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Delivery of small molecules and drugs to tissues is a mainstay of several tissue engineering strategies. Next generation treatments focused on localized drug delivery offer a more effective means in dealing with refractory healing when compared to systemic approaches. Here we describe a novel multidomain peptide hydrogel that capitalizes on synthetic peptide chemistry, supramolecular self-assembly and cytokine delivery to tailor biological responses. This material is biomimetic, shows shear stress recovery and offers a nanofibrous matrix that sequesters cytokines. The biphasic pattern of cytokine release results in the spatio-temporal activation of THP-1 monocytes and macrophages. Furthermore, macrophage–material interactions are promoted without generation of a proinflammatory environment. Subcutaneous implantation of injectable scaffolds showed a marked increase in macrophage infiltration and polarization dictated by cytokine loading as early as 3 days, with complete scaffold resorption by day 14. Macrophage interaction and response to the peptide composite facilitated the (i) recruitment of monocytes/macrophages, (ii) sustained residence of immune cells until degradation, and (iii) promotion of a pro-resolution M2 environment. Our results suggest the potential use of this injectable cytokine loaded hydrogel scaffold in a variety of tissue engineering applications.

In dentistry, the maintenance of a vital dental pulp is of paramount importance because teeth devitalized by root canal treatment may become more brittle and prone to structural failure over time. Advanced carious lesions can irreversibly damage the dental pulp by propagating a sustained inflammatory response throughout the tissue. Although the inflammatory response initially drives tissue repair, sustained inflammation has an enormously destructive effect on the vital pulp, eventually leading to total necrosis of the tissue and necessitating its removal. The implications of tooth devitalization have driven significant interest in the development of bioactive materials that facilitate the regeneration of damaged pulp tissues by harnessing the capacity of the dental pulp for self-repair. In considering the process by which pulpitis drives tissue destruction, it is clear that an important step in supporting the regeneration of pulpal tissues is the attenuation of inflammation. Macrophages, key mediators of the immune response, may play a critical role in the resolution of pulpitis because of their ability to switch to a proresolution phenotype. This process can be driven by the resolvins, a family of molecules derived from fatty acids that show great promise as therapeutic agents. In this review, we outline the importance of preserving the capacity of the dental pulp to self-repair through the rapid attenuation of inflammation. Potential treatment modalities, such as shifting macrophages to a proresolving phenotype with resolvins are described, and a range of materials known to support the regeneration of dental pulp are presented.

Understanding the cellular and molecular mechanisms underlying the self-renewal and differentiation of dental epithelial stem cells (DESCs) that support the unlimited growth potential of mouse incisors is critical for developing novel tooth regenerative therapies and unraveling the pathogenesis of odontogenic tumors. However, analysis of DESC properties and regulation has been limited by the lack of an in vitro assay system and well-documented DESC markers. Here, we describe an in vitro sphere culture system to isolate the DESCs from postnatal mouse incisor cervical loops (CLs) where the DESCs are thought to reside. The dissociated cells from CLs were able to expand and form spheres for multiple generations in the culture system. Lineage tracing indicated that DESC within the spheres were epithelial in origin as evident by lineage tracing. Upon stimulation, the sphere cells differentiated into cytokeratin 14- and amelogenin-expressing and mineral material-producing cells. Compared to the CL tissue, sphere cells expressed high levels of expression of Sca-1, CD49f (also designated as integrin α6), and CD44. Fluorescence-activated cell sorting (FACS) analyses of mouse incisor CL cells further showed that the CD49f(Bright) population was enriched in sphere-forming cells. In addition, the CD49f(Bright) population includes both slow-cycling and Lgr5(+) DESCs. The in vitro sphere culture system and identification of CD49f(Bright) as a DESC marker provide a novel platform for enriching DESCs, interrogating how maintenance, cell fate determination, and differentiation of DESCs are regulated, and developing tooth regenerative therapies.

Background: Understanding of the self-renewal and differentiation of dental epithelial stem cells (DESCs) is important for tooth regeneration therapies. Results: Depletion of FGF signaling suppressed self-renewal and led to differentiation of DESCs. Conclusion: FGF signaling is essential for maintenance of DESCs. Significance: The finding sheds new light on the mechanism by which the homeostasis, expansion, and differentiation of DESCs are regulated. A constant supply of epithelial cells from dental epithelial stem cell (DESC) niches in the cervical loop (CL) enables mouse incisors to grow continuously throughout life. Elucidation of the cellular and molecular mechanisms underlying this unlimited growth potential is of broad interest for tooth regenerative therapies. Fibroblast growth factor (FGF) signaling is essential for the development of mouse incisors and for maintenance of the CL during prenatal development. However, how FGF signaling in DESCs controls the self-renewal and differentiation of the cells is not well understood. Herein, we report that FGF signaling is essential for self-renewal and the prevention of cell differentiation of DESCs in the CL as well as in DESC spheres. Inhibiting the FGF signaling pathway decreased proliferation and increased apoptosis of the cells in DESC spheres. Suppressing FGFR or its downstream signal transduction pathways diminished Lgr5-expressing cells in the CL and promoted cell differentiation both in DESC spheres and the CL. Furthermore, disruption of the FGF pathway abrogated Wnt signaling to promote Lgr5 expression in DESCs both in vitro and in vivo. This study sheds new light on understanding the mechanism by which the homeostasis, expansion, and differentiation of DESCs are regulated.

Implantable devices have improved the lives of many patients, but implant-associated infection remains a serious complication with significant morbidity and mortality. In recent years, the role of biofilm in the development of these infections has become increasingly recognized, and strategies to combat biofilm are beginning to proliferate in the literature. This review will explore how implant-associated infections have historically been treated, where the research currently stands, and the future trends of infection control.

Highlights: Black-Right-Pointing-Pointer Nuclear localization of DMP1 in various cell lines. Black-Right-Pointing-Pointer Non-synchronized cells show either nuclear or cytoplasmic localization of DMP1. Black-Right-Pointing-Pointer Nuclear DMP1 is restricted to the nucleoplasm but absent in the nucleolus. -- Abstract: Dentin matrix protein 1 (DMP1) is highly expressed in odontoblasts and osteoblasts/osteocytes and plays an essential role in tooth and bone mineralization and phosphate homeostasis. It is debatable whether DMP1, in addition to its function in the extracellular matrix, can enter the nucleus and function as a transcription factor. To better understand its function, we examined the nuclear localization of endogenous and exogenous DMP1 in C3H10T1/2 mesenchymal cells, MC3T3-E1 preosteoblast cells and 17IIA11 odontoblast-like cells. RT-PCR analyses showed the expression of endogenous Dmp1 in all three cell lines, while Western-blot analysis detected a major DMP1 protein band corresponding to the 57 kDa C-terminal fragment generated by proteolytic processing of the secreted full-length DMP1. Immunofluorescent staining demonstrated that non-synchronized cells presented two subpopulations with either nuclear or cytoplasmic localization of endogenous DMP1. In addition, cells transfected with a construct expressing HA-tagged full-length DMP1 also showed either nuclear or cytoplasmic localization of the exogenous DMP1 when examined with an antibody against the HA tag. Furthermore, nuclearmore » DMP1 was restricted to the nucleoplasm but was absent in the nucleolus. In conclusion, these findings suggest that, apart from its role as a constituent of dentin and bone matrix, DMP1 might play a regulatory role in the nucleus.« less

Introduction Recent successes in dental pulp engineering indicate that regenerative treatment strategies in endodontics are feasible. Clinically, revascularization procedures render completion of root formation in immature teeth. The generation of a pulp-like tissue after seeding of dental pulp stem cells into dentin discs or cylinders and transplantation in vivo is possible. In this experimental setup, which mimics the situation in the root canal, the pretreatment of dentin might influence cellular behavior at the cell-dentin interface. Thus, the objective of this study was to investigate whether dentin conditioning can determine cell fate. Methods Dental pulp stem cells (DPSCs) were seeded into a growth factor–laden peptide hydrogel, transferred into dentin cylinders, and transplanted subcutaneously into immunocompromised mice. Before cell seeding, dentin cylinders were either pretreated with sodium hypochloride (NaOCl) or conditioned with EDTA. The constructs were explanted after 6 weeks and subjected to histological and immunohistochemical analysis. Results In dentin treated with NaOCl, resorption lacunae were found at the cell-dentin interface created by multinucleated cells with clastic activity. After conditioning with EDTA, DPSCs adjacent to the dentin formed an intimate association with the surface, differentiated into odontoblasts-like cells that expressed dentin sialoprotein, and extended cellular processes into the dentinal tubules. A vascularized soft connective tissue similar to dental pulp was observed inside the dentin cylinder. Conclusions Dentin conditioning considerably influences DPSC fate when seeded in close proximity to dentin. This information might be critical for optimized strategic planning for future regenerative endodontic treatment.

Dentin matrix protein 1 (DMP1), an acidic protein important to the formation of bone and dentin, primarily exists as the processed NH(2)-terminal and COOH-terminal fragments in the extracellular matrix of the two tissues. Previous in vitro studies showed that the substitution of residue Asp(213) by Ala(213) (D213A) at a cleavage site blocked the processing of mouse DMP1 in cells. In this study, we generated transgenic mice expressing mutant D213A-DMP1 (WT/D213A-Tg mice) to test the hypothesis that the proteolytic processing of DMP1 is an activation step essential to osteogenesis. By crossbreeding WT/D213A-Tg mice with Dmp1 knock-out (Dmp1-KO) mice, we obtained mice expressing D213A-DMP1 in a Dmp1-KO background; these mice will be referred to as "Dmp1-KO/D213A-Tg" mice. Biochemical, radiological, and morphological approaches were used to characterize the skeletal phenotypes of Dmp1-KO/D213A-Tg mice compared with wild-type mice, Dmp1-KO mice, and Dmp1-KO mice expressing the normal Dmp1 transgene. Protein chemistry analyses showed that DMP1 was barely cleaved in the bone of the Dmp1-KO/D213A-Tg mice, indicating that D213A substitution effectively blocked the proteolytic processing of DMP1 in vivo. While the expression of the normal Dmp1 transgene completely rescued the phenotypic skeletal changes of the Dmp1-KO mice, the expression of the mutant D213A-Dmp1 transgene failed to do so. These results indicate that the full-length form of DMP1 is an inactive precursor and its proteolytic processing is an activation step essential to the biological functions of this protein in osteogenesis.

Pax9 belongs to the Pax family of transcriptional regulators that are defined by a highly conserved DNA-binding region, the paired domain. Drosophila, mouse and human genetics have shown that Pax proteins play multiple roles in tissue patterning and organogenesis by mediating their functions in a highly tissue-specific manner. Members of the Pax family, Pax9 and Pax1, act synergistically during vertebral formation. However, only Pax9 is essential for tooth formation. Furthermore, mutations of PAX9 are associated with human tooth agenesis. The highly tooth-specific molecular functions of Pax9 suggest that its activity is tightly regulated. Most likely, this occurs through interactions with other protein factors. Among the regulatory molecules that are expressed in dental mesenchyme, the Msx1 homeoprotein is of particular interest. The closely overlapping expression patterns of Pax9 and Msx1 are consistent with a role in epithelial-mesenchymal interactions. To demonstrate that Pax9 interacts with Msx1 physiologically in vivo and in vitro, we performed co-immunoprecipitation and GST interaction assays. Our results indicate that there is a physical association between the two proteins. Our biochemical data, coupled with human genetic studies and expression analysis in a mouse model, indicate a functional relationship between Pax9 and Msx1 during tooth development.

Pax9 is a paired domain-containing transcription factor that plays an essential role in the patterning of murine dentition. In humans, mutations in PAX9 are associated with unique phenotypes of familial tooth agenesis that mainly involve posterior teeth. Among these, a frameshift mutation (219InsG) within the paired domain of PAX9 produces a protein product associated with a severe form of molar agenesis in a single family. The objectives of this study were to gain new insights into the molecular pathogenesis of the 219InsG mutation and its role in tooth agenesis. Here we describe functional defects in DNA binding and transactivation of mutant 219InsGPax9. Although wild type Pax9 binds to the high affinity paired domain recognition sequences, e5 and CD19-2(A-ins), the 219InsGPax9 mutant protein was unable to bind to these cognate DNA-binding sites. In co-transfection assays, wild type Pax9 activated reporter gene transcription although the mutant was transcriptionally inactive. Immunolocalization data show that Pax9 and 219InsGPax9 proteins are synthesized in mammalian cells but that the nuclear localization of the mutant Pax9 protein is altered. Furthermore, transactivation by the full-length Pax9 protein from paired domain binding sites was not impaired by the 219InsGPax9 mutant. The latter did not alter the DNA binding activities of wild type Pax9 in gel mobility shift assays. The combined defects in DNA binding activities and transactivation function of mutant 219InsGPAX9 likely alter the selective activation and/or repression of PAX9 effector genes during odontogenesis. This loss-of-function of PAX9 most likely results in its haploinsufficiency during the patterning of dentition and the subsequent loss of posterior teeth.

Cleidocranial dysplasia (CCD) is an autosomal dominant human disorder that affects development of bones and teeth. The dental disorders in CCD patients include formation of supernumerary teeth, delayed tooth eruption, and lack of formation of cellular cementum in permanent teeth. This disorder involves a mutation in the osteoblast-specific transcription factor Runx2/Cbfa1, leading to haploinsufficiency of the Runx2/Cbfa1 protein. Here, we examined if Runx2/Cbfa1 heterozygous mice (with one functional allele for Runx2/Cbfa1) exhibit similar changes in tooth eruption, and dental cementum formation as in CCD patients. Heads of Runx2/Cbfa1 heterogeneous and wildtype mice aged days 16-35 postnatally were serially sectioned and stained with hematoxylin-eosin or for tartrate resistant acid phosphatase (TRAP) to identify osteoclasts. The results showed that the eruption pattern of the first and second molars in maxilla and mandible in Runx2/Cbfa1 +/- mice was the same as in wildtype animals. No clear difference in distribution or in the (estimated) number of osteoclasts was found. Cellular cement at the apical portions of the molar roots was present in both groups. The data suggests that in the mouse one allele for Runx2/Cbfa1 is sufficient for an undisturbed tooth eruption and an apparently normal formation of the periodontium.

Our laboratory has reported that two major noncollagenous dentin proteins, dentin sialoprotein and dentin phosphoprotein, are specific cleavage products of a larger precursor protein termed dentin sialophosphoprotein (MacDougall, M., Simmons, D., Luan, X., Nydegger, J., Feng, J. Q., and Gu, T. T. (1997) J. Biol. Chem. 272:835-842). To confirm our single gene hypothesis and initiate in vitro promoter studies, we have characterized the structural organization of the mouse dentin sialophosphoprotein gene. This gene has a transcription unit of approximately 9.4 kilobase pairs and is organized into 5 exons and 4 introns. Exon 1 contains a noncoding 5' sequence, and exon 2 contains the transcriptional start site, signal peptide, and first two amino acids of the NH2 terminus. Exons 3 and 4 contain coding information for 29 and 314 amino acids, respectively. The remainder of the coding information and the untranslated 3' region are contained in exon 5. Chromosomal mapping localized the gene to mouse chromosome 5q21 in close proximity to other dentin/bone matrix genes. Computer analysis of the promoter proximal 1.6-kilobase pair sequence revealed a number of potentially important cis-regulatory sequences; these include the recognition elements of AP-1, AP-2, Msx-1, serum response elements, SP-1, and TCF-1. In vitro studies showed that the DSPP promoter is active in an odontoblast cell line, MO6-G3, with basal activity mapped to -95 bp. Two potential enhancer and suppresser elements were identified in the regions between -1447 and -791 bp and -791 and -95 bp, respectively. The structural organization of the dentin sialophosphoprotein gene confirms our finding that both dentin sialoprotein and dentin phosphoprotein are encoded by a single gene with a continuous open reading frame.

We isolated a sialic-rich protein from rat dentin extracts and have named it dentin sialoprotein, DSP (formerly called 95K glycoprotein). DSP is rich in aspartic acid, glutamic acid, glycine and serine, but contains no cysteine or phosphate. The 30% carbohydrate content includes about 9% sialic acid and indicates that several N-glycosides and O-glycosides are present. Sedimentation equilibrium analysis gave a M(r) of 52,570. Based on this molecular weight we calculated that DSP contains about 350-amino acids and 75 monosaccharides. With automated Edman degradation the sequence of the first 8-amino acids was shown to be: Ile-Pro-Val-Pro-Gln-Leu-Val-Pro. The initial 3 residues of this sequence are identical to the first 3 in human osteopontin (OPN) and are closely similar to the Leu-Pro-Val sequences of OPN from other species, as well as at the beginning of bone acidic glycoprotein-75 (BAG-75). On Western immunoblots, purified polyclonal antibodies reacted only with DSP in dentin extracts and with none of the proteins from bone. Similarly, immunolocalization experiments showed the presence of DSP in dentin but not in enamel or alveolar bone. Along with immunohistochemical localization data reported elsewhere, these observations suggest that DSP may be an important marker for cells in the odontoblast lineage.