Research Article Comparison of the Effect of Velvet Antler from...

Research ArticleComparison of the Effect of Velvet Antler from DifferentSections on Longitudinal Bone Growth of Adolescent Rats

Hye Kyung Kim1 Myung-Gyou Kim2 and Kang-Hyun Leem2

1Department of Food amp Biotechnology Hanseo University Seosan 31962 Republic of Korea2College of Korean Medicine Semyung University Jecheon 27136 Republic of Korea

Correspondence should be addressed to Kang-Hyun Leem hkkim1454gmailcom

Received 12 April 2016 Revised 16 May 2016 Accepted 19 May 2016

Academic Editor Yuewen Gong

Copyright copy 2016 Hye Kyung Kim et alThis is an open access article distributed under theCreative CommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The aim of this study was to compare the effectiveness of velvet antler (VA) from different sections for promoting longitudinalbone growth in growing rats VA was divided into upper (VAU) middle (VAM) and basal sections (VAB) An in vivo study wasperformed to examine the effect on longitudinal bone growth in adolescent rats In addition in vitro osteogenic activities wereexamined using osteoblastic MG-63 cells VA promoted longitudinal bone growth and height of the growth plate in adolescent ratsBone morphogenetic protein-2 (BMP-2) in growth plate of VA group was highly expressed compared with control The anaboliceffect of VA on bone was further supported by in vitro study VA enhanced the proliferation differentiation and mineralization ofMG-63 cells The mRNA expressions of osteogenic genes such as collagen alkaline phosphatase and osteocalcin were increasedby VA treatment These effects of in vivo and in vitro study were decreased from upper to basal sections of VA In conclusion VAtreatment promotes longitudinal bone growth in growing rats through enhanced BMP-2 expression osteogenic activities and bonematrix gene expressions In addition present study provides evidence for the regional differences in the effectiveness of velvet antlerfor longitudinal bone growth

1 Introduction

Longitudinal bone growth which determines the size andshape of the body frame proceeds at a rate distinct fromthat of muscle and other tissues and is controlled by specificmechanisms During childhood and adolescence the longbones grow at the ends of the bones which occur at thegrowth plate by endochondral ossification of epiphyseal plate[1] Growth plate chondrocyte proliferation and hypertrophylead to formation of new cartilage chondrogenesis andremodeling of the newly formed cartilage into bone tissueresulting in longitudinal bone growth [2]

The current methods for increasing final height includetreatment with a growth hormone alone or in combinationwith an analog of gonadotropin-releasing hormone [3] How-ever it is generally too expensive and also has harmful sideeffects such as prepubertal gynecomastia arthralgia edemabenign intracranial hypertension insulin resistance andleukemia [4] On that account there has been an increasingawareness of the benefits of natural products and many

plant-derived medicines have been reported However theresearches on animal medicines or functional foods are stillrelatively few although animal medicines have been provenof various important components such as proteins peptidesfatty acids glycosaminoglycans prostaglandins vitaminsminerals dietary fiber essential oils and carotenoids [5 6]which can be used in the prevention and treatment of variousdiseases

Velvet antler (VA) the immature antler of male deer istraditionally used for thousands of years in Asian countriessuch as Korea China Taiwan and Mongolia It is currentlyestimated that the global production of velvet antlers is nearto 1300 tonsyear which is still rapidly growing to meetthe requirements of medicinal markets [7] As a typicaltraditional animal medicine VA has pharmacological effectsto improve immune system physical strength and sexualfunction [7 8] VA also has been reported to possess bonestrengthening activity and has been used in treating arthritisosteoporosis and fracture in animal model or human clinicaltrials [9ndash12]

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2016 Article ID 1927534 9 pageshttpdxdoiorg10115520161927534

2 Evidence-Based Complementary and Alternative Medicine

A whole VA stick is usually divided into four portionswith the value decreasing from the tip to the base Chemicalanalyses of VA revealed that there are regional differencesin chemical composition the contents of proteins and lipidsdecrease downward from the tip to the base while thoseof ash calcium and collagen increase [13] suggesting thedegree of calcification increased from tip to base section ofVA During the ossification of deer antler the total collagencontent was found to be increased Typically the antleris cut off near the base after it is about two-thirds of itspotential full size between 55 and 65 days of growth beforeany significant calcification occurs Traditionally the marketvalues of antlers are downgraded with increasing degree ofcalcification However which parts of VA are suitable forpreventing andmanaging bone health especially bone growthhad not been clarified

The objective of the present study was to compare theeffect of VA from different sections on longitudinal bonegrowth of adolescent rats and elucidate the underlyingmech-anisms for the effect

2 Materials and Methods

21 Materials VA obtained from farmed Elk deer 75 daysafter casting was kindly provided by the Animal GeneticResources Station (National Institute of Animal ScienceSouth Korea) VA was divided into upper (VAU) middle(VAM) and basal (VAB) sections (Figure 1)The criteria usedfor dividing the antler into three sections is defined as followsupper section top 15 cm of the part of the main beam to the2nd division of a deerrsquos antlers from its head middle sectionthe upper half of the remaining main beam and base sectionthe lower half of the remaining main beam Total part of eachsectionwas slicedwith a bone slicer freeze-dried and groundinto powderThe powderedVAwas immersed in 70 ethanolfor 3 days filtered concentrated by vacuum evaporationand finally subjected to freeze drying Respective yields ofsections fromupper to basal were 34 (VAU) 29 (VAM)and 17 (VAB)

22 Animals Longitudinal bone growth was determined in3-week-old male Sprague-Dawley rats (Samtako Co OsanKorea) The experimental procedures were performed inaccordance with protocols approved by the Institutional Ani-mal Care and Use Committee Semyung University (smecae15-09-01)The animals were housed under controlled temper-ature and lighting conditionsThe ratswere randomly dividedinto four groups (119899 = 8) vehicle (control distilled water)and upper (VAU) middle (VAM) and basal (VAB) sectionsof antler extract (100mgkg) were orally administered dailyvia stomach tube for 5 consecutive days Treatment dose wasdecided from the preliminary study On the sixth day all ratswere sacrificed under chloral hydrate anesthesia for tissueanalysis

23 Measurement of Longitudinal Bone Growth To measurethe effect on longitudinal bone growth rate calcein wasused as a fluorescence marker to label the bone line on thesurface of the tibia Calcein plays the role of fluorescent

VAU

VAM

VAB

Figure 1 Sections of velvet antler Fresh velvet antler was dividedinto upper (VAU) middle (VAM) and basal (VAB) sections

dye under ultraviolet illumination Calcein (10mgkg Sigma-Aldrich St Louis MO USA) was injected intraperitoneally24 h before sacrifice The dissected tibias were fixed in01M phosphate buffered formalin for 2 h decalcified anddehydrated by immersing in 30 sucrose for 1 day at 4∘CDehydrated bone was sectioned longitudinally at sagittalsections of proximal part with a thickness of 40120583m using asliding microtome (HM440E Zeiss Germany) Bone growthwas measured by measuring the gap between fluorescentline formed by calcein and the epiphyseal end line of thegrowth plate at three different locations using a fluorescentmicroscope (BX60Olympus Tokyo Japan) and the averageswere obtained

24 Measurement of Growth Plate Height Cresyl violet stain-ing (Sigma-Aldrich) was used to stain the chondrocytes in thegrowth plate of the samples Each tibia sample was sectionedlongitudinally at a thickness of 40 120583m using a sliding micro-tome as described above and stained with cresyl violet Thegrowth plate height was measured at three different locationsand the averages were obtained

25 Bone Morphogenetic Protein-2 Expression For the detec-tion of bonemorphogenetic protein-2 (BMP-2) in the growthplate the dehydrated tibia sections were incubated overnightin 1 Triton X-100 containing goat BMP-2 antibody (SantaCruz Biotechnology diluted 1 500) at room temperatureThen sections were incubated with anti-goat antibody (Vec-tor Laboratories Burlingame CA USA diluted 1 200) for60min and stained with 005 33-diaminobenzidine (SigmaChemical Co) containing 003 hydrogen peroxide

26 Cell Culture Human osteoblast-like MG-63 cells wereobtained from the American Type Culture Collection(Rockville MD USA) Cells were cultured at 37∘C in a 5CO2atmosphere in Dulbeccorsquos Modified Eaglersquos Medium

(DMEM Gibco MD USA) containing 10 heat-inactivated

Evidence-Based Complementary and Alternative Medicine 3

Table 1 Oligonucleotide sequences of osteogenic genes

Primer Direction Sequence

Collagen (COL) Forward 51015840-GCG GCT CCC CAT TTT TAT ACC-31015840

Reverse 51015840-GCT CTC CTC CCA TGT TAA ATA GCA-31015840

Alkaline phosphatase (ALP) Forward 51015840-AAA CCG AGA TAC AAG CAC TCC CAC-31015840

Reverse 51015840-TCC GTC ACG TTG TTC CTG TTC AG-31015840

Osteocalcin (OCN) Forward 51015840-CCA TGA GAG CCC TCA CAC TCC TC-31015840

Reverse 51015840-GCT TGG ACA CAA AGG CTG CAC-31015840

Osteopontin (OPN) Forward 51015840-AGG CTG ATT CTG GAA GTT CTG AGG-31015840

Reverse 51015840-GAC TTA CTT GGA AGG GYC TGT GGG-31015840

GAPDH Forward 51015840-TCA TCA ATG GAA ATC CCA TCA CC-31015840

Reverse 51015840-TGG ACT CCA CGA CGT ACT CAG C-31015840

FBS (Invitrogen Grand island NY USA) and 100UmLpenicillinstreptomycin (Sigma-Aldrich) Bone marrow cellsprepared from the femur of male ICR mice were culturedin 120572-modified minimal essential medium (120572-MEM Gibco)containing 10 heat-inactivated FBS After 3 days of culturefloating cells were removed and attached cells were used asosteoclast precursors

27 Osteoblasts Proliferation The effects of VA from differentsections on proliferation of MG-63 cell were determinedby a colorimetric immunoassay kit (Roche DiagnosticsMannheim Germany) which is based on quantitating bro-modeoxyuridine (BrdU) incorporation into the newly syn-thesized DNA of replicating cells MG-63 cells were seeded(5000 cellswell) in 96-well plates containing DMEM with10 FBS and allowed to adhere overnight The media werereplaced with DMEM containing VA samples (10 50 and100 120583gmL) and incubated for 24 h Subsequently BrdU wasadded to each well and reincubated for 2 h After 14 h ofincubation at 37∘C labeling media was removed cells werefixed and the cells with BrdU label in the DNA were locatedwith peroxidase-conjugated anti-BrdU antibody solutionThen bound anti-BrdU with substrate was colorimetricallymeasured with a microplate reader (BioTek Inc WinooskiVT USA) at 450 nm

28 Alkaline Phosphatase Activity Alkaline phosphatase(ALP) activity was measured using enzymatic assay MG-63 cells incubated in DMEM were seeded in 12-well plates(50000 cellswell) and incubated for 24 h The mediumwas changed to osteogenic medium (DMEM with 10mM120573-glycerophosphate 5 nM dexamethasone and 50 120583gmLascorbic acid) VA samples (50 and 100 120583gmL) were addedto the cells and incubated for another 24 h The 50 and100 120583gmL concentrations were chosen since the maximumeffect and plateau had been gained under those two concen-trations in cell proliferation assay results The cells were thenrinsed with PBS and lysed in 300 120583L of 02 of Triton X-100The cell lysates were centrifuged at 13000 rpm for 5min Thesupernatant of the lysate was used for the measurement ofALP activity by measuring the release of p-nitrophenol fromp-nitrophenylphosphate at pH 98

29 Collagen Content Collagen synthesis was measuredusing picrosirius red method [14] MG-63 cells were seeded

in 12-well plates (50000 cellswell) and allowed to adhereovernight The medium was changed to osteogenic mediumVA samples (100 120583gmL) were added to each well and incu-bated for 7 days Cells were fixed with Bouinrsquos fluid andstained with 01 Sirius red (Direct Red 80 Sigma-Aldrich)in a saturated aqueous solution of picric acid for 30minStained dye was dissolved and the absorbance was measuredat 540 nm

210 Calcium Deposition The formation of calcium phos-phate was determined by alizarin red-S assay [15] MG-63cells were seeded and incubated as described above in thecollagen assay process After 7 days cells were fixed with10 formaldehyde and stained with 2 of alizarin red-S (pH42 Sigma-Aldrich) at room temperatureThe stained alizarinred-S was extracted with 10 acetic acid and the amount ofcalcium deposition was quantified using the absorbance ofextracted alizarin red-S at 405 nm

211 Real-Time PCR Real-time PCR was performed toanalyze relative gene expression MG-63 cells were seededin 6-well plates (100000 cellswell) and allowed to adhereovernight The medium was changed to osteogenic mediumVA sample (100 120583gmL)was added to eachwell and incubatedfor 24 h Total RNA was extracted using RNeasy ProtectMini Kit (Qiagen Valencia CA USA) and cDNA wassynthesized from mRNA using QuantiTect Reverse Tran-scription Kit (Qiagen) Real-time PCR was performed usingQuantiTect SYBR Green PCR Kit (Qiagen) according tothe manufacturerrsquos protocol The PCR primer sequences areshown in Table 1 Analyses were performed using Rotor-GeneQ (Qiagen) and gene expression valueswere calculatedbased on the comparative ΔΔCT method according to themanufacturerrsquos protocol

212 Osteoclastogenesis Assay For induction of osteoclas-togenesis osteoclast precursor cells prepared as describedabove were further cultured for 4 days with RANKL(50 ngmL) and M-CSF (30 ngmL) in the presence of VAsamples Osteoclasts were identified by staining tartrate-resistant acid phosphatase (TRAP) a marker enzyme ofosteoclasts [16] using commercial kit (TRAP 387-A KitSigma-Aldrich) TRAP-positive multinucleated cells withge3nuclei were counted as osteoclasts


Control VAU150120583m

(a)Control VAU VAM VAB

400

600

800

1000

A

BA

A

Bone

gro

wth

(120583m

)

(b)

Figure 2 Effects of velvet antler on longitudinal bone growth (a) Fluorescence photomicrographs of longitudinal sections of the proximaltibias of control and VAU (100mgkg) treated rats (b) Numerical values of longitudinal bone growthThe arrow between the fluorescent lineformed by calcein (lower arrow) and the epiphyseal end line of the growth plate (upper arrow) indicates the bone growth during the 24 hperiod VAU upper section of VA VAM middle section of VA VAB basal section of VA The data are expressed as means plusmn SD The barswith a different letter are significantly different from each other at the level of 119875 lt 005

213 Statistical Analysis The data were expressed as mean plusmnSD One-way ANOVA followed by Tukeyrsquos multiple compar-ison test was performed for statistical analysis (GraphPadPrism ver 6) and 119875 values of less than 005 (119875 lt 005)indicated significant differences All in vitro experimentswere performed with triplicate independent samples

3 Results and Discussion

31 Effects on Cell Longitudinal Bone Growth To determinethe growth per day instead of the total growth over a periodexact methods for measurement are necessary Recently theuse of tetracycline or calcein as intravital markers of thegrowth process to label mineralizing bone in the rat hasbeen reported [17 18] In the present study calcein wasused to label newly formed bone for the determination oflongitudinal bone growth Calcein binds to free calcium andgets deposited in newly deposited bone causing stainingand fluorescence under ultraviolet illumination The effectsof the different sections of VA on the rate of longitudi-nal bone growth from the proximal tibia in the rat aredepicted in Figure 2 The fluorescent line corresponds tothe injection of calcein which binds with calcium in thenewly formed bone (Figure 2(a) lower arrow) The arrowbetween the fluorescent line formed by calcein (Figure 2(a)lower arrow) and epiphyseal end line of the growth plate(Figure 2(a) upper arrow) indicates the length of the bonegrowth during 24 h period Longitudinal bone growth wassignificantly increased by VAU treatment compared with thecontrol (Figure 2(a)) Figure 2(b) shows the numerical valuesof the longitudinal bone growth Longitudinal bone growthin control rat was 6174 plusmn 342 120583mday and administrationof VAU significantly increased the longitudinal bone growthto 7186 plusmn 486 120583mday (119875 lt 005) VAU VAM and VABcaused 164 96 and 27 increase in longitudinal bonegrowth respectively compared with control suggesting thatthe effect decreases downward fromupper section to the baseAlthough VAM caused slight increase in longitudinal bone

growth statistical significance was not observedThe presentstudy provides first evidence for the effectiveness as well asregional differences of VA on longitudinal bone growth inadolescent rats

Recently Tseng et al [19] compared the antiosteoporoticactivity of VA from different sections using ovariectomy-induced osteoporosis animal model and suggest that theupper and middle sections of VA were equally effective inprotecting bones from an estrogen-deficient state Howeverthe effects on proliferation and mineralization of osteoblastsMC3T3-E1 cells were decreased downward from uppersection to the base [19] supporting the results of the presentstudy In addition they reported that the level of insulin-like growth factor 1 (IGF-1) was decreased downward fromupper section to base section Longitudinal bone growth isa function of growth plate chondrocyte proliferation andhypertrophy and IGF-1 is reputed to augment longitudinalbone growth by stimulating growth plate chondrocyte prolif-eration [20] Therefore these data support the results of thepresent study and one of the possible mechanisms may beexplained by the IGF-1 level in each section of VA

32 Effects on Growth Plate Heights The growth plate whichis located at the distal end of the bone is the main locationwhere longitudinal bone growth occurs owing to the stimula-tion of chondrocyte proliferationThegrowth plate consists offour distinctive histological zones beginning with the restingzone and extending through the proliferative hypertrophicand ossification zones (Figure 3(a)) The topmost layer theresting zone contains chondrocytes that serve as stem-likecells for the growth plate with the potential to generate clonesof rapidly proliferating chondrocytes which are located inthe proliferative zone in the second layer of the growthplate [21] The proliferative zone is the driving force behindbone elongation Over time as longitudinal growth proceedsproliferative cells close to the hypertrophic zone undergoterminal differentiation During this process they stop pro-liferating and physically enlarge to become hypertrophic


Control VAU

Resting zone

Proliferative zone

Hypertrophic zone

Ossification zone200120583m


300

400

500

600

700

800

A

B BA

Gro

wth

pla

te h

eigh

t (120583

m)

(b)

Figure 3 Effects of velvet antler on growth plate height (a) Cresyl violet stained sections of tibial growth plates of control and VAU(100mgkg) treated rats (b) Numerical values of growth plate height Upper arrow indicates the resting zone where quiescent chondrocytesare waiting for the proliferation of the growth plates and lower arrow indicates the ossification zone where cartilaginous matrix begins tocalcify and replace with mineralized bone tissue VAU upper section of VA VAM middle section of VA VAB basal section of VAThe dataare expressed as means plusmn SD Values not sharing a common alphabet are significantly different from each other at the level of 119875 lt 005

chondrocytes composing the third hypertrophic layer ofthe growth plate In the ossification zone the chondrocyteseventually die and are transformed into bone matrix wherelongitudinal bone growth occurred [22]

Since the synchronized processes of chondrocyte pro-liferation and cartilage ossification in growth plate lead tolongitudinal bone growth [23] the rate of longitudinal bonegrowth is regulated by the rate of chondrocyte proliferationof the growth plate [24] In the present study the rateof chondrocyte proliferation was determined by measuringthe height of the proximal tibia growth plate (Figure 3(a))The height of the growth plate in the control group was5112 plusmn 216 120583m and administration of 100mgkg VAUand VAM significantly increased the height of the growthplate to 5674 plusmn 234 120583m and 5662 plusmn 206 120583m respectively(Figure 3(b))The effect of VAU (110) was similar to that ofVAM (108) while VAB caused 63 increase whichwas notstatistically significant compared with control These resultssuggest that the effect of VAM was slightly lower but notstatistically significant compared with VAU and the effectdecreases downward from upper section to the base

33 Effects on BMP-2 Expression BMPs play important rolesin regulating growth plate chondrogenesis and longitudinalbone growth Of the various forms of BMP BMP-2 plays animportant role in the development of the epiphyseal growthplate [25] BMP-2 stimulates chondrocyte proliferation in theproliferative zone of the growth plate and also causes anincrease in chondrocyte hypertrophy [26] In this contextit has been suggested that alterations in the expression orproduction of BMP-2 can modulate the proliferation andactivity of bone forming cells

In the present study protein expression of BMP-2 ingrowth plate was highly expressed in hypertrophic andossification zones compared to resting and proliferative zones(Figure 4(a)) As expected BMP-2 expressions particularly inhypertrophic and ossification zones of the growth plates wereincreased in the VAU and VAM treated groups compared

with the control group (Figures 4(a) and 4(b)) Numericalvalues of the BMP-2 expression were increased by 258247 and 105 with VAU VAM and VAB treatmentrespectively compared with control (Figure 4(b)) The effecton BMP-2 expression in growth plate which is relatedto the bone growth and formation was also decreaseddownward from upper section to the base Furthermore theeffect of VAM was similar to VAU The results of the invivo experiment suggest that treatment with VA increaseslongitudinal bone growth rate by promoting chondrocyteproliferation and chondrogenesis through the upregulationof BMP-2 expression in growth plate and the effect decreasesdownward from upper section to the base

34 Effects on MG63 Cell Osteogenesis Bone regeneration isregulated by a fine balance of biochemical and cellular eventsthat ultimately stimulate osteoblasts to produce new tissuein particular new extracellular matrix composed mainlyof collagen The collagen matrix is then mineralized viaALP activity which induces formation of calcium phosphatecrystal seeds Bellows et al [27] reported that ALP is one ofthe osteoblast phenotype markers and an essential enzymefor mineralization Hence osteoblast proliferation collagencontent and ALP activity as early differentiation markersand cellular calcium content as a late marker of differenti-ation were examined to investigate the effects of VA on thedifferentiation of MG-63 cells First the effect of differentsections of VA on osteoblast proliferation was examinedby BrdU incorporation As shown in Figure 5(a) MG-63cell proliferation was significantly and dose-dependentlyincreased with VAU and VAM treatment (119875 lt 005) MG-63cell proliferation reached peak value at 50120583gmL VAU treat-ment exhibiting 119of the basal value whileVAM treatmentreached peak at 100 120583gmL exhibiting 111 of the basal valueVAB at the highest concentration (100 120583gmL) significantly(119875 lt 005) increased cell proliferation compared to thecontrol exhibiting 106 of the basal value Effects of VAU andVAMon increasing cell proliferationwere significantly higher


Resting zone

Proliferative zone

Hypertrophic zone

Ossification zone

Control VAU

200120583m

(a)

BMP-

2 ex

pres

sion

()

Control VAU VAM VAB

60

80

100

120

140

A

B B

A

(b)

Figure 4 Effects of velvet antler on BMP-2 expression (a) Immunohistochemical localizations of bone morphogenetic protein-2 (BMP-2)in the growth plates of control and VAU (100mgkg) treated rats (b) Densitometric results of immunohistochemistry VAU upper sectionof VA VAM middle section of VA VAB basal section of VA Data are shown as a percentage of the control value (means plusmn SD) Values notsharing a common alphabet are significantly different from each other at the level of 119875 lt 005

than those of control at 50 and 100 120583gmL concentrationTherefore the effect of VA on ALP activity in MG-63 cellswas further determined at 50 and 100 120583gmL concentrationVAU treatment significantly increased the ALP activity anearly-stage osteoblasts differentiation marker at 100 120583gmLconcentration (119875 lt 005 Figure 5(b)) comparedwith controlVAM and VAB failed to affect ALP level Hence collagencontent (Figure 5(c)) and calcium deposition (Figure 5(d))were determined at 100 120583gmL of VA concentration Colla-gen synthesis was significantly increased with VAU (112)and VAM (62) treatment compared with control andthe increased collagen synthesis was significantly higherthan VAB Cellular calcium deposition was examined usingAlizarin red-S staining Significant increases in mineraliza-tion were found with VAU and VAM treatment compared tothe control and VAB group Mineralization was significantlyincreased to 118 and 107 of the control by VAU and VAMtreatment respectivelyThe anabolic effect of VA on bonewasfurther supported by the findings in this study demonstratingthat VA enhanced the proliferation differentiation andmineralization of osteoblastic cells Furthermore the effectsof VAU were greater than the effect of VAM suggestingthat the effects were decreased from the upper section tothe base section Lee et al [28] reported that VA waterextract enhanced osteoblasts proliferation and mineraliza-tion Furthermore Tseng et al [19] reported that VAU andVAMdose-dependently increased osteoblasts proliferation aswell as mineralization supporting the results of the presentstudy

35 Effects on Osteogenic Gene Expression It is knownthat preosteoblastic cells produce proteins of the extracel-lular matrix including collagen at first and then to succes-sively produce alkaline phosphatase (ALP) and osteopon-tinosteocalcin during mineralization phase [29] Accord-ingly there is increased expression of osteogenic genes inosteoblasts during the bone formation process and these

genes play roles in extracellularmatrix formation andmineraldeposition Collagen (COL) is a primary gene product ofosteoblasts during bone matrix formation and comprises 85ndash90 of the total organic bone matrix [30] Noncollagenousmatrix proteins also have a great importance in regulating ofossification and bone remodeling The most abundant non-collagenous protein produced by osteoblasts is osteocalcin(OCN) a phosphorylated glycoprotein which has high affin-ity for binding ionic calcium and physiologic hydroxyapatite[31] Osteopontin (OPN) is an osteoblast-derived heavilyglycosylated protein of the bone matrix which is expressedat late stages of differentiation and its appearance closelycorrelates with the appearance of mineral [31] In order toexamine the anabolic effect of VA the mRNA expressions ofCOL ALP OCN and OPN in MG-63 cells were measuredby real-time PCR (Figure 6) All sections of the VA (VAUVAM and VAB) treatment caused marked upregulation ofCOL and OCN mRNA levels Moreover the effects of VAMand VAB were not significantly different from the effectof VAU in COL and OCN expressions (119875 lt 005) Theeffects of VAU VAM and VAB on COL mRNA expressionswere increased 77- 60- and 58-fold respectively whilethose of OCN expressions were increased 225- 202- and150-fold respectively The expressions of ALP mRNA weresignificantly increased with VAU (37-fold) and VAM (35-fold) treatment compared with control and VAB group (12-fold)OPNmRNAexpressionswere not altered by all sectionsof VA These data suggest that VA may play role at earlystage as well as late stage of osteoblast differentiation andVA-induced COL ALP and OCN gene expression couldacceleratemineralization subsequently leading to an increasein the extent of calcium deposition Furthermore the effectson osteogenic gene expressions were decreased from uppersection to basal section of VA and the effect of VAM wassimilar to VAU Lee et al [28] reported that VA increasedmRNA expression of bone sialoprotein which is related tobone mineralization


Cell

pro

lifer

atio

n (

)

VAU VAM VAB

60

80

100

120

140

010

50100

A A

B B

AA

B B

A A AB

(a)

VAU VAM VAB

60

80

100

120

140

Alk

alin

e pho

spha

tase

()

050

100

A A AA B

AA

A A

(b)

60

80

100

120

140

Col

lage

n sy

nthe

sis (

)

Control VAU VAM VAB

A

B

AC

(c)

60

80

100

120

140

Control VAU VAM VAB

Calc

ium

dep

osit

()

A

B

CA

(d)

Figure 5 Effects of velvet antler on cell proliferation (a) alkaline phosphatase activity (b) collagen synthesis (c) and calcium deposit (d) inMG-63 human osteoblast-like cells VAU upper section of VA VAM middle section of VA VAB basal section of VA Data are shown as apercentage of the control value (means plusmn SD) of the three cultures Values not sharing a common alphabet are significantly different fromeach other at the level of 119875 lt 005

36 Effects on Osteoclastogenesis Osteoclasts are the onlycell type capable of resorbing mineralized bone To exam-ine the effect of VA on osteoclast differentiation bonemarrow-derived osteoclast precursor cells were cultured inosteoclastogenic media with different parts of VA extractTRAP-positive multinuclear osteoclasts were generated inresponse to stimulation fromM-CSF andRANKLThe resultsshowed that the addition of VA did not affect osteoclastformation (data not shown) Bacillus-fermented VA extracthas been reported to inhibit osteoclast differentiation [32]which does not support the results of the present studyHowever Lee et al [28] suggested that VA fermented withCordyceps militaris contain more sialic acid than nonfer-mented VA and the stimulatory effects on osteoblasticcell proliferation and ALP production were increased byfermentationTherefore the effect of nonfermentedVA in thepresent study may not be strong enough to induce osteoclastdifferentiation

4 Conclusions

In conclusion results of the present study suggest thatVA promotes longitudinal bone growth in adolescent ratsthrough at least in part MG-63 cell osteogenesis and

the effect was decreased downward from upper section tobasal section VA stimulated the proliferation differentiationand mineralization of osteoblasts through upregulation ofosteogenic gene expressions These results support the stim-ulating nature of VA toward the function of osteoblastic cellsAlthough the molecular mechanisms underlying osteoblastdifferentiation remain to be defined the results of the presentstudy suggest that BMP-2 which plays an important rolein regulating osteoblast differentiation and subsequent boneformation might participate in these mechanisms Further-more the present study provides strong evidence for theregional differences in the effectiveness of VA in longitudinalbone growth However further studies are needed to eluci-date the bioactive chemical constituents associated with theseeffects

Thepresent study has some limitationsOnly a single doseof VA was used in the in vivo study The dose for traditionaluse of VA is usually 1 gday taken all at once or dividedthroughout the day Therefore the dose of 100mgkgdaygiven to the experimental rats in this study was equivalentto the traditional human dosing regimen of 1 gday based onbody surface area conversion where animal dose is equal tohuman equivalent dose times 62 assuming that an adult personrsquosweight is 60 kg [33]


COL

mRN

A le

vel (

fold

)

Control VAU VAM VAB0

5

10

15

A

B

B

B

(a)

0

2

4

6

A

B B

A

ALP

mRN

A le

vel (

fold

)

Control VAU VAM VAB(b)

OCN

mRN

A le

vel (

fold

)


50

100

A

B BB

(c)

OPN

mRN

A le

vel (

fold

)


05

10

15

20

A

A

AA

(d)

Figure 6 Effects of velvet antler on osteogenic gene expression in MG-63 cells Real-time PCR was used to measure mRNA levels followingVA treatment Expression of GAPDHwas used to normalize all samples (a) Collagen (COL) (b) Alkaline phosphatase (ALP) (c) Osteocalcin(OCN) (d)Osteopontin (OPN) Data are shown as a fold of the control value (meansplusmn SD) of the three cultures Values not sharing a commonalphabet are significantly different from each other at the level of 119875 lt 005

Competing Interests

The authors declare that there are no competing interestsregarding the publication of this paper

Acknowledgments

This study was supported by the ldquoCooperative ResearchProgram for Agriculture Science ampTechnologyDevelopment(Project no PJ010181)rdquo of the Rural Development Adminis-tration Republic of Korea

References

[1] J P Iannotti ldquoGrowth plate physiology and pathologyrdquo Ortho-pedic Clinics of North America vol 21 no 1 pp 1ndash17 1990

[2] A C Guyton Text Book of Medical Physiology WB SaundersPhiladelphia Pa USA 10th edition 2000

[3] E W Leschek S R Rose J A Yanovski et al ldquoEffect of growthhormone treatment on adult height in peripubertal childrenwith idiopathic short stature a randomized double-blindplacebo-controlled trialrdquo The Journal of Clinical Endocrinologyand Metabolism vol 89 no 7 pp 3140ndash3148 2004

[4] F M Souza and P F Collett-Solberg ldquoAdverse effects of growthhormone replacement therapy in childrenrdquo Arquivos Brasileirosde Endocrinologia eMetabologia vol 55 no 8 pp 559ndash565 2011

[5] D ChenW-J Yao X-L Zhang et al ldquoEffects of Gekko sulfatedpolysaccharide-protein complex on human hepatoma SMMC-7721 cells Inhibition of proliferation and migrationrdquo Journal ofEthnopharmacology vol 127 no 3 pp 702ndash708 2010

[6] G-F Ge C-H Yu B Yu Z-H Shen D-L Zhang andQ-F Wu ldquoAntitumor effects and chemical compositionsof Eupolyphaga sinensis Walker ethanol extractrdquo Journal ofEthnopharmacology vol 141 no 1 pp 178ndash182 2012

[7] A Gilbey and J D Perezgonzalez ldquoHealth benefits of deerand elk velvet antler supplements a systematic review ofrandomised controlled studiesrdquo New Zealand Medical Journalvol 125 no 1367 pp 80ndash86 2012

[8] Z Sui L Zhang Y Huo and Y Zhang ldquoBioactive componentsof velvet antlers and their pharmacological propertiesrdquo Journalof Pharmaceutical and Biomedical Analysis vol 87 pp 229ndash2402014

[9] S J Jo J H Kim J-W Kim et al ldquoComparative studies onvelvet deer antler and ossified deer antler on the contentsof bioactive components and on the bone mineral densityimproving activity for oophorectomized ratrdquo Natural ProductSciences vol 19 no 4 pp 303ndash310 2013


[10] S-H Tseng H-C Sung L-G Chen et al ldquoEffects of velvetantler with blood on bone in ovariectomized ratsrdquo Moleculesvol 17 no 9 pp 10574ndash10585 2012

[11] P Ghosh R Roubin and M M Smith ldquoRationale for the useof antler cartilage products and genes obtained from their cellsto treat arthritis and repair cartilage defects following jointinjuryrdquo in Antler Science and Product Technology J S Sim HH Sunwoo R J Hudson and B T Jeon Eds pp 112ndash134University of Alberta Edmonton Canada 2001

[12] L-Z Zhang J-L Xin X-P Zhang Q Fu Y Zhang and Q-LZhou ldquoThe anti-osteoporotic effect of velvet antler polypeptidesfrom Cervus elaphus Linnaeus in ovariectomized ratsrdquo Journalof Ethnopharmacology vol 150 no 1 pp 181ndash186 2013

[13] B Jeon S Kim S Lee et al ldquoEffect of antler growth period onthe chemical composition of velvet antler in sika deer (Cervusnippon)rdquoMammalian Biology vol 74 no 5 pp 374ndash380 2009

[14] H Puchtler F S Waldrop and L S Valentine ldquoPolarizationmicroscopic studies of connective tissue stained with picroSirius red F3BArdquo Beitrage zur Pathologie vol 150 no 2 pp 174ndash187 1973

[15] H Puchtler S N Meloan and M S Terry ldquoOn the history andmechanism of alizarin and alizarin red S stains for calciumrdquoJournal of Histochemistry and Cytochemistry vol 17 no 2 pp110ndash124 1969

[16] C Minkin ldquoBone acid phosphatase tartrate-resistant acidphosphatase as amarker of osteoclast functionrdquoCalcified TissueInternational vol 34 no 1 pp 285ndash290 1982

[17] M H Guskuma E Hochuli-Vieira F P Pereira et al ldquoBoneregeneration in surgically created defects filled with autogenousbone an epifluorescencemicroscopy analysis in ratsrdquo Journal ofApplied Oral Science vol 18 no 4 pp 346ndash353 2010

[18] G R Mundy ldquoGrowth factors as potential therapeutic agentsin osteoporosisrdquo Instructional Course Lectures vol 46 pp 495ndash498 1997

[19] S-H Tseng C-H Sung L-G Chen et al ldquoComparison ofchemical compositions and osteoprotective effects of differentsections of velvet antlerrdquo Journal of Ethnopharmacology vol 151no 1 pp 352ndash360 2014

[20] J Wang J Zhou and C A Bondy ldquoIgf1 promotes longitudinalbone growth by insulin-like actions augmenting chondrocytehypertrophyrdquoThe FASEB Journal vol 13 no 14 pp 1985ndash19901999

[21] V Abad J L Meyers M Weise et al ldquoThe role of the restingzone in growth plate chondrogenesisrdquo Endocrinology vol 143no 5 pp 1851ndash1857 2002

[22] G J Breur B A VanEnkevort C E Farnum andN JWilsmanldquoLinear relationship between the volume of hypertrophic chon-drocytes and the rate of longitudinal bone growth in growthplatesrdquo Journal of Orthopaedic Research vol 9 no 3 pp 348ndash359 1991

[23] M Weise S De-Levi K M Barnes R I Gafni V Abadand J Baron ldquoEffects of estrogen on growth plate senescenceand epiphyseal fusionrdquo Proceedings of the National Academy ofSciences of the United States of America vol 98 no 12 pp 6871ndash6876 2001

[24] S Bass P D Delmas G Pearce E Hendrich A Tabenskyand E Seeman ldquoThe differing tempo of growth in bone sizemass and density in girls is region-specificrdquo Journal of ClinicalInvestigation vol 104 no 6 pp 795ndash804 1999

[25] J M Wozney and V Rosen ldquoBone morphogenetic protein andbonemorphogenetic protein gene family in bone formation and

repairrdquo Clinical Orthopaedics and Related Research no 346 pp26ndash37 1998

[26] F De Luca K M Barnes J A Uyeda et al ldquoRegulation ofgrowth plate chondrogenesis by bone morphogenetic protein-2rdquo Endocrinology vol 142 no 1 pp 430ndash436 2001

[27] C G Bellows J E Aubin and J NM Heersche ldquoInitiation andprogression of mineralization of bone nodules formed in vitrothe role of alkaline phosphatase and organic phosphaterdquo Boneand Mineral vol 14 no 1 pp 27ndash40 1991

[28] H-S LeeM K Kim Y-K Kim et al ldquoStimulation of osteoblas-tic differentiation and mineralization in MC3T3-E1 cells byantler and fermented antler using Cordyceps militarisrdquo Journalof Ethnopharmacology vol 133 no 2 pp 710ndash717 2011

[29] J E Aubin F Liu L Malaval and A K Gupta ldquoOsteoblast andchondroblast differentiationrdquo Bone vol 17 no 2 supplement 1pp S77ndashS83 1995

[30] Y Sasano J-X Zhu S Kamakura S Kusunoki I Mizoguchiand M Kagayama ldquoExpression of major bone extracellu-lar matrix proteins during embryonic osteogenesis in ratmandiblesrdquo Anatomy and Embryology vol 202 no 1 pp 31ndash372000

[31] B Sommer M Bickel W Hofstetter and A WetterwaldldquoExpression of matrix proteins during the development ofmineralized tissuesrdquo Bone vol 19 no 4 pp 371ndash380 1996

[32] S-W Choi S-H Moon H J Yang et al ldquoAntiresorptiveactivity of bacillus -fermented antler extracts inhibition ofosteoclast differentiationrdquo Evidence-Based Complementary andAlternative Medicine vol 2013 Article ID 748687 9 pages 2013

[33] S Reagan-Shaw M Nihal and N Ahmad ldquoDose translationfrom animal to human studies revisitedrdquo The FASEB Journalvol 22 no 3 pp 659ndash661 2008

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


A whole VA stick is usually divided into four portionswith the value decreasing from the tip to the base Chemicalanalyses of VA revealed that there are regional differencesin chemical composition the contents of proteins and lipidsdecrease downward from the tip to the base while thoseof ash calcium and collagen increase [13] suggesting thedegree of calcification increased from tip to base section ofVA During the ossification of deer antler the total collagencontent was found to be increased Typically the antleris cut off near the base after it is about two-thirds of itspotential full size between 55 and 65 days of growth beforeany significant calcification occurs Traditionally the marketvalues of antlers are downgraded with increasing degree ofcalcification However which parts of VA are suitable forpreventing andmanaging bone health especially bone growthhad not been clarified

The objective of the present study was to compare theeffect of VA from different sections on longitudinal bonegrowth of adolescent rats and elucidate the underlyingmech-anisms for the effect

2 Materials and Methods

21 Materials VA obtained from farmed Elk deer 75 daysafter casting was kindly provided by the Animal GeneticResources Station (National Institute of Animal ScienceSouth Korea) VA was divided into upper (VAU) middle(VAM) and basal (VAB) sections (Figure 1)The criteria usedfor dividing the antler into three sections is defined as followsupper section top 15 cm of the part of the main beam to the2nd division of a deerrsquos antlers from its head middle sectionthe upper half of the remaining main beam and base sectionthe lower half of the remaining main beam Total part of eachsectionwas slicedwith a bone slicer freeze-dried and groundinto powderThe powderedVAwas immersed in 70 ethanolfor 3 days filtered concentrated by vacuum evaporationand finally subjected to freeze drying Respective yields ofsections fromupper to basal were 34 (VAU) 29 (VAM)and 17 (VAB)

22 Animals Longitudinal bone growth was determined in3-week-old male Sprague-Dawley rats (Samtako Co OsanKorea) The experimental procedures were performed inaccordance with protocols approved by the Institutional Ani-mal Care and Use Committee Semyung University (smecae15-09-01)The animals were housed under controlled temper-ature and lighting conditionsThe ratswere randomly dividedinto four groups (119899 = 8) vehicle (control distilled water)and upper (VAU) middle (VAM) and basal (VAB) sectionsof antler extract (100mgkg) were orally administered dailyvia stomach tube for 5 consecutive days Treatment dose wasdecided from the preliminary study On the sixth day all ratswere sacrificed under chloral hydrate anesthesia for tissueanalysis

23 Measurement of Longitudinal Bone Growth To measurethe effect on longitudinal bone growth rate calcein wasused as a fluorescence marker to label the bone line on thesurface of the tibia Calcein plays the role of fluorescent

VAU

VAM

VAB

Figure 1 Sections of velvet antler Fresh velvet antler was dividedinto upper (VAU) middle (VAM) and basal (VAB) sections

dye under ultraviolet illumination Calcein (10mgkg Sigma-Aldrich St Louis MO USA) was injected intraperitoneally24 h before sacrifice The dissected tibias were fixed in01M phosphate buffered formalin for 2 h decalcified anddehydrated by immersing in 30 sucrose for 1 day at 4∘CDehydrated bone was sectioned longitudinally at sagittalsections of proximal part with a thickness of 40120583m using asliding microtome (HM440E Zeiss Germany) Bone growthwas measured by measuring the gap between fluorescentline formed by calcein and the epiphyseal end line of thegrowth plate at three different locations using a fluorescentmicroscope (BX60Olympus Tokyo Japan) and the averageswere obtained

24 Measurement of Growth Plate Height Cresyl violet stain-ing (Sigma-Aldrich) was used to stain the chondrocytes in thegrowth plate of the samples Each tibia sample was sectionedlongitudinally at a thickness of 40 120583m using a sliding micro-tome as described above and stained with cresyl violet Thegrowth plate height was measured at three different locationsand the averages were obtained

25 Bone Morphogenetic Protein-2 Expression For the detec-tion of bonemorphogenetic protein-2 (BMP-2) in the growthplate the dehydrated tibia sections were incubated overnightin 1 Triton X-100 containing goat BMP-2 antibody (SantaCruz Biotechnology diluted 1 500) at room temperatureThen sections were incubated with anti-goat antibody (Vec-tor Laboratories Burlingame CA USA diluted 1 200) for60min and stained with 005 33-diaminobenzidine (SigmaChemical Co) containing 003 hydrogen peroxide

26 Cell Culture Human osteoblast-like MG-63 cells wereobtained from the American Type Culture Collection(Rockville MD USA) Cells were cultured at 37∘C in a 5CO2atmosphere in Dulbeccorsquos Modified Eaglersquos Medium

(DMEM Gibco MD USA) containing 10 heat-inactivated

























400

600

800

1000

A

BA

A

Bone

gro

wth

(120583m

)

(b)









Control VAU

Resting zone

Proliferative zone

Hypertrophic zone



300

400

500

600

700

800

A

B BA

Gro

wth

pla

te h

eigh

t (120583

m)

(b)









Resting zone

Proliferative zone

Hypertrophic zone

Ossification zone

Control VAU

200120583m

(a)

BMP-

2 ex

pres

sion

()

Control VAU VAM VAB

60

80

100

120

140

A

B B

A

(b)






Cell

pro

lifer

atio

n (

)

VAU VAM VAB

60

80

100

120

140

010

50100

A A

B B

AA

B B

A A AB

(a)

VAU VAM VAB

60

80

100

120

140

Alk

alin

e pho

spha

tase

()

050

100

A A AA B

AA

A A

(b)

60

80

100

120

140

Col

lage

n sy

nthe

sis (

)

Control VAU VAM VAB

A

B

AC

(c)

60

80

100

120

140

Control VAU VAM VAB

Calc

ium

dep

osit

()

A

B

CA

(d)



4 Conclusions





COL

mRN

A le

vel (

fold

)


5

10

15

A

B

B

B

(a)

0

2

4

6

A

B B

A

ALP

mRN

A le

vel (

fold

)


OCN

mRN

A le

vel (

fold

)


50

100

A

B BB

(c)

OPN

mRN

A le

vel (

fold

)


05

10

15

20

A

A

AA

(d)


Competing Interests


Acknowledgments


References









































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of








































400

600

800

1000

A

BA

A

Bone

gro

wth

(120583m

)

(b)









Control VAU

Resting zone

Proliferative zone

Hypertrophic zone



300

400

500

600

700

800

A

B BA

Gro

wth

pla

te h

eigh

t (120583

m)

(b)









Resting zone

Proliferative zone

Hypertrophic zone

Ossification zone

Control VAU

200120583m

(a)

BMP-

2 ex

pres

sion

()

Control VAU VAM VAB

60

80

100

120

140

A

B B

A

(b)






Cell

pro

lifer

atio

n (

)

VAU VAM VAB

60

80

100

120

140

010

50100

A A

B B

AA

B B

A A AB

(a)

VAU VAM VAB

60

80

100

120

140

Alk

alin

e pho

spha

tase

()

050

100

A A AA B

AA

A A

(b)

60

80

100

120

140

Col

lage

n sy

nthe

sis (

)

Control VAU VAM VAB

A

B

AC

(c)

60

80

100

120

140

Control VAU VAM VAB

Calc

ium

dep

osit

()

A

B

CA

(d)



4 Conclusions





COL

mRN

A le

vel (

fold

)


5

10

15

A

B

B

B

(a)

0

2

4

6

A

B B

A

ALP

mRN

A le

vel (

fold

)


OCN

mRN

A le

vel (

fold

)


50

100

A

B BB

(c)

OPN

mRN

A le

vel (

fold

)


05

10

15

20

A

A

AA

(d)


Competing Interests


Acknowledgments


References









































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















400

600

800

1000

A

BA

A

Bone

gro

wth

(120583m

)

(b)









Control VAU

Resting zone

Proliferative zone

Hypertrophic zone



300

400

500

600

700

800

A

B BA

Gro

wth

pla

te h

eigh

t (120583

m)

(b)









Resting zone

Proliferative zone

Hypertrophic zone

Ossification zone

Control VAU

200120583m

(a)

BMP-

2 ex

pres

sion

()

Control VAU VAM VAB

60

80

100

120

140

A

B B

A

(b)






Cell

pro

lifer

atio

n (

)

VAU VAM VAB

60

80

100

120

140

010

50100

A A

B B

AA

B B

A A AB

(a)

VAU VAM VAB

60

80

100

120

140

Alk

alin

e pho

spha

tase

()

050

100

A A AA B

AA

A A

(b)

60

80

100

120

140

Col

lage

n sy

nthe

sis (

)

Control VAU VAM VAB

A

B

AC

(c)

60

80

100

120

140

Control VAU VAM VAB

Calc

ium

dep

osit

()

A

B

CA

(d)



4 Conclusions





COL

mRN

A le

vel (

fold

)


5

10

15

A

B

B

B

(a)

0

2

4

6

A

B B

A

ALP

mRN

A le

vel (

fold

)


OCN

mRN

A le

vel (

fold

)


50

100

A

B BB

(c)

OPN

mRN

A le

vel (

fold

)


05

10

15

20

A

A

AA

(d)


Competing Interests


Acknowledgments


References









































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Control VAU

Resting zone

Proliferative zone

Hypertrophic zone



300

400

500

600

700

800

A

B BA

Gro

wth

pla

te h

eigh

t (120583

m)

(b)









Resting zone

Proliferative zone

Hypertrophic zone

Ossification zone

Control VAU

200120583m

(a)

BMP-

2 ex

pres

sion

()

Control VAU VAM VAB

60

80

100

120

140

A

B B

A

(b)






Cell

pro

lifer

atio

n (

)

VAU VAM VAB

60

80

100

120

140

010

50100

A A

B B

AA

B B

A A AB

(a)

VAU VAM VAB

60

80

100

120

140

Alk

alin

e pho

spha

tase

()

050

100

A A AA B

AA

A A

(b)

60

80

100

120

140

Col

lage

n sy

nthe

sis (

)

Control VAU VAM VAB

A

B

AC

(c)

60

80

100

120

140

Control VAU VAM VAB

Calc

ium

dep

osit

()

A

B

CA

(d)



4 Conclusions





COL

mRN

A le

vel (

fold

)


5

10

15

A

B

B

B

(a)

0

2

4

6

A

B B

A

ALP

mRN

A le

vel (

fold

)


OCN

mRN

A le

vel (

fold

)


50

100

A

B BB

(c)

OPN

mRN

A le

vel (

fold

)


05

10

15

20

A

A

AA

(d)


Competing Interests


Acknowledgments


References









































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Resting zone

Proliferative zone

Hypertrophic zone

Ossification zone

Control VAU

200120583m

(a)

BMP-

2 ex

pres

sion

()

Control VAU VAM VAB

60

80

100

120

140

A

B B

A

(b)






Cell

pro

lifer

atio

n (

)

VAU VAM VAB

60

80

100

120

140

010

50100

A A

B B

AA

B B

A A AB

(a)

VAU VAM VAB

60

80

100

120

140

Alk

alin

e pho

spha

tase

()

050

100

A A AA B

AA

A A

(b)

60

80

100

120

140

Col

lage

n sy

nthe

sis (

)

Control VAU VAM VAB

A

B

AC

(c)

60

80

100

120

140

Control VAU VAM VAB

Calc

ium

dep

osit

()

A

B

CA

(d)



4 Conclusions





COL

mRN

A le

vel (

fold

)


5

10

15

A

B

B

B

(a)

0

2

4

6

A

B B

A

ALP

mRN

A le

vel (

fold

)


OCN

mRN

A le

vel (

fold

)


50

100

A

B BB

(c)

OPN

mRN

A le

vel (

fold

)


05

10

15

20

A

A

AA

(d)


Competing Interests


Acknowledgments


References









































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Cell

pro

lifer

atio

n (

)

VAU VAM VAB

60

80

100

120

140

010

50100

A A

B B

AA

B B

A A AB

(a)

VAU VAM VAB

60

80

100

120

140

Alk

alin

e pho

spha

tase

()

050

100

A A AA B

AA

A A

(b)

60

80

100

120

140

Col

lage

n sy

nthe

sis (

)

Control VAU VAM VAB

A

B

AC

(c)

60

80

100

120

140

Control VAU VAM VAB

Calc

ium

dep

osit

()

A

B

CA

(d)



4 Conclusions





COL

mRN

A le

vel (

fold

)


5

10

15

A

B

B

B

(a)

0

2

4

6

A

B B

A

ALP

mRN

A le

vel (

fold

)


OCN

mRN

A le

vel (

fold

)


50

100

A

B BB

(c)

OPN

mRN

A le

vel (

fold

)


05

10

15

20

A

A

AA

(d)


Competing Interests


Acknowledgments


References









































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















COL

mRN

A le

vel (

fold

)


5

10

15

A

B

B

B

(a)

0

2

4

6

A

B B

A

ALP

mRN

A le

vel (

fold

)


OCN

mRN

A le

vel (

fold

)


50

100

A

B BB

(c)

OPN

mRN

A le

vel (

fold

)


05

10

15

20

A

A

AA

(d)


Competing Interests


Acknowledgments


References









































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Date post:	07-Jul-2020
Category:	Documents
Upload:	others
View:	1 times
Download:	0 times

Research Article Comparison of the Effect of Velvet Antler from...

Documents