**7. Conclusions**

Cell culture has a bright future. It is a fundamental and core component of tissue engineering and regenerative medicine. In this context the majority of studies carried out to date have used the well-established method of growing cells on 2-D plastic or glass substrates. There is

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increasing use of 3-D cell cultures in research areas as diverse as drug discovery, cancer biology, regenerative medicine and basic life science research. There are many methods to facilitate the growth of 3-D cellular structures including nanoparticle facilitated magnetic levitation, gels, beads and solid matrices, self-assembling scaffolds and hanging drop plates. The culture of mammalian cells for toxicity testing or drug screening is likely to increase in the future. The biotechnology, pharmaceutical and cosmetic industries urgently need 3-D cell culture models that more accurately mimic living tissues and organs. This kind of technology will gradually increase in importance because of the vast array of natural and combinatorial products that require screening and the increased pressure from regulatory authorities to reduce animal testing. Most importantly, the pressure to reduce animal testing will stimulate scientists to create more robust biomimetic culture models and culture systems that may eventually eliminate the need for using animals for antibody production and vaccine devel‐ opment. There are many exciting and emerging areas that have not been reviewed in this chapter. This chapter has focused on biomimetic models of joint and muscle. Since joint tissues contain a diverse number of cell types, we have focused our efforts on *in vitro* models of chondrocytes and skeletal muscle cells.

is not due to cell proliferation but rather is due to appositional growth, which includes the transition of perichondral cells into chondrocytes. These conclusions are based on the almost complete absence of mitotic figures in cartilage and perichondrium and on the continuous

The synovial joint contains several important tissue components. These are articular cartilage, synovium, subchondral bone and fat pads (adipose tissue). Developing *in vitro* models that include all of these tissues is a major challenge. Co-culture models involve growing distinct cell types in a combined culture environment. The mixing of different cell types in culture is normally avoided, although it is becoming increasingly apparent that this approach can be used to model the cellular interactions that occur *in vivo*. Some studies of cellular interactions may actually require direct cell-cell contact in a 2-dimensional model where two different cell types are grown in mixture. Other studies may require proximity between the two cell types without any actual cell-cell contact. In this scenario keeping the two types apart can allow investigators to study the effect of one cell type on another. In a synoviocyte-chondrocyte coculture model synoviocytes grown on a filter insert may be stimulated with pro-inflammatory cytokines or other inflammatory mediators (i.e. phorbol esters such as phorbol myristate acetate (PMA), reactive oxygen species such as hydrogen peroxide or a combination of iron and ascorbic acid to simulate inflammation-like radical attacks) before bringing the "activated" cells in contact with chondrocytes. The use of cell culture inserts controls the physical contact and also the duration of that contact. It is also possible to grow different cell types on either side of the membrane, allowing a simulation of tissue behaviour to be built and studied. A coculture model of rat chondrocytes and a rabbit synoviocyte-like cell line (HIG-82, [158]), has been used to show that chondrocytes establish protective mechanisms against reactive oxygen species by interacting with synoviocytes [159]. This co-culture system presents a model to study mechanisms of inflammation in articular joints under well-defined conditions [159]. With the financial support of the National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs) we have established a novel co-culture model of primary equine synoviocytes and primary equine chondrocytes for using equine joints derived

. We have not fully exploited this equine model system. However, since

chondrocytes and synoviocytes are the main cell types present in articular joints, such coculture models have been used for metabolic studies [160] and have the capacity to recapitulate

Cell culture has a bright future. It is a fundamental and core component of tissue engineering and regenerative medicine. In this context the majority of studies carried out to date have used the well-established method of growing cells on 2-D plastic or glass substrates. There is

the inflammatory and catabolic events that occur in an arthritic joint.

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increase in nodule size during cultivation [156].

524 Regenerative Medicine and Tissue Engineering

*6.4.2. Co–cultures of chondrocytes and synoviocytes*

from the abattoir9

**7. Conclusions**

It is becoming clear that OA is a disease of the entire joint rather than any single component and deterioration of associated skeletal muscle masses around an affected joint is a wellreported phenomena. Changes in the highly adaptive skeletal muscle may precede changes seen in other tissue types so analysis of the transcriptome/metabolome could have great value in prognosis and diagnosis. It is possible to conduct such studies in humans although such experiments are logistically complicated, requiring appropriate subject numbers that adhere to complicated inclusion and exclusion criteria and expensive consumables. It could however be argued that a more beneficial approach would be to utilise a "pre-clinical model" to further refine and develop hypotheses before introduction in the human being. This approach would clearly also decrease the need for animal work.

A number of animal models of arthritis have been developed to study arthritic disease pathogenesis and evaluate the efficacy of candidate anti-inflammatory and anti-arthritic drugs for clinical development [161-167]. These animal models may involve injection of inflamma‐ tory agents into the joint, surgical creation of joint instability or surgical replication of joint trauma. Such models not only cause a considerable amount of pain and suffering but also none of them have a proven track record of predictability in human disease. Consequently, there is an acute need for developing novel and alternative *in vitro* models that mimic the biology of cartilage [168-172]. Many studies have used articular cartilage and synovial tissues to establish *in vitro* models of the joint. In our laboratories we have developed explant models of articular cartilage inflammation, co-cultures of primary synoviocytes and chondrocytes as well as 3 dimensional high-density cultures of chondrocytes and MSCs [152, 153, 173-175]. We have also determined the potential of phytochemicals as stimulators of chondrogenic differentiation. These culture models of MSCs, fully differentiated cells and co-cultures have potential as ''proof of principle'' ethically acceptable ''3Rs'' models for engineering biomimetic models of articular cartilage *in vitro* for the purpose of replacing and reducing the use of animals in arthritis research. This approach could help reduce the number of laboratory animals used in arthritis research and may provide a realistic alternative to experimental animals.

Articular cartilage damage is a persistent and increasing problem as the ageing population expands and treatments to achieve biological repair have been challenging [176]. Cartilage tissue engineering has been around for over 20 years. However, none of the approaches available so far have been able to achieve the consistency, effectiveness and reliability that are required for clinical applications. Tissue engineering of a mechanically resilient cartilage construct that meets the structural and functional criteria for effective functional integration into a defect site in the host is a difficult endeavour [177]. One of the fundamental weaknesses of all the models available to date is that none of them possess the normal zonal organization of chondrocytes that is seen *in vivo* (i.e. superficial, middle, deep and calcified zones) and the local composition of extracellular matrix in each zone. This structural organisation is a prerequisite for normal cartilage function and the success of any future clinical applications. The currently available 3-D models produce fairly homogeneous populations of cells without the ability to achieve any zonal organization *in vitro* [176]. The ability to produce a construct that recapitulates the zonal and structural architecture of the original tissue is currently lacking. Even the mechanically stable scaffolds that have been created so far do not allow regeneration of a sufficiently large mass of structurally and functionally competent cartilage construct especially if they were constructed and seeded with 2-D passaged (monolayer) chondrocytes in combination with a biomimetic carrier or scaffold [177]. This is one reason why future studies must begin with 3-D cultured chondrocytes maintained in a physiologically relevant microenvironment that replicates the ionic, osmotic and biomechanical milieu of cartilage. The 3-D and microenvironmental impact on cell phenotype is a significant factor creating cartilage constructs within biomimetic scaffold constructs [177].

In summary, it may be difficult to imagine research being done without animal models but it is worth pointing out that *in vitro* models of joint and musculoskeletal tissues have been around for several decades and new researchers are increasingly adopting them for work on muscu‐ loskeletal diseases. Therefore, it is our optimistic view that in future many animal models could potentially be replaced with biomimetic and animal sparing alternatives in a variety of research disciplines and applications.
