Leucine rich amelogenin peptide prevents ovariectomy-induced bone loss in mice.

Amelogenins, major extra cellular matrix proteins of developing tooth enamel, are predominantly expressed by ameloblasts and play significant roles in the formation of enamel. Recently, amelogenin has been detected in various epithelial and mesenchymal tissues, implicating that it might have distinct functions in various tissues. We have previously reported that leucine rich amelogenin peptide (LRAP), one of the alternate splice forms of amelogenin, regulates receptor activator of NF-kappa B ligand (RANKL) expression in cementoblast/periodontal ligament cells, suggesting that the amelogenins, especially LRAP, might function as a signaling molecule in bone metabolism. The objective of this study was to identify and define LRAP functions in bone turnover. We engineered transgenic (TgLRAP) mice using a murine 2.3kb α1(I)-collagen promoter to drive expression of a transgene consisting of LRAP, an internal ribosome entry site (IRES) and enhanced green fluorescent protein (EGFP) to study functions of LRAP in bone formation and resorption. Calvarial cell cultures from the TgLRAP mice showed increased alkaline phosphatase (ALP) activity and increased formation of mineralized nodules compared to the cells derived from wild-type (WT) mice. The TgLRAP calvarial cells also showed an inhibitory effect on osteoclastogenesis in vitro. Gene expression comparison by quantitative polymerase chain reaction (Q-PCR) in calvarial cells indicated that bone formation makers such as Runx2, Alp, and osteocalcin were increased in TgLRAP compared to the WT cells. Meanwhile, Rankl expression was decreased in the TgLRAP cells in vitro. The ovariectomized (OVX) TgLRAP mice resisted bone loss induced by ovariectomy resulting in higher bone mineral density in comparison to OVX WT mice. The quantitative analysis of calcein intakes indicated that the ovariectomy resulted in increased bone formation in both WT and TgLRAP mice; OVX TgLRAP appeared to show the most remarkably increased bone formation. The parameters for bone resorption in tissue sections showed increased number of osteoclasts in OVX WT, but not in OVX TgLRAP over that of sham operated WT or TgLRAP mice, supporting the observed bone phenotypes in OVX mice. This is the first report identifying that LRAP, one of the amelogenin splice variants, affects bone turnover in vivo.

Design and preclinical evaluation of nanostars for the passive pretargeting of tumor tissue.

INTRODUCTION: Pretargeting strategies that do not rely on the expression of molecular targets have expanded imaging and therapy options for cancer patients. Nanostars with designed multivalency and which highly accumulate in tumor tissue via the enhanced permeability and retention (EPR) effect may therefore be the ideal vectors for the development of a passive pretargeting approach. METHODS: Nanostars were synthesized, consisting of 7-8 center-cross-linked arms that were modified with trans-cyclooctene (TCO) using poly(ethylene glycol) (PEG) linkers of 12 or 106 monomer units or without linker. The bioorthogonal click reaction with radiofluorinated 2,2'-(7-(2-(tetrazine-poly(ethyleneglycol)11-amino)-2-oxoethyl)-1,4,7-triazonane-1,4-diyl)diacetic acid ([18F]F-Tz-PEG11-NODA) or 2,2'-(7-(2-(tetrazine-amino)-2-oxoethyl)-1,4,7-triazonane-1,4-diyl)diacetic acid ([18F]F-Tz-NODA) was measured by ex vivo biodistribution studies and positron emission tomography (PET) in mice bearing tumors with high EPR characteristics. Bioorthogonal masking was performed using a tetrazine-functionalized dextran polymer (Tz-DP). RESULTS: Highest tumor accumulation of [18F]F-Tz-PEG11-NODA was observed for nanostars functionalized with TCO without linker, with a tumor uptake of 3.2 ± 0.4%ID/g and a tumor-to-muscle ratio of 12.8 ± 4.2, tumor-to-large intestine ratio of 0.5 ± 0.3 and tumor-to-kidney ratio of 2.0 ± 0.3, being significantly higher than for nanostars functionalized with TCO-PEG12 (P < 0.05) or TCO-PEG106 (P < 0.05). Tumor uptake and tumor-to-tissue ratios did not improve upon bioorthogonal masking with Tz-DP or when using a smaller, more lipophilic tetrazine([18F]F-Tz-NODA). CONCLUSIONS: A pretargeting strategy was developed based on the passive delivery of TCO-functionalized nanostars. Such a strategy would allow for the imaging and treatment of tumors with apparent EPR characteristics, with high radioactive tumor doses and minimal doses to off-target tissues.

Delivery of polymeric nanostars for molecular imaging and endoradiotherapy through the enhanced permeability and retention (EPR) effect.

Expression levels of biomarkers are generally unknown at initial diagnosis. The development of theranostic probes that do not rely on biomarker availability would expand therapy options for cancer patients, improve patient selection for nanomedicine and facilitate treatment of inoperable patients or patients with acquired therapy resistance. Herein, we report the development of star polymers, also known as nanostars, that allow for molecular imaging and/or endoradiotherapy based on passive targeting via the enhanced permeability and retention (EPR) effect. Methods: We synthesised a star copolymer, consisting of 7-8 centre-cross-linked arms that were modified with Gd3+ for magnetic resonance imaging (MRI), and functionalised either with 89Zr for in vivo quantification and positron emission tomography (PET) imaging, or with 177Lu for endoradiotherapy. 1H longitudinal relaxivities were determined over a continuum of magnetic field strengths ranging from 0.24 mT - 0.94 T at 37 °C (nuclear magnetic relaxation dispersion (NMRD) profile) and T1-weighted MRI contrast enhancement was visualized at 3 T and 7 T. PET imaging and ex vivo biodistribution studies were performed in mice bearing tumours with high EPR (CT26) or low EPR (BxPC3) characteristics. Therapy studies were performed in mice with high EPR tumours and mean absorbed organ doses were estimated for a standard human model. Results: The star copolymer with Gd3+ displayed a significantly superior contrast enhancement ability (T1 = 0.60 s) compared to the standard clinical contrast agent Gadovist (T1 = 1.0 s). Quantification of tumour accumulation using the radiolabelled nanostars in tumour-bearing mice demonstrated an exceptionally high uptake in tumours with high EPR characteristics (14.8 - 21.7 %ID/g). Uptake of the star polymers in tumours with low EPR characteristics was significantly lower (P<0.001), suggesting passive tumour accumulation of the nanostars via the EPR effect. Survival of mice treated with high dose 177Lu-labelled star polymers was significantly higher than survival of mice treated with lower therapy doses or control mice (P=0.001), demonstrating the utility of the 177Lu-labelled star polymers as platforms for endoradiotherapy. Conclusion: Our work highlights the potential of star polymers as probes for the molecular imaging of cancer tissue or for the passive delivery of radionuclides for endoradiotherapy. Their high functionalisability and high tumour accumulation emphasises their versatility as powerful tools for nanomedicine.

Surgical management of negative dysphotopsia.

PURPOSE: To evaluate curative and preventative surgical strategies for negative dysphotopsia. SETTING: Private practice, Los Angeles, California, USA. DESIGN: Retrospective case series. METHODS: Patients with self-reported chronic negative dysphotopsia had corrective surgery as the therapeutic group. Second eye surgery, in cases with negative dysphotopsia in the previously operated eye, comprised the preventative group. Chronologically, several surgical strategies were used, including bag-to-bag intraocular lens (IOL) exchange, reducing posterior chamber depth, piggyback secondary IOL placement, bag-to-sulcus IOL exchange, and reverse optic capture. The primary outcome measure was improvement of negative dysphotopsia by 3 months postoperatively. RESULTS: The therapeutic group comprised 40 eyes of 37 patients; 76.6% of causative IOLs were acrylic and 23.4% were silicone and all were bag-fixated. There were 21 eyes in the preventative group of which 11 were second eyes from the therapeutic group; the remaining 10 did not require surgery for the symptomatic eye. Successful outcomes for each surgical strategy were as follows: bag-to-bag IOL exchange (0/5), a reduction in posterior chamber depth with iris suture fixation of the bag-haptic complex (0/1), piggyback secondary IOL (8/11), secondary reverse optic capture (21/22), ciliary sulcus posterior chamber IOL exchange (7/8), and primary reverse optic capture (21/21). CONCLUSIONS: Negative dysphotopsia was associated with acrylic or silicone IOLs of either square- or round-edge design. Negative dysphotopsia was reduced, eliminated, or prevented when the IOL optic overlaid the anterior capsulotomy rather than when the capsule edge overlaid the optic. Bag-to-sulcus IOL exchange and reverse optic capture were highly successful in managing or preventing negative dysphotopsia.

Photoactivation of endogenous latent transforming growth factor-ß1 directs dental stem cell differentiation for regeneration.

Rapid advancements in the field of stem cell biology have led to many current efforts to exploit stem cells as therapeutic agents in regenerative medicine. However, current ex vivo cell manipulations common to most regenerative approaches create a variety of technical and regulatory hurdles to their clinical translation, and even simpler approaches that use exogenous factors to differentiate tissue-resident stem cells carry significant off-target side effects. We show that non-ionizing, low-power laser (LPL) treatment can instead be used as a minimally invasive tool to activate an endogenous latent growth factor complex, transforming growth factor-ß1 (TGF-ß1), that subsequently differentiates host stem cells to promote tissue regeneration. LPL treatment induced reactive oxygen species (ROS) in a dose-dependent manner, which, in turn, activated latent TGF-ß1 (LTGF-ß1) via a specific methionine residue (at position 253 on LAP). Laser-activated TGF-ß1 was capable of differentiating human dental stem cells in vitro. Further, an in vivo pulp capping model in rat teeth demonstrated significant increase in dentin regeneration after LPL treatment. These in vivo effects were abrogated in TGF-ß receptor II (TGF-ßRII) conditional knockout (DSPP(Cre)TGF-ßRII(fl/fl)) mice or when wild-type mice were given a TGF-ßRI inhibitor. These findings indicate a pivotal role for TGF-ß in mediating LPL-induced dental tissue regeneration. More broadly, this work outlines a mechanistic basis for harnessing resident stem cells with a light-activated endogenous cue for clinical regenerative applications.

TGF-ß regulates enamel mineralization and maturation through KLK4 expression.

Transforming growth factor-ß (TGF-ß) signaling plays an important role in regulating crucial biological processes such as cell proliferation, differentiation, apoptosis, and extracellular matrix remodeling. Many of these processes are also an integral part of amelogenesis. In order to delineate a precise role of TGF-ß signaling during amelogenesis, we developed a transgenic mouse line that harbors bovine amelogenin promoter-driven Cre recombinase, and bred this line with TGF-ß receptor II floxed mice to generate ameloblast-specific TGF-ß receptor II conditional knockout (cKO) mice. Histological analysis of the teeth at postnatal day 7 (P7) showed altered enamel matrix composition in the cKO mice as compared to the floxed mice that had enamel similar to the wild-type mice. The µCT and SEM analyses revealed decreased mineral content in the cKO enamel concomitant with increased attrition and thinner enamel crystallites. Although the mRNA levels remained unaltered, immunostaining revealed increased amelogenin, ameloblastin, and enamelin localization in the cKO enamel at the maturation stage. Interestingly, KLK4 mRNA levels were significantly reduced in the cKO teeth along with a slight increase in MMP-20 levels, suggesting that normal enamel maturation is regulated by TGF-ß signaling through the expression of KLK4. Thus, our study indicates that TGF-ß signaling plays an important role in ameloblast functions and enamel maturation.

A method for rapid demineralization of teeth and bones.

Tooth and bone specimen require extensive demineralization for careful analysis of cell morphology, as well as gene and protein expression levels. The LacZ gene, which encodes the ß-galactosidase enzyme, is often used as a reporter gene to study gene-structure function, tissue-specific expression by a promoter, cell lineage and fate. This reporter gene is particularly useful for analyzing the spatial and temporal gene expression pattern, by expressing the LacZ gene under the control of a promoter of interest. To analyze LacZ activity, and the expression of other genes and their protein products in teeth and bones, it is necessary to carry out a complete demineralization of the specimen before cutting sections. However, strong acids, such as formic acid used for tooth demineralization, destroy the activities of enzymes including those of ß-galactosidase. Therefore, most protocols currently use mild acids such as 0.1 M ethylene diamine tetra-acetic acid (EDTA) for demineralization of tooth and bone specimen, which require a longer period of treatment for complete demineralization. A method by which hard tissue specimens such as teeth and bones can be rapidly, but gently, decalcified is necessary to save time and effort. Here, we report a suitable method for rapid demineralization of mouse teeth in 0.1M EDTA at 42˚C without any loss of ß-galactosidase activity.

Dentin sialoprotein and dentin phosphoprotein have distinct roles in dentin mineralization.

Dentin sialophosphoprotein (DSPP), a major non-collagenous matrix protein of odontoblasts, is proteolytically cleaved into dentin sialoprotein (DSP) and dentin phosphoprotein (DPP). Our previous studies revealed that DSPP null mice display a phenotype similar to human autosomal dominant dentinogenesis imperfecta, in which teeth have widened predentin and irregular dentin mineralization resulting in sporadic unmineralized areas in dentin and frequent pulp exposure. Earlier in vitro studies suggested that DPP, but not DSP, plays a significant role in initiation and maturation of dentin mineralization. However, the precise in vivo roles of DSP and DPP are far from clear. Here we report the generation of DPPcKO mice, in which only DSP is expressed in a DSPP null background, resulting in a conditional DPP knockout. DPPcKO teeth show a partial rescue of the DSPP null phenotype with the restored predentin width, an absence of irregular unmineralized areas in dentin, and less frequent pulp exposure. Micro-computed tomography (micro-CT) analysis of DPPcKO molars further confirmed this partial rescue with a significant recovery in the dentin volume, but not in the dentin mineral density. These results indicate distinct roles of DSP and DPP in dentin mineralization, with DSP regulating initiation of dentin mineralization, and DPP being involved in the maturation of mineralized dentin.