Short-term administration of small molecule phenamil induced a protracted osteogenic effect on osteoblast-like MC3T3-E1 cells

Abstract

Sustained administration (21-day treatment) of the small molecule phenamil has been proposed as an alternative osteogenic factor when used in conjunction with a biodegradable scaffold for in vitro osteogenesis. While promising, the major issue associated with small molecules is non-specific cytotoxicity. The aim of this study was to minimize the side-effects from small-molecule drugs by reducing the frequency of administration. Toward this goal, we investigated whether a shorter phenamil treatment is sufficient to induce in vitro osteogenesis. We compared the effects of short-term (12 h) and continuous treatments of phenamil on osteoblastic differentiation and mineralization. Alkaline phosphatase (ALP) and osteopontin (OPN) activity were used as markers for osteoblastic differentiation. Measurement of the calcium content of the extracellular matrix was used as the hallmark for in vitro bone formation after 21 days of culture. Our findings revealed that both short and continuous phenamil treatment triggers osteoblastic differentiation and mineralization of MC3T3-E1 cells on a biodegradable polymeric scaffold composed of polylactic-co-glycolic acid (PLAGA) at the same time points. In addition, in order to fabricate a phenamil-loaded PLAGA scaffold, the small molecule phenamil was physically absorbed onto the surface of scaffolds and the bioactivity of the loaded scaffolds was evaluated. Furthermore, biochemical analysis indicated that short phenamil treatment of cells was accompanied by upregulation in protein expression of integrin α5, p125(FAK) and phosphorylation of CREB. These effects may contribute to the downstream signalling cascade necessary for osteogenesis, and such responses may account for our observed protracted osteogenic differentiation in vitro. Copyright © 2013 John Wiley & Sons, Ltd.

Delivery of Phenamil Enhances BMP-2-Induced Osteogenic Differentiation of Adipose-Derived Stem Cells and Bone Formation in Calvarial Defects

“…9 Phenamil is a derivative of the FDA-approved diuretic amiloride, an inhibitor of ion transporters. 14,19 A previous study reported that phenamil induced osteoblastic differentiation and mineralization in bone marrow-derived mesenchymal stem cells (MSCs).…”

“…14,[16][17][18][19] These results suggest that phenamil can work additively or synergistically with BMP-2 to increase bone regeneration.…”

“…[20][21][22][23] Our recent study demonstrated that controlled BMP-2 delivery can be achieved by apatite coating and BMP-2-loaded scaffolds enhance osteogenesis of adipose-derived stem cells (ASCs) in vitro and promote bone regeneration in rodent models. 24 Although previous studies have demonstrated that phenamil can induce osteogenic differentiation by enhancing BMP activity, and combined application of phenamil with BMP-2 can enhance osteogenesis in vitro, 14,[16][17][18][19] no single study reported the ability of phenamil to promote bone regeneration and how phenamil delivery influences BMP-2-mediated bone formation in vivo.…”

Enhanced Osteogenesis of Adipose-Derived Stem Cells by Regulating Bone Morphogenetic Protein Signaling Antagonists and Agonists

“…The small molecule phenamil, a derivative of the U.S. Food and Drug Administration-approved diuretic amiloride, is currently attracting attention in bone regeneration due to its osteogenic efficacy through the BMP–Smad signaling pathways [25]. Phenamil induces osteogenic differentiation in mouse bone marrow stromal or preosteoblastic cells and human dental pulp cells [26, 27]. We recently reported the osteogenic activity of phenamil in vitro and bone formation in vivo in a calvarial defect model, both alone and in combination with BMP-2 utilizing a reduced BMP-2 dose [28].…”

Delivery of small molecules for bone regenerative engineering: preclinical studies and potential clinical applications

“…In general, small molecules are small enough to penetrate nontarget cells and trigger unwanted signaling cascades [84]. Thus, the major concern associated with small-molecule therapeutics is their nonspecific adverse effects [10,11,85–87]. In addition, a lack of an effective delivery strategy for small molecules is another issue, given that one of the objectives of small molecule-based bone regenerative engineering is to develop an engineered scaffold system that provides adequate doses of the small molecule and/or acts as a structural support for infiltrating cells [11,17,60].…”