10.59987/ads/2014.2.66-68
The ability of platelet-derived growth factor (PDGF), insulin-like growth factor-1 (IGF-1) and platelet-rich plasma (PRP) to increase the rate of osseointegration of endosseous implants and to improve the quality of bone remodeling on the surface of titanium, has been investigated in an experimental intraosseous defect model by an histological and immunohistochemical evaluation. The results from this study demonstrate that rabbits treated with the combination PDGF/IGF-1 showed a higher positive effect on bone regeneration than PRP-treated or controls.
application/pdf
1971-1441
119
2
en
A. Polimeni 
https://creativecommons.org/licenses/by-nc-nd/4.0
Annali di stomatologia
dental implants
Effect of PDGF, IGF-1 and PRP on the implant osseointegration. An histological and immunohistochemical study in rabbits
Text.Serial.Journal
https://www.annalidistomatologia.eu/ads
5
Original Article
2022-08-16
2022-08-16
2014-06-01
2023-12-04
66-68
Ann Stomatol (Roma)
A. Polimeni 
2014
10.59987/ads/2014.2.66-68
66
1971-1441
2
dental implants
en
68
Effect of PDGF, IGF-1 and PRP on the implant osseointegration. An histological and immunohistochemical study in rabbits
https://www.annalidistomatologia.eu/ads/article/download/119/102
5
                                                                                                   Original article



    Effect of PDGF, IGF-1 and PRP on the implant
    osseointegration. An histological and
    immunohistochemical study in rabbits




                                                                                                                      li
                                                                  na
    Emanuela Ortolani, MD1                                   tissues. In fact, platelets play an important role in
    Massimiliano Guerriero, MD2                              many processes by releasing many factors, such as




                                                                io
    Antonella Coli, MD3                                      platelet-derived growth factor (PDGF), insulin-like
    Amalia Di Giannuario, BD4                                growth factor-1 (IGF-1), transforming growth factor
    Gianluca Minniti, DDS5                                   (TFG) and vascular endothelial growth factor (VEGF),
    Antonella Polimeni, MD, DDS6                             that control cell proliferation, differentiation, chemo-




                                                             az
                                                             taxis and tissue morphogenesis. The clinical use of
                                                             platelet-rich plasma (PRP) has been shown to en-
    1 General Dentistry and Emergency Care Unit George       hance soft and bone tissue healing and PRP has
      Eastman Dental Hospital, Rome, Italy                   been used in implantology to supply growth factors to




                                                          rn
    2 Laboratory of Pathology, A. Cardarelli Hospital,       sites of surgery, in order to increase the rate of bone
      Campobasso, Italy                                      deposition and quality of bone regeneration (1,2).
    3 Institute of Pathology, Sacro Cuore Cattolica Uni-     Once it is released from platelets, PDGF can improve
      versity, Rome, Italy                                   new bone deposition by promoting collagen synthesis




                                                       te
    4 Istituto Superiore di Sanità (ISS), Rome, Italy        and increasing the deposition of bone matrix (3,4). It
    5 Private practice, Rome, Italy                          has been shown that PDGF, which binds to specific
    6 Department of Oral and Maxillo-facial Sciences, Pe-    surface receptors, has important functions in many
                                                    In
      diatric Dentistry Unit, Sapienza University of Rome,
      Italy
                                                             physiological processes including wound healing and
                                                             angiogenesis, also playing a synergistic role with oth-
                                                             er factors, such as IGF-1 (5,6). In the present work
                                                             we carried out a study in an experimental animal
    Corresponding author:                                    model to evaluate the effect of PDGF, IGF-1 and
                                       ni
    Emanuela Ortolani                                        PRP on the integration of bone implants placed in the
    General Dentistry and Emergency                          femurs of rabbits. The influence of the treatment on
    Care Unit George Eastman Dental Hospital, Rome           the process of implant osseointegration was evaluat-
    Viale Regina Elena, 287/B                                ed by histological and immunohistochemical exami-
                           o

    00161 Rome, Italy                                        nation performed on bone samples taken at different
    E-mail: emanuelaortolani@tiscali.it                      time after implantation.
                       izi



    Summary                                                  Materials and methods
          Ed




    The ability of platelet-derived growth factor            All procedures were in accord with the Italian laws
    (PDGF), insulin-like growth factor-1 (IGF-1) and         (DL 27/1/92 n.116) and the directives of the Council
    platelet-rich plasma (PRP) to increase the rate of       of European Communities (886/609 CE). The study
    osseointegration of endosseous implants and to           was performed on eight healthy male breeding rab-
    improve the quality of bone remodeling on the            bits “Fauve de Bourgogne”, weighing 2.5 to 3 Kg. Ex-
    surface of titanium, has been investigated in an         perimental subjects were randomly assigned to 2
    C




    experimental intraosseous defect model by an             groups for evaluating osseointegration and bone re-
    histological and immunohistochemical evalua-             generation capability around dental implants: one
CI




    tion. The results from this study demonstrate that       group treated with PDGF/IGF-1, and the other one
    rabbits treated with the combination PDGF/IGF-1          treated with PRP. The animals were anesthetized
    showed a higher positive effect on bone regener-         with an intravenous injection of penthobarbital (30
    ation than PRP-treated or controls.                      mg/kg). Anesthesia was maintained, when needed,
                                                             with additional penthobarbital. Additionally, a local
    Key words: PDGF, IGF-1, PRP, growth factors,             anesthetic (lidocaine 0.5 ml/adrenaline) was applied
©




    bone formation, dental implants.                         subcutaneously. After the incision of the skin and the
                                                             subcutaneous tissues, planes were dissected in order
                                                             to expose the femur. Then, using a low speed drill
    Introduction                                             with continuous irrigation, bone defects were per-
                                                             formed. Before implantation, bone defects were filled
    The release of growth factors by platelets can con-      with PDGF/IGF-1 combination or with PRP. In each
    tribute to cell proliferation and healing of damaged     animal one intraosseous defect performed in the con-

    66                                                                          Annali di Stomatologia 2014; V (2): 66-68
    Effect of PDGF, IGF-1 and PRP on the implant osseointegration. An histological and immunohistochemical study in rabbits


    trolateral femur was simultaneously filled with methyl-       sites showed only a focal and discontinuous deposi-
    cellulose gel vehicle to serve as control.                    tion of fibroblasts around the implants.
    The combination of PDGF and IGF-1 (both supplied              This fibrous tissue was composed of spindle cells
    by Sigma Chemicals, Milan, Italy, and diluted at a            with elongated or oval nuclei and numerous mitotic
    concentration of 4 μg/μL) was added immediately be-           figures (indicating high proliferative activity) (Fig. 1a).




                                                                                                                              li
    fore use to 30 μl of vehicle, which consisted of a 2%         In this phase, the first deposition of bone was ob-
    methylcellulose gel solution (Methocel, Fluka, Milan,         served below the cortical bone in proximity of the




                                                                    na
    Italy). To obtain PRP, an average 5 mL autologous             neck of the hole, consisting of osteoid matrix trabecu-
    blood sample was retrieved, and 3,8 % sodium citrate          lae, bordered by osteoblasts. These trabeculae of os-
    was immediately added to blood as anticoagulant.              teoid appear not yet joined neither to the cortical
    Then, the whole blood was centrifuged for 15 minutes          bone adjacent neither to the implant.




                                                                  io
    at 100 g. The supernatant was carefully removed and
    30 μl of PRP (containing an average of 50000-70000
    platelets) were added to 20 μl of methylcellulose gel         7th to 12th day
    vehicle (Methocel).




                                                               az
    A total volume of 50 μl of methylcellulose gel contain-       All histological samples taken from the animals treat-
    ing PDGF-IGF1 or PRP was applied in the hole, and             ed with PDGF/IGF-1 exhibited significant bone regen-
    the implant placement followed. The dental implants,          eration in the neck hole adherent to the cortical bone,
    measuring 3.3 mm in diameter and 8 mm in length,              through a process of endosteal bone formation. On




                                                            rn
    were positioned in each femoral metaphysis. After             the other hand, bone formation appeared uneven in
    this procedure, the muscle planes and the superficial
    tissues were sutured. For preventing infection, and




                                                         te
    antibiotic therapy (rocefin, 20 mg/kg) was adminis-
    tered via intramuscular injection after surgery.
    After 4, 7 and 12 days from implant insertion, the rab-
    bits were euthanized by an intravenous injection of
                                                      In
    penthobarbital solution. Their femurs were dissected,
    and a segment of metaphysis about 1.5 cm in length
    comprising the implant was obtained for histological
    and immunohistochemical study.
    After 24 hours of fixation in buffered formalin and ad-
                                                ni
    ditional 36 hours of decalcification with a commercial
    EDTA-hydrochloric acid mixture (Surgipath Decalcifi-
    er II, Leica Biosystem, USA) the implant was “gently”
    removed and the bone segment was cut longitudinal-
                              o

    ly with a plane passing through the hole. The bone
    fragments were embedded in paraffin and 4 µm con-
                          izi


    secutive sections were cut and numbered.                       a
    For conventional histology staining with hematoxylin-                                                                     b
    eosin or Van Gieson to assess the morphological de-
    tail of osseointegration was performed. For immuno-
           Ed




    histochemistry, deparaffinized sections were incubat-
    ed for 10 minutes with methanol containing 10% H2O2
    to block the activity of endogenous peroxidases. The
    sections were then incubated with polyclonal rabbit
    anti-PDGFR-α and monoclonal mouse anti PDGFR-β
    antibodies (Santa Cruz Biotechnology, at cat. sc-338
    C




    and sc-6252 dilution of 1:200), anti-VEGF and anti-
    VEGFR antibodies.
CI




    After washing, the sections were incubated with
    streptavidin-biotin detection system (LSAB, Dako). Fi-
    nally, the reaction was developed with diaminobenzi-
    dine solution.
                                                                  Figure 1. Histological analysis of the implant site treated
©




                                                                  with PDGF and IGF-1. 1a) At 4th day, the implant sites
    Results                                                       treated with PRP or PDGF/IGF1 showed abundant cap-like
                                                                  deposition of fibroblastic tissue around the implants; this fi-
    4th day                                                       brous tissue was composed of spindle cells with elongated
                                                                  or oval nuclei and numerous mitotic figures. 1b) At the 12th
    The implant sites treated with PRP or PDGF/IGF1               day, a substantial amount of well formed, significantly more
    showed abundant cap-like deposition of fibroblastic           abundant woven bone in subjects treated with PDGF/IGF1
    tissue around the implants. In contrast, the control          than in animals treated with PRP and controls, was found.

    Annali di Stomatologia 2014; V (2): 66-68                                                                                 67
    E. Ortolani et al.


    animals PRP-treated. Scarce bone deposition was
    observed in control sites. In the animals treated with
    PDGF/IGF1 numerous islands of cartilage leading to
    endochondral ossification were observed around im-
    plants, and by the 12th day, in the same group of ani-




                                                                                                                                  li
    mals, a substantial amount of well formed, woven
    bone significantly more abundant than in animals




                                                                      na
    treated with PRP and controls, was found (Fig. 1b).


    Immunohistochemistry




                                                                    io
    After immunohistochemistry with antibodies against
    PDGFR-β, presence of scattered positive blast-like
    cells was observed in both treated and untreated ani-




                                                                 az
    mals (Fig. 2). Also megakaryocytes appeared to be
                                                                  Figure 2. Near the implant site at 4th day from surgery, im-
    labelled, but lymphocytes were generally negative. In         munohistochemical staining for PDGFR-b showed scat-
    addition, a fraction of chondroblasts and osteoblasts         tered cells in the reparative tissue rich in fibroblast.
    in osteoid matrix were also positive.




                                                              rn
    After immunohistochemistry with antibodies against
    PDGFR-α, no significative results were seen.                  ing and bone formation, expressed receptors for
    A significant expression of VEGF and VEGFR-2 in               PDGF-β. Therefore, from our data, showing presence
    animals sacrificed at 4th and 7th day was observed,




                                                           te
                                                                  of cells expressing PDGF receptors in foci of osteoin-
    with a strong expression present in the cytoplasm of          tegration, add further evidence for usefulness of
    osteoblasts bordering the osteoid matrix. Then, the           growth factors addition in site of implant surgery.
    expression rapidly decreased in the animals sacri-
                                                        In
    ficed at 12th day. A widespread, although weaker pos-
    itivity was observed in the cytoplasm of fibroblast-like      References
    cells forming the fibrous callus. A similar, although
    more discrete positivity, was observed after immuno-           1. Torres J, Tamimi F, Alkhraisat MH, et al. Platelet-rich plas-
    histochemistry with anti-VEGFR2 antibody.                         ma may prevent titanium-mesh exposure in alveolar ridge
                                           ni
                                                                      augmentation with anorganic bovine bone. J Clin Periodontol.
                                                                      2010;37:943-951.
                                                                   2. Dohan DM, Choukroun J, Diss A, et al. Platelet-rich fibrin
    Discussion and Conclusions                                        (PRF): A second-generation platelet concentrate. Part I: Tech-
                             o

                                                                      nological concepts and evolution. Oral Surg Oral Med Oral
    Recent studies have suggested that a number of                    Pathol Oral Radiol Endod. 2006;101:E37-44.
    growth factors, including PDGF, promote tissue repair          3. Chang PC, Seol YJ, Cirelli JA, et al. PDGF-B gene therapy
                         izi


    and regeneration, including bone formation; more-                 accelerates bone engineering and oral iplant osseointegra-
    over, the efficacy of growth factor therapies in peri-            tion. Gene Ther. 2010;17:95-104.
    odontology and implantology has been well charac-              4. Hollinger JO, Hart CE, Hirsch SN, Lynch S, Friedlaender GE.
    terized in a variety of in vitro and in vivo studies (7,8).       Recombinant human platelet-derived growthfactor: Biology
           Ed




    The present study, focused on the histological char-              and clinical applications. J Bone Joint Surg Am. 2008;90 (sup-
                                                                      pl 1):48-54.
    acteristics of the early osseointegration of implants,
                                                                   5. Lynch SE, Nixon JC, Colvin RB, Antoniades HN. Role of
    with or without addition of PRP and PDGF/IGF-1 in                 platelet-derived growth factor in wound healing: Synergis-
    combination, in an animal model, demonstrate a high-              tic effects with other growth factors: Proc Natl Acad Sci U
    er bone regeneration rate in growth factors-treated               S A. 1987; 84:7696-7700.
    group than in PRP or control group. Our results show
    C




                                                                   6. Tokunaga A, Oya T, Ishii Y, et al. PDGF receptor beta is a
    a greater new bone deposition in animals in which                 potent regulator of mesenchymal stromal cell function. J Bone
    PDGF/IGF-1 was added to bone defect, when com-                    Miner Res. 2008;23:1519-1528.
CI




    pared to animals treated with PRP or controls.                 7. Yun JH, Han SH, Choi SH, et al. Effects of bone marrow-
    Interestingly, our immunohistochemical demonstra-                 derived mesenchymal stem cells and platelet-rich plasma on
                                                                      bone regeneration for osseointegration of dental implants:
    tion that various types of cells present in reparative
                                                                      Preliminary study in canine three-wall intrabony defects. J
    foci express receptors for these molecules, in particu-
                                                                      Biomed Mater Res B Appl Biomater. 2013 Dec 5. doi:
    lar PDGFR-β, support the experimental evidence of                 10.1002/jbm.b.33084.
    positive therapeutic effects of PDGF on osseointegra-
©




                                                                   8. Kaigler D, Avila G, Wisner-Lynch L, et al. Platelet-derived
    tion. In normal bone marrow around the implant,                   growth factor applications in periodontal and peri-implant bone
    many blast cells, also including osteoprogenitors,                regeneration. Expert Opin Biol Ther. 2011;11:375-85. doi:
    chondrocytes and osteoblasts involved in tissue heal-             10.1517/14712598.2011.554814.




    68                                                                                   Annali di Stomatologia 2014; V (2): 66-68
