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Evaluation of healing processes of intraosseous defects with and without guided bone regeneration and platelet rich plasma. An animal study
Annals of Anatomy 194 (2012) 549–555
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Evaluation of healing processes of intraosseous defects with and without guided bone regeneration and platelet rich plasma. An animal study b ˙ Marzena Dominiak a,∗ , Katarzyna Łysiak-Drwal a , Leszek Solski b , Bogusława Zywicka , b a,c Zbigniew Rybak , Tomasz Gedrange a
Department of Oral Surgery, Silesian Piast Medical University, Wrocław, Poland Department of Experimental Surgery and Biomaterials Research, Silesian Piast Medical University, Wrocław, Poland c Department of Orthodontics, Dresden Technology University, Germany b
a r t i c l e
i n f o
Article history: Received 16 October 2011 Received in revised form 18 June 2012 Accepted 18 July 2012
Keywords: Intraosseous defects Bone grafting Bovine bone mineral Guided bone regeneration Platelet rich plasma
a b s t r a c t Background: In most cases, the natural healing of intrabony defects only leads to restoration of tissue continuity without differentiation and function. However, repair is not regarded to be an optimal treatment method, as confirmed in many clinical cases. Thus it is important to choose a surgical procedure which makes it possible to achieve restitution ad integrum of the bone structure. The choice of the GBR technique is crucial, in terms of the clinical conditions and limitations resulting from the use of a particular material. Objective: The objective of this study has been the analysis of effectiveness of selected surgical treatment techniques of intrabony defects in rabbits. Materials and methods: Research was conducted on 36 white rabbits. The operation technique was a criterion of division into 3 groups: BG/BOC (Bio-Oss Collagen® + Bio-Gide Perio® ), BOC/PRP (Bio-Oss Collagen® + PRP), C (control group). Qualitative and quantitative histopathological evaluation was carried out after 1, 3, 6 and 12 months. Results: The highest value of the bone surface area 31.9% (SD 1.8) was achieved in BOC/BG group three months after the implantation, while the lowest was revealed in C – group – 12.5% (SD 1.32) one month following the procedure. Conclusions: Upon quantitative histological assessment, the bone tissue presented the most intensive osteogenesis within one month from the application of BOC/PRP, whereas this was observed after the application of BOC/BG in later stages. The application of two regenerative methods influenced the rate, quality and overall treatment of intraosseus defects. © 2012 Elsevier GmbH. All rights reserved.
1. Introduction The process of healing in intraosseous defects leads to connective tissue (so-called connective tissue scar) formation in lieu of the proper, new bone formation. In clinical conditions healing may not assure the proper size of the alveolar process; it may also impede the differential diagnosis of cicatrices, including the treatment of pathological processes in bone tissue, as well as induce the occurrence of non-specific pain sensations, both in the operated area and its immediate vicinity (Dominiak and Łysiak, 2005). The proper shape and function of damaged tissue may now be restored owing to achievements in tissue engineering – a reconstructive biology that draws on the advances in medicine, surgery, physiology, cell and molecular biology, and polymer chemistry. For
∗ Corresponding author at: Wroclaw Medical University, Krakowska 26 st., 50-425 Wroclaw, Poland. Tel.: +48 717840251; fax: +48 717840253. E-mail address: [email protected] (M. Dominiak). 0940-9602/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.aanat.2012.07.007
clinical purposes, terms of guided tissue regeneration (GTR) and narrower guided bone regeneration (GBR) have been introduced (Nyman et al., 1982; Gottlow et al., 1986; Dahlin et al., 1989; Buser et al., 1993; Taguchi et al., 2005). In 1998, the so-called Lynch triad was designed, comprising three indispensable factors for ensuring undisturbed bone regeneration: structure (carrier), cells filling the base (cells stimulated by growth factors) and extracellular matrix material (ECM), i.e. signal molecules mediating the healing process. According to the principal assumption of tissue engineering, the implanted carrier, essential for initiation of the GTR and/or GBR processes, must be enriched with growth factors, cytokines or autogenous cells (Nyman et al., 1982). Despite dynamic growth in the field of tissue engineering, autogenic bone continues to be considered one of best regenerative materials. One significant limitation to the foregoing method, though, refers to the common difficulty in obtaining adequate quantities of material. Consequently, alternative solutions are being sought based primarily on the application of xenogenic and alloplastic materials, barrier membranes, as well as on the using of
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polypeptide growth factors and morphogenetic proteins (Nyman et al., 1982; Gottlow et al., 1986; Dahlin et al., 1989; Buser et al., 1993; Taguchi et al., 2005). The prevailing opinion suggests that the most effective method of treating intraosseous defects is the combined therapeutic approach involving bone augmentation using biomaterial with the simultaneous application of a barrier membrane for coverage. This method is based on the accumulation of regenerative potential of both materials and mechanical sustainment of these materials in the defect (Dietrich et al., 2003; Rankow and Krasner, 1996; Tseng et al., 1995; Abramowitz et al., 1994). However, a disadvantage of this method lies in its limitations resulting from the processing of xenogenic materials, from which proteins are eliminated which, among others, include growth factors locally activating osteogenesis (Kim et al., 2001). In cases of poor blood supply to the intraosseous defect area, these materials exhibit osteoconductive properties enabling the proliferation of newly formed bone tissue (including the inflow of growth factors carried in the blood). In other cases the application of xenogenic materials does not assure proper regeneration, necessitating the implementation of innovative treatment solutions. A supporting method for xenogenic osteoconductors is the use of an adjunctive method in which xenogenic osteoconductives are combined with autogenic bone or polypeptide growth factors (PRP) obtained from the patient’s full venous blood in centrifuge systems (Marx et al., 1998; Oyama et al., 2004; Kassolis et al., 2000; Jensen et al., 2005; Terheyden et al., 2002; Roldan et al., 2004; Wojtowicz et al., 2002). The majority of publications, however, describe the healing process in intraosseous defects following the application of platelet concentrate on autoor allogenic carriers (Wojtowicz et al., 2002; Indovina and Block, ˙ et al., 2005). Only a few evalu2002; Schwarz et al., 2007; Zywicka ate the effectiveness of bone healing in which xenogenic material – deproteinised bovine-derived xenograft (BDX) - served as the carrier. The relatively small number of direct comparative studies on the results of defect healing after implementation of BDX itself and in conjunction with PRP; and conflicting findings confirm that the subject-matter is still unresolved and requires further detailed research. The aim of this study was a quantitative and qualitative histological assessment of the healing process of iatrogenically induced intraosseous defects on animal models with and without the application of selected techniques of bone regeneration after 1, 3, 6 and 12 months. In the course of the research, healing of intraosseus defects allowed to follow the course of spontaneous healing was compared to healing of defects after having applied two bone regeneration methods; i.e. augmentation with xenograft Bio-Oss Collagen covered with Bio-Guide Perio barrier membrane or augmentation with Bio-Oss Collagen in connection with platelet rich plasma. 2. Materials and methods The studies were conducted on 36 New Zealand White (NZW) rabbits, (2.5–3 kg in weight) with the approval of the First Local Committee on Animal Research and Ethics of Wrocław Medical University, Poland under protocol number 30/2005. Procedural technique served as a criterion for the division into 3 groups: I. BG/BOC (12 rabbits) – defects were filled with xenograft BioOss Collagen® and covered by collagenous membrane Bio-Gide Perio® immobilized by restorable pins Resor-Pin® . II. BOC/PRP (12 rabbits) – defects were filled with xenograft BioOss Collagen® in connection with platelet rich plasma (PRP). III. C (12 rabbits) – defects were treated without regeneration procedures (CG – control group).
In the first group, the classic guided bone regeneration method was used. It is based on connecting the activity of the biomaterial and the barrier membranes, which influences the accumulation of regenerative potential of both materials, as well as mutually influencing the mechanical sustaining of both materials in a defect. The barrier membrane is supported by the material which makes it impossible for it to decay. Moreover, it stabilizes the material and ensures its protection in the area where the material is applied. In clinical practice, it allows for the application of biomaterial in surplus, i.e. above the entrance to the defect, which is beneficial due to the presence of limited biomaterial decline in the process of healing-in. The barrier membrane has an isolating function preventing the penetration of unwanted cells inside the defect. What is more, it limits inflammation of the alveolar socket that may lead to the differentiation of mesenchymal cells into a fibroblastic instead of an osteoblastic line; i.e. the proliferation of non-osteogenic connective tissue into the defect. In the second group, venous blood from the marginal ear vein at the amount of 4 ml was taken before the operation in order to obtain platelet rich plasma (MPW Instruments® Warszawa, Poland). The process of obtaining PRP consisted of two centrifugation steps: the first (10 min, 2400 rpm) and the second (15 min, 3600 rpm). Finally, resuspended thrombolytic concentrate was composed of a layer of maximum concentration of platelets (buffy coat) suspended in 0.5 ml of platelet poor plasma (PPP). In order to initiate the process of coagulation 10% calcium chloride (several drops) and 10–15 drops of thrombin was applied (Biomed® Lublin, Poland). After application of the biomaterial into the defect a layer of fibrinous adhesive tissue glue dressing was applied to the surface of the wound. The procedure was performed using general anesthesia Xylazine (5 mg/kg of body weight, i.m. Sedazin Biowet® , Puławy and Ketamine 35 mg/kg of body weight, i.m. Vetaketam® – Vetagro). After full basic sleep was achieved, a general and disinfected incision (the greater trochanter of the femur) was treated with a series of injections of 1% lignocaine solution. After the incision and preparation of periosteum using manual tools, a bone defect with a 5 mm diameter and 10 mm depth was drilled. The same procedure was carried out on both legs. After bone defects had been drilled and, if required, biomaterials applied, the operated area was marked using two 3-mm long titan pins. Next, muscles and subcutaneous tissues were sutured together in layers. On the 10th day following the operation, all rabbits had their sutures removed. Postoperative examination included clinical observations of the general health of animals and the course of postoperative wound healing. Special attention was paid to the mobility of limbs in the hip joint and their shape and size. Rabbit autopsies were performed 1, 3, 6, and 12 months after the operation. Before the autopsies, rabbits had been intravenously given pentobarbital (Morbital® – Biowet, Puławy, Poland) in a maximum dose up to 80 mg/kg of body weight, applied in doses fractionated according to a given impact; i.e. until respiratory and cardiac arrest occurred. During ongoing sections, special attention was paid to the bone appearance in the postoperative area, as well as to the muscles adjoining and covering the operative field. Bone tissue was fixed at room temperature for 72 h in 10% formaldehyde in phosphate buffer. Bones were decalcified in solutions of formic and hydrochloric acid, and dipped in 96% alcohol. Next, femoral fragments were cut longitudinally and cross-wisely, dehydrated in acetone (56 ◦ C), dipped in xylene at room temperature, and embedded in paraffin blocks. Microtome sections (4 m) were prepared (Leica 2025) and stained with haematoxylin and eosin (H&E) as well as by using Van Gieson’s method (VG) diversifying the connective tissue stroma; afterwards they were embedded in Canada balsam thinned with xylene. Histological preparations
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Fig. 1. BG/BOC group. 1 month observation. Traberculae of bone sponge located in major part at the edges of graft. BM – bone marrow, BT – bone tissue, CT – connective tissue, M – material, Stain VG, magnification 120×.
Fig. 2. BG/BOC group. 3 months observation. Young traberculae of bone sponge around the biomaterial granules visible in the centre of the graft. BM – bone marrow, BT – bone tissue, CT – connective tissue, M – material, Stain VG, magnification 120×.
were assessed under the Axioskop 20 (Zeiss® – Germany) microscope. The histological picture was photographically documented with the application of Zeiss Axiovision 4® software for archiving and analyzing the pictures. Qualitative assessment was carried out using all available system magnifications, quantitative assessment was made on the basis of images obtained at magnification 56×. Quantitative measurements were carried out using the AxioVision 4 Module AutoMeasure software (Zeiss® – Germany) for the most representative group of preparations according to protocol used by Su-Gwan et al. (2001). Qualitative assessment included: (1) type of tissue filling the intraosseous defect, (2) amount of biomaterials resorption, (3) method of integrating the xenogenic material into surrounding tissues filling the intraosseous defect and 4. presence of cells indicating the presence of an inflammatory process. The quantitative assessment included: first, the relative area of newly formed bone and, second, the surface of relative non-resorbed carrier Bio-Oss Collagen® . The examined relative surfaces were presented as a percentage of the total surface area of the test for each group. Due to the small number of samples, it was impossible to perform statistical analysis for quantitative evaluation between the groups. Evaluation of preparations was conducted jointly by the surgeon and a histologist from the Histological Laboratory of Experimental Surgery, Department of Biomaterials Research of the Medical University in Wrocław, Poland.
surface of the unresorbed material Bio-Oss Collagen® was 48.73% (SD 3.26) (Table 1). 3 months – in observed intraosseous defects molecules of resorbing biomaterial were found and fragments of the barrier membranes were visible locally. Emerging young bony trabeculae were observed not only on the periphery of the defects, but also in their central parts (Fig. 2). At the interface biomaterial/bone direct joints of structures were visible, without the presence of inflammatory cells. Minor amounts of fibrous connective tissue were observed locally. The mean relative value of bone defect tissue surface averaged to 31.9% (SD 1.80), while the surface of non-resorbed material Bio-Oss Collagen® was 22.96% (SD 1.56) (Table 1). 6 months – in all cases, the presence of biomaterial with varying fragmentation and changes in color and structure were observed. In none of the examined cases were remains of the guided barrier membrane detected. In bone defects, spongy bone trabeculae (lamellar and woven) were found surrounded by bone marrow (Fig. 3). In none of the cases was inflammation detected around the biomaterial particles or in their nearest neighborhood. Bone osteoblasts or layers of woven bone were observed on the border of the material. Due to the highly fragmented biomaterial its quantitative assessment was abandoned. The mean relative value of bone defect averaged out 23.35% (SD 2.13) (Table 1). 12 months – in all cases, the presence of very fragmented biomaterial was observed. In none of the examined cases were remains of the barrier membrane detected. The material appeared in the
3. Results Autopsies were conducted on 32 rabbits. In all animals, skin wounds healed by primary adhesion. Operated limbs were of normal size, shape, mobility in the hip joint, and the condition of the operated limb muscles showed the correct muscle tension. 3.1. BOC/BG group 1 month – in observed intraosseous defects, molecules of resorbing biomaterial as well as the presence of barrier membranes were observed. In the central part of the defect biomaterial particles were surrounded by bands of fibrous connective tissue, on the edges, however, by newly formed trabeculae of spongy bone (Fig. 1). In none of the cases was inflammation detected around the biomaterial particles or in their nearest neighborhood. Along the new bone trabeculae located at the entrance to the defect canal, the presence of linear accumulation of osteoblasts was indicated. The mean relative value of bone defect averaged out to 16.9% (SD 1.49), while the
Fig. 3. BG/BOC group. 6 months observation. Fragmentation and change of biomaterial pigmentation. Young bone trabeculae around the biomaterial granules. BM – bone marrow, BT – bone tissue, M – material, Stain VG, magnification 120×.
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Table 1 Relative values average of surface of bone tissue and biomaterial in treated bone defects in 1, 3, 6, 12 months observations. I group – BOC/BG
1 month 3 months 6 months 12 months
II group – BOC/PRP
III group – C
Bone
Biomaterial
Bone
Biomaterial
Bone
16.9% (SD 1.49) 31.9% (SD 1.8) 23.35% (SD 2.13) 22.18% (SD 1.26)
48.73% (SD 3.26) 22.96% (1.56) – –
29.58% (SD 0.75) 27.88% (SD 3.07) 22.59% (SD 0.53) 21.76% (SD 1.24)
35.44% (SD 2.47 20.1% (SD 2.87) – –
12.5% (SD 1.32) 17.74% (SD 0.87) 14.2% (SD 1.72) 16.1% (SD 1.71)
form of irregular and dark bands surrounded by trabeculae and bone marrow. (Fig. 4) The bone located in the immediate vicinity of the implant had a woven or lamellar bone structure with marked Haversian canals. At some points in the bone marrow, fragments of biomaterial were found, limited from the outside by a thin, fibrous, glazing connective tissue band, and by cell-rich tissue centers from the implant side. Similarly to the observation after 6 months, the measurement of the unresorbed biomaterial was not conducted. The mean relative value of the bone defect averaged 22.18% (SD 1.26) (Table 1). 3.2. BOC/PRP group 1 month – in the defect, the presence of young bone trabeculae were located around the biomaterial. The newly formed bone trabeculae were seen not just on the outskirts of bone defects, but also in the central parts thereof (Fig. 5). A linear accumulation of osteoblasts was observed along most of the newly formed bone trabeculae. Areas in which biomaterial granule was surrounded by connective fiber tissue with a large number of blood vessels were also observed. The mean relative value of bone defect tissue surface averaged 29.58% (SD 0.75), while the surface of non-resorbed material Bio-Oss Collagen® was 35.44% (SD 2.47) (Table 1). No inflammation was detected around the biomaterial particles or in its nearest vicinity in any of the observed cases. 3 months – a lesser activity of osteoblasts was observed compared to that of the 1st month. At the interface, a biomaterial/bone direct joint of structures was observed without the presence of inflammatory cells. Minor amounts of fibrous connective tissue were locally observed between biomaterial granules (Fig. 6). The mean relative value of bone defect tissue surface was 27.88% (SD 3.07), while the surface of non-resorbed material Bio-Oss Collagen® was 20.1% (SD 2.87) (Table 1). 6 months – on the implantation side, spongy bone tissue with remnants of biomaterial was visible which was surrounded by young bone trabeculae or forming trabeculae. In all cases, the
Fig. 4. BG/BOC group. 12 months observation. Irregular, dark-pigmentation of biomaterial bands surrounded by young bone trabeculae and marrow. BM – bone marrow, BT – bone tissue, M – material, Stain VG, magnification 120×.
Fig. 5. BOC/PRP group. 1 month observation. Young bone trabeculae around the biomaterial granules in the centre of graft. BM – bone marrow, BT – bone tissue, BV – blood vessels, CT – connective tissue, M – material, Stain VG, magnification 120×.
presence of biomaterial characterized by varying degrees of fragmentation was observed as well as changes in its color and structure. There were places with preserved structure, as well as fragmented centers, strongly basophilic (change of color to the intense purple in both stains) (Fig. 7). Due to the highly fragmented biomaterial, its quantitative assessment was not possible. The biomaterial was surrounded by lamellar bone tissue and in the immediate vicinity of the interface biomaterial/bone accumulated woven bone was observed. Isolated cases were found in which biomaterial was surrounded by connective tissue. In few cases of the biomaterial resorption area, accumulations of osteoblasts (from the site of a bone) were detectable and, at the site of the material, inflammatory cells were observed .The mean relative value of the bone defect tissue surface was calculated at 22.59% (SD 0.53). 12 months – there was significant fragmentation of the biomaterial, with characteristics as described after 6 months of
Fig. 6. BOC/PRP group. 3 months observation. Formation of young bone trabeculae around the biomaterial granules. BM – bone marrow, BT – bone tissue, M – material, Stain VG, magnification 120×.
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Fig. 7. BOC/PRP group. 6 months observation. Young bone trabeculae and trabeculae in formation phase around the strongly basophilous biomaterial fragments. BM – bone marrow, BT – bone tissue, M – material, Stain VG, magnification 120×.
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Fig. 9. Control group. 1 month observation. Trabeculae of new forming bone surrounded by marrow with a large amount of blood vessels. BM – bone marrow, BT – bone tissue, BV – blood vessels. Stain HE, magnification 120×.
implantation. The quantitative assessment of the remaining material was not conducted. Biomaterial remnants were surrounded by lamellar bone tissue or fibrous connective tissue with characteristic glazing (Fig. 8). In one case, the evidence of massive bone trabeculae, joined with numerous fibrous bands and remnants of bone biomaterial was provided. In the direct neighborhood of resorbing biomaterial cell rich connective tissue and numerous blood vessels were observed. The mean relative value of the bone tissue surface represented 21.76% (SD 1.24) of the defect. 3.3. C group 1 month – intraosseous defects were filled mainly with bone marrow and a large number of blood vessels. The occurrence of few young bone trabeculae was detected (Fig. 9). The mean relative area of these trabeculae was determined at 12.5% (SD 1.32) of the operated defect area (Table 1). 3 months – intraosseous defects were mainly filled with bone marrow, in which newly formed bone trabeculae were observed. The area of the defect was less vascularized in comparison to those of 1 month period (Fig. 10). Input canals leading to defects were covered with dense bone tissue. The mean relative value of bone defect tissue surface averaged 17.74% (SD 0.87) (Table 1). 6 months – thin bone trabeculae surrounded by bone marrow were in the center of the defects (Fig. 11). The mean relative value of the surface of bone tissue defect was calculated at 14.2% (SD 1.72)
Fig. 8. BOC/PRP group. 12 months observation. Massive bone trabeculae connecting with biomaterial remainders, fibrous connective tissue bands, BM – bone marrow, BT – bone tissue M – material, Stain VG, magnification 120×.
Fig. 10. Control group. 3 months observation. Canal of entry to defect covered with compact tissue. BM – bone marrow, BT – bone tissue, Stain VG, magnification 120×.
(Table 1). Input canals from the top of the defects were filled with cartilage tissue, lamellar and woven bone tissue with lots of blood vessels. Few places of the restricted bone rebuilding were detected. In one case, the defect was filled by a block of connective tissue and woven bone tissue of rectangular shape
Fig. 11. Control group. 6 months observation. Filling of the bone defects by bone marrow and plexiform bone tissue. BT – bone tissue. BM – bone marrow, Stain HE, magnification 120×.
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Fig. 12. Control group. 12 months observation. Bone trabeculae surrounded by marrow. BM – bone marrow, BT – bone tissue. Stain HE, magnification 120×.
12 months – in the defect, visible sponge bone tissue was seen with single thin bone trabeculae and bone marrow (Fig. 12). The average relative amount of bone defect tissue surface accounted for 16.1% (SD 1.71) (Table 1). 4. Discussion On the basis of histological studies performed in all observational periods, the experimental group demonstrated an increase in the relative surface area of bone trabeculae filling intraosseous defects as compared with the reference group. In the early phase of observation; i.e. after 1 and 3 months the defects left for spontaneous healing were mainly filled with bone marrow, within which single bone trabeculae appeared. Within the third month, progressive conversion of the tissue, being a covering of the entrance of the root canal with tight bone tissue was observed. In the groups in which methods of guided bone regeneration were applied, the creation of new bone trabeculae around and inside biomaterial granules was observed as early as 1 month following the operation. Similar histological observations after 1 month were described by Indovina and Block (2002) and Schwarz et al. (2007). Unlike the reference group, in the experimental groups, a linear accumulation of osteoblasts was observed along newly created bone trabeculae, which proved the intensity of the osteogenic processes. The characteristics of the first period of the observation; i.e. after 1 and 3 months, referred to more regular distribution of osteoblasts clusters within the defects in the BOC/PRP group as compared with BOC/BG group. In group II, accumulations were observed particularly in the vicinity of the barrier membrane. It was reflected in the quantity and in the presentation of the bone trabeculae location. In the BOC/BG group, bone trabeculae were observed mainly on the edges of the defects and in the BOC/PRP group also in their center. Therefore, a thesis can be presented that, in the first period of the observation, the dynamics of the regeneration processes was higher with the use of Bio-Oss Collagen® with polypeptide growth factors than with the use of a barrier membrane. It seems that, in the BOC/BG group, the creation of new bone trabeculae occurred in defects areas with good perfusion, namely mainly at their peripheries. In the central part of the defect, the perfusion was insufficient, so the inflow of blood growth factors and indirectly osteoconductive activities of biomaterial were limited. Such a relationship was not observed in case of the defects treated by usage of platelet rich plasma which is a natural source of active growth factors required in the process of bone tissue regeneration. The research results obtained in the early stage of the observations were comparable to the results of other authors, such as Ezirganli and Polat
(2011), Rokn et al. (2011) and Heberer et al. (2011). Similarly to own results, the results of Ezirganli and Polat (2011) indicate a beneficial influence of regenerative methods on the healing of intraosseous defects. Although the authors did not confirm statistically significant differences between spontaneous healing and the GBR method (Bio-Oss + tytanium barrier membrane) after 1 month subsequent to the operation, better healing of the aforementioned GBR method compared with spontaneous healing was observed after 3 months subsequent to the operation. The observed difference was statistically significant. However, Rokn et al. (2011) in their histological studies conducted on rabbits after 4 and 8 weeks, did not demonstrate any statistically significant differences in the amount of bone fill between the control group and GBR group with the usage of Bio-Oss. Entirely different results in comparison to our results and the results of the aforementioned researchers were obtained by Heberer et al. (2011). These authors assessed the healing process of ungrafted and grafted extraction sockets in humans after 12 weeks. This descriptive study demonstrated that bone formation in Bio-Oss Collagen – grafted human extraction sockets was lower than bone formation in ungrafted sockets. It seems that such distinct results in comparison to our results may be the consequence of morphological disparities in the treated bone defects, the differences in operation technique and the method of the retrieval of the material for histological study. In our studies, the treated bone defects were iatrogenic, limited by trabecular bone tissue that ensured optimal blood supply to the operated area. The defects treated by Heberer et al. (2011) were four-wall defects, limited by the compact plate of the alveolar socket that causes a much poorer blood supply, and therefore hinders growth factors that positively influence the regenerative processes. Another significant difference was the operative technique. In our studies, the closed system (flaptechnique) was used, as well as additional materials that could enhance the effectiveness of the regeneration processes (barrier membrane and platelet rich plasma). In the study of Heberer et al. (2011), only the osteoconductive properties of the material placed in the defect and left for healing with the use of the opened system were used. The method of preparing the material before its application into the defect also differed. In our study, unlike that of Heberer et al. (2011), no forming method using a blade was used. The preparation of the material consisted of soaking it in blood to obtain a soft consistency and modeling it on a sterile plate before applying it into a treated defect. It seems that our method enables easier penetration of the blood into the biomaterial, increases its plasticity and limits close compression in the defect. The last issue is the site of retrieval of material for histological study. In our material, tissue contents filling the whole treated bone defect was determined, and in the study of Heberer et al. (2011), the material was retrieved from the central part of the defect (diameter of 2 mm); i.e. the area with potentially the lowest blood supply in which regenerative processes may take the longest. After 3 months following the operation, a lesser presence of linear accumulations of osteoblasts along bone trabeculae was observed while the presence of singular osteogenic cells was more often confirmed around the granules of biomedical material in the BOC/PRP group. Most likely the dynamics of bone-forming processes slowed down then. In order to explain these observations, studies of Marx et al. (1998) on the mechanisms of polypeptide growth factor activity might be useful. They have shown that the activity of these factors is maintained for 7–10 days (Marx et al., 1998). According to Hatakeyama et al. (2008) the factor which is the most important in the assessment of the effectiveness of PRP is the early estimation of the healing process. Hence the authors propose to do the assessment after only 15 days following the operation. According to Arm et al. (1996) the secretion of polypeptide growth factors from an absorbed biomaterial can occur even after 40 days, with a maximum between the 10th and 20th day. On the basis of
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our observations it seems that after 3 months, growth factors from the PRP are inactive, while the regenerative functions have been taken over by the implanted material. Our results shown a favorable effect of applying polypeptide growth factors in the early period of bone healing. Our studies are in accordance with the observations of Aghaloo et al. (2002). According to those authors, connecting Bio-Oss biomaterial with PRP significantly increased the formation of new bones in all periods of observation (after 1, 2 and 4 months). In contrast to our results, studies by Roldan et al. (2004) shows a negative effect of platelet concentrate on Bio-Oss® carrier for bone regenerative processes. The decreased formation of newly formed bone observed by the authors was explained by the lack of osteoblasts in bovine bone derivate material and lack of an osteoinductive effect of PRP. Fur˝ thermore, studies by Furst et al. (2003) have shown that neither reduced nor increased bone formation was observed after the joint application of Bio-Oss® and PRP materials in comparison to Bio˝ Oss® alone (Furst et al., 2003). As opposed to the studies of Roldan ˝ et al. (2003) remain, in the opinion of Andrade et al. (2004) and Furst et al. (2008) a significant effect on the effectiveness of the process of tissue healing after applying polypeptide growth factors have numerous factors modifying the properties of PRP. It is believed that the most important factor that influences bone regeneration process after using PRP is the concentration of blood platelets in the applied concentrate. The concentration of the platelet concentrate depends on the technique of platelet separation, the value of relative centrifuge force (RCF), time of acceleration and deceleration of the centrifuge and the selection of appropriate activators of fibrinogen and anticoagulants. Furthermore, the crucial element for a successful therapy is to keep the optimal concentration of platelets after giving PRP to a tissue without further major changes in the concentration of platelets. Therefore, it seems that the method of obtaining PRP applied in our research has to be recognized as optimal because it allowed the demonstration of a beneficial effect of PRP on the process of healing bone. 5. Conclusions Upon quantitative histological assessment, the animals presented the most intensive osteogenesis within one month after the application of xenogenic material with platelet rich plasma, whereas in the later stages, it was observed after the application of xenogenic material and resorbable collagen membrane. The application of two regenerative methods influenced the rate, quality and overall treatment of intraosseous defects. References Abramowitz, P., Rankow, H., Trope, M., 1994. Multidisciplinary approach to apical surgery in conjunction with the loss of buccal cortical plate. Oral Surg. Oral Med. Oral Pathol. 77, 502–506. Aghaloo, T.L., Moy, P.K., Freymiller, E.G., 2002. Evaluation of platelet rich plasma (PRP) in combination with xenograft materials in the rabbit cranium: a pilot study. J. Oral. Maxillofac. Surg. 60, 1176–1181. Andrade, M.G.S., De Fraitas Brandao, C.J., Neves Sa, S.A., Bittencourt, T.H.B., Sadigursky, M., 2008. Evaluation of factors that can modify platelet rich plasma properties. Oral Surg. Oral Med. Oral Pathol. Oral Radiol. Endod. 105, e5–e12. Arm, D.M., Tencer, S.D., Bain, S.D., Celino, D., 1996. Effect of controlled release of platelet derived growth factor from a porous hydroxyapatite implant on bone ingrowth. Biomaterials 17, 703–709. Buser, D., Dula, K., Belser, U., Hirt, H.P., Berthold, H., 1993. Localized ridge augmentation using guided bone regeneration: surgical procedure in the maxilla. Int. J. Periodontics Restorative Dent. 13, 29–45.
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Oral Implants Res. 10, 14. ˝ G., Gurber, R., Tangl, S., Zechner, W., Haas, R., Mailath, G., Sanroman, F., Furst, Watzek, G., 2003. Sinus grafting with autogenous platelet-rich plasma and bovine hydroxyapatite: a histomorphometric study in minipigs. Clin. Oral. Implants Res. 14, 500–508. Gottlow, J., Nyman, S., Lindhe, J., Karring, T., Wennström, T., 1986. New attachment formation in the human periodontium by guided tissue regeneration: case report. J. Clin. Periodontol. 13, 604–616. Hatakeyama, M., Belletti, M.E., Zanetta-Barbosa, D., Dechichi, P., 2008. Radiographic and histomorphometric analysis of bone healing using autogenous graft associated with platelet-rich plasma obtained by 2 different methods. Oral Surg. Oral Med. Oral Pathol. Oral Radiol. Endod. 105, e13–e18. Heberer, S., Al-Chawaf, B., Jablonski, C., Nelson, J.J., Lage, H., Nelson, K., 2011. Healing of ungrafted and grafted extraction sockets after 12 weeks: a prospective clinical study. Int. J. Oral Maxillofac. Implants 26 (March–April (2)), 385–392. Indovina, A., Block, M.S., 2002. Comparison of 3 bone substitutes in canine extraction sites. J. Oral Maxillofac. Surg. 60, 53–58. Jensen, T.B., Rahbek, O., Overgard, S., Soballe, K., 2005. No effect of platelet rich plasma with frozen or processed bone allograft around noncemented implants. Int. Orthop. 29, 67–72. Kassolis, J.D., Rosen, P.S., Reynols, M.A., 2000. Alveolar ridge and sinus augmentation utilizing platelet-rich plasma in combination with freeze-dried bone allograft: case series. J. Periodontol. 17, 1654–1661. Kim, E.S., Park, E.J., Choung, P.H., 2001. Platelet concentration and its effect on bone formation in calvarial defects: an experimental study in rabbits. J. Prosthet. Dent. 86, 428–433. Marx, R.E., Carlson, E.R., Eichstaed, R.M., Schimmele, S.R., Strauss, J.E., Georgeff, K.R., 1998. Platelet rich plasma. Growth factor enhancement for bone grafts. Oral Surg. Oral Med. Oral Pathol. 85, 638–646. Nyman, S., Gottlow, J., Karting, T., Linde, J., 1982. The regenerative potential of the periodontal ligament. An experimental study in the monkey. J. Clin. Periodontol. 9, 257–265. Oyama, T., Nishimoto, S., Tsugawa, T., Sbimizu, F., 2004. Efficacy of platelet rich plasma in alveolar bone grafting. J. Oral Maxillofac. Surg. 62, 555–558. Rankow, H., Krasner, P., 1996. Endodontic applications of guided tissue regeneration in endodontic surgery. J. Endod. 22, 34–43. Roldan, C.J., Jepsen, S., Miller, J., Freitag, S., Rueger, D.C., Acil, Y., Terheyden, H., 2004. Bone formation in the presence of platelet rich plasma vs. bone morphogenic protein-7. Bone 34, 80–90. Rokn, A.R., Khodadoostan, M.A., Reza Rasouli Ghahroudi, A.A., Montahhary, P., Kharrazi Fard, M.J., Bruyn, H.D., Afzalifar, R., Soolar, E., Soolari, A., 2011. Bone formation with two types of grafting materials: a histologic and histomorphometric study. Open Dent. J. 5, 96–104. Schwarz, F., Herten, M., Ferrari, D., Wieland, M., Schmitz, L., Engelhardt, E., Becker, J., 2007. Guided bone regeneration at dehiscence-type defects using biphasic hydroxyapatite + beta tricalcium phosphate (Bone Ceramic® ) or a collagencoated natural bone mineral (Bio-Oss Collagen® ): an immunohistochemical study in dogs. Int. J. Oral Maxillofac. Surg. 36, 1198–1206. Su-Gwan, K., Hak-Kyun, K., Sung-Chul, L., 2001. Combined implantation of particulate dentine, plaster of Paris, and a bone xenograft (Bio-Oss® ) for bone regeneration in rats. J. Craniomaxill. Surg. 29, 282–288. Taguchi, Y., Amizuka, N., Nokadate, M., Ohnishi, H., Fujii, N., Oda, S., Maeda, T., 2005. A histological evaluation for guided bone regeneration induced by a collagenous membrane. Biomaterials 26, 6158–6166. Terheyden, H., Roldan-Ossa, J.C., Miller, J., Jepsen, S., Acil, Y., 2002. Platelet rich plasma in bone regeneration. Preliminary results of two experimental studies. Implantologie 10, 195–205. 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Annals of Anatomy journal homepage: www.elsevier.de/aanat
Evaluation of healing processes of intraosseous defects with and without guided bone regeneration and platelet rich plasma. An animal study b ˙ Marzena Dominiak a,∗ , Katarzyna Łysiak-Drwal a , Leszek Solski b , Bogusława Zywicka , b a,c Zbigniew Rybak , Tomasz Gedrange a
Department of Oral Surgery, Silesian Piast Medical University, Wrocław, Poland Department of Experimental Surgery and Biomaterials Research, Silesian Piast Medical University, Wrocław, Poland c Department of Orthodontics, Dresden Technology University, Germany b
a r t i c l e
i n f o
Article history: Received 16 October 2011 Received in revised form 18 June 2012 Accepted 18 July 2012
Keywords: Intraosseous defects Bone grafting Bovine bone mineral Guided bone regeneration Platelet rich plasma
a b s t r a c t Background: In most cases, the natural healing of intrabony defects only leads to restoration of tissue continuity without differentiation and function. However, repair is not regarded to be an optimal treatment method, as confirmed in many clinical cases. Thus it is important to choose a surgical procedure which makes it possible to achieve restitution ad integrum of the bone structure. The choice of the GBR technique is crucial, in terms of the clinical conditions and limitations resulting from the use of a particular material. Objective: The objective of this study has been the analysis of effectiveness of selected surgical treatment techniques of intrabony defects in rabbits. Materials and methods: Research was conducted on 36 white rabbits. The operation technique was a criterion of division into 3 groups: BG/BOC (Bio-Oss Collagen® + Bio-Gide Perio® ), BOC/PRP (Bio-Oss Collagen® + PRP), C (control group). Qualitative and quantitative histopathological evaluation was carried out after 1, 3, 6 and 12 months. Results: The highest value of the bone surface area 31.9% (SD 1.8) was achieved in BOC/BG group three months after the implantation, while the lowest was revealed in C – group – 12.5% (SD 1.32) one month following the procedure. Conclusions: Upon quantitative histological assessment, the bone tissue presented the most intensive osteogenesis within one month from the application of BOC/PRP, whereas this was observed after the application of BOC/BG in later stages. The application of two regenerative methods influenced the rate, quality and overall treatment of intraosseus defects. © 2012 Elsevier GmbH. All rights reserved.
1. Introduction The process of healing in intraosseous defects leads to connective tissue (so-called connective tissue scar) formation in lieu of the proper, new bone formation. In clinical conditions healing may not assure the proper size of the alveolar process; it may also impede the differential diagnosis of cicatrices, including the treatment of pathological processes in bone tissue, as well as induce the occurrence of non-specific pain sensations, both in the operated area and its immediate vicinity (Dominiak and Łysiak, 2005). The proper shape and function of damaged tissue may now be restored owing to achievements in tissue engineering – a reconstructive biology that draws on the advances in medicine, surgery, physiology, cell and molecular biology, and polymer chemistry. For
∗ Corresponding author at: Wroclaw Medical University, Krakowska 26 st., 50-425 Wroclaw, Poland. Tel.: +48 717840251; fax: +48 717840253. E-mail address: [email protected] (M. Dominiak). 0940-9602/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.aanat.2012.07.007
clinical purposes, terms of guided tissue regeneration (GTR) and narrower guided bone regeneration (GBR) have been introduced (Nyman et al., 1982; Gottlow et al., 1986; Dahlin et al., 1989; Buser et al., 1993; Taguchi et al., 2005). In 1998, the so-called Lynch triad was designed, comprising three indispensable factors for ensuring undisturbed bone regeneration: structure (carrier), cells filling the base (cells stimulated by growth factors) and extracellular matrix material (ECM), i.e. signal molecules mediating the healing process. According to the principal assumption of tissue engineering, the implanted carrier, essential for initiation of the GTR and/or GBR processes, must be enriched with growth factors, cytokines or autogenous cells (Nyman et al., 1982). Despite dynamic growth in the field of tissue engineering, autogenic bone continues to be considered one of best regenerative materials. One significant limitation to the foregoing method, though, refers to the common difficulty in obtaining adequate quantities of material. Consequently, alternative solutions are being sought based primarily on the application of xenogenic and alloplastic materials, barrier membranes, as well as on the using of
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polypeptide growth factors and morphogenetic proteins (Nyman et al., 1982; Gottlow et al., 1986; Dahlin et al., 1989; Buser et al., 1993; Taguchi et al., 2005). The prevailing opinion suggests that the most effective method of treating intraosseous defects is the combined therapeutic approach involving bone augmentation using biomaterial with the simultaneous application of a barrier membrane for coverage. This method is based on the accumulation of regenerative potential of both materials and mechanical sustainment of these materials in the defect (Dietrich et al., 2003; Rankow and Krasner, 1996; Tseng et al., 1995; Abramowitz et al., 1994). However, a disadvantage of this method lies in its limitations resulting from the processing of xenogenic materials, from which proteins are eliminated which, among others, include growth factors locally activating osteogenesis (Kim et al., 2001). In cases of poor blood supply to the intraosseous defect area, these materials exhibit osteoconductive properties enabling the proliferation of newly formed bone tissue (including the inflow of growth factors carried in the blood). In other cases the application of xenogenic materials does not assure proper regeneration, necessitating the implementation of innovative treatment solutions. A supporting method for xenogenic osteoconductors is the use of an adjunctive method in which xenogenic osteoconductives are combined with autogenic bone or polypeptide growth factors (PRP) obtained from the patient’s full venous blood in centrifuge systems (Marx et al., 1998; Oyama et al., 2004; Kassolis et al., 2000; Jensen et al., 2005; Terheyden et al., 2002; Roldan et al., 2004; Wojtowicz et al., 2002). The majority of publications, however, describe the healing process in intraosseous defects following the application of platelet concentrate on autoor allogenic carriers (Wojtowicz et al., 2002; Indovina and Block, ˙ et al., 2005). Only a few evalu2002; Schwarz et al., 2007; Zywicka ate the effectiveness of bone healing in which xenogenic material – deproteinised bovine-derived xenograft (BDX) - served as the carrier. The relatively small number of direct comparative studies on the results of defect healing after implementation of BDX itself and in conjunction with PRP; and conflicting findings confirm that the subject-matter is still unresolved and requires further detailed research. The aim of this study was a quantitative and qualitative histological assessment of the healing process of iatrogenically induced intraosseous defects on animal models with and without the application of selected techniques of bone regeneration after 1, 3, 6 and 12 months. In the course of the research, healing of intraosseus defects allowed to follow the course of spontaneous healing was compared to healing of defects after having applied two bone regeneration methods; i.e. augmentation with xenograft Bio-Oss Collagen covered with Bio-Guide Perio barrier membrane or augmentation with Bio-Oss Collagen in connection with platelet rich plasma. 2. Materials and methods The studies were conducted on 36 New Zealand White (NZW) rabbits, (2.5–3 kg in weight) with the approval of the First Local Committee on Animal Research and Ethics of Wrocław Medical University, Poland under protocol number 30/2005. Procedural technique served as a criterion for the division into 3 groups: I. BG/BOC (12 rabbits) – defects were filled with xenograft BioOss Collagen® and covered by collagenous membrane Bio-Gide Perio® immobilized by restorable pins Resor-Pin® . II. BOC/PRP (12 rabbits) – defects were filled with xenograft BioOss Collagen® in connection with platelet rich plasma (PRP). III. C (12 rabbits) – defects were treated without regeneration procedures (CG – control group).
In the first group, the classic guided bone regeneration method was used. It is based on connecting the activity of the biomaterial and the barrier membranes, which influences the accumulation of regenerative potential of both materials, as well as mutually influencing the mechanical sustaining of both materials in a defect. The barrier membrane is supported by the material which makes it impossible for it to decay. Moreover, it stabilizes the material and ensures its protection in the area where the material is applied. In clinical practice, it allows for the application of biomaterial in surplus, i.e. above the entrance to the defect, which is beneficial due to the presence of limited biomaterial decline in the process of healing-in. The barrier membrane has an isolating function preventing the penetration of unwanted cells inside the defect. What is more, it limits inflammation of the alveolar socket that may lead to the differentiation of mesenchymal cells into a fibroblastic instead of an osteoblastic line; i.e. the proliferation of non-osteogenic connective tissue into the defect. In the second group, venous blood from the marginal ear vein at the amount of 4 ml was taken before the operation in order to obtain platelet rich plasma (MPW Instruments® Warszawa, Poland). The process of obtaining PRP consisted of two centrifugation steps: the first (10 min, 2400 rpm) and the second (15 min, 3600 rpm). Finally, resuspended thrombolytic concentrate was composed of a layer of maximum concentration of platelets (buffy coat) suspended in 0.5 ml of platelet poor plasma (PPP). In order to initiate the process of coagulation 10% calcium chloride (several drops) and 10–15 drops of thrombin was applied (Biomed® Lublin, Poland). After application of the biomaterial into the defect a layer of fibrinous adhesive tissue glue dressing was applied to the surface of the wound. The procedure was performed using general anesthesia Xylazine (5 mg/kg of body weight, i.m. Sedazin Biowet® , Puławy and Ketamine 35 mg/kg of body weight, i.m. Vetaketam® – Vetagro). After full basic sleep was achieved, a general and disinfected incision (the greater trochanter of the femur) was treated with a series of injections of 1% lignocaine solution. After the incision and preparation of periosteum using manual tools, a bone defect with a 5 mm diameter and 10 mm depth was drilled. The same procedure was carried out on both legs. After bone defects had been drilled and, if required, biomaterials applied, the operated area was marked using two 3-mm long titan pins. Next, muscles and subcutaneous tissues were sutured together in layers. On the 10th day following the operation, all rabbits had their sutures removed. Postoperative examination included clinical observations of the general health of animals and the course of postoperative wound healing. Special attention was paid to the mobility of limbs in the hip joint and their shape and size. Rabbit autopsies were performed 1, 3, 6, and 12 months after the operation. Before the autopsies, rabbits had been intravenously given pentobarbital (Morbital® – Biowet, Puławy, Poland) in a maximum dose up to 80 mg/kg of body weight, applied in doses fractionated according to a given impact; i.e. until respiratory and cardiac arrest occurred. During ongoing sections, special attention was paid to the bone appearance in the postoperative area, as well as to the muscles adjoining and covering the operative field. Bone tissue was fixed at room temperature for 72 h in 10% formaldehyde in phosphate buffer. Bones were decalcified in solutions of formic and hydrochloric acid, and dipped in 96% alcohol. Next, femoral fragments were cut longitudinally and cross-wisely, dehydrated in acetone (56 ◦ C), dipped in xylene at room temperature, and embedded in paraffin blocks. Microtome sections (4 m) were prepared (Leica 2025) and stained with haematoxylin and eosin (H&E) as well as by using Van Gieson’s method (VG) diversifying the connective tissue stroma; afterwards they were embedded in Canada balsam thinned with xylene. Histological preparations
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Fig. 1. BG/BOC group. 1 month observation. Traberculae of bone sponge located in major part at the edges of graft. BM – bone marrow, BT – bone tissue, CT – connective tissue, M – material, Stain VG, magnification 120×.
Fig. 2. BG/BOC group. 3 months observation. Young traberculae of bone sponge around the biomaterial granules visible in the centre of the graft. BM – bone marrow, BT – bone tissue, CT – connective tissue, M – material, Stain VG, magnification 120×.
were assessed under the Axioskop 20 (Zeiss® – Germany) microscope. The histological picture was photographically documented with the application of Zeiss Axiovision 4® software for archiving and analyzing the pictures. Qualitative assessment was carried out using all available system magnifications, quantitative assessment was made on the basis of images obtained at magnification 56×. Quantitative measurements were carried out using the AxioVision 4 Module AutoMeasure software (Zeiss® – Germany) for the most representative group of preparations according to protocol used by Su-Gwan et al. (2001). Qualitative assessment included: (1) type of tissue filling the intraosseous defect, (2) amount of biomaterials resorption, (3) method of integrating the xenogenic material into surrounding tissues filling the intraosseous defect and 4. presence of cells indicating the presence of an inflammatory process. The quantitative assessment included: first, the relative area of newly formed bone and, second, the surface of relative non-resorbed carrier Bio-Oss Collagen® . The examined relative surfaces were presented as a percentage of the total surface area of the test for each group. Due to the small number of samples, it was impossible to perform statistical analysis for quantitative evaluation between the groups. Evaluation of preparations was conducted jointly by the surgeon and a histologist from the Histological Laboratory of Experimental Surgery, Department of Biomaterials Research of the Medical University in Wrocław, Poland.
surface of the unresorbed material Bio-Oss Collagen® was 48.73% (SD 3.26) (Table 1). 3 months – in observed intraosseous defects molecules of resorbing biomaterial were found and fragments of the barrier membranes were visible locally. Emerging young bony trabeculae were observed not only on the periphery of the defects, but also in their central parts (Fig. 2). At the interface biomaterial/bone direct joints of structures were visible, without the presence of inflammatory cells. Minor amounts of fibrous connective tissue were observed locally. The mean relative value of bone defect tissue surface averaged to 31.9% (SD 1.80), while the surface of non-resorbed material Bio-Oss Collagen® was 22.96% (SD 1.56) (Table 1). 6 months – in all cases, the presence of biomaterial with varying fragmentation and changes in color and structure were observed. In none of the examined cases were remains of the guided barrier membrane detected. In bone defects, spongy bone trabeculae (lamellar and woven) were found surrounded by bone marrow (Fig. 3). In none of the cases was inflammation detected around the biomaterial particles or in their nearest neighborhood. Bone osteoblasts or layers of woven bone were observed on the border of the material. Due to the highly fragmented biomaterial its quantitative assessment was abandoned. The mean relative value of bone defect averaged out 23.35% (SD 2.13) (Table 1). 12 months – in all cases, the presence of very fragmented biomaterial was observed. In none of the examined cases were remains of the barrier membrane detected. The material appeared in the
3. Results Autopsies were conducted on 32 rabbits. In all animals, skin wounds healed by primary adhesion. Operated limbs were of normal size, shape, mobility in the hip joint, and the condition of the operated limb muscles showed the correct muscle tension. 3.1. BOC/BG group 1 month – in observed intraosseous defects, molecules of resorbing biomaterial as well as the presence of barrier membranes were observed. In the central part of the defect biomaterial particles were surrounded by bands of fibrous connective tissue, on the edges, however, by newly formed trabeculae of spongy bone (Fig. 1). In none of the cases was inflammation detected around the biomaterial particles or in their nearest neighborhood. Along the new bone trabeculae located at the entrance to the defect canal, the presence of linear accumulation of osteoblasts was indicated. The mean relative value of bone defect averaged out to 16.9% (SD 1.49), while the
Fig. 3. BG/BOC group. 6 months observation. Fragmentation and change of biomaterial pigmentation. Young bone trabeculae around the biomaterial granules. BM – bone marrow, BT – bone tissue, M – material, Stain VG, magnification 120×.
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Table 1 Relative values average of surface of bone tissue and biomaterial in treated bone defects in 1, 3, 6, 12 months observations. I group – BOC/BG
1 month 3 months 6 months 12 months
II group – BOC/PRP
III group – C
Bone
Biomaterial
Bone
Biomaterial
Bone
16.9% (SD 1.49) 31.9% (SD 1.8) 23.35% (SD 2.13) 22.18% (SD 1.26)
48.73% (SD 3.26) 22.96% (1.56) – –
29.58% (SD 0.75) 27.88% (SD 3.07) 22.59% (SD 0.53) 21.76% (SD 1.24)
35.44% (SD 2.47 20.1% (SD 2.87) – –
12.5% (SD 1.32) 17.74% (SD 0.87) 14.2% (SD 1.72) 16.1% (SD 1.71)
form of irregular and dark bands surrounded by trabeculae and bone marrow. (Fig. 4) The bone located in the immediate vicinity of the implant had a woven or lamellar bone structure with marked Haversian canals. At some points in the bone marrow, fragments of biomaterial were found, limited from the outside by a thin, fibrous, glazing connective tissue band, and by cell-rich tissue centers from the implant side. Similarly to the observation after 6 months, the measurement of the unresorbed biomaterial was not conducted. The mean relative value of the bone defect averaged 22.18% (SD 1.26) (Table 1). 3.2. BOC/PRP group 1 month – in the defect, the presence of young bone trabeculae were located around the biomaterial. The newly formed bone trabeculae were seen not just on the outskirts of bone defects, but also in the central parts thereof (Fig. 5). A linear accumulation of osteoblasts was observed along most of the newly formed bone trabeculae. Areas in which biomaterial granule was surrounded by connective fiber tissue with a large number of blood vessels were also observed. The mean relative value of bone defect tissue surface averaged 29.58% (SD 0.75), while the surface of non-resorbed material Bio-Oss Collagen® was 35.44% (SD 2.47) (Table 1). No inflammation was detected around the biomaterial particles or in its nearest vicinity in any of the observed cases. 3 months – a lesser activity of osteoblasts was observed compared to that of the 1st month. At the interface, a biomaterial/bone direct joint of structures was observed without the presence of inflammatory cells. Minor amounts of fibrous connective tissue were locally observed between biomaterial granules (Fig. 6). The mean relative value of bone defect tissue surface was 27.88% (SD 3.07), while the surface of non-resorbed material Bio-Oss Collagen® was 20.1% (SD 2.87) (Table 1). 6 months – on the implantation side, spongy bone tissue with remnants of biomaterial was visible which was surrounded by young bone trabeculae or forming trabeculae. In all cases, the
Fig. 4. BG/BOC group. 12 months observation. Irregular, dark-pigmentation of biomaterial bands surrounded by young bone trabeculae and marrow. BM – bone marrow, BT – bone tissue, M – material, Stain VG, magnification 120×.
Fig. 5. BOC/PRP group. 1 month observation. Young bone trabeculae around the biomaterial granules in the centre of graft. BM – bone marrow, BT – bone tissue, BV – blood vessels, CT – connective tissue, M – material, Stain VG, magnification 120×.
presence of biomaterial characterized by varying degrees of fragmentation was observed as well as changes in its color and structure. There were places with preserved structure, as well as fragmented centers, strongly basophilic (change of color to the intense purple in both stains) (Fig. 7). Due to the highly fragmented biomaterial, its quantitative assessment was not possible. The biomaterial was surrounded by lamellar bone tissue and in the immediate vicinity of the interface biomaterial/bone accumulated woven bone was observed. Isolated cases were found in which biomaterial was surrounded by connective tissue. In few cases of the biomaterial resorption area, accumulations of osteoblasts (from the site of a bone) were detectable and, at the site of the material, inflammatory cells were observed .The mean relative value of the bone defect tissue surface was calculated at 22.59% (SD 0.53). 12 months – there was significant fragmentation of the biomaterial, with characteristics as described after 6 months of
Fig. 6. BOC/PRP group. 3 months observation. Formation of young bone trabeculae around the biomaterial granules. BM – bone marrow, BT – bone tissue, M – material, Stain VG, magnification 120×.
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Fig. 7. BOC/PRP group. 6 months observation. Young bone trabeculae and trabeculae in formation phase around the strongly basophilous biomaterial fragments. BM – bone marrow, BT – bone tissue, M – material, Stain VG, magnification 120×.
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Fig. 9. Control group. 1 month observation. Trabeculae of new forming bone surrounded by marrow with a large amount of blood vessels. BM – bone marrow, BT – bone tissue, BV – blood vessels. Stain HE, magnification 120×.
implantation. The quantitative assessment of the remaining material was not conducted. Biomaterial remnants were surrounded by lamellar bone tissue or fibrous connective tissue with characteristic glazing (Fig. 8). In one case, the evidence of massive bone trabeculae, joined with numerous fibrous bands and remnants of bone biomaterial was provided. In the direct neighborhood of resorbing biomaterial cell rich connective tissue and numerous blood vessels were observed. The mean relative value of the bone tissue surface represented 21.76% (SD 1.24) of the defect. 3.3. C group 1 month – intraosseous defects were filled mainly with bone marrow and a large number of blood vessels. The occurrence of few young bone trabeculae was detected (Fig. 9). The mean relative area of these trabeculae was determined at 12.5% (SD 1.32) of the operated defect area (Table 1). 3 months – intraosseous defects were mainly filled with bone marrow, in which newly formed bone trabeculae were observed. The area of the defect was less vascularized in comparison to those of 1 month period (Fig. 10). Input canals leading to defects were covered with dense bone tissue. The mean relative value of bone defect tissue surface averaged 17.74% (SD 0.87) (Table 1). 6 months – thin bone trabeculae surrounded by bone marrow were in the center of the defects (Fig. 11). The mean relative value of the surface of bone tissue defect was calculated at 14.2% (SD 1.72)
Fig. 8. BOC/PRP group. 12 months observation. Massive bone trabeculae connecting with biomaterial remainders, fibrous connective tissue bands, BM – bone marrow, BT – bone tissue M – material, Stain VG, magnification 120×.
Fig. 10. Control group. 3 months observation. Canal of entry to defect covered with compact tissue. BM – bone marrow, BT – bone tissue, Stain VG, magnification 120×.
(Table 1). Input canals from the top of the defects were filled with cartilage tissue, lamellar and woven bone tissue with lots of blood vessels. Few places of the restricted bone rebuilding were detected. In one case, the defect was filled by a block of connective tissue and woven bone tissue of rectangular shape
Fig. 11. Control group. 6 months observation. Filling of the bone defects by bone marrow and plexiform bone tissue. BT – bone tissue. BM – bone marrow, Stain HE, magnification 120×.
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Fig. 12. Control group. 12 months observation. Bone trabeculae surrounded by marrow. BM – bone marrow, BT – bone tissue. Stain HE, magnification 120×.
12 months – in the defect, visible sponge bone tissue was seen with single thin bone trabeculae and bone marrow (Fig. 12). The average relative amount of bone defect tissue surface accounted for 16.1% (SD 1.71) (Table 1). 4. Discussion On the basis of histological studies performed in all observational periods, the experimental group demonstrated an increase in the relative surface area of bone trabeculae filling intraosseous defects as compared with the reference group. In the early phase of observation; i.e. after 1 and 3 months the defects left for spontaneous healing were mainly filled with bone marrow, within which single bone trabeculae appeared. Within the third month, progressive conversion of the tissue, being a covering of the entrance of the root canal with tight bone tissue was observed. In the groups in which methods of guided bone regeneration were applied, the creation of new bone trabeculae around and inside biomaterial granules was observed as early as 1 month following the operation. Similar histological observations after 1 month were described by Indovina and Block (2002) and Schwarz et al. (2007). Unlike the reference group, in the experimental groups, a linear accumulation of osteoblasts was observed along newly created bone trabeculae, which proved the intensity of the osteogenic processes. The characteristics of the first period of the observation; i.e. after 1 and 3 months, referred to more regular distribution of osteoblasts clusters within the defects in the BOC/PRP group as compared with BOC/BG group. In group II, accumulations were observed particularly in the vicinity of the barrier membrane. It was reflected in the quantity and in the presentation of the bone trabeculae location. In the BOC/BG group, bone trabeculae were observed mainly on the edges of the defects and in the BOC/PRP group also in their center. Therefore, a thesis can be presented that, in the first period of the observation, the dynamics of the regeneration processes was higher with the use of Bio-Oss Collagen® with polypeptide growth factors than with the use of a barrier membrane. It seems that, in the BOC/BG group, the creation of new bone trabeculae occurred in defects areas with good perfusion, namely mainly at their peripheries. In the central part of the defect, the perfusion was insufficient, so the inflow of blood growth factors and indirectly osteoconductive activities of biomaterial were limited. Such a relationship was not observed in case of the defects treated by usage of platelet rich plasma which is a natural source of active growth factors required in the process of bone tissue regeneration. The research results obtained in the early stage of the observations were comparable to the results of other authors, such as Ezirganli and Polat
(2011), Rokn et al. (2011) and Heberer et al. (2011). Similarly to own results, the results of Ezirganli and Polat (2011) indicate a beneficial influence of regenerative methods on the healing of intraosseous defects. Although the authors did not confirm statistically significant differences between spontaneous healing and the GBR method (Bio-Oss + tytanium barrier membrane) after 1 month subsequent to the operation, better healing of the aforementioned GBR method compared with spontaneous healing was observed after 3 months subsequent to the operation. The observed difference was statistically significant. However, Rokn et al. (2011) in their histological studies conducted on rabbits after 4 and 8 weeks, did not demonstrate any statistically significant differences in the amount of bone fill between the control group and GBR group with the usage of Bio-Oss. Entirely different results in comparison to our results and the results of the aforementioned researchers were obtained by Heberer et al. (2011). These authors assessed the healing process of ungrafted and grafted extraction sockets in humans after 12 weeks. This descriptive study demonstrated that bone formation in Bio-Oss Collagen – grafted human extraction sockets was lower than bone formation in ungrafted sockets. It seems that such distinct results in comparison to our results may be the consequence of morphological disparities in the treated bone defects, the differences in operation technique and the method of the retrieval of the material for histological study. In our studies, the treated bone defects were iatrogenic, limited by trabecular bone tissue that ensured optimal blood supply to the operated area. The defects treated by Heberer et al. (2011) were four-wall defects, limited by the compact plate of the alveolar socket that causes a much poorer blood supply, and therefore hinders growth factors that positively influence the regenerative processes. Another significant difference was the operative technique. In our studies, the closed system (flaptechnique) was used, as well as additional materials that could enhance the effectiveness of the regeneration processes (barrier membrane and platelet rich plasma). In the study of Heberer et al. (2011), only the osteoconductive properties of the material placed in the defect and left for healing with the use of the opened system were used. The method of preparing the material before its application into the defect also differed. In our study, unlike that of Heberer et al. (2011), no forming method using a blade was used. The preparation of the material consisted of soaking it in blood to obtain a soft consistency and modeling it on a sterile plate before applying it into a treated defect. It seems that our method enables easier penetration of the blood into the biomaterial, increases its plasticity and limits close compression in the defect. The last issue is the site of retrieval of material for histological study. In our material, tissue contents filling the whole treated bone defect was determined, and in the study of Heberer et al. (2011), the material was retrieved from the central part of the defect (diameter of 2 mm); i.e. the area with potentially the lowest blood supply in which regenerative processes may take the longest. After 3 months following the operation, a lesser presence of linear accumulations of osteoblasts along bone trabeculae was observed while the presence of singular osteogenic cells was more often confirmed around the granules of biomedical material in the BOC/PRP group. Most likely the dynamics of bone-forming processes slowed down then. In order to explain these observations, studies of Marx et al. (1998) on the mechanisms of polypeptide growth factor activity might be useful. They have shown that the activity of these factors is maintained for 7–10 days (Marx et al., 1998). According to Hatakeyama et al. (2008) the factor which is the most important in the assessment of the effectiveness of PRP is the early estimation of the healing process. Hence the authors propose to do the assessment after only 15 days following the operation. According to Arm et al. (1996) the secretion of polypeptide growth factors from an absorbed biomaterial can occur even after 40 days, with a maximum between the 10th and 20th day. On the basis of
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our observations it seems that after 3 months, growth factors from the PRP are inactive, while the regenerative functions have been taken over by the implanted material. Our results shown a favorable effect of applying polypeptide growth factors in the early period of bone healing. Our studies are in accordance with the observations of Aghaloo et al. (2002). According to those authors, connecting Bio-Oss biomaterial with PRP significantly increased the formation of new bones in all periods of observation (after 1, 2 and 4 months). In contrast to our results, studies by Roldan et al. (2004) shows a negative effect of platelet concentrate on Bio-Oss® carrier for bone regenerative processes. The decreased formation of newly formed bone observed by the authors was explained by the lack of osteoblasts in bovine bone derivate material and lack of an osteoinductive effect of PRP. Fur˝ thermore, studies by Furst et al. (2003) have shown that neither reduced nor increased bone formation was observed after the joint application of Bio-Oss® and PRP materials in comparison to Bio˝ Oss® alone (Furst et al., 2003). As opposed to the studies of Roldan ˝ et al. (2003) remain, in the opinion of Andrade et al. (2004) and Furst et al. (2008) a significant effect on the effectiveness of the process of tissue healing after applying polypeptide growth factors have numerous factors modifying the properties of PRP. It is believed that the most important factor that influences bone regeneration process after using PRP is the concentration of blood platelets in the applied concentrate. The concentration of the platelet concentrate depends on the technique of platelet separation, the value of relative centrifuge force (RCF), time of acceleration and deceleration of the centrifuge and the selection of appropriate activators of fibrinogen and anticoagulants. Furthermore, the crucial element for a successful therapy is to keep the optimal concentration of platelets after giving PRP to a tissue without further major changes in the concentration of platelets. Therefore, it seems that the method of obtaining PRP applied in our research has to be recognized as optimal because it allowed the demonstration of a beneficial effect of PRP on the process of healing bone. 5. Conclusions Upon quantitative histological assessment, the animals presented the most intensive osteogenesis within one month after the application of xenogenic material with platelet rich plasma, whereas in the later stages, it was observed after the application of xenogenic material and resorbable collagen membrane. The application of two regenerative methods influenced the rate, quality and overall treatment of intraosseous defects. References Abramowitz, P., Rankow, H., Trope, M., 1994. Multidisciplinary approach to apical surgery in conjunction with the loss of buccal cortical plate. Oral Surg. Oral Med. Oral Pathol. 77, 502–506. Aghaloo, T.L., Moy, P.K., Freymiller, E.G., 2002. Evaluation of platelet rich plasma (PRP) in combination with xenograft materials in the rabbit cranium: a pilot study. J. Oral. Maxillofac. Surg. 60, 1176–1181. Andrade, M.G.S., De Fraitas Brandao, C.J., Neves Sa, S.A., Bittencourt, T.H.B., Sadigursky, M., 2008. Evaluation of factors that can modify platelet rich plasma properties. Oral Surg. Oral Med. Oral Pathol. Oral Radiol. Endod. 105, e5–e12. Arm, D.M., Tencer, S.D., Bain, S.D., Celino, D., 1996. Effect of controlled release of platelet derived growth factor from a porous hydroxyapatite implant on bone ingrowth. Biomaterials 17, 703–709. Buser, D., Dula, K., Belser, U., Hirt, H.P., Berthold, H., 1993. Localized ridge augmentation using guided bone regeneration: surgical procedure in the maxilla. Int. J. Periodontics Restorative Dent. 13, 29–45.
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