2.3.1. CCs, FPMSC and BMMSC isolation

Following assessment of the intrinsic chondrogenic and osteoinductive properties of the tri-layered scaffold using rBMMSCs, a more translationally relevant cell source was then used in order to develop the optimal cell seeding approach for the tri-layered scaffold. Goats are a favourable large animal model for assessment of osteochondral defect repair [43] and our ultimate focus was to test the solution devised in this paper in vivo in a caprine model [9] thus the use of goat CCs, BMMSCs and FPMSCs was explored here. Euthanasia of a skeletally mature male goat was performed, and chondrocytes, FPMSCs and BMMSCs were harvested under Ethics licence approved by the Animal Research Ethics Subcommittee (AREC-P-12-71-Brama). The stifle joint was carefully opened under sterile conditions, and the infrapatellar fat pad was removed in its entirety, placed in PBS and stored at 4 ​°C. The knee joint was then disarticulated and the cartilage surface exposed. The cartilage was harvested from the distal femur with a 6 ​mm punch biopsy and the cartilage strips placed in PBS with antibiotic supplementation (100 U/ml penicillin G and 100 ​μg/ml streptomycin sulphate, and 50 ​μl of Amphotericin B; Gibco). The distal femur was then opened and the bone marrow was removed using a sterile spatula and placed directly into a 50 ​ml falcon tube, 5 ml/tube with 10 ​ml of expansion medium [Gibco DMEM ​+ ​GlutaMAX (61965, Gibco) ​+ ​10% FBS (Foetal Bovine Serum, (Labtech, UK) ​+ ​10 ​ml (100 U/ml penicillin G and 100 ​μg/ml streptomycin sulphate; Sigma)].

The chondrocytes were isolated from the harvested cartilage tissue using a rapid-isolation technique using collagenase and plastic adherence [25,33,44,45]. The cartilage pieces were removed from the antibiotic supplemented PBS and the cartilage was then sliced to pieces less than 1 ​mm in size, prior to rinsing again with antibiotic supplemented PBS and placing in sterile 50 ​ml tubes. Collagenase (Worthington, {"type":"entrez-nucleotide","attrs":{"text":"LS004176","term_id":"1321650548","term_text":"LS004176"}}LS004176 Collagenase Type CLS-2) was dissolved in medium [Gibco DMEM ​+ ​GlutaMAX (61965, Gibco)] (350 U/ml) (8 ​ml/g cartilage), and sterile filtered and rotated in the collagenase solution (2 ​h, 37 ​°C) prior to passing through a 40 ​μm cell strainer. Collected cartilage particles were then crushed using a pipette tip and added to 8 ​ml/g collagenase solution rotating for a further 1 ​h (37 ​°C). The sieved medium was added to stopping medium [Gibco DMEM ​+ ​GlutaMAX (61965, Gibco) ​+ ​10% FBS (Foetal Bovine Serum, (Labtech, UK))] of equal volume and mixed. The crushed cartilage was then removed from the rotator and stopping medium of equal volume added and mixed. This was again strained in a 40 ​μm cell strainer and centrifuged at 650 ​g for 10 ​min. The supernatant was discarded, and the pellet re-suspended in standard expansion medium [Gibco DMEM ​+ ​GlutaMAX (61965, Gibco) ​+ ​10% FBS (Foetal Bovine Serum, (Labtech, UK) ​+ ​10 ​ml (100 U/ml penicillin G and 100 ​μg/ml streptomycin sulphate)] and counted using a trypan blue exclusion test. The chondrocytes were then plated into T175 flasks at a density of 875 ​× ​10³ ​cells/flask and expanded to 90% confluency.

The FPMSCs were isolated as per previously published protocols [25,33,44]. The fat pad digestion was carried out using collagenase dissolved in medium (750 U/ml, 4 ​ml/g of tissue). As previously, the tissue was minced and placed into sterile 50 ​ml falcon tubes. 4 ​ml of collagenase solution was added per gram of tissue. The tubes were rotated in a tube rotator at 37 ​°C for 3–4 ​h. Two volumes of stopping medium were added to the tissue collagenase mixture. The solution was then passed through a sieve (150 ​μm) into fresh sterile tubes and centrifuged for 10 ​min at 650 ​g. The floating fat fraction containing the adipocytes was aspirated off and discarded. The pellet was resuspended in fresh standard expansion medium and then passed through a 40 ​μm strainer into a fresh tube. Further medium was added, and the suspension centrifuged once more at 650 ​g for 5 ​min. The pellet was resuspended and counted using trypan blue.

The BMMSCs were isolated as previously described [44]. Briefly, the medium/marrow solution was titrated with a 16 ​G needle to break up any clumps, then the volume was made up to 40 ​ml using expansion medium and mixed to form a homogenous solution. The tube was then centrifuged twice at 650 ​g for 5 ​min, with the supernatant removed and discarded each time. The cell pellet was then resuspended in 10 ​ml and triturated using a 16 ​G needle prior to passing through a 40 ​μm strainer into a fresh and topping up to 20 ​ml. In order to separate the mononuclear cells, 20 ​ml of Lymphoprep™ (Axis-Shield, Norway) was placed in a 50 ​ml falcon tube and the 20 ​ml suspension added gently on top. The solution then underwent centrifugation for 20 ​min at 650 ​g with deceleration set to 0 and acceleration set to 1. When the centrifugation was complete the mononuclear cell layer residing at the interface of the Lymphoprep™ and the cell suspension was removed and cells were counted using trypan blue.

The ability of the isolated FPMSCs and BMMSCs to differentiate down osteogenic, chondrogenic and adipogenic routes was then assessed to confirm their tripotentiality. To assess osteogenic potential, cells were cultured at a density of 1 ​× ​10⁵ ​cell per well in 6 well plates (n ​= ​6) in osteogenic media [10% foetal bovine serum (FBS; Hyclone, Fisher Scientific, Ireland), 90% DMEM ​+ ​GlutaMAX (Gibco) supplemented with antibiotics (100 U/ml penicillin G and 100 ​μg/ml streptomycin sulphate; Gibco) dexamethasone 0.4 ​μg/ml (Sigma), β-glycerol phosphate 0.01 ​M (Sigma) and ascorbic acid 6.9 ​mg/ml (Sigma)] opposite negative controls in standard expansion media for a total of 21 days followed by staining with alizarin red (Sigma) staining for calcium. To assess chondrogenisis cell pellets with 250,000 ​cells per pellet were cultured in a chondrogenic media [10% foetal bovine serum (FBS; Hyclone, Fisher Scientific, Ireland), 90% DMEM ​+ ​GlutaMAX (Gibco) supplemented with antibiotics (100 U/ml penicillin G and 100 ​μg/ml streptomycin sulphate; Gibco) with 10 ​ng/ml of TGF-β3 (R&D Systems) Dexamethasone 0.4 ​μg/ml (Sigma), ascorbic acid 0.5 ​mg/ml (Sigma), lineloic acid 47 ​μg/ml (Sigma), and ITS 1X; (Sigma)] for 7 days and assessed for sulphated GAG levels using Blyscan Sulphated Glycosaminoglycan (sGAG) assay (Biocolour Ltd, UK). Lastly, to assess adipogenic ability cells were seeded at a density of 1 ​× ​10⁵ ​cell per well in 6 well plates (n ​= ​6) and then cultured in adipogenic media [10% foetal bovine serum (FBS; Hyclone, Fisher Scientific, Ireland), 90% DMEM ​+ ​GlutaMAX (Gibco) supplemented with antibiotics (100 U/ml penicillin G and 100 ​μg/ml streptomycin sulphate; Gibco) dexamethasone 20 ​μg/ml, (Sigma) 0.5 ​mM IBMX (Sigma) 50 ​μM indomethacin (Sigma)] opposite negative controls in standard expansion media for 21 days. The presence of vacuoles observed using optical microscopy indicated adipogenesis of the MSCs. Cell viability was assessed using trypan blue exclusion.