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Feb 22, 2018 - Background and Objective: Transfection of cementum protein 1 (CEMP1) into human gingival fibroblasts (HGFs) notably increases cell ...
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

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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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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