|Year : 2015 | Volume
| Issue : 4 | Page : 151-155
Using PRP and human amniotic fluid combination for osteogenesis in rabbit socket preservation
Amir Hossein Moradi1, Ali Kamalinejad2, Nasrin Jalilian3, Shantia Kazemi4, Mozafar Khazaei5
1 Department of Periodontics, School of Dentistry, Kermanshah University of Medical Sciences, Kermanshah, Iran
2 Student Research Committee, School of Medicine, Kermanshah University of Medical Sciences, Kermanshah, Iran
3 Department of Gynecology and Obstetrics, School of Medicine, Kermanshah University of Medical Sciences, Kermanshah, Iran
4 Advanced Periodontology Department, Ostrow School of Dentistry, University of Southern California, Los Angeles, USA
5 Fertility and Infertility Research Center, Kermanshah University of Medical Sciences, Kermanshah, Iran
|Date of Web Publication||27-Nov-2015|
Fertility and Infertility Research Center, Kermanshah University of Medical Sciences, Kermanshah
Source of Support: None, Conflict of Interest: None
Introduction: Platelet-rich plasma (PRP) is used as an adjunct treatment during periodontal grafting surgery because of its capability of enhancing healing process. Amniotic fluid is a rich source of growth factors and hyaluronic acid (HA) and a good point to study its properties of wound healing and bone formation. The aim of this study was to evaluate the osteogenic properties of a combination of amniotic fluid and PRP in rabbit's dental socket preservation. Materials and Methods: The study population consisted of 24 healthy male laboratory rabbits (average weight 3,125 ± 185 gr) that were randomly allocated into four groups. PRP for the first group, human amniotic fluid (HAF) for the second group, a combination of PRP and HAF (PRHA) for the third group was used. In the fourth (control) group, no biomaterial was used. In each group, half of the rabbits were sacrificed at 4 weeks following surgery and the rest were sacrificed after 8 weeks. Histological analysis of biopsies of the sockets was performed using hematoxylin and eosin (H&E) staining. Data were analyzed using Statistical Package for the Social Sciences (SPSS) software (version 16) and P-value <0.05 was considered significance. Results: All three experimental groups showed positive effect on bone formation in terms of area of trabecular bone and number of osteocytes and also vessel formation. Socket preservation using HAF and PRHA showed the highest impact on bone formation. Socket preservation using HAF also had the highest impact on vessel formation. Conclusion: PRHA and HAF appear to be useful for enhancing bone formation. Since there was no difference between HAF and PRHA, it seems beneficial to use HAF due to its simplicity of application.
Keywords: Amniotic fluid, hyaluronic acid (HA), platelet-rich plasma (PRP)
|How to cite this article:|
Moradi AH, Kamalinejad A, Jalilian N, Kazemi S, Khazaei M. Using PRP and human amniotic fluid combination for osteogenesis in rabbit socket preservation. Dent Hypotheses 2015;6:151-5
|How to cite this URL:|
Moradi AH, Kamalinejad A, Jalilian N, Kazemi S, Khazaei M. Using PRP and human amniotic fluid combination for osteogenesis in rabbit socket preservation. Dent Hypotheses [serial online] 2015 [cited 2019 Jul 16];6:151-5. Available from: http://www.dentalhypotheses.com/text.asp?2015/6/4/151/170642
| Introduction|| |
Alveolar ridge forms during the tooth eruption and the presence of dental elements maintains the alveolar bone shape and density.  Tooth loss results in constant and gradual resorption of the residual ridge.  Periodontal disease, endodontic lesions, facial trauma, and tooth loss are among the contributing factors of bone resorption. , Bone loss and subsequent ridge atrophy poses a challenge to the oral rehabilitation process, accordingly regenerative interventions would be demanded. , Bone regeneration and healing is a cellular process, mediated by biological factors and biochemical agents. A recent translational approach to clinical challenges in regeneration is focusing on applying natural growth factors to accelerate bone healing and to decrease bone maturation period.
Socket preservation after tooth extraction is known to have a great impact on the esthetic outcomes of the prosthesis; however, it is still an unresolved problem ,,,,, Socket preservation basically prevents hard and soft tissue collapse that results in minimal or even no need for augmentation procedures in the future. ,,,,,,
Human amniotic fluid is a rich resource of nutrients and growth factors essential to fetus development such as endothelial growth factor (EGF), transforming growth factor (TGF-β), insulin like growth factor (IGF) (I and II), and fibroblast growth factor (FGF). , It also contains high levels of hyaluronic acid (HA) and hyaluronic stimulating activator (HASA), dermatan sulfate, chondroitin 4 and 6 sulfate, and heparan sulfate. 
Platelet-rich plasma (PRP) contains a number of growth factors such as three isomers of platelet-derived growth factor (PDGFaa, PDGFbb, and PDGFab), TGF-β (TGF-β1 and TGF-β2), vascular EGF (VEGF), and IGFs. , Additionally, the presence of basic-FGF (b-FGF) in α-granules of platelet has also been reported. , PRP is an autologous source of growth factors that have been used to regenerate peri-implant bone and to promote healing at bone graft site and soft tissue with better epithelialization. ,,,,,,
Some of the advantages of PRP includes decreased bleeding at the recipient sites during the operation and postoperation; increased initial stability of the graft at the recipient sites due to its cohesive and adhesive nature; faster vascularization of the healing tissue by delivering growth factors; and, moreover if it is used in combination with bone replacement materials, it can induce soft- and hard- tissue regeneration. ,,,,,
Yet socket preservation is a challenging and technique-sensitive procedure and the results could be unpredictable at times. The aim of this study was to clinically and histologically evaluate the osteogenic effects of PRP, HAF, and their combination in dental socket of rabbit models as grafting biomaterials.
| Materials and Methods|| |
The method of this experimental study was interventional among of population containing 24 healthy male laboratory rabbits with average weight of 3,125 ± 185 gr. All animals were allocated randomly into four groups based on biomaterial-type to be places later as in first group of PRP, second group of HAF, third group of the combination of PRP and HAF, and fourth group of control. Animals were kept in individual cages during the project. Natural condition of day/night light was provided and the animals were fed commercial laboratory animal fodder and green food as nutritional supplement. The Ethical Committee of the Kermanshah University of Medical Sciences approved this work on animal model.
Amniotic fluid preparation
Thirty milliliter of amniotic fluid was obtained during C-section from healthy mothers who were ruled out for infections, inflammatory conditions, and metabolism disorders. The collected fluid was centrifuged for 10 min at 2,500 rpm for separating the serum; the obtained serum was stored at -20°C until use.
Rabbit PRP was obtained by drawing 8 mL of autologous blood from auricular marginal vein into the cell PRP kit that was purchased from the market. Obtained blood was centrifuged at 1,200 g for 10 min, PPP phase was removed and PRP was harvested according to the manufacturer's instruction.
Prior to the procedure, all animals were generally anesthetized with intramuscular injection of Ketamine HCl (35 mg/kg) and xylazine (5 mg/kg). An intramuscular injection of penicillin G (Darou Pakhsh, Tehran, Iran) (60,000 U/kg) was given before procedure as prophylaxis. After scrubbing the surgery site with 10% iodine, for local anesthesia field block injection of lidocaine 2% with 1:100,000 epinephrine was applied.
An intersulcular and circumferential incision was introduced around the lower incisor tooth with scalpel number 15 with the aim of releasing the upper attached ligaments and mucosa. After complete luxation with luxant instruments, tooth was removed automatically with no excessive force. Removal of pulp with tooth was checked and in reminisced cases it was removed by curettage. Sockets got full coverage by gaining the surrounding gingiva with single interrupted absorbable suture; , distances between sutures were designed to create a small entrance for passing a 18 gauge blunt needle of biomaterial carrier into the socket. All recipients received 1.5 mL of grafting biomaterials instilled directly into the socket through the provided gateway. For the combination group (PRHA), equal amounts of PRP and HAF were mixed and applied. In control group, no biomaterial was applied after getting full coverage of surgical site. To maintain the biomaterials in socket, a sterile wet gas was packed on surgery site for 10 min, and the head of animal was kept little upright during the anesthesia procedure.
Animals received a diet of pasty chew for 2 days after the surgery, and 1 gr of Acetaminophen mixed in drinking water were given as an analgesic on the day of the surgery and the next day. For prevention of infection intramuscular injection of penicillin G (Darou Pakhsh, Tehran, Iran) (50,000 U/kg) was given for 5 days.
Histological and morphological analysis
Three rabbits of each group were killed with 4 weeks intervals on the week 4th and 8th after the surgery. Euthanasia was performed by intravenous overdose of pentobarbital (45 mg/kg), and for ensuring of the death of animal bilateral tracheotomy also were performed. Alveolar process was excised as an en block, fixed in 10% neutral buffered formalin for 48 h, and then decalcified in 10% ethylene diamine tetraacetic acid (EDTA). Tissue processing, including dehydration, clearing, impregnation, and embedding, was done through graded ethanol, xylol, and paraffin.
Six nonconsecutive sections with 5-μm thickness separated by 30-μm distance were cut in the coronal plane from each sample and stained with hematoxylin and eosin (H&E) method. From each section three image (10×) were obtained using microscope (Olympus, USA) in conjunction with a digital camera connected to the computer to calculate percentage of trabecular bone area with dividing the trabecular bone area by the total area using Motic Images Plus 0.2 software (Moticam 2000, Spain) and manually quantifying of blood vessels. The magnifications of 40× were assessed for visual counting of osteocyte and empty osteocyte lacunae. For histological evaluation of healing process, Emery's histopathological healing criteria were used, which are explained in [Table 1].
Results of evaluating 720 images were analyzed with Statistical Package for the Social Sciences (SPSS) version 16 software (SPSS Inc., Chicago, IL) by using of ANOVA test and Tukey's analyses. And P < 0.05 was considered significant.
| Results|| |
Histological analysis based on Emery's histopathological healing criteria showed no significant difference between the groups on the 4 th and 8th weeks after tooth extraction. (P = 0.238). Statistical analyses for bone formation activities according to area percentage of bone trabeculae, osteocyte formation [Figure 1], and vessel formation in all treated and control sockets presented as significantly higher results on 8th week after extraction compared with the results on 4 th week (P < 0.001) [Table 2].
|Figure 1: Histological aspect of dental socket 4 weeks after extraction in controls (A), Amniotic (B), PRP (C), and combination (D) groups ×10|
Click here to view
The mean amounts of bone trabeculae after 4 and 8 weeks of tooth extraction revealed that PRHA and human amniotic fluid treated sockets had no significant difference (42.33% ± 2.29% and 43.28% ± 2.29%; P = 0.497) compared to each other, but were significantly higher than PRP treated and control sockets (P < 0.05); amount of bone trabeculae in PRP treated sockets was significantly increased compared to control sockets (25.59% ± 1.05% vs. 13.71% ± 2.21%; P < 0.05).
PRHA and HAF treated sockets showed significantly enhanced numbers of osteocyte formation (628 ± 11.76 and 631 ± 56.5) compared with other two groups, also amount of osteocyte formation for PRP treated sockets was significantly more than control sockets (573.33% ± 7.45% vs. 264.67% ± 6.75%; P < 0.05). Vessel formation was significantly enhanced in HAF treated sockets (92% ± 7.07%; P < 0.05); also PRHA treated sockets had significantly enhanced amount of vessel formation compared with PRP treated and control sockets (62.33% ± 18.99% vs. 81.33% ± 15.91% and 55.33% ± 3.14%; P < 0.05).
| Discussion|| |
This study aimed to evaluate clinical and histological changes following the use of PRP, HAF, and PRHA in socket preservation in rabbits after 4 and 8 weeks. Human amniotic fluid contains high amount of HA that is known to have positive effect on bone regeneration by stimulating migration, adhesion, proliferation, and cell differentiation. ,,,,
The overall findings from this study demonstrated that human amniotic fluid alone and in combination with PRP had most significant influence on enhancing bone formation in rabbit's dental socket preservation. The significant difference that was found between PRP alone versus combination of HAF and PRP might be due to the conjunction of FGF and presented HA in HAF. As reported by Lisignoli et al., human mesenchymal stem cells differentiation in HA scaffold is significantly enhanced by introducing b-FGF. 
Also, the presence of a potent growth factor like TGF-β1 along with HA should be taken into account. It is believed to accelerate bone healing by stimulating the proliferation and differentiation of mesenchymal stem cells, increase of osteoblast mitosis, inhibiting type II collagen synthesis but promoting type I collagen synthesis.  TGF- β1 (TGF-β1) is found in human amniotic fluid only during the late stage of gestation.  Histological evaluation of femur fracture healing in rat models showed enhanced bone formation in PRP group compared with the control group. The results of immunohistochemistry and Western blotting in their study demonstrated that PRP accelerates bone healing via modulating the expression of TGF-β1 and bone morphogenetic protein-2 (BMP-2). 
In the present study, sockets that were preserved using HAF alone showed significantly higher number of blood vessels formation that can be explained by the presence of HA and FGFs. It is hypothesized that HA enhances blood vessel formation via its ability of migration and proliferation of endothelial cells. ,, Mendes et al.  found significantly increased number of vessel formation in sockets treated by HA in rat models, they also reported that HA treated socket demonstrated more pronounced bone deposition. b-FGF is known to be a potent mitogen for several cell types, including vascular endothelial cells. 
Human amniotic fluid also contains high concentration of HASA that stimulates the wound to increase endogenous HA production.  Therefore, by applying HAF in the healing site both endogenous and exogenous HA will increase. The histological and radiological evaluation showed application of HAF significantly enhances bone formation and bone density in calvarias defects of rabbit model.  In the present study, HAF played a significant role both when applied alone or in combination with PRP that can be explained by its high concentration of HA, HASA, and other growth factors. Moreover, bone formation by means of osteocyte and bone trabeculae formation was significantly increased in PRP treated sockets compared to the control sockets. Since there was no significant difference between PRHA and HAF in terms of bone deposition and HAF alone was sufficient for enhancing bone formation in dental sockets, the authors suggest using HAF alone for socket preservation that eliminates the need for blood withdrawing and might be psychologically more comfortable for many patients.
| Conclusion|| |
PRHA and HAF are useful for enhancing bone formation. Since there was no difference between HAF and PRHA, it seems beneficial to use HAF instead of PRHA for socket preservation. However, the current available evidence is scarce and inconclusive to claim for the superiority of HAF over PRHA in socket preservation.
The authors thank the Fertility and Infertility Research Center Staff for the support.
Financial support and sponsorship
This work is a dentistry thesis with grant number: 93155 in the Kermanshah University of Medical Sciences.
Conflicts of interest
Mozafar Khazaei has editorial involvement with Dent Hypotheses.
| References|| |
Reddy MS, Geurs NC, Wang IC, Liu PR, Hsu YT, Jeffcoat MK, et al
. Mandibular growth following implant restoration: Dose Wolff's law apply to residual ridge resorption? Int J Periodontics Restorative Dent 2002; 22:315-21.
Zarb G, Hobkrik J, Eckert S, Jacob R. Prosthodontic Treatment for Edentulous Patients: Complete Denture and Implant-supported Prosthesis. 13 th
ed. St. Louis: Mosby; 2013.
Marcus SE, Drurry TF, Brown LJ, Zion GR. Tooth restoration and tooth loss in the permanent dentition of adults: United States,1988-1991. J Dent Res 1996;75:684-95.
Mecall RA, Rosenfeld AL. Influence of residual ridge resorption patterns on implant fixture placement and tooth position. 1. Int J Periodontics Restorative Dent 1991;11:8-23.
Darby I, Chen ST, Buser D. Ridge preservation techniques for implant therapy. Int J Oral Maxillofac Implants 2009; 24(Suppl):260-71.
Kesmas S, Swasdison S, Yodsanga S, Sessirisombat S, Jansisyanont P. Esthetic alveolar ridge preservation with calcium phosphate and collagen membrane: Preliminary report. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2010; 110:e24-36.
Underwood MA, Gilbert WG, Sherman MP. Amniotic fluid: Not just fetal urine anymore. J Perinatol 2005;25:341-8.
Hill DJ, Tevaarwerk GJ, Arany E, Kilkenny D, Gregory M, Langford KS, et al
. Fibroblast growth factor-2 (FGF-2) is present in maternal and cord serum, and in the mother is associated with a binding protein immunologically related to the FGF receptor-1. J Clin Endocrinol Metab 1995;80:1822-31.
Dahl L, Hopwood JJ, Laurent UB, Lilja K, Tengblad A. The concentration of hyaluronate in amniotic fluid. Biochem Med 1983;30:280-3.
Mazzucco L, Balbo V, Cattana E, Guaschino R, Borzini P. Not every PRP-gel is burn equal. Evaluation of growth factor availability for tissues through four PRP-gel preparations: Fibrinet, RegenPRP-kit, Plateltex and one manual procedure. Vox Sang 2009;97:110-8.
Weibrich G, Kleis WK, Hafner G, Hitzler WE. Growth factor levels in platelet-rich plasma and correlations with donor age, sex, and platelet count. J Craniomaxillofac Surg 2002;30: 97-102.
Landesberg R, Roy M, Glickman RS. Quantification of growth factor levels using a simplified method of platelet-rich plasma gel preparation. J Oral Maxillofac Surg 2000;58:297- 301.
van den Dolder J, Mooren R, Vloon AP, Stoelinga PJ, Jansen JA. Platelet-rich plasma: Quantification of growth factor levels and effect on growth and differentiation of rat bone marrow cells. Tissue Eng 2006;12:3067-73.
Simman R, Hoffmann A, Bohincs RJ, Peterson WC, Russ AJ. Role of platelet-rich plasma in acceleration of bone fracture healing. Ann Plast Surg 2008;61:337-44.
Arosarena OA, Collins WL. Bone regeneration in the rat mandible with bone morphogenetic protein-2: A comparison of two carriers. Otolaryngol Head Neck Surg 2005;132:592-7.
Grigolo B, Roseti L, Fiorini M, Fini M, Giavaresi G, Aldini NN, et al
. Transplantation of chondrocytes seeded on a hyaluronan derivative (hyaff-11) into cartilage defects in rabbits. Biomaterials 2001;22:2417-24.
Lisignoli G, Fini M, Giavaresi G, Nicoli AN, Toneguzzi S, Acchini A. Osteogenesis of large segmental radius defects enhanced by basic fibroblast growth factor activated bone marrow stromal cells grown on non-woven hyaluronic acid-based polymer scaffold. Biomaterials 2002;23:1043-51.
Toole BP, Wight TN, Tammi MI. Hyaluronan-cell interactions in cancer and vascular disease. J Biol Chem 2002; 277:4593-6.
Moseley R, Leaver M, Walker M, Waddington RJ, Parsons D, Chen WY, et al
. Comparison of the antioxidant properties of HYAFF1-11p75, AQUACEL and hyaluronan towards reactive oxygen species in vitro
. Biomaterials 2002;23:2255-64.
Thaller SR, Dart A, Tesluk H. The effects of insulin-like growth factor-1 on critical-size calvarial defects in Sprague-Dawley rats. Ann Plast Surg 1993;31:429-33.
Sandor GK, Lindholm TC, Clokie CML. Bone regeneration of the craniomaxillofacial and dento-alveolar skeletons in the framework of tissue engineering. In: Ashammakhi N, Ferreti P, editors. Topics in Tissue Engineering. Finland: University of Oulu; 2003.
Giavaresi G, Torricelli P, Fornasari PM, Giardino R, Barbucci R, Leone G. Blood vessel formation after soft-tissue implantation of hyaluronan-based hydrogel supplemented with copper ions. Biomaterials 2005;26:3001-8.
Savani RC, Cao G, Pooler PM, Zaman A, Zhou Z, Delisser HM. Differential involvement of the hyaluronan (HA) receptors CD44 and receptor for HA-mediated motility in endothelial cell function and angiogenesis. J Biol Chem 2001;276:36770-8.
Slevin M, Kumar S, Gaffney J. Angiogenic oligosaccharides of hyaluronaninduce multiple signaling pathways affecting vascular endothelial cell mitogenic and wound healing responses. J Biol Chem 2002;277:41046-59.
Mendes RM, Silva GA, Lima MF, Calliari MV, Almeidia AP, Alves JB, et al
. Sodium hyaluronate accelerates the healing process in tooth sockets of rats. Arch Oral Biol 2008; 53:1155-62.
Crossley DA. Management of rabbit and rodent tooth elongation. Dacross Services 2003.
Vittorio C, Margarita G. Rabbit and Rodent Dentistry Handbook. Lake Worth, Florida, USA: Zoological Education Network Inc.; 2005.
Montesano R, Vassali JD, Baird A, Guillemin R, Orci L. Basic fibroblast growth factor induces angiogenesis in vitro
. Proc Natal Acad Sci U S A 1986;83:7297-301.
Mountain RE, Glasby MA, Sharp JF, Murray JA. A morphological comparison of interposed freeze-thawed skeletal muscle autografts and interposed nerve autografts in the repair of the rat facial nerve. Clin Otolaryngol Allied Sci 1993;18:171-7.
Karaçal N, Koºucu P, Cobanglu U, Kutlu N. Effect of human amniotic fluid on bone healing. J Surg Res 2005;129: 283-7.
[Table 1], [Table 2]