Dataset Information

Osterix-mCherry Expression Allows for Early Bone Detection in a Calvarial Defect Model.

ABSTRACT: The process of new bone formation following trauma requires the temporal recruitment of cells to the site, including mesenchymal stem cells, preosteoblasts, and osteoblasts, the latter of which deposit minerals. Hence, bone repair, a process that is assessed by the extent of mineralization within the defect, can take months before it is possible to determine if a treatment is successful. Here, a fluorescently tagged Osterix, an early key gene in the bone formation cascade, is used as a predictive measure of bone formation. Using a calvarial defect model in mice, the ability to noninvasively track the Osterix transcription factor in an Osterix-mCherry mouse model is evaluated as a measure for bone formation following treatment with recombinant human Bone-Morphogenetic-Protein 2 (rhBMP-2). Two distinct delivery materials are utilized, an injectable nanocomposite hydrogel and a collagen sponge, that afford distinct release kinetics and it is found that cherry-fluorescent protein can be detected as early as 2 weeks following treatment. Osterix intensity correlates with subsequent bone formation and hence can serve as a rapid screening tool for osteogenic drugs or for the evaluation and optimization of delivery platforms.

SUBMITTER: Strecker SE

PROVIDER: S-EPMC7393777 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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Osterix-mCherry Expression Allows for Early Bone Detection in a Calvarial Defect Model.

Strecker Sara E SE Unterman Shimon S Charles Lyndon F LF Pivovarchick Dmitry D Maye Peter F PF Edelman Elazer R ER Artzi Natalie N

Advanced biosystems 20191104 12

The process of new bone formation following trauma requires the temporal recruitment of cells to the site, including mesenchymal stem cells, preosteoblasts, and osteoblasts, the latter of which deposit minerals. Hence, bone repair, a process that is assessed by the extent of mineralization within the defect, can take months before it is possible to determine if a treatment is successful. Here, a fluorescently tagged Osterix, an early key gene in the bone formation cascade, is used as a predictiv ...[more]

PMID: 32648681

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Project description:Mesenchymal stem cells sheets have been verified as a promising non-scaffold strategy for bone regeneration. Alveolar bone marrow mesenchymal stem cells, derived from neural crest, have the character of easily obtained and strong multi-differential potential. However, the bone regenerative features of alveolar bone marrow mesenchymal stem cells sheets in the craniofacial region remain unclear. The purpose of the present study was to compare the osteogenic differentiation and bone defect repairment characteristics of bone marrow mesenchymal stem cells sheets derived from alveolar bone (alveolar bone marrow mesenchymal stem cells) and iliac bone (Lon-bone marrow mesenchymal stem cells) in vitro and in vivo. Histology character, osteogenic differentiation, and osteogenic gene expression of human alveolar bone marrow mesenchymal stem cells and Lon-bone marrow mesenchymal stem cells were compared in vitro. The cell sheets were implanted in rabbit calvarial defects to evaluate tissue regeneration characteristics. Integrated bioinformatics analysis was used to reveal the specific gene and pathways expression profile of alveolar bone marrow mesenchymal stem cells. Our results showed that alveolar bone marrow mesenchymal stem cells had higher osteogenic differentiation than Lon-bone marrow mesenchymal stem cells. Although no obvious differences were found in the histological structure, fibronectin and integrin ?1 expression between them, alveolar-bone marrow mesenchymal stem cells sheet exhibited higher mineral deposition and expression levels of osteogenic marker genes. After being transplanted in the rabbit calvarial defects area, the results showed that greater bone volume and trabecular thickness regeneration were found in bone marrow mesenchymal stem cells sheet group compared to Lon-bone marrow mesenchymal stem cells group at both 4?weeks and 8?weeks. Finally, datasets of bone marrow mesenchymal stem cells versus Lon-bone marrow mesenchymal stem cells, and periodontal ligament mesenchymal stem cells (another neural crest derived mesenchymal stem cells) versus umbilical cord mesenchymal stem cells were analyzed. Total 71 differential genes were identified by overlap between the 2 datasets. Homeobox genes, such as LHX8, MKX, PAX9, MSX, and HOX, were identified as the most significantly changed and would be potential specific genes in neural crest mesenchymal stem cells. In conclusion, the Al-bone marrow mesenchymal stem cells sheet-based tissue regeneration appears to be a promising strategy for craniofacial defect repair in future clinical applications.

Project description:BackgroundRunt-related transcription factor 2 (RUNX2) haploinsufficiency causes cleidocranial dysplasia (CCD) which is characterized by supernumerary teeth, short stature, clavicular dysplasia, and osteoporosis. At present, as a therapeutic strategy for osteoporosis, mesenchymal stem cell (MSC) transplantation therapy is performed in addition to drug therapy. However, MSC-based therapy for osteoporosis in CCD patients is difficult due to a reduction in the ability of MSCs to differentiate into osteoblasts resulting from impaired RUNX2 function. Here, we investigated whether induced pluripotent stem cells (iPSCs) properly differentiate into osteoblasts after repairing the RUNX2 mutation in iPSCs derived from CCD patients to establish normal iPSCs, and whether engraftment of osteoblasts derived from properly reverted iPSCs results in better regeneration in immunodeficient rat calvarial bone defect models.MethodsTwo cases of CCD patient-derived induced pluripotent stem cells (CCD-iPSCs) were generated using retroviral vectors (OCT3/4, SOX2, KLF4, and c-MYC) or a Sendai virus SeVdp vector (KOSM302L). Reverted iPSCs were established using programmable nucleases, clustered regularly interspaced short palindromic repeats (CRISPR)/Cas-derived RNA-guided endonucleases, to correct mutations in CCD-iPSCs. The mRNA expressions of osteoblast-specific markers were analyzed using quantitative reverse-transcriptase polymerase chain reaction. iPSCs-derived osteoblasts were transplanted into rat calvarial bone defects, and bone regeneration was evaluated using microcomputed tomography analysis and histological analysis.ResultsMutation analysis showed that both contained nonsense mutations: one at the very beginning of exon 1 and the other at the initial position of the nuclear matrix-targeting signal. The osteoblasts derived from CCD-iPSCs (CCD-OBs) expressed low levels of several osteoblast differentiation markers, and transplantation of these osteoblasts into calvarial bone defects created in rats with severe combined immunodeficiency showed poor regeneration. However, reverted iPSCs improved the abnormal osteoblast differentiation which resulted in much better engraftment into the rat calvarial bone defect.ConclusionsTaken together, these results demonstrate that patient-specific iPSC technology can not only provide a useful disease model to elucidate the role of RUNX2 in osteoblastic differentiation but also raises the tantalizing prospect that reverted iPSCs might provide a practical medical treatment for CCD.

Dataset Information

Osterix-mCherry Expression Allows for Early Bone Detection in a Calvarial Defect Model.

Publications

Osterix-mCherry Expression Allows for Early Bone Detection in a Calvarial Defect Model.

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