New injectable self-assembled hydrogels that promote angiogenesis through a bioactive degradation product.

Hydrogels used in regenerative medicine are often designed to allow cellular infiltration, degradation, and neovascularization. Low molecular weight hydrogels (LMWHs), formed by self-assembly via non-covalent interactions, are gaining significant interest because they are soft, easy to use and injectable. We propose LMWHs as suitable body implant materials that can stimulate tissue regeneration. We produced four new LMWHs with molecular entities containing nucleic acid and lipid building blocks and analyzed the foreign body response upon subcutaneous implantation into mice. Despite being infiltrated with macrophages, none of the hydrogels triggered detrimental inflammatory responses. Most macrophages present in the hydrogel-surrounding tissue acquired an immuno-modulatory rather than inflammatory phenotype. Concomitantly, no fibrotic capsule was formed after three weeks. Our glyconucleolipid LMWHs exhibited different degradation kinetics in vivo and in vitro. LMWHs with high angiogenic properties in vivo, were found to release glyconucleoside (glucose covalently linked to thymidine via a triazole moiety) as a common by-product of in vitro LMWH degradation. Chemically synthesized glyconucleoside exhibited angiogenic properties in vitro in scratch assays with monolayers of human endothelial cells and in vivo using the chick chorioallantoic membrane assay. Collectively, LMWHs hold promise as efficient scaffolds for various regenerative applications by displaying good biointegration without causing fibrosis, and by promoting angiogenesis through the release of a pro-angiogenic degradation product. STATEMENT OF SIGNIFICANCE: The main limitations of biomaterials developed in the field of tissue engineering remains their biocompatibility and vascularisation properties. In this context, we developed injectable Low Molecular Weight Hydrogels (LMWH) exhibiting thixotropic (reversible gelation) and thermal reversible properties. LMWH having injectability is of great advantage since it allows for their delivery without wounding the surrounding tissues. The resulting gels aim at forming scaffolds that the host cells colonize without major inflammation, and that won't be insulated by a strong fibrosis reaction. Importantly, their molecular degradation releases a product (a glycosyl-nucleoside conjugate) promoting angiogenesis. In this sense, these LMWH represent an important advance in the development of biomaterials promoting tissue regeneration.

Development of bone screw using novel biodegradable composite orthopedic biomaterial: from material design to in vitro biomechanical and in vivo biocompatibility evaluation.

A novel composite biomaterial for bone-soft tissue fixation applications was developed. MgO-Silk-PCL, Silk-PCL and MgO-PCL composites were prepared with variable filler concentrations (0, 10, 20 and 30% w/w of MgO nanoparticles and 0%, 5%, 10%, 20% and 30% of degummed silk fiber) in PCL polymer. The highest mechanical properties were obtained with 10% MgO and 20% Silk composite (MSP) wih 1.7× better tensile strength and 7.5× tensile modulus over PCL. It exhibited good cell viability, adhesion and hemocompatibility, increased cell proliferation and differentiation. MgO filler contributed more in increasing tensile strength, whereas silk fiber towards modulus, imparting a synergistic effect on mechanical performance. Prototype bone screws were molded using the MSP composite in a custom-designed mold. It showed significantly increased degradation (2.7 fold after 60 days) in PBS attributable to binary filler phase as compared to PCL. In vivo biosafety studies of MgO-silk-PCL composite screw in SD rats by subcutaneous implantation showed moderate inflammation at 2 weeks which subsided after 4th week. No toxic effect was seen in histopathology of vital organs and in blood parameters. Composite screw showed 2× pull-out strength of PCL in synthetic bone, therefore a potential candidate for bone-soft fixation applications like resorbable orthopedic screws for ACL reconstruction.

Prolonged delivery of BMP-2 by a non-polymer hydrogel for bone defect regeneration.

Bone morphogenetic protein 2 (BMP-2) is a potent inducer of bone formation that is currently used in a limited number of clinical indications to treat extensive bone loss. Extending the field of applications of this molecule requires design of the delivery system to protect the protein from early degradation and allow a slow long-term release. This study describes the use of a non-polymer hydrogel, based on the self-assembly of small amphiphilic glycosyl-nucleolipids into micellar structures, as a new type of delivery system for BMP-2. BMP-2 was readily encapsulated in glycosyl-nucleosyl-fluorinated (GNF)-based gels and slowly released in vitro, while maintaining its osteogenic activity. When hydrogel pieces containing fluorophore-tagged BMP-2 were deposited onto a calvaria defect in mouse, the signal detected in living mice gradually decreased and was still detectable at 3 weeks. Gel-embedded protein promoted significant calvarial bone defect regeneration at 8 weeks after surgery. In contrast, when a solution of BMP-2 without hydrogel was injected into the defects, the fluorescence signal decreased rapidly and no significant bone formation was observed. The unique property of the GNF-based hydrogel as an injectable delivery system for low doses of BMP-2 was revealed in a subcutaneous model of ectopic bone formation. Injected BMP-2-laden GNF hydrogel blocks elicited the formation of cancellous bone, showing all the typical features of remodeling bone that contains bone marrow. These results show that this GNF-based hydrogel is an easy-to-use, efficient delivery system for BMP-2 and osteogenic material to support bone regeneration.

Magnesium oxide nanoparticle-loaded polycaprolactone composite electrospun fiber scaffolds for bone-soft tissue engineering applications: in-vitro and in-vivo evaluation.

The objective of the present investigation was to assess the potential of magnesium oxide nanoparticle (MgO NP)-loaded electrospun polycaprolactone (PCL) polymer composites as a bone-soft tissue engineering scaffold. MgO NPs were synthesized using a hydroxide precipitation sol-gel method and characterized using field emission gun-scanning electron microscopy/energy-dispersive x-ray spectroscopy (FEG-SEM/EDS), field emission gun-transmission electron microscopy (FEG-TEM), and x-ray diffraction (XRD) analysis. PCL and MgO-PCL nanocomposite fibers were fabricated using electrospinning with trifluoroethanol as solvent at 19 kV applied voltage and 1.9 ml h-1 flow rate as optimized process parameters, and were characterized by FEG-TEM, FEG-SEM/EDS, XRD, and differential scanning calorimetry analyses. Characterization studies of as-synthesized nanoparticles revealed diffraction peaks indexed to various crystalline planes peculiar to MgO particles with hexagonal and cubical shape, and 40-60 nm size range. Significant improvement in mechanical properties (tensile strength and elastic modulus) of nanocomposites was observed as compared to neat polymer specimens (fourfold and threefold, respectively), due to uniform dispersion of nanofillers along the polymer fiber length. There was a remarkable bioactivity shown by nanocomposite scaffolds in immersion test, as indicated by formation of surface hydroxyapatite layer by the third day of incubation. MgO-loaded electrospun PCL mats showed enhanced in-vitro biological performance with osteoblast-like MG-63 cells in terms of adhesion, proliferation, and marked differentiation marker activity owing to greater surface roughness, nanotopography, and hydrophilicity facilitating higher protein adsorption. In-vivo subcutaneous implantation study in Sprague Dawley rats revealed initial moderate inflammatory tissue response near implant site at the second week timepoint that subsided later (eighth week) with no adverse effect on vital organ functionalities as seen in histopathological analysis supported by serum biochemical and hematological parameters which did not deviate significantly from normal physiological range, indicating good biocompatibility in-vivo. Thus, MgO-PCL nanocomposite electrospun fibers have potential as an efficient scaffold material for bone-soft tissue engineering applications.

Beneficial effects of garlic (Allium sativum Linn) on rats fed with diets containing cholesterol and either of the oil seeds, coconuts or groundnuts.

Feeding of 2% cholesterol diet increased lipid parameters in serum and tissues of rats during a period of one month. In addition to the above, lipid peroxidation also increased and activities of certain enzymes were significantly altered in the tissues. Similar changes were also observed to a greater extent with diets containing 40% by weight of coconut kernel or groundnut with and without 2% cholesterol. The enzymes studied were HMGCoA reductase, AST, ALT and ALP in tissues and serum as the case may be. In general the atherogenic effects were observed more with groundnut containing diets than those with coconut. Even though the oil from the former is mostly unsaturated and that from the latter is mostly saturated, these analytical criteria do not relate to their atherogenic effects. When 5% garlic was incorporated with any of the high fat diets, the lipid parameters, their peroxidation and alterations in enzyme activities were significantly decreased. These results show that garlic contains some principles that counteract the atherogenicity of the above oil seeds.