The induced pluripotent stem cell (iPSC) is a promising cell source for tissue regeneration. However, the therapeutic value of iPSC technology is limited due to the complexity of induction protocols and potential risks of teratoma formation. A trans‐differentiation approach employing natural factors may allow better control over reprogramming and improved safety. We report here a novel approach to drive trans‐differentiation of human fibroblasts into functional osteoblasts using insulin‐like growth factor binding protein 7 (IGFBP7). We initially determined that media conditioned by human osteoblasts can induce reprogramming of human fibroblasts to functional osteoblasts. Proteomic analysis identified IGFBP7 as being significantly elevated in media conditioned with osteoblasts compared with those with fibroblasts. Recombinant IGFBP7 induced a phenotypic switch from fibroblasts to osteoblasts. The switch was associated with senescence and dependent on autocrine IL‐6 signaling. Our study supports a novel strategy for regenerating bone by using IGFBP7 to trans‐differentiate fibroblasts to osteoblasts.
A panel of 18 recombinant adeno-associated virus (rAAV) variants, both natural and engineered, constitutively expressing Cre recombinase under the cytomegalovirus early enhancer/chicken β actin (CAG) promoter, were screened for their ability to transduce bone in Ai9 fluorescent reporter mice. Transgenic Cre-induced tdTomato expression served as a measure of transduction efficiency and alkaline phosphatase (AP) activity as an osteoblastic marker. Single injections of AAV8, AAV9, and AAV-DJ into midshaft tibial fractures yielded robust tdTomato expression in the callus. Next, the bone cell-specific promoters Sp7 and Col2.3 were tested to restrict Cre expression in an alternate model of systemic delivery by intravenous injection. Although CAG promoter constructs packaged into AAV8 produced high levels of tdTomato in the bone, liver, heart, spleen, and kidney, bone-specific promoter constructs restricted Cre expression to osseous tissues. AAV variants were further tested in vitro in a human osteoblast cell line (hFOB1.19), measuring GFP reporter expression by flow cytometry after 72 h. AAV2, AAV5, and AAV-DJ showed the highest transduction efficiency. In summary, we produced AAV vectors for selective and high-efficiency in vivo gene delivery to murine bone. The AAV8-Sp7-Cre vector has significant practical applications for inducing gene deletion postnatally in floxed mouse models.
Osteogenesis imperfecta (OI) describes a series of genetic bone fragility disorders that can have a substantive impact on patient quality of life. The multidisciplinary approach to management of children and adults with OI primarily involves the administration of antiresorptive medication, allied health (physiotherapy and occupational therapy), and orthopedic surgery. However, advances in gene editing technology and gene therapy vectors bring with them the promise of gene-targeted interventions to provide an enduring or perhaps permanent cure for OI. This review describes emergent technologies for cell-and gene-targeted therapies, major hurdles to their implementation, and the prospects of their future success with a focus on bone disorders.
Fracture repair is a normal physiological response to bone injury. During the process of bony callus formation, a lacuno-canalicular network (LCN) is formed de novo that evolves with callus remodeling. Our aim was the longitudinal assessment of the development and evolution of the LCN during fracture repair. To this end, 45 adult wild type C57BL/6 mice underwent closed tibial fracture surgery. Fractured and intact contralateral tibiae were harvested after 2, 3, and 6 weeks of bone healing (n=15/group). High-resolution micro-computed tomography (µCT) and deconvolution microscopy (DV) approaches were applied to quantify lacunar number density from the calluses and intact bone. On histological sections, Goldner's trichrome staining was used to assess lacunar occupancy, fluorescein isothiocyanate staining to visualize the canalicular network, This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as
Wnt antagonist Dkk1 is a negative regulator of bone formation and Dkk1 heterozygous mice display a high bone mass phenotype. Complete loss of Dkk1 function disrupts embryonic head development. Homozygous Dkk1 mice that were heterozygous for Wnt3 loss of function mutation (termed Dkk1 KO) are viable and allowed studying the effects of homozygous inactivation of Dkk1 on bone formation. Dkk1 KO mice showed a high bone mass phenotype exceeding that of heterozygous mice as well as a high incidence of polydactyly and kinky tails. Whole body bone density was increased in the Dkk1 KO mice as shown by longitudinal dual-energy X-ray absorptiometry. MicroCT analysis of the distal femur revealed up to 3-fold increases in trabecular bone volume and up to 2-fold increases in the vertebrae, compared to wild type controls. Cortical bone was increased in both the tibiae and vertebrae, which correlated with increased strength in tibial 4-point bending and vertebral compression tests. Dynamic histomorphometry identified increased bone formation as the mechanism underlying the high bone mass phenotype in Dkk1 KO mice, with no changes in bone resorption. Mice featuring only Wnt3 heterozygosity showed no evident bone phenotype. Our findings highlight a critical role for Dkk1 in the regulation of bone formation and a gene dose-dependent response to loss of DKK1 function. Targeting Dkk1 to enhance bone formation offers therapeutic potential for osteoporosis.
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