Abstract
The application of appropriate animal models and techniques for the study of osteoporosis is important. Lanyu pigs, a local miniature breed, have been widely used in various biomedical studies in Taiwan. This study aimed to induce bone loss in Lanyu pigs and to examine whether porcine induced pluripotent stem cell (piPSC)-derived osteoblast-like cells could recover bone mass of tibiae via local cell transplantation. piPSCs were directed to differentiate into osteoblast-like cells using osteogenic medium, and differentiated cells expressed osteogenic markers and phenotypes. Twenty mature female Lanyu pigs were divided into four groups, including control (C, 1% calcium diet), treatment 1 (T1, ovariectomy + 1% calcium diet), treatment 2 (T2, ovariectomy + 0.5% calcium diet), and treatment 3 (T3, ovariectomy + 0.5% calcium diet + 1 mg/kg of prednisolone) and were subjected to bone loss induction for twelve months. Micro-CT images revealed that the lowest trabecular bone parameters, such as trabecular bone volume, thickness, separation, number, and total porosity, were detected in the T3 group. The lowest proportions of cortical bone in the proximal metaphysis, proximal diaphysis, and distal diaphysis were also found in the T3 group. These results indicate that ovariectomy, calcium restriction, and prednisolone administration can be applied to induce proper bone loss in Lanyu pigs. After bone loss induction, pigs were subjected to cell transplantation in the left tibiae and were maintained for another six months. Results showed that transplanted piPSC-derived osteoblast-like cells significantly improved trabecular bone structures at transplanted sites and maintained cortical bone structures in the proximal metaphysis. In conclusion, the therapeutic potential of piPSC-derived osteoblast-like cells was confirmed via cell transplantation in the left tibiae of Lanyu pigs. These findings reveal the therapeutic potential of piPSCs for glucocorticoid-induced bone loss in pig models.
Highlights
Osteoporosis is a prevalent health problem characterized by a series of serious bone disorders
The ear tissues were trypsinized by 0.25% (w/v) trypsin-0.02 mM EDTA (Invitrogen, Grand Island, NY, USA) and were maintained in cell culture medium as described previously [20]. pluripotent stem cell (piPSC) expressing green fluorescent protein (GFP) were generated from porcine ear fibroblasts transfected with human OCT4, SOX2, KLF4, and c-MYC genes cloned into lentivirus vectors (TLC-TRE-iPS-II, Tseng Hsiang Life Science LTD, Taipei, Taiwan) and were maintained in embryonic stem cell (ESC) culture medium as described previously [20]
To induce differentiation of osteoblast-like cells, trypsinized piPSCs were maintained in osteogenic medium, containing MEM-α, 10% fetal bovine serum (FBS), 50 μM L-ascorbic acid, 10 mM β-glycerophosphate, 100 nM dexamethasone, 10 nM 1α,25(OH)2D3, and 40 ng/mL bone morphogenetic protein 2 (BMP2)
Summary
Osteoporosis is a prevalent health problem characterized by a series of serious bone disorders. Rodents have the characteristics of anatomical structures, bone remodeling, bone loss caused by estrogen deficiency in common with humans and have been animal models in osteoporosis research for several decades. The genetic and physiological differences to humans caused by body size should be concerned when compared with other larger mammalian species [1]. Pigs have several characteristics in common with humans, such as organ size, anatomical structures, bone remodeling, bone loss caused by estrogen deficiency, and nonseasonal estrous cycle [2,3,4,5,6]. Due to the body size of mini pigs, they are suitable for use in long-term studies and for replacement of rodents in osteoporosis research [1].
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