Author: Dr Daniel Kelly, associate professor in Trinity College Dublin; director of the Trinity Centre for Bioengineering Articular cartilage is a soft hydrated tissue that lines the ends of the bones in our joints. The tissue functions to allow near-frictionless articulation between bones and to help distribute the very high loads that pass through our joints. Injury to articular cartilage is not uncommon and can be very painful, leading to impaired mobility. Furthermore, if such cartilage defects are not successfully treated they can progress, leading to the development of osteoarthritis (OA). In Ireland, over one in five people have some form of arthritis, costing the State €1.6 billion per annum in lost working hours, according to Arthritis Ireland. [login type="readmore"] At present, the main treatment option for OA is surgical replacement of the damaged joint with a metal and polymer prosthesis. The relatively short lifespan of these implants can make them unsuitable for the growing population of younger and more active patients requiring treatment for damaged cartilage. There is therefore a growing need for the development of novel therapies to either prevent the progression of diseases like OA or, following complete joint degeneration, to regenerate the affected tissues. The relatively new field of ‘tissue engineering’ aims to use a combination of cells, materials and appropriate biochemical and/or physical signals to restore function to damaged or diseased tissues. Advances in this field have already led to the development of a cell-based technique known as autologous chondrocyte implantation (ACI) to treat small cartilage defects (1). Clinical studies indicate a durable symptomatic relief and partial structural repair of the damaged tissue (2). However, the technique does not result in the generation of normal hyaline cartilage and is not suitable for treating very large defects or indeed diseases like OA, which is a much greater clinical problem. This motivates the need for more advanced tissue engineering strategies for the regeneration of musculoskeletal tissues such as articular cartilage. In recent years, there has been increased interested in using mesenchymal stem cells (MSCs) to regenerate damaged and diseased tissues such as articular cartilage and bone. These adult stem cells have the capacity to differentiate into (or turn into) specialised cell types, such as those that produce specific ‘mesenchymal’ tissues including  bone, cartilage, muscle, ligament and fat (3). Such cells are found in numerous tissues in the adult body and are believed to act as cellular replacements for differentiated cells (i.e. bone cells or cartilage cells) that die or are injured. MSCs have also been shown to secrete a broad spectrum of biomolecules that may facilitate tissue regeneration following injury (3). In many cases, however, the regenerative capacity of such stem cells may not be sufficient to repair damaged tissues. To address this challenge in the field of orthopaedics, my lab is exploring two different approaches to tissue regeneration that leverages the unique properties of MSCs. The first approach involves the development of implants that act to enhance the capacity of MSCs that migrate to the site of injury to regenerate damaged tissues. The second approach involves isolating MSCs from the body, expanding these in number in vitro, and then using these cells to grow or ‘tissue engineer’ replacement grafts outside the body with functional properties that mimic key aspects of the tissue we are trying to replace or repair. BIOMATERIALS FOR IN VIVO TISSUE REGENERATION The field of tissue engineering relies extensively on the use of hydrogels and porous 3D scaffold biomaterials to provide the appropriate environment for the regeneration of tissues and organs (4). These scaffolds essentially act as a template for tissue formation. They can also be used to deliver cells, genes or growth factors to the site of injury to enhance the body’s endogenous capacity for repair. We have been interested in the development of two classifications of biomaterials for musculoskeletal tissue regeneration – natural hydrogels and extracellular matrix (ECM) derived scaffolds. These biomaterials are intended to be implanted into a defect site, either alone or in combination with cells and/or biomolecules, with the aim of promoting tissue regeneration in vivo.

  • Hydrogels
Hydrogels are water-swollen networks crosslinked by either covalent or physical methods. They are particularly attractive for tissue regeneration because they can be non-invasively injected, fill defects of any size, and can be used to deliver cells and other factors to the site of injury (5). Our lab has been exploring the use of various natural hydrogels for regenerating articular cartilage. We have demonstrated that MSCs embedded into such hydrogels produce a matrix rich in proteoglycans and type II collagen (6,7), the main structural components found in articular cartilage. We have also shown that such hydrogels can be combined with microspheres to control the delivery of growth factors that facilitate cartilage tissue formation (8,9) (see Figure 1). Such systems can be combined with MSCs isolated from within diseased joints, for example from synovial tissue or the infrapatellar fat pad, to engineer cartilage grafts for joint repair (10,11). [caption id="attachment_7213" align="alignright" width="605"] Fig 1A: Viable MSCs (green) surrounding growth factor delivery microspheres that are encapsulated within a fibrin hydrogel. MSCs with this system produce a tissue rich in (1B) proteoglycans and (1C) type II collagen, the main components of articular cartilage. Adapted from Ahearne et al (8).[/caption]
  • ECM-derived scaffolds
[caption id="attachment_7215" align="alignright" width="394"] Fig 1B[/caption] [caption id="attachment_7222" align="alignright" width="390"] Fig 1C[/caption] Scaffolds derived from the ECM of mammalian tissues have been shown to facilitate the repair of many different tissues in preclinical studies and in human clinical applications (12). Such scaffolds contain both the structural (e.g. collagen) and functional (e.g. growth factors) molecules secreted by resident cells within a specific tissue and hence are considered promising biomaterials for tissue engineering applications. In the case of articular cartilage regeneration, we and others (13) have been able to produce porous scaffolds from devitalised mammalian articular cartilage. We have shown that these scaffolds support the chondrogenic differentiation of MSCs (the transformation of an MSC into a cartilage-forming cell) isolated from patients with osteoarthritis. Over 28 days, these cells filled the porous scaffold with a cartilage specific matrix (see Figure 2). TISSUE ENGINEERING MECHANICALLY FUNCTIONAL GRAFTS  From a translational perspective, the ideal approach to tissue repair would be to implant a cell-free or cell-loaded hydrogel or scaffold directly into the site of injury with the aim of promoting or enhancing regeneration in vivo. However, in many cases, and in particular for load-bearing musculoskeletal tissues such as articular cartilage, such scaffolds or hydrogels may not possess the appropriate mechanical (and other) properties to function in vivo and hence they may not facilitate successful regeneration. In such cases, tissue engineers are developing novel approaches to grow more functional grafts in vitro before implanting them into a defect site.
  • Bioreactors
[caption id="attachment_7225" align="alignright" width="113"] Fig 2. Temporal synthesis of proteoglycans (stained with Alcian Blue) within an ECM derived scaffolds seeded with MSCs. After 28 days in culture, the scaffold is filled with a cartilage-specific matrix.[/caption] In the field of tissue engineering, a bioreactor is a device or system which applies different types of mechanical and/or chemical stimuli to cells (14). Our lab has developed a series of bioreactors capable of providing MSCs with a mechanical environment similar to that which they might experience within a synovial joint such as the hip or the knee (see Figure 3A). This serves two useful purposes. Firstly, they can be used as model systems to better understand how MSCs will respond to the type of environmental cues they will experience in the body. Secondly, we can use these bioreactors to tissue engineer more mechanically functional grafts prior to implantation. For example, we have shown that the application of in vivo levels of cyclic hydrostatic pressure (~10 MPa) acts to both promote and maintain a cartilage-like phenotype in MSCs (15). Furthermore, such a mechanical stimulus enhances cartilage-tissue accumulation within MSC seeded hydrogels (see Figure 3B) and hence improves their mechanical properties (16). Other joint-like mechanical cues, such as dynamic compression, can also be applied to engineered tissues to improve their mechanical functionality (17).
  • Engineering complex tissue architectures
[caption id="attachment_7228" align="alignright" width="145"] Fig 3A: A dynamic compression bioreactor used to mechanically stimulate cell-seeded constructs to improve their mechanical functionality[/caption] The composition, structure and organisation of biological tissues are optimized for their specific function. In the case of articular cartilage, the composition and organisation of the tissue varies through its depth in a manner that contributes to its unique mechanical properties. Engineering replacement organs and tissues with such spatial complexity in their composition and organisation remains a major challenge. One approach to engineer such tissues is to recapitulate the gradients in regulatory signals that during development and maturation are believed to drive spatial changes in stem cell differentiation and tissue organisation. As already described, MSC differentiation is known to be influenced by growth factors and mechanical cues, but we have also shown that other factors such as oxygen levels play a key role in regulating the development of engineered cartilage (18). [caption id="attachment_7230" align="alignright" width="98"] Fig 3B: Cartilage tissue engineered using MSCS under unloaded free swelling (FS) conditions or subjected to cyclic hydrostatic pressure (HP). The tissue subjected to hydrostatic pressure contained higher levels of proteoglycans (blue stain) and collagen (red stain)[/caption] By controlling both the oxygen levels and mechanical environment throughout the depth of MSC seeded hydrogels, we have been able to engineer tissues with zonal gradients mimicking certain aspects of native articular cartilage (17). The next stage of this project will explore whether implanting such complex engineered tissues will improve outcomes in pre-clinical models of cartilage injury. Within damaged or diseased synovial joints such as the hip or knee, it is not uncommon for both the articular cartilage and underlying bone to be damaged. This necessitates unique tissue engineering strategies to regenerate multiple different tissues. In the case of joint regeneration, there is often a need to engineer osteochondral tissues, i.e. a cartilaginous tissue with an underlying bony layer. Under appropriate conditions, cartilage tissue that has been engineered using MSCs can undergo a process called endochondral ossification (the replacement of cartilage with bone) when implanted in vivo. We have recently shown that it is possible to engineer osteochondral tissues by promoting endochondral ossification in the bottom layer of an engineered bi-layered tissue and stable cartilage formation in the top layer (19) (see Figure 4).  We are currently exploring the potential of such bi-layered constructs to treat large scale osteochondral defects within the articular surface of load bearing joints. FUTURE OF JOINT REGENERATION [caption id="attachment_7232" align="alignright" width="248"] Fig 4: An engineered tissue consisting of a top layer of cartilage (positive staining for Alcian Blue) and an underlying layer of mineralised tissue (as evidenced by Alizarin Red staining and uCT analysis). Image adapted from Sheehy et al (19)[/caption] While different tissue engineering strategies have been developed to potentially treat small, isolated defects, there is still a pressing need to develop alternatives to total joint arthroplasty for the treatment of degenerative joint diseases like OA. Our vision for the future of joint regeneration is that it will be possible to scale-up the strategies described in this article to tissue engineer biological joint replacement prosthesis. If successful, such a biological implant could be used to regenerate normal articular cartilage and bone within an osteoarthritic joint rather than replacing these tissues with a metallic and polymer implant that fails in the long-term. Dr Daniel Kelly is an associate professor in Trinity College Dublin and director of the Trinity Centre for Bioengineering, where he leads a multidisciplinary orthopaedic tissue engineering and mechanobiology research group. The goal of his lab is to understand how environmental factors regulate the fate of adult stem cells. This research underpins a more translational programme aimed at developing novel mesenchymal stem-cell-based therapies to regenerate damaged and diseased orthopaedic tissues such as articular cartilage and bone. He has authored 80 peer-reviewed journal papers.  Acknowledgements Funding for our lab is provided by Science Foundation Ireland (President of Ireland Young Researcher Award), the European Research Council, the AO Foundation, Enterprise Ireland and the Irish Research Council. References [1]            Brittberg, M et al. New England Journal of Medicine. 331(14):1994 p. 889-895. [2]            Peterson, L et al. Clinical Orthopaedics and Related Research, (374):2000 p. 212-234. [3]            Caplan, AI. J Cell Physiol. 213(2):2007 p. 341-7. [4]            O'Brien, FJ. Materials Today. 14(3):2011 p. 88-95. [5]            Kim, IL et al. Biomaterials. 32(34):2011 p. 8771-8782. [6]            Buckley, CT et al. Journal of Biomechanics. 43(5):2010 p. 920-926. [7]            Vinardell, T et al. Tissue Engineering - Part A. 18(11-12):2012 p. 1161-1170. [8]            Ahearne, M et al. Biotechnol Appl Biochem. 58(5):2011 p. 345-52. [9]            Ahearne, M and Kelly, DJ. Biomedical Materials (Bristol). 8(3):2013 p. [10]         Liu, Y et al. Tissue Engineering - Part A. 18(15-16):2012 p. 1531-1541. [11]         O'Heireamhoin, S et al. Tissue Engineering - Part C: Methods. 19(2):2013 p. 117-127. [12]         Badylak, SF et al. Acta Biomaterialia. 5(1):2009 p. 1-13. [13]         Cheng, NC et al. Tissue Eng Part A. 15(2):2009 p. 231-41. [14]         Martin, I et al. Trends Biotechnol. 22(2):2004 p. 80-6. [15]         Vinardell, T et al. Eur Cell Mater. 23:2012 p. 121-32; discussion 133-4. [16]         Meyer, EG et al. Journal of the Mechanical Behavior of Biomedical Materials. 4(7):2011 p. 1257-1265. [17]         Thorpe, SD et al. PLoS ONE. 8(4):2013 p. [18]         Sheehy, EJ et al. Biochemical and Biophysical Research Communications. 417(1):2012 p. 305-310. [19]         Sheehy, EJ et al. Acta Biomaterialia. 9(3):2013 p. 5484-5492.