Therapeutic effect of Guijiajiao(Colla Carapacis et Plastri)on bone regeneration in rats and zebrafish

INTRODUCTION

Traditional Chinese Medicine has been used to treat various diseases worldwide.The medicines used for bone diseases include Guilu Erxianjiao(tortoise shell and deer horn formula),Yougui Wan,Zuogui Wan,and Yangchin Chienpu Wan.Among them,Guilu Erxianjiao is the most well known and widely used.Guilu Erxianjiao has four ingredients:Lujiao(Cornu Cervi Elaphi),Guijia(Carapax et Plastrum Testudinis),Gouqizi(Fructus Lycii),and Renshen(Radix Ginseng).In Chinese medicine,it is a common prescription to strengthen muscles and bones,and replenishQi,and it has a particularly significant effect in the treatment of osteoporosis.Most literature on the use of Guilu Erxian Jiao for bone disease has used syndrome differentiation as the scientific method to observe its efficacy or the degree of improvement in osteoporosis.

Maoet al1showed that culture medium containing Guilu Erxianjiao significantly increases insulin-like growth factor-1 and its activity in osteoblasts,while significantly inhibiting the activities of osteoclasts,bone resorption activity,and tartrate-resistant acid phosphatase.After 2 months of Guilu Erxianjiao use,Linet al2observed no significant effect on kidney functions,liver functions,or blood factors,and no serious adverse reactions.In addition,Wanget al3conducted a clinical evaluation of the effect of Guilu Erxianjiao on women with menopausal symptoms.Their study demonstrated that administering Guilu Erxianjiao elevates the concentration of estradiol in serum and relieves menopausal symptoms.

At 4 weeks post-surgery,there was no significant difference between control and 0.25 g/kg CCP groups,whereas the 1.25 g/kg CCP group had a thin layer of tissue compared with the other two groups.At 8 weeks post-surgery,there was still no significant difference between control and 0.25 g/kg CCP groups,whereas the 1.25 g/kg CCP group had a thicker layer of tissue over the wound than the control and 0.25 g/kg CCP groups.At 12 weeks post-surgery,recovery was observed in both control and 0.25 g/kg CCP groups,whereas the 1.25 g/kg CCP group had almost completely returned to normal(Figure 8A).Observations were consistent with magnified images of the wounds.Collagen typeⅠis a protein biomarker of osteogenesis.Our results showed no significant difference in the expression of collagen typeⅠbetween control and 0.25 g/kg CCP groups at 4 weeks post-surgery based on immunohistochemical staining,whereas the 1.25 g/kg CCP group had more collage type I expression than the two other groups(Figure 8B).

In vivoandin vitromodels have been used to study influences on bone formation in a previous study.6The differentiation of osteoprogenitor cells into osteoblasts has been studied using various models such as the MC3T3-E1 cell line isolated from mouse skull bone and the MG63 human bone cancer cell line.Most studies of bone formation have focused on maturation and differentiation of osteoblasts.7Osteoblasts are a mixed cell population including both mature and immature cells,but only mature cells have the ability to form bones.During the maturation of osteoblasts,factors such as runt-related transcription factor-2(Runx-2),osterix,and alkaline phosphatase(ALP)serve as osteogenesis markers.Runx-2 regulates the expression of certain genes such as osteocalcin,osteopontin,and collagen typeⅠ.When pre-osteoblasts differentiate into mature osteoblasts,the presence of marker molecules can be observed,such as osteopontin and osteocalcin.8Osteopontin is a type of glycoprotein that acts as a negative regulator of bone formation.9Mature osteoblasts and hypertrophic chondrocytes both synthesize this protein.10In addition to downstream factors such as osteocalcin and Runx-2,upstream factors such as phosphorylated (p)-extracellular signal-regulated kinase(ERK)and p-Janus kinase(JNK)are involved in the maturation and differentiation of osteocytes.

Previous studies have reported that the expression of osteoblastogenic genes and their functions occur via activation of ERK in the mitogen-activated protein kinase(MAPK)pathway,which promotes the differentiation of osteocytes.11In addition,Suzukiet al12have reported that the activity of p38 can be used to control the differentiation of osteoblasts through regulation of ALP.Furthermore,Tetsuya reported that inhibition of specific JNK molecules significantly reduces the expression of late stage osteogenic differentiation proteins,such as osteocalcin,and does not inhibit ALP activity or early markers of osteogenic differentiation,such as osteopontin.13In summary,molecules such as p38,ERK,and JNK play important roles in osteogenic differentiation and maturation,and the effects of CCP on the bone formation mechanisms discussed above have not been studied yet.

This study aimed to investigate the effects of CCP on differentiation and proliferation in MG-63 human osteosarcoma cells,the MAPK pathway,and expression of osteogenic genes.In addition,we explored the osteogenicity of CCP by evaluating skeletal development in zebrafish and skull defects in rats to increase our understanding of the relevant mechanisms in osteogenesis.

MATERIALS AND METHODS

Ethics approval

The use of animals conformed to the Guiding Principles for the Care and Use of Animals published by the American Physiological Society and was approved by the National Sun Yat-Sen University Animal Care and Use Committee.

设第i类生产设备的第k台机器在一个工作日内用于生产第j种零件的时间为xikj(k=1,2,…,pi)．类似地，对每一台设备，被分配用于生产所有零件的工作时间总和为1，即有约束条件:

Preparation of CCP

Red-eared slider(Trachemys scripta elegans)was used in this study.A total of 20 kg tortoise plastron was placed in a copper pot with 14.5 L water.After constant boiling for 16 h,310 mL of the supernatant was collected.A benchtop autoclave(Tomin medical equipment Co.,Ltd.Taipei,Taiwan)was used to sterilize the CCP.The final concentration of CCP solution was 250µg/mL.CCP was ready for use after cooling to room temperature.Sterile phosphate-buffered saline(PBS)was used to dilute CCP to the desired concentrations.

Cell culture

The MG-63 osteosarcoma cell line was obtained from the American Tissue Culture Collection(Rockville,MD,USA).The cells were grown at 37℃in minimum essential medium(Gibco,Grand Island,NY,USA)containing 10%heat-inactivated fetal bovine serum.

Cell viability

土地利用分级是将土地利用程度按照土地自然综合体在社会因素影响下的状况分成若干级［11］。中国资源环境数据库中，从生态学角度将土地利用分级定义为4级，如表2所示。

ALP activity

After incubation with CCP for 3 or 7 d,cells were washed three times with PBS,harvested with scratch,and resuspended in PBS containing 1%Triton X-100.ALP activity was assayed by a spectrophotometric method using para-nitrophenyl phosphate(N7653;Sigma Aldrich,St.Louis,MO,USA)as the substrate.The OD was measured at 405 nm with a microplate reader.The results were normalized to protein levels and expressed as percentages of the control.15

Quantitative real-time polymerase chain reaction(PCR)

Total RNA was extracted from MG-63 cells of each treatment group using TRIzol reagent(Life Technologies,Carlsbad,CA,USA),according to the manufacturer's instructions.RNA was reverse transcribed to single-stranded complementary DNA(cDNA)using the iScript cDNA synthesis kit(Bio-Rad Laboratories,Hercules,CA,USA).Real-time PCR was then performed using iQ SYBR-Green Supermix(Bio-Rad Laboratories)for human Runx-2,osteopontin,and osteocalcin in a Bio-Rad real-time PCR system.The reaction conditions were 95℃for 3 min and then 39 cycles of 95℃for 10 s and 55℃for 30 s,followed by 95℃for 10 s,65℃for 5 s and ending at 95℃.Primers were presented in Table 1.

Mineralization assay

Cells were seeded in 24-well plates containing growth medium supplemented with 50µg/mL L-ascorbic acid and 10 mM β-glycerophosphate.The cells and matrix were washed twice with 20 mM Tris-buffered saline(TBS)containing 0.15 M NaCl(pH 7.4),fixed in ice-cold 75%(vol/vol)ethanol for 30 min,and then air dried.Calcium deposition was determined by quantitative Alizarin red-S staining.Briefly,the ethanol-fixed cells and matrix were stained for 1 h with 40 mM Alizarin red-S(pH 4.2)and then extensively rinsed with water.The bound dye was then completely eluted with 10%(wt/vol)cetylpyridinium chloride and absorbance was measured at 570 nm.Alizarin red-S in samples was quantified according to a standard curve and expressed as micromoles of dye bound per 1×104cells,in which the cell number was determined from replicate cultures using the crystal violet assay.16

将利用GPS实际测量的数据于南方CASS9.0软件中展点，并沿着测量边界点绘制一个闭合的区域作为计算的边界[10]，绘制边界使用“复合线”并要求边界为闭合边界。表面积计算方法与实体表面积计算方法均使用同一个固定的计算边界线以便比较分析两种方法的测量结果，计算结果见表2。

Western blotting

Western blotting was performed as described in our previous studies.17,18Immunoreactive bands were visualized by enhanced chemiluminescence(ECL kit;Millipore,Bedford,MA,USA)and the UVP BioChemi Imaging System.Relative densitometric quantification was performed using LabWorks 4.0 software(UVP,Upland,CA,USA).A monoclonal antibody against β-actin was used as the internal control for protein loading.Relative variations between bands of the variously treated samples and control were calculated using the same image.

Zebrafish maintenance

The AB strain of wild-type zebrafish was used in this study.Embryos were collected after natural spawning,staged according to standard criteria,and raised synchronously at 28.5℃in Hank's buffer(13.7 mM Na-Cl,540 µM KCl,25 µM Na2HPO4,44 µM KH2PO4,300 µM CaCl2,100 µM MgSO4,and 420 µM NaHCO3,pH 7.4).No additional maintenance was required because the embryos receive nourishment from the attached yolk sac.

Table 1 Primers of qPCR for Guijiajiao(Colla Carapaciset Plastri)in MG63 cell line

Notes:qPCR:quantitative real-time polymerase chain reaction;Runx-2:runt-related transcription factor-2.

Reverse 5′-AgAACAAgggggCCgTTA-3′5′-TgTggAATTCACggCTgA-3′5′-AgggTgCCTggAgAggAg-3′Gene name Runx-2 Osteopontin Osteocalcin Forward 5′-CCACCTTAAATggCCTAgCA-3′5′-CTgAAACCCACAgCCACA-3′5′-TgACgAgTTggCTgACCA-3′

Screening of CCP concentrations in zebrafish

Zebrafish larvae at 2 d post-fertilization(dpf)were placed in the wells of a 24-well plate with various concentrations of CCP for 72 h.CCP was changed every 24 h.The control group was Hank's buffer only.During the experiment,zebrafish larval death in each concentration was recorded.

Calcein staining of zebrafish larvae

Zebrafish larvae at 2 dpf were treated with various concentrations of CCP for 3 d in a 24-well plate.At 5 dpf,fish were transferred into 6-cm dishes(eight fish/dish)and bone was stained by a calcein solution.The calcein solution was prepared at 0.2%by dissolving 2 g calcein powder(Sigma Chemical,St.Louis,MO,USA)in 1 L deionized water.The immersion time was 10 min at 7 dpf.After the immersion,the embryos were repeatedly rinsed in fresh water and then allowed to stand for 10 min to allow the excess stain to diffuse from the larval tissue.The embryos were then euthanized in tricaine-methanesulfonate(MS 222)and mounted on depression glass slides with methyl-cellulose(3%).Images were acquired using a Leica Z16 APO microscope(Leica Instruments Inc.,Wetzlar,Germany)combined with a SPOT Idea digital camera system(Diagnostic Instruments,Inc.,Sterling Heights,MI,USA).Each image was acquired at 200 ms,and then fluorescence of calcein staining in images was calculated using NIH ImageJ.The data are presented as the percentage of the relative density of the control group in each experiment.The outline of individual zebrafish was circled using the freehand tool to create regions of interest(ROIs).The average intensity of the same ROI was analyzed in green channels.Then,the ratio was obtained for each region and the average of the ratio was obtained for all ROIs in all images.19

Animals and surgery

All experiments are reported in accordance with ARRIVE guidelines.20Thirty-six 10-week-old male Wistar rats(30-350 g)were maintained in Plexiglas cages in a temperature-controlled(22℃)room under a 12-h light/dark cycle,and provided free access to food and water.The dorsal part of the cranium was shaved and disinfected with povidone-iodine and a sagittal incision of 15 mm was cut over the scalp of the animal.A flap was raised and a bone defect(5 mm in diameter)was created at the middle of the sagittal plane using a slow-speed trephine bur(Stoma,Emmingen-Liptingen,Germany)with saline irrigation to protect host bone from heat damage.The periosteum was repositioned and sutured with a 7-0 Vicryl polyglactin suture(Johnson&Johnson Pty.,Ltd.,Tokyo,Japan),and the skin was sutured with a 4-0 Nesco suture(Alfresa Pharma Co.,Osaka,Japan).The animals were sacrificed at 4,8,and 12 weeks after surgery.

Bone repair area quantification

6 Zhou Y,Fan W,Prasadam I,Crawford R,Xiao Y.Implantation of osteogenic differentiated donor mesenchymal stem cells causes recruitment of host cells.J Tissue Eng Regen Med 2015;9(2):118-126.

Sample collection and hematoxylin and eosin(HE)staining

Rats were sacrificed by inducing deep anesthesia with 2%isoflurane.After obtaining the skulls,the whole skull was fixed in 10%neutral buffered formalin for 3 d and then decalcified for 2 weeks in buffered 12.5%ethylenediaminetetraacetic acid with 10%neutral buffered formalin.The strip of skull bone with the defect in the middle was processed and embedded in paraffin.Using a microtome,5 mm of the samples was cut away to transfer 1 mm-thick sections from the central area of the original defect site onto a glass slide.The sections were stained with HE.

Immunohistochemistry of collagen type I

Each 1µm-thick section from a paraffin-embedded sample was mounted on a slide,deparaffinized with xylene,and dehydrated using a graded ethanol series.Then,endogenous peroxidase was quenched with 3%H2O2for 8 min.Antigens were retrieved by enzymatic digestion with proteinase K(20 mM;Sigma,St.Louis,MO,USA)in PBS for 45 min and then washed with TBS.Non-specific adsorption was minimized by incubating the sections in 5%normal horse serum/PBS for 30 min.The sections were incubated overnight at 4℃with an anti-collagen typeⅠ antibody(1∶100 dilution,catalog No.234167;Calbiochem,USA).Specific labeling was detected with biotin-conjugated anti-rabbit IgG(Vector Laboratories,Burlingame,CA,USA)and avidin-biotin peroxidase in combination with an ABC kit(Vectastain ABC kit;Vector Labs).Finally,the sections were reacted with 3,3′-diaminobenzidine tetrahydrochloride for 1-2 min.Sections were analyzed using a microscope(DM 6000B,Leica Inc.,Wetzlar,Germany)in combination with a digital image output system(idea SPOT;Diagnostic,RT slider SPOT,Hamilton,CA,USA).

纵观高校教育教学督导和评价机制的产生、发展和调整，可以认为所有的学校都非常重视教学工作，教育教学督导和质量评价的机制运行在当时主要依赖于校级的行政管理。这就导致了教育教学督导和评价机制从诞生开始就具有行政管理的性质，这使得督导小组与行政干部彼此分离又相互叠加，从而产生了一些现行的实施特点。

Statistical analysis

All experiments were repeated at least four times.Data are represented as the mean±standard error of the mean.For immunoreactivity data,the intensity of each band is expressed as the relative OD calculated from average control OD values obtained from all controls.Data were analyzed by one-way analysis of variance,and Duncan's method was used to compare multirange differences between groups.AP-value of less than 0.05 was considered as statistically significant.

RESULTS

Amino acid composition of CCP

As shown in Table 2,the CCP contained 18 amino acids:arginine,alanine,glycine,valine,leucine,isoleucine,proline,glutamine,methionine,aspartate,hydroxyproline,phenylalanine,cysteine,lysine,histidine,tyrosine,serine,and threonine.The total content of hydrophobic amino acids was 42.07%.Glutamate(13.32%)and glycine(13.23%)were the major amino acids of CCP.Cysteine and proline were the lowest amino acids of the collagen peptide.

C级供应商：该级别的供应商绩效水平低于B级，处于行业一般水平，当发生采购需求时需对其进行重点监控，例如加强关键工序检查，增加抽样数、提高抽样率等，保证各供应环节的产品和服务质量。

Table 2 Amino acid composition of Guijiajiao(Colla Carapaciset Plastri)

Note:SEM:standard error of the mean.

Content(mg/100 g)Arginine Alanine Glycine Valine Leucine Isoleucine Proline Glutamate Methionine Aspartate Hydroxyproline Phenylalanine Cystine Lysine Histidine Tyrosine Serine Threonine Ca Mg Mean 15.24 73.09 139.07 68.01 78.17 60.52 4.06 140.08 19.33 111.43 74.61 48.73 1.02 36.54 52.62 12.18 60.77 55.63 2.48 1.13 SEM 0.57 2.75 5.22 2.55 2.93 1.72 0.15 5.26 0.76 5.54 2.81 1.84 0.04 1.37 2.30 0.46 2.73 2.27 0.42 0.30

Mineralization of the human osteosarcoma cell line MG-63

After treating MG-63 cells with various concentrations of CCP for 1,7,and 14 d,a significant increase in cell numbers was observed in groups treated with 50,125,and 250µg/mL CCP for 7 and 14 d(Figure 1A).ALP assay results showed that,after treatment with 125 and 250µg/mL CCP for 3 d,ALP activity in MG-63 cells was increased significantly(Figure 1B).Next,an Alizarin red staining was used to evaluate the degree of mineralization.The results showed that 50,125,and 250µg/mL CCP induced a significant increase of mineralization in a dose-dependent manner(Figure1C,1D).

Mineralization-related protein and gene expression

Western blot results showed that the expression of p-Akt was significantly increased by treatment with 125 or 250µg/mL CCP for 2 d(Figure 2A).After treatment with 50-250µg/mL CCP for 2 d,a significant increase was observed in p-ERK expression(Figure 2B).A study has indicated a close relationship between the maturation of osteoblasts and gene expression of osteocalcin,Runx-2,and osteopontin.22Using quantitative real-time PCR,we found that treatment with 125 or 250µg/mL CCP for 2 d significantly increased the mRNA expression of Runx-2,osteopontin,and osteocalcin(Figure 3A).We also analyzed Runx-2,osteopontin,and osteocalcin protein expression after treatment with 50,125,and 250µg/mL CCP for 6 d.The data showed that treatment with 125 and 250µg/mL CCP significantly increased osteopontin and osteocalcin protein expression,but did not affect Runx-2 expression.Based on these results,treatment with CCP significantly promoted mineralization and the expression of osteogenesis-related genes and proteins(Figure 3B).

Osteogenesis in zebrafish larvae

To determine the concentration of CCP in subsequent zebrafish experiments,CCP was administered to zebrafish at 2-4 dpf at concentrations of less than 1.25µg/mL.On the fifth day,there were no significant cases of death(Figure 4).

Osteogenesis in zebrafish larvae

Zebrafish wereadministered 0.0125,0.125,and 1.25µg/mL CCP,as well as 1 and 10 nM vitamin D3for 72 h(between 2 and 4 dpf).Calcein staining and analysis showed that 1.25µg/mL,CCP and 1 and 10 nM vitamin D3significantly increased ventral calcein staining in zebrafish larvae(Figure 5A)and the number of spinal bones(Figure 5B).Additionally,we observed zebrafish administered with 1.25µg/mL CCP over the following days and found significant increases in ventral calcein staining(Figure 6A)and the number of spinal bones(Figure 6B)after treatment with 1.25µg/mL CCP for 72 h(between 2 and 4 dpf)as well as 24 h(6 dpf),48 h(7 dpf),and 72 h(8 dpf)after discontinuation of the treatment.

Skull defect recovery in rats

Figure 1 Effect of CCP on viability of the human osteoblastic cell line MG-63

Doses of 0.0005,0.005,0.05,0.5,5,50,125,and 250 μg/mL Guijiajiao(Colla Carapacis et Plastri)(CCP)were applied to MG-63 cells,followed by culture for 1,7,and 14 d.A:cell viability was measured at 1,7 and 14 d after MG-63 cells were treated with CCP.At days 7 and 14,50,125,and 250 μg/mL CCP significantly increased the viability of MG-63 cells.B:ALP activity was measured at 3 d after MG-63 cells were treated with CCP.C,D:mineralization was measured at 7 d after MG-63 cells were treated with CCP.C1:control,C2:0.5 μg/mL CCP,C3:5 μg/mL CCP,C4:50 μg/mL CCP,C5:125 μg/mL CCP,C6:250 μg/mL CCP at 7 d after treatment.At 7 d after treatment with 50,125,and 250 μg/mL CCP,there was significantly increased mineralization of MG-63 cells.After treatment with 125 and 250 μg/mL CCP for 7 d,there was significantly increased mineralization.One-way analysis of variance was used for statistical analysis of data,and Duncan's method was used to compare multi-range differences between groups.aP＜0.05,compared with the control group.

Figure 2 Effect of CCP on p-AKT and p-ERK expression in MG-63 cells

1:control,2:50 g/mL Guijiajiao(Colla Carapacis et Plastri)(CCP),3:125 g/mL CCP,4:250 g/mL CCP.At 47 h after MG-63 cells were treated with CCP(125 and 250 μg/mL),P+F significant increases in A:p-Akt protein expression after MG-63 cells were treated with CCP for 47 h.A1,A2,A3 protein expression of p-Akt,Akt and quantification results in MG-63 cells of phosphorylated Akt(p-Akt)increased and measure by Western blotting.B:p-ERK protein expression after MG-63 cells were treated with CCP for 47 h.B1,B2,B3 protein expression of phosphorylated extracellular signal-regulated kinase(p-ERK),ERK and quantification results in MG-63 cells of p-ERK protein expression increase and measure by western blotting.One-way analysis of variance was used to analyze the data,and Duncan's method was used to compare multi-range differences between groups.aP＜0.05 compared with the control group.

Administration of 0.25 or 1.25 g/kg CCP promoted skull defect recovery compared with the control group in a dose-dependent manner(Figure 7A).In the quantitative analysis of recovery,a dose of 1.25 g/kg CCP showed significant promotion of skull defect recovery.Although 0.25 g/kg CCP also promoted recovery,this effect was not statistically significant(Figure 7B).

Wound healing in the rat skull defect model

The abovementioned studies demonstrate the therapeutic effects of Guilu Erxianjiao in bone diseases.However,there is currently no research involving relevant tests of the effects of the individual ingredients of Guilu Erxianjiao on bone formation.After identifying the ingredient that promotes bone formation,the mechanisms influencing bone formation can be studied further.According to ancient scriptures,the ingredient most likely to have a bone-forming effect is Guijiajiao(Colla Carapacis et Plastri,CCP).4Breastplates of Testudines consist of CCP.The ancient scripture Qian Jin Yi Fang mentions the effect of CCP on nonclosure of fontanels in infants.5However,no previous literature has discussed the effect of CCP on bone formation.

Serum factors in rats with skull defects

13 Matsuguchi T,Chiba N,Bandow K,et al.JNK activity is essential for Atf4 expression and late-stage osteoblast differentiation.J Bone Miner Res 2009;24(3):398-410.

在上游养殖环节中的诸多影响因素中，毛鸡只重对肉鸡产品的出成和综合售价的影响尤为明显。因此，对养殖端进行养殖水平的约束显得尤为重要，而对于这方面的研究未见报道。本研究针对肉鸡产业链存在的问题，对不同重量的毛鸡进行屠宰分割实验，探究不同毛鸡只重对产品出成和综合售价的影响。以此来探究最佳的毛鸡只重范围，为原料部提供一定的数据指导，培训养殖户注意最佳毛鸡只重养殖，为车间的最大化价值生产提供合格的毛鸡原料，实现肉鸡全产业链的价值共赢。

Figure 4 Toxic effects of CCP in zebrafish

Zebrafish were treated Guijiajiao(Colla Carapacis et Plastri)(CCP)from 2 to 4 d post-fertilization(dpf).The survival rate at 5 dpf of zebrafish treated with 0,0.00125,0.0125,0.125,1.25,12.5,and 125 g/mL CCP from 2 to 4 dpf.The survival rate was observed and recorded.The results showed that 12.5 and 125 μg/mL CCP resulted in significant mortality of zebrafish,and concentrations lower than 12.5 μg/mL showed no significant difference from the control.

DISCUSSION

Summary of our findings

The results of CCP experiments performed in this study show that CCP promotes MG-63 cell proliferation,ALP activity,and mineralization.We also found that CCP activates Akt and ERK signaling pathways and significantly increases the expression of ossification-related genes such as Runx-2,osteocalcin,and osteopontin.Furthermore,two animal models,zebrafish and rat,were used to analyze thein vivoosteogenic effects of CCP.The results revealed promotion of osteogenesis in zebrafish immersed in CCP for 72 h.In addition,an oral dose of 1.25 g/kg CCP in rats significantly improved the condition of skull defects,which was accompanied by an increase in serum ALP.

每个称重传感器由4片电阻应变片组成一个惠斯通电桥。被测物的重量F使弹性元件产生形变，该形变引起电阻应变片的延伸及弹性形变系数ε的变化。如图1所示，A， B 为电阻应变片，粘贴在圆筒的外壁，组成电桥的4个臂。根据虎克定律，弹性形变与外力成正比： ε∝F；根据电阻应变效应理论： 电阻应变片的相对变化ΔR/R与ε成正比，即ΔR/R∝Kε=F，K为电阻应变片灵敏系数。

Efficacy of Chinese herbal medicine to promote osteogenesis

Chinese herbal medicine has been used clinically for osteogenesis for many years.23In recent years,researchers have performed experiments to verify the mechanism of its promotion of osteogenesis.A study conducted by Wanget al24showed that Danggui Buxue Tang(DBT)promotes cell proliferation,differentiation,and mineralizationin vitro.In addition,DBT inhibits osteoclast activity.These results support the use of DBT to accelerate bone fracture recovery and for the treatment of osteoporosis.Furthermore,another study added gelatin to the DBT formulation to form a scaffold that was then used to fill wounds in a rat skull defect model.The results demonstrated significant osteoinductivity and showed that the scaffold also has good biocompatibility and biodegradability.25

The combination of scaffolding and Chinese herbal medicine can also serve as a good bone void filler for bone injuries,26but there are still concerns regarding its clinical use.In addition to DBT,Liet al27indicated that Panax notoginseng saponins(PNS)promotes the expression of osteogenesis-related proteins such as ALP,peroxisome proliferator-activated receptor γ2,receptor activator of nuclear factor κB ligand,and osteoprotegerin in primary mesenchymal stem cells.They also found that the osteogenicity of PNS may be mediated through the regulation of gap junction activity.The CCP used in our study promoted the proliferation,ALP activity,and mineralization of MG-63 osteoblasts.Oral CCP also increased the ALP concentration in animal serum.Another study evaluated osteogenicity associated with a Chinese herbal extractin vitroandin vivo.Penget al28showed that aqueous and ethanol fractions of a mixture of six herbs[Chuanxuduan(Radix DipsaciAsperoidis),Guizhi(RamulusCinnamomi),Jiegumu(Herba Sambuci Williamsii),Sanqi(Radix Notoginseng),Honghua(Flos Carthami),Dahuang(Radix Et Rhizoma Rhei Palmati),and Zhizi(Fructus Gardeniae)]significantly promoted osteogenesis and effectively inhibited the production of large amounts of nitric oxidein vitro.Moreover,the osteogenesis-promoting effects of the aqueous or ethanol fractions have been verified in a rat tibial fracture model.

Compared with previous studies of Chinese herbal medicine,our study further elucidated that the osteogenesis-promoting effects of CCP may be exerted through the induction of genes that promote osteogenesis,such as Runx-2,osteocalcin,and osteopontin(Figure 2C).Among these genes,Runx-2 is an important transcription factor related to osteogenesis.Its activation promotes osteoblast differentiation and the production of many osteogenesis-related proteins,such as ALP,osteocalcin,and osteopontin,which promote mineralization and bone formation.29In addition,we detected the protein expression of Runx-2,osteopontin,and osteocalcin.Treatment with CCP for 6 d significantly increased osteopontin and osteocalcin expression.However,treatment with CCP did not affect Runx-2 expression at 6 d of treatment,but increased Runx-2 mRNA expression at 2 d of treatment.We suggest that CCP promotes nuclear translocation of Runx-2 protein instead of increasing its protein expression.

Role of Akt and ERK in osteogenesis

Figure 5 Effect of CCP on visualization of calcified skeletal structures in zebrafish

Zebrafish were treated with 0.0125,0.125,and 1.25 μg/mL Guijiajiao(Colla Carapacis et Plastri)(CCP)as well as 1 and 10 nM vitamin D3(as a positive control)between 2 and 4 dpf,and calcein staining was performed at 5 dpf.A:ventral calcein expression at 5 dpf of zebrafish treated with 0,0.0125,0.125,1.25 g/mL CCP,1 nMVitamin D and 10 nMVitamin D from 2 to 4 dpf.A1:control;A2:0.0125 g/mL CCP;A3:0.125 g/mL CCP;A4:1.25 g/mL CCP;A5:1 nM Vitamin D;A6:10 nM Vitamin D;A7:quantification results of ventral calcein expression increased after the treatment of CCP and Vitamin D.B:the number of spinal bones at 5 dpf of zebrafish treated with 0,0.0125,0.125,1.25 g/mL CCP,1 nM Vitamin D and 10 nM Vitamin D.B1:control;B2:0.0125 g/mL CCP;B3:0.125 g/mL CCP;B4:1.25 g/mL CCP;B5:1 nM Vitamin D;B6:10 nM Vitamin D;B7:quantification results of the number of spinal bones increased after the treatment of CCP and Vitamin D.Followed by imaging and quantification of ventral staining and the number of spinal bones.The results indicated that 1.25 μg/mL CCP significantly increased ventral bone development.The number 1 to 7 represented the calcification number of vertebrae.One-way analysis of variance was used for data analysis,and Duncan's method was used to compare multi-range differences between groups.aP＜0.05,compared with the control group.

Previous studies have indicated that an increase in ERK phosphorylation promotes the activity of Runx-2 that enhances bone formation of the mouse osteoblastic cell line MC3T3-E1.30Furthermore,the phosphatidylinositide 3-kinase(PI3K)/Akt signaling pathway plays a pivotal role in the differentiation of osteoblasts.31Moreover,Zhanget al32found that activation of thePI3K/Akt signaling pathway promotes Runx-2 expression and mineralization in an osteoblastic cell line.In this study,we found that CCP activated ERK and Akt signaling pathways(Figure 2A,B).Therefore,we suggest that CCP upregulates Runx-2 gene expression via activation of ERK or PI3K/Akt signaling pathways,resulting in an increase of osteogenesis-related factors ALP,osteocalcin,and osteopontin.

Osteogenic effect of CCP in zebrafish

Zebrafish have been used as a model for skeletal development in recent years.33We and other researchers have used this model to explore active compounds in osteogenesis promotion.Pasqualettiet al34observed the number of spinal bones using alizarin red S staining in a zebrafish model and found that strontium has an osteogenesis-promoting effect.They also indicated that zebrafish embryos possess good permeability and visibility that allows convenient observation of skeletal development,thus making zebrafish an excellentin vivomodel for osteogenesis.In addition,Chenet al35used zebrafish as an animal model and showed that deferoxamine prevents iron-induced osteoporosis.Similar to our study,Chenet al35used the number of spinal bones at 10 dpf and ventral calcein staining at 6 dpf to observe bone formation.However,as indicated by Duet al19although both calcein and alcian blue staining in combination with alizarin red S stain calcified bones,calcein was demonstrated to have higher sensitivity and was simpler to use than alizarin red to stain zebrafish bones.In our previous study,we also used calcein staining in zebrafish and successfully screened for compounds that promote osteogenesis or bone markers.36In this study,the experiment was continued to 6,7,and 8 dpf,and the results indicated that 1.25µg/mL CCP did not induce death of zebrafish larvae(data no shown).we further applied calcein staining and found that nontoxic concentrations of CCP increased the number of spinal bones and promoted osteogenesis of the ventral bone in zebrafish(Figure 4).Furthermore,our present results support the use of a traditional rat model to further explore the effects of CCP in promoting osteogenesis.37

Figure 6 Effect of 1.25 μg/mL CCP on calcified skeletal structures in zebrafish at 6,7,and 8 dpf

Zebrafish were administered 1.25 μg/mL Guijiajiao(Colla Carapacis et Plastri)(CCP)between 2 and 4 dpf.A:ventral calcein expression at 6,7 and 8 dpf of zebrafish treated with 0 and 1.25 g/mL CCP from 2 to 4 dpf.A1:Control in 6 dpf;A2:1.25 g/mL CCP in 6 dpf;A3:Control in 7 dpf;A4:1.25 g/mL CCP in 7 dpf;A5:Control in 8 dpf;A6:1.25 g/mL CCP in 8 dpf;A7:quantification results of ventral calcein expression increase after the treatment of CCP.B:The number of spine bones at 6,7 and 8 dpf of zebrafish treated with 0 and 1.25 g/mL CCP from 2 to 4 dpf.B1:Control in 6 dpf;B2:1.25 g/mL CCP in 6 dpf;B3:Control in 7 dpf;B4:1.25 g/mL CCP in 7 dpf;B5:Control in 8 dpf;B6:1.25 g/mL CCP in 8 dpf;B7:quantification results of number of spinal bones increased after the treatment of CCP.Followed by imaging and quantification of ventral expression of bone and the number of spinal bones at 6,7,and 8 dpf.The results indicated that 1.25 μg/mL CCP increased bone development.The number 1 to 10 represented the calcification number of vertebrae.One-way analysis of variance was used for data analysis,and Duncan's method was used to compare multi-range differences between groups.aP＜0.05,compared with the control group at same dpf.

Osteogenesis of CCP in a rat skull defect

Zavattiet al38used a rat skull defect model and found that oral ferutinin and stem cell scaffolding promote bone repair and regeneration.Yanet al39used a rat skull defect as an experimental model and found that injection of an extract of Xiangpu(Typha elephantina Roxb)into defect sites significantly recovers bone defects in a dose-dependent manner.However,the oral route of administration was found to be considerably more advantageous than direct injection at the wound site.In the present study,we also used oral administration to accelerate bone repair and tissue biopsies to confirm its effect.

Effect of CCP on osteogenic serum factors

Clinically,the condition of bone is often represented by serum calcium,phosphorus,ALP,and calcitonin concentrations,40among which ALP most directly reflects osteogenic activity.One study indicated that osteoporosis is accompanied by upregulation of serum ALP.41Elkomyet al42found that a mixture of three herbal medicines,Shuweicao(Herba Salviae Japonicae),Midiexiang(Herba Rosmarini Officinalis),and Dijiao(Herba Thymi Mongolici),inhibits osteoporosis-induced upregulation of ALP and downregulation of calcium concentration in serum.However,Chianget al43found that Lactobacillus paracasei subsp.paracasei(NTU 101)as an additive in mouse feed significantly increases the serum concentration of ALP and has an inhibitory effect on osteoporosis.In addition,another study found that feeding Chinese Herbal Medicines(e.g.,a mixture of angelica,chuanxiong,walnuts,safflower,dragon's blood,ground beetle,Astragalus,Himalayan teasel root,Drynaria,frankincense,myrrh,Danshen(Radix Salviae Miltiorrhizae),pyritum,and rhubarb)that promote osteogenesis increases serum ALP and promotes osteogenesis in chickens.44

Figure 7 CCP promotes bone regeneration in a rat skull defect model

After establishment of the skull defect in rats,the animals were administered Guijiajiao(Colla Carapacis et Plastri)(CCP)at doses of 0.25 and 1.25 g/kg.At weeks 4,8,and 12,the rats were sacrificed,and their skulls were extracted for imaging.A:photos of rat skull at weeks 4,8 and 12 after administration of CCP for 0.25 and 1.25 g/kg.A1:control after lesion for 4 weeks;A2:0.25 g/kg CCP after lesion for 4 weeks;A3:1.25 g/kg CCP after lesion for 4 weeks;A4:control after lesion for 8 weeks;A5:0.25 g/kg CCP after lesion for 8 weeks;A6:1.25 g/kg CCP after lesion for 8 weeks;A7:control after lesion for 12 weeks;A8:0.25 g/kg CCP after lesion for 12 weeks;A9:1.25 g/kg CCP after lesion for 12 weeks.The results showed that 0.25 g/kg CCP showed no significant difference from the control group at week 4,whereas the bone defect in the 1.25 g/kg CCP group was slightly smaller than that in the control group.The results at week 8 showed that the bone defect in the 0.25 g/kg CCP group was slightly smaller than that in the control group,whereas the bone defect in the 1.25 g/kg CCP group was significantly smaller than that in the control group.The 0.25 g/kg CCP group showed no significant difference from the control group,whereas the bone defect in the 1.25 g/kg CCP group was significantly smaller than that in the control group at week 12.B:quantification results of rat skull at weeks 4,8 and 12 after administration of CCP for 0.25 and 1.25 g/kg.Results showed that the 0.25 g/kg CCP group had no significant difference from the control group at week 4,whereas the bone-healing rate of the 1.25 g/kg CCP group was significantly higher than that of the control group.Evaluation at week 8 showed that the bone-healing rate of the 1.25 g/kg CCP group was significantly higher than that of the control group.At week 12,the bone-healing rate of the 0.25 g/kg CCP group showed no significant difference from the control group,whereas the bone-healing rate of the 1.25 g/kg CCP group was significantly higher than that of the control group.One-way analysis of variance was used for the data analysis,and Duncan's method was used to compare multi-range differences between groups.aP＜0.05,compared with the control group.

Figure 8 Hematoxylin and eosin(HE)staining of rat skull defects at 4,8,or 12 weeks after CCP treatment

A:dyeing method of all pictures is the HE stain(×25).A1:control after lesion for 4 weeks;A2:0.25 g/kg Guijiajiao(Colla Carapacis et Plastri)(CCP)after lesion for 4 weeks;A3:1.25 g/kg CCP after lesion for 4 weeks;A4:control after lesion for 8 weeks;A5:0.25 g/kg CCP after lesion for 8 weeks;A6:1.25 g/kg CCP after lesion for 8 weeks;A7:control after lesion for 12 weeks;A8:0.25 g/kg CCP after lesion for 12 weeks;A9:1.25 g/kg CCP after lesion for 12 weeks.At week 4,the 0.25 g/kg CCP group showed no obvious difference from the control group,whereas a thin matrix had formed in the 1.25 g/kg CCP group.At week 8,the 0.25 g/kg CCP group had formed more matrix than the control group,whereas the 1.25 g/kg CCP group had formed a thicker matrix around the defect.At week 12,the 0.25 g/kg CCP group showed no difference from the control group,whereas the 1.25 g/kg CCP group had an even thicker matrix than the other groups.B:dyeing method of all pictures is the immunohistochemical(×25).Immunohistochemistry of collagen type I.B1:control after lesion for 4 weeks;B2:0.25 g/kg CCP after lesion for 4 weeks;B3:1.25 g/kg CCP after lesion for 4 weeks.At week 4,both 0.25 and 1.25 g/kg CCP groups had significant increases in the protein expression of collagen type I at the edge of the wound.

Figure 9 Effect of CCP on serum calcium,phosphorus,ALP,and calcitonin

A:calcium;B:phosphorus.At week 4,calcium and phosphorus levels of the 1.25 g/kg Guijiajiao(Colla Carapacis et Plastri)(CCP)group showed no significant changes compared with those of the control group.C:alkaline phosphatase.However,there was a significant increase in the concentration of alkaline phosphatase(ALP).At week 8,a significant increase in ALP was observed in 0.25 and 1.25 g/kg CCP groups compared with the control group,but there was no significant change in calcium or phosphorus concentrations.At week 12,there were no significant changes in calcium,phosphorus,or ALP concentrations.One-way analysis of variance was used for the data analysis,and Duncan's method was used to compare multi-range differences between groups.aP＜0.05,compared with the control group.

Many studies have reported that osteoporosis caused by ovariectomy is accompanied by an increase in serum ALP concentrations to promote osteogenesis and decrease bone loss.45Administration of osteoporosis medications such as bisphosphonate or Chinese herbal medicine inhibits the activity of osteoclasts,and suppression of osteoclasts leads to a reduction in ALP activity.46In our study,we found that CCP directly increased the activity of ALP in MG-63 osteoblasts.An increase in serum ALP was observed at both 4 and 8 weeks after feeding rats CCP,but there was no significant change in the concentrations of calcium or phosphorus.Based on these results,we propose that the osteogenesis-promoting effects of CCP in the non-osteoporosis model may be related to upregulation of osteoblast activity.Moreover,Hukkanenet al47found that the repair process of tibial fractures in rats is accompanied by expression of large quantities of calcitonin gene-related peptides.Currently,there are clinical cases that use salmon calcitonin in combination with aspirin to treat osteoporosis.48Our results are similar to those of the aforementioned study,because we found that oral administration of CCP(0.25 g/kg)increased serum calcitonin in rats with skull defects.Therefore,we cannot rule out the possibility that high doses of CCP also promote osteogenesis through the calcitonin pathway.

Future directions for the use of CCP

Distinct from traditional studies of Chinese herbal medicine,which explore the treatment of diseases using compound prescriptions involving several medicines,the present study demonstrated that a simple prescription,CCP,promotes osteogenesisin vivoandin vitro.We also found that oral administration induces bone repair in rats with skull defects.CCP is traditional and edible,and its main components include collagen and amino acids.However,it is unclear whether CCP contains other natural osteogenesis-promoting components.Because of its edibility and the effects elucidated in this study,CCP can be immediately developed as an adjuvant treatment for bone repair.

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There was a significant increase of ALP levels in groups administered with 0.25 g/kg and 1.25 g/kg CCP at 4,6,and 8 weeks compared with those in the control group,whereas no significant changes were observed in calcium or phosphorus levels(Figure 9A,B).However,rats administered CCP at 12 weeks post-injury showed no difference in calcium,phosphorus,or ALP levels compared with the control group(Figure 9C).Calcitonin(2.60±0.14)in the 1.25 g/kg CCP group at 12 weeks post-injury was higher than that in 0.25 g/kg CCP and control groups(Figure 9D).

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For the cell survival assay,cells were seeded in 96-well culture dishes(Corning,NY,USA)at an initial density of 1×104cells/well.Cell survival was determined after treatment with alamar blue,a tetrazolium dye that is reduced by living cells to a fluorescent product.This assay is similar to the 3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyl tetrazolium bromide cell viability assay and has been previously validated as an accurate measure of MG-63 cell survival.14Relative protection(%)was calculated as 100×[(optical density(OD)of CCP-treated cells－OD of vehicle-treated cells)/(OD of control cells－OD of vehicle-treated cells)]

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2012年7月13日19时，因连日强降雨，桑植县洪家关乡泉峪村皮家垭、肖家湾两组交界处出现长约25 m、宽约10 m山体滑坡迹象。乡党委、政府主要领导收到山洪灾害预警信息后，立即率领防汛抢险应急队伍赶赴现场，组织受威胁的群众紧急转移，有效避免了人员伤亡。

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