Jan T. Czernuszka

Novel collagen scaffolds with predefined internal morphology made by solid freeform fabrication

Novel collagen scaffolds possessing predefined and reproducible internal channels with widths of 135 microm and greater have been produced. The process employed to make the collagen scaffold utilises a sacrificial mould, manufactured using solid freeform fabrication technology, and critical point drying technique. A computer aided design (CAD) file of the mould to be produced is created. This mould is manufactured using a phase change ink-jet printer. A dispersion of collagen is then cast into the mould and frozen. The mould is dissolved away with ethanol and the collagen scaffold is then critical point dried with liquid carbon dioxide. The effect of processing on the tertiary structure of collagen is assessed by monitoring the wavenumber of the N-H stretching vibration peak using Fourier transform infra-red spectroscopy and it is found that processing does not denature the collagen. Ultraviolet-visual spectroscopy was used to detect the presence of any contamination from the sacrificial mould on the collagen. The ability to use computer aided design and manufacture (CAD/CAM) provides a route to optimise scaffold designs using collagen in tissue engineering applications.

Investigation into the formation and mechanical properties of a bioactive material based on collagen and calcium phosphate

A biomaterial composite was formed by the room-temperature precipitation of calcium phosphate (in the form of brushite) on to collagen. It was found that the addition of 1 mM O-phosphoserine (Ser P) causes the morphology of the brushite crystals to change from large plates to small needles. An increase in the surface coverage and weight fraction of brushite incorporated into the collagen was observed. The mechanical properties of this composite were tested in the wet state. The ultimate tensile strength (UTS) was 45 MPa compared with 34 MPa for the wet collagen. Osteoblastic differentiation was promoted on the surface of the material and new bone formed.

Controlled release of amoxicillin from hydroxyapatite-coated poly(lactic-co-glycolic acid) microspheres

Negatively charged poly(lactic-co-glycolic acid) (PLGA) microspheres with an encapsulated hydrophilic antibiotic (amoxicillin) have been prepared by the solid-in-oil-in-water (s/o/w) method using the anionic surfactant, sodium dodecyl sulfate (SDS). Drug encapsulation efficiency is over 40%. Successful coating of hydroxyapatite (HA) on these negatively charged PLGA microspheres has been achieved by a dual constant composition method in 3-6 h. The HA-coated PLGA microspheres (HPLG) have been characterised by zeta-potential and particle size measurements and the coating has been confirmed to be calcium deficient HA by analysis of X-ray diffraction, Fourier transform infrared spectroscopy and wavelength dispersive spectroscopy. The morphology of HPLG was studied by scanning electron microscopy, and cross sections of HPLG microspheres were prepared and imaged using focused ion beam microscopy. In-vitro drug release experiments in PBS (pH7.4) showed a sustained release profile for at least 31 days with little initial burst release. It shows a triphasic drug release profile commonly observed for biodegradable polymers.

Development of specific collagen scaffolds to support the osteogenic and chondrogenic differentiation of human bone marrow stromal cells

Type I Collagen matrices of defined porosity, incorporating carbonate substituted hydroxyapatite (HA) crystals, were assessed for their ability to support osteo- and chondrogenic differentiation of human bone marrow stromal cells (HBMSCs). Collagen-HA composite scaffolds supported the osteogenic differentiation of HBMSCs both in vitro and in vivo as demonstrated by histological and micro-CT analyses indicating the extensive penetration of alkaline phosphatase expressing cells and new matrix synthesis with localised areas immunologically positive for osteocalcin. In vivo, extensive new osteoid formation of implant origin was observed in the areas of vasculature. Chondrogenic matrix synthesis was evidenced in the peripheral regions of pure collagen systems by an abundance of Sox9 expressing chondrocytes embedded within a proteoglycan and collagen II rich ECM. The introduction of microchannels to the scaffold architecture was seen to enhance chondrogenesis. Tissue specific gene expression and corresponding matrix synthesis indicate that collagen matrices support the growth and differentiation of HBMSCs and suggest the potential of this platform for understanding the ECM cues necessary for osteogenesis and chondrogenesis.

Gradient collagen/nanohydroxyapatite composite scaffold: Development and characterization

This paper reports an in situ diffusion method for the fabrication of compositionally graded collagen/nanohydroxyapatite (HA) composite scaffold. The method is diffusion based and causes the precipitation of nano-HA crystallites in situ. A collagen matrix acts as a template through which calcium ions (Ca(2+)) and phosphate ions (PO4(3-)) diffuse and precipitate a non-stoichiometric HA. It was observed that needle-like prismatic nano-HA crystallites (about 2 x 2 x 20 nm) precipitated in the interior of the collagen template onto the collagen fibrils. Chemical and microstructural analysis revealed a gradient of the Ca to P ratio across the width of the scaffold template, resulting in the formation of a Ca-rich side and a Ca-depleted side of scaffold. The Ca-rich side featured low porosity and agglomerates of the nano-HA crystallites, while the Ca-depleted side featured higher porosity and nano-HA crystallites integrated with collagen fibrils to form a porous network structure.

Extracellular matrix production by adipose-derived stem cells: implications for heart valve tissue engineering.

A key challenge in tissue engineering a heart valve is to reproduce the major tissue structures responsible for native valve function. Here we evaluated human adipose-derived stem cells (ADSCs) as a source of cells for heart valve tissue engineering investigating their ability to synthesize and process collagen and elastin. ADSCs were compared with human bone marrow mesenchymal stem cells (BmMSCs) and human aortic valve interstitial cells (hVICs). ADSCs and BmMSCs were stretched at 14% for 3 days and collagen synthesis determined by [(3)H]-proline incorporation. Collagen and elastin crosslinking was assessed by measuring pyridinoline and desmosine respectively, using liquid chromatography/mass spectrometry. Three-dimensional culture was obtained by seeding cells onto bovine collagen type I scaffolds for 2-20 days. Expression of matrix proteins and processing enzymes was assessed by Real Time-PCR, immunofluorescence and transmission electron microscopy. Stretch increased the incorporation of [(3)H]-proline in ADSCs and BmMSCs, however only ADSCs and hVICs upregulated COL3A1 gene. ADSCs produced collagen and elastin crosslinks. ADSCs uniformly populated collagen scaffolds after 2 days, and fibrillar-like collagen was detected after 20 days. ADSCs sense mechanical stimulation and produce and process collagen and elastin. These novel findings have important implications for the use of these cells in tissue engineering.

Development of biodegradable scaffolds for tissue engineering : a perspective on emerging technology

Abstract The scaffold, a three-dimensional (3D) substrate that serves as a template for tissue regeneration, plays an essential role in tissue engineering. The ideal scaffold should have surface chemistry and microstructure tailored to facilitate cellular attachment, proliferation and differentiation; adequate mechanical strength for handling; and an appropriate biodegradation rate without undesirable byproducts. Research on biopolymer formulation and scaffold fabrication has been intense over the past 10 years. A perspective is provided of important issues related to scaffold development from biodegradable polymers. The mechanical properties and biocompatibility (including biodegradability and bioresorptability) of commonly used biopolymers are reviewed. Scaffold design and fabrication techniques are assessed and compared. Scaffold architecture, including pore size and size distribution, and its effects on cell growth are discussed. The importance of structural hierarchy over a range of length scales is highlighted. Unfortunately, conventional processing techniques cannot yet control both scaffold architecture and surface chemistry. An emerging scaffold fabricating technique using solid free form fabrication (SFF), although currently restricted to relatively symmetrical structures, has been shown to be highly effective in integrating structural architecture with changes in surface chemistry of the scaffolds, and integration of growth factors. Several examples of the application of SFF are presented.

Preparation and characterization of negatively charged poly(lactic-co-glycolic acid) microspheres

Negatively charged poly(lactic-co-glycolic acid) (PLGA) microspheres encapsulated with hydrophilic drugs have been successfully prepared by a solid-in-oil-in-water (s/o/w) solvent evaporation method in the presence of anionic surfactants, sodium dodecyl sulfate (SDS), and dioctyl sodium sulfosuccinate (DSS), and nonionic surfactant polyvinyl alcohol (PVA). The effects of microencapsulation methods, surfactants types, and surfactant concentrations on the properties of microspheres were studied. Amoxicillin (AMX) was chosen as a hydrophilic model drug, and its encapsulation efficiency (EE) and in vitro release profiles were measured. The s/o/w method achieved higher EE of 40% in PLGA microspheres using surfactant SDS compared with the conventional water-in-oil-in-water (w/o/w) method (about 2%). Triphasic release profiles were observed for all PLGA microspheres (s/o/w) with slight drug burst, a slow diffusion-controlled release within the period of about 7 days and followed by the degradation-controlled sustained release for further 30 days. Smaller particle size and surface charge were achieved for s/o/w method than w/o/w method using the same anionic surfactants, and smooth surface and less porous interior matrix. The s/o/w method effectively encapsulated AMX into anionic PLGA microspheres using anionic surfactants, and these negatively charged PLGA microspheres represented an attractive approach for the controlled release of hydrophilic drugs.

The impact of critical point drying with liquid carbon dioxide on collagen–hydroxyapatite composite scaffolds

Collagen-hydroxyapatite composites for bone tissue engineering are usually made by freezing an aqueous dispersion of these components and then freeze-drying. This method creates a foamed matrix which may not be optimum for growing cell colonies larger than a few hundred micrometres due to the limited diffusion of nutrients and oxygen, and the limited removal of waste metabolites. Incorporating a network of microchannels in the interior of the scaffold which may permit the flow of nutrient-rich media has been proposed as a method to overcome these diffusion constraints. A novel three-dimensional printing and critical point drying technique previously used to make collagen scaffolds has been modified to create collagen-hydroxyapatite scaffolds. This study investigates the properties of collagen and collagen-hydroxyapatite scaffolds and whether subjecting collagen and hydroxyapatite to critical point drying with liquid carbon dioxide results in any changes to the individual components. Specifically, the hydroxyapatite component was characterized before and after processing using wavelength-dispersive X-ray spectroscopy, X-ray diffraction and infrared spectroscopy. Critical point drying did not induce elemental, crystallographic or molecular changes in the hydroxyapatite. The quaternary structure of collagen was characterized using transmission electron microscopy and the quarter-staggered array characteristic of native collagen remained after processing. Microstructural characterization of the composites using scanning electron microscopy showed the hydroxyapatite particles were mechanically interlocked in the collagen matrix. The in vitro biological response of MG63 osteogenic cells to the composite scaffolds were characterized using the Alamar Blue, PicoGreen, alkaline phosphate and Live/Dead assays, and revealed that the critical point dried scaffolds were non-cytotoxic.

Synthesis and properties of a novel anisotropic self-inflating hydrogel tissue expander.

The advent of self-inflating hydrogel tissue expanders heralded a significant advance in the reconstructive techniques available for the surgical restoration of a wide variety of soft tissue defects. However, their use in specific applications such as cleft palate surgery is limited on account of their isotropic expansion. An anisotropic self-inflating hydrogel tissue expander has been developed which markedly increases the potential indications for which this restorative tool may be employed. These include complex pediatric soft tissue reconstructions of the palate, nose, ear and digits. Anisotropic expansion in a hydrogel polymer network composed of methyl methacrylate and vinylpyrrolidone has been achieved by annealing the xerogel under a compressive load for a specified time period. By controlling the anisotropic processing conditions and composition we have been able to accurately tailor the ultimate expansion ratio up to 1500%. The expansion rate of the xerogel has also been significantly reduced by encapsulating the polymer within a semi-permeable silicone membrane. The structure and properties of the novel anisotropic hydrogel were characterized by attenuated total reflectance infrared spectroscopy, differential scanning calorimetry, thermogravimetric analysis and small-angle neutron scattering.