Jose Luis Calvo-Guirado ndez Maria P. Ramırez-Ferna nchez Jose E. Mat e-Sa Negri Bruno Pablo Velasquez Piedad N. de Aza
Enhanced bone regeneration with a novel synthetic bone substitute in combination with a new natural crosslinked collagen membrane: radiographic and histomorphometric study
Authors’ affiliations: Jose Luis Calvo-Guirado, Maria P. RamırezFern andez, Jose E. Mat e-S anchez, Negri Bruno, Department of Implant Dentistry, Faculty of Medicine and Dentistry, University of Murcia, Murcia, Spain Pablo Velasquez, Piedad N. de Aza, Bioengineering Institute, Miguel Hernandez University, Elche, Spain
Key words: bone substitute, collagen membrane, critical size defects, guided bone regenera-
Corresponding author: Dr Maria Piedad Ramırez Fernandez Hospital Morales Meseguer 30530 Murcia Spain Tel.: +868888584 Fax: +868884399 e-mail: [email protected]
biphasic calcium phosphate at 15, 30, 45, and 60 days by radiological and histomorphometric
tion, hydroxyapatite, tricalcium phosphate Abstract Objectives: 4Bone is a fully synthetic bioactive bone substitute composed of 60% hydroxyapatite (HA) and 40% beta-tricalcium phosphate (ß-TCP). This study aimed to investigate the effect of resorbable collagen membranes (RCM) on critical size defects in rabbit tibiae filled with this novel analysis. Material and methods: Three critical size defects of 6 mm diameter were created in both tibiae of 20 New Zealand rabbits and divided into three groups according to the filling material: Group A (4Bone), Group B (4Bone plus RCM), and Group C (unfilled control group). At each of the four study periods, five rabbits were sacrificed. Anteroposterior and lateral radiographs were taken. Samples were processed for observation under light microscopy. Results: At the end of treatment, radiological analysis found that cortical defect closure was greater in Group B than Group A, and radiopacity was clearly lower and more heterogeneous in Group A cortical defects than in Group B. There was no cortical defect closure in Group C. Histomorphometric evaluation showed significant differences in newly formed bone and cortical closure in Group B compared with Groups A and C, with the presence of higher density newly formed bone in cortical and medullar zones. Conclusions: Biphasic calcium phosphate functioned well as a scaffolding material allowing bone ingrowth and mineralization. The addition of absorbable collagen membranes enhanced bone gain compared with non-membrane-treated sites. This rabbit study provides radiological and histological evidence confirming the suitability of this new material for guided tissue regeneration of critical defects.
Date: Accepted 21 March 2014 To cite this article: Calvo-Guirado JL, Ramırez-Fernandez MP, Mat e-S anchez JE, Bruno N, Velasquez P, de Aza PN. Enhanced bone regeneration with a novel synthetic bone substitute in combination with a new natural cross-linked collagen membrane: radiographic and histomorphometric study. Clin. Oral Impl. Res. 26, 2015, 454–464 doi: 10.1111/clr.12399
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The ideal conditions for inserting dental implants demand the presence of adequate bone volume and quality at the edentulous site (Zitzmann et al. 2001). Alveolar bone loss can result from tooth extraction, infection, trauma, or pathology, and can prevent implant placement in favorable positions and angulations (Simion et al. 2007). The morphology of a bony defect is a key factor for selecting a method of alveolar ridge augmentation. Current therapies in both traumatology and tissue regeneration dentistry are based on the use of artificial or natural materials, which generate stimulating signals that trigger physiological regeneration (Ersanli et al.
2004; Esposito et al. 2006; Aghaloo & Moy 2007). Materials used for filling bone defects with artificial materials include b-TCP and HA. Bone grafts are incorporated into the host bone and are substituted either completely, in the case of b-TCP, or partially, in the case of HA, while other graft substitutes are not biodegradable and thus remain in the bone unchanged (Galindo-Moreno et al. 2013). Hydroxyapatite (HA) and beta-tricalcium phosphate (b-TCP) are well-known ceramics that possess high tissue compatibility and osteoconductivity (Fan et al. 2007). Several studies have demonstrated the effectiveness of these biomaterials in the field of implant
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Calvo-Guirado et al Enhanced bone regeneration with a novel synthetic bone substitute
dentistry (Gauthier et al. 1999; Horch et al. 2006). Commercial HA/b-TCP has been tested for its suitability as a bone substitute in clinical situations (Ogose et al. 2006). Hydroxyapatite is chemically comparable to biological apatite crystals and is considered the least bioactive of the calcium phosphate ceramics. (Gauthier et al. 1999; LeGeros 2002; LeGeros et al. 2003; Mat e-S anchez de Val et al. 2012). b-TCP has greater bioactivity, greater capacity for dissolution than HA, both in vitro and in vivo (Chazono et al. 2004; Horch et al. 2006; Mat e-S anchez de Val et al. 2014). The balance of a stable component HA, and a more bioactive and resorbable component b-TCP allied to porosity promotes a controlled process of ceramic resorption and bone substitution (Schopper et al. 2005; Teramoto et al. 2005; Frenken et al. 2010). Previous studies of the biphasic material used in this study have shown its osteoconductive capacities. When packed into a bony defect, this material gradually resorbs and is replaced by bone
during the healing process (Kolerman et al. 2012). Bone grafts effectively enhance space provision, and this appears to be the principal mechanism by which biomaterials actually support bone regeneration. When critical defects are treated by bone augmentation procedure with applications of synthetic bone substitute, the site can be covered with a barrier membrane, a technique that has achieved good clinical results (Wang et al. 2002; Park et al. 2008). This is known as guided bone regeneration (GBR). GBR is used as a treatment for alveolar ridge deformities, to repair bone defects in preparation for implant placement, and in the prevention of ridge deformities following tooth extraction (Chiapasco et al. 2006). A variety of biocompatible materials, both non-absorbable and absorbable, have been used as membranes in GBR to inhibit the apical migration of the epithelium and penetration of the gingival connective tissue from the flap and thus allow regeneration of
the alveolar bone (Fiorellini & Nevins 2003). The use of non-resorbable barriers is well established; however, bioabsorbable collagen membranes may simplify the surgical technique and make it more predictable (Llambes et al. 2007). An ideal barrier should be made of a material less susceptible to membrane exposure or that cannot be significantly colonized by periodontopathogenic bacteria when exposed to the oral environment. Furthermore, a membrane that will lead to increases in the width and thickness of keratinized tissue can be advantageous (Schwarz et al. 2008, 2013). Recently, a completely resorbable collagen membrane has been introduced that would appear to be more resistant to bacterial collagenase even when prematurely exposed to the oral environment. However, there has been very little research into the use of resorbable membranes barriers in GBR procedures. Animal-controlled studies are necessary, and experiments to find the best bone-graft materials still continue.
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Fig. 1. (a) A sample of the bone substitute material (4Bone granules). (b) 4Bone is available as granules packaged in syringes. (c) Incorporation of saline solution (0.9%) to the bone-graft material. (d) Pushing the syringe plunger backwards to incorporate all the solution. (e) Pushing the syringe plunger forward to remove excess solution. (f) After hydration with 0.9% saline solution, the graft bone particles were used to fill the defects. (g) A sample of the 4Bone RCM membrane used in the study. (h) RCM is available in a sterile package. (i) Incorporation of saline solution (0.9%) to membrane.
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
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and 60 days after surgery; both tibiae were retrieved in blocks containing the entire graft area for histomorphometric and radiological analysis.(Fig. 3). Radiographic imaging
Two X-rays, antero-posterior and lateral, were taken of each of the bone sections containing the grafts, using the Kodak RVG 6100 Digital Radiography System with X-rays taken at 32 kV, 40 mA using automatic light metering. The images were used to observe the changes in radiopacity at descriptive level in the medullar and cortical areas where the defects had been created.
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Optical microscopy
Histomorphometric analysis
The sample size consisted of 120 specimens (Group A = 40, Group B = 40, and Group C = 40). Specimens were fixed with 10% neutral buffered formalin and decalcified by means of immersion in Osteomolâ Merck KbaA (Germany) containing HCl (10%) and CH2O (4%) for seventeen days, renewing the solution every 24 h. Subsequently, all samples were embedded in paraffin by the usual method, sectioned at 5 lm, and stained using hematoxylin–eosin, Masson’s trichromic and Malaquite Green. All samples were examined under light microscopy (Microphoto FXA, Nikon, Tokyo, Japan).
Histomorphometric evaluations comprised measurements of the areas of bone and xenograft particles in relation to the total measurement area. The central portion of each core was selected to avoid any potential bias; in this way, both the coronal (native host bone) and the apical portion (with a safe margin of 1.5–2 mm) were excluded from analysis. Examinations were performed under a Nikon Eclipse 80i microscope (Teknooptik AB, Huddinge, Sweden) equipped with the EasyImage 2000 system (Teknooptik AB) using 91 to 94 lenses for descriptive evaluation and for taking morphometric measurements. Histomorphometric analysis was performed with a Macintosh computer using the public domain NIH Image program (developed by the US National Institutes of Health and available on the Internet at http://rsb. info.nih.gov/nih-image/).Values for the total percentage of newly formed bone, residual graft material, and non-mineralized connective tissue were then calculated (Fig. 4).
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Statistical analysis
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(d)
The Brunner and Langer’s test (nonparametric repeated-measures analysis of variance) was applied to analyze the cortical defect closure (mm standard deviation) has been performed using R (Statistics Department of the University of Auckland). Mean values and standard deviations were calculated using a descriptive test for new bone, residual material, connective tissue. All histomorphometric parameters were analyzed using descriptive methods (SPSS 19.0; SPSS, Chicago, IL, USA for Windows). The significance level chosen was 5%.
Fig. 3. Macroscopic image of tibiae dissected at (a) 15 days; (b) 30 days; (c) 45 days; (d) 60 days.
Results Radiographic analysis Fifteen days
Fig. 4. Shows the method used to calculate histomorphometric values for new bone, residual material, connective tissue, and cortical closure. Measurements were made manually and data were entered on a spreadsheet.
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Group A: X-rays revealed the graft material to be a cylindrical element, a 6 9 6 mm rectangular structure of great radiopacity, which facilitated its identification within the trabecular bone structure in which it was implanted. This group showed incomplete closure of the cortical defect after 15 days. Group B: This group showed almost complete closure of the cortical defect, the graft material partially filling the interior of the medullar cavity. Group C: The control bone defects showed radio-transparent concave depres-
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and 60 days after surgery; both tibiae were retrieved in blocks containing the entire graft area for histomorphometric and radiological analysis.(Fig. 3). Radiographic imaging
Two X-rays, antero-posterior and lateral, were taken of each of the bone sections containing the grafts, using the Kodak RVG 6100 Digital Radiography System with X-rays taken at 32 kV, 40 mA using automatic light metering. The images were used to observe the changes in radiopacity at descriptive level in the medullar and cortical areas where the defects had been created.
(a)
Optical microscopy
Histomorphometric analysis
The sample size consisted of 120 specimens (Group A = 40, Group B = 40, and Group C = 40). Specimens were fixed with 10% neutral buffered formalin and decalcified by means of immersion in Osteomolâ Merck KbaA (Germany) containing HCl (10%) and CH2O (4%) for seventeen days, renewing the solution every 24 h. Subsequently, all samples were embedded in paraffin by the usual method, sectioned at 5 lm, and stained using hematoxylin–eosin, Masson’s trichromic and Malaquite Green. All samples were examined under light microscopy (Microphoto FXA, Nikon, Tokyo, Japan).
Histomorphometric evaluations comprised measurements of the areas of bone and xenograft particles in relation to the total measurement area. The central portion of each core was selected to avoid any potential bias; in this way, both the coronal (native host bone) and the apical portion (with a safe margin of 1.5–2 mm) were excluded from analysis. Examinations were performed under a Nikon Eclipse 80i microscope (Teknooptik AB, Huddinge, Sweden) equipped with the EasyImage 2000 system (Teknooptik AB) using 91 to 94 lenses for descriptive evaluation and for taking morphometric measurements. Histomorphometric analysis was performed with a Macintosh computer using the public domain NIH Image program (developed by the US National Institutes of Health and available on the Internet at http://rsb. info.nih.gov/nih-image/).Values for the total percentage of newly formed bone, residual graft material, and non-mineralized connective tissue were then calculated (Fig. 4).
(b)
Statistical analysis
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(d)
The Brunner and Langer’s test (nonparametric repeated-measures analysis of variance) was applied to analyze the cortical defect closure (mm standard deviation) has been performed using R (Statistics Department of the University of Auckland). Mean values and standard deviations were calculated using a descriptive test for new bone, residual material, connective tissue. All histomorphometric parameters were analyzed using descriptive methods (SPSS 19.0; SPSS, Chicago, IL, USA for Windows). The significance level chosen was 5%.
Fig. 3. Macroscopic image of tibiae dissected at (a) 15 days; (b) 30 days; (c) 45 days; (d) 60 days.
Results Radiographic analysis Fifteen days
Fig. 4. Shows the method used to calculate histomorphometric values for new bone, residual material, connective tissue, and cortical closure. Measurements were made manually and data were entered on a spreadsheet.
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Group A: X-rays revealed the graft material to be a cylindrical element, a 6 9 6 mm rectangular structure of great radiopacity, which facilitated its identification within the trabecular bone structure in which it was implanted. This group showed incomplete closure of the cortical defect after 15 days. Group B: This group showed almost complete closure of the cortical defect, the graft material partially filling the interior of the medullar cavity. Group C: The control bone defects showed radio-transparent concave depres-
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sions of round or rectangular morphology depending on the image studied. They had clear and regular outlines showing a homogenous density that clearly defined their boundaries (Fig. 5a).
seen radiologically, implying the formation of bone around the graft material. Group C: Some differences were observed when compared with the previous evaluation period; X-ray images revealed linear elements representing irregular trabecular lines that did not follow the axes or load forces of adjacent bone trabeculation (Fig. 5b).
Thirty days
Group A: The radiological radiopacity of this material was lower than that observed for the previous time-period, and the cylindrical form of the grafted material had given way to a more oval and irregular shape. The closure of the cortical defect was still incomplete. Group B: X-rays revealed the cortical as being completely repaired in the filled bone defects, albeit with less density than that of the adjacent cortical bone. Reinforced medullar bone density could be
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Forty-five days
Group A: In the cortical area, the graft area displayed less radiological radiopacity as well as a more oval shape. Irregular borders could be distinguished radiologically. In some areas, there was continuity between the osseous cortex and the implanted material as manifested by the linear images of osseous trabeculae.
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Group B: X-rays revealed the external cortex of the artificial osseous lagoons into which the bone–granulate implant had been introduced; this had a radiopacity similar to that of the adjacent cortex. An increase in medullar radiopacity occurred corresponding to the appearance of trabecular bone. Group C: X-ray images of the control sites showed similar characteristics to the previous study period (Fig. 5c).
Sixty days
Group A: The graft material had a speckled appearance that showed slightly less radiopacity in the medullar zone. Almost complete repair of the osseous defect was also observed, but radiological images showed those trabeculae that reached the implant as being greater in number and radiopacity than for the previous time-period, giving the grafted area a slightly reticular appearance. Group B: It was impossible to distinguish the cortical defect area. No osseous malformations or structural changes to cortical bone development were observed over the study period. Group C: The samples showed a progressive reduction in the size of the cortical defect but in no case was complete closure achieved. The medullar area’s radiographic appearance did not undergo any change (Fig. 5d).
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Histological and histomorphometric analysis The results for cortical defect closure measurement are shown in Table 1. Tables 2–4 show mean values for the total percentages of newly formed bone, residual graft material, and non-mineralized connective tissue at each evaluation period. Fig. 5. Antero-posterior and lateral X-rays of the section of bone containing the implants at: (a) 15 days; (b) 30 days; (c) 45 days; (d) 60 days.
Table 1. Bruner Langer (nonparametric method for dependent or longitudinal data). Statistical results from histomorphometric evaluation of cortical defect closure values for each evaluation period. Median value in (mm standard deviation [SD]) Group A (N = 10) Days
Median value (mm SD)
15 30 45 60
19.87 28.24 59.89 85.46
4.3 2.7 3.4 1.5
Group B (N = 10)
Group C (N = 10)
P value
Median value (mm SD)
0.0735 0.0618 0.5542 0.0619
31.42 45.32 69.98 97.61
2.9* 4.2* 3.4* 2.3*
P value
Median value (mm SD)
0.0108 0.0242 0.0068 0.0056
10.09 15.66 26.47 54.62
2.38 1.54 1.06 0.47
P value 0.0603 0.0715 0.1321 0.5192
The level of significance was set at P < 0.05. *Differences between values achieving statistical significance. Group A: 4Bone. Group B: 4Bone plus RCM membrane. Group C: Unfilled control group.
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Calvo-Guirado et al Enhanced bone regeneration with a novel synthetic bone substitute
Table 2. Statistical results from histomorphometric evaluation of new bone values (% standard deviation [SD]) at different time-periods Group A (N = 10) Days
Mean value (% SD)
15 30 45 60
17.54 23.44 63.67 82.75
2.41 2.37 1.36 0.98
Group B (N = 10)
Group C (N = 10)
P value
Mean value (% SD)
0.0623 0.0811 0.0852 0.0627
30.42 34.51 75.47 91.16
2.29* 1.64* 1.43* 1.02*
P value
Mean value (% SD)
0.0232 0.0147 0.0166 0.0179
8.75 14.53 31.25 39.95
1.66 1.18 3.52 1.57
P value 0.0619 0.0731 0.1361 0.4598
The level of significance was set at P < 0.05. *Differences between values achieving statistical significance. Group A: 4Bone. Group B: 4Bone plus RCM membrane. Group C: Unfilled control group.
Table 3. Statistical results from histomorphometric evaluation of connective tissue values (% standard deviation [SD]) at different time-periods Group A (N = 10) Days
Mean value (% SD)
15 30 45 60
33.55 42.01 23.07 10.83
2.76 1.53 0.52 1.27
Group B (N = 10)
Group C (N = 10)
P value
Mean value (% SD)
0.1954 0.1667 0.2342 0.5631
19.75 29.48 9.31 0.52
2.75 0.63 1.58 0.61
P value
Mean value (% SD)
0.1742 0.2831 0.2156 0.2324
91.25 85.47 68.75 60.05
0.37* 1.43* 1.29* 2.32*
P value 0.0021 0.0134 0.0069 0.0056
The level of significance was set at P < 0.05. *Differences between values achieving statistical significance. Group A: (4Bone). Group B: (4Bone plus RCM membrane). Group C: (Unfilled control group).
Table 4. Statistical results from histomorphometric evaluation of remain material values (% standard deviation [SD]) at different time-periods Group A (N = 10) Days
Mean value (% SD)
15 30 45 60
48.91 34.55 13.26 6.42
1.06 0.19 0.52 0.98
Group B (N = 10)
Group C (N = 10)
P value
Mean value (% SD)
0.0785 0.0652 0.0793 0.0128
49.83 36.01 15.22 8.32
0.21* 0.53* 2.79* 0.26*
P value
Mean value (% SD)
P value
0.0115 0.0264 0.0127 0.2008
0 0 0 0
0.6448 0.5068 0.8123 0.7062
The level of significance was set at P < 0.05. *Differences between values achieving statistical significance. Group A: 4Bone. Group B: 4Bone plus RCM membrane. Group C: Unfilled control group.
Fifteen days
Group A: The grafted defects showed numerous particles of 4Bone granules surrounded by highly vascularized granulation tissue in the cortical defect; defects had undergone incomplete closure. Group B: The membrane was fully intact and maintained the defect space. It appeared intensely stained due to a network of thick fiber bundles apparently consisting of collagen type III. The inner part of the membrane was in direct contact with newly formed bone and the marrow space (Fig 6a). Significant new bone formation was observed on the surface of some bone particles. Specimens exhibited histological patterns of biomaterial granules
with osteogenic activity predominant on the bone walls. The greatest concentrations of immature bone in the outer areas were seen in both the grafted cortical and medullar zones; in this area, small spaces were observed between graft material granules and between these and the cortex. Group C: A slight reduction in the cortical defect was observed; the medullar area was filled by a significant amount of connective tissue and blood vessels without any bone.
Thirty days
Group A: Defects showed immature bone in the cortical zone and in the medullar zone multinucleated giant cells were
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
identified at regular intervals on the surfaces of the graft material, together with signs that an active resorption process was underway. Inside the graft particles, pores of different sizes showed new bone formation coating their inner walls. Group B: The membrane had reduced in thickness, with a slide collapse in the center; capillaries frequently appeared in the interfibrillar meshes. The dark-stained fiber bundles had disappeared while strands of fibers – probably collagen type I – persisted (Fig 6b). The cortical defect could be distinguished as a thinner cortical zone. Spaces between graft material and cortex were observed. Residual graft
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Discussion
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Fig. 6. Histological findings for the membrane in each healing period. (a) Microscope detail of Group B (T.M. 9 250) at 15 days (b) Microscope detail of Group B (T.M. 9 125) at 30 days (c) Microscope detail of Group B (T.M. 9 125) at 45 days (d) Microscope detail of Group B (T.M. 9 250) at 60 days.
material had reduced during this period, and connective tissue was also present, albeit in small quantities, and some new bone formation was observed (Fig 7a). Group C: Progressive, although partial, defect closure was observed, but there was no bone formation in the medullar zone; a fibrous tissue had developed filling the medullar cavity and the cortical defect. The cortical defect was slightly smaller.
Forty-five days
Group A: These spaces had undergone some reduction. Group B: The membrane remained intact, but there was evidence of degradation of the membrane material resulting in the formation of separate elements. The membrane appeared to disintegrate from both surfaces toward the center. (Fig 6c). The defect was no longer visible in the cortical zone; the cortical had further increased in width, and the diameter of the interconnecting trabeculae had also increased. The bone formed in the defect space appeared as a rather loose scaffold of woven bone, with primary bone marrow filling the intertrabecular space whilst in the medullar area, the spaces between granules had decreased and there was a more diffuse
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distribution of residual biomaterial and an increase in osseous remodeling (Fig 7b). Group C: The cortical defect was still visible, sealed by fibrous tissue.
Sixty days
Group A: Cortical bone was slightly thicker in the graft area. The medullar zone showed graft particles that were embedded in newly formed bone, which occasionally bridged the particles with branches of woven bone. Multinucleated giant cells were still present on the particle surfaces (Fig. 8a). Group B: Resorption of the material had taken place, which altered the internal structure, staining properties, shape, and integrity of the membrane (Fig 6d). Significant complete bone reparation of the cortex was observed, manifested as well-organized trabecular bone with a presence of mature bone formation that was not differentiable from the adjacent cortex. The medullar zone also showed mature bone formation in continuity with the cortex (Figs 7c and 8b). Group C: A reduction in cortical defect size was observed, and there was soft tissue closure by dense connective tissue with abundant blood vessels but without any bone formation in the medullar zone (Fig. 8c).
The present study evaluated the clinical outcome of regeneration procedures in critical defects in rabbit tibiae with the use of particulate 4Bone with or without the addition of a resorbable collagen membrane RCM. The outcomes were different for the three study groups, pointing to a positive histological effect resulting from use of the membrane in combination with the bone-graft material. Ideally, a bone substitute biomaterial is eliminated slowly after implantation in the patient, but at a rate of biodegradation, that allows the mechanical strength of the graft to be maintained during healing, balancing the resorption rate of the biomaterial with the patient’s ability to form new bone (Kanczler & Oreffo 2008). Among the formulations reported in the literature, particulate biphasic calcium phosphate biomaterials are especially attractive as they have an increased surface area, are osteoconductive, and can fill any defect size or shape (Ara ujo et al. 2010a,b). The implantation of these graft ceramic particles presented significantly more bone formation compared with the control samples in our study. The present rabbit study supports other observations of this graft’s high osteoconductivity and resorbability (Pekkan et al. 2012). The growth of bone inside biomaterial particles could be related not only to pore sizes but also to the biomaterial’s intrinsic characteristics. This graft material’s porosity is designed to accommodate the required biological exchanges particularly for bone ingrowth and mineralization. Indeed, the graft’s high porosity facilitated the resorption process as the pores’ external and internal surface areas were exposed to the medium. Previous studies of biphasic materials with the same HA/ß-TCP ratio as the material used in the present study have noted that porosity and pore interconnectivity have an effect on the material’s rate of biodegradation (Jones et al. 2009); this also influences new bone formation, vascularization, and graft stability (Calvo-Guirado et al. 2012, 2013). In the present study, the resorbed graft particles were gradually substituted by newly formed bone during bone remodeling processes in a similar way to that observed by Schopper et al. 2005. The resorption of these graft particles may have been helped by the small size of the HA crystallites and by the enzymes secreted by multinucleated giant cells, which were observed in close proximity to the granules. The texture of this graft
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Calvo-Guirado et al Enhanced bone regeneration with a novel synthetic bone substitute
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(c) Fig. 7. (a) Microscope detail of Group B (T.M. 9 250) at 30 days. Monolayers of osteoblasts were seen actively secreting osteoid that bridged the gaps between grafted particles and the surrounding newly formed woven bone. (b) Microscope detail of Group B (T.M. 9 250) at 45 days. Grafted bone presents an irregular surface due to osteoclastic activity with greater quantities of immature connective tissue. Higher magnification revealed newly formed bone surrounding and replacing portions of the synthetic graft particles. (c) Microscope detail of Group B (T.M. 9 250) at 60 days. In this magnified view, new bone is seen forming along pore surfaces with the mineralized graft. Bone formation is in close contact with granules accompanying the particles’ resorptive process. Remodeling of trabecular bone can be seen at this period.
serves as an osteoconductive scaffold for osteoblastic cells and stimulates the deposition of bone matrix. The particles osseointegrate, and the resorbed biomaterial is slowly
replaced by newly formed bone. The presence of canals, resorptive trails, and vessels within these graft particles was observed in all biopsies at 60 days. In the majority of cases with
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
the presence of Haversian and Volkmann’s canals, graft particles were recolonized by vessels through preexisting canals. Bone regeneration is a coordinated process involving the connection between blood vessels and bone cells. Resorption of 4Bone material has also been observed in bone cores from humans, with evidence of diverse active multinucleated cells on the biomaterial (Kolerman et al. 2012). In this study, the biomaterials were seen to be biocompatible, osteoconductive, and reabsorbable. However, the diverse reagent chemistry described for the preparation of biphasic calcium phosphates, allied with the various implantation models, has resulted in great variability among other reported in vivo outcomes. (Ooms et al. 2003). Bone regeneration may be adversely affected by a lack of primary wound closure during the healing period. The application of physical barriers for regenerating bone defects aims to allow sufficient growth of bone tissue to permit implant placement in sites where bone volume would otherwise be insufficient. (Ezirganlı et al. 2013). Collagen membranes used as barriers in GBR have often been found to produce significant amounts of new attachment. In all Group B specimens with 4Bone and RCM membrane, a significant decrease in the osseous defect was achieved. This outcome is in agreement with data reported by other authors (Oh et al. 2003). In the present study, this appeared to have certain benefits in that Group B specimens generated the highest percentages of trabecular bone volume and promoted bone growth without soft tissue interference in comparison with the other groups. Similarly, other authors have reported advantages in using membranes combined with mineralized cancellous grafts, such as a more favorable healing response and the prevention of soft tissue invasion (Carpio et al. 2000; Chiapasco et al. 2006; Jovanovic et al. 2007). Yet, their positive benefits should be carefully balanced against the significant risk of infection, added time and cost, and the potential need for return visits from patients, who may develop complications (Rossa et al. 2006). Simion et al. (2007), in a study with similar results to the present one, found that the barrier membrane placed over the area to be augmented creates a protected space, which stabilizes blood clots and excludes soft tissue penetration. In this way, the protected space can be populated by slow-migrating osteogenic cells resulting in new bone formation. Nevertheless, it is not yet under-
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(c)
Fig. 8. (a) Panoramic image of Group A (H. E. at 923) at 60 days: the perforation in the cortical bone is almost filled by neoformed or immature osseous tissue in the outer areas, with the rest of the residual biomaterial in the cortical area in a phase of resorption and substitution. The ingrowth tissue is mostly fibrous tissue with minimal bone. (b) Panoramic image of Group B(H. E. at 923) at 60 days: complete bone reparation of the cortex of the implant orifice, manifested as well-organized trabecular bone with a presence of mature osseous bone that could not be differentiated from the adjacent cortex. The image also shows trabecular bone formation in the vicinity and in continuity with the cortex, as well as the junction between the newly formed cortical bone and adjacent cortex. (c) Panoramic image of Group C (H.E. at 923) at 60 days: the perforation made in the cortical bone was incompletely filled. Newly formed trabecular bone with large marrow spaces was observed mostly in the peripheral areas of the defects.
stood exactly how factors such as the membrane’s constituents, morphology, adherence capacity, protein-binding capacity, the substances released during degradation, surface texture, size of perforations and the duration of the barrier’s functional life might influence GBR outcomes (Bornstein et al. 2005, 2007). The structural integrity of implanted bioabsorbable barrier membranes needs to be preserved over a sufficient time to allow maturation of newly formed bone within the membrane-protected space (Machtei 2001; Nemcovsky & Artzi 2002). Collagen membrane degradation can be altered by increasing its structural integrity. The recently introduced collagen barrier, RCM, is apparently more resistant to animal and bacterial collagenase even when prematurely exposed to the oral environment. All the characteristics of RCM assayed, including its biocompatibility and biodegradability, have been verified. The barrier consists of a cross-linked collagen membrane, which enables a longer degradation time. The present study found that it remained intact at the GBR site, allowing bone regeneration to occur successfully. In this way, the results clearly demonstrate that a complete selective formation of bone within a defined bone defect can be accomplished by preventing cells not derived from the surrounding bone from repopulating the defect area. The regeneration had already occurred after 60 days, while in the non-membrane-treated group, a higher degree of osteoclastic activity and less osteogenic activity were observed. These results are in agreement with earlier findings in which complete bone regeneration of large perforating bone defects in rat mandibles was
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obtained using a similar surgical technique (Nolff et al. 2010). Natural non-cross-linked collagen membranes are sufficient to enable effective and predictable bone regeneration (Becker et al. 2009; Bornstein et al. 2009). The positive findings obtained in the present work must be analyzed carefully; with cross-linked membranes, vascularization is less and/or much slower, and tissue integration is diminished (Rothamel et al. 2004, 2005; Schwarz et al. 2013). Furthermore, Rothamel et al. (2005) observed a foreign body reaction with different crosslinked membranes in a rat model. However, the use of cross-linked membranes in the present study produced better defect closure and bone formation in comparison with the other groups. The cross-linking of formaldehyde and porcine type I and III collagen in the membrane used in the present study was a possible reason for its increased biocompatibility. The present findings suggest that collagen membranes have a direct effect on bone formation, although the ideal time-period that the membrane should retain its barrier function to maximize the healing results has still not been determined precisely. In guided tissue regeneration processes, a membrane integrity duration of only 4– 6 weeks has been advocated for periodontal regeneration in contrast to a prolonged period of at least 6 months recommended for GBR procedures. It is therefore likely that the duration of the intact membrane is a key issue for the formation and maturation of new bone in protected membrane defects (Park et al. 2008; Fotek et al. 2009). The present results showed that at the end of the study time, all of the cortical defects
at Group B test sites were filled with newly formed bone of a uniform thickness. The membrane still existed, but there was evidence of degradation of the membrane material. The bone grew predictably all the way up to the reference point at Group A test sites (but with large variations), and only half way at control sites. As in other studies, significantly more bone formation was observed around defects protected by the barrier membrane compared with control defects (Cordaro et al. 2002). In this way, the results of the present study clearly demonstrate that complete selective bone formation within a defined bone defect can be accomplished by preventing cells not derived from the surrounding bone from repopulating the defect area. (Walsh et al. 2008). New bone formation was also seen in Group A non-membrane-treated areas but to a much lesser extent than in Group B membrane-treated areas with significant difference. While at Group B sites, only boneforming cells were allowed to migrate into the wound, at Group A sites, granulation tissue derived from the soft tissue cover flap could also invade the wound area, thereby competing with the bone-forming cells. The amount of newly formed bone in this situation may be dependent on the differences in the proliferation rate of the different types of cells and the size of the available space. This may explain the pronounced variation in new bone formation observed at Group A sites as well as the finding that only ten of the 40 Group A sites showed a bone coverage reasonably close to that obtained at Group B membrane sites, which presented practically complete bone coverage. Histological examination of the material showed that the newly
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Calvo-Guirado et al Enhanced bone regeneration with a novel synthetic bone substitute
formed bone was stained darker than the original tibial bone, presumably representing a more active phase of osteogenesis; this has also been observed in others studies (Chazono et al. 2004). The present study found that GBR provided a biologic principle for the development of reconstructive surgical techniques aimed at generating bone tissue of sufficient volume for the placement of titanium implants. Although this was an animal study, it may be surmised
that the technique is also applicable to humans in a clinical setting.
Conclusions The test materials used for GBR fulfilled expectations successfully. 4Bone granules functioned well as a scaffolding material, allowing mineralized tissue formation. The RCM absorbable membranes used in associa-
tion with the new synthetic graft material not only functioned well as a barrier membrane for GBR, but also showed a potential to increase tissue generation as the addition of the absorbable membranes enhanced bone gain compared with non-membrane-treated sites. Within the limits of this study, it is concluded that this technique achieved predictable clinical outcomes.
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Ogose, A., Kondo, N., Umezu, H., Hotta, T., Kawashima, H., Tokunaga, K., Ito, T., Kudo, N., Hoshino, M., Gu, W. & Endo, N. (2006) Histological assessment in grafts of highly purified beta-tricalcium phosphate (OSferion) in human bones. Biomaterials 27: 1542–1549. Oh, T.J., Meraw, S.J., Lee, E.J., Giannobile, W.V. & Wang, H.L. (2003) Comparative analysis of collagen membranes for the treatment of implant dehiscence defects. Clinical Oral Implants Research 14: 80–90. Ooms, E.M., Wolke, J.G.C., van de Heuvel, M.T., Jeschke, B. & Jansen, J.A. (2003) Histological evaluation of the bone response to calcium phosphate cement implanted in cortical bone. Biomaterials 24: 989–1000. Park, S.-H., K-w, L., Oh, T.-J., Misch, C.E., Shotwell, J. & Wang, H.-L.. (2008) Effect of absorbable membranes on sandwich bone augmentation. Clinical Oral Implant Reseach 19: 32–41. Pekkan, G., Aktas, A. & Pekkan, K. (2012) Comparative radiopacity of bone graft materials. Journal of Cranio-Maxillo-Facial Surgery 40: 1–4. Rossa, M.L., Lima, L.A., Pustiglioni, F.E., Hespanhol, A.M., Kon, S., Grigolli Filho, J. & Novo, N.F. (2006) SEM analyses of bacterial contamination of e-ptfe membranes and GTR clinical results. Journal of the International Academy of Periodontology 8: 115–124. Rothamel, D., Schwarz, F., Sager, M., Herten, M., Sculean, A. & Becker, J. (2005) Biodegradation of differently cross-linked collagen membranes: an experimental study in the rat. Clinical Oral Implants Research 16: 369–378. Rothamel, D., Schwarz, F., Sculean, A., Herten, M., Scherbaum, W. & Becker, J. (2004) Biocompatibility of various collagen membranes in cultures of human PDL fibroblasts and human osteoblastlike cells. Clinical Oral Implants Research 15: 443–449. Schopper, C., Ziya-Ghazvini, F., Goriwoda, W., Moser, D., Wanschitz, F., Spassova, E., Lagogiannis, G., Auterith, A. & Ewers, R. (2005) HA/TCP
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Enhanced bone regeneration with a novel synthetic bone substitute in combination with a new natural crosslinked collagen membrane: radiographic and histomorphometric study
Authors’ affiliations: Jose Luis Calvo-Guirado, Maria P. RamırezFern andez, Jose E. Mat e-S anchez, Negri Bruno, Department of Implant Dentistry, Faculty of Medicine and Dentistry, University of Murcia, Murcia, Spain Pablo Velasquez, Piedad N. de Aza, Bioengineering Institute, Miguel Hernandez University, Elche, Spain
Key words: bone substitute, collagen membrane, critical size defects, guided bone regenera-
Corresponding author: Dr Maria Piedad Ramırez Fernandez Hospital Morales Meseguer 30530 Murcia Spain Tel.: +868888584 Fax: +868884399 e-mail: [email protected]
biphasic calcium phosphate at 15, 30, 45, and 60 days by radiological and histomorphometric
tion, hydroxyapatite, tricalcium phosphate Abstract Objectives: 4Bone is a fully synthetic bioactive bone substitute composed of 60% hydroxyapatite (HA) and 40% beta-tricalcium phosphate (ß-TCP). This study aimed to investigate the effect of resorbable collagen membranes (RCM) on critical size defects in rabbit tibiae filled with this novel analysis. Material and methods: Three critical size defects of 6 mm diameter were created in both tibiae of 20 New Zealand rabbits and divided into three groups according to the filling material: Group A (4Bone), Group B (4Bone plus RCM), and Group C (unfilled control group). At each of the four study periods, five rabbits were sacrificed. Anteroposterior and lateral radiographs were taken. Samples were processed for observation under light microscopy. Results: At the end of treatment, radiological analysis found that cortical defect closure was greater in Group B than Group A, and radiopacity was clearly lower and more heterogeneous in Group A cortical defects than in Group B. There was no cortical defect closure in Group C. Histomorphometric evaluation showed significant differences in newly formed bone and cortical closure in Group B compared with Groups A and C, with the presence of higher density newly formed bone in cortical and medullar zones. Conclusions: Biphasic calcium phosphate functioned well as a scaffolding material allowing bone ingrowth and mineralization. The addition of absorbable collagen membranes enhanced bone gain compared with non-membrane-treated sites. This rabbit study provides radiological and histological evidence confirming the suitability of this new material for guided tissue regeneration of critical defects.
Date: Accepted 21 March 2014 To cite this article: Calvo-Guirado JL, Ramırez-Fernandez MP, Mat e-S anchez JE, Bruno N, Velasquez P, de Aza PN. Enhanced bone regeneration with a novel synthetic bone substitute in combination with a new natural cross-linked collagen membrane: radiographic and histomorphometric study. Clin. Oral Impl. Res. 26, 2015, 454–464 doi: 10.1111/clr.12399
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The ideal conditions for inserting dental implants demand the presence of adequate bone volume and quality at the edentulous site (Zitzmann et al. 2001). Alveolar bone loss can result from tooth extraction, infection, trauma, or pathology, and can prevent implant placement in favorable positions and angulations (Simion et al. 2007). The morphology of a bony defect is a key factor for selecting a method of alveolar ridge augmentation. Current therapies in both traumatology and tissue regeneration dentistry are based on the use of artificial or natural materials, which generate stimulating signals that trigger physiological regeneration (Ersanli et al.
2004; Esposito et al. 2006; Aghaloo & Moy 2007). Materials used for filling bone defects with artificial materials include b-TCP and HA. Bone grafts are incorporated into the host bone and are substituted either completely, in the case of b-TCP, or partially, in the case of HA, while other graft substitutes are not biodegradable and thus remain in the bone unchanged (Galindo-Moreno et al. 2013). Hydroxyapatite (HA) and beta-tricalcium phosphate (b-TCP) are well-known ceramics that possess high tissue compatibility and osteoconductivity (Fan et al. 2007). Several studies have demonstrated the effectiveness of these biomaterials in the field of implant
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Calvo-Guirado et al Enhanced bone regeneration with a novel synthetic bone substitute
dentistry (Gauthier et al. 1999; Horch et al. 2006). Commercial HA/b-TCP has been tested for its suitability as a bone substitute in clinical situations (Ogose et al. 2006). Hydroxyapatite is chemically comparable to biological apatite crystals and is considered the least bioactive of the calcium phosphate ceramics. (Gauthier et al. 1999; LeGeros 2002; LeGeros et al. 2003; Mat e-S anchez de Val et al. 2012). b-TCP has greater bioactivity, greater capacity for dissolution than HA, both in vitro and in vivo (Chazono et al. 2004; Horch et al. 2006; Mat e-S anchez de Val et al. 2014). The balance of a stable component HA, and a more bioactive and resorbable component b-TCP allied to porosity promotes a controlled process of ceramic resorption and bone substitution (Schopper et al. 2005; Teramoto et al. 2005; Frenken et al. 2010). Previous studies of the biphasic material used in this study have shown its osteoconductive capacities. When packed into a bony defect, this material gradually resorbs and is replaced by bone
during the healing process (Kolerman et al. 2012). Bone grafts effectively enhance space provision, and this appears to be the principal mechanism by which biomaterials actually support bone regeneration. When critical defects are treated by bone augmentation procedure with applications of synthetic bone substitute, the site can be covered with a barrier membrane, a technique that has achieved good clinical results (Wang et al. 2002; Park et al. 2008). This is known as guided bone regeneration (GBR). GBR is used as a treatment for alveolar ridge deformities, to repair bone defects in preparation for implant placement, and in the prevention of ridge deformities following tooth extraction (Chiapasco et al. 2006). A variety of biocompatible materials, both non-absorbable and absorbable, have been used as membranes in GBR to inhibit the apical migration of the epithelium and penetration of the gingival connective tissue from the flap and thus allow regeneration of
the alveolar bone (Fiorellini & Nevins 2003). The use of non-resorbable barriers is well established; however, bioabsorbable collagen membranes may simplify the surgical technique and make it more predictable (Llambes et al. 2007). An ideal barrier should be made of a material less susceptible to membrane exposure or that cannot be significantly colonized by periodontopathogenic bacteria when exposed to the oral environment. Furthermore, a membrane that will lead to increases in the width and thickness of keratinized tissue can be advantageous (Schwarz et al. 2008, 2013). Recently, a completely resorbable collagen membrane has been introduced that would appear to be more resistant to bacterial collagenase even when prematurely exposed to the oral environment. However, there has been very little research into the use of resorbable membranes barriers in GBR procedures. Animal-controlled studies are necessary, and experiments to find the best bone-graft materials still continue.
(a)
(b)
(c)
(d)
(e)
(f)
(g)
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(i)
Fig. 1. (a) A sample of the bone substitute material (4Bone granules). (b) 4Bone is available as granules packaged in syringes. (c) Incorporation of saline solution (0.9%) to the bone-graft material. (d) Pushing the syringe plunger backwards to incorporate all the solution. (e) Pushing the syringe plunger forward to remove excess solution. (f) After hydration with 0.9% saline solution, the graft bone particles were used to fill the defects. (g) A sample of the 4Bone RCM membrane used in the study. (h) RCM is available in a sterile package. (i) Incorporation of saline solution (0.9%) to membrane.
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
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and 60 days after surgery; both tibiae were retrieved in blocks containing the entire graft area for histomorphometric and radiological analysis.(Fig. 3). Radiographic imaging
Two X-rays, antero-posterior and lateral, were taken of each of the bone sections containing the grafts, using the Kodak RVG 6100 Digital Radiography System with X-rays taken at 32 kV, 40 mA using automatic light metering. The images were used to observe the changes in radiopacity at descriptive level in the medullar and cortical areas where the defects had been created.
(a)
Optical microscopy
Histomorphometric analysis
The sample size consisted of 120 specimens (Group A = 40, Group B = 40, and Group C = 40). Specimens were fixed with 10% neutral buffered formalin and decalcified by means of immersion in Osteomolâ Merck KbaA (Germany) containing HCl (10%) and CH2O (4%) for seventeen days, renewing the solution every 24 h. Subsequently, all samples were embedded in paraffin by the usual method, sectioned at 5 lm, and stained using hematoxylin–eosin, Masson’s trichromic and Malaquite Green. All samples were examined under light microscopy (Microphoto FXA, Nikon, Tokyo, Japan).
Histomorphometric evaluations comprised measurements of the areas of bone and xenograft particles in relation to the total measurement area. The central portion of each core was selected to avoid any potential bias; in this way, both the coronal (native host bone) and the apical portion (with a safe margin of 1.5–2 mm) were excluded from analysis. Examinations were performed under a Nikon Eclipse 80i microscope (Teknooptik AB, Huddinge, Sweden) equipped with the EasyImage 2000 system (Teknooptik AB) using 91 to 94 lenses for descriptive evaluation and for taking morphometric measurements. Histomorphometric analysis was performed with a Macintosh computer using the public domain NIH Image program (developed by the US National Institutes of Health and available on the Internet at http://rsb. info.nih.gov/nih-image/).Values for the total percentage of newly formed bone, residual graft material, and non-mineralized connective tissue were then calculated (Fig. 4).
(b)
Statistical analysis
(c)
(d)
The Brunner and Langer’s test (nonparametric repeated-measures analysis of variance) was applied to analyze the cortical defect closure (mm standard deviation) has been performed using R (Statistics Department of the University of Auckland). Mean values and standard deviations were calculated using a descriptive test for new bone, residual material, connective tissue. All histomorphometric parameters were analyzed using descriptive methods (SPSS 19.0; SPSS, Chicago, IL, USA for Windows). The significance level chosen was 5%.
Fig. 3. Macroscopic image of tibiae dissected at (a) 15 days; (b) 30 days; (c) 45 days; (d) 60 days.
Results Radiographic analysis Fifteen days
Fig. 4. Shows the method used to calculate histomorphometric values for new bone, residual material, connective tissue, and cortical closure. Measurements were made manually and data were entered on a spreadsheet.
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Group A: X-rays revealed the graft material to be a cylindrical element, a 6 9 6 mm rectangular structure of great radiopacity, which facilitated its identification within the trabecular bone structure in which it was implanted. This group showed incomplete closure of the cortical defect after 15 days. Group B: This group showed almost complete closure of the cortical defect, the graft material partially filling the interior of the medullar cavity. Group C: The control bone defects showed radio-transparent concave depres-
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Calvo-Guirado et al Enhanced bone regeneration with a novel synthetic bone substitute
and 60 days after surgery; both tibiae were retrieved in blocks containing the entire graft area for histomorphometric and radiological analysis.(Fig. 3). Radiographic imaging
Two X-rays, antero-posterior and lateral, were taken of each of the bone sections containing the grafts, using the Kodak RVG 6100 Digital Radiography System with X-rays taken at 32 kV, 40 mA using automatic light metering. The images were used to observe the changes in radiopacity at descriptive level in the medullar and cortical areas where the defects had been created.
(a)
Optical microscopy
Histomorphometric analysis
The sample size consisted of 120 specimens (Group A = 40, Group B = 40, and Group C = 40). Specimens were fixed with 10% neutral buffered formalin and decalcified by means of immersion in Osteomolâ Merck KbaA (Germany) containing HCl (10%) and CH2O (4%) for seventeen days, renewing the solution every 24 h. Subsequently, all samples were embedded in paraffin by the usual method, sectioned at 5 lm, and stained using hematoxylin–eosin, Masson’s trichromic and Malaquite Green. All samples were examined under light microscopy (Microphoto FXA, Nikon, Tokyo, Japan).
Histomorphometric evaluations comprised measurements of the areas of bone and xenograft particles in relation to the total measurement area. The central portion of each core was selected to avoid any potential bias; in this way, both the coronal (native host bone) and the apical portion (with a safe margin of 1.5–2 mm) were excluded from analysis. Examinations were performed under a Nikon Eclipse 80i microscope (Teknooptik AB, Huddinge, Sweden) equipped with the EasyImage 2000 system (Teknooptik AB) using 91 to 94 lenses for descriptive evaluation and for taking morphometric measurements. Histomorphometric analysis was performed with a Macintosh computer using the public domain NIH Image program (developed by the US National Institutes of Health and available on the Internet at http://rsb. info.nih.gov/nih-image/).Values for the total percentage of newly formed bone, residual graft material, and non-mineralized connective tissue were then calculated (Fig. 4).
(b)
Statistical analysis
(c)
(d)
The Brunner and Langer’s test (nonparametric repeated-measures analysis of variance) was applied to analyze the cortical defect closure (mm standard deviation) has been performed using R (Statistics Department of the University of Auckland). Mean values and standard deviations were calculated using a descriptive test for new bone, residual material, connective tissue. All histomorphometric parameters were analyzed using descriptive methods (SPSS 19.0; SPSS, Chicago, IL, USA for Windows). The significance level chosen was 5%.
Fig. 3. Macroscopic image of tibiae dissected at (a) 15 days; (b) 30 days; (c) 45 days; (d) 60 days.
Results Radiographic analysis Fifteen days
Fig. 4. Shows the method used to calculate histomorphometric values for new bone, residual material, connective tissue, and cortical closure. Measurements were made manually and data were entered on a spreadsheet.
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Group A: X-rays revealed the graft material to be a cylindrical element, a 6 9 6 mm rectangular structure of great radiopacity, which facilitated its identification within the trabecular bone structure in which it was implanted. This group showed incomplete closure of the cortical defect after 15 days. Group B: This group showed almost complete closure of the cortical defect, the graft material partially filling the interior of the medullar cavity. Group C: The control bone defects showed radio-transparent concave depres-
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sions of round or rectangular morphology depending on the image studied. They had clear and regular outlines showing a homogenous density that clearly defined their boundaries (Fig. 5a).
seen radiologically, implying the formation of bone around the graft material. Group C: Some differences were observed when compared with the previous evaluation period; X-ray images revealed linear elements representing irregular trabecular lines that did not follow the axes or load forces of adjacent bone trabeculation (Fig. 5b).
Thirty days
Group A: The radiological radiopacity of this material was lower than that observed for the previous time-period, and the cylindrical form of the grafted material had given way to a more oval and irregular shape. The closure of the cortical defect was still incomplete. Group B: X-rays revealed the cortical as being completely repaired in the filled bone defects, albeit with less density than that of the adjacent cortical bone. Reinforced medullar bone density could be
(a)
Forty-five days
Group A: In the cortical area, the graft area displayed less radiological radiopacity as well as a more oval shape. Irregular borders could be distinguished radiologically. In some areas, there was continuity between the osseous cortex and the implanted material as manifested by the linear images of osseous trabeculae.
(b)
(c)
Group B: X-rays revealed the external cortex of the artificial osseous lagoons into which the bone–granulate implant had been introduced; this had a radiopacity similar to that of the adjacent cortex. An increase in medullar radiopacity occurred corresponding to the appearance of trabecular bone. Group C: X-ray images of the control sites showed similar characteristics to the previous study period (Fig. 5c).
Sixty days
Group A: The graft material had a speckled appearance that showed slightly less radiopacity in the medullar zone. Almost complete repair of the osseous defect was also observed, but radiological images showed those trabeculae that reached the implant as being greater in number and radiopacity than for the previous time-period, giving the grafted area a slightly reticular appearance. Group B: It was impossible to distinguish the cortical defect area. No osseous malformations or structural changes to cortical bone development were observed over the study period. Group C: The samples showed a progressive reduction in the size of the cortical defect but in no case was complete closure achieved. The medullar area’s radiographic appearance did not undergo any change (Fig. 5d).
(d)
Histological and histomorphometric analysis The results for cortical defect closure measurement are shown in Table 1. Tables 2–4 show mean values for the total percentages of newly formed bone, residual graft material, and non-mineralized connective tissue at each evaluation period. Fig. 5. Antero-posterior and lateral X-rays of the section of bone containing the implants at: (a) 15 days; (b) 30 days; (c) 45 days; (d) 60 days.
Table 1. Bruner Langer (nonparametric method for dependent or longitudinal data). Statistical results from histomorphometric evaluation of cortical defect closure values for each evaluation period. Median value in (mm standard deviation [SD]) Group A (N = 10) Days
Median value (mm SD)
15 30 45 60
19.87 28.24 59.89 85.46
4.3 2.7 3.4 1.5
Group B (N = 10)
Group C (N = 10)
P value
Median value (mm SD)
0.0735 0.0618 0.5542 0.0619
31.42 45.32 69.98 97.61
2.9* 4.2* 3.4* 2.3*
P value
Median value (mm SD)
0.0108 0.0242 0.0068 0.0056
10.09 15.66 26.47 54.62
2.38 1.54 1.06 0.47
P value 0.0603 0.0715 0.1321 0.5192
The level of significance was set at P < 0.05. *Differences between values achieving statistical significance. Group A: 4Bone. Group B: 4Bone plus RCM membrane. Group C: Unfilled control group.
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Calvo-Guirado et al Enhanced bone regeneration with a novel synthetic bone substitute
Table 2. Statistical results from histomorphometric evaluation of new bone values (% standard deviation [SD]) at different time-periods Group A (N = 10) Days
Mean value (% SD)
15 30 45 60
17.54 23.44 63.67 82.75
2.41 2.37 1.36 0.98
Group B (N = 10)
Group C (N = 10)
P value
Mean value (% SD)
0.0623 0.0811 0.0852 0.0627
30.42 34.51 75.47 91.16
2.29* 1.64* 1.43* 1.02*
P value
Mean value (% SD)
0.0232 0.0147 0.0166 0.0179
8.75 14.53 31.25 39.95
1.66 1.18 3.52 1.57
P value 0.0619 0.0731 0.1361 0.4598
The level of significance was set at P < 0.05. *Differences between values achieving statistical significance. Group A: 4Bone. Group B: 4Bone plus RCM membrane. Group C: Unfilled control group.
Table 3. Statistical results from histomorphometric evaluation of connective tissue values (% standard deviation [SD]) at different time-periods Group A (N = 10) Days
Mean value (% SD)
15 30 45 60
33.55 42.01 23.07 10.83
2.76 1.53 0.52 1.27
Group B (N = 10)
Group C (N = 10)
P value
Mean value (% SD)
0.1954 0.1667 0.2342 0.5631
19.75 29.48 9.31 0.52
2.75 0.63 1.58 0.61
P value
Mean value (% SD)
0.1742 0.2831 0.2156 0.2324
91.25 85.47 68.75 60.05
0.37* 1.43* 1.29* 2.32*
P value 0.0021 0.0134 0.0069 0.0056
The level of significance was set at P < 0.05. *Differences between values achieving statistical significance. Group A: (4Bone). Group B: (4Bone plus RCM membrane). Group C: (Unfilled control group).
Table 4. Statistical results from histomorphometric evaluation of remain material values (% standard deviation [SD]) at different time-periods Group A (N = 10) Days
Mean value (% SD)
15 30 45 60
48.91 34.55 13.26 6.42
1.06 0.19 0.52 0.98
Group B (N = 10)
Group C (N = 10)
P value
Mean value (% SD)
0.0785 0.0652 0.0793 0.0128
49.83 36.01 15.22 8.32
0.21* 0.53* 2.79* 0.26*
P value
Mean value (% SD)
P value
0.0115 0.0264 0.0127 0.2008
0 0 0 0
0.6448 0.5068 0.8123 0.7062
The level of significance was set at P < 0.05. *Differences between values achieving statistical significance. Group A: 4Bone. Group B: 4Bone plus RCM membrane. Group C: Unfilled control group.
Fifteen days
Group A: The grafted defects showed numerous particles of 4Bone granules surrounded by highly vascularized granulation tissue in the cortical defect; defects had undergone incomplete closure. Group B: The membrane was fully intact and maintained the defect space. It appeared intensely stained due to a network of thick fiber bundles apparently consisting of collagen type III. The inner part of the membrane was in direct contact with newly formed bone and the marrow space (Fig 6a). Significant new bone formation was observed on the surface of some bone particles. Specimens exhibited histological patterns of biomaterial granules
with osteogenic activity predominant on the bone walls. The greatest concentrations of immature bone in the outer areas were seen in both the grafted cortical and medullar zones; in this area, small spaces were observed between graft material granules and between these and the cortex. Group C: A slight reduction in the cortical defect was observed; the medullar area was filled by a significant amount of connective tissue and blood vessels without any bone.
Thirty days
Group A: Defects showed immature bone in the cortical zone and in the medullar zone multinucleated giant cells were
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
identified at regular intervals on the surfaces of the graft material, together with signs that an active resorption process was underway. Inside the graft particles, pores of different sizes showed new bone formation coating their inner walls. Group B: The membrane had reduced in thickness, with a slide collapse in the center; capillaries frequently appeared in the interfibrillar meshes. The dark-stained fiber bundles had disappeared while strands of fibers – probably collagen type I – persisted (Fig 6b). The cortical defect could be distinguished as a thinner cortical zone. Spaces between graft material and cortex were observed. Residual graft
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Discussion
(a)
(b)
(c)
(d)
Fig. 6. Histological findings for the membrane in each healing period. (a) Microscope detail of Group B (T.M. 9 250) at 15 days (b) Microscope detail of Group B (T.M. 9 125) at 30 days (c) Microscope detail of Group B (T.M. 9 125) at 45 days (d) Microscope detail of Group B (T.M. 9 250) at 60 days.
material had reduced during this period, and connective tissue was also present, albeit in small quantities, and some new bone formation was observed (Fig 7a). Group C: Progressive, although partial, defect closure was observed, but there was no bone formation in the medullar zone; a fibrous tissue had developed filling the medullar cavity and the cortical defect. The cortical defect was slightly smaller.
Forty-five days
Group A: These spaces had undergone some reduction. Group B: The membrane remained intact, but there was evidence of degradation of the membrane material resulting in the formation of separate elements. The membrane appeared to disintegrate from both surfaces toward the center. (Fig 6c). The defect was no longer visible in the cortical zone; the cortical had further increased in width, and the diameter of the interconnecting trabeculae had also increased. The bone formed in the defect space appeared as a rather loose scaffold of woven bone, with primary bone marrow filling the intertrabecular space whilst in the medullar area, the spaces between granules had decreased and there was a more diffuse
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distribution of residual biomaterial and an increase in osseous remodeling (Fig 7b). Group C: The cortical defect was still visible, sealed by fibrous tissue.
Sixty days
Group A: Cortical bone was slightly thicker in the graft area. The medullar zone showed graft particles that were embedded in newly formed bone, which occasionally bridged the particles with branches of woven bone. Multinucleated giant cells were still present on the particle surfaces (Fig. 8a). Group B: Resorption of the material had taken place, which altered the internal structure, staining properties, shape, and integrity of the membrane (Fig 6d). Significant complete bone reparation of the cortex was observed, manifested as well-organized trabecular bone with a presence of mature bone formation that was not differentiable from the adjacent cortex. The medullar zone also showed mature bone formation in continuity with the cortex (Figs 7c and 8b). Group C: A reduction in cortical defect size was observed, and there was soft tissue closure by dense connective tissue with abundant blood vessels but without any bone formation in the medullar zone (Fig. 8c).
The present study evaluated the clinical outcome of regeneration procedures in critical defects in rabbit tibiae with the use of particulate 4Bone with or without the addition of a resorbable collagen membrane RCM. The outcomes were different for the three study groups, pointing to a positive histological effect resulting from use of the membrane in combination with the bone-graft material. Ideally, a bone substitute biomaterial is eliminated slowly after implantation in the patient, but at a rate of biodegradation, that allows the mechanical strength of the graft to be maintained during healing, balancing the resorption rate of the biomaterial with the patient’s ability to form new bone (Kanczler & Oreffo 2008). Among the formulations reported in the literature, particulate biphasic calcium phosphate biomaterials are especially attractive as they have an increased surface area, are osteoconductive, and can fill any defect size or shape (Ara ujo et al. 2010a,b). The implantation of these graft ceramic particles presented significantly more bone formation compared with the control samples in our study. The present rabbit study supports other observations of this graft’s high osteoconductivity and resorbability (Pekkan et al. 2012). The growth of bone inside biomaterial particles could be related not only to pore sizes but also to the biomaterial’s intrinsic characteristics. This graft material’s porosity is designed to accommodate the required biological exchanges particularly for bone ingrowth and mineralization. Indeed, the graft’s high porosity facilitated the resorption process as the pores’ external and internal surface areas were exposed to the medium. Previous studies of biphasic materials with the same HA/ß-TCP ratio as the material used in the present study have noted that porosity and pore interconnectivity have an effect on the material’s rate of biodegradation (Jones et al. 2009); this also influences new bone formation, vascularization, and graft stability (Calvo-Guirado et al. 2012, 2013). In the present study, the resorbed graft particles were gradually substituted by newly formed bone during bone remodeling processes in a similar way to that observed by Schopper et al. 2005. The resorption of these graft particles may have been helped by the small size of the HA crystallites and by the enzymes secreted by multinucleated giant cells, which were observed in close proximity to the granules. The texture of this graft
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Calvo-Guirado et al Enhanced bone regeneration with a novel synthetic bone substitute
(a)
(b)
(c) Fig. 7. (a) Microscope detail of Group B (T.M. 9 250) at 30 days. Monolayers of osteoblasts were seen actively secreting osteoid that bridged the gaps between grafted particles and the surrounding newly formed woven bone. (b) Microscope detail of Group B (T.M. 9 250) at 45 days. Grafted bone presents an irregular surface due to osteoclastic activity with greater quantities of immature connective tissue. Higher magnification revealed newly formed bone surrounding and replacing portions of the synthetic graft particles. (c) Microscope detail of Group B (T.M. 9 250) at 60 days. In this magnified view, new bone is seen forming along pore surfaces with the mineralized graft. Bone formation is in close contact with granules accompanying the particles’ resorptive process. Remodeling of trabecular bone can be seen at this period.
serves as an osteoconductive scaffold for osteoblastic cells and stimulates the deposition of bone matrix. The particles osseointegrate, and the resorbed biomaterial is slowly
replaced by newly formed bone. The presence of canals, resorptive trails, and vessels within these graft particles was observed in all biopsies at 60 days. In the majority of cases with
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
the presence of Haversian and Volkmann’s canals, graft particles were recolonized by vessels through preexisting canals. Bone regeneration is a coordinated process involving the connection between blood vessels and bone cells. Resorption of 4Bone material has also been observed in bone cores from humans, with evidence of diverse active multinucleated cells on the biomaterial (Kolerman et al. 2012). In this study, the biomaterials were seen to be biocompatible, osteoconductive, and reabsorbable. However, the diverse reagent chemistry described for the preparation of biphasic calcium phosphates, allied with the various implantation models, has resulted in great variability among other reported in vivo outcomes. (Ooms et al. 2003). Bone regeneration may be adversely affected by a lack of primary wound closure during the healing period. The application of physical barriers for regenerating bone defects aims to allow sufficient growth of bone tissue to permit implant placement in sites where bone volume would otherwise be insufficient. (Ezirganlı et al. 2013). Collagen membranes used as barriers in GBR have often been found to produce significant amounts of new attachment. In all Group B specimens with 4Bone and RCM membrane, a significant decrease in the osseous defect was achieved. This outcome is in agreement with data reported by other authors (Oh et al. 2003). In the present study, this appeared to have certain benefits in that Group B specimens generated the highest percentages of trabecular bone volume and promoted bone growth without soft tissue interference in comparison with the other groups. Similarly, other authors have reported advantages in using membranes combined with mineralized cancellous grafts, such as a more favorable healing response and the prevention of soft tissue invasion (Carpio et al. 2000; Chiapasco et al. 2006; Jovanovic et al. 2007). Yet, their positive benefits should be carefully balanced against the significant risk of infection, added time and cost, and the potential need for return visits from patients, who may develop complications (Rossa et al. 2006). Simion et al. (2007), in a study with similar results to the present one, found that the barrier membrane placed over the area to be augmented creates a protected space, which stabilizes blood clots and excludes soft tissue penetration. In this way, the protected space can be populated by slow-migrating osteogenic cells resulting in new bone formation. Nevertheless, it is not yet under-
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(a)
(b)
(c)
Fig. 8. (a) Panoramic image of Group A (H. E. at 923) at 60 days: the perforation in the cortical bone is almost filled by neoformed or immature osseous tissue in the outer areas, with the rest of the residual biomaterial in the cortical area in a phase of resorption and substitution. The ingrowth tissue is mostly fibrous tissue with minimal bone. (b) Panoramic image of Group B(H. E. at 923) at 60 days: complete bone reparation of the cortex of the implant orifice, manifested as well-organized trabecular bone with a presence of mature osseous bone that could not be differentiated from the adjacent cortex. The image also shows trabecular bone formation in the vicinity and in continuity with the cortex, as well as the junction between the newly formed cortical bone and adjacent cortex. (c) Panoramic image of Group C (H.E. at 923) at 60 days: the perforation made in the cortical bone was incompletely filled. Newly formed trabecular bone with large marrow spaces was observed mostly in the peripheral areas of the defects.
stood exactly how factors such as the membrane’s constituents, morphology, adherence capacity, protein-binding capacity, the substances released during degradation, surface texture, size of perforations and the duration of the barrier’s functional life might influence GBR outcomes (Bornstein et al. 2005, 2007). The structural integrity of implanted bioabsorbable barrier membranes needs to be preserved over a sufficient time to allow maturation of newly formed bone within the membrane-protected space (Machtei 2001; Nemcovsky & Artzi 2002). Collagen membrane degradation can be altered by increasing its structural integrity. The recently introduced collagen barrier, RCM, is apparently more resistant to animal and bacterial collagenase even when prematurely exposed to the oral environment. All the characteristics of RCM assayed, including its biocompatibility and biodegradability, have been verified. The barrier consists of a cross-linked collagen membrane, which enables a longer degradation time. The present study found that it remained intact at the GBR site, allowing bone regeneration to occur successfully. In this way, the results clearly demonstrate that a complete selective formation of bone within a defined bone defect can be accomplished by preventing cells not derived from the surrounding bone from repopulating the defect area. The regeneration had already occurred after 60 days, while in the non-membrane-treated group, a higher degree of osteoclastic activity and less osteogenic activity were observed. These results are in agreement with earlier findings in which complete bone regeneration of large perforating bone defects in rat mandibles was
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obtained using a similar surgical technique (Nolff et al. 2010). Natural non-cross-linked collagen membranes are sufficient to enable effective and predictable bone regeneration (Becker et al. 2009; Bornstein et al. 2009). The positive findings obtained in the present work must be analyzed carefully; with cross-linked membranes, vascularization is less and/or much slower, and tissue integration is diminished (Rothamel et al. 2004, 2005; Schwarz et al. 2013). Furthermore, Rothamel et al. (2005) observed a foreign body reaction with different crosslinked membranes in a rat model. However, the use of cross-linked membranes in the present study produced better defect closure and bone formation in comparison with the other groups. The cross-linking of formaldehyde and porcine type I and III collagen in the membrane used in the present study was a possible reason for its increased biocompatibility. The present findings suggest that collagen membranes have a direct effect on bone formation, although the ideal time-period that the membrane should retain its barrier function to maximize the healing results has still not been determined precisely. In guided tissue regeneration processes, a membrane integrity duration of only 4– 6 weeks has been advocated for periodontal regeneration in contrast to a prolonged period of at least 6 months recommended for GBR procedures. It is therefore likely that the duration of the intact membrane is a key issue for the formation and maturation of new bone in protected membrane defects (Park et al. 2008; Fotek et al. 2009). The present results showed that at the end of the study time, all of the cortical defects
at Group B test sites were filled with newly formed bone of a uniform thickness. The membrane still existed, but there was evidence of degradation of the membrane material. The bone grew predictably all the way up to the reference point at Group A test sites (but with large variations), and only half way at control sites. As in other studies, significantly more bone formation was observed around defects protected by the barrier membrane compared with control defects (Cordaro et al. 2002). In this way, the results of the present study clearly demonstrate that complete selective bone formation within a defined bone defect can be accomplished by preventing cells not derived from the surrounding bone from repopulating the defect area. (Walsh et al. 2008). New bone formation was also seen in Group A non-membrane-treated areas but to a much lesser extent than in Group B membrane-treated areas with significant difference. While at Group B sites, only boneforming cells were allowed to migrate into the wound, at Group A sites, granulation tissue derived from the soft tissue cover flap could also invade the wound area, thereby competing with the bone-forming cells. The amount of newly formed bone in this situation may be dependent on the differences in the proliferation rate of the different types of cells and the size of the available space. This may explain the pronounced variation in new bone formation observed at Group A sites as well as the finding that only ten of the 40 Group A sites showed a bone coverage reasonably close to that obtained at Group B membrane sites, which presented practically complete bone coverage. Histological examination of the material showed that the newly
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Calvo-Guirado et al Enhanced bone regeneration with a novel synthetic bone substitute
formed bone was stained darker than the original tibial bone, presumably representing a more active phase of osteogenesis; this has also been observed in others studies (Chazono et al. 2004). The present study found that GBR provided a biologic principle for the development of reconstructive surgical techniques aimed at generating bone tissue of sufficient volume for the placement of titanium implants. Although this was an animal study, it may be surmised
that the technique is also applicable to humans in a clinical setting.
Conclusions The test materials used for GBR fulfilled expectations successfully. 4Bone granules functioned well as a scaffolding material, allowing mineralized tissue formation. The RCM absorbable membranes used in associa-
tion with the new synthetic graft material not only functioned well as a barrier membrane for GBR, but also showed a potential to increase tissue generation as the addition of the absorbable membranes enhanced bone gain compared with non-membrane-treated sites. Within the limits of this study, it is concluded that this technique achieved predictable clinical outcomes.
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