Abstract
This study aimed on the development of serum-free conditions for clinical application of articular cartilage tissue engineering. Based on genipin-crosslinked collagen type II (COL II) scaffold and quantitative polymerase chain reaction (qPCR) analysis, 7 kinds of growth factors (GFs) and low-molecular weight chondroitin sulphate type C (LMWCSC) were chosen for the investigation. First, adapted from 2-level fractional factorial design and steepest ascent method, we selected a set of GFs and made a designed serum-free medium (dSFM) formulation which contained EGF 8.31 ng/ml, TGF-β1 20.00 ng/ml, BMP-2 28.85 ng/ml, PDGF-bb 8.11ng/ml, and VEGF 12.69 ng/ml. This dSFM could highly upregulate gene expression level of COL II, the unique marker of normal chondrocyte. From other parameter such as biochemical assay and histocytochemistry observation we also confirmed the better quality of engineered cartilage as compared to serum-containing group. In addition, we found this dSFM could maintain the induced human umbilical cord blood-derived mesenchymal stem cell (hUMSC) based on gene expression and biosynthetic rate. Next, we investigate the effect of LMWCSC on chondrocyte. We prepared it and found this small molecule could help chondrocyte to redifferentiate more rapidly. Meanwhile, several degradative genes were inhibited by LMWCSC. These data indicate that supplement with LMWCSC into medium are potent for promoting excreting ECM of normal cartilage as well as protecting from degradative damage, thus further development is promising. In summary, the choice of serum-free components into medium formulation could help cells develop normally and clinical application of cartilage tissue engineering.