32.6 Histomorphometrical analysis
3.4 Histomorphometry and statistical analysis
As shown in Figure 6, the C+ group demonstrated a significantly higher alveolar bone height compared to the EMP group. The results of the bone area measurement showed that the C+ group had significantly more new bone formation in the defect area than the FGF2 and EMP groups. The O+ group also had significantly more new bone formation than EMP group. For epithelial downgrowth, no statistical differences were found between any of the groups, including the empty control. Finally, for functional ligament length, both the FGF2 and C+ groups had significantly higher scores than the O+ group.
4
Discussion
The overall objective of this study was to explore the most optimal differentiation approach for MSCs to achieve periodontal regeneration, using an intra-bony periodontal defect rat model. For this purpose, MSCs were seeded on a scaffold and pre-cultured in different conditions, i.e. retention of multilineage differentiation
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potential (FGF2), an osteogenic differentiation approach (O+) and a chondrogenic differentiation approach (C+). The in vitro histological results confirmed that cells were distributed throughout the scaffolds. Cartilage was formed in the C+ group, and in vitro calcification occurred in all experimental groups under osteogenic culture condition. The in vivo results demonstrated evident differences. Especially the C+ approach resulted in the regeneration of both alveolar bone and periodontal ligament, whereas FGF2 only contributed to periodontal ligament regeneration and O+ to alveolar bone regeneration.
Regarding our study design, several remarks can be made. A small animal model was applied, whereas a larger animal model would provide higher comparability with the human situation (e.g. metabolic rate, defect size) and thus would facilitate the translation from bench to bedside. Also, it was inherent to the different differentiation protocols, that the culturing time were not equal. As a necessary step for chondrogenic differentiation approach, cells were grown in CM for 4 weeks. This longer culturing time may influence the performance of C+ samples, although the low serum chondrogenic culture condition would principally not stimulate the cell proliferation. Still, it is the purpose of this study to compare different approaches for periodontal regeneration. A final technical remark should address the scaffold degradation, as in all groups scaffold remnants were visible. Thus, it seems necessary to further investigate the degradation capacity in vivo. The PLGA/PCL wet-electrospun scaffold is a highly porous scaffold, which on beforehand was proved to be suitable for MSC loading and (endochondral)
bone formation[22]. Still, compared to previous studies using gelatin cell carriers[5], the
degradation rate of the PLGA-PCL scaffold was much slower, and abundant presence of multinucleated giant cells was seen, potentially interfering with new tissue formation. Regarding our results in the FGF2 group, only periodontal ligament formation was found to be significantly higher than in the O+ group. This corroborates with another study, which also showed that bone marrow MSCs can gain characteristics of PDL cells,
after co-culturing with periodontal ligament-derived cells[24]. However, for bone tissue
regeneration, the induction from the microenvironment was apparently inadequate. In the O+ group, the reverse process happened, i.e. only new bone formation was seen, but without evident ligament formation. In literature it has been postulated that only the establishment of bony support allows the transfer of functional loading, which subsequently is important for ligament formation. Thus, it seems likely that the time in our current set-up was too short for such a process, and longer time points for analyses should be regarded. Alternatively, the lack of ligament formation can be due to the induction protocol, using 10 days of osteogenic pre-culture. MSCs at the earlier stages have a strong growth potential, but the proliferative capacity decreases when
most cells have reached osteogenic differentiation[19,25]. Thus, it can also be argued
that most cells were already terminally differentiated. As a consequence, proliferation rates or differentiation capacity upon implantation were too low to achieve ligament formation.
The reason to investigate chondrogenic differentiation is mostly related to cell survival. Previous studies were already able to show green fluorescent protein (GFP)- positive cementoblasts, osteoblasts, osteocytes and fibroblasts in periodontal tissue
regeneration studies[10]. On the other hand, other recent investigations, tracking GFP-
positive PDL cells in rat calvarial critical-sized defects, underline that cell numbers decrease significantly after 4 weeks, with single or no positive cells detectable after
10 weeks[26]. An enhanced cell survival can explain the positive effects in the current
C+ group, as chondrocytes are known to be able to survive with limited nutrition and oxygen. The C+ group exhibited formation of both hard and soft periodontal tissues,
which corroborates our earlier findings upon subcutaneous implantation[22]. For the
effectiveness of the chondrogenic approach, also several other explanations can be found in literature. First, the in vitro results showed that C+ samples were more resilient to handling due to the large amounts of generated extracellular matrix (ECM) after pre-culture. Earlier investigations have shown that such abundant ECM also can actively sequester growth factors and cytokines, attract cells from the implant site,
and thus inherently favours growth and differentiation[27]. Second, the chondrocytes
produce specific matrix metalloproteinases, initiating remodelling and thus leading to mineralization of the hypertrophic cartilage. Finally, the chondrocytes are shown
to attract blood vessels by releasing VEGF[28], which also could accelerate tissue
regeneration in the periodontium.
5
Conclusion
Based on our findings, and within the limitations of this study, it can be concluded that the chondrogenic differentiation approach is a useful strategy for regeneration of alveolar bone and periodontal ligament, in the currently used rat model. Larger periodontal defect models and scaffold with higher degradation rate will be necessary to examine the full potential of the chondrogenic differentiation approach in periodontal regeneration in further investigations.
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References
1 Needleman IG, Worthington HV, Giedrys-Leeper E, Tucker RJ. Guided tissue
regeneration for periodontal infra-bony defects. Cochrane Database Syst Rev 2006(2):CD001724.
2 Kawaguchi H, Hirachi A, Hasegawa N, Iwata T, Hamaguchi H, Shiba H, Takata
T, Kato Y, Kurihara H. Enhancement of periodontal tissue regeneration by transplantation of bone marrow mesenchymal stem cells. Journal of
Periodontology 2004;75(9):1281-1287.
3 Hasegawa M, Yamato M, Kikuchi A, Okano T, Ishikawa I. Human periodontal
ligament cell sheets can regenerate periodontal ligament tissue in an athymic rat model. Tissue Eng 2005;11(3-4):469-478.
4 Yang Y, Rossi FM, Putnins EE. Periodontal regeneration using engineered bone
marrow mesenchymal stromal cells. Biomaterials 2010;31(33):8574-8582.
5 Yu N, Oortgiesen DA, Bronckers AL, Yang F, Walboomers XF, Jansen JA. Enhanced
periodontal tissue regeneration by periodontal cell implantation. J Clin
Periodontol 2013;40(7):698-706.
6 Huang GT, Gronthos S, Shi S. Mesenchymal stem cells derived from dental tissues
vs. those from other sources: their biology and role in regenerative medicine.
J Dent Res 2009;88(9):792-806.
7 Hynes K, Menicanin D, Gronthos S, Bartold PM. Clinical utility of stem cells for
periodontal regeneration. Periodontol 2000 2012;59(1):203-227.
8 Lin NH, Gronthos S, Bartold PM. Stem cells and future periodontal regeneration.
Periodontol 2000 2009;51:239-251.
9 Li H, Yan F, Lei L, Li Y, Xiao Y. Application of autologous cryopreserved bone
marrow mesenchymal stem cells for periodontal regeneration in dogs. Cells
Tissues Organs 2009;190(2):94-101.
10 Hasegawa N, Kawaguchi H, Hirachi A, Takeda K, Mizuno N, Nishimura M, Koike C, Tsuji K, Iba H, Kato Y, Kurihara H. Behavior of transplanted bone marrow-derived mesenchymal stem cells in periodontal defects. J Periodontol 2006;77(6):1003- 1007.
11 Tsumanuma Y, Iwata T, Washio K, Yoshida T, Yamada A, Takagi R, Ohno T, Lin K, Yamato M, Ishikawa I, Okano T, Izumi Y. Comparison of different tissue-derived stem cell sheets for periodontal regeneration in a canine 1-wall defect model.
Biomaterials 2011;32(25):5819-5825.
12 Scotti C, Tonnarelli B, Papadimitropoulos A, Scherberich A, Schaeren S, Schauerte A, Lopez-Rios J, Zeller R, Barbero A, Martin I. Recapitulation of endochondral bone formation using human adult mesenchymal stem cells as a paradigm for developmental engineering. Proc Natl Acad Sci U S A 2010;107(16):7251-7256.
13 Harada N, Watanabe Y, Sato K, Abe S, Yamanaka K, Sakai Y, Kaneko T, Matsushita T. Bone regeneration in a massive rat femur defect through endochondral ossification achieved with chondrogenically differentiated MSCs in a degradable scaffold. Biomaterials 2014;35(27):7800-7810.
14 Mooney DJ, Vandenburgh H. Cell delivery mechanisms for tissue repair. Cell Stem
Cell 2008;2(3):205-213.
15 Tsutsumi S, Shimazu A, Miyazaki K, Pan H, Koike C, Yoshida E, Takagishi K, Kato Y. Retention of multilineage differentiation potential of mesenchymal cells during proliferation in response to FGF. Biochem Biophys Res Commun 2001;288(2):413- 419.
16 Bianchi G, Banfi A, Mastrogiacomo M, Notaro R, Luzzatto L, Cancedda R, Quarto R. Ex vivo enrichment of mesenchymal cell progenitors by fibroblast growth factor 2.
Exp Cell Res 2003;287(1):98-105.
17 Liechty KW, MacKenzie TC, Shaaban AF, Radu A, Moseley AM, Deans R, Marshak DR, Flake AW. Human mesenchymal stem cells engraft and demonstrate site-specific differentiation after in utero transplantation in sheep. Nat Med 2000;6(11):1282-1286.
18 Zhou J, Shi S, Shi Y, Xie H, Chen L, He Y, Guo W, Wen L, Jin Y. Role of bone marrow-derived progenitor cells in the maintenance and regeneration of dental mesenchymal tissues. J Cell Physiol 2011;226(8):2081-2090.
19 Sikavitsas VI, van den Dolder J, Bancroft GN, Jansen JA, Mikos AG. Influence of the in vitro culture period on the in vivo performance of cell/titanium bone tissue- engineered constructs using a rat cranial critical size defect model. J Biomed
Mater Res A 2003;67(3):944-951.
20 Mauney JR, Volloch V, Kaplan DL. Role of adult mesenchymal stem cells in bone tissue engineering applications: current status and future prospects. Tissue Eng 2005;11(5-6):787-802.
21 Farrell E, Both SK, Odorfer KI, Koevoet W, Kops N, O’Brien FJ, Baatenburg de Jong RJ, Verhaar JA, Cuijpers V, Jansen J, Erben RG, van Osch GJ. In-vivo generation of bone via endochondral ossification by in-vitro chondrogenic priming of adult human and rat mesenchymal stem cells. BMC Musculoskelet Disord 2011;12:31. 22 Yang W, Yang F, Wang Y, Both SK, Jansen JA. In vivo bone generation via the
endochondral pathway on three-dimensional electrospun fibers. Acta Biomater 2013;9(1):4505-4512.
23 Oortgiesen DA, Plachokova AS, Geenen C, Meijer GJ, Walboomers XF, van den Beucken JJ, Jansen JA. Alkaline phosphatase immobilization onto Bio-Gide(R) and Bio-Oss(R) for periodontal and bone regeneration. J Clin Periodontol 2012;39(6):546-555.
periodontal regeneration via MSCs approach
87
3
24 Kramer PR, Nares S, Kramer SF, Grogan D, Kaiser M. Mesenchymal stem cells acquire characteristics of cells in the periodontal ligament in vitro. J Dent Res 2004;83(1):27-34.
25 Castano-Izquierdo H, Alvarez-Barreto J, van den Dolder J, Jansen JA, Mikos AG, Sikavitsas VI. Pre-culture period of mesenchymal stem cells in osteogenic media influences their in vivo bone forming potential. J Biomed Mater Res A 2007;82(1):129-138.
26 Tour G, Wendel M, Moll G, Tcacencu I. Bone repair using periodontal ligament progenitor cell-seeded constructs. J Dent Res 2012;91(8):789-794.
27 Benders KE, van Weeren PR, Badylak SF, Saris DB, Dhert WJ, Malda J. Extracellular matrix scaffolds for cartilage and bone regeneration. Trends Biotechnol 2013;31(3):169-176.
28 Gawlitta D, Farrell E, Malda J, Creemers LB, Alblas J, Dhert WJ. Modulating endochondral ossification of multipotent stromal cells for bone regeneration.