Author: Tariq Mesallati, postgraduate student, Trinity Centre for Bioengineering and winner of the 2014 Engineers Ireland Biomedical Research Medal (supervisor: Daniel J Kelly, associate professor of bioengineering, Trinity College Dublin) Approximately 714,000 people in Ireland currently suffer from the degenerative disease osteoarthritis (OA), with 18% of patients less than 55 years old. Treatment options for OA are limited to surgical replacement of the diseased joint with a metal and polyethylene prosthesis. While this procedure is well established, it is not without its limitations and failures are not uncommon. Joint replacement prostheses also have a finite lifespan, making them unsuitable for the growing population of younger and more active patients requiring treatment for OA. Tissue engineering has been defined as the use of a combination of cells, engineering and materials methods, and suitable biochemical and physico-chemical factors to improve or replace biological functions. In recent years, there has been increased interest in the use of cell and tissue engineering-based therapies for the treatment of focal cartilage defects. While significant progress has been made in this field, realising an efficacious therapeutic option for the treatment of OA remains elusive and is considered to be one of the greatest challenges in the field of orthopaedic medicine.

Implant for joint resurfacing

[caption id="attachment_16914" align="alignright" width="352"]Tariq Tariq Mesallati (left) receiving his medal which was sponsored by Depuy[/caption] The overall aim of this work was to tissue engineer a scaled-up, anatomically accurate biological implant suitable for partial or total joint resurfacing. Given that OA affects multiple tissues in the diseased joint, including the articular cartilage and underlying subchondral bone, the first objective of this study was to develop a scalable approach to simultaneously engineer both of these tissue types within an osteochondral construct (i.e. an engineered tissue composed of a cartilage and bone region). Mesenchymal stem cells, or MSCs, are multi-potent stromal cells that can differentiate into a variety of specialised cell types, including bone forming cells, cartilage forming cells (called chondrocytes) and fat forming cells. It is well documented that cartilage tissue generated using bone marrow-derived mesenchymal stem cells (BMSCs) have an inherent tendency to undergo endochondral ossification (the replacement of cartilage with bone) to form new bone tissue. Endochondral ossification is a naturally occurring process in the body beginning with stem cells differentiating into chondrocytes which lay down a cartilaginous template. As these chondrocytes undergo endochondral ossification, blood vessels are attracted into the template and the cartilage is remodelled into bone. This tendency of BMSCs to undergo endochondral ossification is a major limitation for cartilage tissue engineering, but it can be leveraged for the regeneration of large bone defects. The first objective of this study was to tissue engineer osteochondral constructs (consisting of cartilage and bone) by spatially regulating endochondral ossification within bi-layered MSC laden hydrogels. Another key challenge in developing a biological implant for the treatment of degenerative joint diseases is engineering articular cartilage of sufficient scale to resurface an entire joint. This is particularly challenging in the context of OA, as only a limited number of therapeutically useful (cartilage forming) chondrocytes (CCs) can be isolated from diseased joints. Furthermore, the expansion of CCs in vitro to obtain sufficient numbers of cells can lead to de-differentiation of cells towards a more fibroblast-like phenotype. Mesenchymal stem cells (MSCs) can be used as an alternative to CCs for cartilage tissue engineering. MSCs possess the ability to proliferate extensively in vitro while maintaining their multipotent differentiation potential making them an almost ideal cell type for engineering scaled-up cartilaginous constructs large enough to resurface an entire joint. However, as outlined previously, cartilage tissue engineered using MSCs has been shown to undergo endochondral ossification in vivo (in the body). Previous studies have demonstrated that a co-culture of CCs and MSCs enhances cartilage matrix synthesis and suppresses the process of endochondral ossification. A second objective of this study was to investigate if a co-culture of CCs and BMSCs could be used to engineer a layer of phenotypically stable articular cartilage as part of an osteochondral construct in vivo. The final objective of the study was to scale-up the proposed approach in order to tissue engineer an anatomically shaped osteochondral construct which could potentially replace an entire diseased joint. We hypothesised that this would be possible by combining scaled-up, anatomically shaped BMSC seeded alginate tissues (formed from moulds fabricated by rapid prototyping) with self-assembled cartilage tissue (formed through a co-culture of chondrocytes and BMSCs).

Results

Bi-layered cartilaginous tissues were engineered in the lab by seeding either BMSCs (bottom layer) or chondrocytes (top layer) into bi-layered constructs (Fig. 1). It was demonstrated that BMSCs could support endochondral bone formation in the bottom layer of bi-layered constructs in vivo. [caption id="attachment_16900" align="aligncenter" width="640"]New Picture Figure 1: Schematic of experimental design. Self-assembled chondral layers were combined with alginate hydrogels in custom built agarose moulds (blue moulds in figure) to form osteochondral constructs. CC, chondrocyte; BMSC, bone marrow-derived mesenchymal stem cell[/caption] Following subcutaneous implantation of chondrogenically primed, tissue engineered osteochondral constructs, the bottom osseous region (BMSC laden alginate hydrogels) of bi-layered constructs (that was soft and cartilage-like before implantation) appeared hard and calcified. X-ray micro computed tomography (µCT) analysis demonstrated extensive mineralisation in the alginate layers with H&E staining revealing the formation of bony-like tissue in this bottom hydrogel layer. It was next demonstrated that a phenotypically stable layer of articular cartilage could be engineered over this bony tissue using a mixture of chondrocytes (CCs) and BMSCs. This co-culture of CCs and BMSCs (1:4 ratio) was found to dramatically reduce mineralisation of the chondral (cartilage) phase of the engineered graft in vivo. Co-culture also led to the development of thicker, more homogeneous and more morphologically stable cartilaginous constructs in vivo (compared to corresponding stem cell only groups) that better integrated with the underlying osseous layer. It also led to more robust cartilage tissue formation (indicated by increased sGAG and type II collagen accumulation) in the top chondral layer in vivo. In the final part of the study, scaled up BMSC-seeded alginate constructs (~2 cm diameter) mimicking the geometry of the femoral and tibial components of a partial knee replacement prosthesis were generated from moulds fabricated by rapid prototyping (Fig. 2). Briefly, this involved firstly scanning unicondylar knee replacement prostheses in a 3D laser scanner. The files were manipulated in SolidWorks, and imported into a stratasys dimension FDM (fused deposition modelling) machine. This allowed for the creation of acrylonitrile butadiene styrene (ABS) moulds, from which large MSC-seeded alginate constructs were fabricated (mimicking the shape of the femoral condyle and the tibial plateau). [caption id="attachment_16902" align="aligncenter" width="640"]New Picture Figure 2: Fabrication of scaled-up, anatomically shaped bone marrow derived mesenchymal stem cell (BMSC) seeded alginate constructs in the shape of the femoral condyle and the tibial plateau[/caption] These scaled-up constructs were covered by a self-assembled layer (~2 cm diameter) of engineered cartilaginous tissue (formed through BMSC & CC co-culture). After six weeks of in vitro culture, these scaled-up osteochondral constructs were implanted subcutaneously for a further eight weeks. After eight weeks in vivo, a layer of phenotypically stable articular cartilage remained on the surface of these scaled-up, anatomically shaped engineered implants, which resembled native articular cartilage (Fig. 3). The chondral layer of these scaled-up constructs stained strongly for sGAG and type II collagen (an indication of robust cartilage formation). [caption id="attachment_16903" align="aligncenter" width="407"]New Picture Figure 3: Scaled-up osteochondral construct in the shape of the tibial plateau post-implantation. (A) Macroscopic image and µCT scan of construct. (B) Aldehyde Fuchsin staining with strong sGAG production in self-assembled chondral layer. (C) Picro-sirius red staining for collagen. (D) Collagen immunohistochemistry of anatomic osteochondral construct with strong type II and weak types I and X collagen staining of top cartilage layer (indicative of stable cartilage formation[/caption] [caption id="attachment_16905" align="aligncenter" width="480"]New Picture Figure 4: Analysis of scaled-up osseous alginate layer of osteochondral construct post-implantation. H&E staining of alginate layer and collagen types I and X immunohistochemistry of same. Arrows indicate blood vessel-like structures[/caption] There was also evidence of mineralisation and immature bone development in the underlying osseous alginate layer (Figs. 3, 4). µCT analysis confirmed the deposition of mineral within the osseous region of the scaled-up constructs. H&E staining provided evidence of immature bone formation. Blood vessel structures were detected in these H&E stained samples. Figure 4: Analysis of scaled-up osseous alginate layer of osteochondral construct post-implantation. H&E staining of alginate layer and collagen types I and X immunohistochemistry of same. Arrows indicate blood vessel-like structures. Overall, this study provides a framework for tissue engineering biological joint replacement prostheses for regenerating damaged/diseased joints. A number of challenges remain, including confirmation of efficacy of this approach within a load bearing orthotopic environment and implementation of this approach using diseased human MSCs. If these challenges can be overcome, however, it opens up the potential of a therapeutic solution for the millions of people suffering from OA worldwide. Funding was provided by the Irish Research Council for Science, Engineering and Technology, the Science Foundation Ireland President of Ireland Young Researcher Award and a European Research Council Starter Grant.