29 research outputs found
Marine-inspired enzymatic mineralization of dairy-derived whey protein isolate (WPI) hydrogels for bone tissue regeneration
Whey protein isolate (WPI) is a by-product from the production of cheese and Greek yoghurt comprising β-lactoglobulin (β-lg) (75%). Hydrogels can be produced from WPI solutions through heating; hydrogels can be sterilized by autoclaving. WPI hydrogels have shown cytocompatibility and ability to enhance proliferation and osteogenic differentiation of bone-forming cells. Hence, they have promise in the area of bone tissue regeneration. In contrast to commonly used ceramic minerals for bone regeneration, a major advantage of hydrogels is the ease of their modification by incorporating biologically active substances such as enzymes. Calcium carbonate (CaCO3) is the main inorganic component of the exoskeletons of marine invertebrates. Two polymorphs of CaCO3, calcite and aragonite, have shown the ability to promote bone regeneration. Other authors have reported that the addition of magnesium to inorganic phases has a beneficial effect on bone-forming cell growth. In this study, we employed a biomimetic, marine-inspired approach to mineralize WPI hydrogels with an inorganic phase consisting of CaCO3 (mainly calcite) and CaCO3 enriched with magnesium using the calcifying enzyme urease. The novelty of this study lies in both the enzymatic mineralization of WPI hydrogels and enrichment of the mineral with magnesium. Calcium was incorporated into the mineral formed to a greater extent than magnesium. Increasing the concentration of magnesium in the mineralization medium led to a reduction in the amount and crystallinity of the mineral formed. Biological studies revealed that mineralized and unmineralized hydrogels were not cytotoxic and promoted cell viability to comparable extents (approximately 74% of standard tissue culture polystyrene). The presence of magnesium in the mineral formed had no adverse effect on cell viability. In short, WPI hydrogels, both unmineralized and mineralized with CaCO3 and magnesium-enriched CaCO3, show potential as biomaterials for bone regeneration
Pectin coatings on titanium alloy scaffolds produced by additive manufacturing:Promotion of human bone marrow stromal cell proliferation
Ti6Al4V is a popular biomaterial for load-bearing implants for bone contact, which can be fabricated by additive manufacturing technologies. Their long-term success depends on their stable anchoring in surrounding bone, which in turn depends on formation of new bone tissue on the implant surface, for which adhesion and proliferation of bone-forming cells is a pre-requisite. Hence, surface coatings which promote cell adhesion and proliferation are desirable. Here, Ti6Al4V discs prepared by additive manufacturing (EBM) were coated with layers of pectins, calcium-binding polysaccharides derived from citrus (C) and apple (A), which also contained alkaline phosphatase (ALP), the enzyme responsible for mineralization of bone tissue. Adhesion and proliferation of human bone marrow stromal cells (hBMSC) were assessed. Proliferation after 7 days was increased by A-ALP coatings and, in particular, by C-ALP coatings. Cell morphology was similar on coated and uncoated samples. In conclusion, ALP-loaded pectin coatings promote hBMSC adhesion and proliferation
Degradation, Bioactivity, and Osteogenic Potential of Composites Made of PLGA and Two Different Sol–Gel Bioactive Glasses
We have developed poly(l-lactide-co-glycolide) (PLGA) based composites using sol–gel derived bioactive glasses (S-BG), previously described by our group, as composite components. Two different composite types were manufactured that contained either S2—high content silica S-BG, or A2—high content lime S-BG. The composites were evaluated in the form of sheets and 3D scaffolds. Sheets containing 12, 21, and 33 vol.% of each bioactive glass were characterized for mechanical properties, wettability, hydrolytic degradation, and surface bioactivity. Sheets containing A2 S-BG rapidly formed a hydroxyapatite surface layer after incubation in simulated body fluid. The incorporation of either S-BG increased the tensile strength and Young’s modulus of the composites and tailored their degradation rates compared to starting compounds. Sheets and 3D scaffolds were evaluated for their ability to support growth of human bone marrow cells (BMC) and MG-63 cells, respectively. Cells were grown in non-differentiating, osteogenic or osteoclast-inducing conditions. Osteogenesis was induced with either recombinant human BMP-2 or dexamethasone, and osteoclast formation with M-CSF. BMC viability was lower at higher S-BG content, though specific ALP/cell was significantly higher on PLGA/A2-33 composites. Composites containing S2 S-BG enhanced calcification of extracellular matrix by BMC, whereas incorporation of A2 S-BG in the composites promoted osteoclast formation from BMC. MG-63 osteoblast-like cells seeded in porous scaffolds containing S2 maintained viability and secreted collagen and calcium throughout the scaffolds. Overall, the presented data show functional versatility of the composites studied and indicate their potential to design a wide variety of implant materials differing in physico-chemical properties and biological applications. We propose these sol–gel derived bioactive glass–PLGA composites may prove excellent potential orthopedic and dental biomaterials supporting bone formation and remodeling
Enzymatic, urease-mediated mineralization of gellan gum hydrogel with calcium carbonate, magnesium-enriched calcium carbonate and magnesium carbonate for bone regeneration applications.
Mineralization of hydrogel biomaterials is considered desirable to improve their suitability as materials for bone regeneration. Calcium carbonate (CaCO3) has been successfully applied as a bone regeneration material, but hydrogel‐CaCO3 composites have received less attention. Magnesium (Mg) has been used as a component of calcium phosphate biomaterials to stimulate bone‐forming cell adhesion and proliferation and bone regeneration in vivo, but its effect as a component of carbonate‐based biomaterials remains uninvestigated. In the present study, gellan gum (GG) hydrogels were mineralized enzymatically with CaCO3, Mg‐enriched CaCO3 and magnesium carbonate to generate composite biomaterials for bone regeneration. Hydrogels loaded with the enzyme urease were mineralized by incubation in mineralization media containing urea and different ratios of calcium and magnesium ions. Increasing the magnesium concentration decreased mineral crystallinity. At low magnesium concentrations calcite was formed, while at higher concentrations magnesian calcite was formed. Hydromagnesite (Mg5(CO3)4(OH)2.4H2O) formed at high magnesium concentration in the absence of calcium. The amount of mineral formed and compressive strength decreased with increasing magnesium concentration in the mineralization medium. The calcium:magnesium elemental ratio in the mineral formed was higher than in the respective mineralization media. Mineralization of hydrogels with calcite or magnesian calcite promoted adhesion and growth of osteoblast‐like cells. Hydrogels mineralized with hydromagnesite displayed higher cytotoxicity. In conclusion, enzymatic mineralization of GG hydrogels with CaCO3 in the form of calcite successfully reinforced hydrogels and promoted osteoblast‐like cell adhesion and growth, but magnesium enrichment had no definitive positive effect
Modification of heat-induced whey protein isolate hydrogel with highly bioactive glass particles results in promising biomaterial for bone tissue engineering
This study deals with the design and comprehensive evaluation of novel hydrogels based on whey protein isolate (WPI) for tissue regeneration. So far, WPI has been considered mainly as a food industry by-product and there are very few reports on the application of WPI in tissue engineering (TE). In this work, WPI-based hydrogels were modified with bioactive glass (BG), which is commonly used as a bone substitute material. Ready-to-use, sterile hydrogels were produced by a simple technique, namely heat-induced gelation. Two different concentrations (10 and 20% w/w) of sol–gel-derived BG particles of two different sizes (2.5 and <45 µm) were compared. µCT analysis showed that hydrogels were highly porous with almost 100% pore interconnectivity. BG particles were generally homogenously distributed in the hydrogel matrix, affecting pore size, and reducing material porosity. Thermal analysis showed that the presence of BG particles in WPI matrix reduced water content in hydrogels and improved their thermal stability. BG particles decreased enzymatic degradation of the materials. The materials underwent mineralization in simulated biological fluids (PBS and SBF) and possessed high radical scavenging capacity. In vitro tests indicated that hydrogels were cytocompatible and supported MG-63 osteoblastic cell functions
Phenolic plant extract enrichment of enzymatically mineralized hydrogels
Hydrogel mineralization with calcium phosphate (CaP) and antibacterial activity are desirable for applications in bone regeneration. Mineralization with CaP can be induced using the enzyme alkaline phosphatase (ALP), responsible for CaP formation in bone tissue. Incorporation of polyphenols, plant-derived bactericidal molecules, was hypothesized to provide antibacterial activity and enhance ALP-induced mineralization. Three phenolic rich plant extracts from: (i) green tea, rich in epigallocatechin gallate (EGCG) (herafter referred to as EGCG-rich extract); (ii) pine bark and (iii) rosemary were added to gellan gum (GG) hydrogels and subsequently mineralized using ALP. The phenolic composition of the three extracts used were analyzed by ultra-high-performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MSn). EGCG-rich extract showed the highest content of phenolic compounds and promoted the highest CaP formation as corroborated by dry mass percentage meassurements and ICP-OES de-termination of mass of elemental Ca and P. All three extracts alone exhibited antibacterial activity in the following order EGCG-rich > PI > RO, respectively. However, extract-loaded and mineralized GG hydro-gels did not exhibit appreciable antibacterial activity by diffusion test. In conclusion, only the EGCG-rich extract promotes ALP-mediated mineralization
Injectable gellan gum-based nanoparticles-loaded system for the local delivery of vancomycin in osteomyelitis treatment
Infection spreading in the skeletal system
leading to osteomyelitis can be prevented by the prolonged
administration of antibiotics in high doses. However systemic
antibiotherapy, besides its inconvenience and often
low efficacy, provokes numerous side effects. Thus, we
formulated a new injectable nanoparticle-loaded system for
the local delivery of vancomycin (Vanc) applied in a
minimally-invasive way. Vanc was encapsulated in poly(Llactide-
co-glycolide) nanoparticles (NPs) by double-emulsification.
The size (258 ± 11 nm), polydispersity index
(0.240 ± 0.003) and surface potential (-25.9 ± 0.2 mV)
of NPs were determined by dynamic light scattering and
capillary electrophoresis measurements. They have a
spherical morphology and a smooth topography as
observed using atomic force microscopy. Vanc loading and
encapsulation efficiencies were 8.8 ± 0.1 and
55.2 ± 0.5 %, respectively, based on fluorescence spectroscopy
assays. In order to ensure injectability, NPs were
suspended in gellan gum and cross-linked with ; also a
portion of dissolved antibiotic was added to the system.
The resulting system was found to be injectable (extrusion
force 11.3 ± 1.1 N), reassembled its structure after
breaking as shown by rheology tests and ensured required
burst release followed by sustained Vanc delivery. The
system was cytocompatible with osteoblast-like MG-63
cells (no significant impact on cells’ viability was detected). Growth of Staphylococcus spp. reference strains
and also those isolated from osteomyelitic joints was
inhibited in contact with the injectable system. As a result
we obtained a biocompatible system displaying ease of
application (low extrusion force), self-healing ability after
disruption, adjustable drug release and antimicrobial
properties
Mineralization of gellan gum hydrogels with calcium and magnesium carbonates by alternate soaking for bone regeneration applications
Mineralization of hydrogels is desirable prior to applications in bone regeneration. CaCO3 is a widely used bone regeneration material and Mg, when used as a component of calcium phosphate biomaterials, has promoted bone‐forming cell adhesion and proliferation and bone regeneration. In this study, gellan gum (GG) hydrogels were mineralized with carbonates containing different amounts of calcium (Ca) and magnesium (Mg) by alternate soaking in, firstly, a calcium and/or magnesium ion solution and, secondly, a carbonate ion solution. This alternate soaking cycle was repeated five times. Five different calcium and/or magnesium ion solutions, containing different molar ratios of Ca to Mg ranging from Mg‐free to Ca‐free were compared. Carbonate mineral formed in all sample groups subjected to the Ca:Mg elemental ratio in the carbonate mineral formed was higher than in the respective mineralizing solution. Mineral formed in the absence of Mg was predominantly CaCO3 in the form of a mixture of calcite and vaterite. Increasing the Mg content in the mineral formed led to the formation of magnesian calcite, decreased the total amount of the mineral formed and its crystallinity. Hydrogel mineralization and increasing Mg content in mineral formed did not obviously improve proliferation of MC3T3‐E1 osteoblast‐like cells or differentiation after 7 days