An Experiment Study On Heparanase Stimulating Osteogenesis And Bone Fracture Healing

Objective and BackgroundOsteoporosis and associated fractures are important cause of mortality and morbidity. Heparanase (HPSE), an angiogenic factor, specifically degrades ignali sulfate proteoglycans ubiquitously associated with the cell surface and extracellular matrix (ECM). However, its anabolic function in bone remodeling remains unclear. This study aim:1. to determine:the effects of HPSE in stimulation of osteoblasts (Obs);2. to compare the difference of HPSE in stimulation of osteoblasts proliferation, differentiation, and further promote bone matrix mineralization;3. to observe the effect of HPSE in the process of fracture healing in mice fracture model, and further investigate the mechanism of HPSE in stimulating of fracture healing.Methods1. HPSE was prepared from human platelets (extraction and purification in TORU of Australian national university). Osteoblasts were isolated from normal human bone specimens from7healthy individuals and trebecular bone specimens from7patients with osteoporosis at diagnosis. Primary cell culture of human osteoblasts was performed in vitro. A heparanase stock solution (400μg/mL) was initially made with culture medium (α-MEM) and sterilized with a0.22μm filter. The cells were seeded into25cm2-flasks for alkaline phosphatase (ALP) assay and flow cytometry,96-well plates for ATPLite assay, and6-well plates for Alizarin Red-S Assay at the density of4000cells/cm2. After48-hour culture, the cells were treated with heparanase at the final concentration of a series of dilutions,6μg/mL,3μg/mL, lug/mL,0.1μg/mL,0.01μg/mL, and0μg/mL control. After treatment, cells were cultured for another3,5or14days, depending on different experiments. At7-day culture, culture media were changed and fresh heparanase were added. In addition, osteogenic protein-1(OP-1,0.2ug/mL) was used instead of heparanase as a positive control.2. Thirty-six balb/C mice with8-10weeks were used in this experiment. Eighteen mice were randomly assigned into three HPSE treatment groups and the other18mice were randomly assigned into three control groups. The model of mice with fractured femurs was established in all of mice. In HPSE treatment groups,10ug/mL HPSE were injected into fracture sites and the same dose of normal saline were injected into fracture sites in control groups. The bone samples were collected at7d,14d and28d after operations. The sizes of callus were detected by anterior-posterior and lateral X-rays. All bone samples were sent to the university of Sydney and scanned and the trabecular bones were reconstructed with Micro-CT, the parameters, such as total volume, bone volume, etc were measured. Trabecular bones were measured in a distal femoral metaphyseal segment, the trabecular number (Tb.N), the trabecular thickness (Tb.Th), the trabecular separation (Tb.Sp) and the trabecular pattern factor (Tb.Pf) were measured to assess the effect of HPSE in the process of fracture healing. The bone samples were cut into5um thick sections and stained with hematoxylin and eosin (H&E) and evaluated qualitatively of bone callus under light microscopy. The mice osteoblasts were isolated from specimens in HPSE treatment group and control group at the7days after operations. The mice osteoblasts were cultured in vitro. HPSE were added into culture system and the effects of HPSE on84osteogenetic related genes were assessed by RT2-qPCR.Results1. Upon observation by the bright field microscopy, cells both with and without exposure to heparanase exhibited the same normal osteoblast phenotype, suggesting that cell morphology is not altered by the presence of heparanase. Heparanase-treated osteoblasts were consistently increased in cell numbers rather than in cell size in comparison to the non-treatment controls. Osteoblasts derived from patients with osteoporosis were much more sensitive to heparanase stimulation than those derived from healthy individuals, as a greater increase in cell number was observed in the former cell cultures. Although the increased level in cell proliferation is much higher in healthy subjects compared to osteoporotic subjects under the same level of heparanase stimulation, heparanase ignaling significantly (p<0.05) stimulated cell proliferation in all subjects (n=14) at5-day culture, indicating that heparanase stimulated cell proliferation in a dose-dependent manner. The increased level in ALP activity is much higher in osteoporotic subjects than healthy subjects under the same level of heparanase stimulation, heparanase certainly significantly increased ALP activity in all subjects at5-day culture, indicating that heparanase stimulated ALP activity in a dose-dependent manner. Quantitative analysis of calcium levels from the measurement of the amount of AR-S bound to mineralized nodules was performed.2. The extent of mineralized nodules was markedly increased compared with the non-treatment control in both osteoporotic and healthy subjects. Although the increased level in calcium content was much higher in osteoporotic subjects than in healthy subjects obtained with identical heparanase concentrations. The data from ELISA assay and flow cytometry suggests that the stimulatory effects of heparanase on human osteoblasts are related to histone modification, predominately by phosphorylation at S28of histone H3rather than phosphorylation at S10or acetylation of histone H3.In the mice fracture model, Soft X-ray images showed the formation of external bony callus was at the fracture site of two groups at1week post-fracture. The external bony callus continued to format and fracture line was not clear in two groups at2week post-fracture. The fracture line was disappeared and the fracture was union by X-rays at4week post-fracture. The averaged maximal diameter of callus in HPSE treatment group was larger than that in control group by anterior-posterior X-rays at1week post-fracture (3.26±0.13mm VS2.51±0.12mm, P=0.0321). But the averaged maximal diameter of callus measured by anterior-posterior X-rays at2and4weeks post-fracture was not different between the two groups. The results measured by lateral X-rays at1,2and4week post-fracture were also not different between the two groups. Sections obtained from the HPSE treatment group at1week post-fracture showed more hyaline cartilage-like region and more spicules of new bone in the fracture gap. There were more blood vessels among the bone matrix of the callus in the HPSE treatment group than the control group. Two weeks after fractures, the callus was completely occupied with cartilage between new bone at the periphery and chondrocytes at the center. Healing continued with a complete bridging of the fracture with new bone was evident4weeks after fracture. According to the results of Micro-CT, the average BV/TV in the treatment group and control group were9.24±0.9%and4.64±0.7%at1week post-fracture, respectively. There was significantly different between the two groups. The data from the3D reconstruction of trabecular bone, the averaged Tb.N in treatment group and control group were0.28±0.081/mm and0.1326±0.021/mm, respectively. The averaged Tb.Sp were1.67±0.12mm and2.36±0.14mm, respectively; the average Tb.Pf were-2.90±0.811/mm and4.97±1.641/mm, respectively. The averaged Tb.N, Tb.SP and Tb.Pf were significantly different between the treatment group and control group at1week post-fracture. At2weeks post-fracture, the averaged BV/TV and Tb.N in the treatment group and control group were9.24±0.4%and7.36±0.2%,0.32±0.021/mm and0.28±0.031/mm, respectively. They were significantly different between the two groups, but the averaged Tb.SP, Tb.Pf, and Tb.Th were not different between the two groups at2week post-fracture. The averaged BV. TV, BV/TV, Tb.N, Tb.Sp, Tb.Pf and Tb.Th were not different between the treatment and control groups at4weeks post-fracture. Heparanase regulated the expression of some osteogenic genes in treated specimens, which showed up-expression after treatment, such as ALPL. BMP2. CSF2, FGFR2, and SMAD2, suggesting that HPSE was universal participation in the stimulation of osteogensis. Seventeen genes, such as BMP6, were up-regulation, and eight genes, such as RUNX2, were down-regulation after treatment with HPSE.Conclusions1. HPSE can promote the proliferation, differentiation and bone matrix mineralization of osteoblasts derived from healthy and osteoporotic subjects in the culture system in vitro. 2. With fractured femur model in mice, we have obtained preliminary evidence that HPSE promotes fracture healing at the early stage.3. HPSE regulates bone ignaling likely via TGFβ1/SMAD ignaling pathway. These findings support a novel therapeutic use of heparanase as an anabolic agent in the prevention and treatment of osteoporosis and fractures.