1、Designation:F310614Standard Guide forin vitro Osteoblast Differentiation Assays1This standard is issued under the fixed designation F3106;the number immediately following the designation indicates the year oforiginal adoption or,in the case of revision,the year of last revision.A number in parenthes
2、es indicates the year of last reapproval.Asuperscript epsilon()indicates an editorial change since the last revision or reapproval.1.Scope1.1 This document provides guidance on how to conduct invitro osteoblast differentiation assays with progenitor stemcells including mesenchymal stromal cells.1.2
3、This document describes the roles of various osteogenicsupplements that are added to the cell culture medium of anosteoblast differentiation assay to encourage and support thedifferentiation of progenitor cells into matrix-producing osteo-blasts.1.3 This document provides recommendations for the con
4、-centrations of osteogenic supplements that may prevent theprecipitation of artifactual mineral deposits that are not directlyproduced by osteoblasts,nor correlated with osteoblastic geneexpression of the cells.1.4 This standard does not purport to address all of thesafety concerns,if any,associated
5、 with its use.It is theresponsibility of the user of this standard to establish appro-priate safety and health practices and determine the applica-bility of regulatory limitations prior to use.2.Referenced Documents2.1 ASTM Standards:2F2312 Terminology Relating to Tissue Engineered MedicalProductsF2
6、997 Practice for Quantification of Calcium Deposits inOsteogenic Culture of Progenitor Cells Using FluorescentImage Analysis3.Terminology3.1 Unless provided otherwise in 3.2,terminology shall bein conformance with Terminology F2312.3.2 Definitions:3.2.1 calcium deposits,na calcium phosphate-containi
7、ngsubstance synthesized in cell cultures during mineralization orosteoblast differentiation assays that may be directly producedby osteoblasts or precipitated out of the solution without cellparticipation.3.2.2 mineralizedmatrix,nacalciumphosphate-containing substance produced by cells typically in
8、theosteoblast,odontoblast,and calcifying chondrocyte lineages,which is composed of crystals of calcium phosphate andcontains collagen Type I and other non-collagenous proteins.3.2.3 osteoblasts,nsecretory mononuclear cells that willinitiate the formation of a matrix containing characteristicproteins
9、,such as collagen,and non-collageneous proteins suchas bone sialoprotein and osteocalcin,that will mineralize in thepresence of a calcium and phosphate source.4.Significance and Use4.1 This guidance document describes the components andconditions used for in vitro osteoblast differentiation assaysth
10、at can be used to screen for the osteogenic capability ofprogenitor stem cells from various human or animal sources,including mixed tissue-derived connective tissue progenitorpopulations,or cell populations that may be selectively isolatedor manipulated through culture expansion,processing,trans-fec
11、tion or genetic modification.4.2 The osteoblast differentiation assay may be referred toas an osteogenesis assay or a mineralization assay.4.3 It is important to carefully select the components andconditions used for in vitro osteoblast differentiation assayssince high amounts of osteogenic medium c
12、omponents canlead to dystrophic,pathologic or artifactual calcium-basedprecipitates that do not indicate differentiation of the cells inculture to functional osteoblasts(1).3For example,when highconcentrations of beta-glycerophosphate are used in the me-dium to function as a substrate for the enzyme
13、 alkalinephosphatase secreted by the cells,there is a marked increase infree phosphate,which then precipitates with Ca+ions in themedia to form calcium phosphate crystals independently of thedifferentiation status of the progenitor cell(2,3).4.4 Alkaline phosphatase production is an early event asso
14、-ciated with osteoblast differentiation but it can also be stimu-lated in other cell types by the addition of the osteogenic1This test method is under the jurisdiction ofASTM Committee F04 on Medicaland Surgical Materials and Devices and is the direct responsibility of SubcommitteeF04.43 on Cells an
15、d Tissue Engineered Constructs for TEMPs.Current edition approved Oct.1,2014.Published February 2015.DOI:10.1520/F3106-14.2For referenced ASTM standards,visit the ASTM website,www.astm.org,orcontact ASTM Customer Service at serviceastm.org.For Annual Book of ASTMStandards volume information,refer to
16、 the standards Document Summary page onthe ASTM website.3The boldface numbers in parentheses refer to the list of references at the end ofthis standard.Copyright ASTM International,100 Barr Harbor Drive,PO Box C700,West Conshohocken,PA 19428-2959.United States1 supplement dexamethasone to the medium.Alkaline phos-phatase enhances the formation of calcified deposits prior totheir natural occurrence in bone that typically coincides withbone sialoprotein and osteocalcin expression by mineralizedmat
