Hydroxyapatite-collagen nanoparticles reinforced polyanhydride based injectable paste for bone substitution: effect of dopant addition in vitro.

The present study focuses on the synthesis and characterization of hydroxyapatite-collagen nanoparticles incorporated polyanhydride paste and investigating its bone regeneration capacity in vitro. Photocrosslinkable polyanhydride paste was prepared after synthesizing methacrylate derivatives of 1,6-bis(p-carboxyphenoxy)hexane (MCPH) and sebacic acid dimethacrylate (MSA). These multifunctional monomers, namely 45 wt% MSA, 45 wt% MCPH in addition to 10 wt% poly(ethylene glycol)diacrylate (PEGDA) were photopolymerized under ultraviolet light (365 nm) to produce highly crosslinked polyanhydride networks using camphroquinone (CQ)/ethyl 4-(dimethylamino)benzoate [4-EDMAB] for light initiated crosslinking and benzoyl peroxide (BPO)/dimethyl toludine (DMT) for chemically initiated crosslinking. Separately, using the co-precipitation process, (1 wt%) Si, (1 wt%) Sr, and (0.5 + 0.5) wt% Si/Sr was doped into hydroxyapatite-collagen nanoparticles in size range between 50 and 70 nm. Si, Sr, and both Si/Sr doped hydroxyapatite-collagen nanoparticles to the extent 10 wt% were added to polyanhydride monomer mixture containing 40 wt% MSA, 40 wt% MCPH and 10 wt% PEGDA and subsequently photopolymerized as previously mentioned. Incorporation of hydroxyapatite-collagen nanoparticles to the extent of 10 wt% into polyanhydride matrix enhanced compressive strength of the hardened paste from 30 to 49 MPa. Mesenchymal stem cells obtained from the human umbilical cord were cultured onto pure polyanhydride and hydroxyapatite-collagen added scaffold to assess their cellular proliferation and differentiation capacity to bone cell. MTT assay showed that mesenchymal stem cell proliferation was significantly higher in Si/Sr binary doped hydroxyapatite-collagen-polyanhydride sample as compared to other samples. Again from immunocytochemistry study using confocal images suggested that expression of osteocalcin, a marker indicating differentiation into osteoblast, was the highest in Si/Sr binary doped hydroxyapatite-collagen-polyanhydride sample against the other samples studied in this case. This study thus summarizes the development of photocurable biocomposites containing polyanhydride and Si, Sr doped hydroxyapatite-collagen nanoparticles that exhibited tremendous promise to regenerate bone tissues in complex-shaped musculoskeletal defect sites.

Photo-crosslinked alginate nano-hydroxyapatite paste for bone tissue engineering.

In this study, methacrylation of alginate was carried out by reacting sodium alginate with methacrylic anhydride in the presence of sodium hydroxide. Separately synthesized nano-hydroxyapatite (nano-HAp) powder was surface functionalized using mercaptopropionic acid and ethylene glycol methacrylate phosphate (EGMP) in the presence of azobisisobutyronitrile benzene as a free radical initiator in a nitrogen atmosphere. Methacrylated alginate solution was mixed with the required amount of surface-functionalized HAp nanoparticles in the presence of 0.05% Irgacure 2959 as a photoinitiator and was placed at the centre of a 8 kW UV light source (265 nm) to prepare photo-crosslinked bone paste. X-ray diffraction analysis indicated that surface functionalization did not alter phase purity of HAp nanopowder in the prepared paste. The graft polymerization of EGMP on the surface of HAp was confirmed by the presence of the 1732 cm-1 band, which belongs to C=-O stretching of EGMP, in addition to the characteristic peaks of nano-HAp and alginate in the composite paste. The storage and loss moduli of all the prepared pastes increased non-linearly with time up to 100 s, demonstrating their pseudo plastic behaviour. The rate of release of bone morphogenetic protein 2 (BMP-2) was significantly faster in the first few days, and the release curve gradually levelled off prior to slowing down up to 22 d. Mesenchymal stem cell adhesion studies revealed that cells could attach to the paste material and stretch over the surface of the material after 14 d of incubation. MTT assay showed that prepared paste materials were conducive to attachment and proliferation of mesenchymal stem cells. Immunocytochemical analysis revealed that the addition of surface-functionalized nano-HAp and BMP-2 to alginate hydrogel enhanced the osteogenic potential of the prepared paste. The results indicate that the newly developed photo-crosslinked paste may be physically and biologically suitable for application as a bone filler.

Investigating the mechanical, physiochemical and osteogenic properties in gelatin-chitosan-bioactive nanoceramic composite scaffolds for bone tissue regeneration: In vitro and in vivo.

The aim of this work was to compare the efficacy of gelatin-chitosan based bone scaffolds after incorporation of three different bioactive nanoparticles such as hydroxyapatite (HAp), ß­tricalcium phosphate (ß-TCP) and 58s bio active glass by evaluating its physicochemical, mechanical and osteogenic properties. Gelatin-chitosan based scaffolds made of gelatin-chitosan (GC) and GC composites containing 30 wt% HAp, ß-TCP and 58s bioactive glass nanoparticles were fabricated using freeze drying technique. The porosity and compressive strength of all the prepared scaffolds were evaluated. The average pore size of all the prepared composite scaffolds was in the range between 90 and 125 µm. Most frequent pore size in GCT 30 scaffold was the highest of 120 µm whereas that for GCH 30 was the lowest of 96 µm as suggested by Hg porosimetry analysis. GCH30 scaffolds showed the highest average compressive strength of 3.45 MPa as opposed to 2.24 MPa exhibited by GCB 30, with high degree of interconnected porosity appropriate for cellular colonization. To study the effect of different bioceramic phases on MSCs differentiation, scaffolds were cell cultured for up to 14 days in osteogenic medium. GCB30 scaffold showed higher capacity to proliferate MSCs cultured onto it as compared to other composite scaffolds. Degree of differentiation of MSCs into osteoblast was higher in GCB30 scaffolds than in the GCH30 and GCT30 composite scaffold as evident from higher amount of RUNX2 and osteocalcin expression in the former up to 14 days of cell culture. Inclusion of 58s bioactive glass particles showed positive effects on cell differentiation. In coherence with the in vitro appearance, histomorphometric analysis and fluorochrome study in a rabbit tibia model showed a significantly greater amount of new bone formation in GCB30 compared to other composite scaffolds. The results demonstrated that the prepared GCB30 scaffold could be a better candidate as bone substitute material for its higher bioactivity in bone tissue regeneration.

Preparation and characterization of gelatin-chitosan-nanoß-TCP based scaffold for orthopaedic application.

The primary aim of this study was to fabricate gelatin/chitosan/ß-TCP (GCT) composite scaffold to improve its compressive mechanical behaviour and in-vivo biocompatibility with predictable degradation rate. Beta tricalcium phosphate (ß-TCP) powder was synthesized in size range between 70-100 nm using aqueous precipitation route at a fixed Ca/P molar ratio of 1.5:1 at pH 10 and after subsequent heat treatment of as precipitated powder at 800 °C for 4 hours. The composite scaffolds were fabricated using solid-liquid phase separation of the slurry containing gelatin, chitosan, ß-tricalcium phosphate in varying proportion and subsequent lyophilisation of the phase separated mixture. The prepared scaffolds exhibited high porosity (>80%) with pore sizes ranging between 78-382 µm as determined using Hg-porosimetry. SEM result revealed that incorporation of ß-TCP to the extent of 30 wt% resulted in well-shaped and uniformly distributed interconnected pores of average pore size of 120 ±â€¯18.6 µm in it. Compressive strength of the scaffolds was increased from 0.8 MPa to 2.45 MPa on increase in ß-TCP content from 10 wt%-30 wt% in the prepared scaffold. Human Umbilical Cord derived mesenchymal stem cells (MSCs) exhibited higher degree of lamellopodia and fillopodia extensions and better spreading behaviour onto GCT30 scaffold. MTT assay and immunocytochemistry studies with cultured MSCs revealed that GCT30 scaffolds were more conducive to MSC's proliferation and differentiation into osteoblast lineage. In vivo implantation of GCT30 scaffold subcutaneously into mice did not indicate any significant inflammatory reaction, but ongoing vascularization.

MgAl- Layered Double Hydroxide Nanoparticles for controlled release of Salicylate.

Layered double hydroxides (LDHs), have been known for many decades as catalyst and ceramic precursors, traps for anionic pollutants, and additives for polymers. Recently, their successful synthesis on the nanometer scale opened up a whole new field for their application in nanomedicine. Here we report the efficacy of Mg1-xAlx (NO3)x (OH)2 LDH nanoparticles as a carrier and for controlled release of one of the non-steroidal anti-inflammatory drugs (NSAID), sodium salicylate. Mg1-xAlx (NO3)x (OH)2.nH2O nanoparticles were synthesized using co-precipitation method from an aqueous solution of Mg(NO3)2.6H2O and Al(NO3)3.9H2O. Salicylate was intercalated in the interlayer space of Mg-Al LDH after suspending nanoparticles in 0.0025(M) HNO3 and 0.75 (M) NaNO3 solution and using anion exchange method under N2 atmosphere. The shift in the basal planes like (003) and (006) to lower 2θ value in the XRD plot of intercalated sample confirmed the increase in basal spacing in LDH because of intercalation of salicylate into the interlayer space of LDH. FTIR spectroscopy of SA-LDH nano hybrid revealed a red shift in the frequency band of carboxylate group in salicylate indicating an electrostatic interaction between cationic LDH sheet and anionic drug. Differential thermal analysis of LDH-SA nanohybrid indicated higher thermal stability of salicylate in the intercalated form into LDH as compared to its free state. DLS studies showed a particle size distribution between 30-60 nm for pristine LDH whereas salicylate intercalated LDH exhibited a particle size distribution between 40-80nm which is ideal for its efficacy as a superior carrier for drugs and biomolecules. The cumulative release kinetic of salicylate from MgAl-LDH-SA hybrids in phosphate buffer saline (PBS) at pH7.4 showed a sustained release of salicylate up to 72h that closely resembled first order release kinetics through a combination of drug diffusion and dissolution of LDH under physiological conditions. Also the cytotoxicity tests performed revealed the less toxic nature of the nanohybrid as compared to the bare SA drug.

Preparation and Evaluation of Gelatin-Chitosan-Nanobioglass 3D Porous Scaffold for Bone Tissue Engineering.

The aim of the present study was to prepare and characterize bioglass-natural biopolymer based composite scaffold and evaluate its bone regeneration ability. Bioactive glass nanoparticles (58S) in the size range of 20-30 nm were synthesized using sol-gel method. Porous scaffolds with varying bioglass composition from 10 to 30 wt% in chitosan, gelatin matrix were fabricated using the method of freeze drying of its slurry at 40 wt% solids loading. Samples were cross-linked with glutaraldehyde to obtain interconnected porous 3D microstructure with improved mechanical strength. The prepared scaffolds exhibited >80% porosity with a mean pore size range between 100 and 300 microns. Scaffold containing 30 wt% bioglass (GCB 30) showed a maximum compressive strength of 2.2 ± 0.1 MPa. Swelling and degradation studies showed that the scaffold had excellent properties of hydrophilicity and biodegradability. GCB 30 scaffold was shown to be noncytotoxic and supported mesenchymal stem cell attachment, proliferation, and differentiation as indicated by MTT assay and RUNX-2 expression. Higher cellular activity was observed in GCB 30 scaffold as compared to GCB 0 scaffold suggesting the fact that 58S bioglass nanoparticles addition into the scaffold promoted better cell adhesion, proliferation, and differentiation. Thus, the study showed that the developed composite scaffolds are potential candidates for regenerating damaged bone tissue.

Development of gelatin-chitosan-hydroxyapatite based bioactive bone scaffold with controlled pore size and mechanical strength.

Hydroxyapatite-chitosan/gelatin (HA:Chi:Gel) nanocomposite scaffold has potential to serve as a template matrix to regenerate extra cellular matrix of human bone. Scaffolds with varying composition of hydroxyapatite, chitosan, and gelatin were prepared using lyophilization technique where glutaraldehyde (GTA) acted as a cross-linking agent for biopolymers. First, phase pure hydroxyapatite-chitosan nanocrystals were in situ synthesized by coprecipitation method using a solution of 2% acetic acid dissolved chitosan and aqueous solution of calcium nitrate tetrahydrate [Ca(NO3)2,4H2O] and diammonium hydrogen phosphate [(NH4)2H PO4]. Keeping solid loading constant at 30 wt% and changing the composition of the original slurry of gelatin, HA-chitosan allowed control of the pore size, its distribution, and mechanical properties of the scaffolds. Microstructural investigation by scanning electron microscopy revealed the formation of a well interconnected porous scaffold with a pore size in the range of 35-150 µm. The HA granules were uniformly dispersed in the gelatin-chitosan network. An optimal composition in terms of pore size and mechanical properties was obtained from the scaffold with an HA:Chi:Gel ratio of 21:49:30. The composite scaffold having 70% porosity with pore size distribution of 35-150 µm exhibited a compressive strength of 3.3-3.5 MPa, which is within the range of that exhibited by cancellous bone. The bioactivity of the scaffold was evaluated after conducting mesenchymal stem cell (MSC) - materials interaction and MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay using MSCs. The scaffold found to be conducive to MSC's adhesion as evident from lamellipodia, filopodia extensions from cell cytoskeleton, proliferation, and differentiation up to 14 days of cell culture.