Cartilage Tissue Engineering By Electrospun PCL Scaffolds Seeded With Rabbit Chondrocytes Under Flow Perfusion Culture In Vitro

Articular cartilage has a poor replicative capacity but a relatively highincidence of damage and deterioration from sports injury and osteoarthritis.Tissue engineering strategies, which incorporate biomaterials scaffolds, seedcells, and bioactive factors to regenerate functional cartilage tissue, have beena promising alternative. Electrospun polymer scaffolds are promisingcandidates for tissue engineering applications due to their nonwoven nano-and micro-fiber mesh structure, which mimics the extracellular cellmatrix(ECM), imparts a large surface-to-volume ratio and microporous3Dstructure. Mechanical forces are essential for growth and development ofarticular cartilage. The ability of seed cells to produce ECM is poor whencultured in static. Dynamic culture condition provide mechanical stimulus tocells, and the role of mechanical stimulation on the chondrogenesis has beenmuch appreciated recently. In flow perfusion bioreactor, culture mediumcontinuously flow through the interconnected porous network of the seededscaffold and provide shear stress force, mimicking mechanical environment ofnative joint. Since the particular microstructure and poor mechanicalproperties, references using electrospun meshes in flow perfusion conditionare scarce.Using electrospun scaffolds in flow-perfusion bioreactor for cartilageengineering was studied in recent years, and some results were encouraging.But the two main problems still need be further explored: how to design aflow-perfusion bioreactor particularly matching electrospun scaffolds most,and how to achieve the best quality of engineering constructs by modifyingthe microstructure of electrospun scaffold and perfusion flow rate. Having allthese issues in mind, microfiber porous electrospun PCL meshes werefabricated in this study, and a specialized flow perfuion bioreactor was fabricated on our own. We aimed to determine the effect of perfusion cultureon proliferation and ECM biosynthesis of rabbit chondrocytes in PCLelectrospun scaffolds, and provide basis and experience for future deepresearch.Objective: The investigate the ability of perfusion bioreactor on our ownand electrospun PCL scaffolds on favoring chondrocyte proliferation andextracellular matrix biosynthesis for cartilage tissue engineering.Methods: Nonwoven PCL microfiber mats were fabricated, andcontra-aperture cylindrical glass equipment as perfusion bioreactor on our ownwas designed and manufactured. Primary chondrocytes were isolated from theknee joints of two months old New Zealand white rabbits and seeded ontoscaffolds. Constructs were harvested at3,7, and14day, both from thebioreactor and static groups, for SEM analysis, biochemical assays, real-timePCR and Histology analysis.Results: SEM observations of PCL meshes show a random distributionand solid surface. PCL electrospun meshes were composed of microfiberswith diameters of1.67±0.76μm and pores with diameters of17.65±7.11μm.Custom-made flow perfusion bioreactor had good sealing with no biologicaltoxicity, and the perfusion rate could be controlled accuratly. SEM showedbetter proliferation and matrix production of cells in bioreactor, whichmaintained round in shape, the typical morphology of chondrocytes. At7daysof culture, DNA content in perfusion culture was11.41±4.06μg/mL, whichwas higher than static culture3.67±1.08μg/mL (P<0.05). At3days and7daysof culture, GAG content in perfusion culture was0.6097±0.1504μg/mL and1.9289±0.6256μg/mL, which was higher than static culture0.1253±0.0377μg/mL and0.1669±0.0566μg/mL (P<0.01). At14days of culture, GAGcontent in perfusion culture was1.2666±0.3763μg/mL, which was higherthan static culture0.3566±0.0855μg/mL (P<0.05). At3days and7days ofculture, COL content in perfusion culture was0.084±0.0208μg/mL and0.1133±0.028μg/mL, which was higher than static culture0.0402±0.0033μg/mL and0.0433±0.0142μg/mL (P<0.05). GAG synthetic activity (GAG/DNA) in bioreactor showed higher than in static, and significantdifferences were found at3days (P<0.01),7days (P<0.05) and14days(P<0.05). Real-time PCR showed higher gene expression of aggrecan andcollagen II to the values obtained in static culture (P<0.05), and highercollagen type II to collagen type I gene expression ratio (col II/col I)(P<0.05). HE and safranin O staining showed a better cell infiltration and a denseconcentration of staining from the bioreactor samples, and cartilage cavitieswere formed.Conclusion: Electrospinning technology and a perfusion bioreactor wereeffectively united, and we demonstrated that our custom-made flow perfusionbioreactor is suitable for culturing rabbit chondrocytes on electrospun PCLmicrofiber meshes. Culturing in the bioreactor supported the attachment andproliferation of rabbit chondrocytes and ECM production. The method shouldbe extensively studied, but is a promising method for cartilage tissueengineering.