Cryoreservation of alginate-fibrin beads involving bone marrow derived mesenchymal stromal cells by vitrification

Application of cell-–biomaterial systems in regenerative medicine can be facilitated by their successful low temperature preservation. Vitrification, which avoids ice crystal formation by amorphous solidification, is an emerging approach to cryopreservation. Developing vitrification strategy, effective cryopreservation of alginate–fibrin beads with porcine mesenchymal stromal cells has been achieved in this study. The cell–biomaterial constructs were pre-cultured for 20 days before cryopreservation, allowing for cell proliferation and construct stabilization. Ethylene glycol (EG) was employed as the basic cryoprotectant for two equilibration solutions. Successful cryopreservation of the constructs was achieved using vitrification solution composed of penetrating (EG MW 62 Da) and non-penetrating (sucrose MW 342 Da) cryoprotectants. Stepwise procedure of introduction to and removal of cryoprotectants was brief; direct plunging into liquid nitrogen was applied. Cell viability, evaluated by combining live/death staining and confocal laser microscopy, was similar for both control and vitrified cells in the beads. No detectable damage of microstructure of cryopreserved beads was found as shown by scanning electron microscopy. Both osteogenically induced control and vitrified cells in the constructs were equally capable of mineral production and deposition. There was no statistically significant difference in metabolic activity and proliferation between both groups during the entire culture period. Our study leads to the conclusion that the developed cryopreservation protocol allowed to maintain the integrity of the beads while preserving the ability of the pig bone marrow derived mesenchymal stromal cells to proliferate and subsequently differentiate; demonstrating that vitrification is a promising approach for cryopreser-vation of “ready-to-use” cell–biomaterial constructs.

Bioactive inorganic materials/alginate composite microspheres with controllable drug-delivery ability

Alginate microspheres are considered a promising material as a drug carrier in bone repair due to excellent biocompatibility, but their main disadvantage is low drug entrapment efficiency and non-controllable release. The aim of this study was to investigate the effect of incorporating mesoporous bioglass (MBG), non-mesoporous bioglass (BG) or hydroxyapatite (HAp) into alginate microspheres on their drug-loading and release properties. X-ray diffraction (XRD), transmission electron microscopy (TEM), scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), and atomic emission spectroscopy (AES) were used to analyse the composition, structure and dissolution of bioactive inorganic materials and their microspheres. Dexamethasone (DEX)-loading and release ability of four microspheres were tested in phosphate buffered saline with varying pHs. Results showed that the drug-loading capacity was enhanced with the incorporation of bioactive inorganic materials into alginate microspheres. The MBG/Alginate microspheres had the highest drug loading ability. DEX release from alginate microspheres correlated to the dissolution of MBG, BG and HAp in PBS, and that the pH was an efficient factor in controlling the DEX release; a high pH resulted in greater DEX release, whereas a low pH delayed DEX release. In addition, MBG/alginate, BG/alginate and HAp/alginate microspheres had varying apatite-formation and dissolution abilities, which indicate that the composites would behave differently with respect to bioactivity. The study suggests that microspheres made of a composite of bioactive inorganic materials and alginate have a bioactivity and degradation profile which greatly improves their drug delivery capacity, thus enhancing their potential applications as bioactive filler materials for bone tissue regeneration.

An alginate-based hybrid system for growth factor delivery in the functional repair of large bone defects

The treatment of challenging fractures and large osseous defects presents a formidable problem for orthopaedic surgeons. Tissue engineering/regenerative medicine approaches seek to solve this problem by delivering osteogenic signals within scaffolding biomaterials. In this study, we introduce a hybrid growth factor delivery system that consists of an electrospun nanofiber mesh tube for guiding bone regeneration combined with peptide-modified alginate hydrogel injected inside the tube for sustained growth factor release. We tested the ability of this system to deliver recombinant bone morphogenetic protein-2 (rhBMP-2) for the repair of critically-sized segmental bone defects in a rat model. Longitudinal [mu]-CT analysis and torsional testing provided quantitative assessment of bone regeneration. Our results indicate that the hybrid delivery system resulted in consistent bony bridging of the challenging bone defects. However, in the absence of rhBMP-2, the use of nanofiber mesh tube and alginate did not result in substantial bone formation. Perforations in the nanofiber mesh accelerated the rhBMP-2 mediated bone repair, and resulted in functional restoration of the regenerated bone. [mu]-CT based angiography indicated that perforations did not significantly affect the revascularization of defects, suggesting that some other interaction with the tissue surrounding the defect such as improved infiltration of osteoprogenitor cells contributed to the observed differences in repair. Overall, our results indicate that the hybrid alginate/nanofiber mesh system is a promising growth factor delivery strategy for the repair of challenging bone injuries.

In situ preparation and protein delivery of silicate/alginate composite microspheres with core-shell structure

It is predicted that with increased life expectancy in the developed world, there will be a greater demand for synthetic materials to repair or regenerate lost, injured or diseased bone (Hench & Thompson 2010). There are still few synthetic materials having true bone inductivity, which limits their application for bone regeneration, especially in large-size bone defects. To solve this problem, growth factors, such as bone morphogenetic proteins (BMPs), have been incorporated into synthetic materials in order to stimulate de novo bone formation in the center of large-size bone defects. The greatest obstacle with this approach is that the rapid diffusion of the protein from the carrier material, leading to a precipitous loss of bioactivity; the result is often insufficient local induction or failure of bone regeneration (Wei et al. 2007). It is critical that the protein is loaded in the carrier material in conditions which maintains its bioactivity (van de Manakker et al. 2009). For this reason, the efficient loading and controlled release of a protein from a synthetic material has remained a significant challenge. The use of microspheres as protein/drug carriers has received considerable attention in recent years (Lee et al. 2010; Pareta & Edirisinghe 2006; Wu & Zreiqat 2010). Compared to macroporous block scaffolds, the chief advantage of microspheres is their superior protein-delivery properties and ability to fill bone defects with irregular and complex shapes and sizes. Upon implantation, the microspheres are easily conformed to the irregular implant site, and the interstices between the particles provide space for both tissue and vascular ingrowth, which are important for effective and functional bone regeneration (Hsu et al. 1999). Alginates are natural polysaccharides and their production does not have the implicit risk of contamination with allo or xeno-proteins or viruses (Xie et al. 2010). Because alginate is generally cytocompatible, it has been used extensively in medicine, including cell therapy and tissue engineering applications (Tampieri et al. 2005; Xie et al. 2010; Xu et al. 2007). Calcium cross-linked alginate hydrogel is considered a promising material as a delivery matrix for drugs and proteins, since its gel microspheres form readily in aqueous solutions at room temperature, eliminating the need for harsh organic solvents, thereby maintaining the bioactivity of proteins in the process of loading into the microspheres (Jay & Saltzman 2009; Kikuchi et al. 1999). In addition, calcium cross-linked alginate hydrogel is degradable under physiological conditions (Kibat PG et al. 1990; Park K et al. 1993), which makes alginate stand out as an attractive candidate material for the protein carrier and bone regeneration (Hosoya et al. 2004; Matsuno et al. 2008; Turco et al. 2009). However, the major disadvantages of alginate microspheres is their low loading efficiency and also rapid release of proteins due to the mesh-like networks of the gel (Halder et al. 2005). Previous studies have shown that a core-shell structure in drug/protein carriers can overcome the issues of limited loading efficiencies and rapid release of drug or protein (Chang et al. 2010; Molvinger et al. 2004; Soppimath et al. 2007). We therefore hypothesized that introducing a core-shell structure into the alginate microspheres could solve the shortcomings of the pure alginate. Calcium silicate (CS) has been tested as a biodegradable biomaterial for bone tissue regeneration. CS is capable of inducing bone-like apatite formation in simulated body fluid (SBF) and its apatite-formation rate in SBF is faster than that of Bioglass® and A-W glass-ceramics (De Aza et al. 2000; Siriphannon et al. 2002). Titanium alloys plasma-spray coated with CS have excellent in vivo bioactivity (Xue et al. 2005) and porous CS scaffolds have enhanced in vivo bone formation ability compared to porous β-tricalcium phosphate ceramics (Xu et al. 2008). In light of the many advantages of this material, we decided to prepare CS/alginate composite microspheres by combining a CS shell with an alginate core to improve their protein delivery and mineralization for potential protein delivery and bone repair applications

Cell density alters matrix accumulation in two distinct fractions and the mechanical integrity of alginate-chondrocyte constructs

Chondrocyte density in articular cartilage is known to change with the development and growth of the tissue and may play an important role in the formation of a functional extracellular matrix (ECM). The objective of this study was to determine how initial chondrocyte density in an alginate hydrogel affects the matrix composition, its distribution between the cell-associated (CM) and further removed matrix (FRM) fractions, and the tensile mechanical properties of the developing engineered cartilage. Alginate constructs containing primary bovine chondrocytes at densities of 0, 4, 16, and 64 million cells/ml were fabricated and cultured for 1 or 2 weeks, at which time structural, biochemical, and mechanical properties were analyzed. Both matrix content and distribution varied with the initial cell density. Increasing cell density resulted in an increasing content of collagen and sulfated-glycosaminoglycan (GAG) and an increasing proportion of these molecules localized in the CM. While the equilibrium tensile modulus of cell-free alginate did not change with time in culture, the constructs with highest cell density were 116% stiffer than cell-free controls after 2 weeks of culture. The equilibrium tensile modulus was positively correlated with total collagen (r2 = 0.47, p < 0.001) and GAG content (r2 = 0.68, p < 0.001), and these relationships were enhanced when analyzing only those matrix molecules in the CM fraction (r2 = 0.60 and 0.72 for collagen and GAG, respectively, each p < 0.001). Overall, the results of this study indicate that initial cell density has a considerable effect on the developing composition, structure, and function of alginate–chondrocyte constructs.

The potential for production of freeze-dried oral vaccines using alginate hydrogel microspheres as protein carriers

Oral administration of dry vaccine formulations is acknowledged to offer major clinical and logistical benefits by eliminating the cold chain required for liquid preparations. A model antigen, bovine serum albumin (BSA) was encapsulated in alginate microspheres using aerosolisation. Hydrated microspheres 25 to 65 μm in size with protein loading of 3.3 % w/w were obtained. Environmental scanning electron microscopy indicated a stabilizing effect of encapsulated protein on alginate hydrogels revealed by an increase in dehydration resistance. Freeze drying of alginate microspheres without use of a cryoprotectant resulted in fragmentation and subsequent rapid loss of the majority of the protein load in simulated intestinal fluid in 2 h, whereas intact microspheres were observed following freeze-drying of BSA-loaded microspheres in the presence of maltodextrin. BSA release from freeze-dried preparations was limited to less than 7 % in simulated gastric fluid over 2 h, while 90 % of the protein load was gradually released in simulated intestinal fluid over 10 h. SDS-PAGE analysis indicated that released BSA largely preserved its molecular weight. These findings demonstrate the potential for manufacturing freeze-dried oral vaccines using alginate microspheres.

The in vivo fate of nanoparticles and nanoparticle-loaded microcapsules after oral administration in mice: Evaluation of their potential for colon-specific delivery

Anti-cancer drug loaded-nanoparticles (NPs) or encapsulation of NPs in colon-targeted delivery systems shows potential for increasing the local drug concentration in the colon leading to improved treatment of colorectal cancer. To investigate the potential of the NP-based strategies for colon-specific delivery, two formulations, free Eudragit® NPs and enteric-coated NP-loaded chitosan–hypromellose microcapsules (MCs) were fluorescently-labelled and their tissue distribution in mice after oral administration was monitored by multispectral small animal imaging. The free NPs showed a shorter transit time throughout the mouse digestive tract than the MCs, with extensive excretion of NPs in faeces at 5 h. Conversely, the MCs showed complete NP release in the lower region of the mouse small intestine at 8 h post-administration. Overall, the encapsulation of NPs in MCs resulted in a higher colonic NP intensity from 8 h to 24 h post-administration compared to the free NPs, due to a NP ‘guarding’ effect of MCs during their transit along mouse gastrointestinal tract which decreased NP excretion in faeces. These imaging data revealed that this widely-utilised colon-targeting MC formulation lacked site-precision for releasing its NP load in the colon, but the increased residence time of the NPs in the lower gastrointestinal tract suggests that it is still useful for localised release of chemotherapeutics, compared to NP administration alone. In addition, both formulations resided in the stomach of mice at considerable concentrations over 24 h. Thus, adhesion of NP- or MC-based oral delivery systems to gastric mucosa may be problematic for colon-specific delivery of the cargo to the colon and should be carefully investigated for a full evaluation of particulate delivery systems.

Alginate encapsulation of shoot tips and nodal segments for short-term storage and distribution of the eucalypt Corymbia torelliana × C. citriodora

A protocol was developed for short-term preservation and distribution of the plantation eucalypt, Corymbia torelliana × C. citriodora, using alginate-encapsulated shoot tips and nodes as synthetic seeds. Effects of sowing medium, auxin concentration, storage temperature and planting substrate on shoot regrowth or conversion into plantlets were assessed for four different clones. High frequencies of shoot regrowth (76–100%) from encapsulated explants were consistently obtained in hormone-free half- and full-strength Murashige and Skoog (MS) sowing media. Conversion into plantlets from synthetic seeds was achieved on half-strength MS medium by treating shoot tips or nodes with 4.9–78.4 μM IBA prior to encapsulation. Pre-treatment with 19.6 μM IBA provided 62–100% conversion, and 95–100% of plantlets survived after acclimatisation under nursery conditions. Synthetic seeds containing explants pre-treated with IBA were stored for 8 weeks much more effectively at 25°C than at 4°C, with regrowth frequencies of 50–84% at 25°C compared with 0–4% at 4°C. To eliminate the in vitro culture step after encapsulation, synthetic seeds were allowed to pre-convert before sowing directly onto a range of ex vitro non-sterile planting substrates. Highest frequencies (46–90%) of plantlet formation from pre-converted synthetic seeds were obtained by transferring shoot tip-derived synthetic seeds onto an organic compost substrate. These plantlets exhibited almost 100% survival in the nursery without mist irrigation. Pre-conversion of non-embryonic synthetic seeds is a novel technique that provides a convenient alternative to somatic embryo-derived artificial seeds.

Dual Drug Delivery Microcapsules via Layer-by-Layer Self-Assembly

The integration of hydrophobic and hydrophilic drugs in the polymer microcapsule offers the possibility of developing a new drug delivery system that combines the best features of these two distinct classes of material. Recently, we have reported the encapsulation of an uncharged water-insoluble drug in the polymer membrane. The hydrophobic drug is deposited using a layer-by-layer (LbL) technique, which is based on the sequential adsorption of oppositely charged polyelectrolytes onto a charged substrate. In this paper, we report the encapsulation of two different drugs, which are invariably different in structure and in their solubility in water. We have characterized these dual drug vehicular capsules by confocal laser scanning microscopy, atomic force microscopy, visible microscopy, and transmission electron microscopy. The growth of a thin film on a flat substrate by LbL was monitored by UV−vis spectra. The desorption kinetics of two drugs from the thin film was modeled by a second-order rate model.