| Abstract|| |
Background: Autogenous bone graft is the gold standard for repair of bone defects. However, osteoprogenitor stem cells are suggested as an alternative treatment. Aims: To quantitatively compare bone formation in autogenous bone graft and tissue-engineered graft using digital radiography densitometry software in canine alveolar cleft model. Setting and Design: This experimental study on animal models was conducted in Isfahan University of Medical Sciences. Methods: Mesenchymal stem cells (MSCs) were obtained from subcutaneous adipose tissue of 4 dogs. Undifferentiated cells were incubated with a hydroxyapatite/beta-tricalcium phosphate scaffold in an osteogenic medium for 21 days. A maxillary defect simulating human alveolar cleft was created from the alveolar crest to nasal floor with 15 mm width bilaterally. Two months later, the defect was filled with autogenous bone graft harvested from tibia on one side and tissue-engineered graft from MSCs on the other side. Digital radiography was performed on days 15, 30, 45, 60, 75, and 90 after grafting. Radiographic density was calculated by the mean numeric value of pixels of the desired area ranging from 0 (darkest) to 255 (lightest) by associated software. Statistical Analysis Used: The data were analyzed by Statistical Package for the Social Sciences using descriptive statistics and two-way repeated-measure analysis of variance test (α = 0.05). Results: Mean density measured for autogenous bone graft group was 110.72, 82.70, 75.76, 93.57, 100.22, and 100.32 in days 15, 30, 45, 60, 75, 90, respectively and 120.7, 87.62, 83.72, 92.02, 92.30, and 93.77 in stem cell group. Although the time lapse was a significant factor in two groups (P = 0.01), the results indicated that the difference between two groups was not statistically significant (P = 0.942). Conclusion: Tissue-engineering can be used as an alternative method in reconstruction of bony defects with predictable clinical outcomes.
Keywords: Bone, digital radiography, stem cells, tissue engineering
|How to cite this article:|
Shahnaseri S, Sheikhi M, Hashemibeni B, Mousavi SA, Soltani P. Comparison of autogenous bone graft and tissue-engineered bone graft in alveolar cleft defects in canine animal models using digital radiography. Indian J Dent Res 2020;31:118-23
|How to cite this URL:|
Shahnaseri S, Sheikhi M, Hashemibeni B, Mousavi SA, Soltani P. Comparison of autogenous bone graft and tissue-engineered bone graft in alveolar cleft defects in canine animal models using digital radiography. Indian J Dent Res [serial online] 2020 [cited 2021 May 11];31:118-23. Available from: https://www.ijdr.in/text.asp?2020/31/1/118/281796
| Introduction|| |
Bone defects in the maxilla and mandible are caused by congenital deformities, dental diseases, trauma, and maxillofacial procedures. Complete reconstruction of the defect is the ideal goal in the regenerative treatment of these conditions. Autogenous bone graft is considered as the gold standard for repair of bone defects. However autogenous bone graft may cause short- and long-term complications in donor site. Tissue engineering of bone is a promising approach for bone reconstruction and regeneration.,, In tissue engineering, cells, the extracellular matrix, and growth factors are combined to design novel graft materials, which can induce tissue regeneration and repair based on natural healing potential.
Autogenous bone grafts can be harvested mostly from the iliac crest and ribs and contain cortical and cancellous bone, providing osteogenic cells required for bone formation and integration in the defect area. However, disadvantages of this technique include limitation in size of graft, requiring a donor area, possibility of secondary hematoma and infection, and dysesthesia and paralysis in the donor area., The amount of bone formation in autogenous bone graft depends primarily on the number of cells survived in transplantation procedure, which reach the recipient area. Osteogenesis is continued by angiogenesis and proliferation of fibroblasts within the graft and the recipient site. Fibroblasts and other mesenchymal cells are differentiated into osteoblasts, which produce the newly formed bone material.
Osteoprogenitor stem cells offer new possibilities in regenerating different tissues. It has been demonstrated that adipose-derived stem cells can lead to osteogenesis of long bones when used with certain biomaterials. Moreover, studies have reported successful application of stem cells in repair of alveolar bone defects.,,,,,, Some studies suggest that the stem cell technique in reconstruction of bone defects can lead to less morbidity compared to autogenous bone grafting.
Evaluation of bone healing process needs a sensitive and appropriate diagnostic tool. Histologic examination can show the exact amount and type of the bone formed in the area in research studies. However, noninvasive techniques are needed in clinical cases. Therefore, radiographic examinations may be a useful technique for evaluation of bone formation and healing with time.,
Bone regeneration can be monitored by measurements on standard and reproducible radiographs. Moreover, sensitive and accurate methods are required for detection of changes in the early stages of bone healing. Digital radiography seems to be a good technique for these purposes as it can provide image-enhancement and reproducible radiographs. Studies have shown that the process of bone regeneration is detectable on digital radiographs.
The aim of the present study was quantitative comparison of bone formation in autogenous bone grafting and tissue-engineered bone from adipose-derived stem cells using digital radiography densitometry software in alveolar cleft defects in canine animal models.
| Methods|| |
Institutional Animal Care and Use Committee of the medical university approved this study. The Regional Bioethics Committee approved the study (#388489, registered in October 2016). The experiments was performed on four adult mongrel dogs (mean age 18 month) weighing 20–30 kg. General anesthesia was obtained by Ketamine (20 mg/kg) and Rampone (2 mg/kg). Thereafter, two of the three incisors of each animal were removed bilaterally and a 15 mm wide defect was created from the alveolar crest to the nasal floor. Nasal mucosa was sutured to oral mucosa. Endotracheal tube number 8 was placed in the ends of the defect and secured to the canine teeth to preserve the defect space. The tube was filled with self-cure methyl-methacrylate and was held in place for 2 months as a tolerable stent for the animal till the complete oro-nasal fistula was formed [Figure 1]a,[Figure 1]b,[Figure 1]c.
|Figure 1: Bilateral maxillary alveolar cleft creation and space maintained by the stent (a). After 2 month of alveolar cleft creation & noticeable established oroantral fistula (b and c)|
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Ethical considerations were followed as the surgeries were performed under general anesthesia and sterile condition. Following the surgeries analgesic and antibiotic medications were administered. Moreover, the canines and molars which are the main functional teeth of the animals were maintained to allow the normal masticatory function. The general living condition of the animals was controlled in all steps to be appropriate.
Twenty gram subcutaneous fat was obtained from each animal and transferred to cell culture laboratory. The adipose tissue specimens were then cut into small pieces and washed with phosphate-buffered saline (PBS; Gibco, Thermo Fisher Scientific Inc., MA, USA). Afterwards, the processing was followed by adding 1.5 mg of collagenase type I (Sigma-Aldrich Chemie, Taufkirchen, Germany) per gram of adipose tissue and incubating with continuous shaking for 1 h at 37°C. Multiple centrifugations and washing steps were applied prior to the removal of red blood cells using lysis buffer, allowing the separation of stromal cells from floating adipocytes. The separated stromal cells were then counted by a hemocytometer and plated in tissue culture flasks (3000 cells/cm2) containing Dulbecco's modified Eagle's medium (DMEM; Gibco, Thermo Fisher Scientific Inc., MA, USA) supplemented with 1% penicillin–streptomycin (Gibco, Thermo Fisher Scientific Inc., MA, USA) and 10% fetal bovine serum (FBS; Dainippon Pharmaceutical, Osaka, Japan). The plates were incubated with 5% carbon dioxide at 37°C. The nonadherent cells were discarded and the medium was changed after 24 h. Culture media were replaced every 2–3 days. Moreover, when the cultures reached about 80% confluency, trypsinization and replating was performed.
Flow cytometrical analysis and the differentiation ability of the cell was used for MSC characterization. In one tube 1 × 105 cells were stained with phycoerythrin (PE) conjugated monoclonal antibody to CD44 (ab58754; ABCAM Antibodies, Cambridge Science Park, Cambridge, UK) and fluorecin isothiocyanate conjugated (FITC) monoclonal antibody to CD90 (ab22541; ABCAM Antibodies, Cambridge Science Park, Cambridge, UK). The specimen was analyzed by FACS caliber 488 (Becton Dickenson, CA, USA) after incubation at room temperature for 15 min, showing a distinct population of CD44 and CD90 positive cells. The result of the differentiation assay along with the result of FACS analysis confirms that actual MSCs were transplanted [Figure 2]a and [Figure 2]b.
|Figure 2: (a) Results of flow cytometry show 99% of isolated cells express CD44 marker positively. (b) Results of flow cytometry show 91% of isolated cell express CD 90 marker positively|
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In-vitro osteogenic differentiation
In-vitro osteogenic differentiation was obtained using confluent passage 3 culture. 5 × 106 cells were incubated with 3 mm × 3mm × 3 mm HA/TCP (Ceraform, Teknimed, Bigorre, France) (60% HA and 40% b-TCP with a mean pore size of 200–800 mm). Specific osteogenic medium consisting of 50 mml ascorbic acid 2-phosphate (Sigma-Aldrich Chemie, Taufkirchen, Germany), 100 nmg dexamethasone (Sigma-Aldrich Chemie, Taufkirchen, Germany), and 10 mml b-glycerophosphate (Sigma-Aldrich Chemie, Taufkirchen, Germany) was used for incubation at 37°C and 5% carbon dioxide for 21 days [Figure 3].
Cell differentiation was evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of osteogenic gene expression. Osteocalcin and collagen I were largely produced after 21 days in an osteoinductive medium. The selected housekeeping gene was GAPDH [Figure 4].
|Figure 4: Reverse transcription polymerase chain reaction analysis. Osteocalcin and collagen I expression after 3 weeks|
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Implantation of scaffold/MSCs constructs and autograft
After two months, the second surgery was performed under general anesthesia and the stents were removed. Two weeks were allowed for local inflammation to subside. Elevation of the flap, the defect space was filled with 10 cm3 autogenous bone graft harvested from tibia on one side and bone formed by tissue engineering technique from adipose-derived stem cells on the other side. The defect site was then monitored by serial radiography on 15, 30, 45, 60, 75, and 90 postoperative days by digital radiography system (SIGNUS, Alzenau, Germany) using size 2 charge coupled device (CCD) sensors and parallel technique with film holders. Tube parameters were set on 70 KVP, 10 mA, and 0.40 s. Images were viewed in Dr. Suni software (Suni Medical Imaging Inc., CA, USA). Bone density was measured by Digora for Windows densitometry software (ZUBNI RENDGEN DR. LAUC Ltd., Varaždin, Croatia).
In this study, bone density measured using densitometer software was considered as the indicator of healing of the defect space by either autogenous bone grafting or tissue engineering techniques. This density was calculated by the mean numeric value of pixels of the desired area ranging from 0 (darkest) to 255 (lightest) [Figure 5].
|Figure 5: Digital parallel radiographs of the region (a) before operation, (b) immediately after making the defect, (c) day 15 (d) day 30, (e) day 45, (f) day 60, (g) day 75|
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Moreover, bone regeneration was also evaluated histologically 60 days after graft placement by trephine bur. The specimens were prepared and stained by Masson's trichrome [Figure 6].
|Figure 6: Histological evaluation of bone regeneration in autogenously reconstructed defects|
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Data was analyzed by descriptive, Mann–Whitney and two-way repeated-measure analysis of variance (ANOVA) test using Statistical Package for the Social Sciences 22 (SPSS, IBM, NY, USA).
| Results|| |
Mean density measured by digital radiography for autogenous bone graft group was 110.72 (SD = 1.80), 82.70 (SD = 12.75), 75.76 (SD = 16.20), 93.57 (SD = 19.86), 100.22 (SD = 38.16), and 100.32 (SD = 41.17) in days 15, 30, 45, 60, 75, and 90, respectively. These amounts were 120.75 (SD = 7.73), 87.62 (SD = 25.58), 83.72 (SD = 27.01), 92.02 (SD = 35.20), 92.30 (SD = 34.43), and 93.77 (SD = 29.73) for adipose-derived stem cell group [Table 1].
|Table 1: Mean numeric value of pixels of the defect area in autografted and stem-cell grafted sides 15, 30, 45, 60, 75, and 90 days after implantation|
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It is worth noting that in about two-thirds of the cases, the stem cell group, numbers were larger than the autogenous group [Figure 7]. With Mann–Whitney test, these differences were not significant. In days 15, 30, 45, 60, 75, 90, P values were 0.083, 0.564, 0.663, 0.564, 0.564, and 0.564, respectively.
|Figure 7: Diagram comparing osteogenesis by autogenous bone graft and adipose-derived stem cell techniques|
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The analysis of data with two-way, repeated-measured ANOVA indicated that the difference was not statistically significant between bone formation in autogenous bone grafting and stem cell tissue engineering (P value = 0.942).
| Discussion|| |
In this experimental radiographic study, the canine model was used as its bony structure was similar to humans.
We used digital radiography to compare bone regeneration of alveolar cleft defect model after using autogenous bone graft and tissue engineered bone from adipose-derived stem cells. Although the superimposition of adjacent structures can potentially reduce the accuracy of differential diagnosis in oral cavity, the high resolution of intraoral radiographs makes it possible to examine minor changes of bone structure in time.,,,,
So far, few studies have been carried out to compare radiographic criteria of various bone grafting methods.
In a study by Ihan-Hern et al. in 2008, self-healing of isolated human mandibular bony defect was measured by digital radiography while the bone densities after 2, 6, and 12 months were reported to be 7%, 27%, and 46% respectively.
The mean bone densities in our study intervals were all higher due to canine accelerated proliferation rate and also the dense HA/TCP scaffolds. Hibi et al. in 2006 treated a patient with alveolar cleft using tissue engineered bone from bone marrow-derived stem cells. Consecutive CT scans revealed that 80% of the cleft space was filled by the regenerated bone after 9 months, which was comparable with the success of our study.
In a study performed by Wolf et al. in 1997, bilateral alveolar defects were created in dogs. Their study aimed to compare radiographic density of bone regeneration in cases treated with recombinant human bone morphogenetic protein delivered in poly-lactide-co-glicolide and autogenous blood, poly-lactide-co-glicolide and autogenous blood without bone morphogenetic protein, autograft, and untreated group. They concluded that by 4 months bone morphogenetic protein and poly-lactide-co-glicolide groups were equivalent to autografted side.
By comparing the bone densities of two grafting materials just after implantation, the densities of the grafts were close to and even more than the contiguous bone, which were probably due to the inherent densities of the autograft and HA/TCP scaffolds. A descending trend was observed until day 45 in stem cell groups which seemed to be due the resorption of scaffolds and death of cells groups due to lack of blood supply. This decrease was less in the autografted group and lasted for approximately 30 days. After this period, the density was increased and reached the density of surrounding normal bone in nearly 2 months. Radiographically, bone formed by autogenous bone grafting was homogenous. However, tissue engineering technique led to formation of bone with cotton-wool radiographic appearance.
Based on the findings of the present study, bone density measured by the digital radiography was not significantly different in autogenous and tissue-engineered bone graft groups. Although the radiographic densitometry did not represent the exact bone density of the region, however, they could be useful for comparative evaluation of different regions and different materials when the exposure setting is similar. In our study use of digital radiography software helped to monitor bone regeneration process with time. Moreover, this study presented stem cell tissue engineering technique as a comparable method to conventional one.
In conclusion, the present study showed that tissue-engineering technique can be used for reconstruction of bone defects in patients unable to undergo autogenous bone grafting when large size of the defect limits the size of the autograft, as an appropriate and clinically predictable technique.
Financial support and sponsorship
This study was financially supported by Deputy of Research of Isfahan University of Medical Sciences.
Conflicts of interest
There are no conflicts of interest.
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School of Dentistry, Isfahan University of Medical Sciences, Hezar-Jarib Ave., Isfahan
Source of Support: None, Conflict of Interest: None
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