Gloria Gronowicz

Functionalized silk-based biomaterials for bone formation

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.

Use of Type I Collagen Green Fluorescent Protein Transgenes to Identify Subpopulations of Cells at Different Stages of the Osteoblast Lineage

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. The 2.3‐kb promoter expressed the GFP mRNA in the bone and tail tendon with no detectable mRNA elsewhere. The pattern of fluorescence was evaluated in differentiating calvarial cell (mouse calvarial osteoblast cell [mCOB]) and in marrow stromal cell (MSC) cultures derived from the transgenic mice. The pOBCol3.6GFPtpz‐positive cells first appeared in spindle‐shaped cells before nodule formation and continued to show a strong signal in cells associated with bone nodules. pOBCol2.3GFPemd fluorescence first appeared in nodules undergoing mineralization. Histological analysis showed weaker pOBCol3.6GFPtpz‐positive fibroblastic cells in the periosteal layer and strongly positive osteoblastic cells lining endosteal and trabecular surfaces. In contrast, a pOBCol2.3GFPemd signal was limited to osteoblasts and osteocytes without detectable signal in periosteal fibroblasts. These findings suggest that Col1a1GFP transgenes are marking different subpopulations of cells during differentiation of skeletal osteoprogenitors. With the use of other promoters and color isomers of GFP, it should be possible to develop experimental protocols that can reflect the heterogeneity of cell differentiation in intact bone. In primary culture, this approach will afford isolation of subpopulations of these cells for molecular and cellular analysis.

miR-29 modulates WNT signaling in human osteoblasts through a positive feedback loop

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.

Estrogen Prevents Glucocorticoid-Induced Apoptosis in Osteoblasts in Vivo and in Vitro*

The ability of estrogen to prevent glucocorticoid-induced apoptosis in osteoblasts was studied both in vitro and in vivo. Glucocorticoid treatment for 72 h produced a dose-dependent increase in the number of apoptotic cells, determined by acridine orange/ethidium bromide staining, with a maximal response of 31 ± 2% and 26 ± 3% with 100 nm corticosterone in primary rat and mouse osteoblasts, respectively. Simultaneous administration of varying concentrations of 17β-estradiol and 100 nm corticosterone decreased apoptotic osteoblasts in a dose-dependent manner, with a maximal decrease of 70% with 0.01 nm 17β-estradiol. Terminal deoxynucleotidyltransferase-mediated deoxy-UTP-biotin nick end labeling also demonstrated glucocorticoid-induced DNA fragmentation that was inhibited by estrogen. Estrogen was shown to inhibit apoptosis induced by lipopolysaccharide treatment. As early as 6 h, Western blots demonstrated a dose-dependent decrease in the Bcl-2/Bax ratio, which reached a minimum of 0.18 in osteoblasts tr...

MINERALIZATION AND THE EXPRESSION OF MATRIX PROTEINS DURING IN VIVO BONE DEVELOPMENT

Abstract. 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.

Primary hyperparathyroidism caused by parathyroid-targeted overexpression of cyclin D1 in transgenic mice

The relationship between abnormal cell proliferation and aberrant control of hormonal secretion is a fundamental and poorly understood issue in endocrine cell neoplasia. Transgenic mice with parathyroid-targeted overexpression of the cyclin D1 oncogene, modeling a gene rearrangement found in human tumors, were created to determine whether a primary defect in this cell-cycle regulator can cause an abnormal relationship between serum calcium and parathyroid hormone response, as is typical of human primary hyperparathyroidism. We also sought to develop an animal model of hyperparathyroidism and to examine directly cyclin D1s role in parathyroid tumorigenesis. Parathyroid hormone gene regulatory region--cyclin D1 (PTH--cyclin D1) mice not only developed abnormal parathyroid cell proliferation, but also developed chronic biochemical hyperparathyroidism with characteristic abnormalities in bone and, notably, a shift in the relationship between serum calcium and PTH. Thus, this animal model of human primary hyperparathyroidism provides direct experimental evidence that overexpression of the cyclin D1 oncogene can drive excessive parathyroid cell proliferation and that this proliferative defect need not occur solely as a downstream consequence of a defect in parathyroid hormone secretory control by serum calcium, as had been hypothesized. Instead, primary deregulation of cell-growth pathways can cause both the hypercellularity and abnormal control of hormonal secretion that are almost inevitably linked together in this common disorder.

Human Parathyroid Hormone 1–34 Reverses Bone Loss in Ovariectomized Mice

The experimental work characterizing the anabolic effect of parathyroid hormone (PTH) in bone has been performed in nonmurine ovariectomized (OVX) animals, mainly rats. A major drawback of these animal models is their inaccessibility to genetic manipulations such as gene knockout and overexpression. Therefore, this study on PTH anabolic activity was carried out in OVX mice that can be manipulated genetically in future studies. Adult Swiss‐Webster mice were OVX, and after the fifth postoperative week were treated intermittently with human PTH(1–34) [hPTH(1–34)] or vehicle for 4 weeks. Femoral bones were evaluated by microcomputed tomography (μCT) followed by histomorphometry. A tight correlation was observed between trabecular density (BV/TV) determinations made by both methods. The BV/TV showed >60% loss in the distal metaphysis in 5‐week and 9‐week post‐OVX, non‐PTH‐treated animals. PTH induced a ∼35% recovery of this loss and a ∼40% reversal of the associated decreases in trabecular number (Tb.N) and connectivity. PTH also caused a shift from single to double calcein‐labeled trabecular surfaces, a significant enhancement in the mineralizing perimeter and a respective 2‐ and 3‐fold stimulation of the mineral appositional rate (MAR) and bone formation rate (BFR). Diaphyseal endosteal cortical MAR and thickness also were increased with a high correlation between these parameters. These data show that OVX osteoporotic mice respond to PTH by increased osteoblast activity and the consequent restoration of trabecular network. The Swiss‐Webster mouse model will be useful in future studies investigating molecular mechanisms involved in the pathogenesis and treatment of osteoporosis, including the mechanisms of action of known and future bone antiresorptive and anabolic agents.

The in vitro response of human osteoblasts to polyetheretherketone (PEEK) substrates compared to commercially pure titanium

Polyetheretherketone (PEEK) is used as an alternative to titanium in medical devices. Previous in vitro studies examining PEEK have differed in their choice of polymer variant [PEEK or carbon-fiber reinforced PEEK (CFR-PEEK)], source of polymer (some of which are no longer available or for implantation) and cell type. While all studies demonstrated favorable cytocompatibility of the PEEK material, no studies are available which reflect the current state of the art of the material. Here, we use different forms of the only implantable grade PEEK available. These are compared with commercially pure titanium (cpTi) Grade 1 using a human primary osteoblast model. Sample materials were presented as industrially relevant surfaces. Machined or injection molded PEEK and CFR-PEEK were evaluated along with polished (Ra=0.200microm) and rough (Ra=0.554microm) cpTi. Osteoblast adhesion at 4h on injection molded variants of PEEK (Ra=0.095microm) and CFR-PEEK (Ra=0.350microm) material was comparable to titanium. Machined variants of PEEK (Ra=0.902microm) and CFR-PEEK (Ra=1.106microm) materials were significantly less. Proliferation at 48h determined by [(3)H]-thymidine incorporation was the greatest on the smoothest of all materials, the injection molded unfilled PEEK, which was significantly higher than the rough titanium control. The machined unfilled PEEK had the lowest DNA synthesis. RT-PCR for alkaline phosphatase, Type I collagen and osteocalcin normalized to glyceraldehyde-3-phosphate dehydrogenase revealed different patterns of mRNA levels. High mRNA levels for Type I collagen showed that CFR-PEEK stimulated osteoblast differentiation, whilst injection molded unfilled PEEK was less differentiated. Machined unfilled PEEK had comparable message levels of bone matrix proteins as rough titanium. All material variants permitted a degree of mineralization. Scanning electron microscopy at 3 days and 2 weeks in differentiation medium showed that human osteoblasts were well spread on all the different substrates. The varied response reported here at different time points during the study suggests that material formulation (unfilled PEEK or CFR-PEEK), subjection to industrial processing, surface roughness and topography may all influence the cellular response of osteoblasts to PEEK. Thus, differences in human osteoblast responses were found to the various samples of PEEK, but implantable grade PEEK, in general, was comparable in vitro to the bone forming capacity of rough titanium.

An in vitro model for mineralization of human osteoblast-like cells on implant materials.

An in vitro mineralizing cell-implant system was developed to study osteoblast attachment, secretion of extracellular (ECM) matrix proteins and mineralization. Saos-2 cells were plated on Tivanium (Tiv, Ti-6A1-4V), Zimaloy (Zim, Co-Cr-Mo) and glass disks. The cells were cultured in alpha-MEM medium with 10% fetal bovine serum and 50 microg ml(-1) ascorbic acid. The cultures were analyzed for calcification and for mRNA expression for ECM proteins after 1, 2, 4 and 6 weeks. Calcium content was significantly higher in cells on Tiv, less on Zim and least on glass disks. With the addition of 3 mm beta-glycerophosphate (beta-GP), the cell layer was more calcified on Zim than on Tiv and all substrates had three times more calcium than cultures without beta-GP. All subsequent experiments were performed without beta-GP. Phalloidin immunofluorescence microscopy of the actin-based cytoskeleton at 2 weeks demonstrated nodules composed of multilayered, cobblestone-appearing osteoblasts overlying calcified matrix which was stained with calcein. On Tiv, calcified nodules were connected in a trabecular-like pattern while on Zim, calcification was dispersed throughout the cell layer. Northern blots for alkaline phosphatase, bone sialoprotein, osteocalcin and alpha1(I) procollagen mRNAs were performed at different time points. The amount and pattern of calcification as well as the expression of ECM-mRNAs differed on each implant material. The results indicate that Tiv stimulates the production of more ECM proteins and mineralized matrix than Zim or glass in this osteoblast-like cell/implant culture.

Transcriptional induction of prostaglandin G/H synthase-2 by basic fibroblast growth factor.

In serum-free mouse osteoblastic MC3T3-E1 cells, basic fibroblastic growth factor (bFGF) induced mRNA and protein for prostaglandin G/H synthase-2 (PGHS-2), the major enzyme in arachidonic acid (AA) conversion to prostaglandins. mRNA accumulation peaked at 1 h with bFGF 1 nM. In cells stably transfected with a 371-bp PGHS-2 promoter-luciferase reporter, bFGF stimulated luciferase activity, which peaked at 2-3 h with bFGF 1-10 nM. In the presence of exogenous AA, bFGF stimulated PGE2 production, which paralleled luciferase activity. In serum-free neonatal mouse calvarial cultures, bFGF stimulated PGE2 production in the absence of exogenous AA. bFGF stimulated PGHS-2 mRNA accumulation, which peaked at 2-4 h and then decreased; there were later mRNA elevations at 48 and 96 h that were inhibited by indomethacin. In both MC3T3-E1 cells and neonatal calvariae, bFGF produced smaller and slower increases in PGHS-1 mRNA levels than for PGHS-2. bFGF stimulated bone resorption in mouse calvariae with a maximal increase of 80% at 1 nM. Stimulation was partially inhibited by nonsteroidal anti-inflammatory drugs. We conclude that bFGF rapidly stimulates PGE2 production in osteoblasts, largely through transcriptional regulation of PGHS-2, and that prostaglandins mediate some of bFGFs effects on bone resorption.