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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.

Biofabrication is enriching the tissue engineering field with new ways of producing structurally organized complex tissues. Among the numerous bioinks under investigation, hyaluronic acid (HA) and its derivatives stand out for their biological relevance, cytocompatibility, shear-thinning properties, and potential to fine-tune the desired properties with chemical modification. In this paper, we review the recent advances on bioinks containing HA. The available literature is presented based on subjects including the rheological properties in connection with printability, the chemical strategies for endowing HA with the desired properties, the clinical application, the most advanced preclinical studies, the advantages and limitations in comparison with similar biopolymer-based bioinks, and future perspectives.

International audience; A platform of enzymatically-crosslinked Collagen/Tyramine hyaluronan derivative (Col/HA-Tyr) hydrogels with tunable compositions and gelation conditions was developed to evaluate the impact of the preparation conditions on their physical, chemical and biological properties. At low HA-Tyr content, hydrogels exhibited a fibrillar structure, with lower mechanical properties compared to pure Col hydrogels. At high HA-Tyr and Horse Radish Peroxydase (HRP) content, a microfibrillar network was formed beside the banded Col fibrils and a synergistic effect of the hybrid structure on mechanical properties was observed. These hydrogels were highly resistant against enzymatic degradation while keeping a high degree of hydration. Unlike HA-Tyr hydrogels, encapsulation of human dermal fibroblasts within Col/HA-Tyr hydrogels allowed for high cell viability. These results show that high HA-Tyr and HRP concentrations are required to positively impact the physical properties of hydrogels while preserving collagen fibrils. Those Col/HA-Tyr hydrogels appear promising for novel tissue engineering applications following a biomimetic approach.

Morphogenesis, a complex process, ubiquitous in developmental biology and many pathologies, is based on self-patterning of cells. Spatial patterns of cells, organoids, or inorganic particles can be forced on demand using acoustic surface standing waves, such as the Faraday waves. This technology allows tuning of parameters (sound frequency, amplitude, chamber shape) under contactless, fast and mild culture conditions, for morphologically relevant tissue generation. We call this method Sound Induced Morphogenesis (SIM). In this work, we use SIM to achieve tight control over patterning of endothelial cells and mesenchymal stem cells densities within a hydrogel, with the endpoint formation of vascular structures. Here, we first parameterize our system to produce enhanced cell density gradients. Second, we allow for vasculogenesis after SIM patterning control and compare our controlled technology against state-of-the-art microfluidic culture systems, the latter characteristic of pure self-organized patterning and uniform initial density. Our sound-induced cell density patterning and subsequent vasculogenesis requires less cells than the microfluidic chamber. We advocate for the use of SIM for rapid, mild, and reproducible morphogenesis induction and further explorations in the regenerative medicine and cell therapy fields.

Biofabrication via three-dimensional printing (3DP) is expanding our capabilities of producing tissue engineering constructs for regenerative medicine, personalized medicine, and engineered tissue models of disease and diagnostics. Hydrogel-based materials for extrusion-based printing have been introduced; nevertheless, it is still challenging to combine into a single biomaterial all the requirements of an ink. These inks need to flow for extrusion under low shear, yet have immediate shape retention after deposition, provide a biochemical environment similar to that of physiological extracellular matrix, and a curing mechanism avoiding cell damage. This work introduces a simple and versatile tyramine-modified hyaluronan material (HA-Tyr) for extrusion-based printing, featured by (i) single component yet two distinct cross-linking mechanisms, allowing (ii) shear-thinning tuning independently of the postprinting curing; (iii) no rheological additives or sacrificial components; (iv) curing with visible light for shape stability; (v) possibility to postfunctionalize; and (vi) preservation of hyaluronan structure owing to low modification degree. The ink is based on a hydroxyphenol hyaluronan derivative, where the shear thinning properties are determined by the enzymatic cross-linking, while the final shape fixation is achieved with visible light in the presence of Eosin Y as photosensitizer. The two cross-linking mechanisms are totally independent. A universal rheologically measurable parameter giving a quantitative measure of the "printability" was introduced and employed for identifying best printability range within the parameter space in a quantitative manner. 3DP constructs were postfunctionalized, and cell-laden constructs were produced. Due to its simplicity and versatility, HA-Tyr can be used for producing a wide variety of 3D printing constructs for tissue engineering applications.

Hyaluronic acid (HA) is a non-sulfated glycosaminoglycan. Ubiquitous in the human body, this natural polymer is widely used in the biomedical research thanks to its unique chemical, physical and biological properties [1-3]. Over forty years of use in clinics makes it one of the most successfully naturally-derived polymers in the medical field. The versatility of the HA processing and its unique biological interaction with cells, make it an important building block for the development of new biofunctional materials. HA biomedical applications are related to its physicochemical and biological properties. The first biomedical applications of HA have been as aid in eye surgery [4] and as viscosupplement in osteoarthritis [5]. Not surprising, both applications are connected with its viscoelastic properties, which can be modulated with concentration, molecular weight or chemical modification for creating semi-synthetic derivatives giving physical or covalent gels [6, 7]. The HA chemical groups available for modifications are carboxyl, hydroxyl and N-acetyl group. Chemical alterations are numerous and enable the synthesis of a wide range of HA derivatives targeting applications in the field of tissue engineering and regenerative medicine [8-10]. It is possible to create HA derivatives retaining the cyto- and bio-compatibility of the pristine HA, while having modified mechanics, degradation and interactions with biologics such as cells and proteins. Still, HA derivatives synthesis methods that employ more simple and controlled chemistries, with less toxic by-products to ensure biological biocompatibility of the conjugation and further chemical functionalization are required. This is especially true for the delivery of active biological agents and cells through advanced fabrication technologies. One of the most widespread chemical modification on the HA carboxyl group is the amide formation by carbodiimide chemistry [11]. This is usually performed in presence of 1-ethyl-3-[3-(dimethylamino)-propyl]-carbodiimide (EDC) and N-hydroxylsuccinamide (NHS). This carbodiimide conjugation presents the advantage of being performed in water with no significant cleavage of the HA chains, however, the reaction requires high quantities of reagents and it is strongly pH-dependent [10]. An alternative for the activation of the carboxyl groups is the use of the triazine-chemistry. 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) has shown to promote the grafting of amino groups on HA showing higher efficiency and better control compared to the carbodiimide-mediated reaction [12, 13]. The simplicity of the DMTMM chemistry prompted the interest in investigating a range of HA derivatives for applications in drug delivery, tissue engineering and biofabrication. In this thesis, I have researched on two physical gels, namely a thermoresponsive HA derivatives and short-alkyl derivatives, and a covalent gel (tyramine-modified HA). Specifically, the thermoresponsive hydrogel, where the HA carboxyl group was functionalized with poly(N-isopropylacrylamide), was combined with beta tricalcium phosphate particles. The non- covalent network of the thermoresponsive HA is exploited to obtain a better cohesion of the formulation together with a modulation of drugs delivery for bone tissue engineering. A second physical gel network was obtained grafting short-alkyl moieties to the HA. Here, a low degree of modification with small molecules such as propylamine and butylamine was able to drastically change the viscoelastic properties of HA, indicating profound modification of its structure. This same chemistry was employed to develop a new bioink for 3D printing, where the crosslinking density of a covalent tyramine-modified gel was optimized and controlled with a double crosslinking mechanism. The aim of this thesis was to introduce a series of new HA derivatives for biomedical applications. These derivatives embrace a range of gelation mechanisms and applications as illustrated in the following outline: In Chapter 2 an introduction to HA and its chemical, physical and biological properties are provided. The chemical modifications on the main functional groups and the possible crosslinking strategies for hydrogel creation are described. Finally, the main applications of HA in the field of musculoskeletal repair and regeneration are reported. In Chapter 3 DMTMM is employed for grafting propylamine and butylamine to HA. The impact on the HA viscoelastic properties after the conjugation of these short-alkyl moieties is particularly relevant at low degree of substitution and opens a variety of possibilities in applications such as drug delivery and 3D printing. Additionally, a parametric study on the DMTMM reaction conditions shows the reproducibility of this chemistry and the accurate control over a wider range of degrees of substitutions compared to the traditional carbodiimide modification [14]. Chapter 4 describes the preparation of a thermoresponsive poly(N isopropylacrylamide) HA derivative (HApN) combined with beta-tricalcium phosphate particles (βTCP) as bone substitute and drug delivery system. The asset of this novel composite is the improved cohesion at body temperature that the HApN gives, thus giving the possibility to address bone defects. A range of composite formulations is tested as an injectable or putty formulation: the handling properties and the injectability of the composite are analysed as function of the particle size and the HA molecular weight and concentration. The most promising formulations are tested as drug delivery system for the recombinant human bone morphogenetic protein-2 (rhBMP-2) and dexamethasone (DEX) as models of hydrophilic and small hydrophobic drugs, respectively [15]. In Chapter 5 a simple and versatile HA derivative is introduced as an effective biofunctional ink for extrusion-based 3D printing. HA is modified with tyramine functional groups via DMTMM chemistry. A double-crosslinking mechanism consisting in an enzymatic crosslinking that allows good extrudability followed by a visible-light crosslinking is implemented to ensure the stability of the 3D printed constructs. The ink is still available after printing for a functionalization with cell-adhesive motives for improving the construct-cell interaction. Chapter 6 explores the possibility to 3D print viable cells encapsulated in a tyramine- modified HA and to obtain 3D constructs directly on cartilage tissue. The viscoelastic properties of the cell-laden ink are investigated targeting low shear stress for high cell viability and good extrudability. The influence of the photoinitiator and visible light- photocrosslinking on the cell viability is assessed on three different cell types and the printing of the cartilage-adhesive bioink on a piece of cartilage tissue is shown. Chapter 7 focuses on the future perspectives for the HA derivatives in the biomedical field and the need of developing a new generation of derivatives due to the constant expansion of new technologies.

Extrusion-based three-dimensional bioprinting relies on bioinks engineered to combine viscoelastic properties for extrusion and shape retention, and biological properties for cytocompatibility and tissue regeneration. To satisfy these conflicting requirements, bioinks often utilize either complex mixtures or complex modifications of biopolymers. In this paper we introduce and characterize a bioink exploiting a dual crosslinking mechanism, where an enzymatic reaction forms a soft gel suitable for cell encapsulation and extrusion, while a visible light photo-crosslinking allows shape retention of the printed construct. The influence of cell density and cell type on the rheological and printability properties was assessed correlating the printing outcomes with the damping factor, a rheological characteristic independent of the printing system. Stem cells, chondrocytes and fibroblasts were encapsulated, and their viability was assessed up to 14 days with live/dead, alamar blue and trypan blue assays. Additionally, the impact of the printing parameters on cell viability was investigated. Owing to its straightforward preparation, low modification, presence of two independent crosslinking mechanisms for tuning shear-thinning independently of the final shape fixation, the use of visible green instead of UV light, the possibility of encapsulating and sustaining the viability of different cell types, the hyaluronan bioink here presented is a valid biofabrication tool for producing 3D printed tissue-engineered constructs.

Hyaluronan (HA) is widely used in the clinical practice and in biomedical research. Through chemical modification, HA shear-thinning properties, essential for injectability and additive manufacturing, can be optimized. In this study, we employed 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) for grafting propylamine and butylamine to HA. A parametric study was performed to identify the optimal reaction conditions. Results showed that DMTMM amidation gives reproducible and accurate control over a range of degrees of substitution (DS) from 1% to 50% and proved reliable to tune viscoelasticity. At DS = 3.0% for HA-propylamine and 3.7% for HA-butylamine a maximum for storage modulus and pseudoplasticity was found, whereas above or below this DS, rheological features go back to baseline values of pristine HA. Due to their singular rheological profiles, these derivatives are valuable biomaterials candidates for preparing bioinks and hydrogels for drug delivery and regenerative medicine. (C) 2016 Elsevier Ltd. All rights reserved.

Summary Background/Objective Advanced synthetic biomaterials that are able to reduce or replace the need for autologous bone transplantation are still a major clinical need in orthopaedics, dentistry, and trauma. Key requirements for improved bone substitutes are optimal handling properties, ability to fill defects of irregular shape, and capacity for delivering osteoinductive stimuli. Materials and methods In this study, we targeted these requirements by preparing a new composite of β-tricalcium phosphate (TCP) and a thermoresponsive hyaluronan (HA) hydrogel. Dissolution properties of the composite as a function of the particle size and polymeric phase molecular weight and concentration were analysed to identify the best compositions. Results Owing to its amphiphilic character, the composite was able to provide controlled release of both recombinant human bone morphogenetic protein-2 and dexamethasone, selected as models for a biologic and a small hydrophobic molecule, respectively. Conclusion The TCP–thermoresponsive HA hydrogel composite developed in this work can be used for preparing synthetic bone substitutes in the form of injectable or mouldable pastes and can be supplemented with small hydrophobic molecules or biologics for improved osteoinductivity.