Functionally graded PLGA-nano apatite-lauric acid biocomposite membrance for potential clinical applications

Bone healing is a challenge in orthopaedics and dentistry. An occlusive membrane is used for the reconstruction of bone defects in guided bone regeneration (GBR) technique. Infection is the major cause for GBR membrane failure in which multiple antibiotics have been used to prevent bacterial colonisation in regenerative clinical practice. An anti-infective membrane with alternative antimicrobial agent to substitute antibiotics is paramount to overcome the incidence of bacterial resistance and side-effects. In this study, a composite membrane was developed by incorporating lauric acid (LA), a naturally derived antimicrobial substance. Poly(lactic-co-glycolic acid) (PLGA) based composite membrane was successfully fabricated using a combination of solvent casting-thermally induced phase separation (TIPS)-solvent leaching technique. The triple-layered membrane structure was attained via solvent casting of the composite solutions which then immediately phase separated by freezing at -18±1°C for 24 h. Then, the solvent in phase separated membrane was removed by immersing in precooled water at 3±1°C for 26 h, after which the membrane was air dried at 25°C for 3 days. The triple-layered construct of the PLGA composite membrane was developed with a gradient structure of LA and non-stoichiometric nanoapatite (NAp), to deliver the antimicrobial and osteconductive properties, respectively. The surface morphology and phase composition of the membrane were examined using scanning electron microscopy (SEM) and X-ray diffraction (XRD), respectively. The resulting graded membrane consisted of small pore size layer-1 containing 10wt% NAp + 1-3wt% LA, an intermediate labyrinth layer-2 with 20-50wt% NAp + 1wt% LA, and a large pore size layer-3 containing 30-100wt% NAp without LA. The existence of chemical interaction between PLGA, NAp and LA was identified using Fourier transform infrared spectrophotometry (FTIR) analysis. The synergistic effects of 10-30wt% NAp and 1wt% LA in dry membranes demonstrated higher tensile strength (0.61±0.17 MPa) and elastic modulus (23.15± 6.19 MPa). However, a more pliable behavior with a decrease in elastic modulus (12.50± 4.32MPa) was observed in 3wt% LA added membrane compared to the pure PLGA (20.17±2.21 MPa). The addition of LA resulted in a plasticizing effect at 3wt% due to weak intermolecular interactions in PLGA chains, caused by LA (-OH) and PLGA (C-O) bondings. These results were corroborated by the FTIR peak shift (1-3 cm-1) and glass transition temperature (Tg) reduction as detected using differential scanning calorimeter (DSC). The composite membrane retained its structural integrity with only 22% weight loss after incubation for 24 weeks in phosphate buffered saline (PBS), which indicates its potential use as a physical barrier. The 1-3wt% LA loaded composite membranes had good cell viability toward mouse fibroblasts and showed increased bacterial reduction with increased LA loadings against S. aureus. These results demonstrate the potential of LA loaded biocomposite membrane to provide anti-infective surfaces, useful in clinical applications

In vitro degradation of triple layered poly (lactic-co-glycolic acid) composite membrane composed of nanoapatite and lauric acid for guided bone regeneration applications

Bone healing has been a great challenge in orthopaedic and dentistry fields. In one of the ways for overcoming this, a barrier membrane is used in guided bone regeneration (GBR) applications to cover and aid the healing of bone defects. In this study, lauric acid (LA) and nanoapatite (NAp) were incorporated into poly(lactic-co-glycolic acid) (PLGA) matrices to form triple layered composite membranes for potential use in GBR applications. LA and NAp were added to introduce antimicrobial and bioactive properties, respectively, to the composite membrane. The membranes were fabricated using a combined techniques of solvent casting - thermally induced phase separation (TIPS) - solvent leaching in a single step. In vitro degradation behaviour of the new composite membrane system was studied for 24 weeks in phosphate buffer saline (PBS) at 37 °C; pH = 7.4, to match the bone healing period in GBR applications. Immersion of membrane samples was carried out at pre-determined time intervals of 1, 2, 4, 8, 12, 16, 20 and 24 weeks. Physical changes such as weight loss and water uptake were measured after each time period and relatively monitored pH changes in post-immersed PBS solutions. Scanning electron microscopy with energy dispersive X-ray spectroscopy (SEM-EDS) was used to assess morphology changes and presence of NAp particles on the membrane surfaces after withdrawing from PBS. The entire weight loss for the membranes was only up to 22% over 24 weeks of incubation, which suggests its structural integrity and potential use as a physical barrier in GBR applications

Chemical and thermal stability of multiple ions doped nonstoichiometric nanoapatite heat-treated in CO2 and air atmospheres

Nanostructured apatite has been widely used as a bone substitute material due to its close resemblance to human bone mineral. To further mimic biological apatites, multiple ions doped nonstoichiometric nanoapatite has been studied. A nanosized apatite (NAp-2) containing Mg (1.09 wt%), Na (0.15 wt%), K (0.008 wt%) and CO3 2- (5.18 wt%) was synthesized by a wet precipitation technique. The presence of these ions in NAp-2 was detected using ICP. Broad diffraction peaks of XRD results indicated the presence of nanocrystalline phase-pure NAp-2. The primary particle size of the resulted powder was ~ 20 nm, typical of bone crystal size, estimated using Scherrer's equation. Based on CHN results, the NAp-2 powders showed a total loss of 51 and 78% of carbonate ions when heat-treated at 900°C in both CO2 and air atmospheres, respectively. This indicates that the heat-treatment in CO2 flux has reduced the carbonate ions lost from the NAp-2. A highly crystalline HA phase was formed in the ionic doped NAp-2 without secondary phases, indicating a thermal stability of this powder at 900°C in CO2 and air atmospheres. Thus, this study demonstrated that a phase-pure multiple ions doped nanoapatite was synthesized using a wet precipitation technique

Antibacterial properties of guided bone regeneration membrane for periodontal applications

Guided Bone Regeneration (GBR) membrane is used as a barrier to prevent soft tissue ingrowth and to encourage bone regeneration through cellular exclusion. This study aims to assess antibacterial properties of recently developed three-layered poly(lactic-co-glycolic acid) (PLGA) /lauric acid (LA) composite membrane towards Staphylococcus aureus. One of the outmost layers of three-layered membrane was incorporated with 1-3 wt% of LA. The composite membrane was developed using thermally induced phase separation/solvent leaching technique. SEM results shows formation of PLGA matrix with smaller pores by the addition of 1 wt% LA compared with pure PLGA membrane. Samples of 1.7 cm diameter disk containing 1, 2 and 3 wt% of lauric acid were tested and pure membrane without lauric acid was used as a control. Results showed that the zones of inhibition were 2.3 cm and 2.5 cm for the 2 wt% and 3 wt% LA-containing membranes, respectively. However, 1 wt% LA-containing membrane observed has no inhibition at all, indicating that increasing concentration of LA has significant inhibition against Staphylococcus aureus. The 3 wt% LA composition will be used in the mechanically optimized membranes for degradation studies in future work

Antibacterial efficacy of triple-layered poly(Lactic-co-glycolic acid)/nanoapatite/ lauric acid guided bone regeneration membrane on periodontal bacteria

A guided bone regeneration (GBR) membrane has been extensively used in the repair and regeneration of damaged periodontal tissues. One of the main challenges of GBR restoration is bacterial colonization on the membrane, constitutes to premature membrane degradation. Therefore, the purpose of this study was to investigate the antibacterial efficacy of triple-layered GBR membrane composed of poly(lactic-co-glycolic acid) (PLGA), nanoapatite (NAp) and lauric acid (LA) with two types of Gram-negative periodontal bacteria, Fusobacterium nucleatum and Porphyromonas gingivalis through a disc diffusion and bacterial count tests. The membranes exhibited a pattern of growth inhibition and killing effect against both bacteria. The increase in LA concentration tended to increase the bactericidal activities which indicated by higher diameter of inhibition zone and higher antibacterial percentage. It is shown that the incorporation of LA into the GBR membrane has retarded the growth and proliferation of Gram-negative periodontal bacteria for the treatment of periodontal disease

Triple-layered PLGA/nanoapatite/lauric acid graded composite membrane for periodontal guided bone regeneration

This paper discusses the successful fabrication of a novel triple-layered poly(lactic-co-glycolic acid) (PLGA)-based composite membrane using only a single step that combines the techniques of solvent casting and thermally induced phase separation/solvent leaching. The resulting graded membrane consists of a small pore size layer-1 containing 10 wt% non-stoichiometric nanoapatite (NAp) + 1-3 wt% lauric acid (LA) for fibroblastic cell and bacterial inhibition, an intermediate layer-2 with 20-50 wt% NAp + 1 wt% LA, and a large pore size layer-3 containing 30-100 wt% NAp without LA to allow bone cell growth. The synergic effects of 10-30 wt% NAp and 1 wt% LA in the membrane demonstrated higher tensile strength (0.61 MPa) and a more elastic behavior (16.1% elongation at break) in 3 wt% LA added membrane compared with the pure PLGA (0.49 MPa, 9.1%). The addition of LA resulted in a remarkable plasticizing effect on PLGA at 3 wt% due to weak intermolecular interactions in PLGA. The pure and composite PLGA membranes had good cell viability toward human skin fibroblast, regardless of LA and NAp contents

The influence of new wet synthesis route on the morphology, crystallinity and thermal stability of multiple ions doped nanoapatite

The synthesis of multiple ions doped nanoapatite powder was carried out by wet precipitation technique. A newly developed reaction route with self-controlled pH at high reaction temperatures, i.e. 37 & 85±2 C was compared to the conventional synthesis route at 37±2 C. The XRD peaks were very broad, indicating the presence of nanocrystalline apatite. The primary particle size of the powder was in the range of 20-30 nm whereas the fraction of crystallinity was between 0.20 and 0.63. TEM and SEM characterizations confirmed the nanosized primary particles of the apatite samples. The high temperature synthesis at 37 & 85±2 C improved both crystallite size and crystallinity of the as-prepared samples. A highly crystalline HA phase was formed in the ions doped samples without secondary phases, indicating its thermal stability at 900 C in both CO2 and air atmosphere. The in vitro cytocompatibility of the synthesised nanoapatite powders was confirmed by cell viability of human skin fibroblasts