Biologic Biomaterials: Silk - Biomaterials - page 164

and hMSCs were assessed [72,73]. In additional studies, RGD modification of silk fibroin enhanced the
adhesion and proliferation of human tenocytes and supported differentiation based on elevated tran-
script levels for decorin and Col-I [74]. Increased cell density and enhanced differentiation of cells on
RGD-coupled silk matrices were shown, mediated by cell-cell interactions [73]. Surface modification
with parathyroid hormone, which affects the differentiation of osteoblasts in vitro [75] and in vivo [76],
was used to cell responses on the silk fibroin films [72]. Silk fibroin films decorated with bone morpho-
genetic protein-2 (BMP-2) via covalent coupling enhanced osteogenic differentiation of hMSCs [77].
Compared to adsorbed BMP-2, covalently coupled BMP-2 was retained on the surface at a significantly
higher level for a longer period in culture. Within 1 week, 70% of the adsorbed BMP-2 was released
from the film surface. By the end of week 4, only 10% of the adsorbed BMP-2 remained, while 50% of
the coupled BMP-2 was still present. More importantly, both covalently coupled and surface-adsorbed
BMP-2 remained active and enhanced osteogenic differentiation of the bone marrow stromal cells. The
covalently immobilized BMP-2 was more effective than soluble BMP-2 likely due to the slower degrada-
tion and higher protein concentration in the local microenvironment. These studies demonstrated that
the diversity of amino acid side-chain residues contained in silk fibroin provides useful and accessible
options for surface decorations with adhesion ligands and specific growth/morphogen factors where
in most cases, biological activity was retained and in some cases improved. These strategies open up
further options for selective chemical enhancements of the silk fibroin biomaterial to encode functions
related to directing cell and tissue outcomes in a tissue-engineering context.
7.4.4 Silk Fibroin as a 3D Scaffold Matrix
Tissue engineering combines cells and bioactive factors in a defined microenvironment with biomate-
rial scaffolds that are maintained in bioreactors with controlled environmental stimuli for functional
tissue repair and regeneration [78,79]. A key component is the biomaterial scaffold, which acts as a 3D
support. Scaffolds should (1) be biocompatible to the host immune system where the engineered tissue
will be implanted; (2) support cell attachment, migration, cell-cell interactions, cell proliferation, and
differentiation; (3) biodegrade at a controlled rate to match the rate of neotissue growth and facilitate the
integration of engineered tissue into the surrounding host tissue; (4) provide structural support for cells
and neotissue formed in the scaffold during the initial stages of postimplantation; and (5) be versatile in
processing options to alter structure and morphology related to tissue-specific needs (Figure 7.3).
7.4.4.1 Silk Fibroin Porous Sponges
Although silk has been used clinically worldwide as suture material for centuries, only recently has
it been exploited as a scaffold biomaterial for cell culture and tissue engineering in vitro and in vivo .
Porous 3D sponge scaffolds are important for tissue engineering for cell attachment, proliferation, and
migration, as well as for nutrient and waste transport (Figure 7.3). 3D porous sponges have been formed
from regenerated silk fibroin solutions, using both aqueous and solvent approaches and using poro-
gens, gas foaming, and lyophilization [80]. Solvent-based sponges were prepared using salt (e.g., sodium
chloride) or sugar as porogen. Solvents such as 1,1,3,3-hexafluoropropanol (HFIP) do not solubilize salt
or sugar; therefore, pore sizes in the sponges reflect the size of the porogen used in the process [80].
Similarly, a gradient of pore sizes can be generated by stacking porogens of different sizes within a scaf-
fold. Further, sponges with varying porosity can be controlled by stacking variations of salt/HFIP-silk
solutions. Solvent-based porous sponges can also be prepared by addition of a small amount of solvent
(ethanol, methanol, DMSO) into the aqueous silk fibroin solution before processing [81]. As with the
solvent-based scaffolds, aqueous-based porous silk sponges can be prepared using salt crystals as poro-
gens, with control of pore sizes from 300 to 1000 μm, by manipulating the percent silk solution and the
size of the salt crystals. Pore sizes are generally a little smaller than the size of salt crystals utilized in the
process due to the limited solubilization of the surface of the crystals during supersaturation of the silk
solution prior to solidification [82]. The highly porous scaffolds (porosity up to 99%) prepared by salt