Resumo Como os cães são um bom modelo para estudos in vivo, é interessante avaliar o comportamento de fibroblastos gengivais de cão (CGF) in vitro, para que essas células possam ser cultivadas em uma matriz e estudadas in vivo posteriormente. O objetivo do presente estudo foi realizar uma análise morfológica, funcional e bioquímica de CGF, comparando-os a fibroblastos gengivais humanos (HGF), bem como avaliar as alterações dessas características ao longo de várias passagens. Usando fibroblastos gengivais de 3 cães e 3 indivíduos na subcultura (Sub), primeira (P1), terceira (P3), quinta (P5) e sétima (P7) passagens, os seguintes parâmetros foram avaliados: morfologia, espraiamento, adesão, viabilidade e conteúdo de proteína total. Os resultados mostraram não haver diferenças significativas quanto à morfologia e espraiamento, e uma tendência a maior adesão e viabilidade para HGF, quando comparados a CGF. O conteúdo de proteína total foi significativamente maior para HGF. HGF exibiram maior atividade funcional e bioquímica in vitro quando comparados a CGF. Maiores valores na Sub foram observados para ambos, CGF e HGF, em todos os parâmetros avaliados. As diferenças não impedem o uso de CGF na engenharia tecidual, contudo, seu uso é mais apropriado na subcultura ou primeira passagem.
Original Articles • Braz. Dent. J. 24
(2)
• Mar-Apr 2013 • https://doi.org/10.1590/0103-6440201302144 linkcopiar
Morphological, Functional and Biochemical Characterization of Canine Gingival Fibroblasts
Autoria
person Camila Bonvicino Pelegrini
schoolDepartment of Traumatology and Bucomaxillofacial Surgery and Periodontology, Ribeirão Preto Dental School, USP - University of São Paulo, Ribeirão Preto, SP, BrazilUSP - University of São PauloBrazilRibeirão Preto, SP, BrazilDepartment of Traumatology and Bucomaxillofacial Surgery and Periodontology, Ribeirão Preto Dental School, USP - University of São Paulo, Ribeirão Preto, SP, Brazil
person Luciana Prado Maia
schoolDepartment of Traumatology and Bucomaxillofacial Surgery and Periodontology, Ribeirão Preto Dental School, USP - University of São Paulo, Ribeirão Preto, SP, BrazilUSP - University of São PauloBrazilRibeirão Preto, SP, BrazilDepartment of Traumatology and Bucomaxillofacial Surgery and Periodontology, Ribeirão Preto Dental School, USP - University of São Paulo, Ribeirão Preto, SP, Brazil
person Sérgio Luís Scombatti de Souza
schoolDepartment of Traumatology and Bucomaxillofacial Surgery and Periodontology, Ribeirão Preto Dental School, USP - University of São Paulo, Ribeirão Preto, SP, BrazilUSP - University of São PauloBrazilRibeirão Preto, SP, BrazilDepartment of Traumatology and Bucomaxillofacial Surgery and Periodontology, Ribeirão Preto Dental School, USP - University of São Paulo, Ribeirão Preto, SP, Brazil
person Mário Taba Jr
schoolDepartment of Traumatology and Bucomaxillofacial Surgery and Periodontology, Ribeirão Preto Dental School, USP - University of São Paulo, Ribeirão Preto, SP, BrazilUSP - University of São PauloBrazilRibeirão Preto, SP, BrazilDepartment of Traumatology and Bucomaxillofacial Surgery and Periodontology, Ribeirão Preto Dental School, USP - University of São Paulo, Ribeirão Preto, SP, Brazil
person Daniela Bazan Palioto
schoolDepartment of Traumatology and Bucomaxillofacial Surgery and Periodontology, Ribeirão Preto Dental School, USP - University of São Paulo, Ribeirão Preto, SP, BrazilUSP - University of São PauloBrazilRibeirão Preto, SP, BrazilDepartment of Traumatology and Bucomaxillofacial Surgery and Periodontology, Ribeirão Preto Dental School, USP - University of São Paulo, Ribeirão Preto, SP, Brazil
Correspondence: Profa. Dra. Daniela Bazan Palioto, Avenida do Café S/N, 14040-904 Ribeirão Preto, SP, Brasil. Tel:
+55-16-3602-4135. e-mail:
dpalioto@forp.usp.br
+55-16-3602-4135. e-mail:
dpalioto@forp.usp.br
SCIMAGO INSTITUTIONS RANKINGS
Department of Traumatology and Bucomaxillofacial Surgery and Periodontology, Ribeirão Preto Dental School, USP - University of São Paulo, Ribeirão Preto, SP, BrazilUSP - University of São PauloBrazilRibeirão Preto, SP, BrazilDepartment of Traumatology and Bucomaxillofacial Surgery and Periodontology, Ribeirão Preto Dental School, USP - University of São Paulo, Ribeirão Preto, SP, Brazil
Figuras | Tabelas
imageFigure 1. Epifluorescence of human gingival fibroblasts (HGF - A, B, C) and canine gingival fibroblasts (CGF - D, E, F), seeded on glass coverslips at 30 min (A, D), 4 h (B, E) and 24 h (C, F). Green fluorescence shows actin cytoskeleton, and blue fluorescence shows cell nuclei. Note the following: in A and D, the presence of cells in the stage 1 of adhesion; in B and E, cells ranging from stage 1 to 3 of adhesion; in C and F, the larger number of cells attached in advanced stages of spreading (40′ objective). open_in_new

imageFigure 2. Cell adhesion of human gingival fibroblasts (HGF) and canine gingival fibroblasts (CGF) in the subculture (Sub) and passages 1 (P1), 3 (P3), 5 (P5) and 7 (P7) at 30 min (A), 2 h (B) and 4 h (C), expressed as a percentage of the initial number of cells. Data are reported as mean ± standard deviation. Asterisks (*) indicate p<0.05. open_in_new

imageFigure 3. Total cell number of human gingival fibroblasts (HGF) and canine gingival fibroblasts (CGF) in the subculture (Sub) and passages 1 (P1), 3(P3), 5 (P5) and 7 (P7) at days 3 (A), 7 (B) and 10 (C). Data are reported as mean ± standard deviation. Asterisks (*) indicate p<0.05. open_in_new

imageFigure 4. Cell viability of human gingival fibroblasts (HGF) and canine gingival fibroblasts (CGF) in subculture (Sub) and passages 1 (P1), 3 (P3), 5 (P5) and 7 (P7) at days 3 (A), 7 (B) and 10 (C), expressed as absorbance. Data are expressed as mean ± standard deviation. Asterisks (*) indicate p<0.05. open_in_new

imageFigure 5. Total protein content of human gingival fibroblasts (HGF) and canine gingival fibroblasts (CGF) in subculture (Sub) and passages 1 (P1), 3(P3), 5 (P5) and 7 (P7) at days 7 (A), 14 (B) and 21 (C), expressed as micrograms of protein per mililiter (µg/mL). Data are expressed as mean ± standard deviation. Asterisks (*) indicate p<0.05. open_in_new

table_chartTable 1.
Quantitative analysis (mean ± SD) of cell adhesion (%) of HGF and CGF at 30 min, 2 and 4 h
| Passage | HGF | CGF | ||||||
|---|---|---|---|---|---|---|---|---|
| 30 min | 2 h | 4 h | Intragroup Difference | 30 min | 2 h | 4 h | Intragroup Difference | |
| Sub | 19 ± 8 abA | 39 ± 17 aB | 58 ± 21 aB | p<0.001 | 18 ± 6 aA | 27 ± 6 abA | 40 ± 4 adB | p<0.001 |
| P1 | 23 ± 6 aA | 31 ± 6 abA | 54 ± 11 aB | p<0.001 | 20 ± 8 aA | 30 ± 8 aA | 51 ± 16 aB | p<0.001 |
| P3 | 16 ± 6 abcA | 34 ± 13 abB | 46 ± 16 abB | p<0.001 | 13 ± 5 abA | 27 ± 7 abA | 43 ± 8 acB | p<0.001 |
| P5 | 14 ± 4 bdA | 29 ± 6 abB | 45 ± 6 abC | p<0.001 | 9 ± 3 bA | 21 ± 8 abB | 33 ± 8 bdB | p<0.001 |
| P7 | 11 ± 4 cdA | 24 ± 5 bB | 40 ± 6 bC | p<0.001 | 8 ± 3 bA | 20 ± 4 bB | 35 ± 7 cdC | p<0.001 |
| Intergroup difference | p<0.001 | p=0.007 | p=0.007 | p<0.001 | p=0.004 | p<0.001 | ||
-
HGF: Human gingival fibroblasts. CGF: Canine gingival fibroblasts. Kruskal-Wallis one-way analysis of variance on ranks. Dunn's Method, significance level of 5%. Between-groups (a, b, c, d) and intragroup (A, B, C) comparisons - different letters indicate statistically significant difference.
table_chartTable 2.
Quantitative analysis (mean ± SD) of total cell number (x104) of HGF and CGF at days 3, 7 and 10
| Passage | HGF | CGF | ||||||
|---|---|---|---|---|---|---|---|---|
| 3 days | 7 days | 10 days | Intragroup Difference | 3 days | 7 days | 10 days | Intragroup Difference | |
| Sub | 15.1 ± 3.7 aA | 61.0 ± 19.1 aB | 76.0 ± 13.9 abB | p<0.001 | 10,6 ± 2.7 abA | 52.3 ± 21.1 aB | 67.3 ± 18.1 aB | p<0.001 |
| P1 | 15.7 ± 3.6 aA | 59.2 ± 15.3 aB | 87.3 ± 19.4 aC | p<0.001 | 13.5 ± 2.8 aA | 33.4 ± 12.6 abB | 55.2 ± 12.6 aB | p<0.001 |
| P3 | 14.0 ± 2.5 abA | 43.3 ± 12.7 abB | 57.1 ± 13.9 bB | p<0.001 | 10.8 ± 2.3 abA | 33.4 ± 5.5 aB | 47.3 ± 8.9 aB | p<0.001 |
| P5 | 13.3 ± 1.1 abA | 37.5 ± 6.3 bcB | 56.3 ± 12.1 bC | p<0.001 | 8.9 ± 1.1 bA | 21.9 ± 4.9 bB | 35.3 ± 8.1 bC | p<0.001 |
| P7 | 11.7 ± 2.9 bA | 25.4 ± 3.5 cB | 38.1 ± 4.1 cC | p<0.001 | 8.8 ± 1.4 bA | 16.7 ± 3.0 cB | 30.7 ± 2.5 bC | p<0.001 |
| Intergroup difference | p=0.010 | p<0.001 | p<0.001 | p<0.001 | p<0.001 | p<0.001 | ||
-
HGF: Human gingival fibroblasts. CGF: Canine gingival fibroblasts. Kruskal-Wallis one-way analysis of variance on ranks. Dunn's Method, significance level of 5%. Intergroup (a, b, c, d) and intragroup (A, B, C) comparisons - different letters indicate statistically significant difference.
table_chartTable 3.
Quantitative analysis (mean ± SD) of cell viability (absorbance, 570 nm) of HGF and CGF at days 3, 7 and 10
| Passage | HGF | CGF | ||||||
|---|---|---|---|---|---|---|---|---|
| 3 days | 7 days | 10 days | Intragroup difference | 3days | 7 days | 10 days | Intragroup Difference | |
| Sub | 0.24 ± 0.08 abA | 0.47 ± 0.75 aB | 0.64 ± 0.15 aB | p<0.001 | 0.15 ± 0.09 abA | 0.32 ± 0.17 aB | 0.41 ± 0.17 aB | p<0.001 |
| P1 | 0.15 ± 0.05 bcA | 0.38 ± 0.06 aB | 0.56 ± 0.17 aB | p<0.001 | 0.13 ± 0.08 abA | 0.25 ± 0.09 aAB | 0.31 ± 0.08 aB | p<0.001 |
| P3 | 0.15 ± 0.05 cA | 0.36 ± 0.12 aB | 0.52 ± 0.14 aB | p<0.001 | 0.16 ± 0.02 aA | 0.26 ± 0.04 aB | 0.33 ± 0.04 aB | p<0.001 |
| P5 | 0.25 ± 0.03 aA | 0.45 ± 0.13 aB | 0.59 ± 0.22 aB | p<0.001 | 0.14 ± 0.06 abA | 0.19 ± 0.09 abAB | 0.25 ± 0.11 abB | p<0.001 |
| P7 | 0.10 ± 0.01 cA | 0.21 ± 0.04 bB | 0.29 ± 0.05 bB | p<0.001 | 0.08 ± 0.02 bA | 0.13 ± 0.03 bB | 0.17 ± 0.04 bB | p<0.001 |
| Intergroup difference | p<0.001 | p<0.001 | p<0.001 | p=0.004 | p<0.001 | p<0.001 | ||
-
HGF: Human gingival fibroblasts. CGF: Canine gingival fibroblasts. Kruskal-Wallis one-way analysis of variance on ranks. Dunn's Method, significance level of 5%. Intergroup (a, b, c, d) and intragroup (A, B, C) comparisons - different letters indicate statistically significant difference.
table_chartTable 4.
Quantitative analysis (mean ± SD) of total protein content (µg/mL) of HGF and CGF at days 7, 14 and 21
| Passage | HGF | CGF | ||||||
|---|---|---|---|---|---|---|---|---|
| 7 days | 14 days | 21 days | Intragroup Difference | 7 days | 14 days | 21 days | Intragroup Difference | |
| Sub | 72.4 ± 21.5 aA | 91.5 ± 20.8 aAB | 106.4 ± 21.0 abB | p=0.003 | 45.6 ± 08.9 aA | 72.2 ± 12.9 aB | 88.2 ± 16.0 aB | p<0.001 |
| P1 | 66.5 ± 9.5 aA | 95.9 ± 9.6 aB | 125.9 ± 16.3 aC | p<0.001 | 41.0 ± 15.6 abA | 58.1 ± 26.1 abAB | 72.9 ± 27.0 abB | p=0.003 |
| P3 | 46.2 ± 13.6 bA | 81.0 ± 13.6 aB | 98.6 ± 12.7 abB | p<0.001 | 34.3 ± 7.0 abA | 51.2 ± 6.9 abB | 66.5 ± 06.7 abC | p<0.001 |
| P5 | 59.1 ± 15.5 acA | 77.8 ± 17.4 aAB | 95.0 ± 17.4 bB | p<0.001 | 28.6 ± 8.0 bcA | 40.3 ± 9.6 bcAB | 51.2 ± 13.56 bcB | p<0.001 |
| P7 | 32.1 ± 3.8 bA | 55.8 ± 3.1 bB | 73.1 ± 7.3 cC | p<0.001 | 18.6 ± 3.3 cA | 36.2 ± 3.7 cB | 49.7 ± 6.0 cC | p<0.001 |
| Intergroup difference | p<0.001 | p<0.001 | p<0.001 | p<0.001 | p<0.001 | p<0.001 | ||
-
HGF: Human gingival fibroblasts. CGF: Canine gingival fibroblasts. Kruskal-Wallis one-way analysis of variance on ranks. Dunn's Method, significance level of 5%. Intergroup (a, b, c, d) and intragroup (A, B, C) comparisons - different letters indicate statistically significant difference.
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