ProductDescription
FibriCol®collagenisknownasthestandardofallcollagensforpurity(>99%collagencontent),functionality,andthemostnative-likecollagenavailable.FibriCol®isisolatedfrombovinehidessourcedfromtheonlycontrolled,closedherdintheUnitedStates.AdvancedBiomatrix’smanufacturingprocessescomplywithstringentqualitystandardsthathaveproventoyieldunsurpassedlot-to-lotconsistency.
FibriCol®collagenisapproximately97%TypeIcollagenwiththeremainderbeingcomprisedofTypeIIIcollagen.FibriCol®collagenissuppliedatapproximatelya10mg/mLconcentration.TheconcentrationforeachspecificlotisprovidedonaCertificateofAnalysisthatisavailablewiththepurchaseofeachproduct.FibriCol®issolubleatelocollagenin0.01NHCI,therefore,thepHis2.
FibriCol®collagenisidealforapplicationswherehighconcentrationsofcollagenareneeded.FibriCol®collagenisprovidedina20mLvolumeandiscontainedinuser-friendlypackagingforuseandstorage.FibriCol®issterilefilteredandissuppliedasareadytousesolution.
| Parameter,Testing,andMethod | FibriCol®TypeICollagen#5133 |
| SterilizationMethod | Filtration |
| ExtractionMethod | Enzyme-atelocollagen |
| Form | Solution |
| PackageSize | 20mL,100mL |
| StorageTemperature | 2-10°C |
| ShelfLife | Minimumof6monthsfromdateofreceipt |
CollagenConcentration-Biuret | 9.0-11.5mg/mL |
CollagenPurity-SilverStaining | >99.9% |
| pH | 1.9-2.1 |
| KineticGelTest(Minutes) | <40 |
| GelFormationTubeTest(Minutes) | <40 |
Fibrillogenesis(AbsorbanceUnits) | >0.5 |
ElectrophoreticPattern-CoomassieBlue | Characteristic |
| Sterility-USPmodified | Nogrowth |
| Endotoxin-LAL | <1.0EU/mL |
| Osmolality(mOsmoH2O/kg) | <35 |
| CellAttachmentAssay | Pass |
| Source | BovineHide |
| HydrogelYoung"sModulusE(Pa) | Characteristic |
DirectionsforUse
DownloadthefullPDFversionorcontinuereADIngbelow:
3-DGelPreparationProcedure
- Slowlyadd1partofchilled10XPBSor10Xculturemediato8partsofchilledcollagensolutionwithgentlemixing/swirling.
- AdjustpHofmixtureto7.2–7.6.Useofsterile0.1MNaOHisrecommended.MonitorpHadjustmentcarefully(pHmeter,phenolred,orpHpaper).
- Adjustfinalvolumetoatotalof10partswithsterilewater.
- Topreventgelation,maintaintemperatureofmixtureat2-10°C.
- Toformgel,warmto37°C.Forbestresultsallowapproximately90minutesforgelformation
ProductQ&A
WecompletedastudytoshowthatDNAiscompletelydestroyedatpH2,anddemonstratedthatourcollagenproductsdonotcontainDNA.
Thecollagenisfullyhydrolyzed.TheaminoacidanalysisisdoneusingtheWatersAccQ-Tagderivatizationmethod.Duringtheacidhydrolysisstep,asparagine(N)isconvertedtoasparticacid(D)andglutamine(Q)isconvertedtoglutamicacid(E).Tryptophan(W),ifpresent,isdestroyedduringacidhydrolysis.Experimentally,onecandeterminethepicomoles(pmol)ofeachaminoacidperinjecteddetectedusingaminoacidstandards.Fortheconcentrationdetermination,thetotalnumberofpmolofeachaminoacidissummedtogetthetotalpmolofthe18aminoacidsdetected.Thetotalpmolaminoacidsisdividedbythetheoreticalnumberofaminoacidresiduesincollagenbasedonthepublishedsequence.Theresultisthepmolofcollageninjected.Theresultisthenmultipliedbythedilutionand300,000isusedasthecollagenmolecularweighttogettomg/mL.Themolecularweightofcollagenisnotwellagreedupon.
Dilutingwith1XPBS(ratherthanwateror0.01NHCl)wouldhaveaneffectforcoatingpurposes.ItwouldchangethepHofthedilutedcollagensolutionfromacidtoneutralpH.ThepHchangewilltransformthecollagenmoleculesfromamolecularformtoafibrillarform;andthenthenatureofcoatingsurfacewillbechangedfromamonomericcoatingtoafibrillarcoating.
WeusethefollowingantibodiesfromSouthernBiotech:
1.1310-02–GoatAnti-TypeICollagen-FITC
2.1310-08–GoatAnti-TypeICollagen-BIOT
3.7100-05–Streptavidin-HRP
ThemajorcollagenmolecularspeciesinourTypeIcollagenproductsaremonomers(approx.70%),buttherearedimers,trimersandafewpercentagesofoligomerstoo(approx.30%)withsomeminoramountsofcollagenfragments.Thecollagenmonomerisarodshapedmoleculewith300nminlengthand1.5nmindiameter.Thedimer,trimerandoligomerare600nm,900nmandevenlongerinlengthrespectively.Accordingtothecoatingprocedures,thecollagenmoleculesareattachedtothechargedpolystyrenesurfacerandomlybychargeoraffinityinacidconditionsduringthe1-2hrsincubationperiodat37°C,andanyunattachedmaterialsareremovedbyaspirationandrinsing.Therefore,thecoatedsurfaceisasinglelayerofcollagenmonomer,dimer,trimerandoligomermixtures.Thethicknessofthemono-molecularlayerisdependentonhowthosemoleculesareattachedonthesurface.Thecoatingdensitythicknesswouldgenerallybecharacterizedasa1moleculethicknesswhichcouldberangingfromafewnanometerstoafewhundrednanometerswiththewholesurfacebeingcoveredbycollagen.
ThenetchargeofTypeIcollagenproducts’(PureCol®,BovineCollagenandVitroCol®,HumanCollagen)moleculeisdirectlyrelatedtothepH.AtanacidicpH,theaminoacids(zwitterions)alongthecollagenmoleculearepositivelycharged,makingtheentirecollagenmoleculepositive.Attheisoelectricpoint(orzone)ofcollagen,aroundpH7-8,theaminoacidsalongthecollagenmoleculearepositivelyandnegativelycharged,makingthenetchargeofthecollagenmoleculeclosetozero.AtabasicpH,theaminoacidsalongthecollagenmoleculewerenegativelycharged,makingtheentirecollagenmoleculenegative.
Further,thenatureofthechargeofthecollagencoatingsurfacewillbedependentonthetypeofcoatingapplied.ForamonomericcollagencoatingswhenthecollagenisappliedunderanacidicpHcondition,thesurfaceispositivelycharged.IfthesurfaceisrinsedwithpHneutralbufferormediathenitwillchangethechargeofthecollagensurfacenetchargeclosetozero.Fora3Dgelcoating,thecollagenpreparedunderneutralpH;thenetchargeofthecollagensurfaceisclosetozero.
Usingrotaryshadowingtechniqueundertransmissionelectronmicroscopy,itwasfoundthatourcollagen,onaverage,consistsofapproximately80%monomers,13%dimers,trimers,andoligomerswiththeremaining7%collagenfragments.
Yes.ThecollagenmoleculeinPureCol,Nutragen,VitroCol,andallofourotherAtelocollagenproductswerepreparedfromnativecollagenmatrixbypepsintreatmentundercontrolledconditionstoremovethenon-helicalportion,telo-peptides,onlyandthehelicalportionisintact.Inthiscase,theenzymaticactivesitesforMMP(MatrixMetalloproteinase),suchasforMammalianCollagenaseMatrixMetalloproteinase8(MMP-8),onthemoleculewaspreserved.
Thesepepsintreatedcollagenproductsshouldbehaveasnativeintactcollagen.
TGFbetawouldhavebeendigestedwiththepepsinenzymaticdigestionstep.ItwasundetectablebySDSPAGEsilverstainaswell.Wedidn’tdoanyspecificmeasurementsbyELISAhoweverbutpresencesofTGFbetaisnotanticipated.
WeprimarilyusetheBiuretmethod,butwealsouseBCA,AAA,andhydroxyl-prolineassays.
-Collagensolutionsthatarefrozentendtohaveissuesforming3Dhydrogels,andwilllikelynotwork.Thesolutionsshouldstillbegoodfor2Dcoatings.
-Collagensolutionsthatareleftoutatroomtemperatureforextendedperiodsoftimemayshowsignsofdegradation,whichwillaffecttheformationof3Dhydrogels.Itislikelystillfinefor2Dcoatings.
Ourrecommendationisthis:Ifyouareusingtheproductdirectlyforapublication,wehighlysuggestbuyinganewbottleiftheoneyouhavewascompromised.
ProductReferences
ReferencesforFibriCol®:
Berger,AnthonyJ.,etal."Decouplingtheeffectsofstiffnessandfiberdensityoncellularbehaviorsviaaninterpenetratingnetworkofgelatin-methacrylateandcollagen."Biomaterials141(2017):125-135.
Kreimendahl,Franziska,etal."Three-dimensionalprintingandangiogenesis:tailoredagarose-typeIcollagenblendscomprisethree-dimensionalprintABIlityandangiogenesispotentialfortissue-engineeredsubstitutes."TissueEngineeringPartC:Methods23.10(2017):604-615.
Mas-Vinyals,Anna,etal."Improvinglinkinginterfacebetweencollagen-basedhydrogelsandbone-likesubstrates."ColloidsandSurfacesB:Biointerfaces(2019).
Schoppmeyer,Rouven,etal."Light-sheetmicroscopyforthree-dimensionalvisualizationofhumanimmunecells."JoVE(JournalofVisualizedExperiments)136(2018):e57651.
Semina,E.V.,etal."Three-dimensionalmodelofbiomatrixasamethodofstudyingbloodvesselsandnervegrowthintissueengineeringstructures."MoscowUniversityChemistryBulletin71.3(2016):172-177.
Pompilio,Arianna,etal."MyroidesodoratimimusFormsStructurallyComplexandInherentlyAntibiotic-ResistantBiofilminaWound-LikeinvitroModel."FrontiersinmicroBIOLOGy8(2017):2591.
Christian,SherriL.,etal."Collagenoverlayscaninhibitleptinandadiponectinsecretionbutnotlipidaccumulationinadipocytes."PeerJ6(2018):e4641.
Vining,KyleH.,AlexanderStafford,andDavidJ.Mooney."Sequentialmodesofcrosslinkingtuneviscoelasticityofcell-instructivehydrogels."Biomaterials188(2019):187-197.
Kreimendahl,F.,etal."Combinationofvascularizationandciliaformationforthree‐dimensionalairwaytissueengineering."JournalofBiomedicalMaterialsResearchPartA(2019).
Chen,Yin-Quan,etal."Earlystagemechanicalremodelingofcollagensurroundingheadandnecksquamouscellcarcinomaspheroidscorrelatesstronglywiththeirinvasioncapability."Actabiomaterialia84(2019):280-292.
Caliari,StevenR.,andJasonA.Burdick."Apracticalguidetohydrogelsforcellculture."Naturemethods13.5(2016):405.
Szulc,DanielAndrzej,andHai‐LingMargaretCheng."One‐StepLabelingofCollagenHydrogelswithPolydopamineandManganesePorphyrinforNon‐InvasiveScaffoldTrackingonMagneticResonanceImaging."Macromolecularbioscience(2019):1800330.
ProductCertificateofAnalysis
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SafetyDataSheet
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DeclarationofMaterialSource
ProductDisclaimer
ThisproductisforR&Duseonlyandisnotintendedforhumanorotheruses.PleaseconsulttheMaterialSafetyDataSheetforinformationregardinghazardsandsafehandlingpractices.
