Injectable, self-healing hydrogels based on gelatin, quaternized chitosan, and laponite as localized celecoxib delivery system for nucleus pulpous repair.

Utilization of injectable hydrogels stands as a paradigm of minimally invasive intervention in the context of intervertebral disc degeneration treatment. Restoration of nucleus pulposus (NP) function exerts a profound influence in alleviating back pain. This study introduces an innovative class of injectable shear-thinning hydrogels, founded on quaternized chitosan (QCS), gelatin (GEL), and laponite (LAP) with the capacity for sustained release of the anti-inflammatory drug, celecoxib (CLX). First, synthesis of Magnesium-Aluminum-Layered double hydroxide (LDH) was achieved through a co-precipitation methodology, as a carrier for celecoxib and a source of Mg ions. Intercalation of celecoxib within LDH layers (LDH-CLX) was verified through a battery of analytical techniques, including FTIR, XRD, SEM, EDAX, TGA and UV-visible spectroscopy confirmed a drug loading efficiency of 39.22 ± 0.09 % within LDH. Then, LDH-CLX was loaded in the optimal GEL-QCS-LAP hydrogel under physiological conditions. Release behavior (15 days profile), mechanical properties, swelling ratio, and degradation rate of the resulting composite were evaluated. A G* of 15-47 kPa was recorded for the hydrogel at 22-40 °C, indicating gel stability in this temperature range. Self-healing properties and injectability of the composite were proved by rheological measurements. Also, ex vivo injection into intervertebral disc of sheep, evidenced in situ forming and NP cavity filling behavior of the hydrogel. Support of GEL-QCS-LAP/LDH-CLX (containing mg2+ ions) for viability and proliferation (from ~94 % on day 1 to ~134 % on day 7) of NP cells proved using MTT assay, DAPI and Live/Dead assays. The hydrogel could significantly upregulate secretion of glycosaminoglycan (GAG, from 4.68 ± 0.1 to 27.54 ± 1.0 µg/ml), when LHD-CLX3% was loaded. We conclude that presence of mg2+ ion and celecoxib in the hydrogel can lead to creation of a suitable environment that encourages GAG secretion. In conclusion, the formulated hydrogel holds promise as a minimally invasive candidate for degenerative disc repair.

Dexamethasone loaded injectable, self-healing hydrogel microspheresbased on UPy-functionalized Gelatin/ZnHAp physical network promotes bone regeneration.

Biopolymer-based injectable hydrogels provide great potential as bone tissue engineering (BTE) scaffolds on account of biocompatibility, and pore interconnectivity that enables delivery of cells and/or signaling molecules for bone repair. Recently, Gelatin hydrogels based on H-bonds were considered in response to concerns around the chemical crosslinking agents. In this study, a self-healing gelatin hydrogel with remarkable compressive and self-healing properties was prepared via formation of quadruple hydrogen bonds between ureidopyrimidinon functional groups, which were substituted on NH2 groups of gelatin(GelUPy). Degree of substitution controls properties of the resulting hydrogel from a shape- memory hydrogel (100% substitution), to a hydrogel (about 80%), to this self-healing hydrogel (about 40%). We report a strategy that adopts an emulsion synthesis approach to delivery of dexamethasone and Ca/Zn ions from injectable self-healing GelUPy hydrogel (GelUPy-ZnHApUPy-DEX), to induce osteogenic differentiation of adipose-derived stem cells, in vitro, and enhance bone regeneration in a cranial bone defect in a rat model. We show that key properties of the composite hydrogels, including mechanical properties, and release behavior of DEX are a match to the requirements of BTE. Overall, our results demonstrate that this self-healing gelatin approach is a promising strategy to enhance bone regeneration through a minimally invasive procedure.

Engineering atorvastatin loaded Mg-Mn/LDH nanoparticles and their composite with PLGA for bone tissue applications.

The impact of mixing method in conventional co-precipitation synthesis of layered double hydroxides (LDHs), on particle size, size distribution and drug loading capacity is reported. Synthesis of Mg (II)/Mn (III)-LDH nano-platelets was performed at constant pH using three different mixing systems, magnetic stirrer, mechanical mixer, and homogenizer at ambient temperature and a fixed Mg/Mn ratio of 3/1. The LDH characterization results showed that mechanical mixing and homogenization lead to production of very fine LDH nano-platelets (about 90-140 nm), with narrow particle size distribution. Amount of the intercalated drug was determined as about 60% and showed a significant increase in loading capacity of the LDH through homogenization and mechanical mixing compared to that of the magnetic stirring (about 35%). Our results also showed that in LDH preparation via co-precipitation, the mixing system plays a more influential role in particle size, size distribution, and drug loading control, than the mixing speed of each system. Drug loaded-LDH/PLGA composites were prepared via electrospinning to afford a bioactive/osteoinductive scaffold. A remarkable degree of cell viability on the scaffolds (drug-loaded-LDH/PLGA composite) was confirmed using MTT assay. Osteogenic differentiation of human ADMSCs, as shown by alkaline phosphatase activity and Alizarin Red staining assays, indicated that the scaffold with 5% drug loaded LDH(Mn-Mg-LDH/PLGA/AT5%) induced a remarkably higher level of the markers compared to the PLGA scaffold and therefore, it could be a valuable candidate for bone tissue engineering applications.

Vitamin C Loaded Poly(urethane-urea)/ZnAl-LDH Aligned Scaffolds Increase Proliferation of Corneal Keratocytes and Up-Regulate Vimentin Secretion.

A novel poly(urethane-urea) (PUU) based on poly(glycolide-co-ε-caprolactone) macro-diol with tunable mechanical properties and biodegradation behavior is reported for corneal stromal tissue regeneration. Zn-Al layered double hydroxide (LDH) nanoparticles were synthesized and loaded with vitamin C (VC, VC-LDH) and dispersed in the PUU to control VC release in the cell culturing medium. To mimic the corneal stromal EC, scaffolds of the PUU and its nanocomposites with VC-LDH (PUU-LDH and PUU-VC-LDH) were fabricated via electrospinning. Average diameters of the aligned nanofibers were recorded as 325 ± 168, 343 ± 171, and 414 ± 275 nm for the PUU, PUU-LDH, and PUU-VC-LDH scaffolds, respectively. Results of hydrophilicity and mechanical properties measurements showed increased hydrophobicity and reduced tensile strength and elongation at break upon addition of nanoparticles to the PUU scaffold. VC release studies represented that intercalation of the drug in Zn-Al-LDH controlled the burst release and extended the release period from a few hours to 5 days. Viability and proliferation of stromal keratocyte cells on the scaffolds were investigated via AlamarBlue assay. After 24 h, the cells showed similar viability on the scaffolds and the control. After 1 week, the cells showed some degree of proliferation on the scaffolds, with the highest value recorded for PUU-VC-LDH. SEM images of the scaffolds after 24 h and 1 week confirmed good penetration and attachment of keratocytes on all the scaffolds and the cells oriented with the direction of nanofibers. After 1 week, the PUU-VC-LDH scaffold was fully covered by the cells. Immunocytochemistry assay (ICC) was performed to investigate secretion of vimentin protein, ALDH3A1, and α-SMA by the cells. After 24h and 1 week, remarkably higher levels of vimentin and ALDH3A1 and lower level of α-SMA were secreted by keratocytes on PUU-VC-LDH compared to those on the PUU and PUU-LDH scaffolds and the control. Our results suggest that the aligned PUU-VC-LDH is a promising candidate for corneal stromal tissue engineering due to the presence of zinc and vitamin C.

Atorvastatin loaded PLGA microspheres: Preparation, HAp coating, drug release and effect on osteogenic differentiation of ADMSCs.

Polymer/bioceramic composite micro-particles have been used for bone regeneration in order to address weak mechanical properties/bioactivity of polymers and to enable easy filling of irregular bone defects through minimally invasive injection procedure. The purpose of this study was to determine whether injectable apatite-coated atorvastatin (AT) loaded Poly (d,l-lactide-co-glycolide) (PLGA) micro-particles can support osteogenic differentiation of adipose derived mesenchymal stem cells(ADMSCs). Particle preparation conditions (oil-in-water (O/W) emulsion), were carefully adjusted to yield uniform particles of about 20-50 µm in diameter. Taking a solid in oil-in water (S/O/W) emulsion strategy, it became possible to load atorvastatin (10 wt%) in the micro-particles without deformation. The particles were then coated with HAp by incubation in 10X simulated body fluid (SBF). The apatite coating layer was similar to apatite in natural bone, as demonstrated by SEM, XRD, and FTIR analyses. Adipose derived mesenchymal stem cells (ADMSCs), were cultured on the micro-particles and calcium deposition measurement was performed through Alizarin Red assay. Initial cell adhesion did not differ significantly between the samples and the control. The strongest osteogenic differentiation was observed on PLGA-AT-HAp in both the osteogenic and non osteogenic culture media, while PLGA-AT slightly decreased and PLGA-HAp slightly increased osteogenic differentiation of the cells, indicating suitability of PLGA-AT-HAp as an injectable tissue engineering system.