Maxillary lateral incisor agenesis is associated with maxillary form: a geometric morphometric analysis.

BACKGROUND AND OBJECTIVE: Agenesis of the maxillary lateral incisor occurs in up to 4% of all individuals and requires multidisciplinary treatment. Its developmental origins, however, are not fully understood. Earlier studies documented genetic factors contributing to agenesis but also an association with craniofacial morphology. In this study, we assessed the association between maxillary morphology and lateral incisor agenesis by a geometric morphometric approach to disentangle the roles of developmental plasticity and genetic factors. MATERIALS AND METHODS: We quantified the maxillary alveolar ridge by 19 two-dimensional landmarks on cross-sectional images of 101 computed tomography scans. We compared the shape and size of the alveolar ridge across patients with unilateral or bilateral agenesis of maxillary lateral incisors and patients with extracted or in situ incisors. RESULTS: The maxillary alveolar ridge was clearly narrower in patients with agenesis or an extracted incisor compared to the control group, whereas the contralateral side of the unilateral agenesis had an intermediate width. Despite massive individual variation, the ventral curvature of the alveolar ridge was, on average, more pronounced in the bilateral agenesis group compared to unilateral agenesis and tooth extraction. CONCLUSIONS: This suggests that pleiotropic genetic and epigenetic factors influence both tooth development and cranial growth, but an inappropriately sized or shaped alveolar process may also inhibit normal formation or development of the tooth bud, thus leading to dental agenesis. CLINICAL RELEVANCE: Our results indicate that bilateral agenesis of the lateral incisor tends to be associated with a higher need of bone augmentation prior to implant placement than unilateral agenesis.

Effect of Titanium and Zirconia Nanoparticles on Human Gingival Mesenchymal Stromal Cells.

Nano- and microparticles are currently being discussed as potential risk factors for peri-implant disease. In the present study, we compared the responses of human gingival mesenchymal stromal cells (hG-MSCs) on titanium and zirconia nanoparticles (<100 nm) in the absence and presence of Porphyromonas gingivalis lipopolysaccharide (LPS). The primary hG-MSCs were treated with titanium and zirconia nanoparticles in concentrations up to 2.000 µg/mL for 24 h, 72 h, and 168 h. Additionally, the cells were treated with different nanoparticles (25−100 µg/mL) in the presence of P. gingivalis LPS for 24 h. The cell proliferation and viability assay and live−dead and focal adhesion stainings were performed, and the expression levels of interleukin (IL)-6, IL-8, and monocyte chemoattractant protein (MCP)-1 were measured. The cell proliferation and viability were inhibited by the titanium (>1000 µg/mL) but not the zirconia nanoparticles, which was accompanied by enhanced apoptosis. Both types of nanoparticles (>25 µg/mL) induced the significant expression of IL-8 in gingival MSCs, and a slightly higher effect was observed for titanium nanoparticles. Both nanoparticles substantially enhanced the P. gingivalis LPS-induced IL-8 production; a higher effect was observed for zirconia nanoparticles. The production of inflammatory mediators by hG-MSCs is affected by the nanoparticles. This effect depends on the nanoparticle material and the presence of inflammatory stimuli.

Stepwise molecular-genetic examination in aborted fetuses.

OBJECTIVE: The evaluation of quantitative fluorescence PCR (QF-PCR) and single nucleotide polymorphism array (SNP array) analysis for the identification of chromosomal abnormalities in products of conception (POC). MATERIALS AND METHODS: A total of 1,094 POC samples were processed at Gennet in the years 2018-2020. Chromosomal aneuploidies were tested by QF-PCR using a Omnibor set (STR markers 13, 18, 21, X a Y), SAB-I set (STR markers 2, 7, 15, 16, 22), SAB-II set (from November 2019, STR markers 4, 6, 14) followed by SNP array analysis (Illumina) on samples with a negative QF-PCR result. All POC samples were tested for maternal contamination. RESULTS: After exclusion of maternal contamination (32% samples) the total number of 742 POC samples were tested by QF-PCR. Chromosomal aneuploidies were found in 273 POC samples (36.8%). Then, 469 QF-PCR negative POC samples were tested by SNP array analysis. Normal female/male profile was confirmed in 402 samples (85.7%) and chromosomal aneuploidies and chromosomal aberrations (deletion/duplication > 10 Mb) in 51 samples (10.9%). Microdeletion/microduplication was found in 16 POC samples (3.4%), two were classified as pathogenic variants and 14 as variants of unknown significance. In a group of women > 35 years of age, statistically significant increase of the chromosomal abnormalities was confirmed. No statistically significant difference between the in vitro fertilization group and the group of spontaneous conception was found. CONCLUSION: The application of the molecular work-up based on the stepwise use of QF-PCR and SNP array clarifies the cause of the abortion in 43% POC samples. The overall detection rate in the I. trimester was 50.4%.

Salivary MRP-8/14 and the presence of periodontitis-associated bacteria in children with bonded maxillary expansion treatment.

OBJECTIVES: The aim of this study was to investigate changes in saliva concentration of the inflammatory marker MRP-8/14 and the presence of some periodontitis-associated bacteria in patients with mixed dentition treated with a rigid acrylic, bonded maxillary expander. METHODS: Fifteen patients in mixed dentition treated with a bonded palatal expander were enrolled in this longitudinal study. Saliva samples were taken before the therapy, as well as in 2 weeks and 3, 6, 9, and 12 months after the beginning of the therapy. In each sample, the levels of MRP-8/14 were determined by ELISA and the presence of 11 bacteria was detected by PCR followed by DNA-DNA hybridization. RESULTS: Salivary concentration of MRP-8/14 and the amount of Tannerella forsythia, Treponema denticola, and Eikenella corrodens were significantly increased during treatment with bonded maxillary expander. These changes were transient and the maximal levels of MRP-8/14 and periodontitis-associated pathogens were observed 6-9 months after the beginning of the therapy. CONCLUSION: Therapy with bonded maxillary results in higher MRP-8/14 levels and increased prevalence of some periodontitis-associated bacteria, namely T. forsythia, T. denticola, and E. corrodens. The results suggest the detection of salivary MRP-8/14 levels may be a potential tool to reflect the oral health status in children with fixed orthodontic treatment. CLINICAL RELEVANCE: Our data suggest that the treatment with bonded maxillary expander might influence the oral health status and should be accompanied by the careful control of the oral health during the therapy.

Interleukin-1ß Induced Matrix Metalloproteinase Expression in Human Periodontal Ligament-Derived Mesenchymal Stromal Cells under In Vitro Simulated Static Orthodontic Forces.

The periodontal ligament (PDL) responds to applied orthodontic forces by extracellular matrix (ECM) remodeling, in which human periodontal ligament-derived mesenchymal stromal cells (hPDL-MSCs) are largely involved by producing matrix metalloproteinases (MMPs) and their local inhibitors (TIMPs). Apart from orthodontic forces, the synthesis of MMPs and TIMPs is influenced by the aseptic inflammation occurring during orthodontic treatment. Interleukin (IL)-1ß is one of the most abundant inflammatory mediators in this process and crucially affects the expression of MMPs and TIMPs in the presence of cyclic low-magnitude orthodontic tensile forces. In this study we aimed to investigate, for the first time, how IL-1ß induced expression of MMPs, TIMPs and how IL-1ß in hPDL-MSCs was changed after applying in vitro low-magnitude orthodontic tensile strains in a static application mode. Hence, primary hPDL-MSCs were stimulated with IL-1ß in combination with static tensile strains (STS) with 6% elongation. After 6- and 24 h, MMP-1, MMP-2, TIMP-1 and IL-1ß expression levels were measured. STS alone had no influence on the basal expression of investigated target genes, whereas IL-1ß caused increased expression of these genes. In combination, they increased the gene and protein expression of MMP-1 and the gene expression of MMP-2 after 24 h. After 6 h, STS reduced IL-1ß-induced MMP-1 synthesis and MMP-2 gene expression. IL-1ß-induced TIMP-1 gene expression was decreased by STS after 6- and 24-h. At both time points, the IL-1ß-induced gene expression of IL-1ß was increased. Additionally, this study showed that fetal bovine serum (FBS) caused an overall suppression of IL-1ß-induced expression of MMP-1, MMP-2 and TIMP-1. Further, it caused lower or opposite effects of STS on IL-1ß-induced expression. These observations suggest that low-magnitude orthodontic tensile strains may favor a more inflammatory and destructive response of hPDL-MSCs when using a static application form and that this response is highly influenced by the presence of FBS in vitro.

MMPs and TIMPs Expression Levels in the Periodontal Ligament during Orthodontic Tooth Movement: A Systematic Review of In Vitro and In Vivo Studies.

Background: During orthodontic tooth movement (OTM), applied orthodontic forces cause an extensive remodeling of the extracellular matrix (ECM) in the periodontal ligament (PDL). This is mainly orchestrated by different types of matrix metalloproteinases (MMPs) and their tissue inhibitors of matrix metalloproteinases (TIMPs), which are both secreted by periodontal ligament (PDL) fibroblasts. Multiple in vitro and in vivo studies already investigated the influence of applied orthodontic forces on the expression of MMPs and TIMPs. The aim of this systematic review was to explore the expression levels of MMPs and TIMPs during OTM and the influence of specific orthodontic force-related parameters. Methods: Electronic article search was performed on PubMed and Web of Science until 31 January 2021. Screenings of titles, abstracts and full texts were performed according to PRISMA, whereas eligibility criteria were defined for in vitro and in vivo studies, respectively, according to the PICO schema. Risk of bias assessment for in vitro studies was verified by specific methodological and reporting criteria. For in vivo studies, risk of bias assessment was adapted from the Joanna Briggs Institute Critical Appraisal Checklist for analytical cross-sectional study. Results: Electronic article search identified 3266 records, from which 28 in vitro and 12 in vivo studies were included. The studies showed that orthodontic forces mainly caused increased MMPs and TIMPs expression levels, whereas the exact effect may depend on various intervention and sample parameters and subject characteristics. Conclusion: This systematic review revealed that orthodontic forces induce a significant effect on MMPs and TIMPs in the PDL. This connection may contribute to the controlled depletion and formation of the PDLs' ECM at the compression and tension site, respectively, and finally to the highly regulated OTM.

CAD/CAM indirect bonding trays using hard versus soft resin material: a single-blinded in vitro study.

OBJECTIVES: The present in vitro study aimed to evaluate the accuracy of three-dimensional (3D) printed indirect bonding trays consisting of hard or soft resin materials produced using computer-aided design and manufacturing (CAD/CAM). METHODS: Forty-eight dental casts were 3D printed. Four groups based on frontal crowding were defined and divided into hard- and soft-resin groups. After virtual bracket positioning on the digital models, the transfer trays were 3D printed. To evaluate the accuracy of the procedure, measurements were performed using a digital overlay of the virtual (target) bracket position and a post-bonding scan. The horizontal, transverse, and vertical deviations and angular discrepancies were analyzed. The loss rate was evaluated descriptively as a percentage. RESULTS: A total of 553 brackets were bonded using 24 soft and 24 resilient indirect bonding trays. The mean deviations were of 0.05 mm (transversal), 0.05 mm (horizontal), 0.09 mm (vertical), 0.13° (angulation) in the resilient resin group and of 0.01 mm (transversal), 0.08 mm (horizontal), 0.08 mm (vertical), 0.37° (angular) in the soft resin group. The loss rate was 6.9% and 0.7% in the hard and soft resin groups, respectively. Angular deviations were significantly higher in the soft resin group (P = 0.009), whereas the loss rate was considerably higher in the hard resin group (P < 0.001). SIGNIFICANCE: The findings indicate that indirect bonding using CAD/CAM is an accurate procedure in the laboratory setting. Soft resins are considered favorable for loss rate and useability.

3D printed indirect bonding trays: Transfer accuracy of hard versus soft resin material in a prospective, randomized, single-blinded clinical study.

OBJECTIVES: This prospective clinical study aimed to compare transfer accuracy and immediate loss rate of hard versus soft transfer trays utilizing a CAD/CAM workflow. METHODS: We performed virtual bracket placement on intraoral scans of adolescent patients to create individual indirect bonding trays. Orthodontic software (Appliance Designer, 3Shape, Copenhagen, Denmark) was used to design the trays, which were then produced using 3D printing technology. Patients were randomly assigned to the hard or soft resin groups with a 1:1 allocation. Subgroups were determined based on the Little's Irregularity Index and distributed equally. RESULTS: 552 brackets were bonded onto adolescent patients using 46 CAD/CAM indirect bonding trays. The linear mean transfer errors ranged from -0.011 mm (soft) to -0.162 mm (hard) and angularly -0.255° (hard) and -0.243° (soft). No statistically significant differences were found between the subgroups or soft and hard resin groups. However, the transfer accuracy of molar brackets was significantly lower in the transversal and horizontal directions. All mean transfer errors were within the limits of clinical acceptability. The loss rate was 2.4 % in the hard resin group and 2.3 % in the soft resin group. The Intra Observer Correlation was excellent. SIGNIFICANCE: CAD/CAM technology for indirect bracket bonding has been proven reliable in a randomized clinical trial. Both hard and soft resin showed a low rate of immediate loss compared to the current literature. Soft resin was more favorable than hard resin in terms of accuracy and usability. However, the indirect bonding of molar brackets is significantly less accurate than incisor brackets.

Differential gene expression and protein-protein interaction networks of human periodontal ligament stromal cells under mechanical tension.

Orthodontic treatment is based on complex strategies and takes up to years until a desired therapeutic outcome is accomplished, implying long periods of high costs and discomfort for the patient. Choosing the optimal settings for force intensities in the initial phase of orthodontic tooth movement is the key to successful orthodontic treatment. It is known that orthodontic tooth movement is mainly mediated by tensile and compressive forces that are communicated to the alveolar bone via the periodontal ligament. While the revelation of the complex molecular network was already approached by transcriptomic analysis of compressed periodontal ligament cells, the entity of molecular key players activated by tensile forces remains elusive. Therefore, the aim of this study was to assess the effect of mechanical tensile forces on the gene expression profile of human primary periodontal ligament stromal cells, mimicking the initial phase of orthodontic tooth movement. A transcriptomic analysis of tension-treated and untreated periodontal ligament stromal cells yielded 543 upregulated and 793 downregulated differentially expressed genes. Finally, six highly significant genes were found in the transcriptome that are related to biological processes with relevance to orthodontic tooth movement, including apelin, fibroblast growth factor receptor 2, noggin, sulfatase 1, secreted frizzled-related protein 4 and stanniocalcin 1. Additionally, differences of gene expression profiles between individual cell donors showed a high effect size. Closer understanding of the roles of the identified candidates in the initial phase of orthodontic tooth movement could help to clarify the underlying mechanisms, which will be essential for the development of personalized treatment strategies in orthodontics.

Effects of the Saliva of Patients Undergoing Orthodontic Treatment with Invisalign and Brackets on Human Gingival Fibroblasts and Oral Epithelial Cells.

The transient worsening of oral health sometimes accompanies orthodontic treatment (OT), and the extent of this effect might depend on whether the patients are treated with traditional brackets or clear aligners. Saliva is an important tool for monitoring oral health and influences the functional properties of various oral cells. This study aimed to compare the effects of saliva from patients undergoing OT with Invisalign aligners and brackets on human gingival fibroblasts and oral epithelial cells in vitro. Unstimulated saliva was collected from 15 patients treated with Invisalign and 16 patients treated with brackets before and 3 and 6 months after therapy began. The saliva was used to stimulate primary human gingival fibroblasts and the oral epithelial Ca9-22 cell line, and the resulting cell response was investigated. Saliva did not exhibit any toxic effect on investigated cells, as shown by the proliferation/viability assay with the MTT method. In human gingival fibroblasts, saliva increased gene expression of various proinflammatory mediators, such as interleukin (IL)-6, IL-8, and monocyte chemoattractant protein-1, assessed by qPCR. In epithelial cells, saliva increased the production of IL-8 measured by ELISA and decreased gene expression of various proteins involved in the barrier function. During the therapy, the saliva-induced production of IL-8 tended to be decreased, and the saliva-induced decrease in the expression of barrier protein was partially improved. No difference between aligners and brackets was observed in either cell type. Saliva affects the functional properties of oral cells, but this effect is not influenced by the type of OT.

Behaviour of Human Oral Epithelial Cells Grown on Invisalign® SmartTrack® Material.

Invisalign aligners have been widely used to correct malocclusions, but their effect on oral cells is poorly known. Previous research evaluated the impact of aligners' eluates on various cells, but the cell behavior in direct contact with aligners is not yet studied. In the present study, we seeded oral epithelial cells (cell line Ca9-22) directly on Invisalign SmartTrack material. This material is composed of polyurethane and co-polyester and exhibit better mechanical characteristics compared to the predecessor. Cell morphology and behavior were investigated by scanning electron microscopy and an optical cell moves analyzer. The effect of aligners on cell proliferation/viability was assessed by cell-counting kit (CCK)-8 and 3,4,5-dimethylthiazol-2-yl-2,5-diphenyl tetrazolium bromide (MTT) assay and live/dead staining. The expression of inflammatory markers and proteins involved in epithelial barrier function was measured by qPCR. Cells formed cluster-like structures on aligners. The proliferation/viability of cells growing on aligners was significantly lower (p < 0.05) compared to those growing on tissue culture plastic (TCP). Live/dead staining revealed a rare occurrence of dead cells on aligners. The gene expression level of all inflammatory markers in cells grown on aligners' surfaces was significantly increased (p < 0.05) compared to cells grown on TCP after two days. Gene expression levels of the proteins involved in barrier function significantly increased (p < 0.05) on aligners' surfaces after two and seven days of culture. Aligners' material exhibits no cytotoxic effect on oral epithelial cells, but alters their behavior and the expression of proteins involved in the inflammatory response, and barrier function. The clinical relevance of these effects has still to be established.

Cytokines Differently Define the Immunomodulation of Mesenchymal Stem Cells from the Periodontal Ligament.

Human periodontal ligament stem cells (hPDLSCs) play an important role in periodontal tissue homeostasis and regeneration. The function of these cells in vivo depends largely on their immunomodulatory ability, which is reciprocally regulated by immune cells via cytokines, particularly interferon (IFN)-γ, tumor necrosis factor (TNF)-α, and interleukin (IL)-1ß. Different cytokines activate distinct signaling pathways and might differently affect immunomodulatory activities of hPDLSCs. This study directly compared the effect of IFN-γ, TNF-α, or IL-1ß treated primary hPDLSCs on allogenic CD4+ T lymphocyte proliferation and apoptosis in an indirect co-culture model. The effects of IFN-γ, TNF-α, and IL-1ß on the expression of specific immunomodulatory factors such as intoleamine-2,3-dioxygenase-1 (IDO-1), prostaglandin E2 (PGE2), and programmed cell death 1 ligand 1 (PD-L1) and ligand 2 (PD-L2) in hPDLSCs were compared. The contribution of different immunomodulatory mediators to the immunomodulatory effects of hPDLSCs in the indirect co-culture experiments was assessed using specific inhibitors. Proliferation of CD4+ T lymphocytes was inhibited by hPDLSCs, and this effect was strongly enhanced by IFN-γ and IL-1ß but not by TNF-α. Apoptosis of CD4+ T lymphocytes was decreased by hPDLSCs per se. This effect was counteracted by IFN-γ or IL-1ß. Additionally, IFN-γ, TNF-α, and IL-1ß differently regulated all investigated immunomediators in hPDLSCs. Pharmacological inhibition of immunomediators showed that their contribution in regulating CD4+ T lymphocytes depends on the cytokine milieu. Our data indicate that inflammatory cytokines activate specific immunomodulatory mechanisms in hPDLSCs and the expression of particular immunomodulatory factors, which underlies a complex reciprocal interaction between hPDLSCs and CD4+ T lymphocytes.

Does the time-point of orthodontic space closure initiation after tooth extraction affect the incidence of gingival cleft development? A randomized controlled clinical trial.

BACKGROUND: Gingival clefts (GCs) develop frequently during orthodontic space closure and may compromise the treatment outcome. This study assessed whether the time-point of orthodontic space closure initiation, after permanent tooth extraction, affects the incidence of GC. METHODS: In 25 patients requiring bilateral premolar extraction because of orthodontic reasons, one premolar, chosen at random, was extracted 8 weeks before space closure initiation ("delayed movement," DM), whereas the contralateral premolar was extracted 1 week before ("early movement," EM) ("treatment group"). Presence or absence of GC after 3 and 6 months ("time-point") was recorded and any association with various parameters (i.e., treatment group, time-point, gender, jaw, craniofacial growth, gingival biotype, buccal bone dehiscence after extraction, space closure) was statistically assessed. RESULTS: Twenty-one patients contributing with 26 jaws were finally included in the analysis. Overall, GCs were frequent after 3 (DM: 53.9%; EM: 69.2%) and 6 months (DM: 76.9%; EM: 88.5%). EM (P = 0.014) and larger space closure within the study period (P = 0.001) resulted in a significantly higher incidence of GC. Further, there was a tendency for GC development in the presence of buccal bone dehiscence (P = 0.052) and thin gingival biotype (P = 0.054). "Fast movers" (herein cases with a tooth movement ≥1 mm per month) developed a GC in >90% of the cases already after 3 months. "Slow movers" developed a GC in 25% and 70% after 3 months and final evaluation, respectively. CONCLUSIONS: GC development is a frequent finding during orthodontic space closure and seems to occur more frequently with early tooth movement initiation and in "fast movers."

1,25(OH)2D3 Differently Affects Immunomodulatory Activities of Mesenchymal Stem Cells Depending on the Presence of TNF-α, IL-1ß and IFN-γ.

Periodontal ligament-derived mesenchymal stem cells (hPDLSCs) possess immunomodulatory abilities which are strongly enhanced by various inflammatory cytokines. Vitamin D3 has anti-inflammatory effects on hPDLSCs and immune cells. However, no study to date has directly compared the influence of 1,25(OH)2D3 on the immunomodulatory activities of hPDLSCs in the presence of different cytokines. In the present study, the effects of hPDLSCs treated with tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, or interferon (IFN)-γ in the presence of 1,25(OH)2D3 on the proliferation of allogenic CD4+ T lymphocyte or on the functional status of primary CD68+ macrophages were analyzed in coculture models. Additionally, the effects of 1,25(OH)2D3 on TNF-α-, IL-1ß-, and IFN-γ-induced gene expression of some immunomodulatory factors in hPDLSCs were compared. Under coculture conditions, 1,25(OH)2D3 increased or decreased CD4+ T lymphocyte proliferation via hPDLSCs, depending on the cytokine. hPDLSCs primed with 1,25(OH)2D3 and different cytokines affected pro- and anti-inflammatory cytokine expression in macrophages variably, depending on the priming cytokine. With one exception, 1,25(OH)2D3 significantly reduced TNF-α-, IL-1ß-, and IFN-γ-induced expression of all the investigated immunomediators in hPDLSCs, albeit to different extents. These results suggest that 1,25(OH)2D3 influences the immunomodulatory activities of hPDLSCs depending qualitatively and quantitatively on the presence of certain inflammatory cytokines.

Bone-Conditioned Medium Inhibits Osteogenic and Adipogenic Differentiation of Mesenchymal Cells In Vitro.

BACKGROUND AND PURPOSE: Autografts are used for bone reconstruction in regenerative medicine including oral and maxillofacial surgery. Bone grafts release paracrine signals that can reach mesenchymal cells at defect sites. The impact of the paracrine signals on osteogenic, adipogenic, and chondrogenic differentiation of mesenchymal cells has remained unclear. MATERIAL AND METHODS: Osteogenesis, adipogenesis, and chondrogenesis were studied with murine ST2 osteoblast progenitors, 3T3-L1 preadipocytes, and ATDC5 prechondrogenic cells, respectively. Primary periodontal fibroblasts from the gingiva, from the periodontal ligament, and from bone were also included in the analysis. Cells were exposed to bone-conditioned medium (BCM) that was prepared from porcine cortical bone chips. RESULTS: BCM inhibited osteogenic and adipogenic differentiation of ST2 and 3T3-L1 cells, respectively, as shown by histological staining and gene expression. No substantial changes in the expression of chondrogenic genes were observed in ATDC5 cells. Primary periodontal fibroblasts also showed a robust decrease in alkaline phosphatase and peroxisome proliferator-activated receptor gamma (PPARγ) expression when exposed to BCM. BCM also increased collagen type 10 expression. Pharmacologic blocking of transforming growth factor (TGF)-ß receptor type I kinase with SB431542 and the smad-3 inhibitor SIS3 at least partially reversed the effect of BCM on PPARγ and collagen type 10 expression. In support of BCM having TGF-ß activity, the respective target genes were increasingly expressed in periodontal fibroblasts. CONCLUSIONS: The present work is a pioneer study on the paracrine activity of bone grafts. The findings suggest that cortical bone chips release soluble signals that can modulate differentiation of mesenchymal cells in vitro at least partially involving TGF-ß signaling.