Silks are being reassessed as biomaterial scaffolds due to their unique mechanical properties, opportunities for genetic tailoring of structure and thus function, and recent studies clarifying biocompatibility. We report on the covalent decoration of silk films with integrin recognition sequences (RGD) as well as parathyroid hormone (PTH, 1-34 amino acids) and a modified PTH 1-34 (mPTH) involved in the induction of bone formation. Osteoblast-like cell (Saos-2) responses to the decorated silk films indicate that the proteins serve as suitable bone-inducing matrices. Osteoblast-like cell adhesion was significantly increased on RGD and PTH compared to plastic, mPTH, and the control peptide RAD. At 2 weeks of culture, message levels of alkaline phosphatase were similar on all substrates, but by 4 weeks, alkaline phosphatase mRNA was greatest on RGD. At 2 weeks of culture, alpha 1(I) procollagen mRNA was elevated on silk, RGD, RAD, and PTH, and hardly detectable on mPTH and plastic. However, by 4 weeks RGD demonstrated the highest level compared to the other substrates. Osteocalcin message levels detected by RT-PCR were greatest on RGD at both time points. Calcification was also significantly elevated on RGD compared to the other substrates with an increase in number and size of the mineralized nodules in culture. Thus, RGD covalently decorated silk appears to stimulate osteoblast-based mineralization in vitro.
Green fluorescent protein (GFP)-expressing transgenic mice were produced containing a 3.6-kilobase (kb; pOBCol3.6GFPtpz) and a 2.3-kb (pOBCol2.3GFPemd) rat type I collagen (Col1a1) promoter fragment. The 3.6-kb promoter directed strong expression of GFP messenger RNA (mRNA) to bone and isolated tail tendon and lower expression in nonosseous tissues.
Differentiation of human mesenchymal stem cells into osteoblasts is controlled by extracellular cues. Canonical Wnt signaling is particularly important for maintenance of bone mass in humans. Post-transcriptional regulation of gene expression, mediated by microRNAs, plays an essential role in the control of osteoblast differentiation. Here, we find that miR-29a is necessary for human osteoblast differentiation, and miR-29a is increased during differentiation in the mesenchymal precursor cell line hFOB1.19 and in primary cultures of human osteoblasts. Furthermore, the promoter of the expressed sequence tag containing the human miR-29a gene is induced by canonical Wnt signaling. This effect is mediated, at least in part, by two T-cell factor/LEF-binding sites within the proximal promoter. Furthermore, we show that the negative regulators of Wnt signaling, Dikkopf-1 (Dkk1), Kremen2, and secreted frizzled related protein 2 (sFRP2), are direct targets of miR-29a. Endogenous protein levels for these Wnt antagonists are increased in cells transfected with synthetic miR-29a inhibitor. In contrast, transfection with miR-29a mimic decreases expression of these antagonists and potentiates Wnt signaling. Overall, we demonstrate that miR-29 and Wnt signaling are involved in a regulatory circuit that can modulate osteoblast differentiation. Specifically, canonical Wnt signaling induces miR-29a transcription. The subsequent down-regulation of key Wnt signaling antagonists, Dkk1, Kremen2, and sFRP2, by miR-29a potentiates Wnt signaling, contributing to a gene expression program important for osteoblast differentiation. This novel regulatory circuit provides additional insight into how microRNAs interact with signaling molecules during osteoblast differentiation, allowing for fine-tuning of intricate cellular processes.
The in vivo expression of fibronectin, type I collagen, and several non-collagenous proteins was correlated with the development of bone in fetal and early neonatal rat calvariae. Fibronectin was the earliest matrix protein expressed in calvariae, with a peak expression in fetal 16- and 17-day (d) bones. Fibronectin expression coincided with the condensation of preosteoblasts prior to calcification and decreased once bone mineralization commenced. The expression of type I collagen, osteonectin, bone sialoprotein, and alkaline phosphatase mRNAs was found at 17 d. The increase in type I collagen mRNA levels was correlated with a 3.5-fold increase in calcium deposition at 19-20 d. Bone sialoprotein and alkaline phosphatase peaked on fetal 21 d while osteonectin remained at a low level and was localized to the osteoblast layer and the osteocyte lacunae. Osteopontin mRNA levels increased rapidly in neonatal calvariae. After birth, osteonectin and fibronectin were mainly associated with blood vessels. Thus, fibronectin is one of the earliest matrix proteins expressed in calvariae and is rapidly followed by type I collagen, bone sialoprotein, and alkaline phosphatase. Osteocalcin, osteonectin, and osteopontin mRNAs have similar patterns of expression in the developing fetal calvaria, and their synthesis coincided with mineralization.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
This site is protected by reCAPTCHA and the Google Privacy Policy and Terms of Service apply.
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.