الفهرس 
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Abstract
Introduction
Distraction osteogenesis (DO) is technique performed in different craniofacial and cleft lip/palate treatment protocols. The distraction osteogenesis in maxillofacial region is a treatment protocol proposed by McCarthy et al. in 1992 to generate bone and soft tissue volume; to overcome the congenital deficiency present in the mandible and surrounding soft tissue.
Mesenchymal Stem Cells (MSCs) was a protocol advocated by several authors to enhance distracted bone quality. In a recent review published by Yang et al.; he found 16 articles concerned with MSCs application during DO all of them focused on bone microscopic quality. Fewer articles focused on biomechanical properties.
BMAC was used as source of MSCs by Kitoh et al. to enhance and fasten limbs DO. In the current article we used BMAC as source of MSCs due to ethical concern related to use of multiplied cultured MSCs in clinical studies; the aim was to mimic the future clinical trial by just injecting BMAC.
The aim of the current study was to assess the biomechanical properties of DO generated bone enhanced by BMAC because it has a direct effect on clinical outcome of the procedure, decrease of healing time and early distractor removal.
Material and Methods
The surgical protocol followed a recent article published in journal of oral and maxillofacial surgery in 2016.
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1-Animal grouping and housing
The study followed the guideline of research ethical committee code of practice for animal care and housing of the authors institute . 12 goats of average weight (10 kg) were housed in Animal house of Faculty of Medicine. Animals were kept there for at least one week preoperatively for adaptation and observation by a veterinarian before the procedure. Animals were divided into two groups; study group (A) assisted with MSCs in form of (BMAC) and control group (B) non- assisted each consisting of six animals. Animals were marked to identify animals of each group. Animals were allocated in each group randomly (simple randomization technique by pulling cards; similar to a deck of shuffled cards).
2-Operative procedure
A) Anesthetic protocol:
Surgeries were done in aseptic condition in the Faculty of Medicine. Animals were pre-medicated by 1.0 mg/Kg Xylazine IV and intravenous anesthetic 25 mg/kg Sodium Thiopental and maintained by 10 mg/Kg ketamine hydrochloride 1 mg.
B) Surgical protocol
The right submandibular and submental area was shaved prior to procedure. The area was scrubbed with Povidone-iodine 10-12% before the incision. Local anesthetic Articaine 4% with 1:200,000 epinephrine field block was injected to reduce postoperative pain. The incision was done using size 15 Bard Parker surgical blades. A vertical osteotomy was done in the right-side of mandible using low speed hand piece associated with copious irrigation by saline followed by complete osteotomy using chisel and mallet. A stainless-steel mono-directional distractor was fixed in place at both osteotomy ends by two 2.0 mm mini screws.
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An anterior skin puncture was done to allow exit of distractor activation screw through the skin for activation during distraction procedure.
Cefotaxime 100 mg was given once daily for five days post-operative. Diclofenac Potassium Intramuscular injection-controlled pain. Animals sacrifice was performed by injecting an overdose of thiopental and animals were incinerated in faculty of medicine medical incinerator.
3) Mesenchymal stem cell preparation:
The aim of the current article was to assess the clinical significance of MSCs application injected in form of BMAC. The use of MSCs in clinical studies is prohibited in many countries due ethical concerns related to direct use of MSCs. However, the use of BMAC which is rich with MSCs is allowed due to its autologous nature and no external intervention or multiplication of cells and is accepted in clinical practice. Because of the allowance of BMAC in clinical practice in current research BMAC was used as source of MSCs to mimic clinical results.
Stem cells were prepared to be injected in group (A). Bone Marrow was aspirated using wide bore gauge spinal needle from iliac crest to harvest stem cell. Bone marrow was treated by Ficoll protocol to prepare MSCs in form of BMAC. The aspirated bone marrow was mixed with Phosphate Buffered Saline (PBS) in ratio 4:1. The mixture was titrated over Ficoll 400 and centrifuged at 2,000 rpm for 20 minutes. The undifferentiated mesenchymal stem cells aggregate in form of buffy layer which was aspirated by special pipette. The buffy layer was diluted by PBS again and recentrifuged twice to ensure high concentration of MSCs.
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4) Assessment of experiment results:
The buccolingual (bone width) dimension in addition to the bone density were assessed by two observers radiographically by Cone Beam Computed Tomography at fixed parameters (84 Kv, 4 mA and 12 Sec) using Sidexs software (Sirona, Germany).
To standardize the radiographic results; in the midway between the plates of the distractor a coronal section was taken. In the middle of the coronal section a line was drawn joining the buccal cortex and lingual cortex and the bone width measurement was taken. Three readings for bone density were taken from the previously mentioned coronal section. The three reading were taken; at the buccal cortex, at the lingual cortex and midway between buccal and lingual borders. After collecting those three results the average is calculated and tabulated.
Biomechanical testing (three-point bending test) was done immediately after the radiographic examination. All specimens were subjected to three-point bending testing using an electronic material testing machine Lloyd LR5K (Ametek, United Kingdom). The specimens were positioned and fixed on the base plate, and the three-point bending load was applied at rate of two mm/min until the specimen rupture. To avoid potential for distortion of empirical findings the soft tissue was completely dissected prior to the testing procedure and the stability of bone on the base plates was insured prior to the start of the procedure.
Histomorphometry of Masson Trichrome stained samples were done to assess the osteoid bone percentage (indicate rate of bone maturity). Measurements were performed by using the image analyzer system Leica DFC 295 microsystem (Leica, Germany) image analysis software at the Histomorphometry Laboratory.
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Statistical evaluation of the results was done using the Independent Sample-T-Test and P- value was set at (0.05).
Cohen’s d test was used to measures the effective size of the study (0.2 to 0.3 might be a ”small” effect, around 0.5 a ”medium” effect and 0.8 to infinity, a ”large” effect).
Concordance correlation coefficient test was used to assess inter-examiner error. McBride (2005) suggested the following descriptive scale for values of the concordance correlation coefficient (for continuous variables) ( < 0.90 Poor, =0.90 - 0.95 Moderate, =0.95 - 0.99 Substantial and >0.99 Almost perfect)
Following the independent sample-T-Test; correlation coefficient assessment of the results was done to assess the correlation of the bone width on biomechanical properties, bone density with biomechanical properties and last histological bone maturation with bone biomechanical properties.
Results
Assessment of bone width (BW) showed a NS (P = 0.9) increase in bone width in group (A) (mean BW= 5.3 mm, SD =1.9) compared to (B) (mean BW= 5.2 mm, SD=0.8). The sample effective size of this test was 0.07 which is a very small to get significant statistical data; so, we considered this test insignificant. Because of this issue we didn’t perform the correlation of bone width with biomechanical strength.
Assessment of bone radiographic density showed a NS (P = 0.5) increase in bone density in group (A) (mean BD= 163.4, SD = 6.5) compared to (B) (mean BD= 156.7, SD= 21.8). The sample effective size of this test was 0.5 which is a medium effective size to get significant statistical data.
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Assessment of histomorphometry of Mason Trichrome stained sample showed a significant (P = 0.02) increase in (A) (Mean mature bone % = 30.07 %, SD =9.83 %) compared to (B) (Mean mature bone percent % = 15.92 %, SD=7.19 %). The sample effective size of this test was 1.6 which is a large effective size to get significant statistical data.
Assessment of Biomechanical testing (three-point bending test) showed a NS (P = 0.7) increase in bone strength in group (A) (mean BD= 511.7 N, SD = 189.1N) compared to (B) (mean BD= 467.8 N, SD= 201.1 N). The sample effective size of this test was 0.2 which is a small effective size to get significant statistical data.
Correlation coefficient assessment of the bone radiographic density with the biomechanical properties (bone strength) showed a moderate positive correlation (r =0.5), however the p-value between bone strength and radiographic bone density was 0.1. Consequently there is an inconclusive evidence about the significance of the association between the variables.
Correlation coefficient assessment of the histological bone maturation (bone maturation) with the bone biomechanical (bone strength) properties showed a week positive correlation (r =0.3), however the p-value between bone strength and radiographic bone density was 0.4. Subsequently, there is inconclusive evidence about the significance of the association between the variables.
Discussion
The Distraction osteogenesis in clinical practice suffered from two noticeable defect which was volume deficiency and the need for long consolidation period to overcome early weak bone strength. The distraction succeeded in correction of vertical and antero-posterior discrepancy; However, it failed in correction of volume
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Shruthi et al. claimed that the application of MSCs improve bone quality, quantity and fasten healing. The study extends upon previous claims that the MSCs improved bone microscopic quality ; however, the effect of MSCs on bone volume and mechanical properties needed further assessment. Few studies were concerned with evaluation of effect of MSCs on Mandibular distracted bone quality, quantity and bone physical properties.
There was an insignificant (P= 0.9) increase in bone width in study cases compared to control cases, in disagreement with Aykan et al., Ma et al., Sato et al., Takamine et al., Xu et al. and Sun et al. whom all found a statistical significant increase in bone volume after addition of MSCs to distraction site.
There was insignificant (P= 0.5) increase in bone density in study cases compared to control cases, in disagreement with all articles mentioned by Yang et al. and Tee et al. in their systematic reviews whom found a statistical significant increase in bone density after addition of MSCs to distraction site.
There was insignificant (P= 0.7) increase in the biomechanical strength in study cases compared to control cases, in disagreement with Aykan et al., Ma et al., Takamine et al, Xu et al., Sun et al. Sunay et al., Nomura et al. whom found a statistical significant increase in the biomechanical strength after addition of MSCs to distraction site.
In the current study there was a significant (P=0.02) increase in percent of mature bone in histomorphometric assessment. The effect of MSCs on bone microscopic microstructure was confirmed in the two systematic reviews mentioned before concerned with evaluation of effect of MSCs on distracted bone.
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In the current study there was improvement in strength, density, and bone width. However, the improvement was of no statistical significance. Only, histological assessment showed a significant increase in bone maturity.
Moreover, both the correlations between the increase in bone density/ bone strength and the correlation between histological maturation/ bone strength was statistically insignificant (P= 0.1 and P=0.4 respectively).
In the current study MSCs were introduced in form of BMAC. BMAC actually has less concentration of MSCs compared to cultured and multiplied MSCs used in the studies mentioned previously in the discussion section. Maybe the low number of MSCs in BMAC was the cause of non-significant results compared to the significant results of the previously mentioned articles.
In the current study we used BMAC rather than other preparations because of ethical concerns related to use of multiplied, cultured and genetically engineered MSCs in clinical practice till moment. The aim of this study as mentioned before was to mimic the clinical environment of mandibular distraction by injecting BMAC and using experimenting on relatively a large animal rather than rodents used in most of the previous studies.
Moreover, several reports are already published in the field of maxillofacial surgery praising its role of BMAC in clinical practice as Hernández-Alfaro et al. in post resection reconstruction, Manimaran et al. in management of osteoradionecrosis, Duttenhoefer et al. in improvement of dental implant success rate and Wongchuensoontorn et al. in healing of atrophic mandible post trauma.
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Consequently, we recommend further examination to evaluate the effect of MSCs in form of BMAC on distracted bone because of non-significant and non-correlated improvement in the physical properties (biomechanical strength) which have direct effect on clinical outcome compared to histological nature which has no clinical outcome.
Until new observation is published in literature our article still recommends soft tissue augmentation prior to distraction osteogenesis either by fat or fillers to improve volume defects. The article also recommend classic consolidation time till BMACs proves in future article it effect on fastening the bone healing.