Alternatively, once established by the transduced factors, the converted cells may remain stably osteoblast-like even in the absence of sustained expression of the exogenous genes. The repair tissue between the broken bone ends, the fibrocartilaginous callus, is composed of both hyaline and fibrocartilage. The directly converted osteoblasts (dOBs) showed similar gene expression profiles as normal osteoblasts and contributed to bone repair after transplantation into mice with bone defects. Spongy bone makes up most of the structure of short, flat and irregular bones and the inside of the epiphysis of long bones. In experiments performed to assess the epigenetic status of dOBs, we found that in osteoblasts, genomic DNA was mostly unmethylated at CpG dinucleotides at the osteocalcin gene upstream region, whereas in fibroblasts, the CpG sequences were heavily methylated (Fig. Direct conversion of fibroblasts into osteoblasts. 2B), strongly suggesting a crucial role of Osterix in “full conversion”. 4A). 2019 Nov 19;20(22):5805. doi: 10.3390/ijms20225805. The RXOL-transduced cells expressed osteopontin, BSP, and Osterix mRNA at levels between those of osteoblasts and MSC-OBs. Bone fracture repair is a surgery to fix a broken bone using metal screws, pins, rods, or plates to hold the bone in place. Dermal fibroblasts are a good candidate for tendon tissue engineering because they are similar to the tenocytes used for collagen synthesis. (A) After culturing for 14 d, mRNA levels for ALP and osteocalcin genes were quantified by real-time RT-PCR. Cardiac fibroblasts are critical in extracellular matrix homeostasis. Fibroblast growth factor-2 and fracture healing]. (A) RNA was extracted from fibroblasts and RXOL-transduced cells at 21 d after infection. The differentiation of osteoblasts is regulated by various transcription factors, including Runt-related transcription factor 2 [Runx2, also known as core-binding factor subunit α-1 (Cbfα-1)] (4, 5), Osterix (6, 7), Distal-less homeobox 5 (Dlx5) (8), and activation transcription factor 4 (ATF-4) (8). Fibroblasts were cultured in growth media containing different concentrations of recombinant IGFBP7 (125‐1000 ng/mL). Yin et al found that Sox9-positive GFP-negative chondrocytes were present in bone marrow and callus (Fig. Please enable it to take advantage of the complete set of features! To date, however, there have been no reports on the induction of human osteoblasts from another differentiated somatic cell lineage by means of direct reprogramming procedures. The failure of rotator cuffs to heal after repair is an unresolved surgical issue. Using lineage tracing and single-cell transcriptomics, Leavitt et al. ALP staining showed that transduction with RXOL induced ALP activity in ∼80% of the fibroblasts at 14 d after infection, and OL infection also induced this osteoprogenitor marker (Fig. Functions include support, protection, aid in movement, being a reservoir for minerals such as calcium and phosphate, for hematopoiesis, and fat storage. Characteristics of dOBs induced from fibroblasts. Chen K, Zhou Q, Kang H, Yan Y, Qian N, Li C, Wang F, Yang K, Deng L, Qi J. Radiographically, the defect parts in dOB-transplanted femurs were covered with bridging callus formation, which was not seen in the fibroblast-transplanted femurs (Fig. In addition, c-Myc is considered a crucial factor in the direct reprogramming of mouse fibroblasts into neural stem cells (18, 20) and chondrocytes (21). Fibroblasts are critical components of granulation tissue. It may be possible that the continuous expression of transgenes is required for the dOBs to maintain their osteoblast-like phenotypes. dOBs facilitated bone repair in vivo. Fibroblasts have a high proliferative capacity, which does not decline with increasing donor age (43, 45). S1A). FGF6 is involved in muscle regeneration and those events that occur during the healing process. We also assessed the capability of the resulting directly converted osteoblasts (dOBs) to contribute to bone formation in vivo. Moreover, that bone marrow-derived fibroblasts make a substantial contribution to the formation of new connective tissue, including type I collagen, during wound repair. Micro computed tomography (μCT) revealed that the callus was composed of regularly aligned bone trabeculae, with no signs of pseudoarthrosis. Proc Natl Acad Sci USA. Cathepsins K and L are also expressed in RA synovial fibroblasts and contribute to the degradation of bone matrix, including ColI, II IX, and XI and proteoglycans [ 28, 32, 33 ]. Wound Repair Regen. When tissues are damaged, tissue homeostasis must be re-established, and repair mechanisms have to rapidly provide harmonious mechanical tissue organization, a process essentially supported by (myo)fibroblasts. Heat map and hierarchical clustering analysis of the DNA microarray data showing the genes for transcription factors and signaling molecules involved in osteoblast differentiation. Copyright © 2021 National Academy of Sciences. Such ectopic endochondral ossification leads to the failure of bone repair and results in ankylosis. dOBs were induced by transient expression of RXOL. (B) RNA obtained from the indicated cells were analyzed by the GeneChip human Gene 1.0 ST (Affymetrix), consisting of 29,096 probes. The dOBs expressed endogenous Runx2 and Osterix, and did not require continuous expression of the exogenous genes to maintain their phenotype. A functional decline in osteoblasts relative to osteoclasts results in imbalance between bone formation and resorption and may cause osteolytic pathological conditions, such as osteoporosis (9), alveolar bone resorption associated with periodontitis (10), and bone lysing associated with bone tumors, including multiple myeloma (11). It has been demonstrated that transplantation of skin fibroblasts that overexpress bone morphogenetic protein-2 (BMP-2) via adenoviral vectors can successfully repair mouse calvarial bone defects [1]. 1). These results demonstrate that fibroblasts were directly converted into dOBs without passing through an immature pluripotent stage. Bone repair was estimated by macroscopic, radiographic, biomechanical, and histological examinations at 21 d after transplantation. In mammals, however, the repair of most organs is imperfect and results in scar formation. Both fibroblasts and immature chondrogenic cells can differentiate into cells of the chondrogenic and osteogenic lineages. Schlickewei CW, Kleinertz H, Thiesen DM, Mader K, Priemel M, Frosch KH, Keller J. Int J Mol Sci. The resultant osteoblasts facilitated bone repair in vivo after transplantation into immunodeficient mice at the bone defect lesions. Osteogenic medium was used only after the withdrawal of doxycycline. It is involved in the formation of the flat bones of the skull, the mandible, and the clavicles. One of the primary functions of CFBs is maintaining the integrity of the cardiac ECM, a network structure that in addition to providing structural and functional integrity to the heart, also contains a number of cytokines and growth factors that can impact cardiac function and the fate of cardiac cells. Bone, cartilage, fat, and blood are all special forms of connective tissue. Normal human osteoblasts were stained as controls. The relative mRNA levels were calculated as in Fig. Crossref Medline Google Scholar; 17. Based on these findings, RXOL-transduced cells were used in further detailed analyses as representative directly converted osteoblasts (dOBs). Myofibroblasts differentiate, invade and repair injured tissues by secreting and organizing the extracellular matrix and by developing contractile forces. However, their origin and pathways of differentiation remain poorly defined. Because fibroblasts are the critical cell type for early and late wound healing, we hypothesize that, during normal wound healing, fibroblast precursor cells are recruited from both, the resident resting pool in the skin and the bone marrow-derived stem cell populations, which then subsequently migrate into the wound bed and differentiate into mature fibroblasts. Osterix-null mice also lack bone formation (6). Osteoporosis globally causes … This work was supported by grants from the Japan Science and Technology Agency (PRESTO and A-Step); the Japanese Ministry of Education, Culture, Sports, Science and Technology; and the Suzuken Memorial Foundation. These findings strongly suggest successful direct reprogramming of fibroblasts into osteoblasts, which may be applicable to bone regeneration therapy. Tissue injury initiates a complex repair process, which in some organisms can lead to the complete regeneration of a tissue. Pericondrial cells differentiate into osteogenic cells then into osteoblasts. Bone repair was estimated by macroscopic, radiographic, biomechanical, and histological examinations at 21 d after transplantation. RXOL and OL represent human fibroblasts infected with the corresponding retrovirus vectors. Thus, direct reprogramming may provide another option for such patients in whom MSC transplantation might not be an appropriate therapy. 1 roles of macrophages and fibroblasts in wound repair and regeneration Mitogenic and other growth‐promoting factors released after tissue injury from activated platelets and sequestration sites in the ECM collectively produce an initial proliferative response during the early phase of inflammation which sets the stage for how the tissues will be repaired (Werner & Grose, 2003 ). Tailoring the Interface of Biomaterials to Design Effective Scaffolds. Shu et al. Fibroblasts produce collagen fibers that connect the broken bone ends, while osteoblasts start to form spongy bone. This process is seen as part of normal development, tissue repair … In light of recent results, they’re not so sure. Detailed information on cells, retroviral vectors, infection, induction into osteoblasts, cell staining, real-time RT-PCR, immunostaining, DNA microarray analysis, bisulfite sequencing, surgical procedure, cell transplantation, radiographic and histological assessment, and data analysis is provided in SI Materials and Methods. (C) DNA extracted from human gingival fibroblasts, dOBs (RXOL-transduced fibroblasts), and osteoblasts were tested for CpG methylation at the osteocalcin gene upstream region. Astronomers thought they’d finally figured out where gold and other heavy elements in the universe came from. Real-time RT-PCR analysis revealed no detectable expression of these pluripotent stem cell-specific genes in fibroblasts or in RXOL-infected fibroblasts at 7, 14, and 21 d after transduction (Fig. 2005;19(11):1561–1563. The repair tissue between the broken bone ends, the fibrocartilaginous callus, is composed of both hyaline and fibrocartilage. Alizarin Red S staining showed that the RXOL transduction induced apparent calcium deposition on day 14, and the calcified body occupied almost the entire surface of the culture dish on day 28 (Fig. This is because synovial fibroblasts secrete MMPs such as MMP1, 3, 9, 10, and 13, and accelerate bone erosions in the joints [ 28 ]. Macroscopic observation of the femur showed that the defect parts were totally ossified in dOB-transplanted bones, whereas large defects remained unossified in the fibroblast-transplanted bones . Conversely, fibroblasts sometimes undergo mesenchymal–epithelial transition (MET) to produce epithelia. Depending on the type, severity of the fracture and distance between bone fragments, bones may heal directly by building new bone onto the fracture site (direct bone healing or contact healing) or may heal in a process like endochondral bone formation (indirect bone healing). Fibroblasts are the most common cells of connective tissue in animals. 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