Jake E. Barralet

Precipitation casting of polycaprolactone for applications in tissue engineering and drug delivery

Microporous materials have been produced by gradual precipitation from solutions of poly(epsilon-caprolactone) (PCL) in acetone induced by solvent extraction across a semi-permeable PCL membrane which is formed in situ at the polymer solution/non-solvent interface. Microparticulates of hydroxyapatite and inulin polysaccharide, respectively, were incorporated in precipitation cast PCL matrices to illustrate potential applications in hard tissue repair and macromolecular drug release. Microporous PCL and HA filled PCL materials were found to provide a favourable surface for attachment and growth of primary human osteoblasts in cell culture. The in vitro degradation characteristics of microporous PCL and inulin/PCL materials in PBS at 37 degrees C were monitored over 45 months. Microporous PCL demonstrated zero weight loss, minor changes in molecular weight characteristics and a fairly constant indentation resistance of around 1 MN/m2. Inulin-loaded PCL materials exhibited a total weight loss of approximately 17% after 12 months in PBS. The indentation resistance decreased by 50% from an initial value of 28 MN/m2 in the first 2 months and then remained stable. Precipitation cast materials based on PCL are expected to be useful for formulating long-term, controlled release devices for bioactive molecules such as growth factors and hormones and extended-residence supports for cell growth and tissue development.

Evaluation of sodium alginate for bone marrow cell tissue engineering

Sodium alginate has applications as a material for the encapsulation and immobilisation of a variety of cell types for immunoisolatory and biochemical processing applications. It forms a biodegradable gel when crosslinked with calcium ions and it has been exploited in cartilage tissue engineering since chondrocytes do not dedifferentiate when immobilised in it. Despite its attractive properties of degradability, ease of processing and cell immobilisation, there is little work demonstrating the efficacy of alginate gel as a substrate for cell proliferation, except when RGD is modified. In this study we investigated the ability of rat bone marrow cells to proliferate and differentiate on alginates of differing composition and purity. The mechanical properties of the gels were investigated. It was found that high purity and high G-type alginate retained 27% of its initial strength after 12 days in culture and that comparable levels of proliferation were observed on this material and tissue culture plastic. Depending on composition, calcium crosslinked alginate can act as a substrate for rat marrow cell proliferation and has potential for use as 3D degradable scaffold.

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.

Carbonate substitution in precipitated hydroxyapatite: an investigation into the effects of reaction temperature and bicarbonate ion concentration.

Carbonate substitution in the apatite crystal lattice can occur in either the hydroxyl or the phosphate sites, designated as A or B type, respectively, and previous investigations generally have described precipitated carbonate hydroxyapatite as being B type on the basis of infra red and X-ray data. This paper documents the effects of two precipitation variables, namely temperature and bicarbonate ion concentration, on the morphology, phase composition, and calcium, phosphorus, and carbon contents of precipitated carbonate hydroxyapatite. Variations in both temperature and bicarbonate concentration could yield either acicular or spheroidal crystals. X-ray diffraction and infra red spectroscopy indicated the presence of carbonate in the A site for low carbonate contents (< 4 wt%), and at higher carbonate contents (> 4 wt%), the carbonate was located predominantly in the B site. On the basis of these observations and chemical analyses, a new AB carbonate substitution mechanism is proposed that better describes the experimental data than the B-type models used previously.

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.

Bioinorganics and biomaterials: Bone repair

The field of bioinorganics is well established in the development of a variety of therapies. However, their application to bone regeneration, specifically by way of localized delivery from functional implants, is in its infancy and is the topic of this review. The toxicity of inorganics is species, dose and duration specific. Little is known about how inorganic ions are effective therapeutically since their use is often the result of serendipity, observations from nutritional deficiency or excess and genetic disorders. Many researchers point to early work demonstrating a role for their element of interest as a micronutrient critical to or able to alter bone growth, often during skeletal development, as a basis for localized delivery. While one can appreciate how a deficiency can cause disruption of healing, it is difficult to explain how a locally delivered excess in a preclinical model or patient, which is presumably of normal nutritional status, can evoke more bone or faster healing. The review illustrates that inorganics can positively affect bone healing but various factors make literature comparisons difficult. Bioinorganics have the potential to have just as big an impact on bone regeneration as recombinant proteins without some of the safety concerns and high costs.

Dicalcium phosphate cements: Brushite and monetite

Dicalcium phosphate cements were developed two decades ago and ever since there has been a substantial growth in research into improving their properties in order to satisfy the requirements needed for several clinical applications. The present paper presents an overview of the rapidly expanding research field of the two main dicalcium phosphate bioceramics: brushite and monetite. This review begins with a summary of all the different formulae developed to prepare dicalcium phosphate cements, and their setting reaction, in order to set the scene for the key cement physical and chemical properties, such as compressive and tensile strength, cohesion, injectability and shelf-life. We address the issue of brushite conversion into either monetite or apatite. Moreover, we discuss the in vivo behavior of the cements, including their ability to promote bone formation, biodegradation and potential clinical applications in drug delivery, orthopedics, craniofacial surgery, cancer therapy and biosensors.

The stimulation of angiogenesis and collagen deposition by copper.

Copper is known to trigger endothelial cells towards angiogenesis. Different approaches have been investigated to develop vascularisation in biomaterials. The angiogenic and healing potential of copper ions in combination with two major angiogenic factors was examined. A 3D culture system in which, under stimulation by FGF-2 and to a lesser degree with VEGF, endothelial cells assembled into structures resembling to an angiogenic process was used. The combination of CuSO(4) with increasing doses of VEGF or FGF-2 enhanced the complexity of angiogenic networks in a significant manner. In vivo studies were also conducted by incorporating FGF-2 with CuSO(4) in a cylindrical collagen-based scaffold. CuSO(4) enhanced significantly the invasion of microvessel compared to control implants and to 20ng FGF-2+/-CuSO(4). Vascular infiltration was also significantly improved by combination of CuSO(4) with FGF-2, compared to FGF-2 alone (0.2 and 1microg). Nevertheless, in comparison with CuSO(4) alone, there was a significant increase only with 1microg of FGF-2 combined with CuSO(4). Significantly, collagen fiber deposition was enhanced following the combinatory loading in comparison to that with FGF-2 alone but not with CuSO(4) only. Thus, copper associated with growth factors may have synergistic effects which are highly attractive in the fields of tissue engineering (e.g., bone) and biomaterials.

Angiogenesis in Calcium Phosphate Scaffolds by Inorganic Copper Ion Release

Angiogenesis in a tissue-engineered device may be induced by incorporating growth factors (e.g., vascular endothelial growth factor [VEGF]), genetically modified cells, and=or vascular cells. It represents an important process during the formation and repair of tissue and is essential for nourishment and supply of reparative and immunological cells. Inorganic angiogenic factors, such as copper ions, are therefore of interest in the fields of regenerative medicine and tissue engineering due to their low cost, higher stability, and potentially greater safety compared with recombinant proteins or genetic engineering approaches. The purpose of this study was to compare tissue responses to 3D printed macroporous bioceramic scaffolds implanted in mice that had been loaded with either VEGF or copper sulfate. These factors were spatially localized at the end of a single macropore some 7 mm from the surface of the scaffold. Controls without angiogenic factors exhibited only poor tissue growth within the blocks; in contrast, low doses of copper sulfate led to the formation of microvessels oriented along the macropore axis. Further, wound tissue ingrowth was particularly sensitive to the quantity of copper sulfate and was enhanced at specific concentrations or in combination with VEGF. The potential to accelerate and guide angiogenesis and wound healing by copper ion release without the expense of inductive protein(s) is highly attractive in the area of tissue-engineered bone and offers significant future potential in the field of regenerative biomaterials.

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.