Project description:Objective: Craniofacial bone defects caused by injuries and congenital diseases are a formidable challenge to clinicians. Research has shown promise in using bone marrow mesenchymal stem cells (BM-MSCs) from limb bones for craniofacial bone regeneration; yet little is known about the potential of BM-MSCs from craniofacial bones. This study compared BM-MSCs isolated from limb and craniofacial bones in pigs, a preclinical model closely resembling humans. Design: Bone marrow was aspirated from the tibia and mandible of four-month-old pigs (n=4), followed by BM-MSC isolation, culture-expansion and confirmation by flow cytometry. Proliferation rates were compared using population doubling times. Osteogenic differentiation was evaluated by quantifying alkaline phosphatase (ALP) activity. Total mRNA was extracted from freshly isolated BM-MSCs and analyzed to compare gene expressions of tibial and mandibular BM-MSCs using an Affymetrix GeneChip porcine genome array, followed by real-time RT-PCR evaluation of two neural crest markers. Results: BM-MSCs from both locations expressed MSC markers without expression of hematopoietic markers. Mandibular BM-MSCs proliferated significantly faster than tibial BM-MSCs. Without osteogenic inducers, mandibular BM-MSC alkaline phosphatase activities were 3.3-fold greater than those of tibial origin. Microarray analysis identified 383 differentially expressed genes in mandibular and tibial BM-MSCs, including higher expression of cranial neural crest-related genes nestin and BMP-4 in mandibular BM-MSCs, a trend also confirmed by real-time RT-PCR. Among differently expressed genes, only 47 showed greater than 1.5-fold differences in expression. Conclusions: These data indicate that despite many similarities in gene expression, mandibular BM-MSCs express of number of genes differently than tibial BM-MSCs and have a phenotypic profile that may make them advantageous for craniofacial bone regeneration. Bone marrow was aspirated from the mandibular symphyseal region and the tibia of 3 pigs. Mesenchymal stem cells were isolated from the bone marrow and cultured to 80% confluence. Cells were harvested for total RNA extraction and the RNA was analyzed by Affymetrix GeneChip porcine genome array.

Project description:Recently, there is increasing report of bone necrosis exceptionally in jaw bone areas after long term administration of anti-resorptive agent, bisphosphonate (BP). It is still unknown that why BP does not result in osteonecrosis but potentiate fracture repair in axial skeleton such as long bones. Many reports had shown that mandibular bone or calvarial bone exhibits lesser resoption than iliac bone graft for maxillofacial bone defect.(Jackson et al 1986, Koole et al 1989),(Crespi et al 2007) These might be attributed to the embryological difference between the two different type of the bone; intramembranous versus endochondral bone formation. Maxilla and mandibular alveolar and basal bone are originated from neural crest cells and exhibit intramembranous bone formation.(Chai and Maxson 2006) However, axial skeleton such as iliac or tibial bones are originated from mesoderm and undergo endochondral bone formation.(Helms and Schneider 2003) It had been reported that there is distinct phenotype difference in human bone cells from different skeletal origin.(Akintoye et al 2006, Kasperk et al 1997),(Matsubara et al 2005) Akintoye et al.(Akintoye et al 2006) reported that mandibular bone marrow stromal cells (BMSCs) displayed a fibroblast-like morphology similar to iliac BMSCs in histological appearance. However, mandibular bone contained less red marrow and hematopoietic cells, whereas the iliac bone contained more red marrow thereby contributing more to hematopoiesis. This differential capacity of mandibular BMSCs with less hematopoietic stem cells is better than that of iliac BMSCs because mitosis in hematopoietic stem cells is usually stopped. They also reported that maxillofacial BMSCs shows higher proliferation rate and alkaline phosphotase activity.(Akintoye et al 2006, Akintoye et al 2008) However, in vivo response of human BMSCs to osteogenic induction was higher in iliac bone than maxillomandibular bone.(Akintoye et al 2006) Other reports had shown that BMSCs from mandible showed better in vitro and in vivo bone formation capacity compared to tibia from rats.(Aghaloo et al 2010) Matsubara et al. reported that osteogenic potential of human or canine alveolar bone and iliac bone-derived cells (BC) are similar but showed difference in chondrogenic and adipogenic potential.(Matsubara et al 2005) From the previous studies, the site-related difference of mandibular and iliac BC are clear. However, the pattern of the site-specificity are not exactly same among above mentioned studies. These might be attributed to the difference in the experimental subjects (species, age) and study design (in-patient or random comparison, etc). Moreover, underlying genetic mechanism of these skeletal-site dependent difference had not been fully investigated. The investigation of site-specific differences in the gene expression would be help to understand the mechanism of bisphosphonate-related osteonecrosis of the jaw (BRONJ), which recently has become an major issue in dentistry. Considering that bisphosphonate is frequently prescribed to elderly patients who have a higher risk of osteoporosis, it is necessary to conduct a experimental analysis of cells from older-aged donors. It had been suggested that anatomical site-specific difference can be influenced by 1) difference in the regional blood supply, 2) difference in bone composition (lamellar bone versus trabecular bone), 3) difference in physiological strain exerted to bone, 4) difference in gene expression according to skeletal site.(Kasperk et al 1997) In this study, bone cells derived from elderly donors went through microarray analysis under the hypothesis that there would be the differences in gene expressions involved in bone formation or cell proliferation of bone cells from iliac and mandible. The purpose of this study was to investigate the difference in gene expression between the human mandible and iliac bone so that we can understand the genetic difference in the two different type of bones. <<<References>>> 1. Jackson IT, Helden G, Marx R. Skull bone grafts in maxillofacial and craniofacial surgery. J Oral Maxillofac Surg 1986; 44:949-955. 2. Koole R, Bosker H, van der Dussen FN. Late secondary autogenous bone grafting in cleft patients comparing mandibular (ectomesenchymal) and iliac crest (mesenchymal) grafts. J Craniomaxillofac Surg 1989; 17 Suppl 1:28-30. 3. Crespi R, Vinci R, Cappare P, Gherlone E, Romanos GE. Calvarial versus iliac crest for autologous bone graft material for a sinus lift procedure: a histomorphometric study. Int J Oral Maxillofac Implants 2007; 22:527-532. 4. Chai Y, Maxson RE, Jr. Recent advances in craniofacial morphogenesis. Dev Dyn 2006; 235:2353-2375. 5. Helms JA, Schneider RA. Cranial skeletal biology. Nature 2003; 423:326-331. 6. Kasperk C, Helmboldt A, Borcsok Iet al. Skeletal site-dependent expression of the androgen receptor in human osteoblastic cell populations. Calcif Tissue Int 1997; 61:464-473. <<< Important Abstract of Associated reference>>> Kingsmill VJ1, McKay IJ, Ryan P, Ogden MR, Rawlinson SC. Gene expression profiles of mandible reveal features of both calvarial and ulnar bones in the adult rat.J Dent. 2013 Mar;41(3):258-64. doi: 10.1016/j.jdent.2012.11.010. Epub 2012 Nov 23. OBJECTIVES: Limb and mandibular alveolar bone of the mandible are susceptible to disuse osteopenia, whilst skull and mandibular basal bone appear to resist excessive generalised bone loss. We wanted to compare the site-specific transcriptome of anatomically and functionally distinct bones to confirm the composite nature of the mandible at the molecular level. METHODS: Gene expression profiles were obtained for the mandible, ulna, and calvaria of adult male rats using Affymetrix Rat Genome 230 2.0 GeneChips. Ingenuity Pathways Assist generated association maps, and RGD database software identified site-specific pathways. RESULTS: The majority of expressed transcripts (84%) are common to all three sites. The mandible expressed 873 transcripts in common with ulna but not calvaria, and 1014 transcripts in common with calvaria but not ulna. Transcripts in these groups were excluded if they showed significant differential expression (>2-fold) and the remaining mapped genes were filtered for those related to modulation of gene transcription. Analysis of these genes revealed common pathways shared by the mandible and ulna, or mandible and calvaria, which were not shared by the calvaria and ulna. CONCLUSIONS: There were relatively few differences in the expression of genes responsible for the bone formation process per se in different functional skeletal sites. Differential transcription factor expression suggests that it is the regulation of bone formation and not the mechanism of bone formation itself that differs between the skeletal sites. CLINICAL SIGNIFICAN Objectives: The exact genetic difference between the jaw and long bone had not been clearly understood. The purpose of this study was to investigate the difference in gene expression between the human mandible and iliac bone-derived cells. Methods: Primary cells were obtained from mandibular and iliac bone from the 6 healthy, elderly donors (average age 60.2 years) during the reconstruction of mandibular alveolar bone defects. To investigate site-specific difference, within-patient comparison were carried out with cell proliferation and osteoblastic differentiation assay. Gene expression profile of mandible and iliac BC (bone-derived cell) were compared using cDNA microarray analysis using Affymetrix GeneChipM-BM-.. Results: The mandibular BC showed stronger proliferative capacity but weaker osteoblastic differentiation. The comparison of the gene expression profile identified that 82 genes were significantly up-regulated and 66 genes were down-regulated 1.5 fold or greater in mandible compared to iliac BC. The most significantly differentially regulated genes were associated with skeletal system development and morphogenesis (SIX1, MSX1, MSX2, HAND2, PRRX1, OSR2, HOX gene family, PITX2). Microarray analysis revealed that Msx1 was 2.03 fold and Msx2 was 1.99 fold up-regulated in mandible compared to iliac BC (both p<0.01). HOX gene group in mandibular BC was down-regulated (p<0.01). Osteopontin was also 2.84 fold down-regulated in mandible BC (p<0.01). We found that the results of the microarray were reproducible with qRT-PCR. Conclusions: Site-specific difference between jaw and long bone can be explained by the different gene expression pattern, which is primarily related with embryological origin of these two different bones. Human mandible and iliac bone chip was were collected from 6 donors (average 60.2 years, ranged from 56 to 69 years; 3 males, 3 females). Primary cells from this bone chips were utilized to analysis. Therefore 6 sets of iliac and mandibular bone-derived cells were obtained. <<detailed method>> The experimental protocol using human tissue was approved by the Institutional Review Board of Kyungpook National University Hospital (KNUH_10-1093). Informed consent was obtained from each donors who were undergoing iliac bone graft to mandibular alveolar bone defect. To collect osteoblast-like primary cells migrated from bone chips cortical or cortico-cancellous bone was harvested with the similar method described previously. Human mandible and iliac bone chip was were collected from 6 donors (average 60.2 years, ranged from 56 to 69 years; 3 males, 3 females), of healthy general condition without clinical inflammatory lesion in oral cavity. After complete reflection of periosteal layer, the mandibular bone and iliac crest was visualized. Mandibular cortical or cortico-cancellous bone chip was collected by a ronguer during the host bone trimming and decortication procedure for iliac bone graft to edentulous mandible. Similarly, small amount of iliac cortico-cancellous bone chip was collected seperately during the iliac bone harvesting procedure from the same patient. The harvested bone were broke into small pieces and the size of the bone particle became to be 2~4mm in length. The collected bone samples from the two sites were treated with the same method. The bone sample were gently rinsed with Dulbecco's modified Eagle's Medium (DMEM, Lonza Group Ltd., Basel, Switzerland) containing heparin to exclude fat and blood. The particulated bone were then transferred into culture flasks and cultured with DMEM supplemented with 20% fetal bovine serum (FBS, GIBCO-Invetrogen, Carlsbad, CA), 100 U/ml penicillin, 100 mg/ml streptomycin sulphate and 2 mM glutamine incubated at 37M-BM-0C in a humidified atmosphere of 5% CO2 in air. The medium was first changed 2 days after seeding to remove non-adherent cells and the media (DMEM - 20% FBS) was changed every 2 days until the cells migrated out from the explants and reached confluency. Subconfluent primary cells were trypsinized and replated to 75 Cm2 flask. At passage 2, the cells were harvested to extract RNA for cDNA microarray.

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Cell clusters during mandibular bone regeneration [scRNA-seq]

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