IOP PUBLISHING
BIOMEDICAL MATERIALS
doi:10.1088/1748-6041/4/1/015012
Biomed. Mater. 4 (2009) 015012 (6pp)
Viability of osteocytes in bone autografts harvested for dental implantology Bernard Guillaume1, Christine Gaudin2, Sonia Georgeault2, Romain Mallet2, Michel F Basl´e2 and Daniel Chappard2 1
CFI—Coll`ege Franc¸ais d’Implantologie, 6 rue de Rome, 75008 Paris, France INSERM, U 922—LHEA, Facult´e de M´edecine, 49045 Angers C´edex, France
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E-mail:
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Received 2 September 2008 Accepted for publication 17 November 2008 Published 12 December 2008 Online at stacks.iop.org/BMM/4/015012 Abstract Bone autograft remains a very useful and popular way for filling bone defects. In maxillofacial surgery or implantology, it is used to increase the volume of the maxilla or mandible before placing dental implants. Because there is a noticeable delay between harvesting the graft and its insertion in the receiver site, we evaluated the morphologic changes at the light and transmission electron microscopy levels. Five patients having an autograft (bone harvested from the chin) were enrolled in the study. A small fragment of the graft was immediately fixed after harvesting and a second one was similarly processed at the end of the grafting period when bone has been stored at room temperature for a 20 min ± 33 s period in saline. A net increase in the number of osteocyte lacunae filled with cellular debris was observed (+41.5%). However no cytologic alteration could be observed in the remaining osteocytes. The viability of these cells is known to contribute to the success of autograft in association with other less well-identified factors. (Some figures in this article are in colour only in the electronic version)
with various biomechanical and osteoconductive properties. None of them have been found sufficient to restore large bone defects. Injectable calcium phosphate materials have been proposed but their long-term effects could not be detectable in a multicentric orthopedic study [4]. In a recent animal study conducted in this laboratory, an injectable bone paste based on βTCP-hydroxyapatite induced osteoconduction with woven bone apposition but the remodeling process removed it in a second time, making the rationale of such substitutes questionable [5]. In addition, synthetic ceramics are often too brittle to be used in weight-bearing bones or when compressive stresses are too high. In orthopedics, bone is commonly harvested from the iliac crest, as it provides easy access to good quality and quantity cancellous autograft. Other sources are Gerdy’s tubercle of the tibia and the distal parts of the radius or tibia for cancellous bone; autologous cortical bone can be obtained from the fibula [6], ribs and iliac crest. These sites were also widely used in maxillofacial surgery from as early as World War II [7]. However other sites have been favored
Introduction Materials for replacing bone are necessary in a number of reconstruction surgeries of the skeleton caused by traumatic, tumor-resection or congenital defects. The highest demand concerns orthopedic surgery for the treatment of femoral prosthesis loosening where wear debris induces a marked osteolysis. It has been estimated that more than 500 000 bone grafts are done each year in the USA [1]. However, there is also a growing demand in maxillofacial surgery and in implantology to restore a sufficient volume of bone before placing implants in the mandible or the maxillar [2]. Allogenic bone grafts are common in orthopedics but require access to a bone bank in some countries [3]. The high cost of a bone bank, the difficulties of conditioning bone chips of fragments specially adapted to maxillofacial surgery and the potential risk of transmission of viral or prior infections have made allografts seldom used in implantology. Synthetic phosphocalcic materials (β-tricalcium phosphate, hydroxyapatite) are numerous in the market but are associated 1748-6041/09/015012+06$30.00
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© 2009 IOP Publishing Ltd Printed in the UK
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for the reconstruction of maxillofacial bone defects such as parietal [8] or mandibular bone [9]. These observations were based on experimental works in animals showing that membranous bone grafts survive better than endochondral bone after grafting [10, 11]. Parietal or chin bone grafting has been proposed in implantology because access is simpler in the surgical theatre than for other sites. Autologous bone provides superior results to other methods since it is revascularized easily and rapidly incorporated into the recipient site. It is also commonly thought that osteoblasts, osteocytes (OCs) and lining cells of the graft can survive the transplant and favor the osseointegration of the graft [12]. However, this has been seldom documented in the literature. This study was undertaken to survey the ability of bone cells to survive during the transplant process starting from harvesting to implantation.
(A)
Material and methods Patients and surgical protocol Human mandibular bone samples were obtained from five patients under general anesthesia during the time course of grafting for pre-implantation. Each patient has given his/her informed consent to participate in the present study. The surgical protocol aims at increasing the bone wall thickness at the maxilla or mandible in deficient recipient sites prior to the placement of dental implant(s) of standard diameter. A broader bone volume ensures an easier position for the implant’s axis. The grafting zone was operated on first to appreciate and determine the shape, volume and position of the graft. For each patient, the graft was harvested at the chin; a preliminary radiographic assessment has been done to check the axis and the thickness of the donor site. One or two rectangular cortico-cancellous bone samples and corticocancellous cylinders were removed. The intra sulcular incision was made at the level of the dental collars with two side incisions at the mesial first premolars. This full thickness reflection flap is preferred to an incision at the gingival margin because it allows a better coverage flap of the grafting zone without any tension and exposure of the graft (figure 1). The limits of the harvesting graft areas were done with a thin bur for bone to outline the defect margins on the buccal bone plate to a 6 mm depth. The harvested graft was separated with a chisel by progressive cleavage. Chips of cortico-cancellous bone (mostly cortical) were collected. The bone samples were immediately placed in sterile saline at room temperature until used for grafting. One small sample (1 mm × 1 mm from the chin cortex and containing the endosteal surface) was immediately fixed in a glutaraldehyde-based fluid. The margin flaps were closed with interrupted sutures with 5/0 vycril. For each patient, the amount of the graft corresponded roughly to 1.8 cm3 per cortico-cancellous block graft (usual dimensions: 10 mm × 6 mm × 3 mm). The flow chart of the study appears in figure 2. When harvesting bone was achieved, the surgeon began to release the muccoperiosteal flap; the margin recipient bone site was revived with a round bur to obtain a slightly hemorrhagic zone. The site was carved to create a mortise favorable so
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Figure 1. Harvesting the bone graft at the chin. (A) Section of bone with the thin bur. (B) Mobilization of the cortico-cancellous grafts with a chisel.
Figure 2. Flow chart of the study from harvesting to grafting. The specimens collected for histological analysis were taken immediately and at the end of the grafting period.
to create a primary closure and the anchorage of the graft was then stabilized with stainless steel screws (the cancellous bone of the cortico-cancellous harvested graft was placed in direct contact with the patient’s recipient site) (figure 3(A)). The cancellous bone chips were placed on all sides around 2
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For each block, five sections (1 μm in thickness) were obtained semi-serially every 20 μm (this separation ensures that no osteocyte lacuna profile could appear on two consecutive sections). A Leica Ultracut S (Leica– Rueil Malmaison, France) was used with a glass knife and sections were immediately deposited on a glass slide with a drop of distilled water. The sections were dried at 60 ◦ C; then they were incubated for 15 min in periodic acid 44 mM and stained with an extemporaneously prepared solution combining aqueous methylene blue 1% (1 volume) and azure II 1% (1 volume) for 20 min at room temperature. The sections were then thoroughly washed in distilled water and mounted in NeoEntellanTM (Merck) after air drying. On each set of sections prepared per block, the total number of osteocyte lacunae was counted by light microscopy on the five sections. Lacunae were separately classified as containing a normal OC with nucleus and cytoplasm with prolongations, empty lacunae and lacunae containing cellular debris. The results were expressed as a percentage of the total lacunae.
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Transmission electron microscopy Ultrathin sections (80 nm in thickness) were obtained with a diamond knife and transferred onto nickel grids (mesh 100) coated with a film of collodion. Grids were then air-dried, contrasted with sodium metaperiodate, uranyl acetate and lead citrate. Observations were done with a transmission electron microscope JEOL 2011 (JEOL-France) at 120 kV.
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Figure 3. (A) The cortico-cancellous grafts are immobilized in the recipient site by screwing. (B) Clinical aspect of a well-integrated allograft at 6 months.
Statistical analysis Statistical study was done using Systat 11 (Systat software Inc.). Data were expressed as mean ± standard error of the mean (SEM). Significant differences between samples were assessed with nonparametric Mann and Whitney’s U test. Differences were considered as significant when P < 0.05.
and on top of the monocortical block graft. The full thickness flap was then closed to the primary incisions and sutured with 5/0 vicryl. When the last chip was positioned, the surgeon placed a remaining bone sample into a new vial containing the glutaraldehyde fixative. In this way, the effects of storing in physiological saline at room temperature during a mean of 20 min ± 33 s could be explored on paired specimens. Sutures were removed two weeks post-operatively. A CT-scan was performed at 3 and 6 months post-surgery to ensure the bone graft healing. The graft was deemed successful when a dense bony reaction could be seen at the grafted site.
Results The patients were re-examined in the days following the graft, even in the absence of any complication. The absence of prolonged pain, edema, purulent discharge, elimination of sequestrated and devitalized bone fragments in the following months are in favor of a progressive success of graft fixation. The palpation of the grafted site showed an increased, stable and painless relief (figure 3(B)). Radiographs (especially the CT-scans) confirmed adherence of the graft at the receiver site by the absence of radiolucent space between the two zones. The gain in thickness was 5 mm on average and 4 mm implants could be placed with a satisfactory axis. The methylene-blue-azure staining allowed a clear-cut identification of the various types of lacunae. OCs were clearly evidenced by a blue cytoplasm with processes extending into the matrix lacunae. Empty lacunae were devoid of any cellular material while cellular debris could be identified inside or on the margins of the lacunae and corresponded to remnants of necrotic cells. The matrix itself was unstained, or appeared light bluish (figure 4).
Bone microscopy The fixative was prepared in ready-to-use plastic vials that were stored in the refrigerator until use. The fixative was composed of glutaraldehyde 4% in cacodylate buffer, pH 7.4. Samples were fixed for 1 h at 4 ◦ C in the surgical unit then rinsed, stored in cacodylate buffer and sent to the laboratory. The samples were then cut with a clean blade into small pieces suitable for electron microscopy and post-fixed in 1% osmium tetroxide. They were dehydrated through a graded ethanol series and finally embedded in Epon 812 according to standard methods used for TEM. The blocks were stored at room temperature until ready to use. 3
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(A)
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Figure 5. OC count on semi-thin sections. (A) The number of osteocyte lacunae filled with cell debris increased in all patients. (B) Cumulative histogram of the three different types of lacunae at the beginning and the end of the grafting surgery.
Discussion Cell necrosis can occur rapidly when cells are deprived of oxygen and essential metabolites such as glucose [13]. The process differs from apoptosis, which is a particular mechanism induced by various factors (excess or deprivation of hormones, growth factors or cytokines, p53 . . . ). Necrosis, unlike apoptosis, can induce an inflammatory reaction. However, osteoinductive factors released from the graft during the resorptive process, as well as cytokines released locally by the inflammatory phase, are thought to contribute to healing of the graft. It is likely that other factors may play a key role in the excellent success rate of autografts because devitalized allografts can release similar molecules. The presence of osteogenic cells is frequently advocated as the basis requirement for the success of autografts [12]. Cortical bone has a higher fractional bone volume (i.e., percentage of the tissue volume occupied by bone) than cancellous bone and has a lower porosity. Haversian canals are the main reservoir of osteogenic cells together with the endosteal surfaces but the number of osteoblast precursors is reduced
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Figure 4. The different types of osteocyte lacunae in light microscopy. (A) Lacuna filled with a typical osteocyte with a well-defined nucleus. (B) Lacuna containing cellular debris. (C) Empty lacuna. Original magnification ×1000.
OC counts appear in figure 5; counts are presented on the samples studied at the beginning and the end of the grafting surgery. The number of empty lacunae remained unchanged after the period spent in ex vivo conditions. On the other hand, a significant increase in the number of lacunae containing debris was observed (+41.5%) and conversely, the number of lacunae containing an intact osteocyte was reduced. On TEM sections, the OC lacunae were observed and the analysis of the fine cytological details of these cells did not reveal any change (figure 6). Occasionally some osteoblasts were encountered and no gross abnormalities (e.g., recycling membrane) were evidenced. 4
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no data have been presented on the potential changes in viability of bone cells induced by the harvesting and storage procedures used before grafting. Another way to evaluate the viability of the osteocytes in the bone chips would have been to perform a LDH (lactate dehydrogenase) histoenzymatic analysis. However in the present study, the grafts were harvested far from the laboratory and we chose to evaluate bone cell preservation by using morphological EM criteria (absence of mitochondrial or membrane changes). Furthermore, the method necessitates cryosectioning, which is not compatible with EM techniques [17]. In the present study, the number of osteoblasts that could be seen on the cortical morcellized chips was very low, and only OCs were evidenced in all subjects. OCs have a reduced number of organites when compared to osteoblasts. Because OCs reside distant from the blood supply, their metabolic needs are satisfied by a combination of passive diffusion of fluids through the matrix and canaliculi and enhanced diffusion arising when the skeleton is loaded during functional activity [18–20]. Their metabolic demand is probably lower than other high energy spending cells and this can explain why a large fraction of OCs survived the surgical conditions (room temperature, saline, absence of oxygen and metabolites). In normal humans, a noticeable fraction of lacunae are found empty, the fraction is increased in osteoporotic patients [21]. OCs are highly differentiated cells which differ from osteoblasts [22, 23]. In vitro studies on the MLO-Y4 osteocytic cell line have found the possibility of these cells to dedifferentiate into osteoblasts [22]. Furthermore, the importance of OCs in the success of bone autografting was stressed in a recent animal study: bone-grafted particles healed when living OCs were present and failed when OCs have undergone necrosis [24]. Another important factor that could explain the superiority of autograft versus allograft is the total absence of protein denaturation in the former. The 20 min incubation in saline used in this study is probably harmless to the patient’s proteins. On the other hand, protein denaturation is known to occur after deep freezing [25] or autoclaving [26]. Treatment of bone allografts with various chemical processes used to clean bone was recently found to alter some important matrix proteins [27]. This study is the first to report the cellular effects of the limited storage conditions used surgically to preserve morcellized bone chips of the chin. A noticeable proportion of OCs remains healthy without modifications observed at the electron microscopy level; other OCs died by necrosis. The success of autografts could result in the preservation of living OCs together with additional factors such as better protein preservation.
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Figure 6. Transmission electron microscopy of living osteocytes in a graft sample at the beginning (A) and the end (B) of the surgical procedure. A group of osteoblasts is also shown at the end (C). Scale bars: 2 μm.
when compared to cancellous bone [14]. Also, the absence of hematopoietic tissue is recognized to provide fewer stem cells in cortical bone autografts. In orthopedic surgery, cortical bone grafts are favored in loaded areas and they have been found to be more resistant to vascular ingrowth and remodeling [15]. Vascularized cortical grafts (i.e., parts of harvested bone with their vascular pedicle, reanastomosed at the recipient site) have been proposed to provide bone with viable cells and restore a local vascularization inside the graft [16]. Although the method has shown clearly superior results with large bone defects (e.g. >12 cm), it is associated with increased morbidity. The continued vascular supply is thought to allow for faster bone incorporation but clear histopathologic analyses are lacking. To our knowledge,
Acknowledgment The authors thank Mrs Laurence Lechat for secretarial assistance. This work was supported by funds from ‘Pays de la Loire–Bioregos’ and INSERM.
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