Cartilage regeneration with dual-drug-releasing injectable hydrogel/microparticle system: In vitro and in vivo study.

In this study, we developed an injectable in situ forming hydrogel/microparticle system consisting of two drugs, melatonin and methylprednisolone, to investigate the capability of the system for chondrogenesis in vitro and in vivo. The chemical, mechanical, and rheological properties of the hydrogel/microparticle were investigated. For in vitro evaluation, the adipose-derived stem cells might be mixed with hydrogel/microparticles, then cellular viability was analyzed by acridine orange/propidium iodide and 4',6-diamidino-2-phenylindole staining and also dimethylmethylene blue assay were conducted to find the amount of proteoglycan. The real-time polymerase chain reaction for aggrecan, sex-determining region Y-Box 9, collagen I (COL1), and COL2 gene expression was performed after 14 and 21 days. For evaluation of cartilage regeneration, the samples were implanted in rabbit knees with cartilaginous experimental defects. Defects were created in both knees of three groups of rabbits. Group 1 was the control with no injection, and Groups 2 and 3 were loaded with hydrogel/cell and hydrogel/microparticle/cell; respectively. Then, after 3 and 6 months, histological evaluations of the defected sites were carried out. The amount of glycosaminoglycans after 14 and 21 days increased significantly in hydrogels/microparticles loaded with cells. The expression of marker genes was also significant in hydrogels/microparticles loaded with cells. According to histology analysis, the hydrogels/microparticles loaded with cells showed the best cartilage regeneration. Overall, our study revealed that the developed injectable hydrogel/microparticle can be used for cartilage regeneration.

Self-crosslinking effect of chitosan and gelatin on alginate based hydrogels: Injectable in situ forming scaffolds.

Injectable in situ forming hydrogels has great potential in tissue engineering. Simple and easy preparation of these hydrogels with low toxicity and stability is a great advantage. In the present study, two types of self-crosslinking in situ forming alginate based hydrogels with different formulation were synthesized, characterized and compared in order to introduce an optimal injectable scaffold in minimally invasive applications in tissue engineering. To this end, the hydrogels consist of oxidized alginate (AD), polyethylene glycol (PEG) and carboxymethyl chitosan (CMC) or gelatin (GEL) was synthesized. The hydrogels were assessed by many techniques including microscopy, spectroscopy, compressive analysis, injectability, rheological analysis and cell viability to ascertain hydrogel properties. In comparison with AD/PEG-GEL hydrogel, AD/PEG-CMC hydrogel showed a higher compressive modulus, viscosity and injection time, whereas AD/PEG-GEL hydrogel displayed a higher degree of crosslinking. Due to the rheological behavior of AD/PEG-CMC hydrogel, this composition was more suitable for the injectable application. The hydrogels, despite the composition, showed the ability to survive and proliferate mesenchymal stem cells based on cytotoxicity assays. With respect to rheological, degradation time and compressive properties of the above-mentioned hydrogels, AD/PEG-CMC hydrogel could be considered as an appropriate candidate for injectable self-crosslinking application in tissue engineering.

Simultaneous release of melatonin and methylprednisolone from an injectable in situ self-crosslinked hydrogel/microparticle system for cartilage tissue engineering.

Recently, injectable hydrogel/microparticle systems have so considered for tissue engineering and regenerative medicine. In this study, we produced an injectable in situ self-crosslinked hydrogel/microparticle system for simultaneous dual drug delivery. First, melatonin conjugated chitosan microparticle loaded with methylprednisolone (MCC-MP) microparticle was fabricated by the covalent linkage of melatonin to chitosan by N-hydroxysuccinimide (NHS)1-Ethyl-3-(3-dimethylamino propyl)-carbodiimide (EDC) followed by an ionic gelation of MCC and MP using tripolyphosphate (TPP). Second, the hydrogel was prepared by the connection between the aldehyde group of alginate oxide (AD) and the amine group belonging to carboxymethyl chitosan (CMC) via Schiff base reaction. Finally, microparticle was incorporated into the AD-CMC hydrogel to produce a hydrogel/microparticle system. Hydrogel/microparticle was assessed by many techniques including microscopy, spectroscopy, particle size measurements, mechanical analysis, injectability, rheological analyses to ascertain hydrogel/microparticle properties. The biological assays of mesenchymal stem cells (MSCs) culture, 3-(4,5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), acridine orange/propidium iodide (AO/PI), and 4, 6-diamidino-2-phenylindole (DAPI) to assess cell viability and dimethylmethylene blue (DMMB) to evaluate proteoglycan content were done. The release profiles of melatonin and MP showed acceptable release after 60 and 20 days, respectively. The hydrogel/microparticle system has the ability to sustain cells alive. A higher rate of proteoglycan content was observed in hydrogel/microparticle as compared with hydrogel. With appropriate biocompatibility and adequate properties, this system can be a proper alternative for cartilage tissue engineering. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1932-1940, 2018.

Effects of hydrostatic pressure on biosynthetic activity during chondrogenic differentiation of MSCs in hybrid scaffolds.

The objective of this study was to determine the effects of hydrostatic pressure (HP) on the biochemical properties and gene expression of mesenchymal stem cells (MSCs) on scaffolds for cartilage tissue engineering composed of poly(caprolactone) (PCL) poly(vinyl alcohol) (PVA) gelatin (GEL) semi interpenetrating polymer network (semi-IPN). The MSCs were cultured on PCL-PVA-GEL semi-IPN scaffolds in two groups (A and B) for 7 and 21 days, respectively, and then loaded with hydrostatic pressure (5 MPa, 0.5 Hz) for 2 h per day for the period of 7 days and compared with two non-loaded groups (C and D) as controls. DMMB and real-time PCR analysis for assaying cartilage-specific extracellular matrix (ECM) gene markers were carried out. According to the results, there were no significant differences in GAG amounts between the loaded and non-loaded constructs were observed after 14 days. However, significant and considerable increases in the expression amount of type II collagen mRNA levels in group A ( from 2.43 × 10-4 ± 5.32 × 10-5 to 2.09 × 10-3 ± 1.07 × 10-4 time), and in group B (from 3.04 × 10-4 ± 4.31 × 10-5 to 2.08 × 10-3 ± 1.59 × 10-4 time) in comparison with non-loaded groups (C and D) were observed, respectively. Results showed the beneficial role of hydrostatic pressure on the increase of type II collagen mRNA levels in articular cartilage tissue engineering.