Human Umbilical Cord Mesenchymal Stem Cells Over Platelet Rich Fibrin Scaffold for Mandibular Cartilage Defects Regenerative Medicine

Objective: To evaluate the regeneration of mandibular cartilage defect after implantation of human umbilical cord mesenchymal stem cells (hUCMSC) over platelet rich fibrin (PRF) as scaffold. Material and Methods: 20 male Wistar rats were randomly divided into four experimental groups consisting of: a control group featuring untreated mandibular defects (C), experimental groups whose mandibular defects were implanted with hUCMSC (E1), mandibular defects implanted with PRF (E2), mandibular defects implanted with hUCMSC and PRF scaffold (E3). The subjects were sacrificed after six weeks of observation for immunohistochemical examination in order to evaluate the expression of Ki67, Sox9, FGF 18, type 2 collagen, and aggrecan, in addition to histology examination to evaluate chondrocyte number and cartilage thickness. Data was analyzed with univariate analysis (ANOVA). Results: The implantation of hUCMSC and PRF scaffold proved capable of regenerating mandibular cartilage defect through the expression of FGF 18, Sox9, Ki67, chondrosis counts, type 2 collagen, aggrecan, and cartilage thickness. The regeneration were significantly higher in group E3. Conclusion: Human umbilical cord mesenchymal stem cells in platelet rich fibrin scaffold proved capable of regenerating mandibular cartilage defect.


Introduction
Mandibular cartilage defect can be caused by micro and macro trauma [1] resulting in debilitating effects such as prolonged pain, function impairment, chronic inflammation and progressive cartilage degeneration [2]. In fact, mandibular cartilage defect in children may impair mandibular growth [3]. Posttrauma cartilage degeneration occurs because of low regenerative capacity due to the avascular, alimphatic and aneural nature of articular cartilage and chondrocyte in a low turn-over to maintain extracellular matrix [4].
Various methods that had been developed in orthopedics to reconstruct cartilage defects such as subchondral drilling, abrasion, microfracture, mosaicplasty, autologous chondrocyte implantation and matrixassisted autologous chondrocyte implantation, have not yet been proven capable of providing a long and wellfunctioning cartilage in an extensive defect [5]. Autologous chondrocyte injection initiated in 1994 by Brittberg et al. [6] suffered from several disadvantages: limited cell procurement, donor site morbidity and limited potential for proliferation and differentiation [7]. In Oral and Maxillofacial Surgery, reconstruction of cartilage defect using autograft, allograft, and allopalstic material pose several disantantages and complication such as injury to facial nerve and temporomandibular nerve, and Frey's syndrome [1].
Cartilage generation requires a sufficient number of cells to replace damaged chondrocytes.
Mesenchymal stem cells (MSCs) were considered a potential source in cartilage regeneration and engineering because of their high expansion rate [8]. The umbilical cord is a potential source of MSCs with several advantages such as being procured ethically from biological waste and demonstrating a high expansion rate [9].
However, regeneration of mandibular cartilage defects through the implantation of hUCMSC over PRF scaffold remains to be studied.
This study was performed to evaluate regeneration of surgically created mandibular cartilage defects in rat subjects after implantation of hUCMSC over PRF scaffold through expression of FGF 18, Sox9, Ki67, chondrocyte counts, type 2 collagen, aggrecan, and cartilage thickness. FGF 18 regulates chondrocyte proliferation, produces ECM and proteoglican. It attaches to FGFR-3 that cause anabolic effect in cartilage formation [11].

Preparation of Experimental Subjects
This study was conducted following the granting of Ethical Clearance by the Animal Care and Use Characterisation of MSC was conducted prior to implantation by means of immunocytochemical and flowcytometric analysis [12].

Preparation of PRF
After an animal subject had been anaesthetized with Ketamin/Xylazin, 1.5 ml of venous blood were aspirated from its tail. The blood was obtained using a 3 ml disposable syringe without anticoagulant and centrifuged at 3,000 rpm for ten minutes until it separated into three layers, the middle layer being PRF.

Implantation of hUCMSC, PRF and hUCMSC and PRF
Mandibular cartilage defects were created surgically in the anterior portion of the condylar superior surface using a round bur 1mm in diameter [13]. Two million (2 x 10 6 ) hUCMSC pellets were implanted in group E1, 1 mm of PRF was implanted in group E2, while group E3 was implanted with hUCMSC seeded in 1 mm PRF through centrifugation at 3,000 rpm for five minutes. After the defects had been fully covered, the wound was sutured in layers.

Specimens Preparation and Microscopic Examination
The subjects were sacrificed after a 6-week period of observation. The mandible was subsequently exarticulated and fixated with 20% formalin for two days at room temperature and decalcified in 10% Ethylenediaminee tetra-acetic (EDTA) for eight weeks before being embedded in paraffin. Each paraffin block was cut axially to a thickness of 4 μm, deparaffinized and dehydrated. Specimens were then stained with Harris Hematoxylin-Eosin to enable histological examination to be conducted in order to evaluate chondrocyte counts and cartilage thickness. Immunohistochemical examination was undertaken to evaluate expression of FGF18

Expression of Type 2 Collagen, Aggrecan and Cartilage Thickness
Matrix deposition was represented by the expression of type 2 collagen and aggrecan, whereas the regeneration of cartilage was evidenced by cartilage thickness. Microscopic images of cells expressing type 2 collagen and aggrecan, as well as cartilage thickness; the mean and deviation standard values of each matrix deposition and regeneration variable are presented in Figure 3.

Discussion
The implantation of hUCMSC over PRF scaffold underwent proliferation and differentiation into chondrocytes to initiate extracellular matrix deposition. This, in turn, regenerated defective cartilage through a complex process involving various growth factors of FGF18 and transcription factor of Sox9. In this study, the surface markers found were CD45 -, CD73 + , CD90 + , CD105 + , as previously described [14].
The proliferation of hUCMSC and chondocyte differentiation were significantly higher in the E3 group compared to other groups. This may be caused by the nature of PRF being a flexible, elastic and extremely strong fibrin matrix that fulfils the 3-dimensional requirement for biomaterial. In addition, growth factors, platelets and immune concentrate that are required in the healing and immune process were all released [15,16]. Seeding of hUCMSC in PRF in this study was accomplished through centrifugation where PRF had undergone polymerization that reduces thrombin concentration, thus forming tetramolecular and trimolecular bonds or extensive 3D equilateral bonds. This bonding promotes cytokin attachment, cell migration and retains stem cells within the PRF, chemotactically recruited MSC, caused by dense fibrin structure increasing growth factor and resulting in the gradual release of other mediators. This process will confine hUCMSC to the PRF [17,18].
The high proliferation of hUCMSC in PRF scaffold was demonstrated by the strong expression of Ki67 which results from PRF also constituting an extracellular matrix that forms tissue structure, provides regulatory signal for cell proliferation and differentiation through cell-receptor interaction, mediating diffusion of soluble growth factors and reducing mechanical signals [19]. Platelet-derived growth factor contained in PRF might induce cell proliferation through Akt signal transduction that is important in cell proliferation [20,21]. The strong expression of Ki67 was consistent with high chondrocyte counts.
High chondrocyte counts will express significant FGF 18. This study found the highest expression of FGF 18 to be in E3 group compared to the other groups in the experiment that showed no significant difference. Chondrogenic differentiation was indicated by high expression of FGF18, that was a physiological ligand of FGFR3 [22]. Columnar and flat chondrocyte represent the most proliferative cells in cartilage expressing high FGFR3 [23] enhanced chondrogenic differentiation and cartilage production through increased expression of type 2 collagen [24]. High expression of Sox9 caused induction of chondrogenesis through Smad2/3 [25], influencing morphogenesis of the condyle [26]. Bond of Sox9 in chondocyte specific enhancer in intron 1gen pro-α1 type 2 collagen upregulating the synthesis of α1 type 2 collagen, Sox9 maintained its high expression in fully differentiated chondrocyte [27]. This study also showed the highest expression of Sox9 to occur after 6 weeks' implantation of hUCMSC in PRF scaffold. Expression of Sox9 might be regulated by FGFR3 [28], as was indicated by the findings reported here that showed high expression of FGF18, Sox9, and type 2 collagen, as well as aggrecan.
The collagen network in articular cartilage provides mechanical support to tensile forces generated by compression or interstitial swelling, protects chondrocyte, maintains proteoglycan attachment and attaches cartilage to subchondral bone [29]. Type 2 collagen and aggrecan were molecular markers of mature chondrocyte because they were produced by differentiated chondocytes [30]. This study confirmed a significant difference of type II collagen and aggrecan expression in hUCMSC in PRF scaffold compared to other groups consistent with the high expression of Sox9. FGF 18 released in large quantities in E3 group will upregulate Sox9 expression at several stages of chondrocyte differentiation, increase proteoglycan deposition, aggrecan and type II collagen expression and significantly decrease expression of collagen type I [31].
Regeneration of mandibular cartilage defects in hUCMSC in PRF scaffold was indicated by the highest mean of cartilage thickness. The interaction of chondrocyte and extracellular matrix regulates important processes in homeostasis and cartilage repair mediated by integrin signalling. The mechanical nature of scaffold plays an important role in tissue regeneration and at the cellular level, which affects mesenchymal stem cell differentiation [32]. The proliferation of hUMCSC and its differentiation into chondrocyte leads the latter to produce extracellular matrices such as type II collagen and aggrecan. In addition to the growth factor released by PRF, it also provides the mechanical structure required in promoting cartilage thickness. Extracellular matrices provided by PRF not only form tissue structure and function, but also provide regulatory signals for cell proliferation and differentiation through cell-receptor interaction, mediating soluble growth factor diffusion and absorbing mechanical signals [21].

Conclusion
Human umbilical cord mesenchymal stem cells in platelet rich fibrin scaffold proved capable of regenerating mandibular cartilage defect.

Financial Support
None.