Elina Järvinen

Continuous tooth generation in mouse is induced by activated epithelial Wnt/β-catenin signaling

The single replacement from milk teeth to permanent teeth makes mammalian teeth different from teeth of most nonmammalian vertebrates and other epithelial organs such as hair and feathers, whose continuous replacement has been linked to Wnt signaling. Here we show that mouse tooth buds expressing stabilized β-catenin in epithelium give rise to dozens of teeth. The molar crowns, however, are typically simplified unicusped cones. We demonstrate that the supernumerary teeth develop by a renewal process where new signaling centers, the enamel knots, bud off from the existing dental epithelium. The basic aspects of the unlocked tooth renewal can be reproduced with a computer model on tooth development by increasing the intrinsic level of activator production, supporting the role of β-catenin pathway as an upstream activator of enamel knot formation. These results may implicate Wnt signaling in tooth renewal, a capacity that was all but lost when mammals evolved progressively more complicated tooth shapes.

Sustained epithelial β-catenin activity induces precocious hair development but disrupts hair follicle down-growth and hair shaft formation

During embryonic and postnatal development, Wnt/β-catenin signaling is involved in several stages of hair morphogenesis from placode formation to hair shaft differentiation. Using a transgenic approach, we have investigated further the role of β-catenin signaling in embryonic hair development. Forced epithelial stabilization of β-catenin resulted in precocious and excessive induction of hair follicles even in the absence of Eda/Edar signaling, a pathway essential for primary hair placode formation. In addition, the spacing and size of the placodes was randomized. Surprisingly, the down-growth of follicles was suppressed and hair shaft production was severely impaired. Gene and reporter expression analyses revealed elevated mesenchymal Wnt activity, as well as increased BMP signaling, throughout the skin that was accompanied by upregulation of Sostdc1 (Wise, ectodin) expression. Our data suggest that BMPs are downstream of Wnt/β-catenin and that their interplay may be a critical component in establishing correct patterning of hair follicles through the reaction-diffusion mechanism.

The role of the dental lamina in mammalian tooth replacement.

We have applied the ferret, Mustela putorius furo, as a model for tooth replacement. Ferret has a heterodont dentition, which includes all tooth families, and all antemolar teeth are replaced. Compared with mouse, the ferret therefore has a less derived mammalian dentition resembling that of humans. We have studied tooth replacement in serial histological sections in embryonic and young postnatal ferrets. Our observations indicate that the replacement teeth form from the dental lamina that is intimately connected to the lingual aspect of the deciduous tooth enamel organ. It grows as an offshoot from the enamel organ, elongates in cervical direction and later buds to give rise to the replacement tooth. The extent of the dental lamina growth, preceding replacement tooth budding, varied between different teeth. The dynamic gene expression patterns of Sostdc1, Shh and Axin2 brought new insight into the signal networks regulating the tooth replacement process. The distinct expression of Sostdc1 at the interface between the dental lamina and the deciduous tooth is the first indication of a specific tissue identity of the dental lamina. We suggest that the reactivation of a competent dental lamina is pivotal for the replacement tooth formation.

Tinkering with the inductive mesenchyme: Sostdc1 uncovers the role of dental mesenchyme in limiting tooth induction

Like epithelial organs in general, tooth development involves inductive crosstalk between the epithelium and the mesenchyme. Classically, the inductive potential for tooth formation is considered to reside in the mesenchyme during the visible morphogenesis of teeth, and dental mesenchyme can induce tooth formation even when combined with non-dental epithelium. Here, we have investigated induction of mouse incisors using Sostdc1 (ectodin), a putative antagonist of BMP signaling in the mesenchymal induction of teeth. Deletion of Sostdc1 leads to the full development of single extra incisors adjacent to the main incisors. We show that initially, Sostdc1 expression is limited to the mesenchyme, suggesting that dental mesenchyme may limit supernumerary tooth induction. We test this in wild-type incisors by minimizing the amount of mesenchymal tissue surrounding the incisor tooth germs prior to culture in vitro. The cultured teeth phenocopy the extra incisors phenotype of the Sostdc1-deficient mice. Furthermore, we show that minimizing the amount of dental mesenchyme in cultured Sostdc1-deficient incisors causes the formation of additional de novo incisors that resemble the successional incisor development that results from activated Wnt signaling. Finally, Noggin and Dkk1 prevent individually the formation of extra incisors, and we therefore suggest that inhibition of both BMP and Wnt signaling contributes to the inhibitory role of the dental mesenchyme. Considering the role of mesenchyme in tooth induction and the design of tissue engineering protocols, our work may have uncovered how delicate control of tissue quantities alone influences the outcome between induction and inhibition.

Different roles of Runx2 during early neural crest-derived bone and tooth development

We compared gene expression profiles between Runx2 null mutant mice and their wildtype littermates. Most Runx2‐dependent genes in bones were different from those in teeth, implying that the target genes of Runx2 are tissue‐dependent. In vitro experiments determined that Runx2 is a part of the FGF and BMP signaling pathways in tooth and bone development, respectively.

Preparation and characterization of collagen/PLA, chitosan/PLA, and collagen/chitosan/PLA hybrid scaffolds for cartilage tissue engineering

In this study, three-dimensional (3D) porous scaffolds were developed for the repair of articular cartilage defects. Novel collagen/polylactide (PLA), chitosan/PLA, and collagen/chitosan/PLA hybrid scaffolds were fabricated by combining freeze-dried natural components and synthetic PLA mesh, where the 3D PLA mesh gives mechanical strength, and the natural polymers, collagen and/or chitosan, mimic the natural cartilage tissue environment of chondrocytes. In total, eight scaffold types were studied: four hybrid structures containing collagen and/or chitosan with PLA, and four parallel plain scaffolds with only collagen and/or chitosan. The potential of these types of scaffolds for cartilage tissue engineering applications were determined by the analysis of the microstructure, water uptake, mechanical strength, and the viability and attachment of adult bovine chondrocytes to the scaffolds. The manufacturing method used was found to be applicable for the manufacturing of hybrid scaffolds with highly porous 3D structures. All the hybrid scaffolds showed a highly porous structure with open pores throughout the scaffold. Collagen was found to bind water inside the structure in all collagen-containing scaffolds better than the chitosan-containing scaffolds, and the plain collagen scaffolds had the highest water absorption. The stiffness of the scaffold was improved by the hybrid structure compared to plain scaffolds. The cell viability and attachment was good in all scaffolds, however, the collagen hybrid scaffolds showed the best penetration of cells into the scaffold. Our results show that from the studied scaffolds the collagen/PLA hybrids are the most promising scaffolds from this group for cartilage tissue engineering.

The taming of the shrew milk teeth

SUMMARY A characteristic feature of mammalian dentition is the evolutionary reduction of tooth number and replacement. Because mice do not replace teeth, here we used Sorex araneus, the common shrew, as a model to investigate the loss of tooth replacement. Historically, shrews have been reported to initiate the development of several, milk or deciduous teeth but these soon become rudimentary and only the replacement teeth erupt. Shrews thus offer a living example of a derived mammalian pattern where the deciduous tooth development is being suppressed. Based on histological and gene expression analyses of serial sections, we suggest that S. araneus has discernible tooth replacement only in the premolar 4 (P4) position. Both generations of teeth express Shh in the enamel knot and in the inner enamel epithelium. Nevertheless, the deciduous P4 (dP4) is reduced in size during embryogenesis and is eventually lost without becoming functional. Analysis of growth shows that P4 replaces the dP4 in a “double‐wedge” pattern indicative of competitive replacement where the suppression of the deciduous tooth coincides with the initiation of its replacement. Because activator–inhibitor mechanisms have been implicated in adjacent mouse molars and in transgenic mice with continuous tooth budding, we suggest that evolutionary suppression of deciduous teeth may involve early activation of replacement teeth, which in turn begin to suppress their deciduous predecessors.

Expression of the novel Golgi protein GoPro49 is developmentally regulated during mesenchymal differentiation.

The Golgi complex is the major cell organelle responsible for protein glycosylation and secretion. In this article, we show that GoPro49 is a new gene expressed specifically in mesenchymal and cartilaginous tissues during development. The corresponding human homologue was identified in our previous Golgi proteomics study and was shown to localize to the Golgi complex as an EGFP‐fusion protein. Furthermore, we show using in situ hybridization that GoPro49 expression pattern is both restricted and developmentally regulated. It is specific in vertebrae, ribs, and limbs, and in the craniofacial area in nasal septum and dental follicle. In the trunk, GoPro49 expression decreases before final chondrocyte differentiation, while in the craniofacial area expression is still observed in postnatal tissues. This is the first time a Golgi membrane protein is shown to be expressed in a developmentally regulated manner during mesenchymal and cartilage development in mammals. Developmental Dynamics 237:2243–2255, 2008.

Articular cartilage repair with recombinant human type II collagen/polylactide scaffold in a preliminary porcine study

The purpose of this study was to investigate the potential of a novel recombinant human type II collagen/polylactide scaffold (rhCo‐PLA) in the repair of full‐thickness cartilage lesions with autologous chondrocyte implantation technique (ACI). The forming repair tissue was compared to spontaneous healing (spontaneous) and repair with a commercial porcine type I/III collagen membrane (pCo). Domestic pigs (4‐month‐old, n = 20) were randomized into three study groups and a circular full‐thickness chondral lesion with a diameter of 8 mm was created in the right medial femoral condyle. After 3 weeks, the chondral lesions were repaired with either rhCo‐PLA or pCo together with autologous chondrocytes, or the lesion was only debrided and left untreated for spontaneous repair. The repair tissue was evaluated 4 months after the second operation. Hyaline cartilage formed most frequently in the rhCo‐PLA treatment group. Biomechanically, there was a trend that both treatment groups resulted in better repair tissue than spontaneous healing. Adverse subchondral bone reactions developed less frequently in the spontaneous group (40%) and the rhCo‐PLA treated group (50%) than in the pCo control group (100%). However, no statistically significant differences were found between the groups. The novel rhCo‐PLA biomaterial showed promising results in this proof‐of‐concept study, but further studies will be needed in order to determine its effectiveness in articular cartilage repair.

Biotin-dependent functions in adiposity: a study of monozygotic twin pairs

Background:Biotin acts as a coenzyme for carboxylases regulating lipid and amino-acid metabolism. We investigated alterations of the biotin-dependent functions in obesity and the downstream effects of biotin restriction in adipocytes in vitro.Subjects:Twenty-four monozygotic twin pairs discordant for body mass index (BMI). Mean within-pair difference (heavy-lean co-twin, Δ) of BMI was 6.0 kg m–2 (range 3.1–15.2 kg m–2).Methods:Adipose tissue (AT) DNA methylation, gene expression of AT and adipocytes, and leukocytes (real-time quantitative PCR), serum biotin, C-reactive protein (CRP) and triglycerides were measured in the twins. Human adipocytes were cultured in low and control biotin concentrations and analyzed for lipid droplet content, mitochondrial morphology and mitochondrial respiration.Results:The gene expression levels of carboxylases, PCCB and MCCC1, were upregulated in the heavier co-twins’ leukocytes. ΔPCCB (r=0.91, P=0.0046) and ΔMCCC1 (r=0.79, P=0.036) correlated with ΔCRP within-pairs. Serum biotin levels were lower in the heavier (274 ng l–1) than in the lean co-twins (390 ng l–1, P=0.034). ΔBiotin correlated negatively with Δtriglycerides (r=–0.56, P=0.045) within-pairs. In AT, HLCS and ACACB were hypermethylated and biotin cycle genes HLCS and BTD were downregulated (P<0.05). Biotin-dependent carboxylases were downregulated (ACACA, ACACB, PCCB, MCCC2 and PC; P<0.05) in both AT and adipocytes of the heavier co-twins. Adipocytes cultured in low biotin had decreased lipid accumulation, altered mitochondrial morphology and deficient mitochondrial respiration.Conclusions:Biotin-dependent functions are modified by adiposity independent of genetic effects, and correlate with inflammation and hypertriglyceridemia. Biotin restriction decreases lipid accumulation and respiration, and alters mitochondrial morphology in adipocytes.