Alginate hydrogel has a negative impact on in vitro collagen 1 deposition by fibroblasts

Hydrogels have been widely investigated as 3D culture substrates because of their reported structural similarity to the extracellular matrix (ECM). Limited ECM deposition, however, occurs within these materials, so the resulting “tissues” bear little resemblance to those found in the body. Here matrix deposition by fibroblasts encapsulated within a calcium alginate (Ca-alg) hydrogel was investigated. Although the cells transcribed mRNA for coll Iα over a period of 3 weeks, very little collagen protein deposition was observed within the gel by histology or immunohistochemistry (IHC). Although molecular diffusion demonstrated charge dependency, this did not prevent the flux of both positively and negative charged amino acids through the gel, suggesting that the absence of ECM could not be attributed to substrate limitation. The flux of protein, however, was charge-dependent as proteins with a net negative charge passed quickly through the Ca-alg into the medium. The minimal collagen deposition within the Ca-alg was attributed to a combination of rapid movement of negatively charged procollagen through the gel and steric hindrance of fibril formation

Elucidating the biological role of silicon and designing a delivery system to enhance early bone mineralisation

Silicon has been shown to be an important trace element in bone formation and metabolism, and a decrease in silicon in the mammalian diet leads to abnormal bone formation. Consequently, silicon has been incorporated into many biomaterials to enhance bone generation around implants. Despite this, the mechanism of action has still not been elucidated and a therapeutic dosage has not been determined. In this thesis, the optimum concentration of orthosilicic acid (OSA) to enable cell survival and early mineralisation has been identified. It was noted that a dosage of 5µg/ml of OSA enhanced bone nodule formation. The presence of OSA increased the expression of early osteogenic markers such as osteopontin, osteocalcin and type 1 collagen. In addition, increasing OSA concentration resulted in the development of a collagen fibril network of increasing complexity, up to supraphysiological OSA concentrations when the fibril network became fragmented. It was hypothesised that this may assist with mineral deposition. A sustained delivery system was also developed using a combination of PLGA and calcium silicate. A sustained dose of orthosilicic acid ideal for cell survival was released from the PLGA micro-particles containing calcium silicate. As well as providing a source of OSA, the presence of the alkaline degradation products of calcium silicate aided in the neutralisation of the acidic degradation products of PLGA, which might enhance cell viability in the local environment. In addition to influencing cell behaviour, the OSA was shown to have a strong interaction with alginate, modifying its properties and preventing degradation. This finding is of importance as the molecules comprising alginate bear a structural resemblance to the glycosaminoglycans that are found in the majority of tissues

Elucidating the biological role of soluble silicon in early bone mineralisation

The role of silicon in bone biology has been famously reported by Carlisle (1974) and Schwarz et al (1973). They demonstrated that when the levels of silica in an animal’s diet were decreased to a critical level, bone and cartilage deformation resulted. Silicon at 0.5 wt% in vivo has been shown to be present within the active mineralising osteoid regions, that is, the sites undergoing active calcification. This suggested that silicon may play a key role in the metabolism and stabilisation of the connective tissue present in bone and cartilage although its effect on calcification may arise indirectly through its interaction with matrix components. In vitro studies carried out by Reffit et al (2003) demonstrated that type 1 collagen synthesis in human osteoblast-like cells was enhanced in the presence of 10-20 μM orthosilicic acid. Here we examine the evidence for a possible biological mechanism of orthosilicic acid (OSA), the soluble form of silicon influencing early bone mineralisation specifically focusing on its effect on mineral deposition and collagen fibril formation

Synthesis of pure dicalcium silicate powder by the pechini method and characterization of hydrated cement

Calcium silicate (CS) is a main component of Portland cement and is responsible for the strength development. Recent research has shown that dicalcium silicate cement (CSC) is bioactive and is a potential candidate for bone replacement. Traditionally, dicalcium silicate powder is synthesized by a solid state reaction or a sol-gel method. The solid-state reaction, however, usually needs a higher temperature and a longer calcination time. Furthermore, the dicalcium silicate powder made by the sol-gel method is not pure, and contains a significant quantity of CaO which is harmful to the strength and biological properties of the CSC. The Pechini technique is an alternative, low temperature polymeric precursor route for synthesis of high purity powders. In this study, purer CS powder was synthesized via the Pechini method by calcination at 800°C for 3h. DSC-TGA, XRD, SEM were used for characterization of CS powder and the hydrated cement. The DSC-TGA curves showed that the main exothermic peak was at 479°C and the total mass loss was 79.2%. The XRD patterns of CSC after hydration for 7, 14, and 35 days illustrated that dicalcium silicate hydrate (Ca1.5SiO3.5·xH2O, C-S-H) was formed in the hardened CS paste. The XRD peaks on the diffraction pattern of the C-S-H of the day 35 sample were of greater intensity than those at day 7 and day 14. This demonstrates that the hydration speed was slow and complete hydration could take more than one month. Flake-like crystals were observed on scanning electron micrographs following hardening. The degradation study result showed that there was no mass loss of CSC after the samples were soaked into phosphate buffered saline (PBS) for 40 days. The silicon assay revealed that orthosilicic acid could be released from CSC after the samples were soaked in simulated body fluid (SBF). Silicon is known to be critical to skeletal mineralization. The existence of silicon may stimulate the proliferation of bone and activate cells to produce bone. Investigation of cell attachment confirmed that the MC-3T3 cells attached well to the surfaces of CSC after seeding.</jats:p

Modification of alginate degradation properties using orthosilicic acid

Biopolymers such as alginates have been widely researched for clinical use. Their clinical application, however, have been limited due to their unpredictable and often rapid degradation rates. Here we show that the degradation of an alginate hydrogel can be tailored through the addition of orthosilicic acid (OSA). On immersion in aqueous media a negligible quantity of orthosilicic acid was released from the gel matrix. The presence of the OSA within the gel was shown to significantly slow degradation of the alginate hydrogel when immersed in a potent calcium chelator (EDTA) when compared with the control group. Sample degradation was associated with a significant calcium release from the non modified gel; however, the orthosilicic acid modified gel did not release detectable levels of calcium over the same period. This suggests that the orthosilicic acid inhibits degradation of the gel by forming an interaction with the calcium cross-links. A rapid reduction in the storage modulus G’, was observed in alginate made without OSA, however, the G’ exhibited by the orthosilicic acid modified alginate did not reduce significantly (p<0.05) Furthermore, although both the OSA and alginate exhibit negative charges in solution, it is likely that they form weak interactions, this hypothesis was proven by demonstrating the efficacy of OSA for binding the alginate hydrocolloid. The findings of this study are likely to have utility in applications where controlling gel degradation is desirable, such as in cell delivery or in the controlled release of molecules in the body