In naturally occurring and animal models of:

1
Bioengineering Synovial Fluid with
Modulated Hyaluronan Molecular Weight and
Function Brian J Lao Megan E Blewis Barbara L
Schumacher William D Bugbee
Gary S Firestein Robert L Sah Calit2
Department of Bioengineering University of
California-San Diego, La Jolla, CA

• Automated MATLAB Program (Fig. 2)
• - Stage 1 Creates intensity curve after
  correcting for background
• - Stage 2 Intensities standardized to MW
  standard markers
• through calibration curve
• - Stage 3 Intensities normalized and output
  to intensity map
• - Stage 4 MW bins with percentage of total
  intensity calculated

• INTRODUCTION
• In naturally occurring and animal models of
• - osteoarthritis
• - rheumatoid arthritis
• - injury
• the concentration and MW of a key synovial fluid
  lubricant is often decreased1-3
• - hyaluronan (HA), secreted by synoviocytes
• while SF volume concentration of certain
  cytokines are often increased4-7
• - IL-1ß
• - IL-17
• - IL-32
• - TGF-ß1
• - TNF-a
• How SF cytokines regulate synoviocyte-secreted HA
  MW in vivo in health,
• disease, and injury is unknown, and may have
  marked affects on lubricating
• function

• RESULTS
• Automated MATLAB Program
• - split into 4 subprograms encompassing
  different stages
• - output equivalent to that of manual analysis
• - analysis time 5 min/sample vs. 2
  hrs/sample manually
• HA MW Analysis (Fig. 2)
• - IL-1ß and TNF-a applied individually induced
  low-MW (gt60 at lt 1 MDa)
• - IL-1ß TGF-ß1 TNF-a applied in combination
  induced high-MW (gt60 at
• gt 1 MDa and 40 at gt3 MDa)
  resembling profile of normal human SF

Output
Input
Stage 1

• Net Intensity Curve

• Left/right sample lane positions

12
relative intensity

• xfinal

8

• xinit

• Left/right BG lane positions

• xbgfinal

4

• xbginit

0
1800
200
600
1000
1400
y-pixel coordinate
Input
Output
Stage 2
y -0.001589x 4.372577

• Known Molecular weights
• MW Pixel Locations

1400
900
1400
Output
Input
Stage 3

Figure 3. HA MW with individual and combinations
of cytokines (A) HA migration on gel, (B) MW
intensity profiles, and (C) of total HA in
different MW ranges.

• Master curve MWs
• Interpolated intensities from master calibration
  curve

• Intensity map

• DISCUSSION
• Developed automated MATLAB interface for analysis
  of HA MW
• - novel
• - user-friendly
• - efficient
• - able to be customized for special
  applications
• Goals within 1 year -
• - better understanding of HA regulation in
  health, injury, and disease
• - bioengineering synovial fluid with
  modulated HA MW
• Within 10 years -
• - bioengineer whole joints in
  bioreactor with appropriate lubricant
• environment for biological joint
  replacement
• REFERENCES
• 1 Teeple JOR, 26231, 08. 2 Elsaid AR,
  521746, 05. 3 Belcher Ann Rheum Dis, 56299,
  97. 4 Marks Arthroscopy, 211342, 05. 5
  Joosten PNAS, 1033298, 06. 6 Kotake J Clin
  Inv, 1031345, 99. 7 Wei OAC, 610, 98. 8
  Blewis Eur Cell Mater, 1326, 07. 9 Nugent
  OAC, 15566, 07.

Figure 1. Synovial joints may be modeled
theoretically8 by a system of compartments, and
experimentally in a bioreactor9 with appropriate
components.
Input
Stage 4

• User inputted MW ranges for bin

Figure 2. Stages of HA MW analysis to incorporate
into automated MATLAB program begin from an HA MW
agarose gel and finish at normalized intensity
map.
ACKNOWLEDGMENTS Calit2, UCSD, Cartilage Tissue
Engineering Lab