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 Table of Contents  
ORIGINAL RESEARCH
Year : 2014  |  Volume : 1  |  Issue : 3  |  Page : 123-131

Immunohistochemical evaluation: The effects of propolis on osseointegration of dental implants in rabbit's tibia


1 Department of Oral Histology and Biology, College of Dentistry, University of Kufa, Kufa, Najaf, Iraq
2 Department of Oral Histology and Biology, College of Dentistry, Baghdad University, Baghdad, Iraq
3 Department of OMF Surgery, College of Dentistry, University of Kufa, Kufa Najaf, Iraq

Date of Web Publication8-Dec-2014

Correspondence Address:
Abbas Taher
Department of OMF Surgery, College of Dentistry, University of Kufa, Kufa Najaf
Iraq
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/2348-2915.146490

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  Abstract 

Background: Dental implant is an artificial tooth root-fixed into the jaws to hold a replacement tooth or bridge. Functional surface modifications by organic material such as propolis coating seem to enhance early peri-implant bone formation, enhancing the initial cell attachment. The aim of the study was to study the expression of osteocalcin (OC) and type I collagen (COLL1) as bone formation markers in propolis-coated and -uncoated implant in interval periods (1, 2, 4, and 6 weeks). Materials and Methods: Commercially pure titanium (cpTi) implants, coated with propolis protein, were placed in the tibias of 40 New Zealand white rabbits, histological and immunohistochemical tests for detection of expression of OC and COLL1were performed on all the implants of both control and experimental groups for (1, 2, 4, and 6 weeks) healing intervals. Results: Histological finding for coated titanium implant with propolis illustrated an early bone formation, mineralization, and maturation in comparison to control. Immunohistochemical finding showed that positive reaction for OC and COLL1 was expressed by osteoblast cells at implants coated with propolis, indicating that bone formation and maturation was accelerated by adding biological materials as a modification modality of implant surface. Conclusion: The present study concludes that coating of implants with propolis showed increment in osseointegration in short interval period.

Keywords: Biochemical bone markers, collagen I, dental implant, osseointegration, osteocalcin, propolis


How to cite this article:
Al-Molla BH, Al-Ghaban N, Taher A. Immunohistochemical evaluation: The effects of propolis on osseointegration of dental implants in rabbit's tibia. J Dent Res Rev 2014;1:123-31

How to cite this URL:
Al-Molla BH, Al-Ghaban N, Taher A. Immunohistochemical evaluation: The effects of propolis on osseointegration of dental implants in rabbit's tibia. J Dent Res Rev [serial online] 2014 [cited 2019 Jun 25];1:123-31. Available from: http://www.jdrr.org/text.asp?2014/1/3/123/146490


  Introduction Top


Dental implant is an artificial tooth root-fixed into the jaws to hold a replacement tooth or bridge. [1] Titanium is widely used for dental implants because of its biocompatibility, mechanical strength, and plasticity for prosthetic design. [2] Osseointegration is commonly defined as a direct and stable anchorage of an implant by the formation of bony tissue without growth of fibrous tissue at the bone-implant interface. [3] In order to enhance bone formation, implants have been coated with bone specific biomolecules. [4]

Propolis is the most important chemical weapon of bees against pathogenic microorganisms; it has been used as a remedy by humans since ancient times. It is a sticky, resinous substance collected by honeybees from the sap, leaves, and buds of plants, and then mixed with secreted beeswax. [5] In dentistry, propolis has been used for the treatment of aphthous ulcers, candidiasis, acute necrotizing ulcerative gingivitis (ANUG), gingivitis, periodontitis, and pulpitis. [6] The most important constitution of the propolis is the flavonoids. [7] Caffeic acid phenethyl ester (CAPE), one of major component of the honeybee propolis, it stimulates the proliferation of wound epidermis keratinocytes. [8] Application of 5% ethanolic extracts of propolis in the dental cavity of dogs were reduction of the inflammatory reaction and positive performance as tissue reorganization. [6] Duarte et al., [9] reported thatCAPE is effective in osteoarthritis, showed significantly decreased cartilage destruction and reduced loss of matrix proteoglycans. Experimental work in vitro show that the ethanolic extracted propolis able to prevent cartilage destruction where the caffeic acid ester able to glycosaminoglycans (GAGs) release into medium of human cartilaginous tissue cultures, and help of GAGs synthesis in chondrocytes. Honey, bee venom, pollen, and propolis were used to treat arthritis and other inflammatory, autoimmune, and degenerative diseases such as multiple sclerosis. [10] Study by Chai et al., [11] investigated that the attenuation of osteoclastogenesis and induction of osteoclast apoptosis through the inhibition of nuclear factor-kB (key regulator of osteoclast differentiation, activation, and survival) activation by the propolis caffeic acid phenethyl ester (CAPE), this might be useful for the treatment of osteolysis attended with enhanced osteoclast formation and activation. The ipriflavone (available in bee propolis) stimulates the secretion and synthesis of calcitonin from the thyroid, as well as aiding bone formation and density. [12]

Sabir et al., [13] reported the propolis is capable of stimulating the production of transforming growth factor-beta1 (TGF-B1). Al-Molla, [14] showed that propolis enhanced the bone formation when implanted in a defect in the mandible.

Osteocalcin (OC), the g-carboxyglutamic acid-containing protein, which in most species is the predominant noncollagenous protein of bone and dentin, has been postulated to play roles in bone formation and remodeling. [15] OC is secreted solely by osteoblasts and thought to play a role in the body's metabolic regulation and is pro-osteoblastic, or bone building, by nature. It is also implicated in bone mineralization and calcium ion homeostasis. [16] Type I collagen (COLL1) is the major organic component of the mineralized bone matrix. By immunohistochemical staining they could detected its expression in bone matrix. The formation of bone by osteoblastic cells requires the deposition of an extracellular matrix consisting of COLL1 and a variety of noncollagenous proteins, which subsequently mineralizes by the formation of hydroxyapatite crystals. [17]


  Materials and Methods Top


Materials

  • Eighty screw-shaped implants, 3.5 mm in diameter, and a total lengthof 8 mm (threaded part is 5 mm and smooth part is 3 mm)
  • Ethanolic extracted propolis.
  • Antiosteocalcin antibody (ab13418), Abcam, UK
  • Anticollagen-I antibody (ab90395), Abcam, UK
  • Detection Kits System ( ab94740), Abcam, UK
  • Protein block 15 enhancer
  • Naphthol phosphate
  • Fast red chromogen
  • Alkaline-phosphatase (AP)-conjugate
  • Cofactor enhancer.


Methods

FortyNew Zealand rabbits aged 10-12 months were used in this study, they were divided into four groups for (1, 2, 4, and 6 weeks) healing intervals, 10 animals for each period. Animals were generally anesthetized and atraumatic surgical technique was performed to prepare two holes in the tibia; propolis-coated implant was inserted in one hole and uncoated implant (control) placed in the second one. All tissue specimens, samples and controls, were fixed in 10% neutral formalin and processed in a routine paraffin blocks. Each formalin-fixedparaffin-embedded specimen had serial sections were prepared as follows: Five micrometer thickness sections were mounted on clean glass slides for routine hematoxylin and eosin staining (H and E), from each block of the studied sample and the control group for histopathological reexamination. Other four sections of 5μm thickness were mounted on positively charged microscopic slides to obtain a greater tissue adherence for immunohistochemistry. The procedure of the immunohistochemical (IHC) assay adapted by this study was carried out in accordance with the manufacturer's instructions (Abcam, UK).


  Results Top


Histological examination

One week postoperatively


Threads in the marrow space followed the shape of the screw showed bone trabeculae within woven bone, large and more fat cells with small amount in between them appeared in the marrow space, large number of active mitotic progenitor, and osteoblast cells [Figure 1]. In uncoated implants, the marrow space showed large number of fatty tissue with granulation tissue with large blood vessels, no woven bone shown [Figure 2].
Figure 1: Thread show bone trabeculae in marrow space of P -implant at 1week interval, H and E, ×20

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Figure 2: Thread show no bone trabeculae in uncoated implant at 1week interval, H and E, ×20

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Two weeks postoperatively

Microphotograph view of bone section shows thick bone trabeculae with reticulofiber tissue scattered between them and large number of the osteocytes embedded within these trabeculae, raw of osteoblasts and other of osteoclasts arranged on the periphery of the trabeculae is shown in [Figure 3]. In control group, a number ofactive osteoblast and progenitor cells scattered within woven bone, with few thin bone trabeculae involve preosteocytes and osteocyteswere observed [Figure 4].
Figure 3: Thick bone trabeculae with osteonlast and osteoclast cells in P -implant at 2 weeks interval, H and E, ×20

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Figure 4: Thread show bone trabeculae in marrow space in un coated implant at 2 weeks interval, H and E, ×20

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Four weeks postoperatively

Bone thread developed after 4 weeks of implantation shows thick bone trabeculae with active bone forming cells and progenitor cells in between them, osteoid tissue formed at the periphery of the trabeculae [Figure 5], control one show threads formed at the site of the implant with thin bone trabeculae and reticulofiber tissue in between them [Figure 6].
Figure 5: Calcified bone thread in P-coated show thick bone trbeculea at 4 weeks interval, H and E, ×20

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Figure 6: Thread show bone trabeculae in uncoated implant at 4 weeks interval, H and E, ×20

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Six weeks postoperatively

Mature bone shown in [Figure 7], in the thread area of implant coated with propolis, bone deposit around uncoated implant (control) in a form of thread with thick bone trabeculae in control group [Figure 8].
Figure 7: Calcified bone thread in P-coated show thick bone trabeculea at 6 weeks interval, H and E, ×20

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Figure 8: Calcified bone thread in P-coated show thick bone trabeculea at 6 weeks interval, H and E, ×20

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Immunohistochemical examination for OC expression

One week postoperatively


Propolis-coatedimplantsstaining with OC monoclonal antibody showed that OC expression was strong positive in the osteoblasts, osteocytes, and progenitor cells [Figure 9]. Uncoated implants, negative expression of OC monoclonal antibody on uncoated implant in progenitor, fatty cells, and extracellular matrix [Figure 10].
Figure 9: View for strong positive alkaline phosphatase - conjugate red stain for localization of osteocalcin of P-coated implant for 1week interval in osteoblasts, osteocytes, progenitor cells, red stain with counter stain hematoxylin, ×40

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Figure 10: Negative expression alkaline phosphatase - conjugate red stain for localization of osteocalcin of uncoated implant for 1week interval in osteoblasts, osteocytes and progenitor cell, red stain with counter stain hematoxylin, ×40

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Two weeks postoperatively

The immunohistochemical staining with OC monoclonal antibody at 2 weeks of healing period showed that OCexpression was moderate positive in osteoblasts and osteocytes [Figure 11]. Uncoated implantsshowed that OC expression was strong positive in the osteoblasts, osteoclasts, osteocytes, progenitor cells, and extracellular matrix [Figure 12].
Figure 11: View for moderate positive alkaline phosphatase - conjugate red stain for localization of OC in in some osteoblasts, osteocyts of P-coated implant for 2 week interval, red stain with counter stain hematoxylin, ×40

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Figure 12: strong positive expression alkaline phosphatase- conjugate red stain for localization of osteocalcin in osteoblasts, osteocytes, progenitor cells in uncoated implant, red stain with counter stain hematoxylin, ×40

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Fourweeks postoperatively

Propolis-coated implants showed that OC localization was weakly positiveexpression in osteoblasts, progenitor cells [Figure 13]. While the uncoated implants, moderate positive expression in osteoblasts and osteocytes, progenitor cell in 4 weeks interval, but showed weakly positiveexpression in 6 weeks postoperatively [Figure 14].
Figure 13: View for weak positive alkaline phosphatase- conjugate red stain for localization of osteocalcin in osteoblasts and extracellular matrix, of P-coated implant for 4week interval, red stain with counter stain hematoxylin, ×40

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Figure 14: moderate positive expression alkaline phosphatase-conjugate red stain for localization of osteocalcin in osteoblasts, osteocytes in uncoated implant, red stain with counter stain hematoxylin, ×40

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Six weeks postoperatively

Propolis-coated implants showed that OC localization was weakly positiveexpression [Figure 15]. While the uncoated implants, weekly positive expression [Figure 16].
Figure 15: View for negative alkaline phosphatase- conjugate red stain for localization of osteocalcin in thread site of P-coated implant for 6week interval, red stain with counter stain hematoxylin, ×20

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Figure 16: Weak positive expression alkaline phosphatase- conjugate red stain for localization of osteocalcin in osteoblasts, osteocytes in uncoated implant, red stain with counter stain hematoxylin, ×40

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COLL1 expression

One week postoperatively


Propolis-coated implants staining with COLL1 monoclonal antibody showed that COLL1 expression was strong positive in the osteoblasts, osteocytes, and progenitor cells [Figure 17]. For uncoated implants, weak positive expression of COLL1 monoclonal antibody on uncoated implant in osteoblasts, osteocytes, and progenitor cells [Figure 18].
Figure 17: Woven with strong positive alkaline phosphatase- conjugate red stain for localization of type I collagen of P-coated implant for 1week healing period in osteoblasts, osteocytes, progenitor cells and extracellular matrix (pink color), red stain with counter stain hematoxylin, ×20

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Figure 18: View for weak positive alkaline phosphatase- conjugate red stain for localization of type I collagen in thread site of uncoated implant , red stain with counter stain hematoxylin, ×20

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Two weeks postoperatively

Propolis-coated implants, the COLL1 expression was weak positive in osteoblasts and osteocytes [Figure 19]. Uncoated implantsshowed that COLL1 expression was strong positive in the osteoblasts, osteoclasts, osteocytes, progenitor cells, and extracellular matrix [Figure 20].
Figure 19: View for weak positive alkaline phosphatase- conjugate red stain for localization of type I collagen of P-coated implant for 2week healing period in osteoblast, extracellular matrix, red stain with counter stain hematoxylin, ×40

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Figure 20: With strong positive alkaline phosphatase- conjugate red stain for localization of type I collagen in ostecytes, osteoblasts, progenitor cells, red stain with counter stain hematoxylin, ×40

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Four weeks postoperatively

Propolis-coated implants, COLL1 monoclonal antibody at 4 weeks of healing period showed weak positive expression in osteoblasts, progenitor cells, and the extracellular matrix [Figure 21]. In uncoatedimplants; moderate positive expression in osteoblasts, progenitor cell, blood vessels, and extracellular matrix [Figure 22].
Figure 21: View for weak positive alkaline phosphatase-conjugate redstain for localization of type I collagen in P-coated implant for 4weeks healing period in some osteocytes and osteoclasts, cell red stain, with counter stain hematoxylin, × 40

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Figure 22: View for moderate positive alkaline phosphatase-conjugate red stain for localization of COLL1 in uncoated implant for 4 weeks healing period in osteoblasts, progenitor cells, and extracellular matrix (red arrow) red stain with counter stain hematoxylin, ×40

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Six weeks postoperatively

COLL1 localization showed negative expression in osteoblasts and osteocytes [Figure 23]. Uncoated implants, the COLL1 localization was weakly positive expression in osteoblasts at the periphery of the boneand progenitor cells [Figure 24].
Figure 23: View for negative alkaline phosphatase-conjugate red stain for localization of COLL1 in P-coated implant for 6 weeks healing period in thread site, red stain with counter stain hematoxylin, ×20

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Figure 24: View for weak positive IHC redstain for localization of COLL1 in uncoated implant for 6 weeks healing period in osteoblasts and progenitor cells, red stain with counter stain hematoxylin, ×40

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  Discussion Top


The results of this study showed that osseointegration can be obtained when implants are inserted in a living bone and when a suitable biological environment for bone formation is created. The strength of the bond between osseointegrated implant, this result coincideswith finding of Wennerberg and Albrektsson. [18] Successful attachment on artificial surface is prerequisite for inducing new bone formation locally at the site of implantation. Protein-coated surfaces may influence the biocompatibility of implant materials by initiating and supporting osteogenesis. [19]

The uncoated implants, at 1 week duration, embryonic connective tissue with active collagen fiber deposition were showed around implant; this result agreed with finding of Jamil, [20] who reported that within 1 week, embryonic connective tissue with active collagen fiber around the implant which represent organic constitution of bone would be formed. Uncoated Ti implant in rabbit tibia after 2 weeks of implantation shows a number ofactive osteoblast and progenitor cells scattered within woven bone, with few thin bone trabeculae. These finding shave been supported by the results of Niehaus et al., [21] who found more osteons had uptake of bone marker on day 14 than at any other time during the study new bone was visible within the area between the threads of the control screws.

At 4 weeks duration, according to the study conducted by Yoshinari et al., [22] the micrographs of the implant-bone interfaces at 4 weeks after implantation show that bone tissue has grown on the implant surface; while after 6 weeks duration, thickbone trabeculae and large number of bone forming cells on the border of bone trabeculae and this agree with Depprich et al., [23] who found that when the healing period was near to 6 weeks.

The propolis-coated implants, at 1-2 weeks durationthe threads in the marrow space followed the shape of the screw showed bone trabeculae, osteocytes embedded within these trabeculae, raw of osteoblasts, and other of osteoclasts arranged on the periphery of the trabeculae. This result agree with Al-Molla, [14] who showed an increase in bone formation in 1 and 2 weeks in comparison to the control group.

Milot [12] found that ipriflavone (component of propolis) stimulate the secretion and synthesis of calcitonin from the thyroid as well as aiding bone formation. Calcitonin facilitate the storage of calcium in the bone from the blood. [24] Sabir et al., [13] showed that the propolis stimulate the production of TGF-B1. This growth factor increase the rapidity of bone formation. [25] Study by Wiκckiewicz et al., [26] investigated thatthe extracts of propolis formouth rinsing, or toothpastes or gel-based on propolis extract seems to be a promising agent, not only for prophylaxis but also for the treatment of parodontitis/peri-implantitis. Systemic use of propolis may hasten new bone formation at the expanded suture in rats in 12 days of mechanical retention. [27]

At 4 and 6 weeks duration, micrograph view of bone thread shows thick bone trabeculae with active bone-forming cells and progenitor cells in between them and osteocytes within their lacunaealso theosteon appeared. Propolis not only decreased apoptosis but also increased the metabolic activity and proliferation of periodontal ligament (PDL) cells. [28] A great portion of these flavonoid molecules seem to have their effects on bone through an estrogen-like activity and have been therefore termed phytoestrogens. Toker et al., [29] showed propolis significantly reduced the periodontitis-related bone loss, the findings of this study provide morphologic and histological evidence that propolis, when administered systemically prevents alveolar bone loss in the rat model. Also the result agreed with Al-Molla, [14] who showed that the propolis take the higher score in bone healing criteria in comparison to other groups in 4 weeks interval.

OC expression was strong positive in the active mitotic osteoblast, and progenitor cells in all experimental groups at 1 week interval. OC seems to have a role in the early stages of bone formation and some studies by Al-Ghani et al., [16] suggest that it is chemotactic for osteoclast and regulate osteoblast activity too. Our results shows a greater number of positive cells indicate a more rapid tissue reaction on implant surface. Novaes et al., [30] who reported that OC, as one of the important indicators of osteogenic differentiation and bone tissue formation, have been shown to express at higher levels on modified titanium surfaces. In vitro studies demonstrated that mRNA of COLL1 is expressed during the initial period of proliferation and extracellularmatrix biosynthesis, since it is hypothesized that enhanced expression of osteogenic markers in vitro leads to more and more expeditious bone formation at the bone-biomaterial interface in vivo. [31] COLL1 is a bone marker associated with the differentiation of osteocyte, [32] and this agree with the results in this study where the osteocyte express COLL1.

 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10], [Figure 11], [Figure 12], [Figure 13], [Figure 14], [Figure 15], [Figure 16], [Figure 17], [Figure 18], [Figure 19], [Figure 20], [Figure 21], [Figure 22], [Figure 23], [Figure 24]



 

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