Volume 85 • Number 12
Platelet-Rich Plasma Derived From Bone Marrow Aspirate Promotes New Cementum Formation Maria J.H. Nagata,* Nata´lia de Campos,* Michel R. Messora,† Carolina S. Santinoni,* Suely R.M. Bomfim,‡ Stephen E. Fucini,*§ Nata´lia M. Pola,i Adrieli P. Neves,* Juliano M. de Almeida,* Letı´cia H. Theodoro,* and Edilson Ervolino¶
Background: This study evaluates the influence of platelet-rich plasma derived from bone marrow aspirate (PRP-BMA) on the healing of periodontal fenestration defects in rats. Methods: Periodontal fenestration defects were surgically created in the mandibles of 40 rats. The animals were randomly divided into two groups, control and PRP-BMA, in which defects were filled with blood clot or PRP-bma, respectively. Animals were euthanized at either 10 or 30 days post-surgery. Histologic, histometric, and immunohistochemical analyses were performed. Percentage of new bone area (NBA), area of bone trabeculae (ABT), new cementum (NC), and extension of remaining defect were histometrically evaluated. Proliferating cell nuclear antigen (PCNA), bone sialoprotein (BSP), osteocalcin (OCN), and tartrate-resistant acid phosphatase (TRAP) immunohistochemical staining were performed. Immunolabeled cells were quantified. Data were statistically analyzed (analysis of variance; Tukey, P 0.05). Means and standard dethick bone trabeculae throughout the entire extent viations of ERD, NBA, ABT, and NC at both 10 and of the surgical defect (Figs. 2D and 2E), with com30 days post-surgery, along with intergroup and inplete regeneration of the periosteum and absence tragroup comparisons, are presented in Figure 4. of inflammatory infiltrate. Group PRP-BMA showed For all groups, NC values at 10 days post-surgery and thicker bone trabeculae compared with the control ERD values at 30 days post-surgery were zero. With group. regard to the intergroup comparisons, no significant 1706
J Periodontol • December 2014
Nagata, de Campos, Messora, et al.
specificity in the detection of these proteins, as evidenced by the total absence of immunolabeling in the negative controls for the immunohistochemical reactions. The immunolabeled cells had a brownish color that was confined to the nuclear compartment (PCNA), the cytosolic compartment (BSP, OCN), or exclusively the cytoplasm (TRAP). The control and PRP-BMA groups presented positive immunolabels for PCNA, BSP, OCN, and TRAP at 10 and 30 Figure 3. days post-surgery (Fig. 5). PCNAControl group: a delicate network of collagen fibers on the planed root surface at 10 days (A); a large positive cells were observed amount of collagen fibers oriented parallel to the root surface at 30 days (B). PRP-BMA group: a delicate around the newly formed bone, network of collagen fibers on the planed root surface with deposition of cementoid-like matrix (blue in the bone marrow spaces and arrowheads) at 10 days (C); a thin layer of NC (red arrowheads) covering the previously denuded root was the periosteum, in the connecobserved in all 30-day specimens of this group (D); higher magnification of the white-framed box in D shows collagen fibers inserted and oriented obliquely or perpendicularly to the root surface (E). (H&E stain; tive tissue located at the center original magnification ·1,000 [A through C and E], ·250 [D].) CT = connective tissue; DE = dentin; NB = of the defect in those cases new bone; PDL = periodontal ligament. without complete bone regeneration, and in the regenerated periodontal ligament. BSP immunolabeling was observed in osteoblasts and cementoblast-like cells. OCN immunolabeling was observed in osteoblasts. Mature osteoclasts (multinucleated TRAP-positive cells) were observed adjacent to the surface of the newly formed bone. Means and standard deviations of PCNA-positive, BSP-positive, OCN-positive, and TRAP-positive cells at both 10 and 30 days postsurgery, with intergroup and intragroup comparisons, are presented in Table 1. With regard to the intergroup comparisons, the PRPBMA group presented a significantly higher number of PCNA-positive and BSP-positive cells than the Figure 4. control group at both 10 and 30 days post-surgery. Mean percentage values (– SD) of the measurements ERD, NBA, ABT, No significant differences were observed for OCNand NC, with intergroup and intragroup comparisons, at 10 and 30 days. positive and TRAP-positive cells at either 10 or 30 days post-surgery. With regard to the intragroup differences were observed for NBA or ABT at either comparisons, the PRP-BMA group presented a sig10 or 30 days post-surgery or for ERD at 10 days nificantly higher number of PCNA-positive cells at post-surgery. It is important to note that, at 30 days 10 days compared with 30 days post-surgery. No post-surgery, it was not possible to statistically significant differences were observed for BSPcompare NC values between the two groups because positive, OCN-positive, and TRAP-positive cells at the NC value in the control group was zero. With either 10 or 30 days post-surgery. regard to the intragroup comparisons, the values of NBA and ABT in both experimental groups were DISCUSSION significantly higher at 30 days compared with 10 To the best of the authors’ knowledge, this is the first days post-surgery. study to evaluate the use of PRP-BMA to promote periodontal regeneration in rats. Immunohistochemical Analyses A critical component of periodontal regeneration is The immunohistochemical technique used for dethe formation of NC. Cementum has a very limited tecting PCNA, BSP, OCN, and TRAP yielded high self-healing capacity even in the absence of previous 1707
Bone Marrow Aspirate in Periodontal Fenestration Defects
Volume 85 • Number 12
that the positive periodontal regeneration results observed in the PRP-BMA group were due to a significant increase in MSCs and GFs at the surgical site. Although a specific count of MSCs and GFs was not performed in the present study, this hypothesis is supported by the findings of Nishimoto et al.,32 who conducted an in vitro study to analyze the quality of PRP derived from peripheral blood (PRP-PB) and PRP derived from bone marrow aspirate (PRPBMA). The authors observed that the density of platelets and levels of GFs (PDFG and TGFb) in PRP-BMA were the same as in PRP-PB. They also confirmed that PRP-BMA presented a high concentration of stem cells. Therefore, it is plausible to infer that, in the present Figure 5. study, PRP-BMA acted as a PCNA, BSP, OCN, and TRAP immunolabeling of the periodontal defect. Photomicrographs showing PCNApositive cells in control (A) and PRP-BMA (B) groups at 10 days, and control (C) and PRP-BMA (D) groups good source of MSCs and GFs. at 30 days. Photomicrographs showing BSP-positive cells in control (E) and PRP-BMA (F) groups at 10 Studies have shown that adult days, and control (G) and PRP-BMA (H) groups at 30 days. Photomicrographs showing OCN-positive cells MSCs, osteoblasts, fibroblasts, in control (I) and PRP-BMA (J) groups at 10 days, and control (K) and PRP-BMA (L) groups at 30 days. and endothelial cells express Photomicrographs showing TRAP-positive cells in control (M) and PRP-BMA (N) groups at 10 days, and the cell membrane receptors control (O) and PRP-BMA (P) groups at 30 days. CT = connective tissue; NB = new bone; arrowheads = immunolabeled cells. that are specific to the GFs included in PRP.15,37 It has also been suggested that the GFs exposure to the dental biofilm.34 This was confirmed included in PRP may activate several cell types inin the present study, in which all specimens of the volved in wound healing and induce soft tissue control group, at either 10 or 30 days post-surgery, healing and bone regeneration.15,37 It can be sugshowed a total absence of cementum formation. gested that in the PRP-BMA group, there was a On the other hand, all 30-day specimens of the PRPgreater number of MSCs in the surgical site. The BMA group presented significant NC formation with increased level of GFs probably stimulated not only inserted thick collagen fibers oriented obliquely or the proliferation of MSCs but also their differenperpendicularly to the root surface, indicating a tiation into the fibroblastic, osteoblastic, and cefunctional periodontal ligament. Despite differences mentoblastic lineage and the maturation of these in experimental models and methodologies, the recells. This hypothesis is supported by the imsults of this study seem to corroborate the findings of munohistochemical findings of the present study, Simsek et al.,6 who also observed significant NC where the PRP-BMA group presented significantly along with periodontal ligament and alveolar bone higher numbers of PCNA- and BSP-positive cells formation in Class II furcation defects treated by the than the control group at 10 and 30 days postcombination of MSCs/PRP. The MSCs were derived surgery. The significantly higher number of PCNAfrom bone marrow. Those authors observed that Class II positive cells in the PRP-BMA group compared furcation defects treated with PRP alone showed less with the control group at 30 days post-surgery NC formation compared with defects treated by indicates that there was a sustained positive effect MSCs/PRP. of PRP-BMA on cell proliferation in this group. It is well known that MSCs are one of the cell types Also, the number of BSP-positive cells was still present in bone marrow that contribute to the resignificantly higher in the PRP-BMA group than generation of mesenchymal tissues, including bone, in the control group at 30 days post-surgery, incementum, and periodontal ligament.3-5,8 It is possible dicating a sustained positive effect on differentiation 1708
Nagata, de Campos, Messora, et al.
J Periodontol • December 2014
Table 1.
PCNA-Positive, BSP-Positive, OCN-Positive, and TRAP-Positive Cells (mean 6 SD) at 10 and 30 Days, With Intergroup and Intragroup Comparisons Control
PRP-BMA
Variable
10 Days
30 Days
10 Days
30 Days
PCNA
262.40 – 213.82
66.20 – 12.19
567.80 – 155.40*
197.00 – 66.66‡i
BSP
94.75 – 10.56
80.33 – 65.23
321.66 – 55.05†
210.25 – 77.17§
OCN
14.75 – 12.50
8.80 – 9.44
10.17 – 4.54
6.75 – 1.50
TRAP
30.00 – 26.75
28.43 – 10.16
19.71 – 7.72
24.50 – 13.32
*P †P ‡P §P i P
Platelet-Rich Plasma Derived From Bone Marrow Aspirate Promotes New Cementum Formation Maria J.H. Nagata,* Nata´lia de Campos,* Michel R. Messora,† Carolina S. Santinoni,* Suely R.M. Bomfim,‡ Stephen E. Fucini,*§ Nata´lia M. Pola,i Adrieli P. Neves,* Juliano M. de Almeida,* Letı´cia H. Theodoro,* and Edilson Ervolino¶
Background: This study evaluates the influence of platelet-rich plasma derived from bone marrow aspirate (PRP-BMA) on the healing of periodontal fenestration defects in rats. Methods: Periodontal fenestration defects were surgically created in the mandibles of 40 rats. The animals were randomly divided into two groups, control and PRP-BMA, in which defects were filled with blood clot or PRP-bma, respectively. Animals were euthanized at either 10 or 30 days post-surgery. Histologic, histometric, and immunohistochemical analyses were performed. Percentage of new bone area (NBA), area of bone trabeculae (ABT), new cementum (NC), and extension of remaining defect were histometrically evaluated. Proliferating cell nuclear antigen (PCNA), bone sialoprotein (BSP), osteocalcin (OCN), and tartrate-resistant acid phosphatase (TRAP) immunohistochemical staining were performed. Immunolabeled cells were quantified. Data were statistically analyzed (analysis of variance; Tukey, P 0.05). Means and standard dethick bone trabeculae throughout the entire extent viations of ERD, NBA, ABT, and NC at both 10 and of the surgical defect (Figs. 2D and 2E), with com30 days post-surgery, along with intergroup and inplete regeneration of the periosteum and absence tragroup comparisons, are presented in Figure 4. of inflammatory infiltrate. Group PRP-BMA showed For all groups, NC values at 10 days post-surgery and thicker bone trabeculae compared with the control ERD values at 30 days post-surgery were zero. With group. regard to the intergroup comparisons, no significant 1706
J Periodontol • December 2014
Nagata, de Campos, Messora, et al.
specificity in the detection of these proteins, as evidenced by the total absence of immunolabeling in the negative controls for the immunohistochemical reactions. The immunolabeled cells had a brownish color that was confined to the nuclear compartment (PCNA), the cytosolic compartment (BSP, OCN), or exclusively the cytoplasm (TRAP). The control and PRP-BMA groups presented positive immunolabels for PCNA, BSP, OCN, and TRAP at 10 and 30 Figure 3. days post-surgery (Fig. 5). PCNAControl group: a delicate network of collagen fibers on the planed root surface at 10 days (A); a large positive cells were observed amount of collagen fibers oriented parallel to the root surface at 30 days (B). PRP-BMA group: a delicate around the newly formed bone, network of collagen fibers on the planed root surface with deposition of cementoid-like matrix (blue in the bone marrow spaces and arrowheads) at 10 days (C); a thin layer of NC (red arrowheads) covering the previously denuded root was the periosteum, in the connecobserved in all 30-day specimens of this group (D); higher magnification of the white-framed box in D shows collagen fibers inserted and oriented obliquely or perpendicularly to the root surface (E). (H&E stain; tive tissue located at the center original magnification ·1,000 [A through C and E], ·250 [D].) CT = connective tissue; DE = dentin; NB = of the defect in those cases new bone; PDL = periodontal ligament. without complete bone regeneration, and in the regenerated periodontal ligament. BSP immunolabeling was observed in osteoblasts and cementoblast-like cells. OCN immunolabeling was observed in osteoblasts. Mature osteoclasts (multinucleated TRAP-positive cells) were observed adjacent to the surface of the newly formed bone. Means and standard deviations of PCNA-positive, BSP-positive, OCN-positive, and TRAP-positive cells at both 10 and 30 days postsurgery, with intergroup and intragroup comparisons, are presented in Table 1. With regard to the intergroup comparisons, the PRPBMA group presented a significantly higher number of PCNA-positive and BSP-positive cells than the Figure 4. control group at both 10 and 30 days post-surgery. Mean percentage values (– SD) of the measurements ERD, NBA, ABT, No significant differences were observed for OCNand NC, with intergroup and intragroup comparisons, at 10 and 30 days. positive and TRAP-positive cells at either 10 or 30 days post-surgery. With regard to the intragroup differences were observed for NBA or ABT at either comparisons, the PRP-BMA group presented a sig10 or 30 days post-surgery or for ERD at 10 days nificantly higher number of PCNA-positive cells at post-surgery. It is important to note that, at 30 days 10 days compared with 30 days post-surgery. No post-surgery, it was not possible to statistically significant differences were observed for BSPcompare NC values between the two groups because positive, OCN-positive, and TRAP-positive cells at the NC value in the control group was zero. With either 10 or 30 days post-surgery. regard to the intragroup comparisons, the values of NBA and ABT in both experimental groups were DISCUSSION significantly higher at 30 days compared with 10 To the best of the authors’ knowledge, this is the first days post-surgery. study to evaluate the use of PRP-BMA to promote periodontal regeneration in rats. Immunohistochemical Analyses A critical component of periodontal regeneration is The immunohistochemical technique used for dethe formation of NC. Cementum has a very limited tecting PCNA, BSP, OCN, and TRAP yielded high self-healing capacity even in the absence of previous 1707
Bone Marrow Aspirate in Periodontal Fenestration Defects
Volume 85 • Number 12
that the positive periodontal regeneration results observed in the PRP-BMA group were due to a significant increase in MSCs and GFs at the surgical site. Although a specific count of MSCs and GFs was not performed in the present study, this hypothesis is supported by the findings of Nishimoto et al.,32 who conducted an in vitro study to analyze the quality of PRP derived from peripheral blood (PRP-PB) and PRP derived from bone marrow aspirate (PRPBMA). The authors observed that the density of platelets and levels of GFs (PDFG and TGFb) in PRP-BMA were the same as in PRP-PB. They also confirmed that PRP-BMA presented a high concentration of stem cells. Therefore, it is plausible to infer that, in the present Figure 5. study, PRP-BMA acted as a PCNA, BSP, OCN, and TRAP immunolabeling of the periodontal defect. Photomicrographs showing PCNApositive cells in control (A) and PRP-BMA (B) groups at 10 days, and control (C) and PRP-BMA (D) groups good source of MSCs and GFs. at 30 days. Photomicrographs showing BSP-positive cells in control (E) and PRP-BMA (F) groups at 10 Studies have shown that adult days, and control (G) and PRP-BMA (H) groups at 30 days. Photomicrographs showing OCN-positive cells MSCs, osteoblasts, fibroblasts, in control (I) and PRP-BMA (J) groups at 10 days, and control (K) and PRP-BMA (L) groups at 30 days. and endothelial cells express Photomicrographs showing TRAP-positive cells in control (M) and PRP-BMA (N) groups at 10 days, and the cell membrane receptors control (O) and PRP-BMA (P) groups at 30 days. CT = connective tissue; NB = new bone; arrowheads = immunolabeled cells. that are specific to the GFs included in PRP.15,37 It has also been suggested that the GFs exposure to the dental biofilm.34 This was confirmed included in PRP may activate several cell types inin the present study, in which all specimens of the volved in wound healing and induce soft tissue control group, at either 10 or 30 days post-surgery, healing and bone regeneration.15,37 It can be sugshowed a total absence of cementum formation. gested that in the PRP-BMA group, there was a On the other hand, all 30-day specimens of the PRPgreater number of MSCs in the surgical site. The BMA group presented significant NC formation with increased level of GFs probably stimulated not only inserted thick collagen fibers oriented obliquely or the proliferation of MSCs but also their differenperpendicularly to the root surface, indicating a tiation into the fibroblastic, osteoblastic, and cefunctional periodontal ligament. Despite differences mentoblastic lineage and the maturation of these in experimental models and methodologies, the recells. This hypothesis is supported by the imsults of this study seem to corroborate the findings of munohistochemical findings of the present study, Simsek et al.,6 who also observed significant NC where the PRP-BMA group presented significantly along with periodontal ligament and alveolar bone higher numbers of PCNA- and BSP-positive cells formation in Class II furcation defects treated by the than the control group at 10 and 30 days postcombination of MSCs/PRP. The MSCs were derived surgery. The significantly higher number of PCNAfrom bone marrow. Those authors observed that Class II positive cells in the PRP-BMA group compared furcation defects treated with PRP alone showed less with the control group at 30 days post-surgery NC formation compared with defects treated by indicates that there was a sustained positive effect MSCs/PRP. of PRP-BMA on cell proliferation in this group. It is well known that MSCs are one of the cell types Also, the number of BSP-positive cells was still present in bone marrow that contribute to the resignificantly higher in the PRP-BMA group than generation of mesenchymal tissues, including bone, in the control group at 30 days post-surgery, incementum, and periodontal ligament.3-5,8 It is possible dicating a sustained positive effect on differentiation 1708
Nagata, de Campos, Messora, et al.
J Periodontol • December 2014
Table 1.
PCNA-Positive, BSP-Positive, OCN-Positive, and TRAP-Positive Cells (mean 6 SD) at 10 and 30 Days, With Intergroup and Intragroup Comparisons Control
PRP-BMA
Variable
10 Days
30 Days
10 Days
30 Days
PCNA
262.40 – 213.82
66.20 – 12.19
567.80 – 155.40*
197.00 – 66.66‡i
BSP
94.75 – 10.56
80.33 – 65.23
321.66 – 55.05†
210.25 – 77.17§
OCN
14.75 – 12.50
8.80 – 9.44
10.17 – 4.54
6.75 – 1.50
TRAP
30.00 – 26.75
28.43 – 10.16
19.71 – 7.72
24.50 – 13.32
*P †P ‡P §P i P
