Pre-culture of human mesenchymal stromal cells in spheroids facilitates chondrogenesis at a low total cell count upon embedding in biomaterials to generate cartilage microtissues

Material-assisted cartilage tissue engineering has limited application in cartilage treatment due to hypertrophic tissue formation and high cell counts required. This study aimed at investigating the potential of human mesenchymal stromal cell (hMSC) spheroids embedded in biomaterials to study the effect of biomaterial composition on cell differentiation. Pre-cultured (3 days, chondrogenic differentiation media) spheroids (250 cells/spheroid) were embedded in tyramine-modified hyaluronic acid (THA) and collagen type I (Col) composite hydrogels (four combinations of THA (12.5 vs 16.7 mg/ml) and Col (2.5 vs 1.7 mg/ml) content) at a cell density of 5 × 106 cells/ml (2 × 104 spheroids/ml). Macropellets derived from single hMSCs (2.5 × 105 cells, ScMP) or hMSC spheroids (2.5 × 105 cells, 103 spheroids, SpMP) served as control. hMSC differentiation was analyzed using glycosaminoglycan (GAG) quantification, gene expression analysis and (immuno-)histology. Embedding of hMSC spheroids in THA-Col induced chondrogenic differentiation marked by upregulation of aggrecan (ACAN) and COL2A1, and the production of GAGs. Lower THA led to more pronounced chondrogenic phenotype compared to higher THA content. Col content had no significant influence on hMSC chondrogenesis. Pellet cultures showed an upregulation in chondrogenic-associated genes and production of GAGs with less upregulation of hypertrophic-associated genes in SpMP culture compared to ScMP group. This study presents hMSC pre-culture in spheroids as promising approach to study chondrogenic differentiation after biomaterial encapsulation at low total cell count (5 × 106/ml) without compromising chondrogenic matrix production. This approach can be applied to assemble microtissues in biomaterials to generate large cartilage construct. Statement of significance: In vitro studies investigating the chondrogenic potential of biomaterials are limited due to the low cell-cell contact of encapsulated single cells. Here, we introduce the use of pre-cultured hMSC spheroids to study chondrogenesis upon encapsulation in a biomaterial. The use of spheroids takes advantage of the high cell-cell contact within each spheroid being critical in the early chondrogenesis of hMSCs. At a low seeding density of 5·106 cells/ml (2 × 104 spheroids/ml) we demonstrated hMSC chondrogenesis and cartilaginous matrix deposition. Our results indicate that the pre-culture might have a beneficial effect on hypertrophic gene expression without compromising chondrogenic differentiation. This approach has shown potential to assemble microtissues (here spheroids) in biomaterials to generate large cartilage constructs and to study the effect of biomaterial composition on cell alignment and migration.</p

Modulating design parameters to drive cell invasion into hydrogels for osteochondral tissue formation

Background: The use of acellular hydrogels to repair osteochondral defects requires cells to first invade the biomaterial and then to deposit extracellular matrix for tissue regeneration. Due to the diverse physicochemical properties of engineered hydrogels, the specific properties that allow or even improve the behaviour of cells are not yet clear. The aim of this study was to investigate the influence of various physicochemical properties of hydrogels on cell migration and related tissue formation using in vitro, ex vivo and in vivo models. Methods: Three hydrogel platforms were used in the study: Gelatine methacryloyl (GelMA) (5% wt), norbornene hyaluronic acid (norHA) (2% wt) and tyramine functionalised hyaluronic acid (THA) (2.5% wt). GelMA was modified to vary the degree of functionalisation (DoF 50% and 80%), norHA was used with varied degradability via a matrix metalloproteinase (MMP) degradable crosslinker and THA was used with the addition of collagen fibrils. The migration of human mesenchymal stromal cells (hMSC) in hydrogels was studied in vitro using a 3D spheroid migration assay over 48h. In addition, chondrocyte migration within and around hydrogels was investigated in an ex vivo bovine cartilage ring model (three weeks). Finally, tissue repair within osteochondral defects was studied in a semi-orthotopic in vivo mouse model (six weeks). Results: A lower DoF of GelMA did not affect cell migration in vitro (p ​= ​0.390) and led to a higher migration score ex vivo (p ​< ​0.001). The introduction of a MMP degradable crosslinker in norHA hydrogels did not improve cell infiltration in vitro or in vivo. The addition of collagen to THA resulted in greater hMSC migration in vitro (p ​= ​0.031) and ex vivo (p ​< ​0.001). Hydrogels that exhibited more cell migration in vitro or ex vivo also showed more tissue formation in the osteochondral defects in vivo, except for the norHA group. Whereas norHA with a degradable crosslinker did not improve cell migration in vitro or ex vivo, it did significantly increase tissue formation in vivo compared to the non-degradable crosslinker (p ​< ​0.001). Conclusion: The modification of hydrogels by adapting DoF, use of a degradable crosslinker or including fibrillar collagen can control and improve cell migration and tissue formation for osteochondral defect repair. This study also emphasizes the importance of performing both in vitro and in vivo testing of biomaterials, as, depending on the material, the results might be affected by the model used. The translational potential of this article: This article highlights the potential of using acellular hydrogels to repair osteochondral defects, which are common injuries in orthopaedics. The study provides a deeper understanding of how to modify the properties of hydrogels to control cell migration and tissue formation for osteochondral defect repair. The results of this article also highlight that the choice of the used laboratory model can affect the outcome. Testing hydrogels in different models is thus advised for successful translation of laboratory results to the clinical application

Modulating design parameters to drive cell invasion into hydrogels for osteochondral tissue formation

Background: The use of acellular hydrogels to repair osteochondral defects requires cells to first invade the biomaterial and then to deposit extracellular matrix for tissue regeneration. Due to the diverse physicochemical properties of engineered hydrogels, the specific properties that allow or even improve the behaviour of cells are not yet clear. The aim of this study was to investigate the influence of various physicochemical properties of hydrogels on cell migration and related tissue formation using in vitro, ex vivo and in vivo models. Methods: Three hydrogel platforms were used in the study: Gelatine methacryloyl (GelMA) (5% wt), norbornene hyaluronic acid (norHA) (2% wt) and tyramine functionalised hyaluronic acid (THA) (2.5% wt). GelMA was modified to vary the degree of functionalisation (DoF 50% and 80%), norHA was used with varied degradability via a matrix metalloproteinase (MMP) degradable crosslinker and THA was used with the addition of collagen fibrils. The migration of human mesenchymal stromal cells (hMSC) in hydrogels was studied in vitro using a 3D spheroid migration assay over 48h. In addition, chondrocyte migration within and around hydrogels was investigated in an ex vivo bovine cartilage ring model (three weeks). Finally, tissue repair within osteochondral defects was studied in a semi-orthotopic in vivo mouse model (six weeks). Results: A lower DoF of GelMA did not affect cell migration in vitro (p ​= ​0.390) and led to a higher migration score ex vivo (p ​< ​0.001). The introduction of a MMP degradable crosslinker in norHA hydrogels did not improve cell infiltration in vitro or in vivo. The addition of collagen to THA resulted in greater hMSC migration in vitro (p ​= ​0.031) and ex vivo (p ​< ​0.001). Hydrogels that exhibited more cell migration in vitro or ex vivo also showed more tissue formation in the osteochondral defects in vivo, except for the norHA group. Whereas norHA with a degradable crosslinker did not improve cell migration in vitro or ex vivo, it did significantly increase tissue formation in vivo compared to the non-degradable crosslinker (p ​< ​0.001). Conclusion: The modification of hydrogels by adapting DoF, use of a degradable crosslinker or including fibrillar collagen can control and improve cell migration and tissue formation for osteochondral defect repair. This study also emphasizes the importance of performing both in vitro and in vivo testing of biomaterials, as, depending on the material, the results might be affected by the model used. The translational potential of this article: This article highlights the potential of using acellular hydrogels to repair osteochondral defects, which are common injuries in orthopaedics. The study provides a deeper understanding of how to modify the properties of hydrogels to control cell migration and tissue formation for osteochondral defect repair. The results of this article also highlight that the choice of the used laboratory model can affect the outcome. Testing hydrogels in different models is thus advised for successful translation of laboratory results to the clinical application

Enhancing hyaluronan pseudoplasticity via 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride-mediated conjugation with short alkyl moieties

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

Three-Dimensional Printing of a Tyramine Hyaluronan Derivative with Double Gelation Mechanism for Independent Tuning of Shear Thinning and Postprinting Curing

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

3D printing of inorganic-biopolymer composites for bone regeneration

In most cases, bone injuries heal without complications, however, there is an increasing number of instances where bone healing needs major clinical intervention. Available treatment options have severe drawbacks, such as donor site morbidity and limited availability for autografting. Bone graft substitutes containing growth factors would be a viable alternative, however they have been associated with dose-related safety concerns and lack control over spatial architecture to anatomically match bone defect sites. A 3D printing offers a solution to produce patient specific bone graft substitutes that are customized to the patient bone defect with temporal control over the incorporated therapeutics to maximize their efficacy. Inspired by the natural constitution of bone tissue, composites made of inorganic phases, such as nanosilicate particles, calcium phosphate, and bioactive glasses, combined with biopolymer matrices have been investigated as building blocks for the biofabrication of bone constructs. Besides capturing elements of the bone physiological structure, these inorganic/organic composites can be designed for specific cohesivity, rheological and mechanical properties, while both inorganic and organic constituents contribute to the composite bioactivity. This review provides an overview of 3D printed composite biomaterial-inks for bone tissue engineering. Furthermore, key aspects in biomaterial-ink design, 3D printing techniques, and the building blocks for composite biomaterial-inks are summarized.ISSN:1758-5082ISSN:1758-509

Printability and Shape Fidelity of Bioinks in 3D Bioprinting

Three-dimensional bioprinting uses additive manufacturing techniques for the automated fabrication of hierarchically organized living constructs. The building blocks are often hydrogel-based bioinks, which need to be printed into structures with high shape fidelity to the intended computer-aided design. For optimal cell performance, relatively soft and printable inks are preferred, although these undergo significant deformation during the printing process, which may impair shape fidelity. While the concept of good or poor printability seems rather intuitive, its quantitative definition lacks consensus and depends on multiple rheological and chemical parameters of the ink. This review discusses qualitative and quantitative methodologies to evaluate printability of bioinks for extrusion- and lithography-based bioprinting. The physicochemical parameters influencing shape fidelity are discussed, together with their importance in establishing new models, predictive tools and printing methods that are deemed instrumental for the design of next-generation bioinks, and for reproducible comparison of their structural performance

Development of a hyaluronic acid-collagen bioink for shear-induced fibers and cells alignment

: Human tissues are characterized by complex composition and cellular and extracellular matrix (ECM) organization at microscopic level. In most of human tissues, cells and ECM show an anisotropic arrangement, which confers them specific properties.In vitro, the ability to closely mimic this complexity is limited. However, in the last years, extrusion bioprinting showed a certain potential for aligning cells and biomolecules, due to the application of shear stress during the bio-fabrication process. In this work, we propose a strategy to combine collagen (col) with tyramine-modified hyaluronic acid (THA) to obtain a printable col-THA bioink for extrusion bioprinting, solely-based on natural-derived components. Collagen fibers formation within the hybrid hydrogel, as well as collagen distribution and spatial organization before and after printing, were studied. For the validation of the biological outcome, fibroblasts were selected as cellular model and embedded in the col-THA matrix. Cell metabolic activity and cell viability, as well as cell distribution and alignment, were studied in the bioink before and after bioprinting. Results demonstrated successful collagen fibers formation within the bioink, as well as collagen anisotropic alignment along the printing direction. Furthermore, results revealed suitable biological properties, with a slightly reduced metabolic activity at day 1, fully recovered within the first 3 d post-cell embedding. Finally, results showed fibroblasts elongation and alignment along the bioprinting direction. Altogether, results validated the potential to obtain collagen-based bioprinted constructs, with both cellular and ECM anisotropy, without detrimental effects of the fabrication process on the biological outcome. This bioink can be potentially used for a wide range of applications in tissue engineering and regenerative medicine in which anisotropy is required