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Silk Fibroin and Calcium Phosphate 3D Scaffolds Promote in vitro Osteogenesis
chemical, and biological characteristics . Biopolymers in terms of better resorption and regeneration of bone
[11]
including proteins [12,13] and polysaccharides [14,15] have been under physiological circumstances . In addition, the
[37]
used to formulate inks for extrusion printing. introduction of minerals can improve cell adhesion and
Silk fibroin (SF) is a natural protein extracted promote cell proliferation .
[25]
from silks, which can be processed into a variety of Many works have explored the composites/hybrids
morphologies with tunable molecular structures and from SF and calcium phosphate or precursors for bone
mechanical properties, ranging from soft hydrogels to stiff regeneration. In most recent research, SF-based hybrid
thermoplastics [16-18] . SF has been approved by the U.S. scaffolds were fabricated by freeze-drying. Not many
Food and Drug Administration for use as biomaterials works focused on the mineralization on 3D printed SF
for decades. Many researchers have advanced the field of scaffolds with highly porous morphologies and high
using regenerated SF as biomaterials especially in tissue β-sheet conformation content [22,38,39] .
engineering [19-21] . These studies have indicated that SF has In this study, we prepared SF scaffolds through
excellent biocompatibility with many tissues including extrusion-based printing with sodium alginate (SA) as a
bone, low immunogenicity, and inflammation compared thickener; then introduced various calcium phosphates
with other synthetic and natural polysaccharides. Moreover, for hybrid scaffolds through post-mineralization. The
SF possesses excellent properties for stimulating bone relationship between structure and morphology and
repair, because the fibrous structure of SF is mostly similar the mechanical performance SF and hybrid scaffolds
to collagen I (Col I) resulting in accelerated bone healing was studied. Furthermore, a series of in vitro biological
by improving local blood supply and collagen synthesis. experiments, including CCK-8 cytotoxicity test, by
When supplemented with growth factors, SF could q-PCR alkaline phosphatase (ALP) expression test, and
enhance mineralization, among which the effect from the osteogenesis gene (Runx2, OPN, OCN, OSX and Col1a)
functional amino acids with amide groups and hydroxyl expression test, were conducted to prove the excellent
groups and the conformation effect from β-sheets were osteogenesis effect of these mineralized 3D printed SF
believed to be the most significant in regulating mineral scaffolds. The work would provide a convenient strategy
nucleation and formation [22-24] . The challenge for printing to engineer mechanically robust hybrid scaffolds for bone
SF solution is its low viscosity and poor shape fidelity. tissue engineering.
Sodium alginate (SA) produces viscous aqueous solutions
and exhibits shear thinning characteristics, which is 2. Materials and methods
desirable for 3D printing. Mixture inks from SF and SA 2.1. Experimental materials
or alginic acid (ALG) have been studied to fabricate
scaffolds with good cell compatibility [25,26] . The results Ethanol (99.7%), methanol (99.5%), and ammonia
also suggested although SF was expected to enhance the solution (25 – 28%) were purchased from Modern Oriental
stiffness and strength of hydrogel scaffolds. However, the Technology Development Co., Ltd. (Beijing, China).
lack of control over the conformational structure of SF Glacial acetic acid (99.5%) and sodium bicarbonate
could limit the reinforcement effect. The comprehensive (analytically pure) were purchased from Beijing Chemical
properties of 3D-printed SF scaffolds could be Works (Beijing, China). Lithium bromide was purchased
conveniently tuned through changing the porosity, pore from Sigma-Aldrich (St. Louis, MO). Calcium acetate
size in the printing design, to match that of specific tissues. (99%), diammonium hydrogen phosphate (99.9%), and
At present, SF scaffolds can be fabricated to suffice most polyethylene glycol (PEG) were obtained from Shanghai
soft-tissue application requirements [27,28] . Macklin Biochemical Co., Ltd. (Shanghai, China). SA
Bones are the stiffest among all the human (chemically pure) was purchased from Xilong Chemical
anatomical tissues, which is attributed to the high mineral Co., Ltd. (Guangdong, China).
content and a sophisticated collagen-hydroxyapatite (HA) 2.2. Preparation of SF and SA mixture inks
hybrid morphology . Therefore, minerals, particularly
[29]
various forms of calcium phosphates including HA , The preparation of SF aqueous solution was carried out
[30]
tricalcium phosphate [31] (TCP), and monetite (DCP) in accordance with the protocol in ref. . 10 g Bombyx
[40]
, are often introduced to reinforce the 3D-printed
[32] mori cocoon silk (Fuxiang Pharmaceutical Technology
bone scaffolds. HA is the natural mineral crystal that Co., Ltd., Shanghai, China) was boiled in 0.5% NaHCO
3
exhibits a satisfying bone induction effect. Both brushite solution for 45 min and rinsed with distilled water to wash
(DCPD, CaHPO ·2H O) and monetite (DCP/DCPA, off the residual sericin before drying at 45°C for 24 h.
2
4
CaHPO ) can be converted into HA under physiological Then, 14 g dried silk fiber was dissolved in 100 mL 9.3 M
4
conditions [33,34] . DCPD can be further converted into lithium bromide solution at 40°C for 3 h, and dialyzed in
DCP in aqueous solutions or at high temperatures [35,36] . In deionized water for 3 days using a dialysis bag (8 – 14 kDa).
contrast to HA, DCPD and DCP have unique advantages Subsequently, the SF solution was dialyzed in 15% PEG
2 International Journal of Bioprinting (2022)–Volume 8, Issue 4