Orally administrable peptides derived from egg yolk promote skeletal repair and ameliorate degenerative skeletal disorders in mouse models.

Introduction: Aging, genetic mutations, and other pathological conditions cause impairment of skeletal growth and bone metabolism, which affect activities of daily living and quality of life in all life stages. Although several drugs have been used in clinical settings and new drugs have been developed for the treatment of skeletal degenerative disorders such as osteoporosis and genetic disorders such as osteogenesis imperfecta (OI), there is clear demand for development of new drugs, especially orally available anabolic drugs that are applicable for a wide range of skeletal disorders. Methods: To identify therapeutic candidates for skeletal disorders, peptide screening was performed. To validate the identified peptides, we performed a bone histomorphometric analysis with rat bone tissues and in vitro cell proliferation assays of skeletal cells. To understand the metabolism of the peptides, we performed a biochemical analysis, followed by in vitro assays for proliferation and differentiation of skeletal cells. We examined the therapeutic efficacy of the identified peptides with several mouse models representing skeletal disorders including bone fracture, osteoporosis, and osteogenesis imperfecta. In vivo therapeutic effects of the candidate were assessed with radiological analysis and mechanical property tests. Results: We identified the egg yolk-derived functional peptide PF201. PF201 promoted in vivo bone formation in rodents and enhanced proliferation of osteoblasts and chondrocytes in vitro. D2, a metabolite of PF201, was present and circulated after digestion and absorption in the digestive tract. D2 had positive impacts on the proliferation and differentiation of mesenchymal stem cells and preosteoblasts. Oral administration of D2 accelerated bone healing in a mouse fracture model. D2 also improved bone strength and fracture healing under ovariectomy-induced osteoporotic conditions in mice, and D2 showed a therapeutic effect in a mouse OI model. Conclusion: D2 is likely to be a candidate for an orally available therapeutic for a range of skeletal disorders.

Overexpression of miR-125b in Osteoblasts Improves Age-Related Changes in Bone Mass and Quality through Suppression of Osteoclast Formation.

We recently reported an unexpected role of osteoblast-derived matrix vesicles in the delivery of microRNAs to bone matrix. Of such microRNAs, we found that miR-125b inhibited osteoclast formation by targeting Prdm1 encoding a transcriptional repressor of anti-osteoclastogenesis factors. Transgenic (Tg) mice overexpressing miR-125b in osteoblasts by using human osteocalcin promoter grow normally but exhibit high trabecular bone mass. We have now further investigated the effects of osteoblast-mediated miR-125b overexpression on skeletal morphogenesis and remodeling during development, aging and in a situation of skeletal repair, i.e., fracture healing. There were no significant differences in the growth plate, primary spongiosa or lateral (periosteal) bone formation and mineral apposition rate between Tg and wild-type (WT) mice during early bone development. However, osteoclast number and medial (endosteal) bone resorption were less in Tg compared to WT mice, concomitant with increased trabecular bone mass. Tg mice were less susceptible to age-dependent changes in bone mass, phosphate/amide I ratio and mechanical strength. In a femoral fracture model, callus formation progressed similarly in Tg and WT mice, but callus resorption was delayed, reflecting the decreased osteoclast numbers associated with the Tg callus. These results indicate that the decreased osteoclastogenesis mediated by miR-125b overexpression in osteoblasts leads to increased bone mass and strength, while preserving bone formation and quality. They also suggest that, in spite of the fact that single miRNAs may target multiple genes, the miR-125b axis may be an attractive therapeutic target for bone loss in various age groups.

Effects of Helioxanthin Derivative-Treated Human Dental Pulp Stem Cells on Fracture Healing.

Bone defects affect patients functionally and psychologically and can decrease quality of life. To resolve these problems, a simple and efficient method of bone regeneration is required. Human dental pulp stem cells (DPSCs) have high proliferative ability and multilineage differentiation potential. In our previous study, we reported a highly efficient method to induce osteogenic differentiation using DPSC sheets treated with a helioxanthin derivative (4-(4-methoxyphenyl)pyrido[40,30:4,5]thieno[2,3-b]pyridine-2-carboxamide (TH)) in a mouse calvarial defect model. However, the localization of the DPSCs after transplantation remains unknown. Therefore, in this study, we investigated the localization of transplanted DPSCs in a mouse fracture model. DPSCs were collected from six healthy patients aged 18-29 years, cultured in normal medium (NM), osteogenic medium (OM), or OM with TH, and fabricated them into cell sheets. To evaluate the efficacy of fracture healing using DPSCs treated with OM+TH, and to clarify the localization of the transplanted DPSC sheets in vivo, we transplanted OM+TH-treated DPSC sheets labeled with PKH26 into mouse tibiae fractures. We demonstrated that transplanted OM+TH-treated DPSCs sheets were localized to the fracture site and facilitated bone formation. These results indicated that transplanted OM+TH-treated DPSCs were localized at fracture sites and directly promoted fracture healing.

Sandwich-type PLLA-nanosheets loaded with BMP-2 induce bone regeneration in critical-sized mouse calvarial defects.

To overcome serious clinical problems caused by large bone defects, various approaches to bone regeneration have been researched, including tissue engineering, biomaterials, stem cells and drug screening. Previously, we developed a free-standing biodegradable polymer nanosheet composed of poly(L-lactic acid) (PLLA) using a simple fabrication process consisting of spin-coating and peeling techniques. Here, we loaded recombinant human bone morphogenetic protein-2 (rhBMP-2) between two 60-nm-thick PLLA nanosheets, and investigated these sandwich-type nanosheets in bone regeneration applications. The PLLA nanosheets displayed constant and sustained release of the loaded rhBMP-2 for over 2months in vitro. Moreover, we implanted the sandwich-type nanosheets with or without rhBMP-2 into critical-sized defects in mouse calvariae. Bone regeneration was evident 4weeks after implantation, and the size and robustness of the regenerated bone had increased by 8weeks after implantation in mice implanted with the rhBMP-2-loaded nanosheets, whereas no significant bone formation occurred over a period of 20weeks in mice implanted with blank nanosheets. The PLLA nanosheets loaded with rhBMP-2 may be useful in bone regenerative medicine; furthermore, the sandwich-type PLLA nanosheet structure may potentially be applied as a potent prolonged sustained-release carrier of other molecules or drugs. STATEMENTS OF SIGNIFICANCE: Here we describe sandwich-type poly(L-lactic acid) (PLLA) nanosheets loaded with recombinant human bone morphogenetic protein-2 (rhBMP-2) as a novel method for bone regeneration. Biodegradable 60-nm-thick PLLA nanosheets display strong adhesion without any adhesive agent. The sandwich-type PLLA nanosheets displayed constant and sustained release of the loaded rhBMP-2 for over 2months in vitro. The nanosheets with rhBMP-2 markedly enhanced bone regeneration when they were implanted into critical-sized defects in mouse calvariae. In addition to their application for bone regeneration, PLLA nanosheets may be useful for various purposes in combination with various drugs or molecules, because they displays excellent capacity as a sustained-release carrier.

Local administration of a hedgehog agonist accelerates fracture healing in a mouse model.

Bone fracture healing is processed through multiple biological stages including the transition from cartilaginous callus to bony callus formation. Because of its specific, temporal and indispensable functions demonstrated by mouse genetic studies, Hedgehog (Hh) signaling is one of the most potent signaling pathways involved in these processes, but the effect of Hh-signaling activation by small compounds on the repair process had not yet been addressed. Here we examined therapeutic effects of local and one shot-administration of the Hh agonist known as smoothened agonist (SAG) on bone fracture healing in a mouse model. A quantitative analysis with three-dimensional micro-computed tomography showed that SAG administration increased the size of both the cartilaginous callus and bony callus at 14 days after the surgery. A histological analysis showed that SAG administration increased the number of cells expressing a proliferation marker and a chondrocyte marker in cartilaginous callus as well as the cells expressing an osteoblast marker in bony callus. These results indicate that the SAG administration resulted in an enhancement of callus formation during bone fracture healing, which is at least in part mediated by an increase in chondrocyte proliferation in cartilaginous callus and the promotion of bone formation in bony callus. Therapeutic strategies with a SAG-mediated protocol may thus be useful for the treatment of bone fractures.

Gli1 haploinsufficiency leads to decreased bone mass with an uncoupling of bone metabolism in adult mice.

Hedgehog (Hh) signaling plays important roles in various development processes. This signaling is necessary for osteoblast formation during endochondral ossification. In contrast to the established roles of Hh signaling in embryonic bone formation, evidence of its roles in adult bone homeostasis is not complete. Here we report the involvement of Gli1, a transcriptional activator induced by Hh signaling activation, in postnatal bone homeostasis under physiological and pathological conditions. Skeletal analyses of Gli1+/- adult mice revealed that Gli1 haploinsufficiency caused decreased bone mass with reduced bone formation and accelerated bone resorption, suggesting an uncoupling of bone metabolism. Hh-mediated osteoblast differentiation was largely impaired in cultures of Gli1+/- precursors, and the impairment was rescued by Gli1 expression via adenoviral transduction. In addition, Gli1+/- precursors showed premature differentiation into osteocytes and increased ability to support osteoclastogenesis. When we compared fracture healing between wild-type and Gli1+/- adult mice, we found that the Gli1+/- mice exhibited impaired fracture healing with insufficient soft callus formation. These data suggest that Gli1, acting downstream of Hh signaling, contributes to adult bone metabolism, in which this molecule not only promotes osteoblast differentiation but also represses osteoblast maturation toward osteocytes to maintain normal bone homeostasis.