Evidence based anti-osteoporosis effects of Periplaneta americana L on osteoblasts, osteoclasts, vascular endothelial cells and bone marrow derived mesenchymal stem cells

Background Kangfuxin (KFX) is the ethanol extract of Periplaneta americana L, which has been widely used in the Traditional Chinese Medicine for the repair and regeneration of injured organ and tissues with long history. This study is to investigate the influence of KFX in the various cellular activities and evaluate the anti-osteoporosis potential of KFX. Methods The influence of the KFX in the cellular activities, including: 1) migration, osteocalcin secretion of osteoblasts; 2) apoptosis of osteoclasts; 3) migration and tube formation of human umbilical vein endothelial cell (HUVEC); and 4) proliferation, cell cycle regulation and migration of bone marrow mesenchymal stem cells (BMSCs), were investigated systematically. Results KFX was shown to significantly 1) Promote of the migration of osteoblasts, HUVEC, and BMSCs; 2) Increase the secretion of osteocalcin and mineralization of osteoblasts; 3) Accelerate the apoptosis of osteoclasts; 4) Stimulate the proliferation and regulate the cell cycle of BMSCs. Conclusion Taken together, these results provide the evidence for the osteogenesis, anti-osteoporosis and angiogenesis effects of KFX, with the mechanism of activating the bone formation through stimulating the osteoblasts and HUVECs, as well as inhibiting the bone absorption by inhibiting the osteoclasts activities. The KFX was definitely shown a promising bone turnover agent with great potential for anti-osteoporosis treatment.


Background
Osteoporosis, the most frequent bone remodeling disease, is characterized by a low bone mass with susceptibility to fracture, which results in an increased risk of adverse health outcomes including decreased quality of life, disability, recurrent hospitalizations, and death [1,2]. In recent years, this disease has become a major health hazard afflicting over 2000 million people worldwide. Therefore, prevention and early treatment of osteoporosis are important to avoid its complications, especially for those osteopenic postmenopausal women.
There are some anti-osteoporotic drugs that could prevent and treat osteoporosis. However, many of these drugs are associated with serious side effects. For several years, estrogen replacement therapy has been used to prevent osteoporosis in postmenopausal women [3], but studies have also reported that estrogen replacement * Correspondence: lhpeng@zju.edu.cn † Equal contributors 1 therapy were associated with headache and increased cancer risk such as breast cancer, endometrial cancer, and ovarian cancer [4,5]. The other example is alendronate, which is associated with osteonecrosis of jaws, increase in the risks of atypical odd fractures and incidence of oesophageal cancer after long-term consumption [6]. Current therapeutics in clinical use included bisphosphonates, anti-RANKL, parathyroid hormone, and strontium ranelate, which may reduce the risk of first and recurrent fractures [7]. Therefore, there is an urgent and increasing need for effective pharmacotherapy.
Kangfuxin (KFX), the ethanol extract of Periplaneta americana L (Dictyoptera; Blattidae) has been exploited as Traditional Chinese Medicine (TCM) for over hundreds of years against disorders such as hepatitis, trauma, gastric ulcers, burns, and heart diseases. Shen Nong's Herbal Classic has recorded that Periplaneta americana L is a traditional medicinal insect possesses many therapeutic activities, for examples, promoting blood circulation, nourishing yin and myogenic effects. It is widely used in clinic in China to treat various diseases, such as blood stasis of bedsore, peptic ulcer disease, trauma, burns by improving local blood circulation, eliminating edema, reducing the local inflammatory reaction, alleviating exudation and promoting wound healing. Given the aforementioned findings, KFX may have therapeutic effects in bone diseases, for example, osteoporosis. However, the influence of KFX in osteoporosis has not been reported elsewhere. In the bone environment, bone metabolism is a dynamic process of bone remodeling that is dependent on the balance between osteoblast-driven bone formation and osteoclastmediated bone resorption [8]. Meanwhile, angiogenesis, also known as neovascularization, is a process that forms new blood vessels from existing vessels, which is of physiological and pathological importance [9]. To regenerate the osteoporotic defects and the bone defects, the angiogenesis and neovascularization are critical processes which provide nutrients and renewable autologous cells to heal the defect. It is well known that VEGF is an angiogenic growth factor which regulates all the critical steps of angiogenic process [10][11][12][13]. Recent studies also shown that BMSCs played important roles in regulating the bone regeneration process, in which the BMSCs could be mobilized and they exert reparative effects at the site of bone defect [14,15]. Therefore, in the present study, with a commercially available product, Xianlingubao, prepared from Chinese herbal formula extract that has been widely used for the osteoporosis treatment in clinic in China as a positive control (PC), the antiosteoporosis potential of the KFX in the four important cell models, including osteoblast, osteoclast, HUVEC and BMSCs were investigated.

Preparation of KFX
Periplaneta americana L was obtained from the Good Agriculture Practice (GAP) breeding base, Sichuan, China. The dried and powered Periplaneta americana L (200 g) was extracted with 90% ethanol (1.2 L) twice at 80∘C. After filtration through 0.22 μm filter membranes and concentrated under reduced pressure and lyophilized into power at a yield of 10% by weight of the starting materials, it was stored at −20∘C until use. Major active compounds of KFX, uracil and hypoxanthine, were detected and quantified by Ultra Performance Liquid Chromatography (UPLC) with external standards. The quantity of Uracil and hypoxanthine in the extract was 0.2 mg/ml and 0.6 mg/ml, respectively. The whole study was performed with the product of the same batch.

Osteoblast migration assay
Osteoblast migration assays were performed using Culture-Insert u-Dishes as described by the manufacturer (Ibidi).70ul suspension solution of Osteoblasts (5x10 5 cells/ml) were seeded into the inner well of the u-Dish and incubated at 37°C and 5% CO 2 overnight. The supernatant and migration chamber inserts were removed, unattached cells were rinsed off, then osteoblasts were incubated with 2 ml serum-free DMEM medium containing KFX (0.1, 1, 2 mg/ml) and PC substance (0.1 and 1 mg/ml), and the blank control group treated with 2 ml serum-free DMEM medium. Osteoblast migration distance was determined by microscopic imaging at 0, 24 and 48 h after post treatment. The wound healing percentage was analyzed using Image J analysis software and calculated with the following formula: Wound healing% ¼ 100%−ðRemaining cell free area= Initial cell free areaÞ%

Mineralized nodule formation in osteoblast
Levels of mineralized nodule formation were evaluated as previously described [16]. Briefly, osteoblasts were seeded at a density of 5× 10 4 cells/wells in 24-well culture plated and incubated at 37°C with 5% CO 2 overnight to form a confluent monolayer. The grown medium was replaced with medium containing various concentrations of KFX (0.1, 1 and 2 mg/ml) and PC substance ((0.1, 1 and 2 mg/ml) and incubated for 7 days at 37°C and 5% CO 2 . The group incubated with 1 ml medium was the blank control group. After 7 days, calcium deposition was determined using alizarin red-S staining. Briefly, cells were washed with PBS, followed by fixation in ice cold 70% ethanol for at least 1 h. Ethanol was removed and cells were rinsed with water and stained with 40 mM Alizarin-red S (pH 4.2) for 10 min at room temperature. Stained cells were further processed by five rinses with water, followed by a 15-min wash in PBS with rotation to reduce nonspecific Alizarin-red stain. Stained cultures were photographed under a microscope (Eclipse Ti-E/U/S; Nikon).

BGP level in osteoblast
BGP was measured from cell culture medium using mouse osteocalcin ELISA Assay Kit (EAGLE Bioscience, OST31-K01) according to the manufacturer's protocol. Briefly, osteoblasts were seeded at a density of 6.67 × 10 4 cells/ml (3 ml per well) in 6 well culture plate and incubated at 37°C with 5% CO 2 overnight to form a confluent monolayer. Then, the grown medium was replaced with medium containing various concentrations of KFX (0.1, 1 and 2 mg/ml) and PC substance ((0.1, 1 and 2 mg/ml) and incubated for 72 h. At 72 h to take on the 0.5 ml cell supernatant and saved at −20°C. Subsequently, osteocalcin standards and these samples were placed in 96-well ELISA microtiter plates (100ul per well) coated with monoclonal detective antibodies and incubated for 90 min at 37°C. Then the osteocalcin antiserum were placed in 96 well plates (100ul per well) and incubated for 60 min at 37°C, followed by three washes with TBS (0.01 M). After that, 100ul ABC reagent were placed in well plate and incubated for 30 min at 37°C, followed by five washes with TBS (0.01 M). 100ul of TMB solution were added and incubated for 30 min at 37°C and kept in dark place. After adding stop solution, absorbance was measured at 405 nm.
Results are presented as the percentage of change of the activity compared with the untreated controls.

Phosphatase (ALP) level in osteoblast
To determine the secretion of ALP stimulated by KFX, the osteoblast were seeded in 24-well plates at a density of 5 × 10 4 cells/well and cultured in DMEM with various concentrations of KFX (0.1, 1 and 2 mg/ml) and PC substance ((0.1, 1 and 2 mg/ml). the levels of ALP activity was performed according to the manufacturer's instruction (Beyotime, Jiangsu, China). While the levels of ALP activity at day 7 of cell culture were determined by measuring the transformation of p-nitrophenylphosphate into p-nitrophenol. Briefly, the cells were detached from the plates using trypsin/EDTA, and centrifuged for 5 min at 1000 rpm after being washed twice with PBS. The cells were resuspended in lysis buffer with 0.2% NP-40. ALP activity was determined by absorbance at 405 nm using pNPP as the substrate. All experiments were performed in triplicate to obtain the average data.

Osteoclast apoptosis assay
An annexin V-FITC apoptosis detection Kit (Beyotime, C1063) was used for Osteoclast assessing apoptosis. In the process of induction from RAW264.7 to osteoclasts, RAW264.7 cells were pre-incubated in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin. In order to induce RAW264.7 to osteoclasts, RAW264.7 cells (2 × 10 5 cells per well) were cultured in the presence of receptor activator of NF-kB ligand (RANK-L, 100 ng/ml) for 7 days. The culture medium was replaced on days 3 and 5. After 7 days, RAW264.7 cells were induced to osteoclasts. Subsequently, the medium were removed and cells were incubated with a dose for KFX (0.1, 1 and 2 mg/ml) with serum-free medium and PC substance (0.1, 0.5 and 1 mg/ ml) with serum-free medium for 72 h at 37°C and 5% CO 2 . The experiments were performed in triplicate and 3 ml per well. Then the cell apoptosis assay was performed using Annexin V-FITC Apoptosis Detection Kit following the manufacturer' instruction. Osteoclasts were harvested using 0.25% trypsin, washed once in PBS, resuspended and incubated with 200ul Annexin V-FITC and 10ul propidium iodide for 20 min at room temperature in the dark following the manufacturer's instructions, which were analyzed with a flow cytometer (FC500MCL, Bechman coult). Data are expressed as the mean ± standard deviation (SD) from three independent experiments. All the data in each assay were determined by student's t-t test to compare the difference of each tested group with the blank control group.

HUVEC chemotaxis assay
Endothelial cells analyzed with the aid of a transwell. In a word, the endothelial cells suspension 1 × 10 4 cells/

BMSC proliferation assay
The isolation and culture of primary BMSCs was performed according to the method previously described [18,19]. Three-week-old Sprague-Dawley male rats (50-60 g) were supplied by Zhejiang University Experimental Animal Center, China. Briefly, rat femurs were cut away from the epiphysis, and bone marrow was flushed out by using a syringe filled with Dulbecco's modified Eagle's medium (DMEM; Gibco, USA) supplemented with 10% fetal bovine serum (FBS; Hyclone, USA), Lglutamine, penicillin (50 U/ml), and streptomycin (50 U/ml). The cell suspension was placed into 25-cm2 flasks (IWAKI Glass, Japan) and cultured at 37°C under 5% CO2. The medium was changed on day 4 of culture and every 3 days thereafter. When the cells of the first passage became sub-confluent, the cells were detached from the flask by using treatment, for 5 min at 37°C, with phosphate-buffered saline (PBS) containing 0.25% (wt) trypsin and 0.02% (wt) ethylene-diamine tetraacetic acid.
Fourth passage cells at sub-confluence were used for all experiments.
Cell proliferation was determined by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide (MTT) assay [20]. Briefly, BMSCs were seeded at a density of 2.5 × 10 4 cells/wells in 96-well culture plate and incubated at 37°C with 5% CO 2 overnight to form a confluent monolayer. Then, the grown medium was replaced with medium containing various concentrations of KFX (0.01, 0.05, 0.25, 1.25, 6.25 and 31.25 mg/ml) and PC substance (0.01, 0.05, 0.25, 1.25, 6.25 and 31.25 mg/ml) and incubated for 24 h. Subsequently, 40 μl of a 5 mg/ml MTT solution in medium were added to 200ul of medium in culture wells and incubated for 4 h at 37°C. Finally, the MTT containing media was removed and the insoluble purple formazan crystals produced by live cells were dissolved in 100 μL of dimethyl sulfoxide (DMSO, Sigma-Aldrich, Milwaukee, WI). The plate was placed on a rocking shaker for at least 10 min and then the purple DMSO solution in each well was monitored at 570 nm using an enzyme-linked immunometric meter reader. The experiments were performed in sextuplicate.

BMSCs cell cycle analysis
BMSCs were seeded at a density of 1 × 10 5 cells/wells in DMEM complete media in six well tissue culture plate. The old media was changed by starved DMEM media (0.2% FBS) and kept for another 12 h. Starved BMSCs were incubated with: i) KFX (0.05, 1.25 and 6.25 mg/ mL), ii) PC substance (0.05, 1.25 and 6.25 mg/mL) for 24 h. FACS analysis of treated and untreated cells was performed by PI staining in a flow cytometer according to the standard literature. Extent of propidium iodide staining of the gated population was displayed in a histogram and the regions are defined as: G0/G1, S, and G2/ M. The data were analyzed using FCS Express V3 software [21].

BMSCs migration assay
For the cell migration assay, BMSCs suspension 1 × 10 4 cells/well in 100ul, is added to the upper compartment of the 24 transwell plate, (Corning #3422). A dose response for KFX (0.1, 1 and 5 mg/ml) and PC substance (0.1, 1 and 5 mg/ml) are added to the lower chamber in 600ul medium, the group treated with medium as blank control. The transwell plate is then incubated for 24 h at 37°C and 5% CO 2 . Subsequently, the cells remaining in the upper chamber were removed and cells on the under surface of the filters were fixed with 4% paraformaldehyde for 30 min. The migrated cells were stained with 5% crystal violet dye solution for 20 min, washed with PBS, and then photographed under a microscope (Eclipse Ti-E/U/S; Nikon).

Statistical analysis
Data are expressed as mean ± SD from at least 3 independent experiments. Statistical analyses were performed using the Students t-test to analyze differences among groups. P < 0.05 was considered statistically significant.

Effect of KFX on osteoblasts Osteoblasts migration assay
The osteoblasts migration is one of the key steps of bone formation process. Therefore, the migration of osteoblasts was monitored in relation to the closure of a denuded area scratched in a confluent monolayer. From the Fig. 1, after treated with 24 h, both KFX (0.1, 1, 2 mg /mL) and PC substance (0.1 mg/mL) induced the obvious stimulation in the cells migration (p < 0.05), compared to the blank control. As time extended to 48 h, KFX (0.1, 1, 2 mg/mL) displayed a further enhancement in the osteoblasts migration (p < 0.05), consistent with that expressed by PC substance (0.1 mg/mL, p < 0.05). However, the high concentration of PC substance (1 mg/mL) did not show stimulation in cells migration. These results altogether demonstrate that KFX could markedly enhance the migration of osteoblasts within the 0.1-2 mg/mL concentration range, indicating its potential in facilitating the new bone formation.

Alizarin red S staining for mineralization
Calcification occurs at nucleation sites known as matrix vesicles present in the lacunae of mineralizing cartilage. It is important process to accumulate Ca 2+ and inorganic phosphate which serve as nucleating agents for the formation of hydroxyapatite, the main inorganic component of bone. Calcification is widely used to indicate the recovery of bone healing. Therefore, Alizarin red S (ARS) staining, that has been used for decades to evaluate calcium-rich deposits by cells in culture was investigated in the tested osteoblasts. As it was shown in Fig. 2b-d, in the osteoblasts incubated with KFX (0.1, 1, 2 mg/mL), large area of positive staining, indicated the wide formation of calcium nodules were shown. By contrast, in the blank control and PC substance groups (0.1, 1, 2 mg/mL, Fig. 2e-g), much less calcium nodules were observed, which reminded the advantageous effect of KFX in promoting the calcification in osteoblasts for bone regeneration.

Osteocalcin (BGP) level and ALP activity assay
Osteocalcin is a factor in maintaining a normal bone calcification rate and inhibiting the cartilage mineralization rate, whose concentration can reflect the rate of bone formation and osteoblast activity. Increasing evidence has shown that BGP level is closely related to the bone formation. The extent of BGP level was quantified and presented as a histogram in Fig. 3a, it can be see that the PC substance (1, 2 mg/mL) group exhibit lower BGP level than the KFX treated group, with the significance identified at the 2 mg/mL of KFX treated group in contrast to the PC substance group (p < 0.01). The ALP activity of osteoblasts was examined. As shown in Fig.  3b, the quantitative analysis revealed that the ALP activity of the KFX treated group has not significantly increased. ALP activity of the cells cultured in PC substance was higher than that in KFX. This noticeable increased BGP level and no influence on ALP activity give further evidence for the significant stimulation of KFX in promoting calcification and inhibiting cartilage mineralization for the bone repair and regeneration.

Effect of KFX on osteoclast apoptosis
Osteoclasts play a pivotal role in bone resorption and are key factors in many bone disorders such as osteoporosis. Besides decreasing osteoclast formation, accelerating the death rate of osteoclasts is one of the widely used strategies to decrease bone resorption. We so tested if the influence of KFX in the osteoclasts apoptosis. As shown in Fig. 4, we observed the significant osteoclast apoptosis induced by KFX (0.1 mg/mL) or PC substance (1 mg/mL) treatment, compared with the blank control group. These results reminded that KFX can inhibit osteoclastic bone resorption and promote bone turnover, at least in part, by inducing osteoclast apoptosis.

Effect of KFX on endothelial cells (HUVECs) In vitro chemotaxis assay
Angiogenesis, the growth of new capillaries from preexisting blood vessels, is a complex process involving endothelial cell (EC) activation, disruption of vascular basement membranes, and migration and proliferation of ECs. It plays a crucial role in many biological processes including reproduction, development and repair. Migration is one of the preconditions of the angiogenesis process. In this study, the effect of KFX on endothelial cells migration was firstly examined by performing transwell assay. As shown in Fig. 5, endothelial cells treated with KFX (1, 5 mg/mL, Fig. 5b-d) and PC substance (0.1, 1 mg/mL, Fig. 5e, f) were displayed the significant higher migration rates in contrast to the blank control group (Fig. 5a).

Tube formation assay
HUVEC tube-formation assay is one of the simple, but well-established in vitro angiogenesis assays based on the ability of HUVECs to form three-dimensional capillarylike tubular structures, when cultured on a gel of growth factor-reduced basement membrane extracts. During the assay, HUVECs differentiate, directionally migrate to align, branch, and form the tubular polygonal networks of blood vessels. The tube-formation ability of HUVECs upon KFX treatment was investigated. As shown in Fig. 6, the tube formation assay in endothelial cells treated by KFX (Fig. 6b-d) and PC substance (Fig. 6e-g) have showed the obvious tubes, compared with the scarce and randomly distributed HUVECs of blank control, in which, no tube has been formed. These results together with the HUVEC migration assay results provided definite evidence that KFX has the strong effects in activating angiogenesis, which will be supply a combination effect for osteoporosis treatment. Fig. 1 Osteoblasts migration assay. The migration of osteoblasts was measured with wound scratch assay. Osteoblasts were treated with KFX (0.1, 1 and 2 mg/mL), or PC substance (0.1 and 1 mg/mL). Significant migration was observed in cells treated with KFX (0.1, 1 and 2 mg/mL) compared with the blank control group. The quantitative evaluation and statistical analysis of wound closure percentage in wound scratch assay measured by Image J software. Results are expressed as means ± SD of three experiments (*P < 0.05, versus blank control)

Effect of KFX on BMSCs Cell proliferation and cell cycle analysis
BMSCs are known to differentiate into various types of cells, including osteoblasts, fibroblasts, chondroblasts, and adipocytes [22], which has great roles in the bone recovery. In the present study, the influence of the KFX in the proliferation, cell cycle, as well as the migration of BMSCs was investigated. From the Fig. 7  (1 mg/mL), d KFX (2 mg/mL), e PC substance (0.1 mg/mL), f PC substance (1 mg/mL), g PC substance (2 mg/mL). Some calcium nodules were headed with yellow arrows Fig. 3 (a) Osteocalcin concentration, and (b) ALP activity in osteoblasts incubated with KFX and PC substance were determined. KFX treated group with the concentration of 2 mg/mL exhibit higher BGP level than PC substance. Data from three independent assays are presented as the mean ± SD (n = 3). Statistical significance was calculated by using t -test, * represents * p < 0.05,**p < 0.01 proliferation, compared with that of blank control group. The significance was shown within 1.25-31.25 mg/mL by positive control, and at 31.25 mg/mL by KFX.
Cell cycle is a progressive event for the division of mother cells to daughter cells through the pathway of a series of events consisting of G 0 -G 1 , S, G 2 /M phase [23]. Using fluorescence activated cell sorting (FACS), we further tested and observed a significantly fast cell cycle progression in KFX and positive control treated BMSCs than those of blank control (Fig. 8). The FACS result showed the extremely significant decrease of the cell population in G0-G1 phase in BMSCs upon the KFX (0.05, 1.25, 6.25 mg /mL) treatment (p < 0.01). Meanwhile, extremely significant increase in G2/M phase was expressed by the KFX treatment (0.05, 1.25, 1.25 mg/mL) (p < 0.01 or p < 0.05). Also, there is a significant increase in cell cycle population of S phase in BMSCs treated with KFX (0.05-6.25 mg /mL). Positive control treatment (0.05, 1.25, 6.25 mg/mL) also expressed the similar activities in decrease the cell population of G0-G1 phase, while increase the cell population of G2/M and S phases. However, in contrast to the KFX treatment, the efficacy of positive treatment is lower. These cell cycles regulation of KFX also suggested its therapeutic application in antiosteoporosis with the BMSCs as a target.

Cell migration assay
The effect of KFX on BMSCs migration was further tested. As shown in Fig. 9, BMSCs treated with KFX (0.1, 1 mg/mL) were displayed a high migration rate compared with blank control group (Fig. 9a-c, migrating endothelial cells were indicated by yellow arrows). However, positive control treatment was not shown to give a significant influence in the BMSCs migration and few BMSCs have been identified for migration ( Fig. 9e-g). The priority of KFX in the migration of BMSCs will facilitate the recovery of osteoporosis.

Discussion
Post-menopausal osteoporosis is the most common bone disease, associated with low bone mineral density caused by an uncoupling of bone resorption from bone formation such that the activities of osteoclasts far outweigh those of the osteoblasts [24]. Osteoblasts could synthesize new collagenous organic matrix like BGP and regulate mineralization of matrix. During the bone formation period, the osteoblasts will express numerous markers such as BGP and various cytokines. BGP is a non-collagenous proteins present in bone tissue, which is the specific marker of bone formation and mostly used as indicators in evaluation of bone formation and bone turnover, and it can maintain normal bone mineralization rate and inhibit cartilage mineralization rate, thereby maintaining bone mass balance involved in bone remodeling [25,26].
This study is first investigation to define the role of osteoblasts treated with KFX showed a significant migration to the bone pit and lay down the organic matrix to form solid bone, completing the bone remodeling process. And the positive control drug was the Xianlingubao capsule, which is an herbal formula based commercially available product and widely used in clinic for osteoprosis treatment [27]. Alizarin red S staining and BGP level assay are used as a measure of the ability of osteoblasts to mineralize the collagen matrix. The results above demonstrated that osteoblasts treated with KFX possessed significantly higher rate of mineralization compared with other tested groups. Hence, KFX were shown to be able to significantly stimulate bone formation by enhancing osteoblastic migration, increasing osteoblasts activity and inducing the formation of calcified nodules. Osteoclasts are multinucleated cells that branch from the monocyte or macrophage lineage. When  Data are means ± SD of six independent experiments and statistical significance was calculated by using t -test (* represents * p < 0.05,**p < 0.01) osteoclastic bone resorption exceeds osteoblastic bone formation, bone density decreases, which can lead to osteoporosis. In this study, results showed that KFX is efficient in enhancing the apoptosis of osteoclasts and might be act as an promising agent for the treatment of osteoclasts induced bone resorptive disorders. To regenerate the osteoporotic defects and the bone defects with critical size, the angiogenesis and neovascularization are critical processes which provide nutrients and renewable autologous cells to heal the defect. In the present study suggested that KFX have also been shown to enhance the migration of HUVECs, and accelerated Data are means ± SD of three independent experiments and Statistical significance was calculated by using t -test (* p < 0.05,**p < 0.01) Fig. 9 KFX and positive control promote BMSCs migration. The migration properties of BMSCs are measured using a transwell migration assay. a Blank control (untreated), b KFX (0.1 mg/mL), c KFX (1 mg/mL), d KFX (5 mg/mL), e PC substance (0.1 mg/mL), f PC substance (1 mg/mL), g PC substance (5 mg/mL). Migrating BMSCs are indicated by yellow arrows. Scale bar = 500 μm. Obvious migration was observed for BMSCs after treated with KFX (0.1 mg/mL) the angiogenesis process as compared with the blank control group. In addition, the migration assay of HUVECs demonstrated that KFX could induce the migration of HUVECs to bone defect site and then provide nutrients for renewable autologous cells.
Previous studies have shown that endogenous BMSCs played important roles in regulating the bone regeneration process, and it possesses the self-renewal capacity and the capability to differentiate into osteoblasts [14,28,29]. It is realized that the modulation of the in vivo differentiation of BMSCs toward osteoblasts and inhibition of osteoclastogenesis, accompanying with the promotion of the angiogenic activity of endothelial cells (ECs) are essential for osteoporotic bone regeneration [30][31][32]. In the present study, our results showed that the KFX is capable in stimulating the proliferation and migration of BMSCs comparing with the culture medium alone. Based on the findings in the current and previous research, it was concluded that KFX has the effect of promoting the migration of BMSCs to the osteoporotic site. At the same time, it also increases the number of BMSCs during the differentiation of BMSCs into osteoblasts. Therefore, the current findings will be helpful in future studies that aim to study the effectiveness of KFX in treating of osteoporosis.

Conclusions
In this study, the in vitro anti-osteoporosis effects of KFX (Periplaneta americana L ethanol extract) in osteoblasts, osteoclast, HUVEC and BMSCs were comprehensively investigated. It was found that KFX has the significant 1) Acceleration of the migration of osteoblasts, HUVEC, and BMSCs; 2) Increase the secretion of osteocalcin and mineralization of osteoblasts; 3) Apoptosis of osteoclasts; 4) The proliferation and cell cycle regulation in BMSCs. These are the first reported evidence for the osteogenesis, anti-osteoporosis and angiogenesis effects of KFX, with the mechanism disclaimed to be related with activating the osteoblasts and HUVECs, as well as inhibit the activity of osteoclasts. The Periplaneta americana L extract was definitely shown to be a promising bone turnover agent with great potential for anti-osteoporosis treatment.