Nonmulberry Silk Braids Direct Terminal Osteocytic Differentiation through Activation of Wnt-Signaling

Silk polymers can regulate osteogenesis by mimicking some features of the extracellular matrix of bone and facilitate mineralized deposition on their surface by cultured osteoprogenitors. However, terminal differentiation of these mineralizing osteoblasts into osteocytic phenotypes has not yet been demonstrated on silk. Therefore, in this study we test the hypothesis that flat braids of natively (nonregenerated) spun nonmulberry silk <i>A. mylitta</i>, possessing mechanical stiffness in the range of trabecular bone, can regulate osteocyte differentiation within their 3D microenvironment. We seeded human preosteoblasts onto these braids and cultured them under varied temperatures (33.5 and 39 °C), soluble factors (dexamethasone, ascorbic acid, and β-glycerophosphate), and cytokine (TGF-β1). After 1 week, cell dendrites were conspicuously evident, confirming osteocyte differentiation, especially, in the presence of osteogenic factors and TGF-β1 expressing all characteristic osteocyte markers (podoplanin, DMP-1, and sclerostin). <i>A. mylitta</i> silk braids alone were sufficient to induce this differentiation, albeit only transiently. Therefore, we believe that the combinatorial effect of <i>A. mylitta</i> silk (surface chemistry, braid rigidity, and topography), osteogenic differentiation factors, and TGF-β1 were critical in stabilizing the mature osteocytic phenotype. Interestingly, Wnt signaling promoted osteocytic differentiation as evidenced by the upregulated expression of β-catenin in the presence of osteogenic factors and growth factor. This study highlights the role of nonmulberry silk braids in regulating stable osteocytic differentiation. Future studies could benefit from this understanding of the signaling mechanisms associated with silk-based matrices in order to develop 3D <i>in vitro</i> bone model systems

Reduced aggregation is seen on TEM images of PEG-coated PLGA nanoparticles (b) as compared to uncoated nanoparticles (a).

<p>Reduced aggregation is seen on TEM images of PEG-coated PLGA nanoparticles (b) as compared to uncoated nanoparticles (a).</p

Images of rats acquired at 4 h post-injection. A = Anterior, P = Posterior.

<p>Images of rats acquired at 4 h post-injection. A = Anterior, P = Posterior.</p

Cumulative percentage release ¹⁷⁷Lu-DOTATATE from uncoated and PEG-coated (a) PLGA 50∶50 nanoparticles and (b) PLGA 75∶25 nanoparticles.

<p>The bars represent standard deviation. (c) Total release over 21days.</p

SEM image showing morphology; and Size analysis using measurement software on TEM Image and Particle size analyzer for PLGA 50∶50 nanoparticles (a), (b) & (c) and PLGA 75∶25 nanoparticles (d), (e) & (f) respectively.

<p>SEM image showing morphology; and Size analysis using measurement software on TEM Image and Particle size analyzer for PLGA 50∶50 nanoparticles (a), (b) & (c) and PLGA 75∶25 nanoparticles (d), (e) & (f) respectively.</p

Images of rats acquired at 24 h post-injection. A = Anterior, P = Posterior.

<p>Images of rats acquired at 24 h post-injection. A = Anterior, P = Posterior.</p

Mean Particle Size of Nanospheres.

<p>SD – Standard Deviation; PDI – Poly-dispersity index.</p

Distribution of ¹⁷⁷Lu-DOTATATE (Group A); ¹⁷⁷Lu-DOTATATE-PLGA nanoparticles (Group B); and ¹⁷⁷Lu-DOTATATE-PLGA-PEG nanoparticles (Group C) in different organs over 24 h.

<p>The bars represent standard deviation.</p

Chromatograph of ¹⁷⁷LuCl₃ and ¹⁷⁷Lu-DOTATATE in 50% Aqueous Acetonitrile (a); Stability of Labeled compound till 6^th day post-labeling (b).

<p>Chromatograph of ¹⁷⁷LuCl₃ and ¹⁷⁷Lu-DOTATATE in 50% Aqueous Acetonitrile (a); Stability of Labeled compound till 6^th day post-labeling (b).</p

SEM image and particle size distribution of PEG-coated (a) & (b) PLGA 50∶50 and (c) & (d) PLGA 75∶25 Nan particles.

<p>SEM image and particle size distribution of PEG-coated (a) & (b) PLGA 50∶50 and (c) & (d) PLGA 75∶25 Nan particles.</p