Sodium Trimetaphosphate Crosslinked Starch Films Reinforced with Montmorillonite.

Synthetic polymers are the main food packaging material, although they are nonbiodegradable and their recycling process is expensive. A biodegradable, eco-friendly material, with high availability and low cost, such as starch, is a promising solution for the production of films for food packaging. To enhance starch film mechanical and barrier properties, nanoclays have been incorporated within the film matrix. Crosslinking is a well-established method to modify starch properties, but it has not been investigated in combination with nanoclay addition. In the present study, films were developed with starch that was crosslinked through the addition of 5, 15, and 40% wt. sodium trimetaphosphate (STMP) based on dry starch weight. To investigate the interaction between crosslinking and nanoclay addition, montmorillonite (MMT) was added at a 10.5% wt. concentration based on dry starch weight. Experimental data revealed a synergistic effect between STMP crosslinking and MMT addition regarding film thickness, elongation at break, color properties, and opacity. Regarding barrier properties, MMT addition negated the effect of STMP crosslinking, while, in the case of moisture content, it did not alter the effect of STMP crosslinking. Finally, in the case of tensile strength, a synergistic effect followed by a negative interaction was observed. In conclusion, the addition of MMT can potentially enhance, alongside crosslinking, some properties of the films, while other properties are not affected any more than just by crosslinking.

Optimizing the Functional Properties of Starch-Based Biodegradable Films.

A definitive screening design was used in order to evaluate the effects of starch, glycerol and montmorillonite (MMT) concentrations, as well as the drying temperature, drying tray type and starch species, on packaging film's functional properties. Optimization showed that in order to obtain films with the minimum possible thickness, the maximum elongation at break, the maximum tensile strength, as well as reduced water vapor permeability and low opacity, a combination of factors should be used as follows: 5.5% wt starch concentration, 30% wt glycerol concentration on a dry starch basis, 10.5% wt MMT concentration on a dry starch basis, 45 °C drying temperature, chickpea as the starch species and plexiglass as the drying tray type. Based on these results, starch films were prepared, and fresh minced meat was stored in them for 3 days. It was shown that the incorporation of MMT at 10.5% wt on a dry starch basis in the packaging films led to a decreased mesophilic and psychrotrophic bacteria growth factor compared to commercial packaging. When assessed for their biodegradability, the starch films disintegrated after 10 days of thermophilic incubation under simulated composting conditions. Finally, to prove their handling capability during industrial production, the starch films were rewound in a paper cylinder using an industrial-scale rewinding machine.

Capturing the onset of oral processing: Merging of a model food emulsion drop with saliva.

The events occurring before and during the merging of a model liquid food emulsion with saliva have been captured ex vivo using confocal microscopy. In the order of a few seconds, millimeter-sized drops of liquid food and saliva touch and are deformed; the two surfaces eventually collapse, resulting in the merging of the two phases, in a process reminiscent of emulsion droplets coalescing. The model droplets then surge into saliva. Based on this, two distinct stages can be distinguished for the insertion of a liquid food into the oral cavity: A first phase where two intact phases co-exist, and the individual viscosities and saliva-liquid food tribology should be important to texture perception; and a second stage, dominated by the rheological properties of the liquid food-saliva mixture. The importance of the surface properties of saliva and liquid food are highlighted, as they may influence the merging of the two phases.

In vivo evidence of oral vaccination with PLGA nanoparticles containing the immunostimulant monophosphoryl lipid A.

Although oral vaccination has numerous advantages over the commonly used parenteral route, degradation of vaccine and its low uptake in the lymphoid tissue of the gastrointestinal (GI) tract still impede their development. In this study, the model antigen ovalbumin (OVA) and the immunostimulant monophosphoryl lipid A (MPLA) were incorporated in polymeric nanoparticles based on poly(D,L-lactide-co-glycolide) (PLGA). These polymeric carriers were orally administered to BALB/c mice (Bagg albino, inbred strain of mouse) and the resulting time-dependent systemic and mucosal immune responses towards OVA were assessed by measuring the OVA-specific IgG and IgA titers using an enzyme-linked immunosorbent assay (ELISA). PLGA nanoparticles were spherical in shape, around 320 nm in size, negatively charged (around -20 mV) and had an OVA and MPLA payload of 9.6% and 0.86%, respectively. A single immunization with formulation containing (OVA + MPLA) incorporated in PLGA nanoparticles induced a stronger IgG immune response than that induced by OVA in PBS solution or OVA incorporated into PLGA nanoparticles. Moreover, significantly higher IgA titers were generated by administration of (OVA + MPLA)/PLGA nanoparticles compared to IgA stimulated by control formulations, proving the capability of inducing a mucosal immunity. These findings demonstrate that co-delivery of OVA and MPLA in PLGA nanoparticles promotes both systemic and mucosal immune responses and represents therefore a suitable strategy for oral vaccination.

RGD-functionalized bioengineered spider dragline silk biomaterial.

Spider silk fibers have remarkable mechanical properties that suggest the component proteins could be useful biopolymers for fabricating biomaterial scaffolds for tissue formation. Two bioengineered protein variants from the consensus sequence of the major component of dragline silk from Nephila clavipes were cloned and expressed to include RGD cell-binding domains. The engineered silks were characterized by CD and FTIR and showed structural transitions from random coil to insoluble beta-sheet upon treatment with methanol. The recombinant proteins were processed into films and fibers and successfully used as biomaterial matrixes to culture human bone marrow stromal cells induced to differentiate into bone-like tissue upon addition of osteogenic stimulants. The recombinant spider silk and the recombinant spider silk with RGD encoded into the protein both supported enhanced the differentiation of human bone marrow derived mesenchymal stem cells (hMSCs) to osteogenic outcomes when compared to tissue culture plastic. The recombinant spider silk protein without the RGD displayed enhanced bone related outcomes, measured by calcium deposition, when compared to the same protein with RGD. Based on comparisons to our prior studies with silkworm silks and RGD modifications, the current results illustrate the potential to bioengineer spider silk proteins into new biomaterial matrixes, while also highlighting the importance of subtle differences in silk sources and modes of presentation of RGD to cells in terms of tissue-specific outcomes.

Porous silk fibroin 3-D scaffolds for delivery of bone morphogenetic protein-2 in vitro and in vivo.

Bone morphogenetic protein-2 (BMP-2) plays a key role in osteogenesis. Biomaterials used for the sustained delivery of BMP-2 in vivo have shown therapeutic benefits. In the present study, BMP-2 was loaded in porous silk fibroin scaffolds derived from silkworm cocoons (2.4 +/- 0.14 microg per scaffold). The release profile of BMP-2 under dynamic culture conditions (spinner flasks) showed that after 1 week in culture 25% of the initial BMP-2 was retained adsorbed to the scaffold; up to 4 weeks no additional BMP-2 was released. BMP-2 induced human bone marrow stromal cells (hMSCs) to undergo osteogenic differentiation when the seeded scaffolds were cultured in medium supplemented with osteogenic stimulants for 4 weeks, based on elevated alkaline phosphatase activity, calcium deposition, and transcript levels for bone sialoprotein, osteopontin, osteocalcin, BMP-2, and cbfa-1. Micro-computed tomography revealed densely deposited mineral at the center of the scaffolds. In contrast, hMSCs cultured in control scaffolds (no BMP-2) exhibited limited osteogenesis. When implanted in critical sized cranial defects in mice, scaffolds loaded with BMP-2 and seeded with hMSCs resulted in significant bone ingrowth. These results were qualitatively similar to scaffolds loaded with BMP-2 but no hMSCs or with BMP-2 and hMSCs but not pregrown into bone-like tissue. Bone-related outcomes were improved when compared with the scaffold controls implanted without BMP-2. These studies illustrate the potential use of slow degrading silk fibroin 3-D scaffolds loaded with BMP-2, in combination with hMSCs, in osteogenesis studies in vitro and in vivo, and provide a new range of material properties for these applications.

Porosity of 3D biomaterial scaffolds and osteogenesis.

Porosity and pore size of biomaterial scaffolds play a critical role in bone formation in vitro and in vivo. This review explores the state of knowledge regarding the relationship between porosity and pore size of biomaterials used for bone regeneration. The effect of these morphological features on osteogenesis in vitro and in vivo, as well as relationships to mechanical properties of the scaffolds, are addressed. In vitro, lower porosity stimulates osteogenesis by suppressing cell proliferation and forcing cell aggregation. In contrast, in vivo, higher porosity and pore size result in greater bone ingrowth, a conclusion that is supported by the absence of reports that show enhanced osteogenic outcomes for scaffolds with low void volumes. However, this trend results in diminished mechanical properties, thereby setting an upper functional limit for pore size and porosity. Thus, a balance must be reached depending on the repair, rate of remodeling and rate of degradation of the scaffold material. Based on early studies, the minimum requirement for pore size is considered to be approximately 100 microm due to cell size, migration requirements and transport. However, pore sizes >300 microm are recommended, due to enhanced new bone formation and the formation of capillaries. Because of vascularization, pore size has been shown to affect the progression of osteogenesis. Small pores favored hypoxic conditions and induced osteochondral formation before osteogenesis, while large pores, that are well-vascularized, lead to direct osteogenesis (without preceding cartilage formation). Gradients in pore sizes are recommended for future studies focused on the formation of multiple tissues and tissue interfaces. New fabrication techniques, such as solid-free form fabrication, can potentially be used to generate scaffolds with morphological and mechanical properties more selectively designed to meet the specificity of bone-repair needs.

The inflammatory responses to silk films in vitro and in vivo.

Silks have a long history of biomedical use as sutures. Silk can be purified, chemically modified to attach RGD sequences and processed into highly porous scaffolds for tissue engineering. We report biocompatibility studies of silk films (with or without covalently bound RGD) that were seeded with bone-marrow derived mesenchymal stem cells (MSC) and (a) cultured in vitro with human MSC or (b) seeded with autologous rat MSC and implanted in vivo. Controls for in vitro studies included tissue culture plastic (TCP; negative control), TCP with lipopolysaccharide (LPS) in the cell culture medium (positive control), and collagen films; controls for in vivo studies included collagen, PLA and TCP. After 9 h of culture, the expression of the pro-inflammatory Interleukin 1 beta (IL-1beta) and inflammatory cyclooxygenase 2 (COX-2) in human MSC were comparable for silk, collagen and TCP. After 30 and 96 h, gene expression of IL-1beta and COX-2 in MSC returned to the baseline (pre-seeding) levels. These data were corroborated by measuring IL-1beta and prostaglandin E2 levels in culture medium. The rate of cell proliferation was higher on silk films than either on collagen or TCP. In vivo, films made of silk, collagen or PLA were seeded with rat MSCs, implanted intramuscularly in rats and harvested after 6 weeks. Histological and immunohistochemical evaluation of silk explants revealed the presence of circumferentially oriented fibroblasts, few blood vessels, macrophages at the implant-host interface, and the absence of giant cells. Inflammatory tissue reaction was more conspicuous around collagen films and even more around PLA films when compared to silk. These data suggest that (a) purified degradable silk is biocompatible and (b) the in vitro cell culture model (hMSC seeded and cultured on biomaterial films) gave inflammatory responses that were comparable to those observed in vivo.

Engineering cartilage-like tissue using human mesenchymal stem cells and silk protein scaffolds.

Human mesenchymal stem cells (hMSC) derived from bone marrow aspirates can form the basis for the in vitro cultivation of autologous tissue grafts and help alleviate the problems of immunorejection and disease transmission associated with the use of allografts. We explored the utility of hMSC cultured on protein scaffolds for tissue engineering of cartilage. hMSC were isolated, expanded in culture, characterized with respect to the expression of surface markers and ability for chondrogenic and osteogenic differentiation, and seeded on scaffolds. Four different scaffolds were tested, formed as a highly porous sponge made of: 1) collagen, 2) cross-linked collagen, 3) silk, and 4) RGD-coupled silk. Cell-seeded scaffolds were cultured for up to 4 weeks in either control medium (DMEM supplemented with 10% fetal bovine serum) or chondrogenic medium (control medium supplemented with chondrogenic factors). hMSC attachment, proliferation, and metabolic activity were markedly better on slowly degrading silk than on fast-degrading collagen scaffolds. In chondrogenic medium, hMSC formed cartilaginous tissues on all scaffolds, but the extent of chondrogenesis was substantially higher for hMSC cultured on silk as compared to collagen scaffolds. The deposition of glycosaminoglycan (GAG) and type II collagen and the expression of type II collagen mRNA were all higher for hMSC cultured on silk than on collagen scaffolds. Taken together, these results suggest that silk scaffolds are particularly suitable for tissue engineering of cartilage starting from hMSC, presumably due to their high porosity, slow biodegradation, and structural integrity.

Bone morphogenetic protein-2 decorated silk fibroin films induce osteogenic differentiation of human bone marrow stromal cells.

Bone morphogenetic protein (BMP)-2 has a critical role in bone formation and regeneration. Therefore, the ability to immobilize this molecule in certain matrices may be crucial in bone tissue engineering. Using carbodiimide chemistry, BMP-2 was directly immobilized on silk fibroin films. Whereas human bone marrow stromal cells cultured on unmodified silk fibroin films in the presence of osteogenic stimulants exhibited little if any osteogenesis, the same cells cultured on BMP-2 decorated films in the presence of osteogenic stimulants differentiated into an osteoblastic lineage as assessed by their significantly elevated alkaline phosphatase activity, calcium deposition, and higher transcript levels of collagen type I, bone sialoprotein, osteopontin, osteocalcin, BMP-2, and cbfa1. Using cell culture inserts, it was demonstrated that differentiation was induced by the immobilized protein and not by protein released into the culture medium. Comparison with a similar amount of medium-supplemented BMP-2, where no additional protein was added with medium changes, showed that delivery of BMP-2 immobilized on the biomaterial surface was more efficient than soluble delivery. The results illustrate that BMP-2 covalently coupled on silk biomaterial matrices retains biological function in vitro based on the induction of osteogenic markers in seeded bone marrow stromal cells.

Engineering bone-like tissue in vitro using human bone marrow stem cells and silk scaffolds.

Porous biodegradable silk scaffolds and human bone marrow derived mesenchymal stem cells (hMSCs) were used to engineer bone-like tissue in vitro. Two different scaffolds with the same microstructure were studied: collagen (to assess the effects of fast degradation) and silk with covalently bound RGD sequences (to assess the effects of enhanced cell attachment and slow degradation). The hMSCs were isolated, expanded in culture, characterized with respect to the expression of surface markers and ability for chondrogenic and osteogenic differentiation, seeded on scaffolds, and cultured for up to 4 weeks. Histological analysis and microcomputer tomography showed the development of up to 1.2-mm-long interconnected and organized bonelike trabeculae with cuboid cells on the silk-RGD scaffolds, features still present but to a lesser extent on silk scaffolds and absent on the collagen scaffolds. The X-ray diffraction pattern of the deposited bone corresponded to hydroxyapatite present in the native bone. Biochemical analysis showed increased mineralization on silk-RGD scaffolds compared with either silk or collagen scaffolds after 4 weeks. Expression of bone sialoprotein, osteopontin, and bone morphogenetic protein 2 was significantly higher for hMSCs cultured in osteogenic than control medium both after 2 and 4 weeks in culture. The results suggest that RGD-silk scaffolds are particularly suitable for autologous bone tissue engineering, presumably because of their stable macroporous structure, tailorable mechanical properties matching those of native bone, and slow degradation.

Bone tissue engineering using human mesenchymal stem cells: effects of scaffold material and medium flow.

We report studies of bone tissue engineering using human mesenchymal stem cells (MSCs), a protein substrate (film or scaffold; fast degrading unmodified collagen, or slowly degrading cross-linked collagen and silk), and a bioreactor (static culture, spinner flask, or perfused cartridge). MSCs were isolated from human bone marrow, characterized for the expression of cell surface markers and the ability to undergo chondrogenesis and osteogenesis in vitro, and cultured for 5 weeks. MSCs were positive for CD105/endoglin, and had a potential for chondrogenic and osteogenic differentiation. In static culture, calcium deposition was similar for MSC grown on collagen scaffolds and films. Under medium flow, MSC on collagen scaffolds deposited more calcium and had a higher alcaline phosphatase (AP) activity than MSC on collagen films. The amounts of DNA were markedly higher in constructs based on slowly degrading (modified collagen and silk) scaffolds than on fast degrading (unmodified collagen) scaffolds. In spinner flasks, medium flow around constructs resulted in the formation of bone rods within the peripheral region, that were interconnected and perpendicular to the construct surface, whereas in perfused constructs, individual bone rods oriented in the direction of fluid flow formed throughout the construct volume. These results suggest that osteogenesis in cultured MSC can be modulated by scaffold properties and flow environment.

Human bone marrow stromal cell responses on electrospun silk fibroin mats.

Fibers with nanoscale diameters provide benefits due to high surface area for biomaterial scaffolds. In this study electrospun silk fibroin-based fibers with average diameter 700+/-50 nm were prepared from aqueous regenerated silkworm silk solutions. Adhesion, spreading and proliferation of human bone marrow stromal cells (BMSCs) on these silk matrices was studied. Scanning electron microscopy (SEM) and MTT analyses demonstrated that the electrospun silk matrices supported BMSC attachment and proliferation over 14 days in culture similar to native silk fibroin (approximately 15 microm fiber diameter) matrices. The ability of electrospun silk matrices to support BMSC attachment, spreading and growth in vitro, combined with a biocompatibility and biodegradable properties of the silk protein matrix, suggest potential use of these biomaterial matrices as scaffolds for tissue engineering.

Human bone marrow stromal cell and ligament fibroblast responses on RGD-modified silk fibers.

Adhesion, spreading, proliferation, and collagen matrix production of human bone marrow stromal cells (BMSCs) on an RGD-modified silk matrix was studied. Anterior cruciate ligament fibroblasts (ACLFs) were used as a control cell source. Scanning electron microscopy (SEM) and MTT analyses demonstrated that the modified silk matrices support improved BMSC and ACLF attachment and show higher cell density over 14 days in culture when compared with the non-RGD-modified matrices. Collagen type I transcript levels (at day 7) and content (at day 14) was significantly higher on the RGD-modified substrate than on the nonmodified group. The ability of RGD-coupled silk matrices to support BMSC attachment, which leads to higher cell density and collagen matrix production in vitro, combined with mechanical, fatigue, and biocompatibility properties of the silk protein matrix, suggest potential for use of this biomaterial for tissue engineering.

Macrophage responses to silk.

Silk fibers have potential biomedical applications beyond their traditional use as sutures. The physical properties of silk fibers and films make it a promising candidate for tissue engineering scaffold applications, particularly where high mechanical loads or tensile forces are applied or in cases where low rates of degradation are desirable. A critical issue for biomaterial scaffolds is biocompatibility. The direct inflammatory potential of intact silk fibers as well as extracts was studied in an in vitro system. The results indicate that silk fibers are largely immunologically inert in short- and long-term culture with RAW 264.7 murine macrophage cells while insoluble fibroin particles induced significant TNF release. Soluble sericin proteins extracted from native silk fibers did not induce significant macrophage activation. While sericin did not activate macrophages by itself, it demonstrated a synergistic effect with bacterial lipopolysaccharide. The low level of inflammatory potential of silk fibers makes them promising candidates in future biomedical applications.