Materials 2011, 4, 1469-1482; doi:10.3390/ma4081469
The Effect of Synovial Fluid Enzymes on the Biodegradability of
Collagen and Fibrin Clots
Matthew Palmer, Elizabeth Stanford and Martha M. Murray *
Department of Orthopaedic Surgery, Children’s Hospital of Boston, Harvard Medical School,
300 Longwood Ave, Boston, MA 02115, USA; E-Mails: firstname.lastname@example.org (M.P.);
* Author to whom correspondence should be addressed; E-Mail: email@example.com;
Tel.: +1-617-355-7132; Fax: +1-617-730-0459.
Received: 18 July 2011; in revised form: 5 August 2011 / Accepted: 12 August 2011 /
Published: 22 August 2011
Abstract: Recently there has been a great deal of interest in the use of biomaterials to
stimulate wound healing. This is largely due to their ability to centralize high
concentrations of compounds known to promote wound healing at a needed location. Joints
present a unique challenge to using scaffolds because of the presence of enzymes in
synovial fluid which are known to degrade materials that would be stable in other parts of
the body. The hypothesis of this study was that atelocollagen scaffolds would have greater
resistance to enzymatic degradation than scaffolds made of gelatin, fibrin and whole blood.
To test this hypothesis, collagen and fibrin-based scaffolds were placed in matrix
metallopeptidase-1 (MMP-1), elastase, and plasmin solutions at physiologic concentrations,
and the degradation of each scaffold was measured at varying time points. The
atelocollagen scaffolds had a significantly greater resistance to degradation by MMP-1,
elastase and plasmin over the fibrin based scaffolds. The results suggest that
atelocollagen-based scaffolds may provide some protection against premature degradation
by synovial fluid enzymes over fibrin-based matrices.
Keywords: collagen; fibrin; scaffold; fibroblast; enzyme
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Recently there has been a great deal of interest surrounding the use of biomaterials as provisional
scaffolds to stimulate the healing of tissues within joints. Targeted tissues include the anterior cruciate
ligament (ACL) [1-3] and meniscus [4-6]. The biomaterials are designed to act as a substrate that
allows cells to grow into, remodel, and eventually form new tissue [2,6]. Collagen scaffolds are
reasonable biomaterials because of their ability to centralize high concentrations of compounds known
to promote wound healing at a needed location [1,2]. Atelocollagen scaffolds are manufactured by
using a pepsin solution to remove the antigenic ends of the collagen molecule, neutralizing the
collagen, and lyophilizing to form a porous scaffold. Gelatin scaffolds are manufactured by
lyophilizing a 1 to 2% weight by volume gelatin (hydrolysed collagen) suspension, also resulting in a
porous scaffold. However, it is currently unknown how these different processing techniques will
affect the function of the collagen scaffold as a biomaterial. Fibrin scaffolds and blood clot have also
commonly been reported for use in enhancing ligament  and meniscal healing [6,8] with mixed
reports of efficacy. Both collagen and pepsin can be cross-linked in order to increase their mechanical
properties and control their degradation rate [9,10].
Joints present a unique challenge to using scaffolds because of the presence of enzymes in synovial
fluid which are known to degrade materials that would be stable in other parts of the body [11-14].
Synovial fluid is composed of hyaluronan and D-N-acetylglucosamine, as well as many enzymes [15-17],
among them matrix metallopeptidase 1 (MMP-1), elastase and plasmin, found in the synovial fluid at
varying concentrations [18-20]. These three enzymes are hypothesized to affect the healing of
intra-articular injuries [21-28]. MMP-1 is known to be involved with the breakdown of the extracellular
matrix [21-23], as well as with tissue remodeling. It specifically degrades type I, II, and III collagen.
Elastase is a peptidase that breaks down elastin and collagen fibers [19,24,25]. Plasmin is an enzyme that
is present in synovial fluid in increased amounts after injury and degrades fibrin clots [26-28].
How these enzymes affect atelocollagen, gelatin, fibrin and blood-based scaffolds is critical to
define if these materials are to be used in joint tissue engineering. The knee is one of many synovial
joints found in the human body and it is vital to understand how a provisional scaffold will behave
when placed in the synovial fluid . By subjecting these materials to the concentration of enzymes
found in synovial fluid (Table 1), this response can be studied. The structural integrity of the scaffolds
is important when determining the ability of these scaffolds to withstand the joint environment long
enough to participate in ACL repair or repair of other joint injuries. In addition, determining what
effect, if any, the collagen structure will have on cellular proliferation within the scaffold could be
useful for design of collagen-based scaffolds.
Table 1. Enzyme concentrations found in the synovial fluid of the knee and those used in
Concentration in the knee
177 to 279 µg/mL (Gysen, 1985) 
0.041 to 101.8 µg/mL (Kleesiek, 1986) 
23 to 55 µg/mL (Kummer, 1992) 
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In this study, we evaluated identically sized scaffolds of atelocollagen, gelatin, fibrin and blood clot
biomaterials for their ability to withstand degradation by enzymes found in synovial fluid. We
hypothesized that the collagen formulation—i.e., atelocollagen vs. gelatin—as well as the composition
of the scaffold, would significantly affect the ability of the scaffold to facilitate cell proliferation and to
resist degradation by enzymes found in the synovial fluid.
2. Results and Discussion
2.1. MMP-1 Degradation
The atelocollagen scaffolds had a significantly higher resistance to degradation by MMP than all
other groups at 12 and 24 h (Figure 1, p < 0.02 for all comparisons). While the blood clots still retained
almost 40% of their area at 12 h, the gelatin and fibrin clot scaffolds were completely degraded after
12 h. After 24 h in the MMP-1 solution, the only remaining scaffolds with any integrity were the
atelocollagen scaffolds, which had retained 39% of their area, and by 48 h, even the atelocollagen
scaffolds had been degraded (Figure 1A).
Figure 1. Enzymatic degradation of atelocollagen, gelatin, fibrin, and blood clot scaffolds
over a 48 h period by (A) MMP-1; (B) Elastase and (C) Phosphate buffered saline (PBS)
(control). By 24 h in the MMP-1 solution, the only remaining scaffolds with any integrity
were the atelocollagen scaffolds and by 48 h, even the atelocollagen scaffolds had been
degraded. † denotes significantly less degradation of the atelocollagen scaffolds at 12, and
24 h (p < 0.02). * denotes significantly less degradation of the blood clot scaffold than the
fibrin or gelatin at the time points identified (p < 0.001 for all comparisons).
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Figure 1. Cont.
2.2. Elastase Degradation
The atelocollagen scaffolds had significantly greater resistance to elastin than any of the other
scaffolds (p < 0.001 for all comparisons, Figure 1B). Interestingly, the atelocollagen scaffolds
exhibited complete resistance to degradation by the elastase solution throughout the experiment. In
fact, in this group, there was an increase in scaffold area in the first 12 h (117 ± 7%) which remained at
48 h. The gelatin and fibrin clot scaffolds were completely degraded after 12 h (Figure 1B). The blood
clot scaffolds were 63 ± 21%, 38 ± 13% and 15 ± 10% of their original sizes at 12, 24 and 48 h,
respectively, having degraded significantly more than the atelocollagen scaffolds at all time
points (p < 0.001).
2.3. PBS Degradation
Phosphate buffered saline (PBS) was the carrier used to deliver the MMP-1 enzyme and the elastase
enzyme, and thus was also evaluated for its potential to influence scaffold degradation. The
atelocollagen and gelatin scaffolds had minimal change in size in PBS. At 48 h, the atelocollagen
scaffolds were 106 ± 14% and the gelatin scaffolds 96 ± 3% of their respective initial areas. The mean
area of the blood clot and the fibrin clot scaffolds decreased, respectively, to 88 ± 4% and
68 ± 2% of their initial sizes at 12 h and remained stable throughout the remaining time of the
experiment (Figure 1C).
2.4. Plasmin Degradation
Both atelocollagen and gelatin scaffolds had improved resistance to plasmin degradation over the
fibrin and blood clot scaffolds (Figure 2A, p < 0.02 for all comparisons). The two collagen scaffolds
had similar plasmin resistance, with no significant difference in area between these groups at any time
point (Figure 2A; p > 0.05). Blood clot scaffolds were significantly smaller than both the atelocollagen
and gelatin scaffolds at all time points (p ≤ 0.02); whereas the fibrin clot scaffolds were completely
degraded at 12 h.
Materials 2011, 4
Figure 2. Enzymatic degradation of atelocollagen, gelatin, fibrin, and blood clot scaffolds
over a 48 h period by (A) plasmin and (B) water (control). † denotes significantly less
degradation by plasmin of atelocollagen and gelatin scaffolds than the blood clot or fibrin
clot (p < 0.05 for all comparisons).
2.5. Water Degradation
Water was the carrier used to deliver the plasmin enzyme, and thus was also evaluated for its
potential to influence scaffold degradation. The atelocollagen and gelatin scaffolds exhibited slight
decrease in size when subjected to water, with 95 ± 10% and 99 ± 14% of their respective initial areas
at 48 h. The mean area of the blood clot and the fibrin clot scaffolds decreased, respectively, to
88 ± 11% and 62 ± 5% of their initial sizes and remained stable throughout the remaining time of the
experiment (Figure 2B).
2.6. Cell Proliferation
The atelocollagen scaffolds had more cells present than the gelatin scaffolds at 2 days
(0.14 × 106 ± 0.03 × 106 vs. 0.06 × 106 ± 0.06 × 106 cells) and 10 days (0.39 × 106 ± 0.14 × 106 vs.
0.20 × 106 ± 0.06 × 106 cells), p < 0.05 for both days. The atelocollagen and gelatin scaffolds
experienced 2.7 ± 1.1 and 3.1 ± 3.2 times increase in cell number, respectively, from day 2 to day
10 (p = 0.40; Figure 3).
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Figure 3. Cell proliferation within atelocollagen and gelatin scaffolds at 2 and 10 days
after seeding as measured by the MTT assay. Cell numbers in both types of scaffolds
increased between 2 and 10 days. Atelocollagen scaffolds had a higher cell concentration at
2 days. † denotes significantly higher cell number in the atelocollagen scaffolds at 2 days
(p < 0.05).
2.7. Scaffold Contraction
After 9 days in culture, the cell-seeded atelocollagen scaffolds had contracted more than the
cell-seeded gelatin scaffolds (91.7 ± 5.2% of day 1 surface area vs. 101.1 ± 8.5% of day 1 surface area,
p < 0.05). Control scaffolds, which had been cultured without cells, showed similar contraction rates to
those seen in the cell-seeded scaffolds (88.3 ± 7.0% of day 1 surface area vs. 101.1 ± 8.5% of day 1
surface area, p = 0.06).
Figure 4. Contraction of scaffolds cultured for 9 days measured as a percentage of initial
area. The atelocollagen scaffolds had a greater reduction in surface area than the gelatin
scaffolds. A similar trend was seen for cell seeded scaffolds, and non-cell seeded scaffolds.
* denotes significantly greater contraction of the cell seeded atelocollagen scaffold
compared to the cell seeded gelatin scaffold (p < 0.05).
Materials 2011, 4
3. Experimental Section
3.1. Manufacturing of Atelocollagen Scaffolds
The collagen used in this study was derived from bovine knees (Research 87, Boylston, MA, USA).
The tissue was sterilely harvested, minced, and solubilized in an acidic pepsin solution. The resulting
slurry was frozen overnight at −80 °C and lyophilized under full vacuum at −35 °C for three days and
re-hydrated with sterile water to a collagen concentration >10 mg/mL. The same reconstituted
atelocollagen slurry was used in all experiments.
The atelocollagen scaffolds were then made by mixing the atelocollagen slurry with HEPES
Buffer (Cellgro, Mediatech, Inc, Herndon, VA, USA), and neutralizing to a pH of 7.4 using 7.5%
sodium bicarbonate (Cambrex BioScience Walkersville, Inc., Walkersville, MD, USA). The solution
was transferred to a sterile 100 mm petri dish, and incubated for 20 min at 37 °C and 5% CO2 to allow
for gelation. The resulting gel was frozen at −80 °C overnight and lyophilized.
3.2. Gelatin Scaffolds
Commercially available gelatin scaffolds (Surgifoam® sheets, Johnson and Johnson, Binghampton,
NY, USA) of the same thickness of the atelocollagen sheet were obtained, and a sterile one centimeter
diameter circular punch was used to cut out individual scaffolds from both materials. Unlike the
atelocollagen scaffolds which were not cross-linked, the gelatin scaffolds were chemically cross-linked
with an aldehyde during manufacturing.
3.3. Preparation of Fibrin Clot Scaffolds
Fibrin clots were prepared by adding a 500 units/mL thrombin NaCl solution (King Pharmaceuticals,
Bristol, TN) to complete medium (Dulbecco’s Minimum Essential Medium (DMEM) (Cellgro,
Mediatech Inc., Herndon, VA, USA) supplemented with 10% defined fetal bovine serum (FBS)
(Hyclone, Logan, UT) and 1% antibiotic-antimycotic solution (Cellgro, Mediatech Inc., Herndon,
VA, USA), to create a 10 units/mL solution. 715 μL of the thrombin solution was transferred to each
well of a 24-well plate. 285 μL of a 20 mg/mL fibrinogen solution (Sigma-Aldrich, Saint Louis,
Missouri, Product#: F4753) was added to each thrombin aliquot. The plate was mixed on a shaker
table at 60 oscillations/min for 30 seconds. The plate was put into an incubator (37 °C, 5% CO2) for
30 min to allow fibrin clot formation. A sterile one centimeter circular punch was then used to cut
3.4. Preparation of Blood Clot Scaffolds
A total of 300 mL of whole blood was drawn from two hematologically normal volunteers meeting
all criteria of the American Association of Blood Banks (Food and Drug Administration, Center for
Biologics Evaluation and Research). Blood was collected in 60 cc syringes with 10% acid-citrate
dextrose as an anticoagulant at the Immune Disease Institute (Boston, MA, USA). 120 mL of blood
was used for making blood clots while 180 mL was used for making platelet-rich plasma as described
in the next section. A solution of 1000 Units thrombin/mL CaCl2 was used to clot the whole blood.
Materials 2011, 4
100 μL aliquots of the thrombin solution were placed into wells of a 24-well plate. 900 μL of whole
blood was added, and the plate was placed on a shaker table at 60 oscillations/minute for 30 seconds,
and then incubated (37 °C, 5% CO2) for 20 min to complete blood clot scaffold formation. A sterile
one centimeter circular punch was then used to create uniform individual scaffolds.
3.5. Preparation of Enzyme Stock Solutions
The MMP-1 stock solution was made by combining 50 mg of MMP-1 (Worthington Biochemical
Corporation, Lakewood, NJ, product number LS004214) with 200 mL of PBS, yielding a 250 µg/mL
(72.2 units/mL) MMP-1 solution. Aliquots of the solution were then frozen at −80 °C in 50 cc tubes.
The elastase stock solution was made by combining 5 mg of elastase (Worthington Biochemical
Corporation, Lakewood, NJ, product number LS006363) with 50 mL of PBS, yielding a 100 µg/mL
(0.86 units/mL) solution. Aliquots of the solution were then frozen at −80 °C in 50 cc tubes.
The plasmin stock solution was made by combining 1.5 mg of plasmin (Sigma Aldrich Co., St.
Louis, MO, USA, product number P1867) with 37.5 mL of ultrapure water to yield a 40 µg/mL
plasmin (0.416 units/mL) solution. Aliquots of the solution were then frozen at −80 °C in 50 cc tubes.
3.6. Enzyme Degradation Experiment
Enzymatic degradation rate was determined by measuring the change in surface area of the
scaffolds over time. Atelocollagen, gelatin, fibrin clot, and blood clot scaffolds were subjected to each
of the enzyme stock solutions. Experiments were run in triplicate. We also observed the response of
the scaffolds when exposed to the solvents used to make the enzyme stock solutions (PBS for MMP-1
and elastase; water for plasmin). The scaffolds were placed into wells of a 12-well plate and 1.0 mL of
solution was added to each well. The solutions were changed after 12, 24, and 36 h. Digital images
were taken with a Canon Rebel XT camera at 12 h, 24 h, and 48 h. Surface area measurements of the
scaffolds were determined using the NIH Image-J software (v. 1.38×).
3.7. Cell Source
Human ACL explants were obtained from the knee using sterile technique during ACL
reconstruction. After ligament harvest, explants were cultured in completed medium (Dulbecco’s
modified Eagle medium or DMEM) containing 4.5 g/L glucose, 10% fetal bovine serum, and 1%
AB/AM) which was changed two times per week. When primary outgrowth cells were 80% confluent,
they were trypsinized, counted, and suspended in complete medium.
3.8. Measurement of Cell Proliferation in Scaffolds
Cell proliferation was determined by indirectly measuring the change in cell number in the scaffolds
between two and ten days, using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide) assay. Atelocollagen and gelatin scaffolds (n = 6, per experimental group and time point)
were placed into the wells of 12-well plates. The scaffolds were seeded with 2.5 × 105 cells by placing
250 μL of the cell solution on top of each scaffold and incubating (37 °C, 5% CO2) for one hour to
Materials 2011, 4
allow for absorption. After incubation, 0.75 mL of complete medium was added to each well and the
plates were placed back into the incubator. Media was changed every other day.
At 2 and 10 days, the MTT working solution was prepared at a concentration of 1 mg/mL
in complete medium from a sterile stock MTT solution (Sigma-Aldrich, St. Louis, MO, USA,
Cat. #: M5655-500MG). The media was aspirated from each well and 1 mL of the working MTT
solution was added to each well. The plates were incubated for 3 h (37 °C, 5% CO2). Subsequently, the
excess MTT solution was removed and 1 mL of sterile 1X Phosphate Buffer Saline (EMD Chemicals,
Gibbstown, NJ, USA, Cat. #: B10241-34) was added to each well, placed on a agitator table (Fisher
Scientific Clinical Rotator, 100 rpm) and left to rinse at room temperature for 30 min. Rinses were
repeated until the absorbance readings of the wash were less than 0.100. All PBS was then removed
and each scaffold transferred into 3 ml centrifuge tubes. 1mL of a detergent containing 20% aqueous
SDS/formamide (1:1 volume ratio) was added to each tube and incubated overnight in a 37 °C water
bath. The tubes were vortexed for 5 seconds and then centrifuged for 5 min at 250 × g. Aliquots of the
supernatant from each tube (200 µL) were then transferred into a 96-wellplate, and the absorbencies
were measured at 562 nm. The cell proliferation assay was carried out after 2 and 10 days of culture,
and cell proliferation was determined by calculating the change in absorbance from day 2 to day 10.
Measurement of the contraction of the atelocollagen and gelatin cell-seeded scaffolds was also
performed using surface area measurements of the scaffolds at days 1, 2, 5, 6, 7 and 9. Photos of the
scaffolds were taken and the area of each scaffold calculated using Image J. Contraction was recorded
as the percent change in scaffold area compared to day 1.
3.9. Statistical Analysis
All statistical analysis was performed using SPSS version 16.0. For the enzyme degradation
experiments, a mixed model repeated measures ANOVA was utilized. For the cellular proliferation
experiments, a two way ANOVA was used with Fisher’s LSD post hoc test to detect significant
differences between groups. Results were given as mean ± SD. For all experiments, a p-value < 0.05
was deemed statistically significant.
It has been found that after trauma to a joint occurs, the concentration of certain enzymes in
synovial fluid increases. This release of enzymes can lead to more rapid degradation of connective
tissue, as well as degradation of scaffolds used for tissue repair in the joint environment. When using a
scaffold for tissue repair, it is important that the scaffold not dissolve before the surrounding cells can
invade and stabilize it [29-31]. The results here suggest that scaffolds made of atelocollagen may be
better able to resist degradation when compared to gelatin scaffolds or fibrin clots.
This result may help to reconcile several seemingly contradictory reports on the use of biologically
based scaffolds for joint repair. The use of platelet-rich plasma products, which are fibrin-based, has
not demonstrated positive results when used in the synovial environment for ACL repair  or rotator
cuff healing . In contrast, when the fibrin-based materials are used outside of the joint
environment, they appear to have greater success . For example, use of PRFM on the synovial side
of a rotator cuff tendon tear (which is exposed to synovial fluid) has been found to be ineffective ,
Materials 2011, 4
whereas the use of a different fibrin-based product on the bursal side of the tendon (outside of the
joint) has shown early efficacy . In addition, animal studies have demonstrated that while the use
of fibrin based scaffolds alone is ineffective in ACL repair  and intra-articular rotator cuff
repair , the use of collagen-based scaffolds is effective in stimulating ACL repair [35-37] and
rotator cuff repair [38,39].
The fibrin and blood clot scaffolds decreased in size during the first 12 h of culture in PBS and
water, and then had no significant further change. Due to the lack of further change on exposure to
these solutions, it is unlikely these initial decreases in size were due to degradation of the scaffolds;
rather it is more likely these changes were due to an initial contraction of the scaffolds. In the case of
the blood clot scaffolds, this contraction may have been due to the presence of platelets in the scaffold
as platelets are known to have a contractile function [40,41]. For the fibrin scaffolds, the reason for the
initial contraction is less clear. Improved organization of the fibrin fibrils is one possibility .
Taking into account this initial contraction, the difference between the change in size in the carrier and
that in the enzyme solutions is likely due to degradation.
One of the major limitations with all of the scaffolds tested here was their degradation in the MMP-1
solution by 48 h. The length of time required for a scaffold to be present in a wound site before it is
stabilized sufficiently by invasion of surrounding cells is as yet unknown. Previous studies have
demonstrated that immature animals have a more rapid cellular invasion of an ACL wound site than
adult animals , likely due to the observed increased cellular proliferation and migration speed in
these animals when compared with adults [44,45]. Thus, one possible reason for the improved
functional ACL healing noted in immature animals  may be that the immature cells are able to
stabilize the atelocollagen implants before the synovial enzymes degrade it, while the slower adult
cells are not able to populate the scaffold before it degrades. Use of a scaffold material with greater
resistance to synovial degradation may be necessary to obtain functional healing in the adult knee.
Another strategy would be to remove or neutralize the enzymes in the adult animals. Arthroscopy
results in washing out of the enzymes and replacement with saline, and this act alone may help tip the
balance in favor of scaffold preservation. What effect an additional synovectomy may have on the
re-accumulation of the enzymes is also as yet unknown, but may be a key factor in the success of
The atelocollagen and gelatin had different manufacturing protocols which may have affected results.
The gelatin is cross-linked during its production, while the atelocollagen is not. However, the addition of
cross-linking should increase the resistance of the atelocollagen to enzymatic degradation [9,10], so if
the atelocollagen were manufactured the same way as the gelatin, one might expect even greater
resistance to degradation, not less.
Tissue engineering within the synovial joint is a challenging task. In this work, we define some of
the basic material responses to elements that are intrinsic to this unique environment; namely, the
presence of enzymatic agents that are not found in significant quantities in other tissues. The results
suggest that atelocollagen-based scaffolds may provide some protection against premature degradation
by synovial fluid enzymes over blood or fibrin clots, but even they may be degraded within 48 h by
MMP-1 at high enough levels in the traumatized knee. Additional in vivo evaluations of these
materials is likely to provide further insight into the use of these biologic materials in tissue
engineering within the joint.
Materials 2011, 4
The atelocollagen scaffolds had a significantly greater resistance to degradation by MMP-1, elastase
and plasmin over the fibrin based scaffolds; while the cellular proliferation was similar in both types of
collagen scaffolds. The atelocollagen scaffolds contracted more than the gelatin scaffolds during
9 days in culture, though this does not appear to be cell mediated, as similar results were seen for the
non-cell seeded scaffolds. The results suggest that atelocollagen-based scaffolds may provide some
protection against premature degradation by synovial fluid enzymes over fibrin-based matrices.
The authors would like to acknowledge the assistance of David Luftman, Sherwin Kevy and May
Jacobson. This project was supported by the NIH grants R01 AR054099 and AR052772 (MMM).
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