Osteolectin increases bone elongation and body length by promoting growth plate chondrocyte proliferation.

Osteolectin is a recently identified osteogenic growth factor that binds to Integrin α11 (encoded by Itga11), promoting Wnt pathway activation and osteogenic differentiation by bone marrow stromal cells. While Osteolectin and Itga11 are not required for the formation of the skeleton during fetal development, they are required for the maintenance of adult bone mass. Genome-wide association studies in humans reported a single-nucleotide variant (rs182722517) 16 kb downstream of Osteolectin associated with reduced height and plasma Osteolectin levels. In this study, we tested whether Osteolectin promotes bone elongation and found that Osteolectin-deficient mice have shorter bones than those of sex-matched littermate controls. Integrin α11 deficiency in limb mesenchymal progenitors or chondrocytes reduced growth plate chondrocyte proliferation and bone elongation. Recombinant Osteolectin injections increased femur length in juvenile mice. Human bone marrow stromal cells edited to contain the rs182722517 variant produced less Osteolectin and underwent less osteogenic differentiation than that of control cells. These studies identify Osteolectin/Integrin α11 as a regulator of bone elongation and body length in mice and humans.

The effect of parathyroid hormone on osteogenesis is mediated partly by osteolectin.

We previously described a new osteogenic growth factor, osteolectin/Clec11a, which is required for the maintenance of skeletal bone mass during adulthood. Osteolectin binds to Integrin α11 (Itga11), promoting Wnt pathway activation and osteogenic differentiation by leptin receptor+ (LepR+) stromal cells in the bone marrow. Parathyroid hormone (PTH) and sclerostin inhibitor (SOSTi) are bone anabolic agents that are administered to patients with osteoporosis. Here we tested whether osteolectin mediates the effects of PTH or SOSTi on bone formation. We discovered that PTH promoted Osteolectin expression by bone marrow stromal cells within hours of administration and that PTH treatment increased serum osteolectin levels in mice and humans. Osteolectin deficiency in mice attenuated Wnt pathway activation by PTH in bone marrow stromal cells and reduced the osteogenic response to PTH in vitro and in vivo. In contrast, SOSTi did not affect serum osteolectin levels and osteolectin was not required for SOSTi-induced bone formation. Combined administration of osteolectin and PTH, but not osteolectin and SOSTi, additively increased bone volume. PTH thus promotes osteolectin expression and osteolectin mediates part of the effect of PTH on bone formation.

The potential bone regeneration effects of leptin- and osteolectin-coated 3D-printed PCL scaffolds: anin vivostudy.

This study investigated the effectiveness of bone regeneration upon the application of leptin and osteolectin to a three-dimensional (3D) printed poly(ϵ-caprolactone) (PCL) scaffold. A fused deposition modeling 3D bioprinter was used to fabricate scaffolds with a diameter of 4.5 mm, a height of 0.5 mm, and a pore size of 420-520 nm using PCL (molecular weight: 43 000). After amination of the scaffold surface for leptin and osteolectin adhesion, the experimental groups were divided into the PCL scaffold (control), the aminated PCL (PCL/Amine) scaffold, the leptin-coated PCL (PCL/Leptin) scaffold, and the osteolectin-coated PCL (PCL/Osteo) scaffold. Next, the water-soluble tetrazolium salt-1 (WST-1) assay was used to assess cell viability. All groups exhibited cell viability rates of >100%. Female 7-week-old Sprague-Dawley rats were used forin vivoexperiments. Calvarial defects were introduced on the rats' skulls using a 5.5 mm trephine bur. The rats were divided into the PCL (control), PCL/Leptin, and PCL/Osteo scaffold groups. The scaffolds were then inserted into the calvarial defect areas, and the rats were sacrificed after 8-weeks to analyze the defect area. Micro-CT analysis indicated that the leptin- and osteolectin-coated scaffolds exhibited significantly higher bone regeneration. Histological analysis revealed new bone and blood vessels in the calvarial defect area. These findings indicate that the 3D-printed PCL scaffold allows for patient-customized fabrication as well as the easy application of proteins like leptin and osteolectin. Moreover, leptin and osteolectin did not show cytotoxicity and exhibited higher bone regeneration potential than the existing scaffold.

Effect of 3D-printed polycaprolactone/osteolectin scaffolds on the odontogenic differentiation of human dental pulp cells.

Cell-based tissue engineering often requires the use of scaffolds to provide a three-dimensional (3D) framework for cell proliferation and tissue formation. Polycaprolactone (PCL), a type of polymer, has good printability, favorable surface modifiability, adaptability, and biodegradability. However, its large-scale applicability is hindered by its hydrophobic nature, which affects biological properties. Composite materials can be created by adding bioactive materials to the polymer to improve the properties of PCL scaffolds. Osteolectin is an odontogenic factor that promotes the maintenance of the adult skeleton by promoting the differentiation of LepR+ cells into osteoblasts. Therefore, the aim of this study was to evaluate whether 3D-printed PCL/osteolectin scaffolds supply a suitable microenvironment for the odontogenic differentiation of human dental pulp cells (hDPCs). The hDPCs were cultured on 3D-printed PCL scaffolds with or without pores. Cell attachment and cell proliferation were evaluated using EZ-Cytox. The odontogenic differentiation of hDPCs was evaluated by alizarin red S staining and alkaline phosphatase assays. Western blot was used to evaluate the expression of the proteins DSPP and DMP-Results: The attachment of hDPCs to PCL scaffolds with pores was significantly higher than to PCL scaffolds without pores. The odontogenic differentiation of hDPCs was induced more in PCL/osteolectin scaffolds than in PCL scaffolds, but there was no statistically significant difference. 3D-printed PCL scaffolds with pores are suitable for the growth of hDPCs, and the PCL/osteolectin scaffolds can provide a more favorable microenvironment for the odontogenic differentiation of hDPCs.

Osteolectin Promotes Odontoblastic Differentiation in Human Dental Pulp Cells.

INTRODUCTION: Osteolectin is a secreted glycoprotein of the C-type lectin domain superfamily, expressed in bone tissues and is reported as a novel osteogenic factor that promotes bone regeneration. However, the effect of osteolectin on human dental pulp cells (hDPCs) has not been reported. Therefore, we aimed to investigate the odontoblastic differentiation of osteolectin in hDPCs and further attempt to reveal its underlying mechanism. METHODS: Cytotoxicity assays were used to detect the cytotoxicity of osteolectin. The odontoblastic differentiation of hDPCs and its underlying mechanisms were measured by the alkaline phosphatase (ALP) activity, mineralized spots formation, and the gene and protein expression of odontoblastic differentiation through ALP staining, Alizarin red S staining, quantitative real-time polymerase chain reaction, and Western blot analysis, respectively. RESULTS: WST-1 assay showed osteolectin at concentrations below 300 ng/ml was noncytotoxic and safe for hDPCs. The following experiment demonstrated that osteolectin could increase ALP activity, accelerate the mineralization process, and up-regulate the odontogenic differentiation markers in both gene and protein levels (P < .05). Osteolectin stimulated the phosphorylation of ERK, JNK, and Protein kinase B (AKT) in hDPCs. Extracellular signal-regulated kinase (ERK), Jun N-terminal kinase (JNK), and AKT inhibitors decreased ALP activity and mineralization capacity and suppressed the expression of dentin sialophosphoprotein and dentin matrix protein-1. CONCLUSION: Osteolectin can promote odontoblastic differentiation of hDPCs, and the whole process may stimulate ERK, JNK, and AKT signaling pathways by increasing p-ERK, p-JNK, and p-AKT signals.

Identification of Fibroblast Activation Protein as an Osteogenic Suppressor and Anti-osteoporosis Drug Target.

Osteogenic suppressors such as Sclerostin not only regulate skeletal development and regeneration but also serve as anti-osteoporosis drug targets. However, very few druggable suppressors have been identified due to limited understanding of the molecular mechanisms governing osteogenesis. Here, we show that fibroblast activation protein (Fap), a serine protease inhibited by the bone growth factor Osteolectin, is an osteogenic suppressor. Genetic deletion of Fap significantly ameliorates limb trabecular bone loss during aging. Pharmacological inhibition of Fap significantly promotes bone formation and inhibits bone resorption in wild-type mice by differentially regulating canonical Wnt and nuclear factor κB (NF-κB) pathways. Pharmacological inhibition of Fap promotes osteoblast differentiation, inhibits osteoclast differentiation, and significantly attenuates osteoporosis in ovariectomized mice. Epistasis analyses in zebrafish show that Osteolectin functions as an endogenous inhibitor of Fap to promote vertebrae mineralization. Taken together, we identify Fap as an important osteogenic suppressor and a potential drug target to treat osteoporosis.

Integrin alpha11 is an Osteolectin receptor and is required for the maintenance of adult skeletal bone mass.

We previously discovered a new osteogenic growth factor that is required to maintain adult skeletal bone mass, Osteolectin/Clec11a. Osteolectin acts on Leptin Receptor+ (LepR+) skeletal stem cells and other osteogenic progenitors in bone marrow to promote their differentiation into osteoblasts. Here we identify a receptor for Osteolectin, integrin α11, which is expressed by LepR+ cells and osteoblasts. α11ß1 integrin binds Osteolectin with nanomolar affinity and is required for the osteogenic response to Osteolectin. Deletion of Itga11 (which encodes α11) from mouse and human bone marrow stromal cells impaired osteogenic differentiation and blocked their response to Osteolectin. Like Osteolectin deficient mice, Lepr-cre; Itga11fl/fl mice appeared grossly normal but exhibited reduced osteogenesis and accelerated bone loss during adulthood. Osteolectin binding to α11ß1 promoted Wnt pathway activation, which was necessary for the osteogenic response to Osteolectin. This reveals a new mechanism for maintenance of adult bone mass: Wnt pathway activation by Osteolectin/α11ß1 signaling.