2012/8/13

Outline
TissueEngineering(TE) Collagen
Adviser:FengHuei Lin,PhD Speaker:JungFeng Lin ChunTingYang YenJye Shyong Date:2012/08/13

ApplicationofCollageninBiomedical PartI ExtractionofCollagentypeI PartII Bradfordproteinbindingassay

NationalTaiwanUniversity InstituteofBiomedicalEngineering,Bioceramics andCompositesLab.

NationalTaiwanUniversity InstituteofBiomedicalEngineering,Bioceramics andCompositesLab.

TissueEngineering
Definition: Afieldthatappliesthe principlesofengineeringand biologicalsciencestocreate substitutesfordamagedtissue.

TissueEngineeringTriad
Hollowfiber Rotatingwall Spinnerflask Direct perfusion
Cell

Primarycells Adultcells EScells Engineeredcells MSCs

Bioreactor

Alginate Collagen Chitosan Gelatin PLGA HA

NationalTaiwanUniversity InstituteofBiomedicalEngineering,Bioceramics andCompositesLab.

Scaffold

Signal

NationalTaiwanUniversity InstituteofBiomedicalEngineering,Bioceramics andCompositesLab.

Medium Growthfacotrs Chemicalcompound Mechanicalstimuli

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Collagen
Collagen isthemainproteinof

connectivetissueinanimalsand oneofthemostabundantprotein inmammals,makingupabout40% ofthetotal. Itisoneofthelong,fibrous structuralproteins whose functionsarequite differentfromthoseofglobular proteinssuchasenzymes.
NationalTaiwanUniversity InstituteofBiomedicalEngineering,Bioceramics andCompositesLab.

MajorscaffoldproteinsofECM Mostabundantproteininmammals,upto30%ofallproteins Responsibleforfunctionalintegrityoftissuessuchascartilage,

skin,tendon
15collagentypespresentinhumantissues Hightensilestrength,equivalenttosteelwhencomparedon

crosssectionalarea,factorofthreegreateron aperweightbasis

NationalTaiwanUniversity InstituteofBiomedicalEngineering,Bioceramics andCompositesLab.

Molecularstructure ofCollagen
Ithasatriplehelixstructurecontaining
proline

CollagenSynthesis
TranslatedfromRNA OHgroupsadd Transportoutofcell Cleavageofaminoterminalandcarboxy

threepolypeptidechainsrangedin righthandedsupercoil
Glycine,Proline,Hydroxyproline

glycine

carbon nitrogen oxygen

terminal
Adistinctivefeatureofcollagenistheregular Selfassemblybeginsspontaneously

arrangementofaminoacids ineachofthethree chainsofthesecollagensubunits.Thesequence oftenfollowsthepattern GlyProXorGlyXHyp, whereXmaybeanyofvariousotheraminoacid residues.

NationalTaiwanUniversity InstituteofBiomedicalEngineering,Bioceramics andCompositesLab.
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NationalTaiwanUniversity InstituteofBiomedicalEngineering,Bioceramics andCompositesLab.

2012/8/13

Collagenoccursinmanyplacesthroughoutthebody.The29 typesofcollagenhavebeenidentifiedanddescribed. Inhumanbody

Collagen
Advantages

CollagenI:skin,tendon,bone,fishscale CollagenII:cartilage CollagenIII:commonlyfoundalongsidetypeI. CollagenIV:formsbasesofcellbasementmembrane

Lowimmunoresponse Biocompatiable Biodegradable Tensilestrength Swellinginacid,alkalineandslats Manufacturable

NationalTaiwanUniversity InstituteofBiomedicalEngineering,Bioceramics andCompositesLab.

NationalTaiwanUniversity InstituteofBiomedicalEngineering,Bioceramics andCompositesLab.

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Application
Membrane (filmsheet) Porous (sponge fiber) Solution Filament Tubular Gel Composite (collagen/ polymer collagen/ceramic)
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Collagen Manufacturing
Prizeofcollagenisexpensive. Amountofcollagenneededfortissueengineeringis

notlow. Selfmanufacturingisnecessary.

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Collagen Manufacturing
Pigskin Dialysis

MaterialI ExtractionofCollagentypeI frompigskin

1. 2. 3.

Extraction

Purity determination

4. 5. 6. 7.

Enzymatic degradation

Concentration

PigSkin Scalpel Acetone 10%and5%Brine(10/5gNaCl in100mlddH2O) Citratebuffersolution(pH=4.5) Pepsin 0.5MHCl (pH=2)

Saltingout

Sterilization

Packing

NationalTaiwanUniversity InstituteofBiomedicalEngineering,Bioceramics andCompositesLab.

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MethodI ExtractionofCollagentypeI frompigskin 8/13

Peeledoffmuscle,fatandallremainsofsurroundingconnective tissue 2. Mince 3. Defatted>withacetonefor30minatR.T.,twice 4. Cleaned>withdeionized water,twice 5. Swollen>Soakedin10%Brine at4 for1hr 6. Swollen>Soakedincitratebuffersolution(pH=4.5)for3hr 7. Digested >withpepsinin0.5M(pH=2)HCl atRTforover night(TheweightratioSkin:pepsin/HCl =10:0.3:50) 8/14 1. Precipitated >with5%NaCl solution
1.
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GelelectrophoresisoftypeI collagen
TodeterminethepurityoftypeIcollagen.

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GelelectrophoresisoftypeI collagen
CompositionofTypeIcollagen:[1(I)]2[2(I)] Thehydrogenbondbetweentwo1peptide:11(I) Thehydrogenbondbetweena1anda2peptide:12(I)

MaterialII Bradfordproteinbinding

assay
Materials 1. Bovineserumalbumin(BSA)(Thermo) 2. Microcentrifugetube 3. BioRadDyeReagent:ddH2O=1:4 4. ELISAreader 5. 96wellplate

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NationalTaiwanUniversity InstituteofBiomedicalEngineering,Bioceramics andCompositesLab.

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MethodII Bradfordproteinbinding

assay
8/13 1. PrepareBSAstandards:50g,25 g,12.5 g,6.25 gand3.125 g 2. Add10 lBSAstandardsin200lBioRadDyeReagentand incubateatroomtemperaturefor5minutes 3. Assayabsorbanceat595nm.

NationalTaiwanUniversity InstituteofBiomedicalEngineering,Bioceramics andCompositesLab.

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NationalTaiwanUniversity InstituteofBiomedicalEngineering,Bioceramics andCompositesLab.

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NationalTaiwanUniversity InstituteofBiomedicalEngineering,Bioceramics andCompositesLab.

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