New Hydrogel Biomaterials for the

Minimally Invasive Treatment of Disc

Degeneration

1
J. Mercuri, 1C. Addington, 2S. Gill,
1
D. Simionescu

Department of Bioengineering, Clemson University, Clemson, SC

1

2
The Steadman Clinic, Vail, CO
 BACKGROUND
Tissue Engineering
 90% of adult spine surgeries are 2
performed due to consequences 1

arising from intervertebral disc

degeneration.1
 Current surgical treatment solutions
are palliative.
3
4
 Tissue engineering aims to
repair, replace, or enhance the
function of a tissue.2
 Requires a suitable biomaterial Figure 1: The paradigmatic approach to tissue
scaffold which will allow for re- engineering: 1) autologous cells are harvested
population with cells and will from the patient, 2) isolated cells are
function mechanically upon expanded in culture and 3) subsequently
implantation. seeded onto biological or synthetic scaffolds
to form healthy tissue prior to 4) re-insertion
into the patient to replace damaged or
diseased tissue.
An et al. Spine. 2004;29(23):2677-2678. 2Nerem et al. Tissue Eng. 1995;1(1):3-13.
1
Adapted from: http://www.bioartificial-organs.net/_images/content/tissue-
engineering-chart_en.gif
RESEARCH HYPOTHESIS
 We hypothesize that a tissue engineered nucleus
pulposus (NP) replacement may serve as an early-stage
intervention for mitigating intervertebral disc
degeneration.
 Moreover the success of such an approach requires the
development of scaffolds that:
 Mimic the biochemical and mechanical characteristics of the human NP.
 Support NP cell or an alternative cell source viability.
 Can be delivered in to the disc via minimally invasive surgical approaches.

 We are currently developing two new hydrogel

biomaterials to be used as scaffolds which exhibit
characteristics tailored towards nucleus pulposus tissue
engineering.
METHODS – EGC Hydrogels
 Elastin-Glycosaminoglycan-Collagen (EGC) hydrogel formation:
 Hyaluronic acid (5mg/ml), chondroitin-6-sulfate (27mg/ml) and soluble elastin
(40mg/ml) were added to a solution of type I collagen (3mg/ml).
 Following gelation at 37oC and subsequent freeze-drying, EGC hydrogels were
chemically stabilized with carbodiimide and pentagalloyl glucose based fixatives
and subjected to partial enzymatic digestion with chondroitinase and
hyaluronidase.
 Partial enzymatic digestion of EGC hydrogels yielded a material with shape-memory
sponge characteristics.

 EGC hydrogel characterization:

 Physical and mechanical properties: Unconfined compression, incremental stress
relaxation testing and water content measurements.
 Cytocompatibility: In vitro culture of porcine nucleus pulposus cells on EGC
hydrogels for 28 days.
METHODS – APNP Hydrogels
 Acellular porcine nucleus pulposus (APNP) hydrogel
formation:
 Porcine nucleus pulposus (NP) was harvested from juvenile pigs and
subjected to a combination of chemical (Triton X-100 and deoxycholic acid
detergents) and physical (ultrasonication) decellularization methods over a
72 hour period.
 Samples were subsequently incubated in a solution of DNase/RNase for 48
hours to remove residual porcine nucleic acid .

 APNP hydrogel characterization:

 Biochemical composition: Immunohistochemistry and biochemical assays
(glycosaminoglycan and hydroxyproline).
 Mechanical properties: Dynamic mechanical analysis and incremental stress
relaxation compression testing.
 Cytocompatibility: In vitro culture of human adipose derived stem cells
(hADSCs) on APNP for 14 days.
RESULTS - EGC Hydrogels
 Physical attributes under
compressive loading:
A B C
 EGC hydrogel height was
reduced by a mean of 76.6%
and water was expressed.

 Immediately after
unloading:
 96.4% ±1.3% of original EGC
hydrogel height was
recovered and water content
was not significantly different
from pre-compression
values.
D E F
Figure 2: Representative EGC hydrogel (white mass) A) prior to
B) during, and C) immediately following compression
illustrating its ability to recover original shape/height upon load
removal. D) Image depicting the formation of an EGC hydrogel
within a silicone mold (orange) resulting in E-F) a nucleus
pulposus-like shape.
RESULTS - EGC Hydrogels
A
 EGC hydrogel
characteristics:
 Equilibrium modulus: 4.73
± 1.05 kPa.
 Percent energy
B C
dissipation (hysteresis):
6.9% ± 5.4%.
 Water content: 94.5% ±
0.6%.
 Maintained nucleus
Figure 3: A) Representative stress relaxation curve of an EGC
pulposus cell viability. hydrogel subjected to increments of 5% strain followed by a 20
minute relaxation period to a total of 25% strain. B) Live/Dead
image of a porcine NP cell seeded EGC hydrogel indicating live
cells (green) after 28 days in culture. C) Alcian blue stained EGC
hydrogel depicting a porous micro-architecture containing
glycosaminoglycan (blue) and elastin (pink).
RESULTS - APNP Hydrogels
 Macroscopic evaluation:
A B
 APNP hydrogels appeared
hydrated, swollen and
gelatinous.
 Microscopic evaluation:
 Histological evaluation
revealed that APNP hydrogels C D
contained glycosaminoglycan,
aggrecan and collagens II, IX,
and XI.
 No evidence of porcine cells,
remnant DNA, or porcine α-gal
Figure 4: A) Representative macroscopic image of an APNP
immunogenic epitope was hydrogel. Histological images of APNP hydrogels stained for
observed. B) glycosaminoglycan (blue), C) aggrecan (brown) and D)
collagen type II (brown). Insert = immunohistochemistry
negative control. Bar = 10 mm.
 RESULTS - APNP Hydrogels
Table 1: Biochemical and mechanical characteristics of the APNP
 APNP hydrogels hydrogel and healthy human NP.

exhibited comparable
characteristics to
healthy human NP.

 APNP hydrogels
decrease hydration
in response to
applied osmotic
pressure similar to
human NP.
1
LeMaitre et al. Biochem Soc Tran. 2007;35(4):652-5.
2
Nguyen et al. J Bone Joint Surg Am.2008;90(4):796-802.
3
Mwale et al. European Cells and materials. 2007;8:58-64. Figure 5: APNP hydrogel hydration with respect to
4
Cloyd et al. Eur Spine J. 2007;16(11):1892-1898.
5
Leahy et al. Journal of Materials Science. 2001;12: 689-692 applied osmotic pressure compared to human NP.
RESULTS - APNP Hydrogels
 APNP hydrogels maintained
human stem cell viability and A B

allowed for cell infiltration

and proliferation in vitro:
 Histology indicated hADSCs
infiltrated the APNP hydrogel.
 hADSC DNA content increased C D
up to 69% from day 7 to 14.

 APNP hydrogel cell-mediated

contraction was observed.
Figure 6: A) Representative macroscopic image of an hADSC
seeded APNP hydrogel in culture. B-C) Live/Dead staining of
hADSCs on APNP hydrogels indicating live cells (green) at
day 7 and 14, respectively. D) Alcian blue stained hADSC
seeded APNP indicating the presence of glycosaminoglycan
(blue) and infiltration of hADSCs (pink). Bar = 5 mm.
CONCLUSIONS – EGC Hydrogels
 EGC hydrogels possess a shape memory characteristic.
 Allows for creating a pre-formed scaffold tailored to effectively fill the
NP region of a patient.
 Enables minimally invasive introduction into the NP region while in a
compressed orientation prior to re-swelling the hydrogel to its original
pre-compressed shape once in position.

 EGC hydrogels also possess a sponge-like property.

 A similar phenomenon is observed within the native nucleus
pulposus which expels water during diurnal loading and reabsorbs
water when in an unloaded state.
 This unique characteristic may also provide a means for mass
transfer of nutrients and waste to and from cells seeded within the
hydrogel.

 EGC hydrogels behave as a viscoelastic solid with a

predominantly elastic characteristic and is conducive to
maintaining NP cell viability.
 CONCLUSIONS – APNP Hydrogels
 Minimally invasive delivery of the APNP hydrogel may be
achieved due to its gel-like consistency.
 Preliminary investigations of syringe injection are on-going.

 APNP hydrogels are comprised of the same biochemical

components and in similar quantities as the human NP.

 APNP function as scaffolds which are conducive to re-

population with a human stem cell source.
 Stem cells have exhibited the ability to differentiate into NP-like cells
and may produce NP tissue in vitro .1,2

 Although mechanical characteristics of the APNP hydrogel

are on the lower end of reported values for human NP, it’s
believed that cell-mediated extracellular matrix production
within the APNP will improve these properties.
Li et al. Connect Tissue Res. 2005;46(2):75-82. 2Lu et al. Biochem Biophys Res Commun. 2007;359(4):991-6.
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