Abstract
The benefits associated with polyhydroxyalkanoates (PHA) in tissue engineering include high immunotolerance, low toxicity, and biodegradability. Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx), a molecule from the PHA family of biopolymers, shares these features. In this study, the applicability of human embryonic stem cells (hESCs), spontaneously differentiated hESCs (SDhESCs), and mesenchymal stem cells (hMSCs) in conjunction with PHBHHx and collagen as a biocompatible replacement strategy for damaged tissues was exploited. Collagen gel contraction was monitored by seeding cells at controlled densities (0, 10(3), 10(4), and 10(5) cells/mL) and measuring length and diameter at regular time intervals thereafter when cultured in a complete medium. Cell viability was measured by trypan blue exclusion assay. Porous PHBHHx tube scaffolds were prepared using a dipping method followed by salt leaching. PHBHHx/collagen composites were generated via syringe injection of collagen/cell mixtures into sterile PHBHHx porous tubes. Reverse transcription polymerase chain reaction was used to determine the fate of cells within PHBHHx/collagen scaffolds with tendon, bone, cartilage, and fat-linked transcript expression being explored at days 0, 5 10, and 20. The capacity of PHBHHx/collagen scaffolds to support differentiation was explored using a medium specific for osteogenic, chondrogenic, and adipogenic lineage generation. Collagen gel tube contraction required initial seeding densities of ≥10(5) hMSCs or SDhESCs in 1.5 mg/mL collagen gel tubes. Gels with a collagen concentration of 3 mg/mL did not display contraction across the examined cell seeding densities. Cell viability was ∼50% for SDhESC and 90% for hMSCs at all cell densities tested in porous PHBHHx tube/3 mg/mL collagen hybrid scaffolds after 20 days in vitro culture. Undifferentiated hESCs did not contract collagen gel tubes and were unviable after 20 days culture. In the absence of additional stimuli, SOX9 was sporadically found, while RUNX2 was not present in both hMSC and SDhESC. Hybrid scaffolds were shown to promote retention of osteogenic, chondrogenic, and adipogenic differentiation by expression of RUNX2, SOX9, and PPARγ genes, respectively, following exposure to the appropriate induction medium. PHBHHx/collagen scaffolds have been successfully used to culture hMSC and SDhESC over an extended period supporting the potential of this scaffold combination in future tissue engineering applications.