The unique calcium chelation property of poly(vinyl phosphonic acid-co-acrylic acid) and effects on osteogenesis in vitro.

There is a clear clinical need for a bioactive bone graft substitute. Poly(vinyl phosphonic acid-co-acrylic acid) (PVPA-co-AA) has been identified as a promising candidate for bone regeneration but there is little evidence to show its direct osteogenic effect on progenitor or mature cells. In this study mature osteoblast-like cells (SaOS-2) and human bone marrow-derived mesenchymal stem cells (hBM-MSCs) were cultured with PVPA-co-AA polymers with different VPA:AA ratio and at different concentrations in vitro. We are the first to report the direct osteogenic effect of PVPA-co-AA polymer on bone cells and, more importantly, this effect was dependent on VPA:AA ratio and concentration. Under the optimized conditions, PVPA-co-AA polymer not only has an osteoconductive effect, enhancing SaOS-2 cell mineralization, but also has an osteoinductive effect to promote hBM-MSCs' osteogenic differentiation. Notably, the same PVPA-co-AA polymer at different concentrations could lead to differential osteogenic effects on both SaOS-2 and hBM-MSCs in vitro. This study furthers knowledge of the PVPA-co-AA polymer in osteogenic studies, which is critical when utilizing the PVPA-co-AA polymer for the design of novel bioactive polymeric tissue engineering scaffolds for future clinical applications. © 2017 The Authors Journal of Biomedical Materials Research Part A Published by Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 168-179, 2018.

Polymer scaffolds with preferential parallel grooves enhance nerve regeneration.

We have modified the surface topography of poly ɛ-caprolactone (PCL) and polylactic acid (PLA) blended films to improve cell proliferation and to guide the regeneration of peripheral nerves. Films with differing shaped grooves were made using patterned silicon templates, sloped walls (SL), V-shaped (V), and square-shaped (SQ), and compared with nongrooved surfaces with micropits. The solvent cast films were tested in vitro using adult adipose-derived stem cells differentiated to Schwann cell-like cells. Cell attachment, proliferation, and cell orientation were all improved on the grooved surfaces, with SL grooves giving the best results. We present in vivo data on Sprague-Dawley rat sciatic nerve injury with a 10-mm gap, evaluating nerve regeneration at 3 weeks across a polymer nerve conduit modified with intraluminal grooves (SL, V, and SQ) and differing wall thicknesses (70, 100, 120, and 210 µm). The SL-grooved nerve conduit showed a significant improvement over the other topographical-shaped grooves, while increasing the conduit wall thickness saw no positive effect on the biological response of the regenerating nerve. Furthermore, the preferred SL-grooved conduit (C) with 70 µm wall thickness was compared with the current clinical gold standard of autologous nerve graft (Ag) in the rat 10-mm sciatic nerve gap model. At 3 weeks postsurgery, all nerve gaps across both groups were bridged with regenerated nerve fibers. At 16 weeks, features of regenerated axons were comparable between the autograft (Ag) and conduit (C) groups. End organ assessments of muscle weight, electromyography, and skin reinnervation were also similar between the groups. The comparable experimental outcome between conduit and autograft, suggests that the PCL/PLA conduit with inner lumen microstructured grooves could be used as a potential alternative treatment for peripheral nerve repair.

The osteoblast and osteoclast responses to phosphonic acid containing poly(ε-caprolactone) electrospun scaffolds.

We describe a novel polymer containing phosphonic acid moieties which has been shown to increase the osteoblast response and decrease osteoclast activity. A 3D scaffold comprising of poly(ε-caprolactone) (PCL) functionalised with poly(vinyl phosphonic acid-co-acrylic acid) (PVPA-AA) was fabricated using electrospinning. PVPA-AA is hypothesised to mimic the action of bisphosphonates, a group of drugs used to treat osteoporosis, and likely to affect bone turnover by increasing the activity of osteoblasts and reducing osteoclast activity. The expression of collagen type I, osteocalcin and alkaline phosphatase has been found to be significantly higher (p≤ 0.001) on PCL/PVPA-AA scaffolds when compared to PCL and tissue culture plastic (TCP). In addition, after 21 days there was a significant increase (p≤ 0.001) in mineralisation on PCL/PVPA-AA substrates. The PCL/PVPA-AA scaffold has been reported to significantly (p≤ 0.001) decrease osteoclast viability, with comparable results to Alendronate, a commercially available bisphosphonate. For the first time, we describe a novel active synthetic bone graft substitute, which has been shown to increase osteoblast proliferation and matrix deposition as well as reducing the number of osteoclast cells by locally induced apoptosis. The combined affects of the novel PVPA-AA polymer on osteoblasts and osteoclasts may lead to active bone repair and healing.

Chemical surface modification of poly-ε-caprolactone improves Schwann cell proliferation for peripheral nerve repair.

Poly-ε-caprolactone (PCL) is a biodegradable and biocompatible polymer used in tissue engineering for various clinical applications. Schwann cells (SCs) play an important role in nerve regeneration and repair. SCs attach and proliferate on PCL films but cellular responses are weak due to the hydrophobicity and neutrality of PCL. In this study, PCL films were hydrolysed and aminolysed to modify the surface with different functional groups and improve hydrophilicity. Hydrolysed films showed a significant increase in hydrophilicity while maintaining surface topography. A significant decrease in mechanical properties was also observed in the case of aminolysis. In vitro tests with Schwann cells (SCs) were performed to assess film biocompatibility. A short-time experiment showed improved cell attachment on modified films, in particular when amino groups were present on the material surface. Cell proliferation significantly increased when both treatments were performed, indicating that surface treatments are necessary for SC response. It was also demonstrated that cell morphology was influenced by physico-chemical surface properties. PCL can be used to make artificial conduits and chemical modification of the inner lumen improves biocompatibility.

Long term peripheral nerve regeneration using a novel PCL nerve conduit.

The gold standard in surgical management of a peripheral nerve gap is currently autologous nerve grafting. This confers patient morbidity and increases surgical time therefore innovative experimental strategies towards engineering a synthetic nerve conduit are welcome. We have developed a novel synthetic conduit made of poly ε-caprolactone (PCL) that has demonstrated promising peripheral nerve regeneration in short-term studies. This material has been engineered to permit translation into clinical practice and here we demonstrate that histological outcomes in a long-term in vivo experiment are comparable with that of autologous nerve grafting. A 1cm nerve gap in a rat sciatic nerve injury model was repaired with a PCL nerve conduit or an autologous nerve graft. At 18 weeks post surgical repair, there was a similar volume of regenerating axons within the nerve autograft and PCL conduit repair groups, and similar numbers of myelinated axons in the distal stump of both groups. Furthermore, there was evidence of comparable re-innervation of end organ muscle and skin with the only significant difference the lower wet weight of the muscle from the PCL conduit nerve repair group. This study stimulates further work on the potential use of this synthetic biodegradable PCL nerve conduit in a clinical setting.

Novel thin-walled nerve conduit with microgrooved surface patterns for enhanced peripheral nerve repair.

Randomly aligned nerve cells in vitro on conventional culture substrata do not represent the complex neuronal network in vivo and neurites growing in uncontrolled manner may form neuroma. It is of great importance to mimic the organised growth pattern of nerve cells in the study of peripheral nerve repair. The aim of this work was to modify and optimize the photolithographic technique in creating a reusable template in the form of a silicon wafer that could be used to produce contact guidance on biodegradable polymer surface for the orientated growth of nerve cells. Micro-grooves (approximately 3 µm in depth) were etched into the silicon template using KOH at increased temperature. The originality of this work lies in the low cost and high efficiency method in producing microgrooves on the surface of biodegradable ultra-thin polymer substrates (50-100 µm), which can be readily rolled up to form clinically implantable nerve conduits. The design of a pattern with small ridge width (i.e., 5 µm) and bigger groove width (i.e., 20 µm) favored the alignment of cells along the grooves rather than on the ridges of the patterns, which minimized the effect of cross growing of neurites between adjacent grooves. Effectively, enhanced nerve regeneration could be anticipated from these patterned conduits.

In vitro evaluation of polyester-based scaffolds seeded with adipose derived stem cells for peripheral nerve regeneration.

To overcome the disadvantages of autografts for peripheral nerve repair, different methods such as artificial nerve conduits have been investigated for an alternative approach. This study demonstrated that solvent casting is a simple but efficient method to create thin polyester-based scaffolds for stem cell delivery. Using poly (ε-caprolactone) and poly (D,L-lactic acid), we produced scaffold films containing heterogenous depressions (pits) on the air surface with a size ranging from 0.5 to 30 µm(2). These scaffolds were moderately hydrophobic; however, they supported the differentiation of adipose derived stem cells (ADSC) into a Schwann cell-like phenotype. The differentiated ADSC (dADSC) expressed S100 protein and glial fibrillary acidic protein and readily adhered to the films and proliferated at a similar rate to those cultured on tissue culture polystyrene. Cells were also positive for proliferating cell nuclear antigen. Furthermore, dADSC retained functional activity and significantly enhanced neurite outgrowth from dorsal root ganglia neurons. This study suggests polymer scaffolds combined with dADSCs could be a promising therapy for peripheral nerve injuries.

Evaluation of critical size defects of mouse calvarial bone: An organ culture study.

Mouse calvarial organ culture has been used widely for the study of bone biology. The purpose of this study was to evaluate the healing potential of neonatal mouse parietal defects in different culture media. The critical size defect (CSD) was also investigated. The parietal bones of neonatal mice were used. Full-thickness, 0.8-mm circular defects were created through the bones from one litter of mice. The bones were divided into three groups: Dulbecco's Modified Eagle Medium (DMEM) group, DMEM/osteogenic medium (OM) group, and OM group. Cultures were analyzed with microcomputed-tomography, dissecting-microscope, phase-contrast-microscope, Von Kossa stain, scanning-electron-microscopy, and energy-dispersive-X-ray. Continuous bone formation of parietal bones was observed in all groups. Defects in the DMEM/OM group showed the highest healing potential and exhibited woven bone formation. Defects in the OM group showed limited bone healing at the defect edge. Defects in the DMEM group showed fibrous healing. The most effective culture medium (DMEM/OM) was used to determine the CSD of mouse calvaria in a separate experiment. Circular defects (diameters: 0.8, 1.0, and 1.5 mm) were made in the parietal bones from another litter of mice. The bones were analyzed with microcomputed-tomography, and phase-contrast-microscopy. The bone filling percentages of different size defects were statistically significant: 1.5-mm defects (4.49%), 1.0-mm defects (47.65%), and 0.8-mm defects (73.45%). In three culture conditions, DMEM/OM was the most effective approach to repair bone defects. A 1.5 mm in diameter, full-thickness parietal defect was found to be the CSD under the DMEM/OM culture conditions.

Physicochemical characterisation of novel ultra-thin biodegradable scaffolds for peripheral nerve repair.

In this study, the physicochemical properties of microporous poly (epsilon-caprolactone) (PCL) films and a composite material made of PCL and polylactic acid (PLA) blend were tested. Fabricated by solvent casting using dichloromethane, these ultra-thin films (60 +/- 5 microm in thickness) have a novel double-sided surface topography, i.e. a porous surface with pores 1-10 microm in diameter and a relatively smooth surface with nano-scaled texture. Porous surfaces were found to be associated with increased protein adsorption and the treatment of these polyester scaffolds with NaOH rendered them more hydrophilic. Differential Scanning Calorimetry (DSC) showed that the incorporation of PLA reduced the crystallinity of the original homopolymer. Chemical changes were investigated by means of Fourier Transform Infrared Spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS). Average surface roughness (Ra), hydrophilicity/hydrophobicity and mechanical properties of these materials were also assessed for the suitability of these materials as nerve conduits.

Montelukast improves asthma control in asthmatic children maintained on inhaled corticosteroids.

BACKGROUND: Because of potential toxicities of inhaled corticosteroid (ICS) use in pediatric asthma, alternative or steroid-sparing therapy is desirable. There are no previous studies evaluating montelukast's steroid-sparing effects in children with asthma. OBJECTIVE: To evaluate whether (1) montelukast as add-on therapy improves asthma symptom control and (2) montelukast provides steroid-sparing effects in children with asthma treated with low to moderate doses of ICS therapy. METHODS: In a double-blind, placebo-controlled trial, 36 children ages 6 to 14 years with symptomatic asthma maintained on a stable low to moderate dose of ICSs were randomly assigned to receive montelukast or matching placebo for 24 weeks after a run-in period of 2 weeks (period I). During the trial, subjects kept daily asthma diary cards and monthly spirometry was performed. After a 4 week add-on period (period II), the subjects completed a 20-week (period III) ICS tapering period based on a predetermined protocol. RESULTS: In period II, the difference in the number of rescue-free days was significantly higher in the montelukast group (P = 0.0001), and the number of rescue-free days per week was also significantly higher in montelukast-treated subjects compared with placebo subjects (P = 0.002). In period III, the percentage reduction in ICS dose was not significant between montelukast and placebo (P = 0.10), but the montelukast group experienced an average 17% decrease in ICS dose and the control group experienced an average 64% increase in ICS dose. CONCLUSIONS: Montelukast treatment significantly increased the number of rescue-free days in symptomatic children with asthma.

Cytotoxicity of glutaraldehyde crosslinked collagen/poly(vinyl alcohol) films is by the mechanism of apoptosis.

Collagen has been investigated as a potential natural biomaterial, because of its occurrence in the extracellular matrix. Collagen requires crosslinking in this context, by reagents that are often cytotoxic. Glutaraldehyde is one such agent that is potentially cytotoxic. The aim of this study was to determine the cause of poor cell attachment and growth on collagen/poly(vinyl alcohol) bioartificial composite films, when crosslinked with glutaraldehyde. Dehydrothermal crosslinking was used as a comparison. Human osteoblasts were observed to undergo apoptosis on glutaraldehyde crosslinked films dependent on concentration of collagen present. Higher collagen content resulted in higher levels of apoptosis with poor cell attachment and spreading of remaining cells. Post-treatment of films with 8% L-glutamic acid prevented the apoptotic response of osteoblasts and allowed attachment and spreading. The addition of 100 nM insulin-like growth factor-1 to the culture medium also prevented apoptosis. Glutaraldehyde toxicity of crosslinked collagen has been demonstrated in this study, the mechanism of which is apoptosis. This study indicates that poor biocompatibility and induction of apoptosis on collagen/poly(vinyl alcohol) films crosslinked by glutaraldehyde are attributed to glutaraldehyde components on the surface of the films (not residual glutaraldehyde), whose effects can be quenched by glutamic acid, and prevented by insulin-like growth factor-1.

Protein adsorption and human osteoblast-like cell attachment and growth on alkylthiol on gold self-assembled monolayers.

Protein adsorption and growth of primary human osteoblasts on self-assembled monolayers of alkylthiols on gold (SAMs) with carboxylic acid and hydroxyl and methyl termini were investigated. Single-component SAMs and SAMs patterned by photolithographic techniques were used. Cell growth on patterned SAMs demonstrated preferences for one pattern region in all combinations of alkylthiols, with the hierarchical preference COOH > OH > CH(3). Patterned SAMs and immunochemistry were used to investigate adsorption of fibronectin and albumin with respect to different alkylthiol termini. Fibronectin adsorption from both pure solution and serum containing cell culture medium (SDMEM) followed the sequence COOH > OH > CH(3). Albumin adsorption from pure solution followed the sequence OH > COOH > CH(3); from SDMEM the sequence was CH(3) > OH > COOH. Cell attachment to SAMs with the above termini, after preadsorption with fibronectin, albumin, or mixtures of fibronectin and albumin, was measured. Attachment was maximal on COOH-terminated SAMs precoated with fibronectin. Attachment to COOH was significantly reduced only when fibronectin was omitted from the protein preadsorption solution. On OH and CH(3) SAMs increasing the proportion of albumin in the solution was sufficient to significantly reduce cell attachment. The distribution vinculin and the integrins alpha(5)beta(1) and alpha(v)beta(3) indicated that focal contact formation by cells varied with alkylthiol termini in the following sequence: COOH > OH > CH(3).