RNA-sequencing reveals positional memory of multipotent mesenchymal stromal cells from oral and maxillofacial tissue transcriptomes.

BACKGROUND: Multipotent mesenchymal stromal cells (MSCs) can be isolated from numerous tissues and are attractive candidates for therapeutic clinical applications due to their immunomodulatory and pro-regenerative capacity. Although the minimum criteria for defining MSCs have been defined, their characteristics are known to vary depending on their tissue of origin. RESULTS: We isolated and characterized human MSCs from three different bones (ilium (I-MSCs), maxilla (Mx-MSCs) and mandible (Md-MSCs)) and proceeded with next generation RNA-sequencing. Furthermore, to investigate the gene expression profiles among other cell types, we obtained RNA-seq data of human embryonic stem cells (ESCs) and several types of MSCs (periodontal ligament-derived MSCs, bone marrow-derived MSCs, and ESCs-derived MSCs) from the Sequence Reads Archive and analyzed the transcriptome profile. We found that MSCs derived from tissues of the maxillofacial region, such as the jaw bone and periodontal ligament, were HOX-negative, while those derived from other tissues were HOX-positive. We also identified that MSX1, LHX8, and BARX1, an essential regulator of craniofacial development, were strongly expressed in maxillofacial tissue-derived MSCs. Although MSCs may be divided into two distinct groups, the cells originated from over the neck or not, on the basis of differences in gene expression profile, the expression patterns of all CD antigen genes were similar among different type of MSCs, except for ESCs. CONCLUSIONS: Our findings suggest that MSCs from different anatomical locations, despite meeting general characterization criteria, have remarkable differences in gene expression and positional memory. Although stromal cells from different anatomical sources are generally categorized as MSCs, their differentiation potential and biological functions vary. We suggested that MSCs may retain an original tissue memory about the developmental process, including gene expression profiles. This could have an important impact when choosing an appropriate cell source for regenerative therapy using MSCs.

The Specific Morphological Features of Alveolar Bone.

OBJECTIVE: The aim of the study was to study the specific morphological features of alveolar bone and compare it to femoral bone in rats. METHODS: Twelve 3-month-old nonpregnant female Sprague-Dawley rats were used in the present study. The left maxillae and femurs of 6 rats were used for micro-computed tomography (micro-CT) scanning. The trabecular bone of the distal femur and the interradicular alveolar bone of the maxillary first molar were reconstructed and analyzed. Another 6 rats were used for histological analysis of trabecular bone and alveolar bone. RESULTS: Micro-CT analysis suggested that the femoral trabecular bone was porous with rod-like trabeculae with a scattered distribution in bone marrow, whereas alveolar bone showed a compact structure with plate-like trabeculae and limited bone marrow. Tissue mineral density, bone mineral density, bone volume fraction, and trabecular thickness were dramatically higher in the alveolar bone compared with that in the trabecular bone. Alveolar bone displayed lower trabecular number and trabecular separation. Histomorphometric analysis showed that alveolar bone was formed of compact bone with wide trabeculae, whereas femurs were composed of loose bone with finer trabeculae. CONCLUSIONS: In comparison to the spongiosa of the distal femur, alveolar bone displays specific morphological features with compact, wide, and highly mineralized trabeculae.

Differences in the developmental origins of the periosteum may influence bone healing.

BACKGROUND AND OBJECTIVE: The jaw bone, unlike most other bones, is derived from neural crest stem cells, so we hypothesized that it may have different characteristics to bones from other parts of the body, especially in the nature of its periosteum. The periosteum exhibits osteogenic potential and has received considerable attention as a grafting material for the repair of bone and joint defects. MATERIAL AND METHODS: Gene expression profiles of jaw bone and periosteum were evaluated by DNA microarray and real-time polymerase chain reaction. Furthermore, we perforated an area 2 mm in diameter on mouse frontal and parietal bones. Bone regeneration of these calvarial defects was evaluated using microcomputed tomography and histological analysis. RESULTS: The DNA microarray data revealed close homology between the gene expression profiles within the ilium and femur. The gene expression of Wnt-1, SOX10, nestin, and musashi-1 were significantly higher in the jaw bone than in other locations. Microcomputed tomography and histological analysis revealed that the jaw bone had superior bone regenerative abilities than other bones. CONCLUSION: Jaw bone periosteum exhibits a unique gene expression profile that is associated with neural crest cells and has a positive influence on bone regeneration when used as a graft material to repair bone defects. A full investigation of the biological and mechanical properties of jaw bone as an alternative graft material for jaw reconstructive surgery is recommended.

A randomized clinical trial evaluating the efficacy of the sandwich bone augmentation technique in increasing buccal bone thickness during implant placement. II. Tomographic, histologic, immunohistochemical, and RNA analyses.

OBJECTIVES: This study aimed to evaluate the biologic and structural phenotypes of the bone regenerated via the sandwich bone augmentation (SBA) technique, on buccal implant dehiscence defects. MATERIAL AND METHODS: Twenty-six patients with one buccal implant dehiscence defect each were randomly assigned to two groups. Both groups received a standardized amount of mineralized cancellous and cortical allogenic bone graft. In the test group, a bovine pericardium membrane was placed over the graft, while no membrane was placed in the control group. After 6 months of healing, a bone core biopsy of the regenerated bone was harvested and processed for histologic, immunohistochemical, mRNA, and micro-computed tomography (µCT) analyses. Of the 26 bone core biopsies, only six cores from the test group and six cores from the control group were suitable for the analysis. RESULTS: Bone volume (BV) in the test group was maintained, but tissue maturation appeared to be delayed. In contrast, tissue maturation appeared to be completed in the control group, but BV was compromised. Micro-CT analysis showed that specimens from the control group were more structured and mineralized compared with those from the test group. Histologic analysis showed more residual graft particles scattered in a loose fibrous connective tissue matrix with sparse bone formation in the test group, while the control group showed obvious vital bone formation surrounding the residual graft particles. Positive periostin (POSTN), sclerostin, and runt-related transcription factor-2 (RUNX2) immunoreactivities were detected in both the control and test groups. However, tartrate-resistant acid phosphatase (TRAP) positive was mostly noted in the control group. There were significant differences in POSTN, RUNX2 and VEGF expressions between the test and control groups. CONCLUSION: These findings indicated that the SBA technique was an effective method in preserving adequate structural volume while promoting new vital bone formation. Use of the collagen barrier membrane has successfully maintained the volumetric dimensions of the ridge but might have slowed down the complete maturation of the outermost layer of the grafted site.

Immunolocalization of FGF-2 and VEGF in rat periodontal ligament during experimental tooth movement.

OBJECTIVE: This article aimed at identifying the expression of fibroblast growth factor-2 (FGF-2) and vascular endothelial growth factor (VEGF) in the tension and pressure areas of rat periodontal ligament, in different periods of experimental orthodontic tooth movement. METHODS: An orthodontic force of 0.5 N was applied to the upper right first molar of 18 male Wistar rats for periods of 3 (group I), 7 (group II) and 14 days (group III). The counter-side first molar was used as a control. The animals were euthanized at the aforementioned time periods, and their maxillary bone was removed and fixed. After demineralization, the specimens were histologically processed and embedded in paraffin. FGF-2 and VEGF expressions were studied through immunohistochemistry and morphological analysis. RESULTS: The experimental side showed a higher expression of both FGF-2 and VEGF in all groups, when compared with the control side (P < 0.05). Statistically significant differences were also found between the tension and pressure areas in the experimental side. CONCLUSION: Both FGF-2 and VEGF are expressed in rat periodontal tissue. Additionally, these growth factors are upregulated when orthodontic forces are applied, thereby suggesting that they play an important role in changes that occur in periodontal tissue during orthodontic movement.

Expression of bone markers and micro-CT analysis of alveolar bone during orthodontic relapse.

OBJECTIVES: To investigate biological changes in alveolar bone occurring during orthodontic relapse. MATERIALS AND METHODS: Rat maxillary first molars were moved mesially for 10 days. After orthodontic tooth movement (OTM), appliances were removed, and the molars were allowed to relapse for one, three, five, seven, 14 or 21 days. Changes in 3D morphometric parameters of bone located mesial to the first molars were evaluated by micro-CT. Total RNA was isolated from the same bone site, and real-time RT-PCR was used to measure the expression of bone formation and resorption markers. RESULTS: One day after appliance removal, the molars relapsed to a mean 73% of the achieved OTM and then steadily relapsed to 93% at 21 days. Tissue mineral density and per cent bone volume increased over the experimental period. Inversely, there was a decrease in total porosity. Gene expression of OCN, Coll-I and ALP decreased during OTM, whilst as the molars relapsed showed tended to increase. Gene expression of RANKL and TRAP increased during OTM. Changes in mRNA expression of H(+)-ATPase were minor. By 21 days post-appliance removal, the remodelling process in rats appeared to have returned to control levels. CONCLUSIONS: Bone tissue reactions on a molecular level are similar during OTM and orthodontic relapse. These findings validate the importance of immediate retention following active OTM.

Immunolocalization of FGF-2 and VEGF in rat periodontal ligament during experimental tooth movement

OBJECTIVE: This article aimed at identifying the expression of fibroblast growth factor-2 (FGF-2) and vascular endothelial growth factor (VEGF) in the tension and pressure areas of rat periodontal ligament, in different periods of experimental orthodontic tooth movement. METHODS: An orthodontic force of 0.5 N was applied to the upper right first molar of 18 male Wistar rats for periods of 3 (group I), 7 (group II) and 14 days (group III). The counter-side first molar was used as a control. The animals were euthanized at the aforementioned time periods, and their maxillary bone was removed and fixed. After demineralization, the specimens were histologically processed and embedded in paraffin. FGF-2 and VEGF expressions were studied through immunohistochemistry and morphological analysis. RESULTS: The experimental side showed a higher expression of both FGF-2 and VEGF in all groups, when compared with the control side (P < 0.05). Statistically significant differences were also found between the tension and pressure areas in the experimental side. CONCLUSION: Both FGF-2 and VEGF are expressed in rat periodontal tissue. Additionally, these growth factors are upregulated when orthodontic forces are applied, thereby suggesting that they play an important role in changes that occur in periodontal tissue during orthodontic movement. .

OBJETIVO: o objetivo desse estudo foi identificar a expressão do fator de crescimento de fibroblastos 2 (FGF-2) e do fator de crescimento vascular endotelial (VEGF) nos lados de tensão e pressão do ligamento periodontal de ratos, durante movimento ortodôntico experimental, em diferentes períodos de tempo. MÉTODOS: uma força ortodôntica de 0,5N foi aplicada no primeiro molar superior direito de 18 ratos Wistar machos, por períodos de 3 (grupo I), 7 (grupo II) e 14 dias (grupo III). O primeiro molar do lado oposto foi utilizado como controle. Os animais foram sacrificados nos períodos de tempo mencionados, sendo a arcada superior removida e fixada. Após a desmineralização, os espécimes foram processados histologicamente e embebidos em parafina. A expressão do FGF-2 e do VEGF foram estudadas por meio de análise imuno-histoquímica. RESULTADOS: o ligamento periodontal dos dentes submetidos à movimentação ortodôntica mostraram maior expressão tanto de FGF-2 quanto de VEGF, em todos os grupos experimentais, quando comparados com os dentes do lado controle (p < 0,05). Diferenças estatisticamente significativas entre os lados de tensão e pressão também foram encontradas nos dentes submetidos à movimentação ortodôntica. CONCLUSÕES: tanto o FGF-2 quanto o VEGF são expressos no tecido periodontal de ratos, e esses fatores de crescimento são aumentados quando forças ortodônticas são aplicadas, sugerindo que esses desempenham um papel importante na reorganização do periodonto durante o movimento ortodôntico. .

From genotype to phenotype: the differential expression of FGF, FGFR, and TGFbeta genes characterizes human cranioskeletal development and reflects clinical presentation in FGFR syndromes.

Mutations in the fibroblast growth factor receptor (FGFR) genes 1, 2, and 3 are causal in a number of craniofacial dysostosis syndromes featuring craniosynostosis with basicranial and midfacial deformity. Great clinical variability is displayed in the pathologic phenotypes encountered. To investigate the influence of developmental genetics on clinical diversity in these syndromes, the expression of several genes implicated in their pathology was studied at sequential stages of normal human embryo-fetal cranial base and facial ossification (n = 6). At 8 weeks of gestation, FGFR1, FGFR2, and FGFR3 are equally expressed throughout the predifferentiated mesenchyme of the cranium, the endochondral skull base, and midfacial mesenchyme. Both clinically significant isoforms of FGFR2, IgIIIa/c and IgIIIa/b, are coexpressed in maxillary and basicranial ossification. By 10 to 13 weeks, FGFR1 and FGFR2 are broadly expressed in epithelia, osteogenic, and chondrogenic cell lineages. FGFR3, however, is maximally expressed in dental epithelia and proliferating chondrocytes of the skull base, but poorly expressed in the osteogenic tissues of the midface. FGF2 and FGF4, but not FGF7, and TGFbeta1 and TGFbeta3 are expressed throughout both osteogenic and chondrogenic tissues in early human craniofacial skeletogenesis. Maximal FGFR expression in the skull base proposes a pivotal role for syndromic growth dysplasia at this site. Paucity of FGFR3 expression in human midfacial development correlates with the relatively benign human mutant FGFR3 midfacial phenotypes. The regulation of FGFR expression in human craniofacial skeletogenesis against background excess ligand and selected cofactors may therefore play a profound role in the pathologic craniofacial development of children bearing FGFR mutations.

p75NTR and Trk receptors are expressed in reciprocal patterns in a wide variety of non-neural tissues during rat embryonic development, indicating independent receptor functions.

The p75 kDa neurotrophin receptor (p75NTR) has been detected in a number of non-neural tissues, especially during development. Reports of Trk receptor transcripts in non-neural tissues raise the possibility that the sites of p75NTR expression during development may correlate with Trk receptor expression. Coexpression of p75NTR with the Trk receptors in developing non-neural tissues would support the hypothesis that there is a cooperative function between the two receptor subclasses. To address these questions, p75NTR was localized relative to the three known Trk receptors in adjacent sections of rat embryos at stages of development when the highest levels of p75NTR have been observed in the muscle, maxillary pad, kidney, and lung. Using in situ hybridization and immunhistochemical analyses, we show here that the Trk receptors are expressed extensively in non-neural tissues during cell differentiation and tissue morphogenesis but in patterns that are generally reciprocal to that of p75NTR. The results indicate p75NTR most likely functions independently of the Trk receptors in most developing non-neural tissues. However, the p75NTR consistently appears in non-neural cells adjacent to those expressing Trk receptors. The reciprocal patterns of expression indicate that the separate activities of the two receptors most likely complement each other in regulating cell-cell interactions important for the innervation of developing non-neural tissues.

Tooth and bone tissue analysis by element mapping spectrometry.

Elemental analyses of the tooth and bone tissues were carried out by X-ray fluorescence element mapping spectrometry, a technique we have recently developed. This analytical system has the advantage of providing data on distribution of element concentration in a given specimen in overhead view without involving destruction of the tissue architecture. This paper briefly introduces the apparatus of this system and presents analytical data obtained for several elements (calcium, iron, arsenic, zinc and strontium) in the tooth and bone tissues of rats bearing tumor induced with lead acetate. Lead distributed in the maxillary incisive bone and femur was demonstrated in rats with lead acetate-induced tumor sacrificed at 42 weeks after injections. Arsenic observed in the femur of control rats was found replaced by lead in the tumor bearing rats. The findings are of interest as viewed with reference to chelating activity of lead.