A handful of studies have exploited antitumor potential of esculetin, a dihydroxy coumarine derivative; the targets to which it binds and the possible downstream mechanism for its cytotoxicity in cancer cells remain to be elucidated. Using pancreatic cancer cell lines as a model system, herein the study was initiated to check the efficacy of esculetin in inhibiting growth of these cancer cells, to decipher mechanism of its action and to predict its direct binding target protein. The cytotoxicity of esculetin was determined in PANC-1, MIA PaCa-2 and AsPC-1 cell lines; followed by an inspection of intracellular levels of ROS and its associated transcription factor, p65-NF-κB. The interaction between transcription factor, Nrf2 and its regulator KEAP1 was studied in the presence and absence of esculetin. The effect of Nrf2 on gene expression of antioxidant response element pathway was monitored by real time PCR. Thereafter, potential binding target of esculetin was predicted through molecular docking and then confirmed in vitro. Esculetin treatment in all three pancreatic cancer cell lines resulted in significant growth inhibition with G1-phase cell cycle arrest and induction of mitochondrial dependent apoptosis through activation of caspases 3, 8 and 9. A notable decrease was observed in intracellular ROS and protein levels of p65-NF-κB in PANC-1 cells on esculetin treatment. Antioxidant response regulator Nrf2 has been reportedly involved in crosstalk with NF-κB. Interaction between Nrf2 and KEAP1 was found to be lost upon esculetin treatment in PANC-1 and MIA Paca-2 cells. Nuclear accumulation of Nrf2 and an upregulation of expression of Nrf2 regulated gene NQO1, observed on esculetin treatment in PANC-1 further supported the activation of Nrf2. To account for the loss of Nrf2-KEAP1 interaction on esculetin treatment, direct binding potential between esculetin and KEAP1 was depicted in silico using molecular docking studies. Pull down assay using esculetin conjugated sepharose beads confirmed the binding between esculetin and KEAP1. We propose that esculetin binds to KEAP1 and inhibits its interaction with Nrf2 in pancreatic cancer cells. This thereby promotes nuclear accumulation of Nrf2 in PANC-1 cells that induces antiproliferative and apoptotic response possibly by attenuating NF-κB.

Biomaterial integration in bone depends on bone remodelling at the bone-implant interface. Optimal balance of bone resorption by osteoclasts and bone deposition by osteoblasts is crucial for successful implantation, especially in orthopaedic surgery. Most studies examined osteoblast differentiation on biomaterials, yet few research has been conducted to explore the effect of different orthopaedic implants on osteoclast development. This review covers, in detail, the biology of osteoclasts, in vitro models of osteoclasts, and modulation of osteoclast activity by different implant surfaces, bio-ceramics, and polymers. Studies show that surface topography influence osteoclastogenesis. For instance, metal implants with rough surfaces enhanced osteoclast activity, while smooth surfaces resulted in poor osteoclast differentiation. In addition, surface modification of implants with anti-osteoporotic drug further decreased osteoclast activity. In bioceramics, osteoclast development depended on different chemical compositions. Strontium-incorporated bioceramics decreased osteoclast development, whereas higher concentrations of silica enhanced osteoclast activity. Differences between natural and synthetic polymers also modulated osteoclastogenesis. Physiochemical properties of implants affect osteoclast activity. Hence, understanding osteoclast biology and its response to the natural microarchitecture of bone are indispensable to design suitable implant interfaces and scaffolds, which will stimulate osteoclasts in ways similar to that of native bone.

Fibrous encapsulation that prevents the direct contact between an implant and the bone can cause implant failure. However, prevention of fibrous encapsulation is difficult because of the lack of effective strategies which can selectively control the growth of fibroblasts and osteoblasts. Because curcumin, an extract from Curcuma longa, was recently found to reduce the formation of fibrous tissue, it is hypothesized that loading curcumin on implant surfaces would be efficacious in inhibiting fibrous encapsulation without adversely affecting the osteoblast functions. To prove this hypothesis, curcumin was loaded on to a titanium surface using poly(dopamine) as an anchor, and the behaviors of fibroblasts and osteoblasts on these curcumin-modified surfaces were investigated. Curcumin was successfully loaded on to titanium and showed a low release after incubation in phosphate-buffered saline for seven days. On the curcumin-modified surfaces, fibroblast proliferation was suppressed, and fibrous marker expressions as well as collagen synthesis were significantly reduced. These reductions were possibly because of the enhancement of fibroblast apoptosis induced by the surface curcumin. In contrast, no significant reduction in osteoblast functions was observed on the curcumin-modified substrates. These findings may provide a promising solution to reduce fibrous encapsulation, and thus may be highly beneficial for orthopaedic applications.

Implant anchorage remains a challenge, especially in porous osteoporotic bone with high osteoclast activity. The implant surface is modified with osteogenic molecules to stimulate osseointegration. Strontium (Sr) is known for its osteogenic and anti-osteoclastogenic effects. In this study, Sr was immobilized on a titanium (Ti) surface using bioinspired polyphenol tannic acid (pTAN) coating as an ad-layer (Ti-pTAN). Two separate coating techniques were employed for comparative analysis. In the first technique, Ti was coated with a tannic acid solution containing Sr (Ti-pTAN-1Stp). In the second method, Ti was first coated with pTAN, before being immersed in a SrCl₂ solution to immobilize Sr on Ti-pTAN (Ti-pTAN-2Stp). Ti-pTAN-1Stp and Ti-pTAN-2Stp augmented the alkaline phosphatase activity, collagen secretion, osteocalcin production and calcium deposition of MC3T3-E1 cells as compared to those of Ti and Ti-pTAN. However, osteoclast differentiation of RAW 264.7, as studied by TRAP activity, total DNA, and multinucleated cell formation, were decreased on Ti-pTAN, Ti-pTAN-1Stp and Ti-pTAN-2Stp as compared to Ti. Of all the substrates, osteoclast activity on Ti-pTAN-2Stp was the lowest. Hence, an economical and simple coating technique using pTAN as an adlayer preserved the dual biological effects of Sr. These results indicate a promising new approach to tailoring the cellular responses of implant surfaces.