Tissue engineering and characterisation
Tissue engineering
Cartilage tissue engineering
Most tissues require a fully developed vascular system to oxygen and nutrients. This makes the tissues difficult to engineer, as they can die in the patient's body before they can develop a blood supply. Articular cartilage tissue, however, does not require a blood supply, acquiring its nutrients and oxygen by diffusion from its surface. Implanted cartilage does, therefore, generally survive well in the patient.
Right: Photomicrograph of engineered cartilage stained to show the specialised pericellular matrix which is important in cell signalling and protecting the chondrocyte under compressive loading.
Cartilage is also a clinically important tissue, as it does not recover well from injury, and its deterioration is associated with debilitating diseases of old age, such as arthritis, which is of growing concern in the developed world. Cartilage occurs as three types within the body:
Elastic cartilage, an example of which is found in the ears.
Hypertrophic cartilage is laid down as a template for bone growth, and this may serve as a useful precursor tissue to bone (which requires a good blood supply) in tissue engineering approaches to orthopaedics. Studies are underway at Sheffield to evaluate this approach to tissue engineering bone for re-constructive surgery.
Hyaline cartilage is found in the ribs, nasal septum and covering the bone ends of joints, such as the knees and hips. In the joints the cartilage is known as articular and is vital for cushioning and lubricating surfaces.
Research at Sheffield has focussed on evaluating the cell biology of engineered cartilage, developed using a variety of different scaffolds and growth media, and comparing this with natural material. The primary cell type in cartilage is the chondrocyte, this is a specialised cell that can survive compression and shear forces within the collagen matrix.
Histological studies have shown that the chondrocyte is embedded in a zone of specialised matrix, forming a chondron. This structure is essential to for the survival of the cell under conditions of load and shear stress. Studies have demonstrated that diseased (arthritic) chondrocytes develop a reduced chondron, and are therefore more susceptible to damage.
Above right: Photomicrograph of engineered cartilage stained to show collagen II (orange ground). Collagen II is a vital support protein secreted by chondrocytes in natural collagen. Its appearance in this engineered tissue is indicative of a good culture regime. Bone tissue engineering
Researchers at Sheffield are pursuing two broad strategies to tackle the challenges presented by the need to engineer a tissue for bone repair. Working with a local company (Ceramisys Ltd.), we are evaluating porous calcium phosphate ceramics for use as a cell support and scaffold for bone tissue engineering. Porous ceramics are seeded with mesenchymal stem cells from the bone marrow and cultured under osteogenic conditions. The resulting constructs are evaluated using a range of techniques including histology, electron microscopy, and microCT (in collaboration with Ralph Muller in Zurich as part of the EXPERTISSUES project). Researchers are also investigating the potential of hypertrophic chondrocytes to generate a tissue engineered construct for bone repair. This approach is attractive as hypertrophic chondrocytes are able to survive with relatively little oxygen (hypoxia) such as an injury site or wound bed (see Cell Sources research pages).
Above: MicroCT 3D reconstruction of pore spaces in a typical calcium phosphate scaffold, the large void (shown in red) is approximately 0.8 µm in diameter.
More information about this project can be found on the EXPERTISSUES project website.
Periodontal tissue engineering
The periodontal ligament forms a sheath about the root of the tooth. It acts as a shock absorber, as well as holding the tooth firmly in place. Due to the embryological development of the tooth root, however, cells in the periodontal ligament cannot re-generate.
Right: A 3-D model of a human molar sectioned to show internal structure, and that of the periodontium. The periodontal ligament is depicted in pale blue.
As a consequence the ligament wears out over time in a process of chronic peridontitis, the most common cause of tooth loss, though it does not commonly manifest itself until later life. Research aims at regenerating the lost connective tissue by growing up large numbers of ligament cells and re-implanting them.
Normally cultured cells must adhere to a surface to survive and continue developing. If only presented with hydrophobic surfaces in the culture tray, however, the cells can develop as clusters in suspension. This process avoids the introduction of exogenous factors in the cell development, and allows us to evaluate the influence of the serum medium, and community effects within the clusters.
Characterisation
Model engineered tissues are characterised using a range of techniques, including histology and immunocytochemistry, biochemical analysis of matrix, electron microscopy and MicroCT (performed in collaboration with Keele University and ETH Zurich).
Right: Photomicrograph of engineered cartilage stained to show proteoglycan (purple). Proteoglycan is also expressed strongly by natural collagen, and is an important component of the chondron, which protects chondrocytes in this tissue. Black inclusions are fibres of the scaffold.
Histology allows us to examine and compare the distribution of a range of indicator matrix components in natural and engineered cartilage, for example:
- Various types of collagen (our interest is principally in types I, II, VI and X)
- Glucosaminoglycans and mineral deposition, important for engineering bone.
- Surface enzymes, such as alkaline phosphotase, which is strongly expressed by osteocytes and hypertrophic chondrocytes.
- Cell surface receptors, eg CD44, the expression of which is a measure of the cells continuing utility for clinical treatments.
Quantitative biochemical analysis allows us to evaluate absolute amounts of glycosaminoglycans, and real time PCR techniques permit us to examine levels of expression for matrix components such as aggrican, collagen I and II.
MicroCT (micro computed tomography) is a useful technique for measuring pore sizes and permeability of solid supports for bone.
Researchers engaged in this work are Richard Ackbar, Prof. Ian Brook, Dr Aileen Crawford, Prof Paul Hatton, Dr. Adrian Jowett, Felora Mirvakily, Ana MacIntosh, Prof. Ralph Muller (ETH Zurich) and Dr Gwen Reilly (Engineering Materials). (PhD project by Sarah Fraser, 2003)