Accepted: 22 February 2018 DOI: 10.1111/jre.12553
ORIGINAL ARTICLE
Cementum protein 1 transfection does not lead to ultrastructural changes in nucleolar organization of human gingival fibroblasts C. E. Villegas-Mercado1,2
| L. T. Agredano-Moreno1,2 | M. Bermúdez3 |
M. L. Segura-Valdez1,2 | H. Arzate4 | E. F. Del Toro-Rangel1,2 | L. F. Jiménez-García1,2
1 Faculty of Sciences, Electron Microscopy Laboratory, National Autonomous University of México (UNAM), Ciudad de Mexico, Mexico 2
Faculty of Sciences, Department of Cell Biology, Cell Nano-Biology Laboratory, National Autonomous University of México (UNAM), Ciudad de Mexico, Mexico 3
School of Higher Education of Zaragoza, National Autonomous University of México (UNAM), Ciudad de Mexico, Mexico 4
Faculty of Dentistry, Periodontal Biology Laboratory, DEPeI, National Autonomous University of Mexico (UNAM), Ciudad de Mexico, Mexico Correspondence Luis Felipe Jiménez-García, Faculty of Sciences, Electron Microscopy Laboratory, National Autonomous University of México (UNAM), Ciudad de Mexico, Mexico. Email:
[email protected] Funding information Consejo Nacional de Ciencia y Tecnología, Grant/Award Number: 180835 and 379385
Background and Objective: Transfection of cementum protein 1 (CEMP1) into human gingival fibroblasts (HGFs) notably increases cell metabolism and results in overexpression of molecules related to biomineralization at transcriptional and protein levels. Therefore, HGF-CEMP1 cells are considered as putative cementoblasts. This represents a significant advance in periodontal research because cementum neoformation is a key event in periodontal regeneration. In addition, it is well known that important changes in cell metabolism and protein expression are related to nucleolar structure and the function of this organelle, which is implicated in ribosome biogenesis. The aim of this study was to determine the effect of transfecting CEMP1 gene in human HGF on the ultrastructure of the nucleolus. Material and Methods: Cells were processed using the conventional technique for transmission electron microscopy, fixed with glutaraldehyde, postfixed with osmium tetraoxide, and embedded in epoxy resin. Semi-thin sections were stained with Toluidine blue and observed by light microscopy. Thin sections were stained with uranyl acetate and lead citrate. For ribonucleoprotein detection, the staining method based on the regressive effect of EDTA was used. In addition, the osmium ammine technique was used for specific staining of DNA. Results: The results obtained in this study suggest that transfection of CEMP1 into HGFs does not produce changes in the general nucleolar ultrastructure because the different components of the organelle are present as fibrillary centers, and dense fibrillar and granular components compared with the control. Conclusion: The transfection of CEMP1 into HGFs allows these cells to perform cementoblast-like functions without alteration of the ultrastructure of the nucleolus, evaluated by the presence of the different compartments of this organelle involved in ribosomal biogenesis. KEYWORDS
cell therapy, cementum protein 1, human gingival fibroblasts, microscopy, nucleolus, ultrastructure
J Periodont Res. 2018;1–7.
wileyonlinelibrary.com/journal/jre
© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
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1 | I NTRO D U C TI O N
assembly, nuclear export pathways, processing of pre-mRNA and noncoding RNAs, modifications of small nuclear ribonucleoproteins
One of the areas currently growing in oral research is periodontal 1
(RNPs), and coordination of the cellular response to stress.16 An es-
biology. Periodontal disease is one of the most common diseases of
tablished role is the nucleolar capacity to adapt its morphology in
the oral cavity. It represents the major cause for tooth loss in adults
response to cellular needs, stimuli, pathologies, viral infections, cel-
2,3
Available periodontal therapies are only capable of re-
lular stress, and even alterations in some epigenetic mechanisms. For
moving the causal agents and stopping progression of the disease.
those reasons, the nucleolus is considered as an important indicator
The biggest hurdle is the limitation to stimulate cementum neofor-
of physiological cell state,15 and the nucleolar ultrastructure can be
mation, which is one of the crucial events required for the regenera-
used as a morphological marker for cell state under physiological,
worldwide.
tion of periodontal structures.
pathological, and experimental conditions.18
4-7
The human cementum protein 1 gene (CEMP1) contains 1 exon,
The literature shows a deficiency of studies focused on the nu-
spans 1.4 kb, and maps to the short arm of chromosome 16 (16p
cleolar ultrastructure of human periodontal cells or transfected cells
13.3).8 CEMP1 protein was first isolated from human cementum and
intended to be used in cell therapy. This prompted us to fulfill such
9
human cementoblastoma-derived conditioned medium. It is com-
a knowledge gap. Consequently, the aim of this study was to de-
posed of 247 amino acids with an initial theoretical molecular mass
termine whether transfection of CEMP1 into healthy HGFs directly
of 25.9 kDa which, after post-translational modifications, increases
affects the nucleolar architecture at the ultrastructural level, and
to 50 kDa.8 The physicochemical characteristics of CEMP1 reveal
hence the cell function.
that it is an alkaline protein with an isoelectric point of 9.73. This protein does not have a signal peptide and its secondary structure comprises 55% β-sheets, 10% α-helix, and 35% random conformation.10 CEMP1 is an intrinsically disordered protein. Intrinsically disordered proteins are characterized by a high percentage of random-coil sec-
2 | M ATE R I A L A N D M E TH O DS 2.1 | Cell culture HGFs were isolated as described by Narayanan and Page.19 HGFs
ondary structures.7 CEMP1 appears to be a key regulator of cementogenesis be-
and HGFs transfected with CEMP1 (HGF-CEMP1) between the
cause it regulates the deposition rate, composition, and morphology
2nd and 5th passages were used for the experiments. The cells
10
Human recombinant CEMP1 has been
were grown in Dulbecco′s modified Eagle′s medium supplemented
shown to promote hydroxyapatite crystal nucleation by triggering
with 10% fetal bovine serum in mineralizing medium (10% fetal
organization of the crystals into parallel arrays, which self-assemble
bovine serum, 10 mmol/L β-glycerophosphate, and 50 μg/mL of
into nanospheres that later form aggregates resembling nanos-
freshly prepared ascorbic acid) in a 5% CO2 and 95% air atmos-
trings. This suggests that organization might facilitate hydroxyapa-
phere at 100% humidity. Untransfected HGFs and HGFs trans-
tite crystal formation and orientation. Studies in vitro demonstrated
fected with CEMP1 cultures were grown for 14 days and processed
the capacity of CEMP1 to promote cell attachment and differenti-
simultaneously.
of hydroxyapatite crystals.
ation of cementoblasts and progenitor cells. It has been shown that CEMP1 induces the differentiation of human periodontal ligament cells in a 3-dimensional culture condition.11 Additionally, CEMP1 is capable of inducing bone healing in critical-sized calvarial defects in vivo.12
2.2 | Construction of a pcDNA40-CEMP1- expressing vector and transfection into HGFs Transfection of HGFs with CEMP1 was performed as described
Transfection of CEMP1 into HGFs (to create the HGF-CEMP1
by Carmona- Rodriguez et al.13 Briefly, the CEMP1 coding region
cell line) gives, as a result, cells with a “mineralizing-like” phenotype.
(GenBank Accession No. NM_001048212) was subcloned into the
These cells are considered as putative cementoblasts because they
pENTR/SD/D vector (Invitrogen, Carlsbad, CA, USA). The construct
increase the rate of proliferation, synthetize mineralized extracel-
pENTR/SD/D-CEMP1 cDNA was then ligated into a pcDNA40(+)
lular matrix (biological- t ype hydroxyapatite), and express higher
vector [CEMP1-pcDNA40(+)] (Invitrogen). The plasmid, pcDNA40-
alkaline phosphatase activity and biomineralization- related and
CEMP1, was transfected into HGFs (to create the HGF-CEMP1 cell
cementum-specific proteins.13 Microarray studies show that CEMP1
line) using Lipofectamine 2000 (Invitrogen). Stable expressing cells
can modulate the expression of several genes related to cellular de-
were selected with G418 at 600 μg/mL (Sigma Chemical Co., St
velopment, cellular growth, cell cycle, and cell death in HGFs trans-
Louis, MO, USA), during 8 weeks.
fected with CEMP1.14 Thus, the effect of CEMP1 in HGFs is known at the transcriptional level but not at the ultrastructural level. The nucleolus is a multifunctional and dynamic organelle that
2.3 | Sample preparation
participates in several vital processes. In addition to ribosomal syn-
Cell pellets of HGFs and the HGF-CEMP1 cell line were processed
thesis, nucleoli have a major role in regulating the cellular cycle,15
for standard transmission electron microscopy. 20 Briefly, all samples
16
control of cell aging, telomer-
were fixed in a mixture of 1.5% glutaraldehyde and 4% paraformal-
ase activity and telomere metabolism,17 signal recognition particle
dehyde, buffered in phosphate-buffered saline (pH 7.2), for 1 hour at
DNA-damage sensing and repair,
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VILLEGAS-MERCADO et al.
room temperature. Half of the samples from both cell lines were also
mounted on gold grids without membrane support. The grids were
postfixed with 1% osmium tetraoxide for 4 hours. All samples were
treated with 5M HCl for 1 hour in a humid chamber and then washed
subsequently dehydrated by incubation in a series of ascending con-
gently and immersed in the osmium ammine staining solution for
centrations of ethanol and embedded in an epoxy resin (Epon 812;
24 hours. 23 Finally, the grids were washed and dried for examination
Electron Microscopy Science, Hatfield, PA, USA).
with a transmission electron microscope at 80 kV.
2.4 | Light microscopy
3 | R E S U LT S
Semi-thin sections (250 nm) were stained using Toluidine blue. 21 Microphotographs were taken in bright field using a light microscope
We used semi-thin sections stained with Toluidine blue to verify the
equipped with a 40× achromatic objective of 0.65 numeric aperture
integrity of processed samples. Nucleoli of both cell lines were vis-
and a Canon camera utilizing the EOS-1000D program. (Canon USA,
ible, by light microscopy, using this technique. We found that HGFs
Inc., Melville, NY, USA)
and the HGF-CEMP1 cell line have nucleoli with similar size, number, and form, as shown in Figure 1. At this level, no differences between
2.5 | Standard transmission electron microscopy Ultrathin sections (50-60 nm) were mounted on formvar-coated cop-
the nucleoli of HGFs before and after transfection of CEMP1 were observed. In addition, this technique helped us to select areas from which ultrathin sections were obtained.
per grids, then stained, using the double-contrast technique, with
In order to analyze the nucleolar ultrastructure, the standard
uranyl acetate for 30 minutes followed by lead citrate for 15 min-
technique for electron microscopy was used, which allowed identifi-
utes. Later, the sections were rinsed with deionized water and dried.
cation of the nucleolar structural components, their distribution, and
Subsequently, they were examined at 80 kV with a JEOL 1010 trans-
their organization. We found that fibrillar centers (FCs), dense fibril-
mission electron microscope (JEOL, Peabody, MA, USA). Depending
lar component (DFC), and granular component (GC) are present and
on the structure of interest, multiple microphotographs were taken
structurally unaltered in both in HGFs and HGF-CEMP1 cells, which
with a magnification range of 5000× to 120 000×.
implies that FCs are surrounded by the DFC and that the DFC is surrounded by the GC. Figure 2 shows nucleoli contrasted using this technique, for HGF (A, C) and HGF-CEMP1 (B, D). As shown in the
2.6 | Preferential staining of RNPs
images at higher magnifications (C, D) the nucleolar ultrastructure
To perform the preferential regressive method for RNPs, 22 sam-
indicates that HGF and HGF-CEMP1 cells are metabolically active
ples without post-fixation of HGF and HGF-CEMP1 were cut into
with full capacity to perform ribosome synthesis. This ultrastructural
ultrathin sections and mounted on formvar-coated copper grids. All
evidence suggests appropriate cellular functioning.
the grids were treated with uranyl acetate for 2 minutes, followed by
The preferential regressive technique using EDTA marks RNPs in
treatment with EDTA for 14 minutes and then with lead citrate for
nucleoli and clusters of interchromatin granules. The results suggest
2 minutes. Thereafter, samples were examined using transmission
that nucleoli of HGF-CEMP1 preserved a normal composition of RNPs.
electron microscopy at 80 kV.
In addition, the organization of nucleolar structural components (FCs, DFC, and GC) was corroborated by this technique (Figure 3B). It is im-
2.7 | Osmium ammine staining of DNA
portant to note that no alterations or disaggregations of the nucleolar components were found in the transfected cells.
The osmium ammine technique is specific for detecting structures
Osmium ammine is a specific staining technique for DNA. As
containing DNA. Sections of about 90- 120 nm thickness were
shown in Figure 4, both cell types presented a greater amount of
(A)
F I G U R E 1 Light microscopy of semi- thin sections of human gingival fibroblasts (HGFs) (A) and HGFs transfected with cementum protein 1 (HGF-CEMP1) (B) stained with Toluidine blue. Nucleoli (arrows) are observed within the nuclei of both cell lines
(B)
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VILLEGAS-MERCADO et al.
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(A)
(B)
(C)
(D)
(A)
F I G U R E 2 Transmission electron microscopy of human gingival fibroblasts (HGFs) (A, C) and HGFs transfected with cementum protein 1 (HGF-CEMP1) (B, D) stained with uranyl acetate-lead citrate. Low and high magnifications show nucleoli (n) within the nuclei (N) containing all nucleolar elements. Cyt, cytoplasm; DFC, dense fibrillar component; FC, fibrillar center; GC, granular component; NE, nuclear envelope
(B)
F I G U R E 3 Transmission electron microscopy of human gingival fibroblasts (HGFs) (A) and HGFs transfected with cementum protein 1 (HGF-CEMP1) (B) after application of the staining method, based on the regressive effect of EDTA, for identifying ribonucleoproteins. Both cell variants display structural components within the nucleoli (n). C, cytoplasm; DFC, dense fibrillar component; FC, fibrillar center; GC, granular component; N, nucleus, NE, nuclear envelope
(A)
(B)
F I G U R E 4 Transmission electron microscopy of human gingival fibroblasts (HGFs) (A) and HGFs transfected with cementum protein 1 (HGF-CEMP1) (B) after treatment with the osmium ammine technique for detecting structures containing DNA. Nucleoli (n) are stain- negative and clumps of compact chromatin (chr) are displayed within nuclei (N) and around nucleoli (arrows). NE, nuclear envelope compact chromatin in close relation with the nuclear envelope
compact chromatin were located within the nucleolus, but de-
and a smaller amount interacting with nucleoli in a perinucleo-
spite this, compact chromatin still preserves the same distribu-
lar arrangement (arrows). In HGF-C EMP1 cells, small fibers of
tion as in HGF.
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VILLEGAS-MERCADO et al.
4 | D I S CU S S I O N
Previous studies have also investigated ultrastructural changes after gene transfection, as in Batra et al. In their study, the mucin 1
In this study, it was proved that transfection of HGFs with CEMP1 in-
gene (MUC1) was transfected into pancreatic cells. However, their
duces no changes in the morphology of their nucleoli. Light and elec-
attention was focused only on the ultrastructural changes in cyto-
tron microscopy were used to analyze the nucleolus before and after
plasm, finding an increase in rough endoplasmic reticulum along with
the transfection. To obtain additional information, different contrast
the appearance of several central granules of various sizes in the
techniques, such as standard staining for transmission electron mi-
Golgi area related to increased secretory activity.32 Barbasian et al.
croscopy, RNP preferential staining, and osmium amine staining,
used nuclear and nucleolar image analysis to search for morpho-
were utilized to evaluate nucleolar aspects, such as the ultrastruc-
metric changes in human breast epithelial cell lines transfected with
tural arrangement of its components and the distribution of DNA
an oncogene. In their study, the nucleolar changes (nucleolar area/
in it. After transfection of CEMP1 into HGFs, the organization and
nuclear area ratio) were related to the tumorigenic phenotype.33
interaction of nucleolar components was preserved. This suggests
Nevertheless, no prior study that employed nucleolar ultrastructure,
that the nucleolar function of ribosomal synthesis is being carried
suggesting that cellular processes are affected after gene transfec-
out normally. More importantly, the ultrastructural characteristics
tion, was found in the literature.
were not those found in cells with damage, stress, or deregulation of
Given the valuable information provided by the nucleolar ultrastructure, analysis of the nucleolar components after CEMP1
cellular processes in which the nucleolus participates. In order to use the nucleolar ultrastructure as a physiological indi-
transfection becomes relevant. We were able to corroborate that
cator, it is indispensable to know the relationship between structure
HGF-CEMP1 cells contain all 3 nucleolar components (FCs, DFCs,
and function within the nucleolus, as well as the normal organization
and GC) as in other cell systems, thus strongly suggesting full capac-
of its components. This is required to discern between structural dif-
ity for ribosomal synthesis. As none of the structural modifications
ferences that could indicate alterations in some cellular processes.
related to DNA damage, cell stress, or alterations in nucleolar epi-
Nucleolar organization is directly related to ribosomal synthesis
genetic mechanisms were found, it can be inferred that transfection
activity. 24 FCs are sites proposed as protein storage sites, needed
of CEMP1 does not cause any such damage in HGFs.
for ribosomal DNA transcription (performed by RNA polymerase 1).
Achieving periodontal regeneration involves many challenges,
FCs are surrounded by DFC. The transition zone between these 2
the most remarkable being: the simultaneous and coordinated pro-
components is where ribosomal DNA transcription takes place. In
duction of mineralized, fibrous, and epithelial tissues;6 restoration
DFC, the initial assembly of ribosomal subunits is performed and it
of alveolar bone height to the cementuo-enamel junction; the re-
contains pre-ribosomal RNA chains associated with the DNA tem-
generation of gingival connective tissue destroyed by inflamma-
plate. These pre-ribosomal RNAs are related to proteins and small
tion; the formation of new acellular extrinsic fiber cementum on
nucleolar RNAs, whereas in the GC, pre-ribosomal complexes that
previously exposed root surfaces; the synthesis of Sharpey’s fibers
have moved away from the pre-RNA synthesis site can be found. It
and their insertion in root surfaces; and the re-establishment of
presents different states of assembly and maturation because GC
the epithelial seal at the coronal portion of the root. 34 As a result
operates as a storage compartment until release of pre-ribosomal
of the degree of complexity involved, it is expected that cell struc-
complexes is required.
25
tures and different inducing molecules should be present, as in the
Several authors have documented the structural changes that
tissues unaffected by periodontal disease. Additionally, it is highly
occur in the nucleolus in response to alterations in cellular pro-
probable that the combination of techniques, such as cell therapy
cesses. One of the main cytological features of cancer cells is nucle-
and guided tissue regeneration, may also be needed. Moreover,
26,27
During apoptosis, nucleolar segregation has
novel biomaterials that function as scaffolds to the cells will be im-
been reported to lead to formation of clusters which contain inter-
portant in order to determine the optimal therapy that can offer
chromatin and perichromatin granules, and perichromatin fibers. 25
the best results.
olar hypertrophy.
In ribosomal DNA transcriptional arrest induced by physiological
So far, various approaches have been explored in periodontal
conditions or low doses of actinomycin D, a segregation of nucle-
regeneration. Much research has been focused on investigating
oli characterized by separation of the nucleolar components occurs.
the potential of dental-d erived postnatal stem cells, 35 periodontal
28
ligament stem cells, 36,37 dental pulp-d erived stem cells, 38,39 stem
In the case of cellular stress caused by pathogens (cardiac diseases),
cells from exfoliated deciduous teeth,40 dental follicle- d erived
an increase in nucleolar size with a higher number of FCs and DFC
stem cells,41-43 dental apical papilla stem cells,44 and dental socket-
Such components remain superimposed but no longer intermingle.
By contrast, in cellular stress caused by DNA
derived stem cells.45 In addition, studies have used induced plu-
damage, a segregation of nucleolar components characterized by
ripotent stem cells 46 for periodontal regeneration.47-49 Literature
the condensation and subsequent separation of both the FC and GC,
suggests that the safest approach to cell therapy is autologous
and the formation of nucleolar caps around the nucleolar remnant,
transplantation. Clinical application of autologous fibroblast cells
occurs.30 When alterations in epigenetic mechanisms that regulate
in patients has been carried out with excellent results in treat-
the expression of ribosomal genes are present, GC segregation and
ment of gingival recession. 50 A study using autologous cell ther-
has been observed.
29
loss of FCs and DFCs have been observed.
31
apy in an animal model has achieved periodontal regeneration;51
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VILLEGAS-MERCADO et al.
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nevertheless, the extraction of 2 healthy dental organs was re-
contained herein are those of the authors and do not necessary re-
quired to obtain the cells required. This study shows that au-
flect those of the sponsors.
tologous cell therapy could achieve periodontal regeneration, although the need for dental extractions to obtain the cells considerably limits its application in patients. This limitation could be avoided by using autologous fibroblasts transfected with CEMP1,
ORCID C. E. Villegas-Mercado
http://orcid.org/0000-0001-5729-4677
an approach similar to that used in the present study. The model of HGFs transfected with CEMP1 is promising because previous studies with CEMP1 have proven its role as a
REFERENCES
local regulator of cell differentiation and in extracellular matrix
1. Slots J. Periodontology: past, present, perspectives. Periodontol 2000. 2013;62:7‐19. 2. Kassebaum NJ, Bernabe E, Dahiya M, Bhandari B, Murray CJ, Marcenes W. Global burden of severe periodontitis in 1990- 2010: a systematic review and meta- regression. J Dent Res. 2014;93:1045‐1053. 3. Pihlstrom BL, Michalowicz BS, Johnson NW. Periodontal diseases. Lancet. 2005;366:1809‐1820. 4. Rios HF, Lin Z, Oh B, Park CH, Giannobile WV. Cell-and gene-based therapeutic strategies for periodontal regenerative medicine. J Periodontol. 2011;82:1223‐1237. 5. Zeichner-David M. Regeneration of periodontal tissues: cementogenesis revisited. Periodontol 2000. 2006;41:196‐217. 6. Magan A, Ripamonti U. Biological aspects of periodontal tissue regeneration: cementogenesis and the induction of Sharpey’s fibres. SADJ. 2013;68:304‐306, 8-12, 14 passim. 7. Arzate H, Zeichner-David M, Mercado-Celis G. Cementum proteins: role in cementogenesis, biomineralization, periodontium formation and regeneration. Periodontol 2000. 2015;67:211‐233. 8. Alvarez-Perez MA, Narayanan S, Zeichner-David M, Rodriguez Carmona B, Arzate H. Molecular cloning, expression and immunolocalization of a novel human cementum-derived protein (CP-23). Bone. 2006;38:409‐419. 9. Alvarez Perez MA, Pitaru S, Alvarez Fregoso O, Reyes Gasga J, Arzate H. Anti- cementoblastoma- derived protein antibody partially inhibits mineralization on a cementoblastic cell line. J Struct Biol. 2003;143:1‐13. 10. Villarreal-Ramirez E, Moreno A, Mas-Oliva J, et al. Characterization of recombinant human cementum protein 1 (hrCEMP1): primary role in biomineralization. Biochem Biophys Res Commun. 2009;384:49‐54. 11. Hoz L, Romo E, Zeichner-David M, et al. Cementum protein 1 (CEMP1) induces differentiation by human periodontal ligament cells under three- dimensional culture conditions. Cell Biol Int. 2012;36:129‐136. 12. Serrano J, Romo E, Bermudez M, et al. Bone regeneration in rat cranium critical-size defects induced by cementum protein 1 (CEMP1). PLoS One. 2013;8:e78807. 13. Carmona-Rodriguez B, Alvarez-Perez MA, Narayanan AS, et al. Human cementum protein 1 induces expression of bone and cementum proteins by human gingival fibroblasts. Biochem Biophys Res Commun. 2007;358:763‐769. 14. Bermudez M, Imaz-Rosshandler I, Rangel-Escareno C, ZeichnerDavid M, Arzate H, Mercado-Celis GE. CEMP1 induces transformation in human gingival fibroblasts. PLoS One. 2015;10:e0127286. 15. Pederson T. The nucleolus. Cold Spring Harb Perspect Biol. 2011;3:pii: a000638. 16. Lam YW, Trinkle-Mulcahy L. New insights into nucleolar structure and function. F1000Prime Rep. 2015;7:48. 17. Pederson T. The plurifunctional nucleolus. Nucleic Acids Res. 1998;26:3871‐3876. 18. Smetana K. The nucleolus through the years. J Appl Biomed. 2011;9:119‐127.
mineralization.9 CEMP1 has the ability to select progenitor cells present in the periodontal ligament, to differentiate toward the cementoblastic phenotype. 52 Moreover, CEMP1 has the ability to select multipotent stem cells to differentiate into various cell phenotypes. 53 Additionally, CEMP1 promotes the migration of STRO1-p ositive cells and provides a possible mechanism for the recruitment of mesenchymal cells through migration toward the CEMP1 signal. 54 HGF-CEMP1 could be used in autologous cell therapy, with the additional advantage of obtaining tissue samples without sacrificing healthy dental organs because a sample of the interdental papilla will provide large numbers of cells in a simple and noninvasive procedure for the patient. Those cells could be expanded in vitro and transfected with CEMP1 to be transformed into putative cementoblasts. Nevertheless, further research is required before this model is suitable for clinical application. This study represents an initial approach, but important information is still required. In addition, biochemical and molecular techniques may be needed to monitor if ribosome biogenesis is altered at that level. Furthermore, determination of the optimal number of cells required to achieve tissue regeneration and the establishment of a suitable scaffold that facilities the permanence of such cells in periodontal defects should be investigated. Future work may emphasize the associated effectiveness of this methodology in animal models with a long-term follow up. If positive results are obtained, then clinical trials can be addressed. It can be concluded that transfection of CEMP1 into HGFs produces no changes in the ultrastructure of the nucleoli, strongly suggesting that ribosomal biogenesis is carried out as in nontransfected cells. This and other aspects of different cell structures may be taken into account for further applications in autologous cell therapy. The results from this work are very promising, thus encouraging us to continue with the next stages of the research.
AC K N OW L E D G E M E N T S This work was supported by the Consejo Nacional de Ciencia y Tecnologia (CONACYT) 180835. DGAPA- UNAM- IT200717. The first author acknowledges CONACYT for the scholarship No. 379385, the Universidad Autónoma de Sinaloa (UAS) for the financial support through the institutional program “Doctores Jóvenes”, and the Academic Writing Office at UNAM for the help provided while revising the manuscript. The ideas, discussions, and conclusions
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19. Narayanan AS, Page RC. Biochemical characterization of collagens synthesized by fibroblasts derived from normal and diseased human gingiva. J Biol Chem. 1976;251:5464‐5471. 20. Vázquez Nin G, Echeverría O. Introducción a la Microscopía Electrónica Aplicada a las Ciencias Biológicas. Mexico City, Mexico: Fondo de la Cultura Económica; 2000:167. 21. Segura-Valdez ML, Chávez R, Agredano-Moreno LT, et al. Microscopy of in situ DNA and RNA-containing structures. In: Méndez-Vilas A, ed. Microscopy: Advances in Scientific Research and Education. Microscopy Book Series N 6. VOL. 1. Badajoz, Spain: Formatex Research Center; 2014:492‐502. 22. Bernhard W. A new staining procedure for electron microscopical cytology. J Ultrastruct Res. 1969;27:250‐265. 23. Spector DL, Goldman RD, Leinwand LA. Cells a laboratory manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 1998:2136. 24. Olson MO, Dundr M. Nucleolus: structure and function. In: eLS. Chichester, UK: John Wiley & Sons Ltd; 2015:1‐9. 25. Biggiogera M, Bottone MG, Scovassi AI, Soldani C, Vecchio L, Pellicciari C. Rearrangement of nuclear ribonucleoprotein (RNP)- containing structures during apoptosis and transcriptional arrest. Biol Cell. 2004;96:603‐615. 26. Derenzini M, Trere D, Pession A, Govoni M, Sirri V, Chieco P. Nucleolar size indicates the rapidity of cell proliferation in cancer tissues. J Pathol. 2000;191:181‐186. 27. Farley KI, Surovtseva Y, Merkel J, Baserga SJ. Determinants of mammalian nucleolar architecture. Chromosoma. 2015;124:323‐331. 28. Hernandez-Verdun D. Nucleolus: from structure to dynamics. Histochem Cell Biol. 2006;125:127‐137. 29. Rosello-Lleti E, Rivera M, Cortes R, et al. Influence of heart failure on nucleolar organization and protein expression in human hearts. Biochem Biophys Res Commun. 2012;418:222‐228. 3 0. Boulon S, Westman BJ, Hutten S, Boisvert FM, Lamond AI. The nucleolus under stress. Mol Cell. 2010;40:216‐227. 31. Hernandez-Hernandez A, Soto-Reyes E, Ortiz R, et al. Changes of the nucleolus architecture in absence of the nuclear factor CTCF. Cytogenet Genome Res. 2012;136:89‐96. 32. Batra SK, Kern HF, Worlock AJ, Metzgar RS, Hollingsworth MA. Transfection of the human Muc 1 mucin gene into a poorly differentiated human pancreatic tumor cell line, Panc1: integration, expression and ultrastructural changes. J Cell Sci. 1991;100(Pt 4):841‐849. 33. Barbisan LF, Russo J, Mello ML. Nuclear and nucleolar image analysis of human breast epithelial cells transformed by benzo[a]pyrene and transfected with the c- Ha- ras oncogene. Anal Cell Pathol. 1998;16:193‐199. 3 4. Grzesik WJ, Narayanan AS. Cementum and periodontal wound healing and regeneration. Crit Rev Oral Biol Med. 2002;13:474‐484. 3 5. Bassir SH, Wisitrasameewong W, Raanan J, et al. Potential for stem cell- b ased periodontal therapy. J Cell Physiol. 2016;231:50‐61. 36. Seo BM, Miura M, Gronthos S, et al. Investigation of multipotent postnatal stem cells from human periodontal ligament. Lancet. 2004;364:149‐155. 37. Liu Y, Zheng Y, Ding G, et al. Periodontal ligament stem cell- mediated treatment for periodontitis in miniature swine. Stem Cells. 2008;26:1065‐1073. 38. Gronthos S, Mankani M, Brahim J, Robey PG, Shi S. Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo. Proc Natl Acad Sci U S A. 2000;97:13625‐13630.
39. Graziano A, d’Aquino R, Cusella-De Angelis MG, et al. Scaffold’s surface geometry significantly affects human stem cell bone tissue engineering. J Cell Physiol. 2008;214:166‐172. 4 0. Miura M, Gronthos S, Zhao M, et al. SHED: stem cells from human exfoliated deciduous teeth. Proc Natl Acad Sci U S A. 2003;100:5807‐5812. 41. Yokoi T, Saito M, Kiyono T, et al. Establishment of immortalized dental follicle cells for generating periodontal ligament in vivo. Cell Tissue Res. 2007;327:301‐311. 42. Park JY, Jeon SH, Choung PH. Efficacy of periodontal stem cell transplantation in the treatment of advanced periodontitis. Cell Transplant. 2011;20:271‐285. 43. Guo W, Chen L, Gong K, Ding B, Duan Y, Jin Y. Heterogeneous dental follicle cells and the regeneration of complex periodontal tissues. Tissue Eng Part A. 2012;18:459‐470. 4 4. Xu L, Tang L, Jin F, et al. The apical region of developing tooth root constitutes a complex and maintains the ability to generate root and periodontium-like tissues. J Periodontal Res. 2009;44:275‐282. 45. Nakajima R, Ono M, Hara ES, et al. Mesenchymal stem/progenitor cell isolation from tooth extraction sockets. J Dent Res. 2014;93:1133‐1140. 46. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006;126:663‐676. 47. Duan X, Tu Q, Zhang J, et al. Application of induced pluripotent stem (iPS) cells in periodontal tissue regeneration. J Cell Physiol. 2011;226:150‐157. 48. Hynes K, Menicanin D, Han J, et al. Mesenchymal stem cells from iPS cells facilitate periodontal regeneration. J Dent Res. 2013;92:833‐839. 49. Yang H, Aprecio RM, Zhou X, et al. Therapeutic effect of TSG-6 engineered iPSC-derived MSCs on experimental periodontitis in rats: a pilot study. PLoS One. 2014;9:e100285. 50. Milinkovic I, Aleksic Z, Jankovic S, et al. Clinical application of autologous fibroblast cell culture in gingival recession treatment. J Periodontal Res. 2015;50:363‐370. 51. Nunez J, Sanz-Blasco S, Vignoletti F, et al. Periodontal regeneration following implantation of cementum and periodontal ligament- derived cells. J Periodontal Res. 2012;47:33‐44. 52. Komaki M, Iwasaki K, Arzate H, Narayanan AS, Izumi Y, Morita I. Cementum protein 1 (CEMP1) induces a cementoblastic phenotype and reduces osteoblastic differentiation in periodontal ligament cells. J Cell Physiol. 2012;227:649‐657. 53. Xu J, Wang W, Kapila Y, Lotz J, Kapila S. Multiple differentiation capacity of STRO-1+/CD146+ PDL mesenchymal progenitor cells. Stem Cells Dev. 2009;18:487‐496. 54. Paula-Silva FW, Ghosh A, Arzate H, Kapila S, da Silva LA, Kapila YL. Calcium hydroxide promotes cementogenesis and induces cementoblastic differentiation of mesenchymal periodontal ligament cells in a CEMP1-and ERK-dependent manner. Calcif Tissue Int. 2010;87:144‐157.
How to cite this article: Villegas-Mercado CE , AgredanoMoreno L. T. , Bermúdez M. , et al. Cementum protein 1 transfection does not lead to ultrastructural changes in nucleolar organization of human gingival fibroblasts. J Periodont Res. 2018:00:1–7. https://doi.org/10.1111/jre.12553
