Abstract

This study was conducted to establish the efficiency of microcomputerized tomography (micro-CT) in detection of trabecular bone remodeling of onlay grafts in a rodent calvaria model, and to compare bone remodeling after onlay grafts with beta-tricalcium phosphate (TCP) or coral calcium carbonate. Ten rats received calvarial onlay blocks—5 with TCP and 5 with coral calcium carbonate. The grafts were fixed with a titanium miniplate screw and were covered with a collagen resorbable membrane. Three months after surgery, the calvaria were segmented, and a serial 3-dimensional micro-CT scan of the calvarium and grafted bone block at 16-micrometer resolution was performed. Image analysis software was used to calculate the percentage of newly formed bone from the total block size. Newly formed bone was present adjacent to the calvarium and screw in all specimens. The mean area of newly formed bone of the total block size ranged from 34.67%–38.34% in the TCP blocks, and from 32.41%–34.72% in the coral blocks. In the TCP blocks, bone remodeling was found to be slightly higher than in the coral blocks. Micro-CT appears to be a precise, reproducible, specimen-nondestructive method of analysis of bone formation in onlay block grafts to rat calvaria.

Introduction

Autogenous bone grafts, especially intramembranous bones, are the current gold standard for the reconstruction of congenital and acquired craniofacial and jawbone defects.13 

However, their volume retention is unpredictable, with high rates of partial or complete resorption of the graft at the recipient site.47 This factor, in addition to the scanty amount of autogenous bone graft available for reconstruction, especially in infants, the difficulty involved in trimming the graft to the desired shape, and the high risk of complications in the donor region and postoperatively811 have prompted attention to alloplastic grafts. Besides biocompatibility, alloplast materials are required to meet some of the criteria of autogenous grafts, that is, bone ingrowth and attachment and migration and distribution of vascular and osteogenic cells within the graft (osteoconduction). To enhance these properties,12 researchers have developed novel 3-dimensional biodegradable scaffolds on which cultured stem cells are loaded before transplantation.

Microcomputerized tomography (micro-CT) was introduced as a nondestructive alternative to histology, with studies showing a high correlation with conventional histomorphometric parameters.13,14 The technique has since been adapted for analysis of the bone architecture in osteoporosis,1518 the implant osseointegration profile,1921 and the effect of parathyroid hormone on bone structure.2225 In a comparative study of early healing of intramembranous and endochondral autogenous bone grafts to rabbit mandibles, findings on micro-CT analysis were found to be compatible with those for computer-assisted morphometry.26 However, the value of the information provided by micro-CT in alloplast grafting has not yet been established.

This study aimed to establish the efficiency of micro-CT in detecting trabecular bone remodeling of onlay grafts in a rodent calvaria model. The second aim was to compare bone remodeling after onlay grafts with beta-tricalcium phosphate (TCP) or coral calcium carbonate in the same model.

Materials and Methods

The study was approved by the Committee for Ethics in Animal Experiments of Rabin Medical Center.

Surgical procedure

Ten adult Fischer inbred isogenic male rats weighing 250–300 g were anesthetized with an intraperitoneal injection of 4 mg/kg chloral hydrate. The cranial surgical site was scrubbed with 10% povidone-iodine solution, and 0.5 mL lidocaine 2% with epinephrine 1∶100 000 was infiltrated into the surgical site for analgesia and hemostasis. A longitudinal skin incision was made along the sagittal suture of the skull, and a pericranial flap was raised, exposing the frontal bone. Two groups of 5 rats each were randomly allocated to receive 1 of 2 onlay block types. The first group received a porous TCP onlay block (Chronos, Synthes, Switzerland), trimmed to an average size of 5 × 5 × 5 mm. The second group received a coral calcium carbonate block (ProOsteon 200R, Biomet Osteobiologics, Parsippany, NJ), trimmed to an average size of 7 × 7 × 5 mm. The blocks were placed over the cranial bone and were fixed with a central-drive titanium miniplate screw of 1.5 mm diameter and 7.0 mm length (Walter-Lorenz, Blomet, Jacksonville, Fla). Care was taken to not disturb the dura. A collagen-resorbable membrane (Mem-Lok, Collagen Matrix Inc, Franklin Lakes, NJ) was applied tightly over the bone block, extending onto the intact bone. The periosteum was sutured over the membrane with resorbable 4-0 Vicryl sutures, and the cutaneous flap was adjusted and sutured with 4-0 Monocryl sutures (Figure 1).

Figure 1.

Surgical method: (a) Block graft and fixed screw in place. (b) Positioning of collagen membrane over graft and screw. (c) Closure of operation site.

Figure 1.

Surgical method: (a) Block graft and fixed screw in place. (b) Positioning of collagen membrane over graft and screw. (c) Closure of operation site.

The rats were housed postoperatively in stainless steel hutches maintained at a temperature of 19°C–25°C, in ventilated air of approximately 55% humidity. Rats were fed standard rat chow with water ad libitum. An elixir of acetaminophen with codeine was added to their drinking water during the first 3 postoperative days, and antibiotics were added for the first 5 days.

Three months after surgery, the rats were killed in a carbon dioxide chamber. The heads were excised, and specimens containing the bone graft, screw, and host segment of the calvarium were sectioned with a bone microsaw (Figure 2). The specimens were maintained in 10% buffered formalin. Micro-CT–assisted analysis was used to estimate the amount of newly formed bone.

Figure 2.

Examples of specimens after killing: (a) Coral block. (b) Beta-tricalcium phosphate (TCP) block.

Figure 2.

Examples of specimens after killing: (a) Coral block. (b) Beta-tricalcium phosphate (TCP) block.

Micro-CT imaging

The calvarium specimens were scanned and analyzed with a desktop micro-CT machine (µCT 40, Scanco Medical, Brüttisellen, Switzerland) at a resolution of 16 µm, a voxel (3-dimensional pixel) size of 163 µm, and an X-ray energy level of 60 KVP. The apparatus consists of a microfocus X-ray tube with a focal spot of 10 µm, a turntable system for mounting and rotating the specimens, and a linear array charge-coupled device (CCD) detector connected to a computer. First, the specimens were placed in the specialized holding tube of the micro-CT, and a preview scan was performed to obtain a low-resolution image of the bone block to check the orientation and identify the upper and lower borders. The upper border of the scan was defined as the head of the screw, and the lower border as the calvarium.

The serial specimen slices were then evaluated with image analysis software (Scion Image for Windows, version 4.0.3.2; Scion Corp, Frederick, Md). The millimetric scale on each image exported from the micro-CT system was used to calibrate the software. The trabecular bone area of the specimen was analyzed in 3 dimensions according to the sagittal, coronal, and axial reconstruction of the calvarium and bone block.

The ideal method for interpretation and analysis of these scanned specimens would have been a digitized differentiation between the originally grafted onlay blocks and the newly formed or remodeled bone in the graft vicinity. However, in our setting, this was not possible because the radiopacity of the grafted block and of the newly formed bone were very similar, to an extent not possible for computerized detection. Therefore, in each slice analyzed, a manual outline of the block size and the newly formed bone was performed. The area defined as newly formed bone included locations in which the porous architecture of the block was substituted by a smooth mosaic bony form (for example, see Figure 3). The total block area was measured from the block interface with the calvarium to the top of the block, and the amount of newly formed bone was calculated relative to the original total block graft size. For each specimen, microtomographic slices were analyzed at constant intervals of 196 µm in 3-dimensional planes to cover the entire length of the sample. All digital analyses were performed by a single operator.

Figure 3.

Axial and coronal reconstructions of beta-tricalcium phosphate (TCP) bone block, demonstrating manual outline of newly formed bone. (Outline of total block size omitted for clarity). Long arrows: location of newly formed bone, outlined by bold black line. Short arrows: grafted TCP block with no bone replacement. Black arrow (coronal): location of calvaria.

Figure 3.

Axial and coronal reconstructions of beta-tricalcium phosphate (TCP) bone block, demonstrating manual outline of newly formed bone. (Outline of total block size omitted for clarity). Long arrows: location of newly formed bone, outlined by bold black line. Short arrows: grafted TCP block with no bone replacement. Black arrow (coronal): location of calvaria.

A nonparametric Mann-Whitney U statistical test was employed to assess the difference between results obtained from the 2 groups.

Results

In the present study, micro-CT was used to calculate the extent of bone remodeling at the calvarium-graft interface in a rodent model of alloplastic grafting. The digital reconstruction made it possible to view the scanned specimens in the sagittal, coronal, and axial planes.

Results showed that trabecular bone filled or replaced a portion of the pores in both the TCP and coral block grafts. The mean values and standard deviations of bone block area, area of newly formed bone, and mean percentage of area formed in the axial, coronal, and sagittal planes are shown in the Table. The range of mean percentage of newly formed bone was slightly higher in the TCP blocks (34.67%–38.34%) than in the coral blocks (32.41%–34.72%). Results did not show a statistically significant difference between bone formation in the 2 onlay block materials examined.

Table

Mean and standard deviation of block size, area of newly formed bone, and percentage of newly formed bone of the total block size*

Mean and standard deviation of block size, area of newly formed bone, and percentage of newly formed bone of the total block size*
Mean and standard deviation of block size, area of newly formed bone, and percentage of newly formed bone of the total block size*

Bone formation was maximal in proximity to the block-screw-cranium intersection. In the coronal and sagittal sections, the percent of bone fill ranged from high in proximity to the screw to low at the periphery. In the axial plane, higher percentages of bone fill were noted in proximity to the cranium-graft interphase (Figure 4).

Figure 4.

Selected reconstructions in 3 dimensions. (a) Beta-tricalcium phosphate (TCP). (b) Coral blocks in the region of the block-screw-cranium interphase. Note: Increased bone formation between threads of screw, circumferential to the screw and at the cranium-block interphase.

Figure 4.

Selected reconstructions in 3 dimensions. (a) Beta-tricalcium phosphate (TCP). (b) Coral blocks in the region of the block-screw-cranium interphase. Note: Increased bone formation between threads of screw, circumferential to the screw and at the cranium-block interphase.

Discussion

The present study compared trabecular bone remodeling of craniofacial alloplast TCP and coral onlay bone grafts, using micro-CT in a rodent model.

Bone tissue engineering is a promising method for reconstruction of bone defects. Bone marrow stem cells possess the ability to generate osteogenic and hematopoietic cells, but a vehicle is needed for delivery of the aspirated cells and for promotion of cell growth in a 3-dimensional structure. Therefore, the osteogenic cells with or without growth factors are seeded onto a porous biological 3-dimensional scaffold,27,28 which is placed and fixed at the graft site. Experimental studies have shown that under these conditions, osteoconduction usually proceeds from the host bone into the scaffold, with new bone emerging in a slow, controlled process of creeping substitution.

The biomaterials used for scaffolding include collagen, polymer composites such as polylactic acid, polyglycolic acid, or poly-DL-lactic-co-glycolic acid,29,30 and ceramics27,31 such as TCP and hydroxyapatite, which have chemical and structural similarity to the anorganic phase of native bone.32 However, most of the scaffolds cannot withstand the mechanical demands,32 are not stably fixed, and shift easily at the surgical site.

At present, qualitative histologic and quantitative histomorphometric analyses of bone remodeling processes are typically performed by light microscopy of serial 2-dimensional bone histologic sections.33 However, only a limited data set can be obtained from these procedures, and the structural properties for a specific location cannot be assessed repetitively.34,35 Furthermore, the destructive nature of the histologic preparation prevents the bone blocks from being used for further experiments, such as biomechanical testing.36 

The main objective of computerized tomography and micro-CT is to provide realistic, 3-dimensional images of the objects being examined, and to make their accurate measurement possible. In the past few years, micro-CT has been applied extensively to quantify the microarchitectural properties of trabecular bone.36,37 It has been used to measure small bones,13 monitor small anesthetized animals,38 characterize bone tissue,39,40 and measure new bone around implants.20 Others have used micro-CT to study the microarchitectural changes that trabecular bone undergoes during osteoporosis in animals15 and patients.16 Micro-CT also offers a sophisticated method for the study of porous structures, such as metallic foams, stone, wood, and polymers and bone biomaterials.41 It does not require specimen preparation, the specimens can be rotated and viewed from any angle, and the procedure is not destructive to the sample. Images can be produced at a resolution of tens of microns, and the results are reproducible.14,42 Micro-CT has been found to be superior to histologic sectioning, planar radiography, and medical computerized tomography.43 It has also proved to be more sensitive than X-ray absorptiometry and bone histomorphometry in detecting changes in bone mass and trabecular microarchitecture in a tibial rat model of disused osteoporosis.15 Mulder et al (2004)44 concluded that the applied micro-CT system is adequate for assessment of the degree and distribution of mineralization in developing bone.

A quantitative structural comparison of micro-CT and conventional histomorphometry yielded significant correlations between the 2 techniques for trabecular morphometry measurements.14,42,45 Micro-CT has been used to quantitatively analyze the cancellous bone architecture of the rat proximal tibia,40 the human transiliac crest bone,14 the humerus and femur from nonhuman primates,43 and the volume, projection, and microarchitecture of cranial bone.46 As yet, no studies have applied micro-CT for quantitative and qualitative analysis and for characterization of different scaffold materials. The extent of bone remodeling and the nature of new bone formation after scaffold grafting have important implications for the success of reconstruction of the craniofacial skeleton.

In the present study, we utilized 3-dimensional micro-CT to detect bone gain and trabecular architectural formation following TCP and coral calcium carbonate block onlay grafting in rat calvaria. We chose to evaluate bone remodeling in onlay block grafts, without enhancing them with growth factors so as not to mask differences between the native properties of the graft materials. The rat calvarium is an efficient model for studying reconstruction of the craniofacial skeleton because the onlay bone graft can be stabilized and placed adjacent to the host bone.

Using micro-CT evaluation after a 3-month healing period revealed no significant resorption of blocks in the vertical, transverse, or horizontal direction. New bone formation was found in both block materials used, in the 3 dimensions analyzed, and the mean newly formed bone ranged from 34.67%–38.34% of original block volume in rats treated with a TCP graft, and from 32.41%–34.72% of original block volume in rats treated with a coral graft. TCP onlay blocks showed slightly higher values of bone formation, but the differences between materials were not statistically significant. New bone produced by the host tissue could be seen adjacent to the calvarial bone. Bone density was increased in the area along the fixation screws, suggesting increased remodeling. We also noted erosion of the outer cortical host bone in the coral blocks. This finding may be due to constant pressure on the cortical bone as applied by the block and screw.

In conclusion, in the present study, utilizing micro-CT, both TCP and coral onlay block grafts to rat calvaria showed new bone formation after 3 months. Of the total size of the block, 32% to 38% was substituted with newly formed bone. A statistically significant difference between the 2 graft materials was not found. Micro-CT is a viable approach for analysis and quantification of onlay graft bone structure in 3 dimensions. It is nondestructive to the specimen and is reproductive and precise, providing reliable indices of the morphometric properties of newly formed bone.

Abbreviations

     
  • micro-CT

    microcomputerized tomography

  •  
  • TCP

    beta-tricalcium phosphate

Acknowledgment

This study was supported (in part) by grant no. 3-00000-5094 from the Chief Scientist Office of the Ministry of Health, Israel.

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