1. TWU Institute for Women’s Health
Denton, Texas
BLUEBERRY POLYPHENOLS DOSE-DEPENDENTLY PROMOTE OSTEOBLAST
DIFFERENTIATION Claire Kozlow, Huanbiao Mo, Parakat Vijayagopal, Shanil Juma
Department of Nutrition and Food Sciences, Texas Woman’s University, Denton, TX 76204
ABSTRACT
INTRODUCTION
STUDY OBJECTIVES
METHODS
RESULTS
SUMMARY AND PLANNED
EXPERIMENTAL WORK
Normal bone homeostasis is maintained by a
balance between bone formation and bone
resorption Conditions in which bone formation by
osteoblasts is decreased relative to the bone
resorptive activity of osteoclasts result in a net
loss of bone mass. Osteoporosis is a skeletal
disorder that is classified by low bone mass and
micro-architectural deterioration of bone tissue.
As a consequence, there is increase in bone
fragility and hence susceptibility to fracture. In
the US, as our aging population expands, severe
bone loss represents a major public health
problem which results in over 1.5 million
fractures a year with an estimated cost of 20
billion dollars annually. The bone forming cells,
osteoblasts, synthesize enzymes and matrix
proteins involved in the formation of mineralized
bone. Inflammatory cytokines such as tumor
necrosis factor (TNF)-α and interleukin (IL)-6
decrease osteoblast activity and stimulate
osteoblasts to produce inflammatory cytokines
such as receptor activator of NF-kB ligand
(RANKL), prostaglandin E2 (PGE2), and IL-1 that
can enhance osteoclast differentiation and
activity. Fruit and vegetable intake, as well as
grains and other plant-derived food, have been
linked to decreased risk of major chronic diseases
including bone loss resulting in osteoporotic
fractures. This effect has been partially attributed
to the polyphenols found in these foods. Thus, it
has been suggested that these compounds may
provide desirable bone health benefits through
their anti-inflammatory and anti-oxidative action
on bone cell metabolism.
Dietary polyphenols present in fruits and vegetables
may play an important role in bone metabolism
through modulation of osteoblasts, the bone-forming
cells. Blueberries contain a rich mixture of
polyphenolic that exhibit anti-inflammatory and anti-
oxidant actions in various tissues. Hence, polyphenols
present in blueberries may directly stimulate
osteoblasts, and favorably alter bone formation.
Utilizing mouse pre-osteoblastic cells, we evaluated
whether blueberry polyphenols (BBP) in a dose-
dependent manner increase markers of bone
formation in presence and absence of an inflammatory
agent. Cells were treated for 7 days with various
doses (0, 10, 50, 100, 200 ug/mL) of BBP and
challenged with tumor necrosis factor-alpha (TNFa).
BBP dose-dependently increased (p<0.05) alkaline
phosphatase, a marker of bone formation and
osteoblast differentiation in presence of TNFa.
However, no change in nitrite levels were observed
with BBP in presence and absence of TNFa. The
effect of BBP on molecular mechanisms involved in
osteoblast differentiation requires further
investigation. Blueberries contain a mixture of different polyphenol
compounds that can be separated into three primary
fractions enriched in either anthocyanins,
proanthocyanidins or hydroxycinnamic esters,
mainly chlorogenic acid. Major components of each
of these fractions have been shown to confer
significant antioxidant activity and can protect cells
against oxidative stress in vitro (Youdim et al., 2000;
Zheng & Wang, 2003).
To examine whether blueberry polyphenols dose-
dependently stimulate osteoblast activity and
differentiation
• The findings of our study indicate the blueberry
polyphenols dose-dependently increases ALP
activity in pre-osteoblast cells. The dosage of
100ug/ml media without TNFα stimulation had
the greatest effect. This dosage also is most
effective in osteoblast differentiation in presence
of TNFα.
• There was no effect on nitrite levels observed with
the doses of blueberry polyphenols in presence or
absence of TNFα.
• Visual observation of ALP staining does indicated
that blueberry polyphenols increases osteoblast
differentiation. This will need to be further
confirmed using alizarin red-S staining and
assessment of mineralized nodule formation.
Figure 2: Based on the standard curve we quantified the
polyphenolic concentration in the lyophilized blueberry extract
using the slope formula shown above
Cell Culture and Treatment
Mouse calvarial preosteoblastic (MC3T3-E1) cells were obtained
from American Type Culture Collection (Rockville, MD) Upon
attainment of experimental conditions, progenitor cells were
dose-dependently treated with BBP and stimulated with TNF- .α
Cell Viability and Nitric Oxide Assay
Nitrite levels will be determined using the MEASURE IT nitrite
assay from Molecular Probes. The nitrite level in each sample
will calculated from a standard curve generated with sodium
nitrite. The CellTiter 96®
colorimetric method assay for
proliferative cells was used to determine the number of viable
cells and doses of BBP to be used in the experiments.
Bone Alkaline Phosphatase (ALP) Activity
ALP was be determined in cell lysate and media based on the
conversion of p-nitrophenyl phosphate to p-nitrophenol by
spectrophotometry at 405 nm using a kit from Bio-Rad.
Cell Staining for ALP
Cells treated with BBP and stimulated with TNF-α were fixed
with formalin fixative and stained with p-nitrophenyl phosphate
for microscopy evaluation of ALP stainiing.
Polyphenol Extraction
Several batches of blueberry polyphenols have been extracted
from freeze dried blueberry powder (generously provided by US
Highbush Blueberry Council) using ethanol. These extracts have
been concentrated and lyophilized using a Freeze Dry System
(Labconco Inc).
Quantification of Blueberry Polyphenols
Lyophilized blueberry polyphenols have been reconstituted and
analyzed for polphenolic content. The total polyphenol content of
the extract has been quantified using the Folin-Calteau assay and
gallic acid as standards. The results of this assay is shown below.
Fig. 1: Proposed Mode of Action on Bone
Progenitor Cells by Blueberry Polyphenols (BBP)
Figure 3: ALP Activity in Osteoprogenitor Cells Treated
with Blueberry Polyphenols and Stimulated with TNFα
Figure 4: ALP Staining of Osteoprogenitor Cells Treated
with Blueberry Polyphenols and Stimulated with TNFα
Supported by a grant from the National Institute of Food and Agriculture at USDA (Number 2009-02942).
Lyophilized Blueberry Powder was generously provided by US Highbush Blueberry Council