Abstract
This in vivo study investigated the in vivo performance of two newly developed synthetic bone substitutes and compared them to commercially available xenografts (Bio-Oss, Geistlich Pharma AG, Switzerland; OsteoGraf, Dentsply, USA). The materials were tested in a rabbit calvaria model, and the bone forming properties were observed at 4 and 8 weeks after implantation by means of histomorphometry and micro computed tomography (micro-CT). Defects without any graft material were used as negative controls. Micro-CT showed that all materials tested presented new bone formation that filled the defects at both time points, whereas the negative control presented less bone formation, with soft tissue infiltration into the defects. Comparable bone fill percentages were observed for histomorphometric and micro-CT results. Even though no statistically significant difference was found quantitatively between all of the bone graft substitute groups, a higher mean decrease in graft material filling the defects, along with higher remodelling activity, was evident for the experimental materials compared to the commercially available xenografts at 8 weeks. The results indicate that the experimental materials possess high degradability, along with osteoconduction comparable to commercially available xenografts.
Introduction
Form and function replacement of lost hard tissue through guided regeneration with either synthetic materials or engineered tissues is of interest to both healthcare practitioners and patients. The main reason for this interest is the avoidance of a second bone autograft acquisition surgical site, which involves complex procedures. Bone autografts have an inherent limited quantity, especially when taken intraorally. Bone allograft and xenograft materials are in general considered to be safe; however, the possibility of potential disease transmission from the cadaveric or animal source cannot be excluded.
Various bone graft materials have been studied on their own or in combination: autografts, xenografts, allografts, and synthetic grafts. Among synthetic materials, calcium- and phosphate-based bioactive ceramics such as hydroxyapatite (HA), β-tricalcium phosphate (β-TCP or Ca 3 (PO 4 ) 2 ), and biphasic blends have been utilized widely in dentistry and orthopedics. However, to date, no synthetic or engineered grafting material has been able to provide receptor healing sites with the biocompatible, osteoconductive, and osteoinductive properties of autologous grafts.
It is known that xenografts and allografts can cause an immune response by the host and may be slow to degrade, as seen with certain bovine bone grafts primarily used in dentistry. These materials have been used successfully in numerous studies, including guided bone regeneration (GBR), sinus augmentation, and socket augmentation. However, while successful in yielding improved bone regeneration in multiple challenging regeneration scenarios, concerns have been raised as to the remodelling/degradation of such materials and how their physicochemical process affects their bone regeneration potential and degradation properties. For instance, it has been described in literature reviews that the physical and chemical configuration of synthetic grafting materials may drastically affect their biological and degradation kinetics. These investigations have led to a plethora of material combinations that include biological factors and different biopolymers, in an attempt to minimize the biological, mechanical, and degradation kinetics/properties of autologous, xenogeneic, and allogeneic grafts.
On the other hand, human- and animal-derived bone graft materials have been investigated extensively concerning their clinical performance and biocompatibility in vivo , in both the short term and long term. However, the literature concerning these biomaterials and tissue engineering science is limited. In fact, the physicochemical characterization of these materials is seldom performed or reported in the literature before they are evaluated in cell cultures or in in vivo or clinical scenarios. Thus, determining the effect of changing these material fabrication parameters to yield different compositions and physical aspects (particle size and porosity), and their effects on in vivo behaviour, is highly desirable for the establishment of a design platform for the next generation of graft materials.
In this study, we used a newly developed bovine-derived microporous HA-based xenograft material, which possesses a unique combination of enlarged surface area and high porosity. The bone-forming properties were compared to commercially available xenografts using a bone defect model. Both the bone regeneration potential and degradation of these materials were evaluated at two time points.
Materials and methods
Four graft materials were placed in a calvarial defect until visually filled; assessments were made after 4 weeks and 8 weeks. There were two exploratory graft materials, EBR-027-500 final and EBR-027-final (Dentsply International, York, PA, USA), and two US Food and Drug Administration (FDA) approved graft materials, Bio-Oss (Geistlich Pharma AG, Switzerland) and OsteoGraf N-300 (Dentsply International, York, PA), which are currently available commercially as xenogeneic bone graft materials (positive control). Results were compared to a negative control (no graft). A total of 20 animals were used, 10 animals per time point, with four defects per animal. The grafting materials and empty defect were interpolated between sites to minimize bias from different implantation sites. The properties of the materials, as provided by the manufacturers, are given in Table 1 ; the two positive control materials have higher and lower porosity values than the experimental materials, hence the need for two positive controls.
| Sample | Sample background | Mean particle size (μm) | BET surface area (m 2 /g) | % Porosity (Hg porosity) a | Total pore area (m 2 /g) |
|---|---|---|---|---|---|
| Bio-Oss | Obtained from market place | – | – | ∼45 b | ∼80 b |
| OsteoGraf N-300 | Obtained from current Dentsply inventory | 300 ± 75 b | ∼1.0 b | 16.0 | 1.60 |
| EBR-027-final | Prototype material run at high sintering temperature | 326 | 53.1020 | 31.3 | 53.71 |
| EBR-027-500 final | Prototype material run at low sintering temperature | 333 | 55.0471 | 31.6 | 57.05 |
a Pores greater than 10 μm were eliminated to exclude the volume resulting from the filling of inter-particle voids. Since the graft material is in granular/powder form, as mercury flows in it creates a packed bed. As pressure is increased mercury will start flowing in, filling the voids first. This will register on the instrument as intrusion, resulting in an over-assessment of the level of porosity in the sample. Excluding the >10 μm pores corrects for this.
b Typical values measured for this product. Actual lot used in the study was not measured.
Surgical procedure
Surgery was performed on 20 New Zealand white rabbits following an Institutional Animal Care and Use Committee (IACUC) approved protocol. As per the standard procedure, animals were anesthetized by intramuscular administration of a combination of ketamine (35 mg/kg), xylazine (5 mg/kg), and acepromazine (0.75 mg/kg). They were monitored until unconscious and then prepared for surgery. The dorsum of the head was exposed for surgery, and two bilateral 8.0-mm diameter defects were made with a trephine immediately proximal to the coronal suture in the parietal bone, without dural involvement.
Once created, collagen membranes (OsteoShield; Dentsply, USA) were placed between the skull and dura mater in order to prevent particle migration, and the defects were filled with one of the graft materials or left empty. The periosteum was closed with a 4-0 Vicryl (Ethicon, Auneau, France) resorbable suture using simple interrupted stitches, and the skin was then closed with 4-0 nylon (Ethicon, Auneau, France) sutures. Animals were euthanized at 4 weeks or 8 weeks postsurgically by means of an anaesthesia overdose.
Determination of bone microarchitecture using micro-CT
The bone defect samples were dehydrated in a series of alcohol solutions ranging from 70% to 100% ethanol. The samples were embedded in a methacrylate-based resin (Technovit 9100; Heraeus Kulzer GmbH, Wehrheim, Germany) in accordance with the manufacturer’s instructions. After the samples were polymerized in resin, the embedded samples were first analyzed by micro computed tomography (micro-CT; μCT 40, SCANCO Medical, Bassersdorf, Switzerland) with a medium scan mode at an integration time of 200 ms. The total scan time per sample was 26 min within the defined volume of interest (VOI). Samples were placed in a 12.3-mm micro-CT specimen holder, and scans were performed using medium resolution (12 μm nominal resolution) to assess the new mineralized structure formed. Data were collected at 55 kVp and 145 μA, and reconstructed images were filtered using a constrained three-dimensional (3D) Gaussian filter to partially suppress noise in the volumes ( σ = 1.2 and support = 1). All micro-CT data were exported in DICOM format, and imported in Amira (Visage Imaging GmbH, Berlin, Germany) for evaluation. x -, y -, and z -axes were realigned in order to properly observe three-dimensionally. Before segmentation, threshold levels for bone and graft material were determined. Threshold determination was repeated for evaluation of intra- and inter-examiner repeatability, and each defect was reconstructed for the evaluation of bone formation throughout the defect. The same resin-embedded samples were thereafter used for non-decalcified histological processing.
Sectioning and staining of embedded specimens
A Buehler IsoMet saw (Buehler, Lake Bluff, IL, USA) was used to obtain histological sections, parallel to the axis of the 8-mm defect, 150 μm thick, which were polished to a 1200 grit finish. A 1-μm polishing compound was used to remove residual scratches, and their final thicknesses were between 50 and 60 μm. A Stevenel’s blue and Van Gieson’s picro fuchsin differential tissue staining protocol (SVG) was used for staining the sections. The SVG stains soft tissue green–blue and mineralized tissue red–orange, while graft material will stain an orange–brown. The stained sections were examined using a histology slide scanning system (Aperio Technologies, Vista, CA, USA) and the amount of bone, soft tissue, and graft material within the defect was quantified by image thresholding (Photoshop, Adobe, San Jose, USA). Polarized light microscopy (Leica DM4000; Leica Microsystems, Wetzlar, Germany) was utilized to observe the collagen alignment. The total amount of bone, soft tissue, and graft material was added and served as the total area of the defect. Since a slight immunogenic response on the surface of the collagen membrane, which slowed the progression of bone formation in the defect side, was noted in the preliminary histological analysis, the area with the collagen membrane was not quantified.
Statistical analysis
The statistical analysis was performed by Kruskal–Wallis test followed by Dunn’s post hoc test for multiple comparisons using computer software NCSS (NCSS LLC, East Kaysville, UT, USA). Statistical significance was set at the 95% level of significance. The parameters evaluated were bone ingrowth %, graft %, and soft tissue % (along with empty spaces) for all groups.
Materials and methods
Four graft materials were placed in a calvarial defect until visually filled; assessments were made after 4 weeks and 8 weeks. There were two exploratory graft materials, EBR-027-500 final and EBR-027-final (Dentsply International, York, PA, USA), and two US Food and Drug Administration (FDA) approved graft materials, Bio-Oss (Geistlich Pharma AG, Switzerland) and OsteoGraf N-300 (Dentsply International, York, PA), which are currently available commercially as xenogeneic bone graft materials (positive control). Results were compared to a negative control (no graft). A total of 20 animals were used, 10 animals per time point, with four defects per animal. The grafting materials and empty defect were interpolated between sites to minimize bias from different implantation sites. The properties of the materials, as provided by the manufacturers, are given in Table 1 ; the two positive control materials have higher and lower porosity values than the experimental materials, hence the need for two positive controls.
| Sample | Sample background | Mean particle size (μm) | BET surface area (m 2 /g) | % Porosity (Hg porosity) a | Total pore area (m 2 /g) |
|---|---|---|---|---|---|
| Bio-Oss | Obtained from market place | – | – | ∼45 b | ∼80 b |
| OsteoGraf N-300 | Obtained from current Dentsply inventory | 300 ± 75 b | ∼1.0 b | 16.0 | 1.60 |
| EBR-027-final | Prototype material run at high sintering temperature | 326 | 53.1020 | 31.3 | 53.71 |
| EBR-027-500 final | Prototype material run at low sintering temperature | 333 | 55.0471 | 31.6 | 57.05 |
a Pores greater than 10 μm were eliminated to exclude the volume resulting from the filling of inter-particle voids. Since the graft material is in granular/powder form, as mercury flows in it creates a packed bed. As pressure is increased mercury will start flowing in, filling the voids first. This will register on the instrument as intrusion, resulting in an over-assessment of the level of porosity in the sample. Excluding the >10 μm pores corrects for this.
b Typical values measured for this product. Actual lot used in the study was not measured.
Surgical procedure
Surgery was performed on 20 New Zealand white rabbits following an Institutional Animal Care and Use Committee (IACUC) approved protocol. As per the standard procedure, animals were anesthetized by intramuscular administration of a combination of ketamine (35 mg/kg), xylazine (5 mg/kg), and acepromazine (0.75 mg/kg). They were monitored until unconscious and then prepared for surgery. The dorsum of the head was exposed for surgery, and two bilateral 8.0-mm diameter defects were made with a trephine immediately proximal to the coronal suture in the parietal bone, without dural involvement.
Once created, collagen membranes (OsteoShield; Dentsply, USA) were placed between the skull and dura mater in order to prevent particle migration, and the defects were filled with one of the graft materials or left empty. The periosteum was closed with a 4-0 Vicryl (Ethicon, Auneau, France) resorbable suture using simple interrupted stitches, and the skin was then closed with 4-0 nylon (Ethicon, Auneau, France) sutures. Animals were euthanized at 4 weeks or 8 weeks postsurgically by means of an anaesthesia overdose.
Determination of bone microarchitecture using micro-CT
The bone defect samples were dehydrated in a series of alcohol solutions ranging from 70% to 100% ethanol. The samples were embedded in a methacrylate-based resin (Technovit 9100; Heraeus Kulzer GmbH, Wehrheim, Germany) in accordance with the manufacturer’s instructions. After the samples were polymerized in resin, the embedded samples were first analyzed by micro computed tomography (micro-CT; μCT 40, SCANCO Medical, Bassersdorf, Switzerland) with a medium scan mode at an integration time of 200 ms. The total scan time per sample was 26 min within the defined volume of interest (VOI). Samples were placed in a 12.3-mm micro-CT specimen holder, and scans were performed using medium resolution (12 μm nominal resolution) to assess the new mineralized structure formed. Data were collected at 55 kVp and 145 μA, and reconstructed images were filtered using a constrained three-dimensional (3D) Gaussian filter to partially suppress noise in the volumes ( σ = 1.2 and support = 1). All micro-CT data were exported in DICOM format, and imported in Amira (Visage Imaging GmbH, Berlin, Germany) for evaluation. x -, y -, and z -axes were realigned in order to properly observe three-dimensionally. Before segmentation, threshold levels for bone and graft material were determined. Threshold determination was repeated for evaluation of intra- and inter-examiner repeatability, and each defect was reconstructed for the evaluation of bone formation throughout the defect. The same resin-embedded samples were thereafter used for non-decalcified histological processing.
Sectioning and staining of embedded specimens
A Buehler IsoMet saw (Buehler, Lake Bluff, IL, USA) was used to obtain histological sections, parallel to the axis of the 8-mm defect, 150 μm thick, which were polished to a 1200 grit finish. A 1-μm polishing compound was used to remove residual scratches, and their final thicknesses were between 50 and 60 μm. A Stevenel’s blue and Van Gieson’s picro fuchsin differential tissue staining protocol (SVG) was used for staining the sections. The SVG stains soft tissue green–blue and mineralized tissue red–orange, while graft material will stain an orange–brown. The stained sections were examined using a histology slide scanning system (Aperio Technologies, Vista, CA, USA) and the amount of bone, soft tissue, and graft material within the defect was quantified by image thresholding (Photoshop, Adobe, San Jose, USA). Polarized light microscopy (Leica DM4000; Leica Microsystems, Wetzlar, Germany) was utilized to observe the collagen alignment. The total amount of bone, soft tissue, and graft material was added and served as the total area of the defect. Since a slight immunogenic response on the surface of the collagen membrane, which slowed the progression of bone formation in the defect side, was noted in the preliminary histological analysis, the area with the collagen membrane was not quantified.
Statistical analysis
The statistical analysis was performed by Kruskal–Wallis test followed by Dunn’s post hoc test for multiple comparisons using computer software NCSS (NCSS LLC, East Kaysville, UT, USA). Statistical significance was set at the 95% level of significance. The parameters evaluated were bone ingrowth %, graft %, and soft tissue % (along with empty spaces) for all groups.
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