Elena Geta Popa

Cell Delivery Systems Using Alginate–Carrageenan Hydrogel Beads and Fibers for Regenerative Medicine Applications

The present work was focused on the development and characterization of new hydrogel systems based on natural origin polymers, namely, alginate and carrageenan, into different formats and with adequate properties to sustain the viability of encapsulated cells, envisioning their application as cell delivery vehicles for tissue regeneration. Different formulations of alginate and carrageenan hydrogels and different processing parameters were considered to determine the best conditions required to achieve the most adequate response in terms of the mechanical stability, cell viability, and functionality of the developed systems. The morphology, size, and structure of the hydrogels and their degradation behavior and mechanical properties were evaluated during this study. In addition to cytotoxicity studies, preliminary experiments were carried out to investigate the ability of alginate--carrageenan beads/fibers to encapsulate chondrocytes. The results obtained indicated that the different formulations, both in the form of beads and fibers, have considerable potential as cell-carrier materials for cell delivery in tissue engineering/regenerative medicine applications.

Marine algae sulfated polysaccharides for tissue engineering and drug delivery approaches.

Biomedical field is constantly requesting for new biomaterials, with innovative properties. Natural polymers appear as materials of election for this goal due to their biocompatibility and biodegradability. In particular, materials found in marine environment are of great interest since the chemical and biological diversity found in this environment is almost uncountable and continuously growing with the research in deeper waters. Moreover, there is also a slower risk of these materials to pose illnesses to humans. In particular, sulfated polysaccharides can be found in marine environment, in different algae species. These polysaccharides don’t have equivalent in the terrestrial plants and resembles the chemical and biological properties of mammalian glycosaminoglycans. In this perspective, are receiving growing interest for application on health-related fields. On this review, we will focus on the biomedical applications of marine algae sulfated polymers, in particular on the development of innovative systems for tissue engineering and drug delivery approaches.

An investigation of the potential application of chitosan/aloe-based membranes for regenerative medicine

A significant number of therapeutics derived from natural polymers and plants have been developed to replace or to be used in conjunction with existing dressing products. The use of the therapeutic properties of aloe vera could be very useful in the creation of active wound dressing materials. The present work was undertaken to examine issues concerning structural features, topography, enzymatic degradation behavior, antibacterial activity and cellular response of chitosan/aloe vera-based membranes. The chitosan/aloe vera-based membranes that were developed displayed satisfactory degradation, roughness, wettability and mechanical properties. A higher antibacterial potency was displayed by the blended membranes. Moreover, in vitro assays demonstrated that these blended membranes have good cell compatibility with primary human dermal fibroblasts. The chitosan/aloe vera-based membranes might be promising wound dressing materials.

Enhancement of osteogenic differentiation of human adipose derived stem cells by the controlled release of platelet lysates from hybrid scaffolds produced by supercritical fluid foaming.

A new generation of scaffolds capable of acting not only as support for cells but also as a source of biological cues to promote tissue regeneration is currently a hot topic of in bone Tissue Engineering (TE) research. The inclusion of growth factor (GF) controlled release functionalities in the scaffolds is a possible strategy to achieve such goal. Platelet Lysate (PL) is an autologous source of GFs, providing several bioactive agents known to act on bone regeneration. In this study, chitosan-chondroitin sulfate nanoparticles loaded with PL were included in a poly(D,L-lactic acid) foam produced by supercritical fluid foaming. The tridimensional (3D) structures were then seeded with human adipose-derived stem cells (hASCs) and cultured in vitro under osteogenic stimulus. The osteogenic differentiation of the seeded hASCs was observed earlier for the PL-loaded constructs, as shown by the earlier alkaline phosphatase peak and calcium detection and stronger Runx2 expression at day 7 of culture, in comparison with the control scaffolds. Osteocalcin gene expression was upregulated in presence of PL during all culture period, which indicates an enhanced osteogenic induction. These results suggest the synergistic effect of PL and hASCs in combinatory TE strategies and support the potential of PL to increase the multifunctionality of the 3D hybrid construct for bone TE applications.

Chondrogenic potential of injectable κ-carrageenan hydrogel with encapsulated adipose stem cells for cartilage tissue-engineering applications

Due to the limited self‐repair capacity of cartilage, regenerative medicine therapies for the treatment of cartilage defects must use a significant amount of cells, preferably applied using a hydrogel system that can promise their delivery and functionality at the specific site. This paper discusses the potential use of κ‐carrageenan hydrogels for the delivery of stem cells obtained from adipose tissue in the treatment of cartilage tissue defects. The developed hydrogels were produced by an ionotropic gelation method and human adipose stem cells (hASCs) were encapsulated in 1.5% w/v κ‐carrageenan solution at a cell density of 5 × 106 cells/ml. The results from the analysis of the cell‐encapsulating hydrogels, cultured for up to 21 days, indicated that κ‐carrageenan hydrogels support the viability, proliferation and chondrogenic differentiation of hASCs. Additionally, the mechanical analysis demonstrated an increase in stiffness and viscoelastic properties of κ‐carrageenan gels with their encapsulated cells with increasing time in culture with chondrogenic medium. These results allowed the conclusion that κ‐carrageenan exhibits properties that enable the in vitro functionality of encapsulated hASCs and thus may provide the basis for new successful approaches for the treatment of cartilage defects. Copyright

Chondrogenic phenotype of different cells encapsulated in κ-carrageenan hydrogels for cartilage regeneration strategies.

Engineering articular cartilage substitutes using hydrogels with encapsulated cells is an approach that has received increasing attention in recent years. Hydrogels based on κ‐carrageenan (κC), a thermoreversible natural‐origin polymer, have been recently proposed as new cell/growth factor delivery vehicles for regenerative medicine. In this work, we report the potential of such hydrogels encapsulating either human‐adipose‐derived stem cells (hASCs), human nasal chondrocytes (hNCs), or a chondrocytic cell line (ATDC5) for cartilage regeneration strategies. The in vitro cellular behavior of the encapsulated cells within κC hydrogel was analyzed after different culturing periods using biochemical assays and histological and real‐time reverse‐transcription PCR analysis. The three types of cells encapsulated in κC hydrogels showed good cellular viability and proliferation up to 21 days of culture, and the cell‐laden hydrogels were positive for specific cartilage markers. In summary, the results demonstrate that hASCs embedded in κC hydrogels proliferate faster and exhibit higher expression levels of typical cartilage markers as compared with hNCs or ATDC5 cells. Based on these data, it is possible to conclude that κC hydrogel provides a good support for culture and differentiation of encapsulated cells and that hASCs may provide an advantageous alternative to primary chondrocytes, currently used in clinical treatments of cartilage defects/diseases.

Silk hydrogels from non-mulberry and mulberry silkworm cocoons processed with ionic liquids

Matrices based on silk fibroin from the non-mulberry silkworm Antheraea mylitta and the mulberry silkworm Bombyx mori have demonstrated good applicability in regenerative medicine. However, the cocoons of A. mylitta are underutilized in part due to their lack of solubility in traditional organic solvents. Therefore, the present work investigates the solubilization and processing of degummed fibers obtained from the cocoons of both silkworm species into hydrogels using ionic liquids (ILs). The developed hydrogels exhibited a rubbery consistency, viscoelastic behavior and rapid degradation in the presence of protease XIV. Scanning electron and confocal microscopy images suggest that human adipose stem cells (hASCs) are able to adhere to and migrate at different levels within the hydrogel structures. Moreover, the MTS assay demonstrated the maintenance of cell metabolic activity for up to 28 days, while DNA quantification showed that hASCs were able to proliferate on the seeded hydrogels. The findings indicate that complete IL removal from the fabricated hydrogels results in a positive hASCs cellular response. Thus the present approach provides a unique opportunity to broaden the processability and application of silk fibroin obtained from A. mylitta cocoons for regenerative medicine, namely cartilage regeneration.

Evaluation of the in vitro and in vivo biocompatibility of carrageenan‐based hydrogels

Carrageenans are highly sulphated galactans, well-known for their thermogelation properties which have been extensively exploited in food and cosmetics industry but poorly explored in the biomedicine field. In this study, we have assessed the in vitro and in vivo biocompatibility of κ-carrageenan hydrogels that have been explored for regenerative medicine and tissue engineering applications. The in vitro cytotoxicity of the materials using a L929 mouse fibroblast cell line was evaluated, and the effect of κ-carrageenan hydrogels on the activation of human polymorphonuclear neutrophils cells (hPMNs) was also evaluated by the quantification of reactive oxygen species by chemiluminescence. Subsequently, the inflammatory/immune reaction to κ-carrageenan hydrogels on subcutaneous implantation was studied in rats. Explants were retrieved after 1 and 2 weeks of implantation for histological and RT-PCR analysis. The cytotoxicity screening revealed that κ-carrageenan hydrogels did not significantly affect L929 metabolic activity. Moreover, hPMNs contact with κ-carrageenan resulted in a reduced and a neglectable signal regarding the detection of superoxide and hydroxyl anions, respectively. The results from the in vivo experiments indicated that κ-carrageenan induce a low inflammatory response. Overall, the data obtained suggest that κ-carrageenan hydrogels are biocompatible and thus can be further studied for their use in target biomedical applications.

Cryopreservation of cell laden natural origin hydrogels for cartilage regeneration strategies

The time span needed for obtaining a functional cartilage substitute using tissue engineering strategies, together with the need for specific patient oriented constructs has stimulated the growing interest for developing “off-the shelf” products. One way to deliver such products is based on long-term storage processes, such as cryopreservation, that will provide the clinical substitute available as needed and could be adapted to an autologous immediate solution for the patient. The aim of this study was to examine the effects of cryopreservation on the chondrogenic differentiation characteristics of human mesenchymal derived stem cells isolated from adipose tissue and encapsulated in κ-carrageenan hydrogels. These bioengineered constructs are anticipated to participate in a cartilage regeneration strategy providing temporary habitation for cell survival, proliferation and production of an extracellular matrix which is expected to replace the hydrogel, enhancing the regeneration of native tissues in clinical settings. The results obtained show that the hydrogels withstand the cryopreservation with dimethyl sulfoxide, maintaining their structural integrity, while assisting cells proliferation and chondrogenic potential after cryopreservation. Thus, cell encapsulation systems of natural based hydrogels seem to be an interesting approach for the preservation of cartilage tissue engineered products.

Seaweed polysaccharide-based hydrogels used for the regeneration of articular cartilage

Abstract This manuscript provides an overview of the in vitro and in vivo studies reported in the literature focusing on seaweed polysaccharides based hydrogels that have been proposed for applications in regenerative medicine, particularly, in the field of cartilage tissue engineering. For a better understanding of the main requisites for these specific applications, the main aspects of the native cartilage structure, as well as recognized diseases that affect this tissue are briefly described. Current available treatments are also presented to emphasize the need for alternative techniques. The following part of this review is centered on the description of the general characteristics of algae polysaccharides, as well as relevant properties required for designing hydrogels for cartilage tissue engineering purposes. An in-depth overview of the most well known seaweed polysaccharide, namely agarose, alginate, carrageenan and ulvan biopolymeric gels, that have been proposed for engineering cartilage is also provided. Finally, this review describes and summarizes the translational aspect for the clinical application of alternative systems emphasizing the importance of cryopreservation and the commercial products currently available for cartilage treatment.