Biologic Biomaterials: Silk - Biomaterials - page 162

from nanometer length scales to macroscopic tissue architectures [40]. The structures generated with
silks, such as via electrospinning, contain similar nanoscale fibers with micro-scale interconnected
pores, resembling the topographic features of the ECM. Nonwoven fibrous silk fibroin nets/mats/mem-
branes can also be fabricated with diameters in the range of micrometers in their native or partially
dissolved forms [41-43]. Similarly, finer meshes with diameters in the range of tens to hundreds of
nanometers can be obtained with electrospun silk fibroin fibers [44-50]. Nonwoven microfibrous nets
support the adhesion, proliferation, and cell-cell interactions of a wide variety of human cell types
including epithelial cells, endothelial cells, glial cells, keratinocytes, osteoblasts, and fibroblasts [43].
A follow-up study using precoating with fibronectin supported in vitro endothelialization, an essential
step for vascularization [42]. After seeding in fibronectin-coated silk fibroin nets, normal structure, pro-
liferative activity, migration, cell-cell interactions, and other phenotypic features were observed in pri-
mary human endothelial cells of macro-/microvascular origin. No alteration in the structural integrity
of the nonwoven nets was observed during cell cultivation. In addition, cytocompatibility of these non-
woven nets to keratinocytes and osteoblasts suggest potential for skin or bone repair. The biocompat-
ibility of these nonwoven microfibrous meshes composed of partially dissolved native silk fibroin fibers
has also been reported [41]. After subcutaneous implant, the nonwoven microfibrous meshes induced a
mild foreign body response without fibrosis. Microarray analysis of 23 proinflammatory genes identi-
fied an increase in migration inhibitory factor transcript level in implantation sites with the silk fibroin
mesh. No appreciable infiltration of lymphocytes was observed within 6 months after implantation,
further suggesting biocompatibility. These silk fibroin mesh implants supported the regeneration of
vascularized reticular connective tissue based on cytokeratins, vimentin, and Col-I detection, and mor-
phological, histological, and immunohistochemical evaluation of the regenerated tissue. Further, the
silk fibroin mesh implants integrated with the surrounding tissue while no apparent degradation was
observed within 6 months of implantation. An in vivo study [51] further identified silk fibroin-based
membranes/meshes as promising materials for skin regeneration. Similar to the above system, micron-
diameter fiber mats, nonwoven nanofibrous nets/mats prepared by electrospinning, can be used for sim-
ilar goals [44,46,47] and support the attachment, spreading, and proliferation of human bone marrow
stromal cells, keratinocytes, and fibroblasts in vitro [47,48,52]. In an in vivo study, the biocompatibility
of silk fibroin nonwoven nanofiber membranes/nets and their effect on guided repair of critical-sized
calvarial bone defects were assessed in a rabbit model [45]. The nanofiber membranes/nets were formed
by electrospinning regenerated silk fibroin solution in 98% formic acid and treated with 50% methanol.
The resulting nonwoven nanofibrous membranes contained randomly deposited fibers with diameters
ranging from 150 to 300 nm. The membranes supported the in vitro attachment, spreading, prolifera-
tion, and differentiation of MC3T3-E1 osteoblast-like cells. When evaluated in vivo in a rabbit calvarial
bone defect model, the silk fibroin nonwoven nanofibrous membranes showed good biocompatibility
and structural stability. The membranes enhanced bone formation over 12 weeks with no evidence of
inflammatory reactions. This study further suggests that nonwoven silk fibroin nanofibrous nets/mats/
membranes have potential for guided regeneration of bones in nonload bearing sites [53-55].
7.4.2 Regenerated Silk Fibroin Films and Coatings
Silk was first evaluated for cellular responses on 2D films in tissue culture wells. The films formed from
silkworm fibroin collected from glands of B. mori and A. pernyi wild silkworms were comparable to
collagen films in terms of supporting the attachment, spreading, and proliferation of murine L-929
fibroblasts [56-57] and the growth of human and animal cell lines [58,59]. Cell attachment to positively
charged residues like arginine near the C-terminus of the nonrepetitive (hydrophilic) regions of the silk
fibroin sequence was identified as an important feature, considering the surface of mammalian cells is
predominantly negatively charged [56,58]. Further observations identified stronger cell adhesion on
films formed by silk fibroins from A. pernyi than those from B. mori [56]. The difference in terms of cell
attachment was attributed to the presence of the tripeptide Arg(R)-Gly(G)-Asp(D), a recognition site