Use of Polyphenol Tannic Acid to Functionalize Titanium with Strontium for Enhancement of Osteoblast Differentiation and Reduction of Osteoclast Activity.

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.

Bioinspired polydopamine and polyphenol tannic acid functionalized titanium suppress osteoclast differentiation: a facile and efficient strategy to regulate osteoclast activity at bone-implant interface.

Osseointegration of metallic implants in porous osteoporotic bone remains a challenge. Surface modification of implants to reduce peri-implant osteoclastic bone resorption was explored in the study. Bioinspired polydopamine (pDOP) and polyphenol tannic acid (pTAN) are nature-derived universal coating systems that have emerged either as a sole coating or ad-layer for biomolecular conjugation on different biomaterials. The effects pDOP and pTAN on osteoclast development have not been reported before. In this study, osteoclast development was investigated on titanium (Ti) substrates coated with pDOP (Ti-pDOP) and pTAN (Ti-pTAN). The results showed that Ti-pDOP and Ti-pTAN coating reduced tartrate-resistant acid phosphatase activity and osteoclast cell number as compared with pristine Ti. Intriguingly, the reduction was higher on Ti-pTAN than on Ti-pDOP. Economical and biocompatible tannic acid serves as a superior coating in decreasing osteoclast activity when compared with that of pDOP coating and could be used to modulate osteoclast activity at bone-implant interfaces.

Beta-cyclodextrin modified mesoporous bioactive glass nanoparticles/silk fibroin hybrid nanofibers as an implantable estradiol delivery system for the potential treatment of osteoporosis.

Osteoporosis, a systemic skeletal disease prevalent in elderly women, is associated with post-menopausal estrogen deficiency. Although systemic administration of exogenous estradiol (E2) reduced fragility fractures, the treatment has adverse effects. Localized delivery technologies of E2 could be utilized to circumvent the systemic adverse effects of systemic administration. In this study, a localized E2 delivery system is developed. Mesoporous bioactive glass nanoparticles (MBGNPs) with inherent osteogenic properties are modified with ß-cyclodextrin (CD-MBGNPs) to enhance their affinity for E2. To ensure mechanical stability and integrity, E2 loaded CD-MBGNPs are further electrospun with silk fibroin (SF) to produce a nanofibrous mesh (E2@CD-MBGNPs/SF). The incorporation of MBGNPs in SF enhances in vitro apatite formation and sustains the constant release of E2. Moreover, osteoblast proliferation and differentiation markers such as alkaline phosphatase activity, collagen 1 and osteocalcin expression of MC3T3-E1 are augmented in CD-MBGNPs/SF and E2@CD-MBGNPs/SF as compared to SF nanofibers. On the other hand, osteoclast DNA, tartrate resistant acid phosphatase activity and multinucleated cell formation are reduced in E2@CD-MBGNPs/SF as compared to CD-MBGNPs/SF and SF. Hence the presence of CD-MBGNPs in SF stimulates osteoblast function whereas E2 incorporation in CD-MBGNPs/SF reduces osteoclast activity. This is the first report to develop CD-MBGNPs/SF as a localized delivery system for hydrophobic molecules such as estradiol to treat osteoporosis.

Development of mesoporous bioactive glass nanoparticles and its use in bone tissue engineering.

The incidence of bone disorders, from trauma, tissue degeneration due to ageing, pathological conditions to cancer, has been increasing. The pursuit for bone graft substitutes to assist in regenerating large bone defects is ever growing as a result of the shortage in conventional autografts and allografts, in addition to the associated risks of disease transmission. However, the use of alloplastic biomaterials is limited in clinical settings, as further investigations are required to address the properties of synthetic grafts to mimic the native bone tissue and deliver desirable biomolecules to facilitate bone regeneration. This review discusses the fundamental structure and properties of bone with the emphasis on organic and inorganic components that are important for the biomaterial design. The main focus will be on the advancement and usage of bioactive glass (BG) for bone tissue engineering due to its similarity to the natural inorganic constituent of bone. The various BG synthetic processes, modifications of composition, as well as the biomolecule delivery will be discussed in great detail. As the properties of BG are tuneable according to clinical needs, it creates a new paradigm in addition to displaying its superior potential for bone tissue engineering and translational medicine in the field of orthopedic surgery. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 2878-2887, 2018.

Modulation of Osteoclast Interactions with Orthopaedic Biomaterials.

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.

Estradiol-Loaded Poly(ε-caprolactone)/Silk Fibroin Electrospun Microfibers Decrease Osteoclast Activity and Retain Osteoblast Function.

Estrogen, a steroid hormone, plays an important role in modulating osteoclast proliferation and development. Estrogen deficiency boosts osteoclast activity, leading to osteoporosis in elderly women. In this study, 17-ß estradiol (E2)-loaded poly(ε-caprolactone) (PCL)/silk fibroin (SF) electrospun microfibers were developed as a proposed localized E2 delivery system to treat osteoporotic fractures. PCL is a synthetic polymer known for its biocompatibility and excellent mechanical properties. The bioactivity of PCL was enhanced by mixing it with a natural SF polymer that has low immunogenicity and inherent bioactivity. Different ratios of PCL/SF blends were electrospun and characterized by scanning electron microscopy, Fourier transform infrared spectroscopy, and water contact angle measurement. PCL and SF at a ratio of 50:50 (PCL50/SF50) augmented cell proliferation of murine preosteoblast MC3T3-E1 cells and murine preosteoclast RAW 264.7 cells. Hence, PCL50/SF50 was selected and mixed with three concentrations of E2 to produce electrospun fiber mesh (0.1% E2@PCL/SF, 1% E2@PCL/SF, and 5% E2@PCL/SF). Sustained release of E2 was obtained for about 3 weeks at higher E2 concentration 5% E2@PCL/SF. An E2-loaded PCL50/SF50 elecrospun microfiber (1% E2@PCL/SF and 5% E2@PCL/SF) reduced tartrate-resistant acid phosphate activity, total DNA, and multinucleated cell formation of osteoclasts. On the other hand, the alkaline phosphatase activity and collagen I expression of osteoblasts were retained on all E2-loaded electrospun microfibers. The E2@PCL/SF system shows potential to be used for localized E2 delivery for the treatment of osteoporotic fractures.

In Vitro Findings of Titanium Functionalized with Estradiol via Polydopamine Adlayer.

To improve orthopedic implant fixation and reduce post-operative complications, osteogenic molecules are delivered locally by immobilizing them on the surface of implants, which will modulate the biology of cell attachment and differentiation on the implant surface. Estradiol, a natural steroid hormone, maintains bone metabolism by decreasing bone resorption. It either directly or indirectly affects osteoclasts. In this work, estradiol was immobilized on a titanium surface by polydopamine adlayer. Immobilization of estradiol was confirmed by X-ray electron spectroscopy (XPS), immunofluorescence staining and enzyme-linked immunosorbent assay (ELISA). Estradiol-modified substrates enhanced alkaline phosphatases activity (ALP) and calcium deposition of osteoblasts. However, these substrates did not decrease tartrate-resistant acid phosphatase (TRAP) activity and actin ring formation of the osteoclast. The scanning electron microscopic (SEM) images of estradiol-modified substrates showed the formation of estradiol crystals, which decreased the potency of immobilized estradiol. Despite having a successful immobilization of estradiol via the polydopamine technique, the bioavailability and potency of coated estradiol is reduced due to crystallization, suggesting that this is not a suitable system for localized estradiol delivery as tested in vitro here. Consequently, other suitable platforms have to be explored for immobilizing estradiol that will prevent crystal formation while preserving the biological activity.

Esculetin induces antiproliferative and apoptotic response in pancreatic cancer cells by directly binding to KEAP1.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Surface modification of titanium with curcumin: a promising strategy to combat fibrous encapsulation.

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.