ASTM F3106-22

Standard Guide for <emph type="bdit"> in vitro</emph> Osteoblast Differentiation Assays

SIGNIFICANCE AND USE
4.1 This guidance document describes the components and conditions used for in vitro osteoblast differentiation assays that can be used to screen for the osteogenic capability of progenitor stem cells from various human or animal sources, including mixed tissue-derived connective tissue progenitor populations, or cell populations that may be selectively isolated or manipulated through culture expansion, processing, transfection, or genetic modification.  
4.2 The osteoblast differentiation assay may be referred to as an osteogenesis assay or a mineralization assay.  
4.3 It is important to carefully select the components and conditions used for in vitro osteoblast differentiation assays since high amounts of osteogenic medium components can lead to dystrophic, pathologic, or artifactual calcium-based precipitates that do not indicate differentiation of the cells in culture to functional osteoblasts (1).4 For example, when high concentrations of beta-glycerophosphate are used in the medium to function as a substrate for the enzyme alkaline phosphatase secreted by the cells, there is a marked increase in free phosphate, which then precipitates with Ca++ ions in the media to form calcium phosphate crystals independently of the differentiation status of the progenitor cell (2, 3).  
4.4 Alkaline phosphatase production is an early event associated with osteoblast differentiation, but it can also be stimulated in other cell types by the addition of the osteogenic supplement dexamethasone to the medium. Alkaline phosphatase enhances the formation of calcified deposits prior to their natural occurrence in bone that typically coincides with bone sialoprotein and osteocalcin expression by mineralized matrix-producing osteoblasts. These kinds of calcified/mineral deposits are thus considered dystrophic, pathologic, or artifactual because they were not initiated by a mature osteoblast. A calcium measurement, such as that described in Practice F2997 for the Quantification of ...
SCOPE
1.1 This document provides guidance on how to conduct in vitro osteoblast differentiation assays with progenitor stem cells including mesenchymal stromal cells.  
1.2 This document describes the roles of various osteogenic supplements that are added to the cell culture medium of an osteoblast differentiation assay to encourage and support the differentiation of progenitor cells into matrix-producing osteoblasts.  
1.3 This document provides recommendations for the concentrations of osteogenic supplements that may prevent the precipitation of artifactual mineral deposits that are not directly produced by osteoblasts, nor correlated with osteoblastic gene expression of the cells.  
1.4 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.5 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

General Information

• Status: Published
• Publication Date: 31-Mar-2022
• ICS: 11.040.40 - Implants for surgery, prosthetics and orthotics
• Technical Committee: F04 - Medical and Surgical Materials and Devices
• Drafting Committee: F04.43 - Cells and Tissue Engineered Constructs for TEMPs

Relations

• Refers: ASTM F2312-11(2020) - Standard Terminology Relating to Tissue Engineered Medical Products
• Effective Date: 01-Feb-2020
• Refers: ASTM F2944-12 - Standard Test Method for Automated Colony Forming Unit (CFU) Assays&mdash;Image Acquisition and Analysis Method for Enumerating and Characterizing Cells and Colonies in Culture
• Effective Date: 01-Mar-2012
• Refers: ASTM F2312-11 - Standard Terminology Relating to Tissue Engineered Medical Products
• Effective Date: 01-Apr-2011
• Refers: ASTM F2312-10 - Standard Terminology Relating to Tissue Engineered Medical Products
• Effective Date: 01-Jun-2010
• Refers: ASTM F2312-04 - Standard Terminology Relating to Tissue Engineered Medical Products
• Effective Date: 01-Nov-2004
• Refers: ASTM F2312-03 - Standard Terminology Relating to Tissue Engineered Medical Products
• Effective Date: 10-Sep-2003

Overview

ASTM F3106-22: Standard Guide for in vitro Osteoblast Differentiation Assays provides comprehensive guidance for designing, conducting, and interpreting laboratory assays that evaluate the osteogenic differentiation capacity of progenitor stem cells. This standard, developed by ASTM International, supports researchers and laboratories in assessing the ability of various human or animal stem cell populations-including mesenchymal stromal cells-to differentiate into osteoblasts, the bone-forming cells. The guide outlines the necessary components, conditions, and recommended practices for achieving reliable and reproducible in vitro osteogenesis and mineralization, while highlighting the importance of distinguishing true differentiation from artifactual mineral deposits.

Key Topics

• Cell Sources: The standard covers the use of mixed tissue-derived progenitor populations, selectively isolated or manipulated cells, osteoprogenitors from bone, and pluripotent stem cells. Species differences in differentiation responses are noted.
• Culture Media and Supplements:
  • Proliferation and differentiation media formulations, including the role of serum (such as fetal bovine serum).
  • Importance of osteogenic supplements like ascorbic acid, beta-glycerolphosphate, dexamethasone, vitamin D3, and BMP-2.
  • Recommendations for supplement concentrations to avoid pathologic or artifactual mineral deposition that does not reflect genuine osteoblast activity.
• Assay Conditions: Guidance on cell seeding density, passage number, and the influence on mineralization outcomes.
• Marker Assessment:
  • Use of colorimetric, staining, and imaging methods to quantify calcium deposits and mineralized matrix.
  • The role of gene and protein expression analysis-specifically for markers like alkaline phosphatase, collagen type I, bone sialoprotein, and osteocalcin-for confirming osteoblast differentiation.
• Interpretation: Advises caution when interpreting mineralization data, as certain compositions or conditions can result in false-positive outcomes not related to osteoblast maturity.

Applications

• Stem Cell Characterization: This guide is vital for screening the osteogenic potential of different progenitor stem cell lines and determining their suitability for bone tissue engineering applications.
• Biomaterial and Drug Testing: The assay supports evaluation of the osteoinductive properties of biomaterials, scaffolds, and osteogenic drugs or growth factors.
• Comparative Analysis: Enables standardization and comparison of results across laboratories, cell sources, and culture conditions.
• Quality Control: Laboratories developing cell-based therapeutic products can use these standardized assays for reproducibility and regulatory compliance.
• Research and Development: Facilitates studies in biomedical research involving bone biology, regenerative medicine, and stem cell differentiation protocols.

Related Standards

Researchers and practitioners using ASTM F3106-22 should be familiar with the following related standards:

• ASTM F2312: Terminology Relating to Tissue Engineered Medical Products
• ASTM F2944: Practice for Automated Colony Forming Unit (CFU) Assays-Image Acquisition and Analysis
• ASTM F2997: Practice for Quantification of Calcium Deposits in Osteogenic Culture of Progenitor Cells Using Fluorescent Image Analysis
• ISO 21709: Biotechnology-Biobanking-Process and Quality Requirements for Establishment, Maintenance and Characterization of Mammalian Cell Lines

Practical Value

Implementing ASTM F3106-22 in laboratory protocols enhances the validity, reliability, and comparability of in vitro osteoblast differentiation assays. By standardizing procedures and emphasizing careful selection and control of assay components, the standard helps minimize experimental artifacts, supports regulatory submissions, and accelerates progress in developing effective cell-based bone therapies and materials.

Standards Content (Sample)

This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the
Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
Designation: F3106 − 22
Standard Guide for
in vitro Osteoblast Differentiation Assays
This standard is issued under the fixed designation F3106; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope F2997 Practice for Quantification of Calcium Deposits in
Osteogenic Culture of Progenitor Cells Using Fluorescent
1.1 This document provides guidance on how to conduct in
Image Analysis
vitro osteoblast differentiation assays with progenitor stem
2.2 ISO Standards:
cells including mesenchymal stromal cells.
ISO 21709 Biotechnology—Biobanking—Process and
1.2 This document describes the roles of various osteogenic
Quality Requirements for Establishment, Maintenance
supplements that are added to the cell culture medium of an
and Characterization of Mammalian Cell Lines
osteoblast differentiation assay to encourage and support the
differentiation of progenitor cells into matrix-producing osteo-
3. Terminology
blasts.
3.1 Unless provided otherwise in 3.2, terminology shall be
1.3 This document provides recommendations for the con-
in conformance with Terminology F2312.
centrations of osteogenic supplements that may prevent the
3.2 Definitions:
precipitationofartifactualmineraldepositsthatarenotdirectly
3.2.1 calcium deposits, n—a calcium phosphate-containing
produced by osteoblasts, nor correlated with osteoblastic gene
substance synthesized in cell cultures during mineralization or
expression of the cells.
osteoblast differentiation assays that may be directly produced
1.4 This standard does not purport to address all of the
by osteoblasts or precipitated out of the solution without cell
safety concerns, if any, associated with its use. It is the
participation.
responsibility of the user of this standard to establish appro-
3.2.2 mineralized matrix, n—a calcium phosphate-
priate safety, health, and environmental practices and deter-
containing substance produced by cells typically in the
mine the applicability of regulatory limitations prior to use.
osteoblast, odontoblast, and calcifying chondrocyte lineages,
1.5 This international standard was developed in accor-
which is composed of crystals of calcium phosphate and
dance with internationally recognized principles on standard-
contains collagen Type I and other non-collagenous proteins.
ization established in the Decision on Principles for the
3.2.3 osteoblasts, n—secretory mononuclear cells that will
Development of International Standards, Guides and Recom-
initiate the formation of a matrix containing characteristic
mendations issued by the World Trade Organization Technical
proteins, such as collagen, and non-collageneous proteins such
Barriers to Trade (TBT) Committee.
as bone sialoprotein and osteocalcin, that will mineralize in the
2. Referenced Documents
presence of a calcium and phosphate source.
2.1 ASTM Standards:
4. Significance and Use
F2312 Terminology Relating to Tissue Engineered Medical
Products
4.1 This guidance document describes the components and
F2944 Practice for Automated Colony Forming Unit (CFU)
conditions used for in vitro osteoblast differentiation assays
Assays—Image Acquisition and Analysis Method for
that can be used to screen for the osteogenic capability of
Enumerating and Characterizing Cells and Colonies in
progenitor stem cells from various human or animal sources,
Culture
including mixed tissue-derived connective tissue progenitor
populations,orcellpopulationsthatmaybeselectivelyisolated
This guide is under the jurisdiction of ASTM Committee F04 on Medical and
or manipulated through culture expansion, processing,
Surgical Materials and Devices and is the direct responsibility of Subcommittee
transfection, or genetic modification.
F04.43 on Cells and Tissue Engineered Constructs for TEMPs.
Current edition approved April 1, 2022. Published April 2022. Originally
4.2 The osteoblast differentiation assay may be referred to
approved in 2014. Last previous edition approved in 2014 as F3106 – 14. DOI:
as an osteogenesis assay or a mineralization assay.
10.1520/F3106-22.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM
Standards volume information, refer to the standard’s Document Summary page on Available fromAmerican National Standards Institute (ANSI), 25 W. 43rd St.,
the ASTM website. 4th Floor, New York, NY 10036, http://www.ansi.org.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
F3106 − 22
4.3 It is important to carefully select the components and 5.2 To assess the extent of differentiation of the progenitor
conditions used for in vitro osteoblast differentiation assays cells after the osteoblast differentiation assay, the calcium
since high amounts of osteogenic medium components can deposits can be quantified using the nondestructive fluorescent
lead to dystrophic, pathologic, or artifactual calcium-based image analysis as described in Practice F2997 or terminated
precipitates that do not indicate differentiation of the cells in and measured directly by conducting a total calcium content
culture to functional osteoblasts (1). For example, when high analysis using one of the many commercial colorimetric kits
concentrations of beta-glycerophosphate are used in the me- available for this purpose. Calcium deposition alone should
dium to function as a substrate for the enzyme alkaline never be used as a measurement of osteogeneic potential.
phosphatase secreted by the cells, there is a marked increase in
5.3 Quantifying the expression of osteogenic genes or
++
free phosphate, which then precipitates with Ca ions in the
proteins is another important measurement to use in conjunc-
media to form calcium phosphate crystals independently of the
tion with measurement of calcified deposits to confirm the
differentiation status of the progenitor cell (2, 3).
presence of osteoblasts. Both gene and protein measurements
should be performed at multiple time points, as many peak and
4.4 Alkaline phosphatase production is an early event asso-
ciated with osteoblast differentiation, but it can also be stimu- then later decrease.
lated in other cell types by the addition of the osteogenic
supplement dexamethasone to the medium. Alkaline phos- 6. In vitro Osteoblast Differentiation Assay Components
phatase enhances the formation of calcified deposits prior to
6.1 Cell Sources:
their natural occurrence in bone that typically coincides with
6.1.1 Mixed tissue-derived connective tissue progenitors
bone sialoprotein and osteocalcin expression by mineralized
from various tissues (for example, from marrow, bone, fat,
matrix-producing osteoblasts. These kinds of calcified/mineral
synovium, periosteum, cartilage, muscle, vascular and perivas-
deposits are thus considered dystrophic, pathologic, or artifac-
cular cells, and cord blood).
tual because they were not initiated by a mature osteoblast. A
6.1.2 Progenitor populations that have been selectively
calciummeasurement,suchasthatdescribedinPracticeF2997
isolated or manipulated through culture expansion, processing,
for the Quantification of Calcium Deposits in Osteogenic
transfection, or genetic modification.
Culture of Progenitor Cells Using Fluorescent ImageAnalysis,
6.1.3 Osteoprogenitors obtained from the pool of cells that
may thus result in a potentially false interpretation of the
grow out of bone chips during explant culture or from
differentiation status of osteoprogenitor cells if used in isola-
collagenase digestions of bone.
tion without gene or protein expression data.
6.1.4 Embryonic stem cells, induced pluripotent stem cells
4.5 In addition to screening for multipotentiality of undif-
or their progeny.
ferentiated stem cells, osteoblast differentiation assays are
6.1.5 It should be noted that optimal conditions for osteo-
useful for assessing the osteoinductivity of cell culture sub-
genesis vary between species, with human cells typically more
strates or biomaterial scaffolds or drugs or biomolecules, such
resistant to differentiation, while rodent are more reproducible.
as cytokines or growth factors.
Outcomes also differ between human and rodent cells, with
rodent cells more likely to form discrete calcified nodules
4.6 In vitro osteoblast differentiation assays are not predic-
particularly when harvested from neonatal tissues by enzy-
tive of in vivo bone formation, but are useful for comparison
matic digestion.
purposes to standardize performance between different types,
sources or passages of progenitor cells, biomaterials, or types
6.2 Media for Osteoblast Differentiation Assays:
and concentrations of biomolecules.
6.2.1 Proliferation Phase Cell Culture Medium—
Dulbecco’s Modified Eagle Medium (DMEM), including low
5. An Overview of the in vitro Osteoblast Differentiation
glucose DMEM, or DMEM F-12 supplemented with 10 %
Assay Procedure
Fetal Bovine Serum (FBS) is commonly used for the prolif-
eration or expansion phase of the osteoblast differentiation
5.1 Briefly, progenitor stem cells are seeded in monolayer
assay. Some cells may have been propagated or expanded in
on treated tissue culture plastic cell culture dishes or plates, or
alpha-MEM and should therefore remain in that medium for
on biomaterial substrates, and allowed to proliferate in prolif-
the proliferation phase, even although it contains ascorbate
eration medium until they reach confluency with bi- or
which can promote osteoblast differentiation.
tri-weekly medium changes. After confluency is reached, cell
6.2.2 Differentiation Phase Cell Culture Medium—For os-
culture medium is then changed from proliferation medium to
teogenic differentiation cultures, α-MEM is commonly used
differentiation medium that contains supplements to promote
during the differentiation phase, although, as mentioned, it can
the osteogeneic differentiation of the progenitor cells and the
be used for proliferation as well. Low-glucose DMEM with
formationofamineralizedmatrix.Thecellsareculturedforup
additional osteogenic supplements has also been used for
to 28 days in total with medium changes bi-weekly or every
human mesenchymal stem cell osteogenic differentiation.
other day. Cells will undergo apoptosis during in vitro miner-
alization (4).
6.3 Fetal Bovine Serum (FBS):
6.3.1 Fetal bovine serum (FBS), previously called fetal calf
serum (FCS), is typically used at 10 % of the medium volume
duringosteoblastdifferentiationassaysinboththeproliferation
The boldface numbers in parentheses refer to the list of references at the end of
this standard. medium and the differentiation medium. Serum composition
F3106 − 22
varies by lot and as such it is important to screen various lots formation/remodeling axis (9). Some studies have provided
to ensure osteogenic potential. Some lots of FBS are more evidenceforthenecessityofdexamethasonein in vitromineral
efficient at promoting cell proliferation while others are better formation, particularly in rat marrow-stromal derived cells
for promoting mineralization; thus, sera suitable for differen- (10), and human mesenchymal stem cells (6). In general,
tiation may not be the same as sera suitable for proliferation. dexamethasone is used at a concentration range from 1 to 100
Several companies offer serum qualified for human mesenchy- nM. 100 nM can be used during the first three weeks, but if
mal stem cells to support either their proliferation or differen- used longer inhibits mineralization; therefore, 10 nM dexam-
tiation. This testing can also be performed “in house” whereby ethasone is preferred throughout the culture time period (11).
serum batches can be compared to current stocks. Any serum The use of dexamethasone with human cells is a matter of
testing should include information about the cell type used for debate with regards to osteocalcin expression, which has been
screening, and serum source and lot number. Use of a standard shown to decrease in osteoblastic cells after dexamethasone
cell line could be incorporated to compare results across serum exposure (12).
lots. Heat inactivation of the serum is not necessary before use
6.4.4 Vitamin D3—1,25-dihydroxyvitamin D3 (vD3) is
inanosteoblastdifferentiationassay,butmayberequiredwhen known to accentuate osteoblast gene expression when added to
growth factor supplements are being tested in this assay in
the differentiation media if cells have already begun to differ-
order to avoid a high background.
entiate to the pre-osteoblast phase (13) in particular osteocal-
cin. It may be added to later stage cultures, for example after
6.4 Osteogenic Supplements:
two weeks, at a dose of 10 nM. It will also stimulate mineral
6.4.1 Ascorbic Acid—In osteogenic cell cultures, ascorbic
deposition.
acid is necessary to promote extracellular matrix protein
6.4.5 Bone Morphogenetic Protein-2 (BMP-2)—BMP-2
production. It has been shown to act as a co-facto
...

This document is not an ASTM standard and is intended only to provide the user of an ASTM standard an indication of what changes have been made to the previous version. Because
it may not be technically possible to adequately depict all changes accurately, ASTM recommends that users consult prior editions as appropriate. In all cases only the current version
of the standard as published by ASTM is to be considered the official document.
Designation: F3106 − 14 F3106 − 22
Standard Guide for
in vitro Osteoblast Differentiation Assays
This standard is issued under the fixed designation F3106; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope
1.1 This document provides guidance on how to conduct in vitro osteoblast differentiation assays with progenitor stem cells
including mesenchymal stromal cells.
1.2 This document describes the roles of various osteogenic supplements that are added to the cell culture medium of an osteoblast
differentiation assay to encourage and support the differentiation of progenitor cells into matrix-producing osteoblasts.
1.3 This document provides recommendations for the concentrations of osteogenic supplements that may prevent the precipitation
of artifactual mineral deposits that are not directly produced by osteoblasts, nor correlated with osteoblastic gene expression of the
cells.
1.4 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility
of the user of this standard to establish appropriate safety safety, health, and healthenvironmental practices and determine the
applicability of regulatory limitations prior to use.
1.5 This international standard was developed in accordance with internationally recognized principles on standardization
established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued
by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
2. Referenced Documents
2.1 ASTM Standards:
F2312 Terminology Relating to Tissue Engineered Medical Products
F2944 Practice for Automated Colony Forming Unit (CFU) Assays—Image Acquisition and Analysis Method for Enumerating
and Characterizing Cells and Colonies in Culture
F2997 Practice for Quantification of Calcium Deposits in Osteogenic Culture of Progenitor Cells Using Fluorescent Image
Analysis
2.2 ISO Standards:
ISO 21709 Biotechnology—Biobanking—Process and Quality Requirements for Establishment, Maintenance and Character-
ization of Mammalian Cell Lines
3. Terminology
3.1 Unless provided otherwise in 3.2, terminology shall be in conformance with Terminology F2312.
This test method guide is under the jurisdiction of ASTM Committee F04 on Medical and Surgical Materials and Devices and is the direct responsibility of Subcommittee
F04.43 on Cells and Tissue Engineered Constructs for TEMPs.
Current edition approved Oct. 1, 2014April 1, 2022. Published February 2015April 2022. Originally approved in 2014. Last previous edition approved in 2014 as
F3106 – 14. DOI: 10.1520/F3106-14.10.1520/F3106-22.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM Standards
volume information, refer to the standard’s Document Summary page on the ASTM website.
Available from American National Standards Institute (ANSI), 25 W. 43rd St., 4th Floor, New York, NY 10036, http://www.ansi.org.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
F3106 − 22
3.2 Definitions:
3.2.1 calcium deposits, n—a calcium phosphate-containing substance synthesized in cell cultures during mineralization or
osteoblast differentiation assays that may be directly produced by osteoblasts or precipitated out of the solution without cell
participation.
3.2.2 mineralized matrix, n—a calcium phosphate-containing substance produced by cells typically in the osteoblast, odontoblast,
and calcifying chondrocyte lineages, which is composed of crystals of calcium phosphate and contains collagen Type I and other
non-collagenous proteins.
3.2.3 osteoblasts, n—secretory mononuclear cells that will initiate the formation of a matrix containing characteristic proteins,
such as collagen, and non-collageneous proteins such as bone sialoprotein and osteocalcin, that will mineralize in the presence of
a calcium and phosphate source.
4. Significance and Use
4.1 This guidance document describes the components and conditions used for in vitro osteoblast differentiation assays that can
be used to screen for the osteogenic capability of progenitor stem cells from various human or animal sources, including mixed
tissue-derived connective tissue progenitor populations, or cell populations that may be selectively isolated or manipulated through
culture expansion, processing, transfection, or genetic modification.
4.2 The osteoblast differentiation assay may be referred to as an osteogenesis assay or a mineralization assay.
4.3 It is important to carefully select the components and conditions used for in vitro osteoblast differentiation assays since high
amounts of osteogenic medium components can lead to dystrophic, pathologic, or artifactual calcium-based precipitates that do not
indicate differentiation of the cells in culture to functional osteoblasts (1). For example, when high concentrations of
beta-glycerophosphate are used in the medium to function as a substrate for the enzyme alkaline phosphatase secreted by the cells,
++
there is a marked increase in free phosphate, which then precipitates with Ca ions in the media to form calcium phosphate
crystals independently of the differentiation status of the progenitor cell (2, 3).
4.4 Alkaline phosphatase production is an early event associated with osteoblast differentiation, but it can also be stimulated in
other cell types by the addition of the osteogenic supplement dexamethasone to the medium. Alkaline phosphatase enhances the
formation of calcified deposits prior to their natural occurrence in bone that typically coincides with bone sialoprotein and
osteocalcin expression by mineralized matrix-producing osteoblasts. These kinds of calcified/mineral deposits are thus considered
dystrophic, pathologic, or artifactual because they were not initiated by a mature osteoblast. A calcium measurement, such as that
described in Practice F2997 for the Quantification of Calcium Deposits in Osteogenic Culture of Progenitor Cells Using
Fluorescent Image Analysis, may thus result in a potentially false interpretation of the differentiation status of osteoprogenitor cells
if used in isolation without gene or protein expression data.
4.5 In addition to screening for multipotentiality of undifferentiated stem cells, osteoblast differentiation assays are useful for
assessing the osteoinductivity of cell culture substrates or biomaterial scaffolds or drugs or biomolecules;biomolecules, such as,as
cytokines or growth factors.
4.6 In vitro osteoblast differentiation assays are not predictive of in vivo bone formation, but are useful for comparison purposes
to standardize performance between different types, sources or passages of progenitor cells, biomaterials, or types and
concentrations of biomolecules.
5. An Overview of the in vitro Osteoblast Differentiation Assay Procedure
5.1 Briefly, progenitor stem cells are seeded in monolayer on treated tissue culture plastic cell culture dishes or plates, or on
biomaterial substrates, and allowed to proliferate in proliferation medium until they reach confluency with bi- or tri-weekly
medium changes. After confluency is reached, cell culture medium is then changed from proliferation medium to differentiation
medium that contains supplements to promote the osteogeneic differentiation of the progenitor cells and the formation of a
The boldface numbers in parentheses refer to the list of references at the end of this standard.
F3106 − 22
mineralized matrix. The cells are cultured for up to 28 days in total with medium changes bi-weekly or every other day. Cells will
undergo apoptosis during in vitro mineralization (4).
5.2 To assess the extent of differentiation of the progenitor cells after the osteoblast differentiation assay, the calcium deposits can
be quantified using the non-destructivenondestructive fluorescent image analysis as described in Practice F2997 or terminated and
measured directly by conducting a total calcium content analysis using one of the many commercial colorimetric kits available for
this purpose. Calcium deposition alone should never be used as a measurement of osteogeneic potential.
5.3 Quantifying the expression of osteogenic genes or proteins is another important measurement to use in conjunction with
measurement of calcified deposits to confirm the presence of osteoblasts. Both gene and protein measurements should be
performed at multiple time points, as many peak and then later decrease.
6. In vitro Osteoblast Differentiation Assay Components
6.1 Cell Sources:
6.1.1 Mixed tissue-derived connective tissue progenitors from various tissues (e.g. (for example, from marrow, bone, fat,
synovium, periosteum, cartilage, muscle, vascular and perivascular cells, and cord blood).
6.1.2 Progenitor populations that have been selectively isolated or manipulated through culture expansion, processing,
transfection, or genetic modification.
6.1.3 Osteoprogenitors obtained from the pool of cells that grow out of bone chips during explant culture or from collagenase
digestions of bone.
6.1.4 Embryonic stem cells, induced pluripotent stem cells or their progeny.
6.1.5 It should be noted that optimal conditions for osteogenesis vary between species, with human cells typically more resistant
to differentiation, while rodent are more reproducible. Outcomes also differ between human and rodent cells, with rodent cells more
likely to form discrete calcified nodules particularly when harvested from neonatal tissues by enzymatic digestion.
6.2 Media for Osteoblast Differentiation Assays:
6.2.1 Proliferation Phase Cell Culture Medium—Dulbecco’s Modified Eagle Medium (DMEM), including low glucose DMEM,
or DMEM F-12 supplemented with 10%10 % Fetal Bovine Serum (FBS) is commonly used for the proliferation or expansion
phase of the osteoblast differentiation assay. Some cells may have been propagated or expanded in alpha-MEM and should
therefore remain in that medium for the proliferation phase, even although it contains ascorbate which can promote osteoblast
differentiation.
6.2.2 Differentiation Phase Cell Culture Medium—For osteogenic differentiation cultures, α-MEM is commonly used during the
differentiation phase, although, as mentioned, it can be used for proliferation as well. Low glucose Low-glucose DMEM with
additional osteogenic supplements has also been used for human mesenchymal stem cell osteogenic differentiation.
6.3 Fetal Bovine Serum (FBS):
6.3.1 Fetal bovine serum (FBS), previously called fetal calf serum (FCS), is typically used at 10%10 % of the medium volume
during osteoblast differentiation assays in both the proliferation medium and the differentiation medium. Serum composition varies
by lot and as such it is important to screen various lots to ensure osteogenic potential. Some lots of FBS are more efficient at
promoting cell proliferation while others are better for promoting mineralization; thus, sera suitable for differentiation may not be
the same as sera suitable for proliferation. Several companies offer serum qualified for human mesenchymal stem cells to support
either their proliferation or differentiation. This testing can also be performed “in house” whereby serum batches can be compared
to current stocks. Any serum testing should include information about the cell type used for screening, and serum source and lot
number. Use of a standard cell line could be incorporated to compare results across serum lots. Heat inactivation of the serum is
not necessary before use in an osteoblast differentiation assay, but may be required when growth factor supplements are being
tested in this assay in order to avoid a high background.
6.4 Osteogenic Supplements:
F3106 − 22
6.4.1 Ascorbic Acid—In osteogenic cell cultures, ascorbic acid is necessary to promote extracellular matrix protein production. It
has been shown to act as a co-factor in the hydroxylation of proline and lysine residues in collagen (5). There are two forms of
this supplement that are typically used in the differentiation medium. The first is ascorbic acid which is typically used at a
concentration of 50 μg/mL. Due to its fast degradation, it is necessary to add this supplement to the cultures daily. The other form
of ascorbic acid is the more stable ascorbic acid-2-phosphate (AA2P) which is added to the culture media typically at a final
concentration of 50 μM (6). If this form is chosen, daily addition is not necessary and can be replenished at the same time as media
change (every 2-4 two to four days). A range of AA2P can be used; however, when used at 500 μM, there may be cellular damage
that reduces proliferation of human mesenchymal stem cells (7). AA2P at 1 mM or higher causes cell death of human mesenchymal
stem cells (6).
6.4.2 Beta-glycerolphosphate—β-glycerolphosphate (β-GP) is a phosphate source required for in vitro mineral deposition and is
added to the differentiation medium at a range of 2-10 mM 2 to 10 mM during osteoblast differentiation assays. There are concerns
that high concentrations of β-GP, such as 10 mM or higher, lead to dystrophic mineralization because of the ability of alkaline
phosphatase produced by osteogenic cells to cleave phosphate groups from β-GP and cause calcium phosphate deposits not
deposited directly by a mature osteoblast (8). This confounds the accuracy of calcium measurements to assess osteoblast
differentiation status. To avoid dystrophic or non-osteoblast mediated mineralization, concentrations of 2.5-4 mM 2.5 to 4 mM are
recommended for this supplement. Dystrophic calcium phosphate deposition within the cultures appears to promote osteogenic
differentiation of the cells and thus 10 mM β-GP continues to be commonly used. When β-GP is used at 10 mM, 10 mM, calcium
content measurements alone may not adequate to distinguish if osteogenic differentiation has occurred.
6.4.3 Dexamethasone—Dexamethasone is a glucocorticoid which can act in both a stimulatory and inhibitory manner on
osteogenic differentiation depending on dose, duration, stage of cell differentiation, and species of responding cell (6).
Glucocorticoids have been shown to be involved in the bone formation/remodeling axis (9). Some studies have provided evidence
for the necessity of dexamethasone in in vitro mineral formation, particularly i
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