There is a need for materials that are well suited for cartilage tissue engineering. Hydrogels have emerged as promising biomaterials for cartilage repair, since, like cartilage, they have high water ...content, and they allow cells to be encapsulated within the material in a genuinely three-dimensional microenvironment. In this study, we investigated the mechanical properties of tissue-engineered cartilage constructs using in vitro culture models incorporating human chondrocytes from osteoarthritis patients. We evaluated hydrogels formed from mixtures of photocrosslinkable gelatin-methacrylamide (Gel-MA) and varying concentrations (0-2%) of hyaluronic acid methacrylate (HA-MA). Initially, only small differences in the stiffness of each hydrogel existed. After 4 weeks of culture, and to a greater extent 8 weeks of culture, HA-MA had striking and concentration dependent impact on the changes in mechanical properties. For example, the initial compressive moduli of cell-laden constructs with 0 and 1% HA-MA were 29 and 41 kPa, respectively. After 8 weeks of culture, the moduli of these constructs had increased to 66 and 147 kPa respectively, representing a net improvement of 69 kPa for gels with 1% HA-MA. Similarly the equilibrium modulus, dynamic modulus, failure strength and failure strain were all improved in constructs containing HA-MA. Differences in mechanical properties did not correlate with glycosaminoglycan content, which did not vary greatly between groups, yet there were clear differences in aggrecan intensity and distribution as assessed using immunostaining. Based on the functional development with time in culture using human chondrocytes, mixtures of Gel-MA and HA-MA are promising candidates for cartilage tissue-engineering applications.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Progress in advancing a system-level understanding of the complexity of human tissue development and regeneration is hampered by a lack of biological model systems that recapitulate key aspects of ...these processes in a physiological context. Hence, growing demand by cell biologists for organ-specific extracellular mimics has led to the development of a plethora of 3D cell culture assays based on natural and synthetic matrices. We developed a physiological microenvironment of semisynthetic origin, called gelatin methacryloyl (GelMA)-based hydrogels, which combine the biocompatibility of natural matrices with the reproducibility, stability and modularity of synthetic biomaterials. We describe here a step-by-step protocol for the preparation of the GelMA polymer, which takes 1-2 weeks to complete, and which can be used to prepare hydrogel-based 3D cell culture models for cancer and stem cell research, as well as for tissue engineering applications. We also describe quality control and validation procedures, including how to assess the degree of GelMA functionalization and mechanical properties, to ensure reproducibility in experimental and animal studies.
Current clinical treatment strategies for the bypassing of small diameter (<6 mm) blood vessels in the management of cardiovascular disease frequently fail due to a lack of suitable autologous ...grafts, as well as infection, thrombosis, and intimal hyperplasia associated with synthetic grafts. The rapid advancement of 3D printing and regenerative medicine technologies enabling the manufacture of biological, tissue-engineered vascular grafts (TEVGs) with the ability to integrate, remodel, and repair in vivo, promises a paradigm shift in cardiovascular disease management. This review comprehensively covers current state-of-the-art biofabrication technologies for the development of biomimetic TEVGs. Various scaffold based additive manufacturing methods used in vascular tissue engineering, including 3D printing, bioprinting, electrospinning and melt electrowriting, are discussed and assessed against the biomechanical and functional requirements of human vasculature, while the efficacy of decellularization protocols currently applied to engineered and native vessels are evaluated. Further, we provide interdisciplinary insight into the outlook of regenerative medicine for the development of vascular grafts, exploring key considerations and perspectives for the successful clinical integration of evolving technologies. It is expected that continued advancements in microscale additive manufacturing, biofabrication, tissue engineering and decellularization will culminate in the development of clinically viable, off-the-shelf TEVGs for small diameter applications in the near future.
Current clinical strategies for the management of cardiovascular disease using small diameter vessel bypassing procedures are inadequate, with up to 75% of synthetic grafts failing within 3 years of implantation. It is this critically important clinical problem that researchers in the field of vascular tissue engineering and regenerative medicine aim to alleviate using biofabrication methods combining additive manufacturing, biomaterials science and advanced cellular biology. While many approaches facilitate the development of bioengineered constructs which mimic the structure and function of native blood vessels, several challenges must still be overcome for clinical translation of the next generation of tissue-engineered vascular grafts.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
The ex vivo engineering of autologous cartilage tissues has the potential to revolutionize the clinical management of joint disorders. Yet, high manufacturing costs and variable outcomes associated ...with tissue-engineered implants are still limiting their application. To improve clinical outcomes and facilitate a wider use of engineered tissues, automated bioreactor systems capable of enhancing and monitoring neotissues are required. Here, we developed an innovative system capable of applying precise uni- or biaxial mechanical stimulation to developing cartilage neotissues in a tightly controlled and automated fashion. The bioreactor allows for simple control over the loading parameters with a user-friendly graphical interface and is equipped with a load cell for monitoring tissue maturation. Applying our bioreactor, we demonstrate that human articular chondrocytes encapsulated in hydrogels composed of gelatin methacryloyl (GelMA) and hyaluronic acid methacrylate (HAMA) respond to uni- and biaxial mechanical stimulation by upregulation of hyaline cartilage-specific marker genes. We further demonstrate that intermittent biaxial mechanostimulation enhances accumulation of hyaline cartilage-specific extracellular matrix. Our study underlines the stimulatory effects of mechanical loading on the biosynthetic activity of human chondrocytes in engineered constructs and the need for easy-to-use, automated bioreactor systems in cartilage tissue engineering.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
The development of hydrogels tailored for cartilage tissue engineering has been a research and clinical goal for over a decade. Directing cells towards a chondrogenic phenotype and promoting new ...matrix formation are significant challenges that must be overcome for the successful application of hydrogels in cartilage tissue therapies. Gelatin–methacrylamide (Gel-MA) hydrogels have shown promise for the repair of some tissues, but have not been extensively investigated for cartilage tissue engineering. We encapsulated human chondrocytes in Gel-MA-based hydrogels, and show that with the incorporation of small quantities of photocrosslinkable hyaluronic acid methacrylate (HA-MA), and to a lesser extent chondroitin sulfate methacrylate (CS-MA), chondrogenesis and mechanical properties can be enhanced. The addition of HA-MA to Gel-MA constructs resulted in more rounded cell morphologies, enhanced chondrogenesis as assessed by gene expression and immunofluorescence, and increased quantity and distribution of the newly synthesized extracellular matrix (ECM) throughout the construct. Consequently, while the compressive moduli of control Gel-MA constructs increased by 26kPa after 8weeks culture, constructs with HA-MA and CS-MA increased by 114kPa. The enhanced chondrogenic differentiation, distribution of ECM, and improved mechanical properties make these materials potential candidates for cartilage tissue engineering applications.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Gelatin‐methacrylamide (gelMA) hydrogels are shown to support chondrocyte viability and differentiation and give wide ranging mechanical properties depending on several cross‐linking parameters. ...Polymer concentration, UV exposure time, and thermal gelation prior to UV exposure allow for control over hydrogel stiffness and swelling properties. GelMA solutions have a low viscosity at 37 °C, which is incompatible with most biofabrication approaches. However, incorporation of hyaluronic acid (HA) and/or co‐deposition with thermoplastics allows gelMA to be used in biofabrication processes. These attributes may allow engineered constructs to match the natural functional variations in cartilage mechanical and geometrical properties.
Gelatin‐methacrylamide (gelMA) hydrogel mechanical properties are predictably tuned by varying the crosslinking conditions. These gels also support chondrocyte survival and function. Furthermore, addition of hyaluronic acid and co‐printing with thermoplastics allows for biofabrication with gelMA. These attributes may allow engineered constructs to match the natural functional variations in the mechanical and geometrical properties of cartilage.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Gelatin methacryloyl (GelMA) hydrogels are a mechanically and biochemically tuneable biomaterial, facilitating chondrocyte culture for tissue engineering applications. However, a lack of ...characterisation and standardisation of fabrication methodologies for GelMA restricts its utilisation in surgical interventions for articular cartilage repair. The purpose of this study was to determine the effects of gelatin source and photoinitiator type on the redifferentiation capacity of monolayer-expanded human articular chondrocytes encapsulated in GelMA/hyaluronic acid methacrylate (HAMA) hydrogels. Chondrocyte-laden hydrogels reinforced with multiphasic melt-electrowritten (MEW) medical grade polycaprolactone (mPCL) microfibre scaffolds were prepared using bovine (B) or porcine-derived (P) GelMA, and photocrosslinked with either lithium acylphosphinate (LAP) and visible light (405 nm) or Irgacure 2959 (IC) and UV light (365 nm). Bulk physical properties, cell viability and biochemical features of hydrogel constructs were measured at day 1 and day 28 of chondrogenic cell culture. The compressive moduli of all groups increased after 28 days of cell culture, with B-IC displaying similar compressive strength to that of native articular cartilage (∼1.5 MPa). Compressive moduli correlated with an increase in total glycosaminoglycan (GAG) content for each group. Gene expression analysis revealed upregulation of chondrogenic marker genes in IC-crosslinked groups, whilst dedifferentiation gene markers were upregulated in LAP-crosslinked groups. mPCL reinforcement correlated with increased accumulation of collagen I and II in B-IC, B-LAP and P-IC groups compared to non-reinforced hydrogels. A reduction in cell viability was noted in all samples at day 28, potentially due to the generation of free radicals during photocrosslinking or cytotoxicity of the photoinitiators. In summary, hydrogel constructs prepared with bovine-derived GelMA and photocrosslinked with Irgacure 2959 and 365 nm light displayed properties most similar to native articular cartilage after 28 days of cell culture. The differences in biological response between investigated construct types emphasises the necessity to characterise and standardise biomaterials before translating
in vitro
tissue engineering research to preclinical applications for articular cartilage injuries.
This work investigates neocartilage formation in bovine and porcine gelatin methacryloyl-based hydrogels photocrosslinked using ultraviolet or visible light photoinitiator systems.
Abstract Biochemically modified proteins have attracted significant attention due to their widespread applications as biomaterials. For instance, chemically modified gelatin derivatives have been ...widely explored as biomaterials and specifically to develop hydrogels for tissue engineering and regenerative medicine applications. Among the reported methods, modification of gelatin with methacrylic anhydride (MA) stands out as a convenient and efficient strategy to introduce functional groups and form hydrogels via photopolymerization. Combination of light-activation of modified gelatin with soft lithography has enabled the materialization of microfabricated hydrogels. So far, this gelatin derivative has been referred to in the literature as gelatin methacrylate, gelatin methacrylamide, or gelatin methacryloyl, with the same abbreviation of GelMA. Considering the complex composition of gelatin and the presence of different functional groups on the amino acid residues, both hydroxyl groups and amine groups can possibly react with methacrylic anhydride during functionalization of the protein. This can also apply to the modification of other proteins, such as recombinant human tropoelastin. Here, we employed analytical methods to quantitatively determine the amounts of methacrylate and methacrylamide groups in MA-modified gelatin and tropoelastin to better understand the reaction mechanism. By combining two chemical assays with instrumental techniques, such as proton nuclear magnetic resonance (1 H NMR) and liquid chromatography tandem-mass spectrometry (LC-MS/MS), our results indicated that while amine groups have higher reactivity than hydroxyl groups and resulted in a majority of methacrylamide groups, modification of proteins by MA can lead to the formation of both methacrylamide and methacrylate groups. It is therefore suggested that the standard terms for GelMA and MeTro should be defined as gelatin methacryloyl and methacryloyl-substituted tropoelastin, respectively, to remain consistent with the widespread abbreviations used in literature.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPUK, ZRSKP
Additive manufacturing of tissues and organs Melchels, Ferry P.W.; Domingos, Marco A.N.; Klein, Travis J. ...
Progress in polymer science,
08/2012, Volume:
37, Issue:
8
Journal Article
Peer reviewed
Additive manufacturing techniques offer the potential to fabricate organized tissue constructs to repair or replace damaged or diseased human tissues and organs. Using these techniques, spatial ...variations of cells along multiple axes with high geometric complexity in combination with different biomaterials can be generated. The level of control offered by these computer-controlled technologies to design and fabricate tissues will accelerate our understanding of the governing factors of tissue formation and function. Moreover, it will provide a valuable tool to study the effect of anatomy on graft performance. In this review, we discuss the rationale for engineering tissues and organs by combining computer-aided design with additive manufacturing technologies that encompass the simultaneous deposition of cells and materials. Current strategies are presented, particularly with respect to limitations due to the lack of suitable polymers, and requirements to move the current concepts to practical application.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPUK
Tissue engineering has offered unique opportunities for disease modeling and regenerative medicine; however, the success of these strategies is dependent on faithful reproduction of native cellular ...organization. Here, it is reported that ultrasound standing waves can be used to organize myoblast populations in material systems for the engineering of aligned muscle tissue constructs. Patterned muscle engineered using type I collagen hydrogels exhibits significant anisotropy in tensile strength, and under mechanical constraint, produced microscale alignment on a cell and fiber level. Moreover, acoustic patterning of myoblasts in gelatin methacryloyl hydrogels significantly enhances myofibrillogenesis and promotes the formation of muscle fibers containing aligned bundles of myotubes, with a width of 120–150 µm and a spacing of 180–220 µm. The ability to remotely pattern fibers of aligned myotubes without any material cues or complex fabrication procedures represents a significant advance in the field of muscle tissue engineering. In general, these results are the first instance of engineered cell fibers formed from the differentiation of acoustically patterned cells. It is anticipated that this versatile methodology can be applied to many complex tissue morphologies, with broader relevance for spatially organized cell cultures, organoid development, and bioelectronics.
Ultrasound standing waves are used to preorganize myoblast populations within collagen‐based hydrogels for the engineering of anisotropic skeletal muscle tissue. The acoustic cell patterning directs myoblast fusion and differentiation to generate oriented myotubes bundled into aligned muscle fibers; a physiologically relevant tissue morphology that yields anisotropic tensile mechanics and enhanced myofibrillogenesis.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
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