Assessing Chondrogenesis of Chondroprogenitors and Chondrocytes on Trilaminar Electrospun Scaffolds

The rapidly emerging field of tissue engineering in oral and maxillofacial surgery holds great potential for functional cartilage tissue substitutes by engineering tissue constructs in-vitro for subsequent implantation in-vivo. Chondrocytes required for cell-based therapies are isolated and expanded in-vitro to produce sufficient numbers of cells for surgical procedures. However, extensive expansion causes progressive de-differentiation of the chondrocytes (1). Human chondrocytes show an inability to retain a chondrogenic potential past 7 population doublings in culture. A chondroprogenitor population has been identified on the surface of bovine articular cartilage and has been described, opening up the possibility that human cartilage could also contain a cartilage progenitor population (2). The main advantage of chondroprogenitor cells over full-depth chondrocytes is the ability to, from a single cell, produce very large cell numbers that retain their phenotype.  Hence it would be feasible to treat much larger defects than is currently possible.

        Herein, we assess in-vitro TGF-β3 induced chondrogenesis of bovine chondroprogenitor and chondrocyte cells on the trilaminar electrospun scaffolds we generated that mimics both the fibre orientation and zonal tensile properties of articular cartilage. Poly(ε-caprolactone) based fibrous constructs were fabricated by a custom-made electrospinning set-up. Bovine cartilage was harvested from the subchondral section of the joint. Cells were isolated by digesting the tissue overnight at 37°C with agitation. After digestion, isolated cells were filtered and plated. Discrete colonies consisting of more than 32 cells were selected as chondroprogenitors using an inverted Olympus IX51 microscope. Chondrocytes and chondroprogenitors were seeded onto fibrous constructs measuring 8mm in diameter. Following adhesion, they were transferred to non-adherent 24 well plates and cultured in 1 ml of chondrogenic differentiation medium. Cell viability in-vitro was assessed using the LIVE/DEAD® Cell Viability assay. Total DNA content using the Quant-iT^™ PicoGreen^® kit and sulfated glycosaminoglycans were determined using the Blyscan Kit. Fibrous constructs were examined histologically by staining for deposited extracellular matrix components of sulfated proteoglycans using 0.1% Alcian Blue, Hematoxylin and Eosinophilic stains for cell nuclei and extraneous extracellular matrix material and Picrosirius Red for collagen. Seeded constructs and acellular control constructs were assessed for compressional properties by performing unconfined uniaxial compression testing using an Instron Model 5540 testing machine.

        All experimental sample groups had a sample size of at least n = 3 for biochemical, histological and mechanical property analyses done on week 5.  Data is presented as average ± standard error mean.  Statistical significance was determined by performing Student’s T-test for all data sets with significance accepted at p-value < 0.05. Histological examination of seeded fibrous constructs revealed a greater distribution and amount of cell nuclei, collagen and proteoglycans throughout the cross-section of chondroprogenitor compared to chondrocyte seeded constructs. Chondroprogenitor seeded constructs exhibited higher compressive moduli (417.5 ± 31.2 kPa) compared to chondrocyte seeded constructs (227.7 ± 20.9 kPa).

        In conclusion, both chondroprogenitors and chondrocytes are viable and able to undergo chondrogenesis on the trilaminar electrospun scaffolds but alter its mechanical properties in different ways. The clinical implication is that with an improved understanding of the different cell types that are viable and factors that affect the mechanical properties of hierarchical scaffolds we are able to tune and tailor the most effective engineered tissue for articular cartilage regeneration.

 1.Williams R, Khan IM, Richardson K, Nelson L, McCarthy HE, Analbelsi T, et al. Identification and clonal characterisation of a progenitor cell sub-population in normal human articular cartilage. PloS one 2010 Oct 14;5(10): pp. e13246.

 2.Khan IM, Bishop JC, Gilbert S, Archer CW. Clonal chondroprogenitors maintain telomerase activity and Sox9 expression during extended monolayer culture and retain chondrogenic potential. Osteoarthritis and cartilage / OARS, Osteoarthritis Research Society 2009 Apr;17(4): pp. 518-528.