British Journal of Oral and Maxillofacial Surgery
Volume 49, Issue 1 , Pages 37-41, January 2011

Osteoconduction of different sizes of anorganic bone particles in a model of guided bone regeneration

  • Xinwen Zhou

      Affiliations

    • Department of Prothodontics, School of Stomatology, Capital Medical University, Beijing, China
    • ,
  • Zhenting Zhang

      Affiliations

    • Department of Prothodontics, School of Stomatology, Capital Medical University, Beijing, China
    • Corresponding Author InformationCorresponding authors. School of Stomatology, Capital Medical University, No. 4 of Tiantan xili, Chongwen District, Beijing 100050, China. Tel.: +86 10 67099225; fax: +86 10 67099310.
    • ,
  • Song Li

      Affiliations

    • Department of Orthodontics, School of Stomatology, Capital Medical University, Beijing, China
    • ,
  • Yuxing Bai

      Affiliations

    • Department of Orthodontics, School of Stomatology, Capital Medical University, Beijing, China
    • ,
  • Hui Xu

      Affiliations

    • Department of Orthodontics, School of Stomatology, Capital Medical University, Beijing, China
    • Corresponding Author InformationCorresponding authors. School of Stomatology, Capital Medical University, No. 4 of Tiantan xili, Chongwen District, Beijing 100050, China. Tel.: +86 10 67099225; fax: +86 10 67099310.

Accepted 1 January 2010. published online 27 January 2010.

Article Outline

Abstract 

The aim of this study was to evaluate the effect of two different sizes of anorganic bone particles (300–500 and 850–1000μm) on the formation of new bone in a model of guided bone regeneration. In both groups, newly formed bone was seen histologically adjacent to the original surface of the skull, and there were outgrowths to the centre of the secluded graft 4 weeks after implantation. Some particles near the surface were in contact with the newly formed bone, and osteoconductive bone growth was present along their surface. Ten weeks after implantation the area created by grafting with small particles seemed to have a denser structure than that created with large particles. Histomorphometric analysis showed a higher density of newly formed bone in the small-particle group than in the large-particle group both 4 and 10 weeks after implantation. The total contact length between newly formed bone and particles and the ratio of bone:space between the particles were also significantly higher in the small-particle group at both time points. We conclude that the size of grafted particles of bone and the spaces between particles are important determinants of osteogenesis during guided bone regeneration.

Keywords: Guided bone regeneration, Anorganic bone, Particle, Rabbit

 

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Introduction 

In recent years, a tissue regenerative technique, guided bone regeneration (GBR), has been developed by which it is possible to regenerate resorbed alveolar bone and also close small bony defects. These procedures require the use of grafts to maintain the augmented space and to promote osteogenesis.1, 2, 3, 4 Many biomaterials have been developed as substitutes for bone, including ceramics, polymers, metals, and organic or non-organic bone substitutes. Among them, anorganic bone has been shown to be an excellent material for bone grafts as it is biocompatible and osteoconductive, and causes no immune reaction. It has facilitated the growth of new bone in numerous experimental studies, and has also been used successfully in humans for the regeneration of defects, augmentation of ridges, raising of the sinus floor, and repair of periodontal defects.5, 6

The size of particles in the bone graft is an important indicator of osteogenic activity.7, 8 However, the effect of anorganic bone particles of different sizes in GBR has not been examined in detail, so we have evaluated the osteoconductive potential of different sizes of anorganic bone particles in GBR histologically and histomorphometrically.

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Material and methods 

Preparation of bone grafts 

The anorganic bone was prepared as described by Matsuda et al.9 The calcium:phosphorus ratio was 2:34, which is close to that of human bone, and similar to that of commercial anorganic bovine bone mineral.10 The anorganic bone was pulverised in a bone mill (TOM, Leibinger, Freiburg, Germany) and sorted into particles of 300–500μm (the small-particle group) and 850–1000μm (the large-particle group) using a standard sieve (Tokyo Screen, Tokyo, Japan).

Surgical techniques 

The study was approved by the Institutional Animal Care and Use Committee of Capital Medical University. Twelve adult male Japanese white rabbits (mean weight 3.0kg) were used, 6 rabbits being studied in each group. The rabbits were anaesthetised with pentobarbitone sodium (0.5mg/kg) given intravenously, and 0.5ml of 1% lignocaine with adrenaline (1:100,000) was injected subcutaneously in each surgical field as local anaesthetic. The frontal bone was exposed via a midsagittal incision through the skin and periosteum. A skin and periosteal flap was raised to expose the skull on both sides of the midline. Under generous irrigation with saline, 9 drill holes were made into the experimental space using a round bur to facilitate bleeding. Two titanium hemispherical domes 5mm high, 0.5mm thick, and 10mm in diameter were placed over the experimental area in each animal and anchored to the bone surface with 2 miniscrews (Ti-SIS pins, SIS-System Trade, Klagenfurt, Austria). Before placement the dome was filled with either small or large particles. The periosteum was then sutured over the borders of the rim with non-resorbable suture material.

Preparation of tissue 

The rabbits were killed 4 and 10 weeks after operation. The experimental sites were resected, fixed in 4% paraformaldehyde, and demineralised in 10% EDTA at 4°C. The specimens were then dehydrated in a graded series of ethanol, embedded in paraffin, and sliced into sections about 3μm thick. The sections were stained with haematoxylin and eosin and examined under light microscopy.

Histomorphometric analysis 

We examined 3 sections at least 30μm apart from each specimen. For each section, four fields were chosen randomly and evaluated under a light microscope at 10× magnification and a semiautomatic computer-assisted digitiser (Cadkey System Corp., Tokyo, Japan). Areas and distances were marked by hand with a mouse and calculated by the computer. The following histomorphometric measurements were made: area of bone (percentage of newly formed bone to total measured area); total length of surface of particles; total length of contact between newly formed bone and particles; degree of contact between bone and particles (percentage of length of contact between newly formed bone and particles to total length of surface of particles); area of space between particles (percentage of space between particles to total measured area); and ratio of bone:space between particles (percentage of area of newly formed bone to area of space between particles). All measurements were made by the same author. To test the reliability of measurement, 9 sections were also measured by a second examiner. Repeated measurements made on the same images resulted in non-significant differences between the measurements.

Statistical analysis 

The significance of differences between groups and implantation times was assessed by two-way analysis of variance (ANOVA) using Tukey's method with the assistance of the Statistical Package for the Social Sciences, version 14.0 (SPSS Inc., Chicago). Probabilities of less than 0.05 were accepted as significant. All data are expressed as mean (SD).

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Results 

Histological findings 

4 weeks after implantation 

In both groups, new bone had formed beneath the domes, the rims of which seemed to have adapted well to the surface of the skull, preventing growth of the surrounding connective tissue into the membrane-secluded space. Newly formed bone was present adjacent to the original skull surface and showed outgrowths to the centre of the secluded graft space, in the top part of which the particles were surrounded by fibrous connective tissue. There were no signs of inflammation (Fig. 1A and B).

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  • Fig. 1. 

    Histological findings 4 weeks after implantation. Newly formed bone is present adjacent to the original skull. Some anorganic bone particles near the skull are in contact with the bone tissue in both groups (SB: the original skull bone; NB: newly formed bone; P: particles; F: fibrous connective tissue). (A) Small-particle group and (B) large-particle group (haematoxylin and eosin, original magnification ×10).

10 weeks after implantation 

In both groups, the secluded graft space was occupied by newly formed bone, connective tissue, and particles of anorganic bone. The created area grafted with the small particles seemed to contain more bone than the area grafted with large particles. In the small-particle group, newly formed bone showed many interconnections, in most parts of the spaces (Fig. 2A). In the large-particle group, the newly formed bone had limited intercommunications. Some central areas contained fibrous tissue with no evidence of ossification. There were no signs of inflammation (Fig. 2B).

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  • Fig. 2. 

    Histological findings 10 weeks after implantation. (A) In the small-particle group, newly formed bone shows many interconnections and is present in most parts of the spaces. (B) In the large-particle group, the newly formed bone shows limited intercommunications and some central areas of the secluded graft space contain fibrous connective tissue without ossification (NB: newly formed bone; P: particles; F: fibrous connective tissue) (haematoxylin and eosin, original magnification ×10).

Histomorphometrical analysis 

Area of bone 

In both groups the area of bone increased with time. There were significant differences between 4 and 10 weeks after implantation (p<0.001). There was significantly more bone area in the small-particle group than in the large-particle group both 4 and 10 weeks after implantation (p<0.01) (Table 1, Fig. 3).

Table 1. Mean (SD) histomorphometric measurements after grafting with the different sizes of deproteinised bone particles.
Group (particle size μm)Time (weeks)Bone area (%)Total length of surface of particles (μm)Total length of contact between bone and particles (μm)Degree of contact between bone and particles (%)Space between particles (%)Ratio of bone:space between particles (%)
300–500415.9 (2.6)1491.1 (205.2)566.8 (85.8)39.2 (6.7)50.3 (2.8)34.6 (2.7)
1026.3 (3.4)1576.9 (157.6)864.1 (105.4)56.8 (7.9)57.5 (4.3)46.7 (4.1)

850–1000410.5 (2.1)678.9 (98.3)295.6 (34.5)43.2 (5.3)41.7 (3.5)25.1 (3.0)
1016.7 (1.5)630.2 (65.7)423.3 (76.6)61.7 (8.1)46.2 (2.8)36.1 (2.2)
Total length of surface of particles 

The total length of the surface of small particles was significantly more than that of the large particles at 4 and 10 weeks after implantation (p<0.001) (Table 1).

Total length of contact between newly formed bone and particles 

In both groups, the total length of contact between newly formed bone and particles increased with time. There were significant differences in both the small-particle (p<0.01) and the large-particle (p<0.05) groups. The length of contact between bone and particles in the small-particle group was significantly greater than that in the large-particle group at 4 and 10 weeks after implantation (p<0.001) (Table 1).

Degree of contact between bone and particles 

In both groups, the degree of contact between bone and particle increased with time, and there were significant differences between 4 and 10 weeks after implantation (p<0.05). The degree of contact did not differ significantly between the groups at any time (Table 1).

Area of space between particles 

The area was significantly greater in the small-particle group both 4 and 10 weeks after implantation (p<0.01) (Table 1).

Ratio of bone:space between particles 

In both groups, the ratio of bone:space between particles significantly increased with time (p<0.05). The ratio of bone:space was significantly higher in the small-particle group both 4 and 10 weeks after implantation (p<0.01) (Table 1).

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Discussion 

Guided bone regeneration is widely used to augment the width of the alveolar ridge before implants are inserted. The success or failure of treatment with osseointegrated implants in edentulous patients depends on the volume and quality of newly formed bone in the augmented space, as adequate primary anchorage is often difficult in low density bone.11, 12

We have presented a model that replicates a clinical one-wall defect. Corresponding preclinical models with hemispheres that provide space for bone substitutes have been established in rats and rabbits.13, 14, 15 In our model the formation of bone is supported by opening the marrow cavity with small drills, which also permit blood to permeate into the augmented area to provide a provisional extracellular matrix for the immigration of repair cells.16

Histologically both sizes of particles induced bony growth, which indicates that the anorganic bone particles have bone-conductive properties. The particles acted as a scaffold to facilitate the formation of bone. Scaffolding is a critical component in tissue engineering because it provides the three-dimensional clues for seeding, migration, and growth of cells, and formation of new tissue.17, 18 Histomorphometric analysis showed a higher density of newly formed bone in the small-particle group than in the large-particle group. The total length of the surface of the small particles was significantly greater than that of the large particles, and the contact length between bone and particles in the small-particle group was significantly greater than that in the large-particle group, so it may be that the size of the particles plays a part in osteogenic activity. The small particles may provide a large surface area around which more bone may form.

With porous graft materials, the size of the pores and the porosity have critical roles in the formation of bone.19, 20 It was claimed that a minimum pore size of 100μm was necessary for bone to grow into the porous materials.21 The size of pore is comparable to the size of the spaces between particles, the spaces between the small particles were significantly larger than those between the large particles. The possible reasons are that the anorganic bone particles were irregularly shaped and were lightly packed into the secluded graft space without compression during operation. The differences in osteogenicity between the two groups may be linked to the specific packing and the macroporosity that was created. The small particles will have created a different macroporosity in the tissue-engineered construct, and this could be favourable for the formation of bone.

This requires a rich blood supply,21 and the larger interparticular spaces may allow the ingrowth of capillaries, as such ingrowth precedes the formation of new bone. The small-particle group showed evidence of more and denser amounts of new bone than the large-particle group. Histomorphometric analysis showed that the ratio of bone:space of the small particles was significantly greater than that of the large particles after implantation. We therefore suggest that the space between the particles is an important factor for osteoconduction. The small particles might provide a better scaffold on which tissue may infiltrate and regenerate than the large particles.

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Acknowledgements 

The study was supported by Beijing scientific and technological new star program (2006B62) and the Scientific Research Foundation for the Returned Overseas Chinese Scholars, Ministry of Education of China.

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PII: S0266-4356(10)00003-3

doi:10.1016/j.bjoms.2010.01.001

British Journal of Oral and Maxillofacial Surgery
Volume 49, Issue 1 , Pages 37-41, January 2011

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