1.1. Tissue Engineering

Reddi defines tissue engineering as “the science of design and manufacture of new tissues for the functional restoration of impaired organs and replacement of lost parts due to disease, trauma or tumours” [12]. Furthermore, a tissue-engineered biological substitute should resemble the native tissue and perform similar biological functions [13]. Three key ingredients for the success of tissue engineering are inductive signals, responsive candidate cells, and an appropriate extracellular matrix (ECM). Inductive signals, such as growth factors, are an essential component to any tissue-engineered substitute since most candidate cells used in tissue engineering have lost their intrinsic differentiation program [13]. Hence, the identification of growth factors or other stimuli controlling the differentiation process is of crucial importance and the cannabinoid system may represent a novel target in this regard. A variety of candidate cells are used in tissue engineering, including chondrocytes, osteoblasts, embryonic stem cells and adult stem cells. The application of adult stem cells in tissue engineering holds great promise since there are no associated ethical problems with their use and they surpass the growth and differentiation capacity of native cells such as chondrocytes. The ECM is comprised of structural and specialized proteins combined with proteoglycans that form a microenvironment for cells to reside in, cells which in turn contribute to its formation. Each of the three ingredients for the success of tissue engineering should encourage cell migration, survival and proliferation, and precursor differentiation which are the three main tenets for the success of any tissue-engineered approach to regeneration [14,15]. Many challenges lie ahead in the path to the use of tissue-engineered approaches in the clinic namely the control of differentiation, immunologic issues, tissue integration and the consistency of engineered tissues. Understanding more about what controls these facets of tissue regeneration will be of the upmost importance for the generation of better tissue-engineered approaches and products.

1.2. Cartilage Disease

Chondrocytes are responsible for maintaining cartilage tissue homeostasis by balancing the biosynthesis and incorporation of extracellular matrix components. Cartilage has poor to no blood supply and derives nutrients from the surrounding synovial fluid [16,17,18]. Therefore, chondrocytes are continually challenged by a hypoxic cellular micro-environment, as low as 1% oxygen during endochondral ossification [19,20,21]. Cartilage-specific extracellular matrix components, such as collagen II and sulphated proteoglycans, are synthesized by chondrocytes in order to maintain cartilage homeostasis [22,23,24]. This balance may be destroyed, however during injury or disease, resulting in a loss of cartilage and joint damage. Since cartilage is poorly supplied by blood vessels, nerves and the lymphatic system, its regenerative capacity is limited which can prove problematic. Damage as a result of the inflammation associated with joint diseases such as osteoarthritis, or traumatic injury can lead to pain and long-term disability. There are many diseases that affect cartilage the majority of which are rare, e.g., polychondritis and achondroplasia. However, changing lifestyles and an ageing global population has led to increased levels of ‘wear and tear’ diseases of cartilage such as intervertebral disc herniation and trauma-induced tears, and ‘failure to repair’ diseases of cartilage such as osteoarthritis. Bio-engineering applications are currently enhancing the treatment of cartilage disease by providing both in situ regeneration and cell and tissue transplantation [15]. However, identifying factors that may enhance the survival and differentiation of transplanted and resident cells warrants investigation.

A cell based tissue engineering approach to cartilage disease has the potential to become the superior treatment choice, providing biological resurfacing of affected joints [25] compared to mechanical approaches such as total joint arthroplasty or in situ cartilage regeneration techniques such as the microfracture technique and autologus chondrocyte transplantation (ACT) [26,27]. Some success is noted with these techniques however, long recovery periods, the slow regeneration of cartilage and lack of recipient response (especially older patients) make the search for better alternatives particularly relevant [28,29,30]. Furthermore, osteoarthritis is an exclusion criteria for ACT and the microfracture technique [31].

6.2. Drug Treatments

MSCs were incubated with drugs or vehicle for the time indicated in each experiment. Δ9-THC and anandamide were obtained from Sigma-Aldrich Company Ltd. Δ9-THC was held under license granted by the Irish Department of Health and Children. Δ9-THC was stored as a 80mM stock solution in ethanol at -20oC and diluted to a final concentration of 1 μM in culture media. Anandamide was stored as a 10 mM stock solution in ethanol at -20oC and diluted to a final concentration of 1 μM in culture media.

6.3. Cell Viability Assay

Cell viability was determined by quantifying the enzymatic conversion of cell permeable calcein AM (Invitrogen, Scotland) to a fluorescent product by active intracellular esterases. Briefly, MSCs were grown on sterile 96 well plates (6 × 103 cells per well) and treated as indicated in each experiment. MSCs were washed in pre-warmed PBS to remove serum from the wells. Calcein AM solution (2 μM in PBS) was applied to each well and incubated in a humidified atmosphere (95% air and 5% CO2) at 37 oC for 1 hour. Following incubation calcein fluorescence at 530 nm was determined using a microplate reader heated to 37oC (Synergy™ HT, BioTek Instruments, USA). Data are presented as mean relative fluorescent intensity units (RFU) ± SEM.

6.4. Cell Migration

For cell migration experiments 50 × 103 cells in s-DMEM were seeded onto the upper chamber of a migration chamber (8 μm Millicell 24 well plate cell culture insert, Millipore, Ireland) and s-DMEM was added to the bottom chamber. Cells were allowed to settle for 48 hours before beginning migration experiments. Chemoattractant media (5 ng/mL TGF-β; + control), anandamide (1 μM) or control non chemoattractant media (DMEM supplemented with 1% BSA, 100U/mL penicillin/streptomycin; 2 mM glutamax; 1 mM L-glutamine and 1% non-essential amino acids) were placed into the bottom chamber and cells were allowed to migrate for 48 hours. Cells remaining on the upper chamber were scrapped off whilst cells that had migrated to the lower chamber were stained using the Rapi Diff II staining kit (Bios Europe, UK). Blinded scorers manually counted cells from 10 random fields at 20 × magnification. Data are presented as mean number of migrated cells ± SEM.

6.5. Real-time PCR for Collagen II

Total RNA was isolated from MSCs using a NucleoSpin® total RNA isolation kit (Macherey-Nagel Inc., Germany) following the manufacturer’s instructions. This protocol included a DNase step in order to remove any genomic DNA contamination. Total RNA concentrations were determined by spectrophotometry (NanoDrop Technologies, USA) and stored at -80oC until required for cDNA synthesis. Total RNA concentrations were adjusted to a standard concentration prior to cDNA synthesis. cDNA was generated from 0.5-1 μg total RNA using High Capacity cDNA Archive kit (Applied Biosystems, Germany) following the manufacturer’s instructions. The resultant cDNA was stored at -20oC until required for real time PCR. Real-time PCR was performed using Taqman Gene expression assays (Applied Biosystems, Germany) on an ABI Prism 7300 instrument (Applied Biosystems, Germany). The assay ID for the collagen II gene was Rn01637085 mL. Rat β-actin (4352340E) was used as an endogenous control. Gene expression was calculated relative to the endogenous control (β-actin) and to the control samples to give a relative quantification (RQ) value.

6.6. Immunofluorescence for Collagen II

Following drug treatment MSCs were fixed in 100% methanol for 5 minutes at -20oC, permeabilized with 0.2% Triton-X100 for 10 min and washed in three changes of PBS at room temperature (RT). Cells were washed 3×5 min in PBS and non-reactive sites were blocked with blocking buffer (2% BSA in PBS with 0.1% Triton × 100 and 20% heat-inactivated horse serum; Vector, USA) for two hours at room temperature (22oC). The cells were incubated overnight with mouse anti-collagen II antibody (1:500 dilution in blocking buffer; Abcam, Cambridge, UK) at 4oC. Cells were washed 3 × 5 min in PBS, incubated with a biotinylated secondary horse anti-mouse IgG (1:50 dilution; Vector, USA) for one hour at room temperature and washed 3 × 5 min in PBS. In a light protected environment, Extravidin FITC (1:50 dilution in PBS; Sigma-Aldrich, England) was added for one hour washed 6 × 5 min in dH2O. Coverslips were mounted onto glass slides in Vectashield mounting medium (Vector, USA) and sealed using nail varnish. Slides were examined at 20 × magnification with a fluorescence microscope (Zeiss, Germany).

This research was funded by Science Foundation Ireland (07/RFP/BIMF126).