1. Effects of Cross-linking on the Mechanical Properties of a Porous Foam Scaffold of Small Intestine Submucosa
Mark R. DiSilvestro1
, Charles Carter1,2
, Jessica Williams 1,2
, J-K Francis Suh3
, Janine M. Orban1
, Prasanna Malaviya1
1
DePuy Orthopaedics, Inc., 700 Orthopaedic Drive, Warsaw, Indiana 46581, USA
2
Department of Bioengineering, University of Toledo, Toledo, Ohio 43606, USA
3
Department of Biomedical University, Tulane University, New Orleans, LA 70118, USA
Introduction:Small intestine submucosa (SIS) is a naturally occurring
biomaterial that is finding increasing use in tissue engineering1-5
. The
success of SIS as a scaffold is due to its biochemical composition
including collagens and growth factors6
. SIS is frequently configured as
a multi-laminate scaffold for resisting tensile stresses during remodeling.
In cartilage tissue engineering, however, it is advantageous to have a
porous 3-D scaffold that allows easy cell infiltration and nutrient
transfer. We have fabricated 3-D SIS scaffolds, with varying pore sizes,
while fully maintaining the biomaterial’s natural biochemical
composition. The biphasic poroviscoelastic (BPVE) model7
, which
assumes the material modeled to be composed of an incompressible,
isotropic, viscoelastic solid phase, and an incompressible, inviscid fluid
phase, has been shown to be efficacious in modeling the compressive
mechanical response of articular cartilage8
. As SIS foam scaffolds also
fulfill the assumptions of the BPVE model, this study examined the
effects of chemical cross-linking on the mechanical properties of 3-D
SIS foam scaffolds determined both by direct measurement and from
BPVE model curve fits. The hypothesis that cross-linking enhances the
compressive mechanical properties of the SIS foam was tested.
Materials and Methods: Foam Fabrication: SIS material was
processed through a communition machine (Comitrol 1700, Urschel
Labs, Valparaiso, IN) to obtain fine fibers (50-200 µm x 1-5 mm)
suspended in RO water. Water was partially driven off via
centrifugation (1500 rpm, 10 min) and the resulting pellet was placed in
6-well plates (7.2 g wet weight/well). The plates were frozen one of
three ways to vary final pore size: (1) slow freezing in an EtOH bath at–
20o
C (large pore size), (2) rapid freezing in a –80o
C freezer (medium
pore size), and (3) flash freezing in a LN2 bath (small pore size). The
plates were lyophilized and the resulting 3-D SIS foam structure was
visualized using SEM. Several foams with medium pore size were
cross-linked (4 hrs, room temp), using 1-ethyl-3(3-diaminopropyl)
carbodiimide (EDC) at either 2.5 mM (low cross-link density, LXL) or
20 mM (high cross-link density, HXL) concentration, and re-lyophilized.
Densities of all samples were used to determine porosity. Compression
Testing: Unconfined compression stress relaxation tests were performed
using a Test Resources 200R to determine equilibrium stress-strain
curves of non-cross-linked (NXL), LXL, and HXL medium pore size
cylindrical foam samples (n=5 per group). Cylindrical geometry was
ensured using a 5 mm biopsy punch, and a scalpel was used to cut the
specimens to 2 mm thickness. Samples were first preconditioned (60%
cyclic strain, 50 cycles @ 1Hz), allowed to relax in PBS overnight, and
then tested in PBS. Each test involved 4 stress relaxation tests at a strain
rate of 0.003 strain/sec in increments of 5% strain (5%, 10%, 15%, 20%
strain). Each specimen was allowed to relax for 30 min at each strain
level while compressive force was recorded (Interface Technologies,
SMT1-2.2, resolution of 0.2 g). A slope of less than 10-5
N/min defined
equilibrium for each test. Direct Permeability Measurements: The
permeability of LXL, and HXL medium pore size cylindrical foam
samples to distilled water at room temperature was measured using a
custom-designed permeability chamber (n=5 per group). The
permeability of plugs was measured with an applied compressive strain
of between 11 and 36%. Direct permeability measurement of NXL
samples was inhibited by specimen integrity. BPVE Modeling: The
stress relaxation tests measured from each specimen between 10-15%
strain and between 15-20% strain were used for BPVE model curve
fitting. The Poisson’s ratio (ν) of all specimens was assumed to be
0.25. The Young’s modulus (E) value was allowed to vary between the
two values calculated from the two relaxation tests for each specimen.
The permeability (κ) was allowed to vary within the range of values
measured directly. Relaxation spectrum magnitude (G), short -term
relaxation time (τS), and long-term relaxation time (τL) were allowed to
vary over large parameter ranges. The best-fit BPVE parameters were
determined using an optimization algorithm8
. Single factor ANOVA
was performed for each model parameter for each pair of groups.
Results: Foam structure: SEM micrographs revealed the interconnected
porous structure of the SIS foam (Figure 1). Varying pore size ranges
and porosities were obtained: large – 500-800 µm (96% porous),
medium – 100-150 µm (90% porous), small – 20-60 µm (65% porous).
Figure 1: SEM of medium pore size SIS foam.
Compression Testing: Increased cross-linking density resulted in higher
equilibrium stresses for the same strain values (Figure 2). The
equilibrium stress-strain curves were found to be linear from 5-20%
(R2
>0.99). It was found that the slope of the stress-strain curves of the
HXL specimens over this region was significantly different than both the
NXL and LXL specimens (p<0.05).
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
0 0.05 0.1 0.15 0.2 0.25
Equilibrium Strain
EquilibriumStress(kPa) NXL
LXL
HXL
Figure 2: Equilibrium stress-strain curves.
Direct Permeability Measurements: Direct permeability measurements
showed no clear trend between groups. The values measured ranged
from 1x10-3
to 1x10-7
m4
/N-s. BPVE Modeling: The BPVE model fit
the entire mechanical response of a given specimen to both stress
relaxation tests modeled simultaneously using a single parameter set.
Parameter NXL (n=5) LXL (n=5) HXL (n=5)
ν 0.25 0.25 0.25
E (kPa) 2.94 ± 0.99* 3.97 ± 1.75* 11.21 ± 5.80
κ (m4
/N-s) x
10-4
3.41 ± 3.48 5.55 ± 2.37 5.47 ± 3.77
G 2.71 ± 0.83* 1.92 ± 0.69 1.14 ± 0.50
τS (sec) 4.01 ± 1.76 3.81 ± 1.85 1.62 ± 1.67
τL (sec) 114.2 ± 127.2 122.5 ± 165.2 294.5 ± 180.0
Table 1: BPVE parameters (mean ± S.D.). * p<0.05 versus HXL
Discussion: 3-D porous SIS foams, with varying pore sizes, were
produced. As evidenced by direct measurement and BPVE modeling, it
has been shown that EDC crosslinking in HXL specimens increases E as
compared to NXL and LXL specimens without significantly changing
other mechanical properties. Future work will investigate the effects of
cross-linking on foams with various pore sizes.
References: 1. Dejardin LM, et al. Am J Sports Med 29:175 (2001). 2.
Cook JL, et al. Am J Sports Med 27:658 (1999). 3. Roeder RA, et al.
Biomed Instrum Technol 35:110 (2001). 4. Sofer M, et al. J Endourol
16:27 (2002). 5. Badylak S, et al. J Surg Res 99:282 (2001). 6. Hodde
J. Tissue Eng 8:295 (2002). 7. Mak. J Biomech Eng 106:123 (1986). 8.
DiSilvestro. J Biomech 34:519 (2001).
©2003 Society For Biomaterials 29th Annual Meeting Transactions
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