Liam M. Grover

Ionic modification of calcium phosphate cement viscosity. Part II: hypodermic injection and strength improvement of brushite cement.

Brushite-forming calcium phosphate cements are of great interest as bone replacement materials because they are resorbable in physiological conditions. However, their short setting times, low mechanical strengths and limited injectability limit broad clinical application. In this study, we showed that a significant improvement of these properties of brushite cement could be achieved by the use of sodium citrate or citric acid as setting retardants, such that workable cement pastes with a powder to liquid ratio of up to 5 could be manufactured. The cement used in this study consisted of an equimolar powder mixture of beta-tricalcium phosphate and monocalcium phosphate hydrate The use of 500 mM-1M retardant solutions as liquid phase enabled initial setting times of 8-12 min. Wet compressive strength were found to be in the range between 12-18 MPa after immersion of uncompacted cement samples in serum for 24 h. A further strength improvement to 32 MPa was obtained by compaction of the cement paste during samples preparation. This is significant because high-temperature processes cannot be used to fabricate hydrated calcium phosphate materials. Cement pastes were injectable through a hypodermic needle at a powder to liquid ratio of 3.3 g/ml when a 1M citric acid was used as liquid phase, thus enabling precise controlled delivery to small defects.

Preparation of macroporous calcium phosphate cement tissue engineering scaffold

Unlike sintered hydroxyapatite there is evidence to suggest that calcium phosphate cement (CPC) is actively remodelled in vivo and because CPC is formed by a low-temperature process, thermally unstable compounds such as proteins may be incorporated into the matrix of the cement which can then be released after implantation. The efficacy of a macroporous CPC as a bone tissue engineering scaffold has been reported; however, there have been few previous studies on the effect of macroporosity on the mechanical properties of the CPC. This study reports a novel method for the formation of macroporous CPC scaffolds, which has two main advantages over the previously reported manufacturing route: the cement matrix is considerably denser than CPC formed from slurry systems and the scaffold is formed at temperatures below room temperature. A mixture of frozen sodium phosphate solution particles and CPC powder were compacted at 106 MPa and the sodium phosphate was allowed to melt and simultaneously set the cement. The effect of the amount of porogen used during processing on the porosity, pore size distribution and compressive strength of the scaffold was investigated. It was found that macroporous CPC could reliably be fabricated using cement:ice ratios as low as 5:2.

In vitro ageing of brushite calcium phosphate cement.

In vivo studies investigating the use of brushite cements have demonstrated mixed results with one or more of dissolution, hydrolysis, fragmentation and long term stability being demonstrated. It has been suggested that sample volume, implant location, and species can affect in vivo behaviour. As few in vitro studies on this cement system have been performed, this study aimed to compare the effects of static and dynamic in vitro ageing protocols on the phase composition, weight loss and mechanical properties of brushite cement. The effects of immersion liquid to cement volume ratio (LCVR) and sample volume on phase composition were investigated and comparative in vitro experiments were also performed in foetal bovine serum. It was determined that the weight loss after 28 days was up to seven times higher in serum than in phosphate buffered saline (PBS) and that fragmentation accounted for most of the weight loss observed. Hydroxyapatite was formed in PBS but not in serum when aged in refreshed media at all LCVRs investigated. This study has highlighted that LCVR, media refresh rate and media composition are critical to brushite cement performance. It appears that brushite cement removal from an implant site may be complex and dependent on physiological processes other than simple dissolution. A better understanding of these processes could provide the means to engineer more precise calcium phosphate cement degradation profiles.

Silver-doped calcium phosphate cements with antimicrobial activity.

There is a current need for the localised delivery of antibiotics in order to treat implant-based bacterial infections. Existing treatments use non-resorbable materials such as poly(methyl methacrylate) beads loaded with antibiotics; unfortunately, as they are not resorbable, these beads require secondary surgery for removal. Calcium phosphate cements have considerable potential for the localised delivery of drugs since they can be resorbed to some extent within the body, eliminating the need for a secondary surgical procedure. Therefore, in this study, the efficacy of both hydroxyapatite and brushite cements in the delivery of silver ions has been investigated. The activity of the Ag(+) released from the cements was assessed against the growth of both Staphylococcus aureus and Staphylococcus epidermidis; the brushite cement exhibited excellent antibacterial properties and also showed an increase in compressive strength of over 30%. In this study we have found that with a few changes in Ag(+) concentration it should be possible to produce a fully resorbable bone replacement material that is combined with an antibacterial scaffold with controlled release over a period of time, which is likely to inhibit bacterial infections associated with implantation procedures.

Encapsulation of fibroblasts causes accelerated alginate hydrogel degradation

Calcium-alginate hydrogel has been widely studied as a material for cell encapsulation for tissue engineering. At present, the effect that cells have on the degradation of alginate hydrogel is largely unknown. We have shown that fibroblasts encapsulated at a density of 7.5 x 10(5) cells ml(-1) in both 2% and 5% w/v alginate remain viable for at least 60 days. Rheological analysis was used to study how the mechanical properties exhibited by alginate hydrogel changed during 28 days in vitro culture. Alginate degradation was shown to occur throughout the study but was greatest within the first 7 days of culture for all samples, which correlated with a sharp release of calcium ions from the construct. Fibroblasts were shown to increase the rate of degradation during the first 7 days when compared with acellular samples in both 2% and 5% w/v gels, but after 28 days both acellular and cell-encapsulating samples retained disc-shaped morphologies and gel-like spectra. The results demonstrate that although at an early stage cells influence the mechanical properties of encapsulating alginate, over a longer period of culture, the hydrogels retain sufficient mechanical integrity to exhibit gel-like properties. This allows sustained immobilization of the cells at the desired location in vivo where they can produce extracellular matrix and growth factors to expedite the healing process.

Preparation and characterisation of nanophase Sr, Mg, and Zn substituted hydroxyapatite by aqueous precipitation.

Hydroxyapatite (HA) substituted with 2 mol% Sr, 10 mol% Mg, and 2 mol% Zn were precipitated under identical alkaline conditions (pH 11) at 20°C from an aqueous solution. As-synthesised materials were confirmed to be phase pure by XRD and samples prepared in air contained surface adsorbed CO2 as observed by FTIR. SEM studies revealed a globular morphology and agglomeration behaviour, typical of precipitated nHA. EDS spectra confirmed nominal compositions and substitution of Sr, Mg and Zn. At the levels investigated cationic doping was not found to radically influence particle morphology. An indication of the potential in-vivo bioactivity of samples was achieved by analysing samples immersed in SBF for up to 28 days by interferometry and complementary SEM micrographs. Furthermore, a live/dead assay was used and confirmed the viability of seeded MC3T3 osteoblast precursor cells on HA and substituted HA substrates up to 7 days of culture.

Temperature dependent setting kinetics and mechanical properties of β-TCP–pyrophosphoric acid bone cement

Brushite (CaHPO4·2H2O) is the product of acidic calcium phosphate cement forming reactions. It has a higher solubility than hydroxyapatite under physiological conditions and is a promising resorbable bone cement candidate. It reacts extremely fast so dilute mixes are required to form cements having setting times with surgical utility, which in turn compromises strength and produces a highly porous material. Pyrophosphate ions have a strong affinity for calcium orthophosphate surfaces, can inhibit their mineralisation and are thought to hydrolyze, forming orthophosphate during bone formation. The effect of replacing the acid orthophosphate component of a brushite cement with pyrophosphoric acid on the cement setting reaction time at temperatures of between 24 °C and 70 °C was determined. The substitution of pyrophosphate for orthophosphate retarded setting and improved mechanical performance of the cement. Pyrophosphate did not inhibit the extent of reaction but did influence microstructure of the brushite crystals. Temperature was found to have a significant (p < 0.01) influence on mechanical performance, and this was attributed to the formation of monetite (CaHPO4) rather than brushite at temperatures of ≥55 °C.

Phase composition, mechanical performance and in vitro biocompatibility of hydraulic setting calcium magnesium phosphate cement

Brushite (CaHPO(4) x 2H(2)O)-forming calcium phosphate cements are of great interest as bone replacement materials because they are resorbable in physiological conditions. However, their short setting times and low mechanical strengths limit broad clinical application. In this study, we showed that a significant improvement of these properties of brushite cement could be achieved by the use of magnesium-substituted beta-tricalcium phosphate with the general formula Mg(x)Ca((3-x))((PO(4))(2) with 0 < x < 3 as cement reactants. The incorporation of magnesium ions increased the setting times of cements from 2 min for a magnesium-free matrix to 8-11 min for Mg(2.25)Ca(0.75)(PO(4))(2) as reactant. At the same time, the compressive strength of set cements was doubled from 19 MPa to more than 40 MPa after 24h wet storage. Magnesium ions were not only retarding the setting reaction to brushite but were also forming newberyite (MgHPO(4) x 3H(2)O) as a second setting product. The biocompatibility of the material was investigated in vitro using the osteoblast-like cell line MC3T3-E1. A considerable increase of cell proliferation and expression of alkaline phosphatase, indicating an osteoblastic differentiation, could be noticed. Scanning electron microscopy analysis revealed an obvious cell growth on the surface of the scaffolds. Analysis of the culture medium showed minor alterations of pH value within the physiological range. The concentrations of free calcium, magnesium and phosphate ions were altered markedly due to the chemical solubility of the scaffolds. We conclude that the calcium magnesium phosphate (newberyite) cements have a promising potential for their use as bone replacement material since they provide a suitable biocompatibility, an extended workability and improved mechanical performance compared with brushite cements.

AN ALGINATE HYDROGEL MATRIX FOR THE LOCALISED DELIVERY OF A FIBROBLAST/KERATINOCYTE CO-CULTURE

There is significant interest in the development of tissue-engineered skin analogues, which replace both the dermal and the epidermal layer, without the use of animal or human derived products such as collagen or de-epidermalised dermis. In this study, we proposed that alginate hydrogel could be used to encapsulate fibroblasts and that keratinocytes could be cultured on the surface to form a bilayered structure, which could be used to deliver the co-culture to a wound bed, initially providing wound closure and eventually expediting the healing process. Encapsulation of fibroblasts in 2 and 5% w/v alginate hydrogel effectively inhibited their proliferation, whilst maintaining cell viability allowing keratinocytes to grow uninhibited by fibroblast overgrowth to produce a stratified epidermal layer. It was shown that the alginate degradation process was not influenced by the presence of fibroblasts within the hydrogel and that lowering the alginate concentration from 5 to 2% w/v increased the rate of degradation. Fibroblasts released from the scaffold were able to secrete extracellular matrix (ECM) and thus should replace the degrading scaffold with normal ECM following application to the wound site. These findings demonstrate that alginate hydrogel may be an effective delivery vehicle and scaffold for the healing of full-thickness skin wounds.

Reversible mitotic and metabolic inhibition following the encapsulation of fibroblasts in alginate hydrogels

Limiting cell proliferation without reducing cell viability for in vivo tissue engineering applications is important in co-culture applications where the growth of one cell type must be inhibited to prevent overgrowth of the scaffold at the expense of another cell type. Also, it is vital for maintaining viability of cells in large constructs before vascularisation occurs. In this study we have shown by means of the Thiazolyl blue (MTT) assay and immuno-staining for proliferating cell nuclear antigen (PCNA) that encapsulating fibroblasts in 2% and 5%w/v calcium-alginate at a density of 7.5 x 10(5)cells/ml as uniformly dispersed entities, enabled cells to maintain viability and caused a reversible mitotic inhibition. Alginate encapsulation also caused reversible metabolic inhibition as demonstrated by the MTT assay and fluorescent staining for mitochondrial membrane potential. Histological evaluation of the alginate constructs containing fibroblasts showed that mitotic and metabolic inhibition was possibly due to cell isolation during the first five weeks of culture. The alginate scaffold degraded with time releasing encapsulated fibroblasts. Upon implantation to a wound site this should ensure that encapsulated cells are able to replace the damaged tissue after sufficient proliferation of the co-cultured cell type or sufficient vascularisation of the construct.