1、Designation: F3106 14Standard 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 par
2、entheses 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
3、cells.1.2 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 f
4、or the con-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 an
5、y, associated 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 Me
6、dicalProductsF2997 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
7、phosphate-containingsubstance 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 mineralized matrix, na calcium phosphate-containing substance produced
8、by cells typically in 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 containi
9、ng characteristicproteins, 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 osteob
10、last differentiation assaysthat 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
11、expansion, processing, trans-fection 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 hig
12、h amounts of osteogenic medium components 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 fun
13、ction as a substrate for the enzyme 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 phosph
14、atase production is an early event asso-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 of ASTM Committee F04 on Medicaland Surgical Materials and Devices and is the direct responsi
15、bility of SubcommitteeF04.43 on Cells and 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
16、Book of ASTMStandards volume information, refer to 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
17、. United States1supplement 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 mineralizedmatrix-producing osteoblasts. These kinds of cal
18、cified/mineraldeposits are thus considered dystrophic, pathologic, or artifac-tual because they were not initiated by a mature osteoblast. Acalcium measurement, such as that described in Practice F2997for the Quantification of Calcium Deposits in OsteogenicCulture of Progenitor Cells Using Fluoresce
19、nt Image Analysis,may thus result in a potentially false interpretation of thedifferentiation status of osteoprogenitor cells if used in isola-tion without gene or protein expression data.4.5 In addition to screening for multipotentiality of undif-ferentiated stem cells, osteoblast differentiation a
20、ssays areuseful for assessing the osteoinductivity of cell culture sub-strates or biomaterial scaffolds or drugs or biomolecules; suchas, cytokines or growth factors.4.6 In vitro osteoblast differentiation assays are not predic-tive of in vivo bone formation, but are useful for comparisonpurposes to
21、 standardize performance between different types,sources or passages of progenitor cells, biomaterials, or typesand concentrations of biomolecules.5. An Overview of the in vitro Osteoblast DifferentiationAssay Procedure5.1 Briefly, progenitor stem cells are seeded in monolayeron treated tissue cultu
22、re plastic cell culture dishes or plates, oron biomaterial substrates, and allowed to proliferate in prolif-eration medium until they reach confluency with bi- ortri-weekly medium changes. After confluency is reached, cellculture medium is then changed from proliferation medium todifferentiation med
23、ium that contains supplements to promotethe osteogeneic differentiation of the progenitor cells and theformation of a mineralized matrix. The cells are cultured for upto 28 days in total with medium changes bi-weekly or everyother day. Cells will undergo apoptosis during in vitro miner-alization (4)
24、.5.2 To assess the extent of differentiation of the progenitorcells after the osteoblast differentiation assay, the calciumdeposits can be quantified using the non-destructive fluorescentimage analysis as described in Practice F2997 or terminatedand measured directly by conducting a total calcium co
25、ntentanalysis using one of the many commercial colorimetric kitsavailable for this purpose. Calcium deposition alone shouldnever be used as a measurement of osteogeneic potential.5.3 Quantifying the expression of osteogenic genes orproteins is another important measurement to use in conjunc-tion wit
26、h measurement of calcified deposits to confirm thepresence of osteoblasts. Both gene and protein measurementsshould be performed at multiple time points, as many peak andthen later decrease.6. In vitro Osteoblast Differentiation Assay Components6.1 Cell Sources:6.1.1 Mixed tissue-derived connective
27、tissue progenitorsfrom various tissues (e.g. from marrow, bone, fat, synovium,periosteum, cartilage, muscle, vascular and perivascular cells,and cord blood).6.1.2 Progenitor populations that have been selectivelyisolated or manipulated through culture expansion, processing,transfection or genetic mo
28、dification.6.1.3 Osteoprogenitors obtained from the pool of cells thatgrow out of bone chips during explant culture or fromcollagenase digestions of bone.6.1.4 Embryonic stem cells, induced pluripotent stem cellsor their progeny.6.1.5 It should be noted that optimal conditions for osteo-genesis vary
29、 between species, with human cells typically moreresistant to differentiation, while rodent are more reproducible.Outcomes also differ between human and rodent cells, withrodent cells more likely to form discrete calcified nodulesparticularly when harvested from neonatal tissues by enzy-matic digest
30、ion.6.2 Media for Osteoblast Differentiation Assays:6.2.1 Proliferation Phase Cell Culture MediumDulbeccos Modified Eagle Medium (DMEM), including lowglucose DMEM, or DMEM F-12 supplemented with 10% FetalBovine Serum (FBS) is commonly used for the proliferation orexpansion phase of the osteoblast di
31、fferentiation assay. Somecells may have been propagated or expanded in alpha-MEMand should therefore remain in that medium for the prolifera-tion phase, even although it contains ascorbate which canpromote osteoblast differentiation.6.2.2 Differentiation Phase Cell Culture MediumFor os-teogenic diff
32、erentiation cultures, -MEM is commonly usedduring the differentiation phase, although, as mentioned, it canbe used for proliferation as well. Low glucose DMEM withadditional osteogenic supplements has also been used forhuman mesenchymal stem cell osteogenic differentiation.6.3 Fetal Bovine Serum (FB
33、S):6.3.1 Fetal bovine serum (FBS), previously called fetal calfserum (FCS), is typically used at 10% of the medium volumeduring osteoblast differentiation assays in both the proliferationmedium and the differentiation medium. Serum compositionvaries by lot and as such it is important to screen vario
34、us lotsto ensure osteogenic potential. Some lots of FBS are moreefficient at promoting cell proliferation while others are betterfor promoting mineralization; thus, sera suitable for differen-tiation may not be the same as sera suitable for proliferation.Several companies offer serum qualified for h
35、uman mesenchy-mal stem cells to support either their proliferation or differen-tiation. This testing can also be performed “in house” wherebyserum batches can be compared to current stocks. Any serumtesting should include information about the cell type used forscreening, and serum source and lot nu
36、mber. Use of a standardcell line could be incorporated to compare results across serumlots. Heat inactivation of the serum is not necessary before usein an osteoblast differentiation assay, but may be required whengrowth factor supplements are being tested in this assay inorder to avoid a high backg
37、round.6.4 Osteogenic Supplements:F3106 1426.4.1 Ascorbic AcidIn osteogenic cell cultures, ascorbicacid is necessary to promote extracellular matrix proteinproduction. It has been shown to act as a co-factor in thehydroxylation of proline and lysine residues in collagen (5).There are two forms of thi
38、s supplement that are typically usedin the differentiation medium. The first is ascorbic acid whichis typically used at a concentration of 50 g/mL. Due to its fastdegradation, it is necessary to add this supplement to thecultures daily. The other form of ascorbic acid is the morestable ascorbic acid
39、-2-phosphate (AA2P) which is added to theculture media typically at a final concentration of 50 M (6).Ifthis form is chosen, daily addition is not necessary and can bereplenished at the same time as media change (every 2-4 days).A range of AA2P can be used; however, when used at 500 M,there may be c
40、ellular damage that reduces proliferation ofhuman mesenchymal stem cells (7). AA2P at 1 mM or highercauses cell death of human mesenchymal stem cells (6).6.4.2 Beta-glycerolphosphate-glycerolphosphate (-GP)is a phosphate source required for in vitro mineral depositionand is added to the differentiat
41、ion medium at a range of 2-10mM during osteoblast differentiation assays. There are con-cerns that high concentrations of -GP, such as 10 mM orhigher, lead to dystrophic mineralization because of the abilityof alkaline phosphatase produced by osteogenic cells to cleavephosphate groups from -GP and c
42、ause calcium phosphatedeposits not deposited directly by a mature osteoblast (8). Thisconfounds the accuracy of calcium measurements to assessosteoblast differentiation status. To avoid dystrophic or non-osteoblast mediated mineralization concentrations of 2.5-4 mMare recommended for this supplement
43、. Dystrophic calciumphosphate deposition within the cultures appears to promoteosteogenic differentiation of the cells and thus 10 mM -GPcontinues to be commonly used. When -GP is used at 10 mM,calcium content measurements alone may not adequate todistinguish if osteogenic differentiation has occurr
44、ed.6.4.3 DexamethasoneDexamethasone is a glucocorticoidwhich can act in both a stimulatory and inhibitory manner onosteogenic differentiation depending on dose, duration, stage ofcell differentiation, and species of responding cell (6). Gluco-corticoids have been shown to be involved in the boneform
45、ation/remodeling axis (9). Some studies have providedevidence for the necessity of dexamethasone in in vitro mineralformation, particularly in rat marrow-stromal derived cells(10), and human mesenchymal stem cells (6). In general,dexamethasone is used at a concentration range from 1-100nM. 100 nM ca
46、n be used during the first three weeks, but ifused longer inhibits mineralization, therefore 10 nM dexam-ethasone is preferred throughout the culture time period (11).The use of dexamethasone with human cells is a matter ofdebate with regards to osteocalcin expression, which has beenshown to decreas
47、e in osteoblastic cells after dexamethasoneexposure (12).6.4.4 Vitamin D31,25-dihydroxyvitamin D3 (vD3) isknown to accentuate osteoblast gene expression when added tothe differentiation media if cells have already begun to differ-entiate to the pre-osteoblast phase (13) in particular osteocal-cin. I
48、t may be added to later stage cultures, for example after2 weeks, at a dose of 10 nM. It will also stimulate mineraldeposition.6.4.5 Bone Morphogenetic Protein-2 (BMP-2)BMP-2may be used to stimulate in vitro osteogenic differentiation ofMSCs when the combination of ascorbic acid, -GP, anddexamethaso
49、me are not sufficient (14).6.4.6 Non-Essential Amino Acids and GlutamineNon-essential amino acids (NEAA) and L-glutamine are twosupplements commonly used in proliferation medium to sup-port progenitor cell expansion. They are not known to influ-ence osteoblast mineralization.6.4.7 Antibiotics or FungicidesThe addition of antibioticsor fungicides to prevent bacterial and fungi growth does notaffect the mineralization or osteogenic potential of cultures.The mostly commonly used antibiotics are penicillin andstreptomycin.6.5 Seeding Density:6.5.1 Cell seeding den
