Biomedical Engineering Reference
In-Depth Information
that found on the native GAG molecules found in the NP. Bovine NP cell-laden
scaffolds composed of a 3% solution of 250 kDa CMC exhibited an equilibrium
Young's modulus of 3.53
0.87 kDa and 60% cell viability after 7 days in culture.
Additionally, histologic analysis indicated that the NP cells had a round morphol-
ogy and resided in lacunae surrounded by pericellular and interterritorial deposits of
chondroitin sulfate proteoglycan.
Yang et al. developed scaffolds derived from fibrin, citing such advantages as
the ability to deliver cells and scaffolds in a minimally invasive fashion while
maintaining the ability to form a scaffold that conforms to the nucleotomized space
[
124
]. NP cells were suspended in fibrinogen concentrate derived from centrifuged
human plasma, which was subsequently clotted via the addition of calcium. Results
suggest a significant twofold increase GAG production, a 326% increase in
aggrecan, and a significantly greater amount of DNA in the fibrin clot scaffolds
as compared to human NP cells cultured in alginate. However, NP cells cultured in
fibrin also expressed increased collagen types I and VI, which could be indicative of
fibrotic phenotype.
Seguin et al. developed NP tissue in vitro on a porous calcium phosphate (CPP)
substrate surface, which could theoretically allow the construct to be anchored to
bone of the vertebral body [
115
]. CPP substrates were formed by sintering CaP
powder, yielding a scaffold with a mean porosity of ~37% and pore size of 27
m
m.
After 2 weeks in culture on CPP, bovine NP cells were able to form a continuous
layer, with subsequent ECM production by 6 weeks resulting in a tissue thickness of
approximately 1.8 mm and a dry weight of 2.5 mg. This matrix stained with
toluidine blue throughout the ECM, with more intense staining in the pericellular
regions indicative of proteoglycan production. The growth rate of newly formed
tissue decreased as time passed (0.6 mg/week at 2 weeks to 0.3 mg/week at
6 weeks), despite a steady increase in Hoescht DNA staining indicative of cell
proliferation. GAG content within the tissue formed on CPP was not significantly
different at 6 weeks from content found in native bovine tissue (218 and 227
m
g/mg
dry weight, respectively). SDS-PAGE illustrated that collagen II was predomi-
nantly found in the formed tissue, whereas unconfined compression testing indi-
cated no significant difference in mechanical properties of in-vitro formed tissue
compared to native bovine NP. Although the CPP appears to be an excellent
substrates on which to develop NP tissue, further investigation of the implanted
construct should be evaluated to determine the potential bone in-growth and
possible over-growth into the formed NP tissue which would be detrimental.
Additionally, this initial work does not take into consideration that the native NP
tissue does not directly interface with bone; instead a cartilaginous endplate would
lie between the two.
The latter issue was addressed more recently by Hamilton et al. who developed a
trilayer composite consisting of CPP, articular cartilage (CEP), and NP tissue [
125
].
By sequential seeding of chondrocytes onto the CPP surface followed 2 weeks later
by seeding NP cells onto the matrix produced by the chondrocytes, the authors were
able to form a tissue composite construct. Although it appeared that the NP cells
were able to maintain a rounded morphology, the interfacial shear load required to
