Biodegradable zinc oxide composite scaffolds promote osteochondral differentiation of mesenchymal stem cells (1)
Zinc is an essential nutrient for many physiological functions including growth, DNA synthesis, immune function, and bone metabolism and it is part of almost 300 enzymes and a large number of proteins. Zinc supplementation in the cell culture medium has been shown to enhance mineralization and osteocalcin gene expression of MC3T3‐E1 osteoblasts.
Zinc oxide (ZnO) is an inorganic compound and is currently listed as generally recognized as safe material by the US Food and Drug Administration. ZnO has been shown to be biodegradable and biocompatible. ZnO nanoparticles exhibit antibacterial properties which could be attractive for tissue engineering applications since it may prevent infection. ZnO nanoparticles can be incorporated into polycaprolactone (PCL), a slow degrading material, to allow for zinc dissolution from a composite scaffold which could be beneficial for both bone and cartilage regeneration. ZnO nanoparticles were combined with PCL to form fibrous scaffolds using the electrospinning technique. Electrospun scaffolds mimic the native fibrous extracellular matrix (ECM) having a high surface area to volume ratio which is beneficial for cell adhesion, growth, and differentiation. In addition to a slow degradation rate, PCL has proven biocompatibility and low glass transition, allowing for mechanical flexibility at room and body temperatures.
Cell number was significantly higher on 5% and 10% ZnO as compared with PCL control (0% ZnO) by days 14 and 28 in the growth medium (GM). Cell number was significantly higher on 2.5% ZnO in GM by Day 28. In chondrogenic induction medium (CCM), no significant differences in cell number were detected between ZnO groups and control. Glycosaminoglycans (GAG) production was significantly higher on 5% and 10% ZnO at days 14 and 28 in GM as compared with PCL control. In CCM, GAG production was significantly higher on 1%, 2.5%, and 5% ZnO composite scaffolds as compared with control at Day 14 but no significant differences were detected between groups at Day 28. Cells cultured on 2.5% ZnO scaffold produced the highest collagen type II as compared with all other groups in CCM at Day 28 (p < .05). Cells cultured on 1% ZnO produced significantly higher collagen type II as compared with control (0% ZnO) and 5% ZnO (p < 0.05). Cells on PCL and 1% ZnO had significantly higher collagen I as compared with other groups in CCM at Day 28 (p < .05). The cells on 2.5% ZnO composite scaffold produced the highest collagen type II with low collagen type I. ELISA was not conducted for samples in GM due to minimal immunostaining for collagen.
ALP activity was significantly higher at Day 21 for 2.5%, 5%, and 10% ZnO composite scaffolds as compared with control in GM. In OS, ALP activity was higher on 1% and 2.5% ZnO groups at Day 14 and on all ZnO groups at Day 21 as compared with control. Total collagen production on ZnO composite scaffolds was quantified using hydroxyproline assay. Cells on 10% ZnO had significantly higher collagen production as compared with PCL control in GM (p < .05). Cells on ZnO composite scaffolds produced more collagen in OS as compared to GM (p < .05). Collagen production was significantly higher on 5% and 10% ZnO in OS as compared with other groups (p < .05).
The slow release of the zinc was observed for all ZnO composite scaffolds with a higher release for the 10% ZnO composite scaffolds. Zinc release was less than 1mg/L for all scaffolds over the course of 28 days of study with 95–98% zinc still remaining in the scaffolds. Collagen type II production is a mature marker of hyaline cartilage. Moreover, chondroadherin, a mature marker of cartilage, plays a role in collagen fibril organization and cell‐matrix interaction. Collagen type I expression is associated with immature chondrocytes. Chondroadherin and collagen type II expression are low in immature cartilage and fibrocartilage and increase with maturation. ALP, an early marker of osteogenesis, increases inorganic phosphate and mineralization. ALP activity increased with increasing ZnO. Zinc is necessary for collagen synthesis and mineralization in bone in humans and animals. The differences in proliferation and differentiation could also be related to the ZnO remaining on the fiber surface as the cells can interact with the zinc on the surface of the fibers. Modification of titanium with zinc increased the focal adhesion of human osteoblast‐like MG‐63 cells. Osteoblast proliferation and adhesion on 5–7wt% ZnO with hydroxyapatite as compared to hydroxyapatite alone due to the dissolution of ZnO and changes in surface wettability.