .......................................................... 676 34.6 Conclusions ......................................................................................................................... 678 Acknowledgments ...................................................................................................................... 678 References ..................................................................................................................................... 678Sowmya et al., 2011; Tamura et al., 2011). This chapter will focus on the advantages of chitin nanobers, nanogels, and nanocomposite scaffolds in drug delivery, tissue engineering, and wound healing in detail.

The research on tissue engineering applications has been progressing to manufacture ideal tissue scaffold biomaterials. In this study, a double-layered electrospun biofiber scaffold biomaterial including Polycaprolactone (PCL)/Collagen (COL) fibrous inner layer and PCL/ Momordica charantia (MC) and Hypericum perforatum (HP) oils fibrous outer layer was developed to manufacture a functional, novel tissue scaffold with the advantageous mechanical and biological properties. The main approach was to combine the natural perspective using medicinal oils with an engineering point of view to fabricate a potential functional scaffold for tissue engineering. Medicinal plants MC and HP are rich in functional oils and incorporation of them in a tissue scaffold will unveil their potential to augment both new tissue formation and wound healing. In this study, a novel double-layered scaffold prototype was fabricated using electrospinning technique with two PCL fiber layers, first is composed of collagen, and second is composed of oils extracted from medicinal plants. Initially, the composition of plant oils was analyzed. Thereafter the biofiber scaffold layers were fabricated and were evaluated in terms of morphology, physicochemistry, thermal and mechanical features, wettability, in vitro bio-degradability. Double-layered scaffold prototype was further analyzed in terms of in vitro biocompatibility and antibacterial effect. The medicinal oils blend provided antioxidant and antibacterial properties to the novel PCL/Oils layer. The results signify that inner PCL/COL layer exhibited advanced biodegradability of 8.5% compared to PCL and enhanced wettability with 11.7° contact angle. Strength of scaffold prototype was 5.98 N/mm2 thanks to the elastic PCL fibrous matrix. The double-layered functional biofiber scaffold enabled 92% viability after 72 h contact with fibroblast cells and furthermore provided feasible attachment sites for the cells. The functional scaffold prototype's noteworthy mechanical, chemical, and biological features enable it to be suggested as a different novel biomaterial with the potential to be utilized in tissue engineering applications.

Tissue engineering (TE) is an emerging technology that combines expertise in life sciences, clinical medicine, and engineering. Current challenges in TE include anticipating and streamlining appropriate regulation with product development. Consequently this study has focused on views of developers, companies, and regulators on biological risks of TE products, aspects of commercial applications, and patentability. Most concerns about TE products focus on risk of cancer formation, infection risk, and rejection risk. Thus, at the present time, product developers should follow guidelines for medicinal products, in order to address product safety at an adequate level. According to the data of the present study, cell and biomaterial products, manipulated cells, and scaffolds appear to be the primary interest for commercial product development. In contrast, producing services were not considered as interesting. Constraints are also imposed by patentability, which stipulates demands for technical performance and highlights ethical issues, which are difficult to address.

Matrices and scaffolds for DNA delivery in tissue engineering

Calcium phosphate ceramics have been widely considered as scaffolds for bone tissue engineering. Selection of the best support for cultured cells, crucial for tissue engineered systems, is still required. We examined three types of calcium phosphate compounds: α-tricalcium phosphate - the most soluble one, carbonate hydroxyapatite - chemically the most similar to the bone mineral and biphasic calcium phosphate - with the best in vivo biocompatibility in order to select the best support for osteoblastic cells for tissue engineered systems. Human osteoblasts were tested in direct contact with both dense samples and 3D scaffolds in either static or dynamic culture. Cell viability, cell spreading, osteogenic cell capacity, and extracellular matrix production were examined. The obtained data indicate that biphasic calcium phosphate is the optimal cell-supporting material. In addition, dynamic culture improved cell distribution in the scaffolds, enhanced production of the extracellular matrix and promoted cells osteogenic capacity. Biphasic calcium phosphate should be recommended as the most suitable matrix for osteogenic cells expansion and differentiation in tissue engineered systems.

Purpose– The purpose of this paper is to review the current status of additive manufacturing (AM) used for tissue engineering (TE) scaffold. AM processes are identified as an effective method for fabricating geometrically complex objects directly from computer models or three-dimensional digital representations. The use of AM technologies in the field of TE has grown rapidly in the past 10 years.Design/methodology/approach– The processes, materials, precision, applications of different AM technologies and their modified versions used for TE scaffold are presented. Additionally, future directions of AM used for TE scaffold are also discussed.Findings– There are two principal routes for the fabrication of scaffolds by AM: direct and indirect routes. According to the working principle, the AM technologies used for TE scaffold can be generally classified into: laser-based; nozzle-based; and hybrid. Although a number of materials and fabrication techniques have been developed, each AM technique is a process based on the unique property of the raw materials applied. The fabrication of TE scaffolds faces a variety of challenges, such as expanding the range of materials, improving precision and adapting to complex scaffold structures.Originality/value– This review presents the latest research regarding AM used for TE scaffold. The information available in this paper helps researchers, scholars and graduate students to get a quick overview on the recent research of AM used for TE scaffold and identify new research directions for AM in TE.

Over the past 20 years, tissue engineering (TE) has evolved into a thriving research and commercial development field. However, applying TE strategies to musculoskeletal (MSK) and craniofacial tissues has been particularly challenging since these tissues must also transmit loads during activities of daily living. To address this need, organizers invited a small group of bioengineers, surgeons, biologists, and material scientists from academia, industry, and government to participate in a two and half-day conference to develop general and tissue-specific criteria for evaluating new concepts and tissue-engineered constructs, including threshold values of success. Participants were assigned to four breakout groups representing commonly injured tissues, including tendon and ligament, articular cartilage, meniscus and temporomandibular joint, and bone and intervertebral disc. Working in multidisciplinary teams, participants first carefully defined one or two important unmet clinical needs for each tissue type, including current standards of care and the potential impact of TE solutions. The groups then sought to identify important parameters for evaluating repair outcomes in preclinical studies and to specify minimally acceptable values for these parameters. The importance of in vitro TE studies was then discussed in the context of these preclinical studies. Where data were not currently available from clinical, preclinical, or culture studies, the groups sought to identify important areas of preclinical research needed to speed the development process. This report summarizes the findings of the conference.

Regenerative medicine and tissue engineering strategies aim to provide tissue substitutes for repair of defects due to disease, trauma, or congenital abnormalities. Biomaterials play a key role in design strategies in tissue engineering functioning to provide a three dimensional environment for cells, present biological cues, and implant in a physiological milieu. Basic research topics including stem cell biology, small molecule therapeutics, and metabolism in combination with biomaterials research in regenerative medicine will be discussed. Finally, translation of key technologies to clinical application in musculoskeletal and soft tissue reconstruction will be presented.

Polyhedral oligomeric silsesquioxane (POSS) is a unique molecule that is composed of an inorganic silica core with eight organic functional arms, which can be used as a nucleus for covalent bonding to create star-shaped block copolymers with improved mechanical and biological properties. In this work, highly porous star-shaped POSS-polycaprolactone-polyurethane (POSS-PCL-PU) films were synthesized as scaffold biomaterials for tissue engineering. These films have an interesting morphology consisting of rough spherulites with filamentous structures spreading out from their centers; the unique nanotopography was shown to be suitable for cell growth. In vitro degradation was monitored for 52 weeks in terms of the weight loss and morphology changes of the films. The degradation profile exhibited a slow initial weight loss of <1% during the first 24 weeks, followed by a rapid weight loss of ~18% in the following 28 weeks. The films demonstrated excellent biocompatibility and cell-substrate affinity, with a high cell viability of >95% and rapid cell proliferation. The high porosity, unique surface nanotopography and excellent biocompatibility of the star-shaped POSS-PCL-PU film make it a great candidate as a tissue engineering scaffold biomaterial. Researchers in Singapore have prepared highly porous films that are very promising as scaffold biomaterials for tissue engineering. They synthesized the films by using star-shaped polyhedral oligomeric silsesquioxane as the starting inorganic core and incorporating polycaprolactone (PCL) and polyurethane (PU). The researchers, who are from the Institute of Materials Research and Engineering and the National University of Singapore, demonstrate that the inorganic-organic nanocomposite films satisfy all the key criteria for a biodegradable scaffold biomaterial — they have an excellent biocompatibility, a high porosity and a controlled and slow degradability. In addition, the scientists show that the material's unique nanotopography, which is produced by filamentous structures branching out from the centres of rough spherulites, promotes cell growth. The combination of organic and inorganic functionalities makes these films superior to conventional PCL-PU films. Highly porous star-shaped polyhedral oligomeric silsesquioxane (POSS) hybrid films were synthesized using POSS as the starting core followed by polycaprolactone (PCL) extension and polyurethane (PU) cross-linking. The unique three-dimensional nanotopography of these films is conducive for cell growth. The combination of favorable factors such as high porosity, reactive surface topography, excellent biocompatibility and biphasic degradation makes the star-shaped POSS-PCL-PU film a great candidate as a tissue engineering scaffold biomaterial.

Osteonecrosis of the femoral head (ONFH) is defined as a tissue disorder and successive subchondral bone collapse resulting from an ischemic process, which may progress to hip osteoarthritis. Cell therapy with multipotent bone marrow mesenchymal stromal cells (BM-MSC) of autologous origin appears to be safe and has shown regenerative potential in previous preclinical and clinical studies. The use of allogeneic cells is far more challenging, but may be a promising alternative to use of autologous cells. Moreover, an optimized dosage of cells from an allogeneic source is needed to obtain off-the-shelf tissue engineering products (TEPs). The purpose of this study was to evaluate the efficacy of a TEP composed of undifferentiated ex vivo expanded BM-MSC of allogeneic origin, combined with bone matrix particles in variable doses. A comparative analysis of TEP's bone regenerative properties against its autologous counterpart was performed in an early-stage ONFH preclinical model in mature sheep. Allogeneic BM-MSC groups demonstrated bone regeneration capacity in osteonecrotic lesions equivalent to autologous BM-MSC groups 6 weeks after treatment. Likewise, stimulation of bone regeneration by a low cell dose of 0.5 × 106 BM-MSC/cm3 was equivalent to that of a high cell dose, 5 × 106 BM-MSC/cm3. Neither local nor systemic immunological reactions nor tumorigenesis were reported, strengthening the safety profile of allogeneic BM-MSC therapy in this model. Our results suggest that low-dose allogeneic BM-MSC is sufficient to promote bone regeneration in femoral head osteonecrotic lesions, and should be considered in translation of new allogeneic cell-based TEPs to human clinics. Impact statement Cell therapy and tissue engineering hold promise as novel regenerative therapies for musculoskeletal diseases, and particularly in bone regeneration strategies. In this article, we report the evaluation of the efficacy of an allogeneic cell-based tissue engineering product (TEP) in an early-stage osteonecrosis of the femoral head preclinical model in skeletally mature sheep. Moreover, we demonstrate its bone regeneration capacity and safety in vivo and its equivalence to autologous counterparts. These findings have important implications for the translation of new allogeneic cell-based TEPs to human clinics.