Gelatin-based Biomaterials Research Articles

Crosslinking gelatin with robust inherent antibacterial natural polymer for wound healing

Gelatin-based biomaterials are widely acknowledged as a promising choice for wound dressings, given their similarity to the extracellular matrix and biocompatibility. However, the challenge of cross-linking gelatin while preserving its biocompatibility and cost-effectiveness persists. This study aimed to enhance the properties of gelatin by incorporating the oxidized lignosulfonate (OLS) biopolymer as an inexpensive and biocompatible natural material. The polyphenolic structure of OLS acts as both a cross-linking agent and an antibacterial component. The OLS/gelatin films were prepared using a casting method with varying weight ratios (0.1, 0.2, 0.3, 0.4, and 0.5 w/w). FTIR analysis confirmed the formation of Schiff-base and hydrogen bonds between gelatin and OLS. The resulting films exhibited enhanced mechanical properties (Young's modulus ∼40 MPa), no cytotoxicity, and excellent cell adhesion and morphology. Antimicrobial tests showed significant activity against Escherichia coli and Staphylococcus aureus, with higher activity against S. aureus (17 mm inhibition zone and 99 % bactericidal rate). In vivo studies in a mouse model demonstrated that the gelatin/0.2OLS dressing significantly improved wound healing, including re-epithelialization, collagen formation, inflammation reduction, and blood vessel density, compared to untreated wounds. These findings suggest that the synthesized novel gelatin/OLS wound dressing has promising healing and antibacterial properties.

Fluorogenic in-situ Labelling of Gelatin Polymer in Aqueous Solution and Hydrogel.

Gelatin polymers made from partially degraded collagen are important biomaterials, but their in-situ analysis suffers from uncontrollable covalent labelling and poor spatial-temporal imaging resolution. Herein, three tetrazolate-tagged tetraphenylethylene fluorophores (TPE-TAs) are introduced for practical fluorogenic labelling of gelatin in aqueous phase and hydrogels. These probes with aggregation-induced emission characteristics offer negligible background and elicit turn-on fluorescence by simply mixing with the gelatin in aqueous phase, giving a detection limit of 0.15 mg/L over a linear dynamic range up to 100 mg/L. This method does not work for collagens and causes minimal interference with gelatin properties. Mechanistic studies reveal a key role for multivalent electrostatic interactions between the abundant basic residues in gelatin (e. g., lysine, hydroxylysine, arginine) and anionic tetrazolate moieties of the lipophilic fluorophore synergistically in spatially rigid macromolecular encapsulation to achieve fluorogenic labelling. The AIE strategy by forming non-covalent fluorophore-gelatin complexes was developed for novel hydrogels that exhibited reversible fluorescence in response to dynamic microstructural changes in the hydrogel scaffold upon salting-in/out treatments, and enabled high spatial-temporal imaging of the fiber network in lyophilized samples. This work may open up avenues for in-situ imaging analysis and evaluation of gelatin-based biomaterials during processes such as in vivo degradation and mineralization.

Gelatin-based biomaterials and gelatin as an additive for chronic wound repair.

Disturbing or disrupting the regular healing process of a skin wound may result in its progression to a chronic state. Chronic wounds often lead to increased infection because of their long healing time, malnutrition, and insufficient oxygen flow, subsequently affecting wound progression. Gelatin-the main structure of natural collagen-is widely used in biomedical fields because of its low cost, wide availability, biocompatibility, and degradability. However, gelatin may exhibit diverse tailored physical properties and poor antibacterial activity. Research on gelatin-based biomaterials has identified the challenges of improving gelatin's poor antibacterial properties and low mechanical properties. In chronic wounds, gelatin-based biomaterials can promote wound hemostasis, enhance peri-wound antibacterial and anti-inflammatory properties, and promote vascular and epithelial cell regeneration. In this article, we first introduce the natural process of wound healing. Second, we present the role of gelatin-based biomaterials and gelatin as an additive in wound healing. Finally, we present the future implications of gelatin-based biomaterials.

Gelatin-based Targeted Delivery Systems for Tissue Engineering.

Gelatin is an attractive material for drug delivery and tissue engineering applications due to its excellent biocompatibility and biodegradability, which has been utilized as cell, drug, and gene carriers. Gelatin is less immunogenic compared to collagen and its precursor and retains informational signals, such as RGD (Arg-Gly-Asp) sequence, thus promoting cell adhesion and proliferation. To tune the mechanical strength and bioactivity, gelatin can be easily modified via chemical reactions and physical methods to obtain various derivatives. Furthermore, gelatin-based biomaterials can be achieved through chemical immobilization of specific molecules and physical combination with other biopolymers. This review focuses on the recent advances of gelatin and its derivatives as biomaterials in the field of drug delivery, including cell scaffolds for tissue engineering applications.

Analysis of gelatin secondary structure in gelatin/keratin-based biomaterials

The possibility of using protein-based materials as cellular scaffold strongly depends on protein conformation, and several attempts have been made by researchers to obtain scaffold with morphology miming the extracellular matrix. It is widely recognized that the secondary structure of proteins affects the mechanical and biological properties of protein-based scaffolds. However, few studies have been published, and an exhaustive explanation is still missing. In this work the study of the gelatin structure in gelatin-based materials and the investigation of the possible correlations between structure, mechanical and biological features is reported.We have examined how the secondary structure of gelatin is affected (i) by the process used to obtain the biomaterials (solvent casting vs. electrospinning), (ii) by the concentration of cross linker (3-(Glycidyloxypropyl)trimethoxysilane) (GPTMS), and (iii) by the raw keratin extract added. Gelatin electrospun materials have shown a content of ordered structure higher than gelatin casted films, likely due to the random coil – α-helix transition occuring during electrospinning. GPTMS gives a decrease of ordered structures in gelatin casted films (random structure increasing from 20% to 60%), while it does not affect the percentage of ordered structure in electrospun samples. In the gelatin/keratin electrospun biomaterials, the presence of keratin produces a decrease of α-helix content from 31% to 2–15% and an increase of β-structures, promoting the conversion from antiparallel to parallel β-sheet. The structure of gelatin affects the mechanical performances of biomaterials. In gelatin/keratin electrospun biomaterials we have found a positive correlation between failure strain and helix conformation and a negative correlation with β-structures. Elastic modulus has opposite correlations. All gelatin-based biomaterials have been tested as scaffold for pre-osteoblastic cells showing good biocompatibility for both casted films and electrospun biomaterials.

Semi-Synthetic Click-Gelatin Hydrogels as Tunable Platforms for 3D Cancer Cell Culture.

Basement membrane extracts (BME) derived from Engelbreth-Holm-Swarm (EHS) mouse sarcomas such as Matrigel® remain the gold standard extracellular matrix (ECM) for three-dimensional (3D) cell culture in cancer research. Yet, BMEs suffer from substantial batch-to-batch variation, ill-defined composition, and lack the ability for physichochemical manipulation. Here, we developed a novel 3D cell culture system based on thiolated gelatin (Gel-SH), an inexpensive and highly controlled raw material capable of forming hydrogels with a high level of biophysical control and cell-instructive bioactivity. We demonstrate the successful thiolation of gelatin raw materials to enable rapid covalent crosslinking upon mixing with a synthetic poly(ethylene glycol) (PEG)-based crosslinker. The mechanical properties of the resulting gelatin-based hydrogels were readily tuned by varying precursor material concentrations, with Young's moduli ranging from ~2.5 to 5.8 kPa. All hydrogels of varying stiffnesses supported the viability and proliferation of MDA-MB-231 and MCF-7 breast cancer cell lines for 14 and 21 days of cell culture, respectively. Additionally, the gelatin-based hydrogels supported the growth, viability, and osteogenic differentiation of patient-derived preosteoblasts over 28 days of culture. Collectively, our data demonstrate that gelatin-based biomaterials provide an inexpensive and tunable 3D cell culture platform that may overcome the limitations of traditional BMEs.

4D bioprintable self-healing hydrogel with shape memory and cryopreserving properties

Four-dimensional (4D) bioprinting is an emerging biofabrication technology that integrates time as a fourth dimension with three-dimensional (3D) bioprinting for fabricating customizable tissue-engineered implants. 4D bioprinted implants are expected to possess self-healing and shape memory properties for new application opportunities, for instance, fabrication of devices with good shape integrity for minimally invasive surgery. Herein, we developed a self-healing hydrogel composed of biodegradable polyurethane (PU) nanoparticles and photo-/thermo-responsive gelatin-based biomaterials. The self-healing property of hydrogel may be associated with the formation of reversible ionomeric interaction between the COO− group of PU nanoparticles and NH3 + group on the gelatin chains. The self-healing hydrogel demonstrated excellent 3D printability and filament resolution. The UV-crosslinked printed hydrogel showed good stackability (>80 layers), structural stability, elasticity, and tunable modulus (1−60 kPa). The shape-memorizable 4D printed constructs revealed good shape fixity (∼95%) and shape recovery (∼98%) through the elasticity as well as forming and collapsing of water lattice in the hydrogel. The hydrogel and the printing process supported the continuous proliferation of neural stem cells (NSCs) (∼3.7-fold after 14 days). Moreover, the individually bioprinted NSCs and mesenchymal stem cells in the adjacent, self-healed filaments showed mutual migration and such interaction promoted the cell differentiation behavior. The cryopreserved (−20 °C or −80 °C) 4D bioprinted hydrogel after awakening and shape recovery at 37 °C demonstrated cell proliferation similar to that of the non-cryopreserved control. This 4D bioprintable, self-healable hydrogel with shape memory and cryopreserving properties may be employed for customized biofabrication.

Gelatin Methacryloyl (GelMA) Nanocomposite Hydrogels Embedding Bioactive Naringin Liposomes.

The development of nanocomposite hydrogels that take advantage of hierarchic building blocks is gaining increased attention due to their added functionality and numerous biomedical applications. Gathering on the unique properties of these platforms, herein we report the synthesis of bioactive nanocomposite hydrogels comprising naringin-loaded salmon-derived lecithin nanosized liposomal building blocks and gelatin methacryloyl (GelMA) macro-sized hydrogels for their embedding. This platform takes advantage of liposomes’ significant drug loading capacity and their role in hydrogel network reinforcement, as well as of the injectability and light-mediated crosslinking of bioderived gelatin-based biomaterials. First, the physicochemical properties, as well as the encapsulation efficiency, release profile, and cytotoxicity of naringin-loaded nanoliposomes (LipoN) were characterized. Then, the effect of embedding LipoN in the GelMA matrix were characterized by studying the release behavior, swelling ratio, and hydrophilic character, as well as the rheological and mechanical properties of GelMA and GelMA-LipoN functionalized hydrogels. Finally, the dispersion of nanoliposomes encapsulating a model fluorescent probe in the GelMA matrix was visualized. The formulation of naringin-loaded liposomes via an optimized procedure yielded nanosized (114 nm) negatively charged particles with a high encapsulation efficiency (~99%). Naringin-loaded nanoliposomes administration to human adipose-derived stem cells confirmed their suitable cytocompatibility. Moreover, in addition to significantly extending the release of naringin from the hydrogel, the nanoliposomes inclusion in the GelMA matrix significantly increased its elastic and compressive moduli and decreased its swelling ratio, while showing an excellent dispersion in the hydrogel network. Overall, salmon-derived nanoliposomes enabled the inclusion and controlled release of pro-osteogenic bioactive molecules, as well as improved the hydrogel matrix properties, which suggests that these soft nanoparticles can play an important role in bioengineering bioactive nanocomposites for bone tissue engineering in the foreseeable future.

Quantification of fractional and absolute functionalization of gelatin hydrogels by optimized ninhydrin assay and 1H NMR

3D cell culture in protein-based hydrogels often begins with chemical functionalization of proteins with cross-linking agents such as methacryloyl or norbornene. An important and variable characteristic of these materials is the degree of functionalization (DoF), which controls the reactivity of the protein for cross-linking and therefore impacts the mechanical properties and stability of the hydrogel. Although 1H NMR has emerged as the most accurate technique for quantifying absolute DoF of chemically modified proteins, colorimetric techniques still dominate in actual use and may be more useful for quantifying fractional or percent DoF. In this work, we sought to develop an optimized colorimetric assay for DoF of common gelatin-based biomaterials and validate it versus NMR; along the way, we developed a set of best practices for both methods and considerations for their most appropriate use. First, the amine-reactive ninhydrin assay was optimized in terms of solvent properties, temperature, ninhydrin concentration, and range of gelatin standards. The optimized assay produced a linear response to protein concentration in a convenient, 96-well plate format and yielded a fractional DoF similar to NMR in most cases. In comparing with NMR, we identified that DoF can be expressed as fractional or absolute, and that fractional DoF can be inaccurate if the amino acid content of the parent protein is not properly accounted for. In summary, the fractional DoF of methacryloyl- and norbornene-functionalized gelatins was quantified by an optimized colorimetric ninhydrin assay and orthogonally by 1H NMR. These methods will be valuable for quality control analysis of protein-based hydrogels and 3D cell culture biomaterials. Graphical abstract.

Engineering and Functionalization of Gelatin Biomaterials: From Cell Culture to Medical Applications.

Health care and medicine were revolutionized in recent years by the development of biomaterials, such as stents, implants, personalized drug delivery systems, engineered grafts, cell sheets, and other transplantable materials. These materials not only support the growth of cells before transplantation but also serve as replacements for damaged tissues in vivo. Among the various biomaterials available, those made from natural biological sources such as extracellular proteins (collagen, fibronectin, laminin) have shown significant benefits, and thus are widely used. However, routine biomaterial-based research requires copious quantities of proteins and the use of pure and intact extracellular proteins could be highly cost ineffective. Gelatin is a molecular derivative of collagen obtained through the irreversible denaturation of collagen proteins. Gelatin shares a very close molecular structure and function with collagen and thus is often used in cell and tissue culture to replace collagen for biomaterial purposes. Recent technological advancements such as additive manufacturing, rapid prototyping, and three-dimensional printing, in general, have resulted in great strides toward the generation of functional gelatin-based materials for medical purposes. In this review, the structural and molecular similarities of gelatin to other extracellular matrix proteins are compared and analyzed. Current strategies for gelatin crosslinking and production are described and recent applications of gelatin-based biomaterials in cell culture and tissue regeneration are discussed. Finally, recent improvements in gelatin-based biomaterials for medical applications and future directions are elaborated. Impact statement In this study, we described gelatin's biochemical properties and compared its advantages and drawbacks over other extracellular matrix proteins and polymers used for biomaterial application. We also described how gelatin can be used with other polymers in creating gelatin composite materials that have enhanced mechanical properties, increased biocompatibility, and boosted bioactivity, maximizing its benefits for biomedical purposes. The article is relevant, as it discussed not only the chemistry of gelatin, but also listed the current techniques in gelatin/biomaterial manufacturing and described the most recent trends in gelatin-based biomaterials for biomedical applications.

Modification and crosslinking of gelatin-based biomaterials as tissue adhesives.

Tissue adhesives have been developed to overcome the difficulties of conventional wound closure techniques (e.g. sutures and staples), such as the potential for collateral damage and difficulty of stopping body fluid and gas. At the same time, it provides advantages such as simpler implementation, less painful, and does not require removal. However, representative adhesives such as cyanoacrylates and fibrin glues are plagued by cytotoxicity and low adhesion. In this study, we choose instead gelatin as the backbone of adhesive, due to its biocompatibility, biodegradability, and low cost. Firstly, catechol-modified gelatin and phenol-modified gelatin were synthesized via an EDC/NHS chemistry. Then, gelatin-based adhesives were prepared via ruthenium-based photochemistry, including photo-crosslinked gelatin (PG), phenol-modified gelatin (PPG), and catechol-modified gelatin (PCG). We also compared the photo-crosslinked adhesives to the recently reported ion-crosslinked catechol-modified gelatin. Our results indicate that gelatin-based adhesives demonstrate lower swelling index, great degradability, and low cytotoxicity. This shows that gelatin-based adhesives demonstrate great potential for wound closure and healing.

A highly versatile adaptor protein for the tethering of growth factors to gelatin-based biomaterials.

In an effort to functionalize collagen/gelatin-based biomaterials with growth factors, we have designed an adaptor protein corresponding to a collagen-binding domain fused to a coil peptide. In our strategy, this adaptor protein captures growth factors being tagged with the partner coil peptide in a specific, stable and oriented manner. We have found that the tethering of the Epidermal Growth Factor preserved its mitogenic and anti-apoptotic activity. In the case of the basic Fibroblast Growth Factor, the captured growth factor remained bioactive although its tethering via this adaptor protein modified its natural mode of interaction with gelatin. Altogether this strategy is easily adaptable to the simultaneous tethering of various growth factors.

Biocompatibility and inflammatory response in vitro and in vivo to gelatin-based biomaterials with tailorable elastic properties

Hydrogels prepared from gelatin and lysine diisocyanate ethyl ester provide tailorable elastic properties and degradation behavior. Their interaction with human aortic endothelial cells (HAEC) as well as human macrophages (Mɸ) and granulocytes (Gɸ) were explored. The experiments revealed a good biocompatibility, appropriate cell adhesion, and cell infiltration. Direct contact to hydrogels, but not contact to hydrolytic or enzymatic hydrogel degradation products, resulted in enhanced cyclooxygenase-2 (COX-2) expression in all cell types, indicating a weak inflammatory activation in vitro. Only Mɸ altered their cytokine secretion profile after direct hydrogel contact, indicating a comparably pronounced inflammatory activation. On the other hand, in HAEC the expression of tight junction proteins, as well as cytokine and matrix metalloproteinase secretion were not influenced by the hydrogels, suggesting a maintained endothelial cell function. This was in line with the finding that in HAEC increased thrombomodulin synthesis but no thrombomodulin membrane shedding occurred. First in vivo data obtained after subcutaneous implantation of the materials in immunocompetent mice revealed good integration of implants in the surrounding tissue, no progredient fibrous capsule formation, and no inflammatory tissue reaction in vivo. Overall, the study demonstrates the potential of gelatin-based hydrogels for temporal replacement and functional regeneration of damaged soft tissue.

Cross‐linked gelatin microspheres with continuously tunable degradation profiles for renal tissue regeneration

Collagen and gelatin-based biomaterials are widely used in tissue engineering applications. Various methods have been reported for the cross-linking of these macromolecules for the purpose of delaying their biodegradation to prolong their in vivo residence (in tissue engineering applications) or tailoring their drug releasing capacity (when used as drug carriers). In this study, a carbodiimide-based cross-linking method, also used in the production of United States Food and Drug Administration-approved products, was employed to obtain differentially cross-linked gelatin beads. The colorimetric determination of the in vitro enzymatic susceptibility of the beads indicated that the resistance to degradation linearly correlated with the concentration of carbodiimide used for the cross-linking reaction. This result was also confirmed in vivo by the histological evaluation of the residence time of orthotopically injected cell-seeded beads. These data would indicate that the production of gelatin-based microbeads with tunable degradation profiles might be applicable toward the development of products that catalyze regeneration of kidney and other solid organs.

Novel glycidyl methacrylated dextran/gelatin nanoparticles loaded with basic fibroblast growth factor: formulation and characteristics

Objective: To develop a novel efficient nanoparticulate carrier loaded with basic fibroblast growth factor (bFGF). Methods: Gelatin and glycidyl methacrylate-derivatized dextran (dex-GMA) were cross-linked and polymerized to form interpenetrating polymeric networks. The properties of the nanoparticles (NPs) were investigated as a function of the degree of dex-GMA substitution and the concentration of gelatin used in the preparation of the hydrogels. The morphology was observed with scanning eletromicroscopy and transmission eletromicroscopy. The swelling, degradation, and entrapment efficiency were also determined by dynamic evaluation methods in vitro. The protein release ratio and in vitro release kinetics were evaluated by routine procedure, and the biological activity of bFGF-loaded NPs was studied by cell proliferation assay, cell attachment, and cell function. Results: The NPs have a particle size of 320 ± 20 nm. bFGF was entrapped in the nanoparticles quantitatively (the encapsulation efficiency, 89.6 ± 0.9%). The bFGF in vitro release kinetics fitted to zero-order and Higuchi equations. Proliferation assay, attachment assay, and western blot showed that bFGF NPs had good biological effects on cultured bone marrow mesenchymal stem cells and could achieve a much longer action time than bFGF solution. Conclusion: These results suggested that a novel biodegradable dex-GMA/gelatin hydrogel NPs loaded with bFGF could be successfully developed from both dextran- and gelatin-based biomaterials.

Novel glycidyl methacrylated dextran (Dex-GMA)/gelatin hydrogel scaffolds containing microspheres loaded with bone morphogenetic proteins: Formulation and characteristics

Novel thermomechanical hydrogel scaffolds containing our previously prepared microspheres loaded with bone morphogenetic proteins (BMP) were successfully generated by radical crosslinking and low dose γ-irradiation from combination of two kind of biomaterials: glycidyl methacrylated dextran (Dex-GMA) and gelatin. The structure of those resulting smart hybrid hydrogels was evaluated by mercury intrusion porosimetry (MIP) and scanning electron microscopy (SEM) analyses, and as a function of the degree of Dex-GMA's substitution (DS), the proportion between Dex-GMA and gelatin, and the initial polyethyleneglycol (PEG) concentration used in the preparation of the hydrogels. The swelling and degradation properties and the temperature-sensitive drug release manner were determined by dynamic evaluation methods in vitro, and the gel content was also calculated. MIP analysis showed that by systematically altering the preparation parameters, the overall networks were clearly macroporous with pore sizes ranging from 5.6 ± 4.2 to 37.7 ± 13.7 μm. As expected, the pore size decreased as DS and initial PEG concentration increased, whereas the opposite was found for the gel content. Moreover, the porosity values ranged from 73.7 ± 12.4% up to 89.6 ±6.3%. The SEM results also showed the inter-connective pores as well as microspheres encased into their porous structure of those hydrogels. The swelling and degradation properties of the resultant hydrogels varied according to the DS of Dex-GMA and initial PEG concentration, while the proportion between Dex-GMA and gelatin had no significant influence on those characterizations. By changing the composition ratio of the two precursors, the phase transition temperature (lower critical solution temperature, LSCT) of the hydrogel scaffolds could also be adjusted to be or near the body temperature, so BMP release from microsphere–hydrogel compounds could be accordingly controlled and the release period could be varied from 18 to more than 28 days. These results demonstrated that a novel temperature-sensitive and biodegradable Dex-GMA/gelatin scaffold containing microspheres loaded with BMP could be successfully developed from both dextran- and gelatin-based biomaterials, which could promisingly satisfy the need, desire, and expectation of both self-regulated drug delivery and tissue-engineering applications.

Mechanical and thermal properties of novel polymerized NDGA–gelatin hydrogels

Nordihydroguaiaretic acid (NDGA), an antioxidant with two functional ortho-catechols from the creosote bush, has been shown to increase the mechanical properties of synthetic collagen fibers, producing biologically based, biocompatible fibers with material properties in uniaxial tensile tests to failure that are comparable to those of native tendon (Koob and Hernandez, Biomaterials 23 (2002) 203; Koob et al., J Biomed Mater Res, 56 (2001) 31; 56 (2001) 40). The NDGA polymerization scheme was applied to gelatin hydrogels to determine whether it could provide a viable approach for producing gelatin based biological materials with advantageous mechanical and thermal properties. NDGA treatment eliminated gelatin solubilization from hydrogels in chaotropic agents and increased the thermal stability of gelatin hydrogels from less than 37°C to over 80°C. NDGA caused a dose dependent increase in the compressive stiffness and fracture load of gels ranging in concentration from 2.5% to 40% gelatin in uniaxial, unconfined compression tests to failure. Maximum fracture load averaged 0.5±0.1 MPa and the compressive modulus averaged 4.4±1.4 MPa for all gelatin concentrations, however, the concentration of NDGA that produced maximum strength and stiffness varied inversely with gelatin concentration. The compressive strength and stiffness of 5% gelatin hydrogels treated with NDGA were independent of temperature up to 52°C. These results indicate that NDGA polymerization renders gelatin hydrogels thermally and mechanically stable and thereby potentially useful for surgical procedures that would benefit from biocompatible, stable and mechanically competent gelatin-based biomaterials.