Targeting of ACSA-Expressing Fibroblast Cells in Tendon Injury and Repair
Mark L. Wang, MD, PhD, Pedro K. Beredijiklian, Ryan Hoffman, Andrzej
Fertala, PhD
To date, methods for the repair of tendon injuries have been somewhat limited, as modes pertaining to surgery, physical therapy, or
injection have been largely utilized; however, these approaches possess several adverse consequences. Previously used in cancer
research, antibody-based artificial collagen-specific anchor (ACSA) has proven effective in the delivery of cells to collagen-rich sites. This
information was used as a foundation to help improve injured tendons experimentally. In this study, targeting of fibroblast cells expressing
ACSA presents a better way of localizing engineered cells to areas of injury—an issue of difficulty seen in past orthopedic experimentation
with similar aims. This approach proves unique in that it reveals a non-surgical method for repairing tendon injuries, thus reducing the
potential for accumulation of excessive scar tissue and improved healing. Due to high similarity with human counterparts, rabbit tendons
were used to study the effects of the ACSA construct to direct cells toward pre-formed injury sites, thus mimicking conditions experienced
in vivo. Scaffolds populated with the ACSA-expressing cells were inserted into injury sites and placed in a bioreactor. Through histological
analysis, it was revealed that ACSA-expressing cells successfully interlaced with native collagen fibers, a feature not seen in the control.
Furthermore, use of a bioreactor will provide supplemental information on the elasticity and tension of experimental tendons. These
results, coupled with those received histologically, should further reveal this method as both functionally and structurally successful in
areas of tendon repair.
Artificial collagen-specific antibody has proven effective in targeting and localization of cells in cancer research.
This information was utilized towards localization of fibroblast cells to collagen-rich sites in rabbit achilles tendons
in vitro. Use of such localization techniques is significant as non-operative methods of therapy for tendon injury
have often faced problems with successful localization of desired cells. This data is significant because in the
normal growth and remodeling phases of collagen-rich areas, fibroblasts are responsible for growth and
subsequent deposition of native collagens to help support, and fortify surrounding tissue. In this study, utilization of
ACSA will help bypass previous difficulties mentioned above, and yield results more indicative of repair one might
expect in native conditions. Furthermore, this unique design set-up will allow for data to help substantiate results in
favor of non-surgical methods of tendon repair. This aspect of research is significant as often times, healing rates
post-operatively are complicated due to the exessive build-up of scar tissue. Since scar-tissue is unable to reach
the same level of elasticity or tensile strength seen initially, procedures aimed at reducing this process will help
allow for long-term improved healing and care of patients.
After harvest, tendons are stored at -80 in PBS
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Rabbit Achilles tendons
0.075% collagenase II
37 degree C water bath
Ham’s F12 medium containing 10% rabbit serum
Centrifuge
37 degree C incubator
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PBS
0.05% trypsin
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0.53 mmol/L ethylenediaminetetraacetic acid (EDTA) •
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Triton X-100 0.5%
CO2
Bioreactor
Hematoxylin
Eosin
NIH3T3 cells—+TELO,DOX
NIH3T3 cells—+TELO,
+DOX
NIH3T3 cells—-TELO,-DOX
NIH3T3 cells—-TELO, -DOX
Doxycycline
Preparing tendon scaffolds:
1.  Remove rabbit tendons from -80°C freezer and thaw to room temperature
2.  Place thawed tendons in 0.05% trypsin and 0.53% EDTA for 24 hours at room temperature
3.  Add Triton X-100 @ 0.5% for 24 hours at room temperature
4.  Repeat treatment with 0.05% trypsin and 0.075% collagenase II for 24 hours at room temperature
5.  Immerse tendons in 20mL of Ham’s F12 medium for 24 hours
Seeding cells on artificial scaffolds:
1.  Place PGA non-woven matrices pre-cut to 5 mm x 5 mm squares in Ham’s F12 medium.
2.  Seed these scaffolds with fibroblast added at a density of 2 X 106 cells/mL.
3.  Incubate cell-scaffold constructs in cell-culture chambers to allow uniform distribution of cells within scaffolds’ cavities.
4.  Place rotator in an incubator at 37°C with 5% CO2 for 24 hours.
5.  Insert cell-scaffold constructs into incisions made in rabbit tendons (see above).
6.  Place tendons into a bioreactor to expose them to a mechanical stimulation; 1.25N over 5 days with frequency of 1 cycle/second in
alternating 1-hr periods of mechanical load and rest.
7.  Collect the tendon-cells-scaffold constructs for histological assays.
C.
D.
E.
Tendon
Clamp
A. Scaffolds were cut from a porous, mesh-like sheet to dimensions of 2mmx2mm. B. Using forceps, scaffolds
were then placed in PBS (clear) and DMEM (red) for 5min each, in this order, respectively. C. Scaffolds were then
placed in circular vessels containing a density of ~2 million cells. Vessels were separated based on the population
of cells--3T3 and TELO. Vessels were then placed on rotators inside of a incubator. D. After incubation for an
appropriate amount of time, using a scalpel, incisions were made in rabbit Achilles tendons to mimic injury. Once
this was completed, using forceps, scaffolds were placed inside injury sites. E. After insertion, tendons were bound
by clamps inside a bioreactor, where repopulation was facilitated in a medium infused chamber. Bioreactors
provided ~1.25N of force, extending tendons in a manner which would increase elasticity and tensile strength while
allowing for maximal repopulation to occur. These attributes helped allow for rabbit tendons to mimic those of
native tendons, structurally, functionally, and compositionally.
A - ACSA sample; B- control sample
stained with hematoxylin and eosin
(H&E)
C, D (corresponding samples
observed in a fluorescence
microscope) - note GFP-positive
green cells expressing the ACSA
construct, Blue color represent cell
nuclei. In all panels white arrows
indicate fibers of scaffolds in which
we seeded the employed cells. I
overlaid an image taken to visualize
fibrillar features of a native tendon
(gray).
Symbols: Te- tendon; Sc- scaffold
with seeded cells.
After histological analysis of both experimental and control rabbit tendons, it was revealed that ACSAlabeled fibroblast cells were successfully targeted to collagen rich sites in vitro. Furthermore, in addition to
localization, fibroblast cells were able to successfully interlace with existing collagen molecules, thus
displaying the potential for growth and development of non-native collagen incorporation in subjects. This
data is compelling as it provides information which can be used towards developing more effective
treatments and therapies for patients experiencing tendon injuries. Furthermore, this mode of treatment
presents a less invasive method of repair which reduces several complications seen in surgical intervention,
such as increased risks for infection, and excessive build up of scar-tissue. With this information, further
research and analysis can be placed into, and used in substantiating similar methods of repair to help
facilitate drastic improvement in the field of Orthopedic Surgery.
Special thanks to Dr. Mark Wang, M.D., P.hD, and Dr. Andrzej Fertala, PhD for their involvement in this study. Further
appreciation is credited to Thomas Jefferson Medical College for their facilities and support throughout experimentation.