Cartilage is a non-vascular connective tissue composed of specialized cells – chondrocytes. The lack of blood vessels results in limited cartilage self-regeneration capabilities. While various options are available to treat osteoarthritis, no current method can restore the mechanical function of the original hyaline cartilage. Therefore, herein we address, hyaline cartilage regeneration issue by engineering a synthetic biocompatible hydrogel scaffold capable to promote chondrogenic differentiation.
In this study, the chemically crosslinked hydrogels consisting of synthetic peptides that have the collagen-like sequence Cys-Gly-(Pro-Lys-Gly)4 (Pro-Hyp-Gly)4 (Asp-Hyp-Gly)4- conjugated with the cell adhesion sequence RGD (CLP-RGD) and crosslinked hydrogels of type I collagen (CA) were used. Both hydrogels were compared as cell growth substrates potentially applicable as implantable biomaterials for cartilage regeneration. For cartilage formation, we used human skeletal muscle-derived stem/progenitor cells (hMDSPCs) set for differentiation towards a chondrogenic lineage by BMP-7 and TGF-ß3 growth factors. Since the precise amount at which the growth factors induce differentiation is unknown, we chose to investigate minimal growth factors dosages, relying on the ability of the two types of hydrogels to retain and release the growth factors into cell culture media. We monitored the amount of growth factors diffusing out of the scaffolds for two weeks by changing the cell culture media and observed almost identical diffusion of the different amount of initially loaded growth factors (150, 100 and 75 ng/scaffold). Growth factors incorporation strategy allowed a sustained release of TGFß3, 6.0 0.3% of the initially loaded amount diffused out after 4 h and 2.7 0.5% already at the second time point (24h) from CA and CLP-RGD substrates. For the BMP-7 growth factor, 13.1 2.3% and 15.75 1.6% of the initially loaded amount diffused out after 4 h, 1.7 0.2% and 2.45 0.3% at the second time point (24 h) from CA and CLP-RGD respectively. We monitored growth factors diffusion out during all incubation period (14 days). In vitro experiments were performed by seeding hMDSPCs onto hydrogels scaffolds loaded with growth factors (75ng/scaffold) and cultured for 28 days. Cartilage formation was monitored by extracellular matrix protein collagen type II deposition (using biochemical ELISA assay) and quantification of glycosaminoglycans genes expression (RT-PCR). We measured collagen type II expression at different time points for 28 days. From CLP-RGD scaffolds onwards, collagen type II was significantly higher expressed than on CA hydrogel irrespective of growth factors present. The scaffolds with immobilized growth factors resulted in higher collagen type II accumulation when compared to the scaffolds alone. The gene expression on CLP-RGD hydrogels (day 28) with growth factors has shown lower collagen type I expression and higher aggrecan expression compared to day 0. However, we also report increased collagen X gene expression on CA hydrogels (with growth factors) that can be expected considering hydrogel scaffold composition which can lead to chondrocytes hypertrophy.
Our results support the potential of the strategy of combining implantable hydrogels functionalized with differentiation factors toward improving cartilage repair via precision tissue engineering.