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Objectives The lack of durability in resin–dentine bonds led to the use of chlorhexidine as MMP-inhibitor to prevent the degradation of hybrid layers. Biomimetic remineralisation is a concept-proven approach in preventing the degradation of resin–dentine bonds. The purpose of this study is to examine the integrity of aged resin–dentine interfaces created with a nanofiller-containing etch-and-rinse adhesive after the application of these two approaches. Methods The more established MMP-inhibition approach was examined using a parallel in vivo and in vitro ageing design to facilitate comparison with the biomimetic remineralisation approach using an in vitro ageing design. Specimens bonded without chlorhexidine exhibited extensive degradation of the hybrid layer after 12 months of in vivo ageing. Results Dissolution of nanofillers could be seen within a water-rich zone within the adhesive layer. Although specimens bonded with chlorhexidine exhibited intact hybrid layers, water-rich regions remained in those hybrid layers and degradation of nanofillers occurred within the adhesive layer. Specimens subjected to in vitro biomimetic remineralisation followed by in vitro ageing demonstrated intrafibrillar collagen remineralisation within hybrid layers and deposition of mineral nanocrystals in nanovoids within the adhesive. Conclusions The impact was realized by understanding the lack of an inherent mechanism to remove water from resin–dentine interfaces as the critical barrier to progress in bonding with the etch-and-rinse technique. The experimental biomimetic remineralisation strategy offers a creative solution for incorporating a progressive hydration mechanism to achieve this goal, which warrants its translation into a clinically applicable technique.

Fluoride-releasing restorative materials are available for remineralization of enamel and root caries. However, dentin remineralization is more difficult than enamel remineralization due to the paucity of apatite seed crystallites along the lesion surface for heterogeneous crystal growth. Extracellular matrix proteins play critical roles in controlling apatite nucleation/growth in collagenous tissues. This study examined the remineralization efficacy of mineral trioxide aggregate (MTA) in phosphate-containing simulated body fluid (SBF) by incorporating polyacrylic acid and sodium tripolyphosphate as biomimetic analogs of matrix proteins for remineralizing caries-like dentin. Artificial caries-like dentin lesions incubated in SBF were remineralized over a 6-week period using MTA or MTA containing biomimetic analogs in the absence or presence of dentin adhesive application. Lesion depths and integrated mineral loss were monitored with micro-computed tomography. Ultrastructure of baseline and remineralized lesions were examined by transmission electron microscopy. Dentin remineralization was best achieved using MTA containing biomimetic analogs regardless of whether an adhesive was applied; dentinal tubules within the remineralized dentin were occluded by apatite. It is concluded that the MTA version employed in the study may be doped with biomimetic analogs for remineralization of unbonded and bonded artificial caries-like lesions in the presence of SBF.

Homology between p63 and p53 has suggested that these proteins might function similarly. However, the majority of data from human tumors have not supported a similar role for p63 in tumor suppression. To investigate this issue, we studied spontaneous tumorigenesis in p63؉͞؊ mice in both WT and p53-compromised backgrounds. We found that p63؉͞؊ mice were not tumor prone and mice heterozygous for both p63 and p53 had fewer tumors than p53؉͞؊ mice. The rare tumors that developed in mice with compromised p63 were also distinct from those of p53؉͞؊ mice. Furthermore, p63؉͞؊ mice were not prone to chemically induced tumorigenesis, and p63 expression was maintained in carcinomas. These findings demonstrate that, in agreement with data from human tumors, p63 plays a markedly different biological role in cancer than p53.mouse model ͉ p53 ͉ tumor suppressor ͉ cancer T he p63 protein is a member of the p53 family that includes p53, p63, and p73, which share extensive homology within the core DNA binding domain (DBD) (reviewed in ref. 1). A well established feature of p53 is that it is frequently lost or inactivated in a variety of human cancers (2). Although some evidence suggests that p63 may perform biological roles analogous to those of p53 (3, 4), p63 is rarely mutated, and p63 expression is often retained in human tumors (reviewed in ref. 5). Although mutations in human p63 have been identified, these mutations cause severe birth anomalies, and tumor susceptibility is not a typical feature of these syndromes (1). In contrast to p53's ubiquitous pattern of expression, p63 is most readily detectable in proliferating cells of stratified squamous epithelia such as the skin, the precursor cell type for the majority of human cancers. Interactions between p63 and p53 and subsequent degradation of p63 can occur when p53 contains tumorderived missense mutations, suggesting that p63 could affect the tumor-suppressive capabilities of p53 (6). Thus, p63's role in modulating tumor-suppressive mechanisms in vivo has not been fully evaluated.To investigate whether p63 functions as a tumor suppressor in the intact organism, we evaluated spontaneous and chemically induced tumorigenesis in mice with decreased levels of p63. In addition, spontaneous tumors were monitored in mice that had reduced levels of p63 in combination with compromised levels of p53 to assess whether combined p63 and p53 deficiency affects tumorigenesis in vivo.Results p63؉͞؊ Mice Are Not Predisposed to Spontaneous Tumors. p53Ϫ͞Ϫ and p53ϩ͞Ϫ mice are highly tumor-prone with the majority of mice developing spontaneous tumors by 10 months and 2 years, respectively (7,8). To determine the impact of compromised p63 on tumorigenesis, we performed a spontaneous tumor study of 153 p63ϩ͞Ϫ mice and sibling controls for Ͼ2 years. Mice lacking p63 die shortly after birth (9, 10), and therefore the effect of germ-line deficiency of p63 in cancer could not be evaluated in p63Ϫ͞Ϫ mice. We had previously disrupted the endogenous p63 locus and generated two distinct p63 null alleles...

p63 is a member of the p53 gene family that is essential for the development of ectodermally derived structures whose morphogenesis is dependent on ectodermal-mesenchymal interactions. Stratified epithelia fail to differentiate and hair follicles, teeth, sweat glands, and mammary primordia are not formed in mice lacking p63 Yang et al., 1999). Although p63-deficient models have been extremely informative for assessing the in vivo role of p63 and for illuminating p63 as a candidate in several human developmental syndromes (Celli et al., 1999;Ianakiev et al., 2000;McGrath et al., 2001), the perinatal lethality of mice that are homozygous for p63 null alleles limits the utility of these models for examining the function of p63 at later stages. We have therefore generated a conditional p63 allele that has an essential region of the gene flanked by loxP sites and tested this allele by crossing it to a strain that expresses Cre during early embryogenesis. Cre excision is efficient and inactivates p63, leading to a phenotype that is indistinguishable from p63 nullizygous mice.The p63 floxed allele (p63 floxN ) was generated by embryonic stem (ES) cell technology using a replacement vector that exchanged the endogenous p63 gene with a modified version that had loxP sites flanking exons encoding the DNA binding domain (DBD) (Schmale and Bamberger 1997;Osada et al., 1998;Yang et al., 1998) (Fig. 1A). Recombinant clones were identified by Southern analysis and the functionality of the integrated loxP sites was assessed by introducing a Creexpressing plasmid into ϩ/p63 floxN ES cells. G418 sibselection was used to assess the presence or absence of the neomycin resistance cassette and Southern analysis using a probe specific for exon 5 was used to identify clones that contained exons encoding the DBD. Clones falling into each of the three possible classes of recombination products were obtained, indicating that all three loxP sites that had been introduced during the gene targeting step were functional.Mice carrying the p63 floxN allele were generated by standard procedures. ϩ/p63 floxN mice were intercrossed to generate p63 floxN /p63 floxN progeny (Fig. 1B). Mice homozygous for the p63 floxN allele were obtained at the expected frequency, were viable and fertile, and were phenotypically indistinguishable from wild-type siblings (Fig. 1C), indicating that introduction of loxP sites and the PGKNeobpA cassette at this site does not perturb the function of the p63 locus. (Bradley, 1987) and mice carrying the floxed allele were generated by crossing chimeras to C57BL/6J-Tyr c-Brd females. ϩ/p63 floxN mice were identified by Southern analysis of BamHI digested DNA using the flanking probe shown in A (solid bar). Wild-type (ϩ/ϩ), ϩ/p63 floxN (fn/ϩ), and p63 floxN /p63 floxN (fn/fn) mice were identified by the presence of endogenous (E) or targeted (T) p63 alleles of 13.3kb and 4.3kb, respectively. C: Dorsal backskin from wild-type and p63 floxN /p63 floxN mice was subjected to single (upper panel) or double (lower two panels) im...