Tissue engineered bone grafts based on biomimetic nanocomposite PLGA/amorphous calcium phosphate scaffold and human adipose-derived stem cells.

For tissue engineering of critical size bone grafts, nanocomposites are getting more and more attractive due to their controllable physical and biological properties. We report in vitro and in vivo behaviour of an electrospun nanocomposite based on poly-lactic-co-glycolic acid and amorphous calcium phosphate nanoparticles (PLGA/a-CaP) seeded with human adipose-derived stem cells (ASC) compared to PLGA. Major findings were that cell attachment, three-dimensional ingrowth and proliferation were very good on both materials. Cell morphology changed from a spindle-shaped fibroblast-like form to a more roundish type when ASC were seeded on PLGA, while they retained their morphology on PLGA/a-CaP. Moreover, we found ASC differentiation to a phenotype committed towards osteogenesis when a-CaP nanoparticles were suspended in normal culture medium without any osteogenic supplements, which renders a-CaP nanoparticles an interesting osteoinductive component for the synthesis of other nanocomposites than PLGA/a-CaP. Finally, electrospun PLGA/a-CaP scaffold architecture is suitable for a rapid and homogenous vascularisation confirmed by a complete penetration by avian vessels from the chick chorioallantoic membrane (CAM) within one week.

Biocompatibility and Bone Formation of Flexible, Cotton Wool-like PLGA/Calcium Phosphate Nanocomposites in Sheep.

BACKGROUND: The purpose of this preliminary study was to assess the in vivo performance of synthetic, cotton wool-like nanocomposites consisting of a biodegradable poly(lactide-co-glycolide) fibrous matrix and containing either calcium phosphate nanoparticles (PLGA/CaP 60:40) or silver doped CaP nanoparticles (PLGA/Ag-CaP 60:40). Besides its extraordinary in vitro bioactivity the latter biomaterial (0.4 wt% total silver concentration) provides additional antimicrobial properties for treating bone defects exposed to microorganisms. MATERIALS AND METHODS: Both flexible artificial bone substitutes were implanted into totally 16 epiphyseal and metaphyseal drill hole defects of long bone in sheep and followed for 8 weeks. Histological and histomorphological analyses were conducted to evaluate the biocompatibility and bone formation applying a score system. The influence of silver on the in vivo performance was further investigated. RESULTS: Semi-quantitative evaluation of histology sections showed for both implant materials an excellent biocompatibility and bone healing with no resorption in the adjacent bone. No signs of inflammation were detectable, either macroscopically or microscopically, as was evident in 5 µm plastic sections by the minimal amount of inflammatory cells. The fibrous biomaterials enabled bone formation directly in the centre of the former defect. The area fraction of new bone formation as determined histomorphometrically after 8 weeks implantation was very similar with 20.5 ± 11.2 % and 22.5 ± 9.2 % for PLGA/CaP and PLGA/Ag-CaP, respectively. CONCLUSIONS: The cotton wool-like bone substitute material is easily applicable, biocompatible and might be beneficial in minimal invasive surgery for treating bone defects.

Two-layer membranes of calcium phosphate/collagen/PLGA nanofibres: in vitro biomineralisation and osteogenic differentiation of human mesenchymal stem cells.

The present study evaluates the in vitro biomedical performance of an electrospun, flexible, anisotropic bilayer with one layer containing a collagen to mineral ratio similar to that in bone. The double membrane consists of a poly(lactide-co-glycolide) (PLGA) layer and an amorphous calcium phosphate (a-CaP)/collagen (Col)/PLGA layer. In vitro biomineralisation and a cell culture study with human mesenchymal stem cells (hMSC) were conducted to characterise such membranes for possible application as biomaterials. Nanofibres with different a-CaP/Col/PLGA compositions were synthesised by electrospinning to mimic the actual composition of bone tissue. Immersion in simulated body fluid and in cell culture medium resulted in the deposition of a hydroxyapatite layer. Incubation of hMSC for 4 weeks allowed for assessment of the proliferation and osteogenic differentiation of the cells on both sides of the double membrane. Confocal laser scanning microscopy was used to observe the proper adhesion of the cells. Calcium and collagen content was proven by Alizarin red S and Sirius red assays. Acute cytotoxic effects of the nanoparticles or the chemicals used in the scaffold preparation could be excluded based on viability assays (alamarBlue and alkaline phosphatase activity). The findings suggest possible application of such double membranes is in treatment of bone defects with complex geometries as wound dressing material.

Light-curable polymer/calcium phosphate nanocomposite glue for bone defect treatment.

Light-curable, methacrylate-based resins are clinically used for complex defect repair in dentistry (Heliobond). The present study investigates how such easy-to-apply polymers may be used on a much broader range of applications, particularly for gluing wet bone. We investigate the significantly improved adhesion of the polymer to wet bone surfaces in a close to in vivo setup using freshly cut cow hip bone as a model. The use of a reactive filler (20 wt.% amorphous, glassy calcium phosphate nanoparticles, a-CaP) allows for combination of the properties of the polymer (strength; light-curing) and the reactive filler (recrystallization of amorphous CaP to hydroxyapatite within minutes). This filler alone has been earlier suggested for use as an injectable bone cement since it reacts under in vivo conditions within 10-15 min. Our study transfers this reactivity into a composite, thus using the reactive CaP phase to establish an improved adhesion of the composite to wet bone surfaces. Additional in vitro bioactivity tests, compressive and tensile strength suggest use of such light-curable nanocomposites for complex-shaped load-bearing implant materials and fracture repair.

In vivo and in vitro evaluation of flexible, cottonwool-like nanocomposites as bone substitute material for complex defects.

The easy clinical handling and applicability of biomaterials has become a focus of materials research due to rapidly increasing time and cost pressures in the public health sector. The present study assesses the in vitro and in vivo performance of a flexible, mouldable, cottonwool-like nanocomposite based on poly(lactide-co-glycolide) and amorphous tricalcium phosphate nanoparticles (PLGA/TCP 60:40). Immersion in simulated body fluid showed exceptional in vitro bioactivity for TCP-containing fibres (mass gain: 18%, 2 days, HAp deposition). Bone regeneration was quantitatively investigated by creating four circular non-critical-size calvarial defects in New Zealand White rabbits. The defects were filled with the easy applicable cottonwool-like PLGA/TCP fibres or PLGA alone. Porous bovine-derived mineral (Bio-Oss) was used as a positive control and cavities left empty served as a negative control. The area fraction of newly formed bone (4 weeks implantation) was significantly increased for TCP-containing fibres compared to pure PLGA (histological and micro-computed tomographic analysis). A spongiosa-like structure of the newly formed bone tissue was observed for PLGA/TCP nanocomposites, whereas Bio-Oss-treated defects afforded a solid cortical bone.

Micro-organism-triggered release of silver nanoparticles from biodegradable oxide carriers allows preparation of self-sterilizing polymer surfaces.

The antimicrobial activity of silver has attracted significant research interest and contributes to an exponentially growing use of this noble metal in commodity products. In this investigation, we describe a general approach to increase the antimicrobial activity of a silver-containing surface by two to three orders of magnitude. The use of 1-2-nm silver particles decorating the surface of 20-50-nm carrier particles consisting of a phosphate-based, biodegradable ceramic allows the triggered release of silver in the presence of a growing microorganism. This effect is based on the organism's requirements for mineral uptake during growth creating a flux of calcium, phosphate, and other ions to the organism. The growing micro-organism dissolves the carrier containing these nutrients and thereby releases the silver nanoparticles. Further, we demonstrate the rapid self-sterilization of polymer surfaces containing silver on calcium phosphate nanoparticles using a series of human pathogens. Colony-forming units (viable bacteria or fungi counts) have been routinely reduced below detection limit and suggest application of these self-sterilizing surfaces in hospital environments, food and pharmaceutical processing, and personal care.

Cotton wool-like nanocomposite biomaterials prepared by electrospinning: in vitro bioactivity and osteogenic differentiation of human mesenchymal stem cells.

The present study evaluates the in vitro biomedical performance of an electrospun, flexible, and cotton wool-like poly(lactide-co-glycolide) (PLGA)/amorphous tricalcium phosphate (ATCP) nanocomposite. Experiments on in vitro biomineralization, applicability in model defects and a cell culture study with human mesenchymal stem cells (hMSC) allowed assessing the application of the material for potential use as a bone graft. Scaffolds with different flame made ATCP nanoparticle loadings were prepared by electrospinning of a PLGA-based composite. Immersion in simulated body fluid showed significant deposition of a hydroxyapatite layer only on the surface of ATCP doped PLGA (up to 175% mass gain within 15 days for PLGA/ATCP 60:40). Proliferation and osteogenic differentiation of hMSC on different nanocomposites were assessed by incubating cells in osteogenic medium for 4 weeks. Proper adhesion and an unaffected morphology of the cells were observed by confocal laser scanning microscopy for all samples. Fluorometric quantification of dsDNA and analysis of ALP activity revealed no significant difference between the tested scaffolds and excluded any acute cytotoxic effects of the nanoparticles. The osteocalcin content for all scaffolds was 0.12-0.19 ng/ng DNA confirming osteogenic differentiation of human mesenchymal stem cells on these flexible bone implants.