Nonmulberry Silk Braids Direct Terminal Osteocytic Differentiationthrough Activation of Wnt-SignalingSwati Midha, Shibu Chameettachal, Emeli Dey, and Sourabh Ghosh*
Department of Textile Technology, IIT Delhi, Hauz Khas, New Delhi-110016, India
*S Supporting Information
ABSTRACT: Silk polymers can regulate osteogenesis bymimicking some features of the extracellular matrix of boneand facilitate mineralized deposition on their surface bycultured osteoprogenitors. However, terminal differentiation ofthese mineralizing osteoblasts into osteocytic phenotypes hasnot yet been demonstrated on silk. Therefore, in this study wetest the hypothesis that flat braids of natively (nonregenerated)spun nonmulberry silk A. mylitta, possessing mechanicalstiffness in the range of trabecular bone, can regulate osteocytedifferentiation within their 3D microenvironment. We seededhuman preosteoblasts onto these braids and cultured themunder varied temperatures (33.5 and 39 °C), soluble factors (dexamethasone, ascorbic acid, and β-glycerophosphate), andcytokine (TGF-β1). After 1 week, cell dendrites were conspicuously evident, confirming osteocyte differentiation, especially, inthe presence of osteogenic factors and TGF-β1 expressing all characteristic osteocyte markers (podoplanin, DMP-1, andsclerostin). A. mylitta silk braids alone were sufficient to induce this differentiation, albeit only transiently. Therefore, we believethat the combinatorial effect of A. mylitta silk (surface chemistry, braid rigidity, and topography), osteogenic differentiationfactors, and TGF-β1 were critical in stabilizing the mature osteocytic phenotype. Interestingly, Wnt signaling promotedosteocytic differentiation as evidenced by the upregulated expression of β-catenin in the presence of osteogenic factors andgrowth factor. This study highlights the role of nonmulberry silk braids in regulating stable osteocytic differentiation. Futurestudies could benefit from this understanding of the signaling mechanisms associated with silk-based matrices in order to develop3D in vitro bone model systems.
Tailoring the 3D architecture, surface chemistry, amino acidsequences, or secondary conformation of silk fibers hasemerged as an effective strategy for the fabrication ofbiomimetic silk-based materials for bone tissue engineering.1
Silk fibroin, the primary component of silk fibers, comprisesunique amino acid sequences (GAGAGS and GAGAGY) inrepeating units which enables the tight packaging of proteinchains into antiparallel β-sheets and renders strength andresilience to the structures, akin to native bone matrix. Of thetwo widely known varieties of silkworm silk, Bombyx mori(mulberry) and Antheraea mylitta (nonmulberry), the latter hasshown exceptional potential in bone regeneration andmineralization both in vitro and in vivo.2 This is mostlyattributed to the presence of an additional Arg-Gly-Asp (RGD)peptide-motif in A. mylitta. However, as the sequence of A.mylitta is only partially known, the existence and exact role ofthis sequence is still debatable.3 Moreover, silk-based tissueengineering mostly relies upon tailoring scaffolds by recon-stitution of dissolved silk fibers into various 3D morphologies.Since A. mylitta silk is difficult to dissolve using standardlaboratory solvents,2 their application has largely been limited.
Apart from the exceptional mechanical properties, silk fibroinprotein is considered a potential matrix for bone tissueengineering due to its striking resemblance to collagen typeI.3 As proof of concept, previously we used silk fibroin fromB.mori silkworm to investigate the role of material properties(chemical, structural, and mechanical) in regulating hydrox-yapatite deposition on silk using simulated body fluid.4
Interestingly, we noticed that B. mori braids, constituting ofnative silk fibers, regulated hydroxyapatite deposition along thec-axis (002), oriented longitudinally parallel to silk fibers,typically found in native bone. This was a very significantfinding as the mechanism was found to be exactly similar to thebiomineralization of collagen type I in bone. On the contrary,the regenerated 3D porous silk scaffold lacked this potential,most likely due to the disruption of amino acid sequences as aresult of dissolution and reconstitution of original proteinassembly.5 This was followed by a comparative in vivoinvestigation on the osteogenic potential of 3D porous B.mori with its nonmulberry counterpart A. mylitta in critical size
Received: January 5, 2017Accepted: March 22, 2017Published: March 22, 2017
calvarial defects of rats.2 Radiographic and histological evidencerevealed complete osseo-integration of implanted defects within6 months, albeit only in A. mylitta scaffolds. On the basis ofthese results, we hypothesized that natively spun fibers ofnonmulberry silk A. mylitta will provide a suitable matrix forbone regeneration studies.In native bone, osteoblasts initiate the synthesis and
deposition of osteoid matrix wherein these cells get entrappedand start transitioning into dendritic osteocytes, characterizedby genotypic expression of dentin matrix protein-1 (Dmp-1)and sclerostin (Sost). Existing strategies to establish suchosteocyte models include the use of commercial cell lines in 2Dmonolayer or animal model testing. However, the signalingcues provided by 3D architectures (biochemical, topographical,and mechanotransduction) that direct cellular differentiationtoward a particular lineage cannot be replicated in conventionalmonolayers. Animal testing, on the other hand, is expensive, isplagued by ethical concerns, and often fails to correlate withhuman physiology.6 To circumvent these issues, 3D culturesystems of polymers and proteins are becoming popular. Todate, in vitro studies on silk reported mineralized matrixdeposition using osteoblast cultures, but we could not find anyreport on complete cellular differentiation and transition intoterminally differentiated osteocytes. Therefore, with an aim todevelop a 3D bone model on A. mylitta braids ex vivo byreproducing the phenotypic and genotypic transition ofpreosteoblasts to dendritic osteocytes, akin to native bone, wewill address the following key points: (i) examining cellularresponses toward precisely fabricated hierarchical complexitiesof such nonmulberry braided structures, (ii) the role ofsubstrate properties in regulating osteocytic differentiation on
silk matrices; and (iii) major signaling pathways directingosteogenic signaling on nonmulberry silk matrices.Osteogenic signaling on silk scaffolds has been poorly
understood so far.3 Jung et al. studied the gene expression of ratbone marrow cells on B. mori silk fibroin proteins whichdemonstrated suppressed Notch-activated genes while upregu-lating the expression of osteogenic markers, thus indicating acritical role of silk fibroin proteins in suppressing Notchsignaling.7 Little is known about the role of silk in driving thekey osteogenic signaling mechanisms. Wnt signaling, one of themain bone signaling pathways, is regulated via β-catenin, atranscription factor known to express in the presence oftransforming growth factor-β1 (TGF-β1).8 TGF-β1, the mostabundant cytokine in human bone (200 mg/kg), plays a centralrole in bone matrix turnover,9 storage of minerals, and thegeneration of hematopoietic cell lineage. Varying theconcentration of TGF-β resulted in varied outcomes in invitro and in vivo studies.10 This poor correlation between theexisting literature on in vitro and in vivo based experimentsfurther emphasizes the inadequacies of the current in vitro 3Dculture systems for bone tissue engineering and demands areliable, long-term sustainable in vitro model to accuratelycapture the physiology of native bone tissue microenviron-ments.11 Such 3D in vitro models will not only facilitate animproved understanding of the fabrication of advancedmaterials but also help in investigating pathological signalingmechanisms and screening of drug candidates for therapeuticapplications.Considering the applications of this in vitro model in bone
differentiation, hFOBs 1.19, a preosteoblastic cell line withsurface markers similar to osteogenic progenitors12 served as anexcellent choice for the study. While the use of cell lines is often
Figure 1. Physical characterization of the A. mylitta braid. (A) Schematic explaining the structural braid hierarchy and nomenclature established; (B−E) SEM micrographs of the braid demonstrating a part of the braided fabric (B,C), two ply yarn (D), and silk fibers (E).
plagued with concerns regarding genetic manipulation andmisrepresentation of human physiology, an interesting traitabout this cell line is that it is immortalized but non-transformed, transfected stably with temperature sensitiveSV40 T-antigen.13 Gene expression analysis showed that at39 °C, hFOBs expressed upregulated levels of bone-relatedmarkers: Cbfa1, parathyroid hormone receptor, and osteocal-cin. Differentiation of hFOBs into mature osteoblasts synthesiz-ing mineralized matrix in 3D culture systems is known,14−16 butno study has so far reported the transition of these cells intoosteocytic phenotype on any material. Keogh et al. culturedhFOBs on collagen-glycosaminoglycan scaffolds and demon-strated early evidence of mineralization only after 35 days invitro.14 Therefore, the underlying mechanisms that triggerterminal differentiation of preosteoblast cells to osteocytes,especially as a function of the underlying substrate, still remainsa question.In this study, we test the hypothesis that nonregenerated,
natively spun A. mylitta silk fibers fabricated into a braidedmorphology can regulate extracellular matrix synthesis andosteocytic transition of cultured cells within the 3D micro-environment. The effect of biomechanical, topographical, andbiological cues were investigated by culturing humanpreosteoblast cells in the presence or absence of bone inducingfactors and TGF-β1. Osteogenic differentiation into matureosteoblasts/osteocytic phenotype was investigated by real-timegene expression profiling from early osteoblasts to terminalosteocytes, monitoring cell morphology and metabolic activityand protein synthesis by confocal microscopy. To the best ofour knowledge, this is the first study reported on nonmulberrysilk braids that maps the entire process of bone differentiationfrom preosteoblasts to stable osteocytes on A. mylitta braids.Beyond the conventional scope of clinical bone graft
substitutes, this nonmulberry silk braid has immensetherapeutic potential for being used as a 3D in vitro modelsystem of bone differentiation for screening the efficacy ofdrugs targeted toward bone disorders.
2. MATERIALS AND METHODS2.1. Source of Silk Yarn. A. mylitta silk fibers of 60 N m (16.7 tex)
count were procured from Starling Silk Mills Pvt. Ltd., Malda, Bengalsuch that silk double yarn (obtained by twisting two single yarnstogether) was produced with a resulting count of 30 N m (33.3 tex) todevelop braided structures, so that the yarns could withstand forcesduring the braiding process.2.1.1. Fabrication of A. mylitta Braids. Flat braids of silk were
produced by passing a fixed number of yarns in a diagonally oppositedirection to the subsequent layer such that each bundle alternates overand under another bundle of yarn. Then, the structure is oriented at anangle to the circumferential axis of the resulting braid on a flat braidingmachine with 34 two ply yarns. Figure 1A schematically illustrates thestructural hierarchy of the flat braid used in the experiment andestablishes the nomenclature used throughout the article. The braidswere subjected to degumming by boiling them in 0.02 M Na2CO3solution for 30 min in order to remove sericin and subsequently driedovernight.17 Next day, dried braid fabrics were manually cut intoscaffolds of smaller dimensions (4 mm × 4 mm) for cell culture.4
2.1.2. Physical Characterization of Braids. 2.1.2.1. Fiber/YarnDiameter. The diameter (d) of each respective fiber and yarn (n = 150each) in the A. mylitta braid was measured using a projectionmicroscope (WeswoxOptik microscope, MP-385A, India) on 40× and10× magnifications, respectively.2.1.2.2. Braid Angle Measurement. Braid angle is the angle
between the tow (bunch of yarns) direction and the axis of the tube.Braid angle was measured from macroscopic images obtained using an
inverted optical microscope (Leica DM2500P, Germany) equippedwith a high resolution digital microscope camera system (LeicaDFC425C, Germany). A total of 12 observations were taken acrossrandom regions in the braided sample.
2.1.2.3. Packing Fraction. Packing fraction is the fraction of yarnvolume in a fabric structure that is occupied by a collective bunch offibers. Packing fraction of silk scaffolds was measured as describedelsewhere.18
= ‐
‐ =
packing fraction total cross sectional area of the fibers
/cross sectional area of yarn volume of fiber in yarn
/volume of yarn
2.1.2.4. Twist Per Inch (tpi). For measuring the twist density, theuntwist−retwist method was used on a EY06 type Eureka PrecisionInstrument. Yarn length of 20 cm (n = 5) was used to measure the tpi.
2.1.2.5. Tensile Strength. The tensile tests of A. mylitta braids (n =3) were carried out by a H5KS Tensile Strength Tester materialstesting machine under ambient conditions, i.e., 28 °C and 70 ± 5%relative humidity. The gauge length used was 75 mm with 300 mm/min cross-head speed.
2.1.2.6. Compression. The compression test was performed using aZwickRoell LTM 1000 machine on braids (n = 3) measuring 10 mm ×4 mm. For measuring the silk braid compression, the Essdial ThicknessGauge was used where different levels of load from 50 gf/cm2 to 2000gf/cm2 were applied, and the consequential change in materialthickness was recorded.
2.2. In Vitro Cell Culture. 2.2.1. Cell culture. Human fetalosteoblast cell line (hFOB 1.19 ATCC, CRL-11372) was purchasedfrom Promochem, Bangalore, India. According to ATCC guidelines,hFOBs cultured at 33.5 °C undergo rapid cell division, whereas celldifferentiation is observed at the temperature of 39 °C.13 The cellswere expanded in standard medium comprising Dulbecco’s modifiedEagle’s medium/nutrient mixture F-12 Ham, 1:1 mixture (cat. no.AL187A, Himedia) with 10% FBS (Biological Industries, cat. no. 04-121-1A), and 3 μg/mL Geneticin (cat. no. 10131035, Invitrogen) at33.5 °C in 95% humidity and 5% CO2.
2.2.2. Cell Seeding on Braids. Scaffolds (4 mm × 4 mm × 0.6 mm)were sterilized in 70% ethanol followed by prewetting in completemedium overnight followed by cell seeding with 1.72 × 105 cells percm2 of the braided construct. After 2 days of incubation in standardmedia, half of the cell-seeded constructs were shifted to differentiationmedium. Differentiation media contained standard media with 10 nMdexamethasone (Sigma-Aldrich, USA), 0.01 M β-glycerol phosphate(Sigma-Aldrich, USA), and 50 μg/mL ascorbic acid-2-phosphate(Sigma-Aldrich, USA) to promote cell differentiation. Media werechanged twice a week. All cell culture condition sets were carried outat both 33.5 and 39 °C.
2.3. Analyzing Cell Behavior. 2.3.1. Quantification of CellAttachment on Braids. The braids (n = 3) seeded with 2 × 105
hFOBs were placed in a 24-well plate with wells precoated with 2%poly(2-hydroxyethyl methacrylate) (Sigma, USA, cat. no. P3932) inorder to avoid cell attachment to the wells. Nonattached cells werecollected by centrifugation, stained with trypan blue, and countedusing a Neubauer chamber to determine % adhesion.
2.3.2. Determining Metabolic Activity. The cellular metabolicactivity was determined using the standard MTT assay. After 1 and 7days, hFOB-laden braids [(n = 3), experiment repeated twice] werestained with tetrazolium MTT salt (1:10 ratio in media) at 37 °C for 4h followed by dissolution using dimethyl sulfoxide. The absorbancewas measured at 560 nm, using an iMark microplate absorbance reader(Biorad). Cell-free braids served as a control for this procedure.
2.3.3. Scanning Electron Microscopy. After 7 days, cell-ladenbraids were fixed, dehydrated with gradient alcohol series, gold coated(EMITECH K550X, UK), and visualized using SEM (model EVO 50,Zeiss, UK). To compute mean pore size, 20 pores were measuredacross 3 images per sample using ImageJ software (NIH, USA).
2.4.4. Real Time-Polymerase Chain Reaction (RT-PCR). For theexperiment, the total RNA was isolated at day 1, 7, and 14 from cells
cultured on braids using an RNeasy minikit (Qiagen) as per themanufacturer’s protocol. RNA concentration was determined using aNanodrop 2000C (Thermo Scientific, Wilmington, USA) spectropho-tometer, and cDNA was synthesized using a first strand cDNASynthesis Kit (ThermoScientific, cat. no. K1612). Real timequantitative PCR was conducted using SYBR Green Master Mix(Quantitect, cat no. 204074) and a rotor gene Q thermocycler(Qiagen). QuantiTect primers (Qiagen) used for gene expressionanalysis included alkaline phosphatase (ALP; cat. no. QT00211582),collagen type 1 alpha (COL1A1; cat. no. QT00037793), Runt-relatedtranscription factor (RUNX2; (cat. no. QT00020517), osteonectin(SPARC; cat. no. QT00018620), osteopontin (SPP1; cat. no.QT01008798), osteocalcin (BGLAP; cat. no. QT00232771),podoplanin (cat. no. QT01015084), DMP1 (cat. no. QT00022078),SOST (cat. no. QT00219968), and β-catenin (Cat. No.QT00077882). Glyceraldehyde-3-phosphate-dehydrogenase(GAPDH, cat. no. QT00079247) was used as the house keepinggene. hFOBs cultured in the form of a 2D monolayer served ascontrols. The reactions were set up in triplicate with the wholeexperiment repeated twice.2.4.5. Immunofluorescence Studies. For immunofluorescence
analysis, hFOB-laden braids were harvested at day 7, fixed,permeabilized, and blocked.17 Staining was performed usingantiosteopontin (10 μg/mL, Millipore) followed by secondary stainingwith goat antimouse IgG antibody-FITC conjugate (1:200, Millipore)for 1 h at RT. Actin staining was performed for 30 min at RT usingrhodamine phalloidin (Sigma, cat. no. P1951), followed by Alexafluor546 (cat. no. A11003, Millipore, MA, USA). Nuclear staining wasperformed with DAPI (Sigma-Aldrich, USA, cat. no. 32670). Forimage capturing, a Leica TCS SP5 (Leica Microsystems) invertedconfocal laser scanning microscope was used.2.5. Statistical Analysis. Data are presented as the mean ±
standard deviation, where n is the number of experimental repeatsconducted. To determine statistical significance of data, Student’s t testwas conducted, and probability values in the range of p < 0.05 werenoted as significant.
3. RESULTS3.1. Structural Hierarchy of Braids. The tpi value of the
yarn was carefully optimized to 6.12 in order to achieve thedesired mechanical properties required to withstand braidingand aid in compaction of the braided structure. The physicalcharacterization of the fibers, yarn (Table 1), and braids (Table
2) was performed. Moreover, the braid depicted a flattenedmorphology comprising several layers of hierarchy (Figure 1B).The measured thickness of the total braid was 0.61 mm. Thecriss-cross braid structure (Figure 1C) comprised two ply yarns
(Figure 1D), with 34 yarns intertwined to make a single layer ofthe braided scaffold. Each yarn (two ply) was further made upof 66 fibers: building blocks of the 3D braided architecture(Figure 1E). Quantitative analysis revealed a network ofuniformly oriented multilayer stack of fibers with a thicknessof 21.9 μm ± 0.2 μm each. Further to this, considering thedimensions of a human osteon (223 μm),19 we decided to useA. mylitta fibers in the 3D braided structure with average poresizes close to this value (i.e., 196 μm) as determined with SEMmicrographs. In addition, a constant braiding angle in the rangeof 35−40° with respect to the subsequent layer (Figure 1A)resulted in the compaction of the structure due to the packingof a large number of such fibers which was possibly a majorreason for the resulting lower packing fraction and pore size ofthe braided fabric. Moreover, some silk fibers appeared loose inthe textile braid structure possibly due to the degree of physicalwear during the braid fabrication and/or degumming process(Figure 1B). This made the material surface relatively rough.The surface roughness of yarn as evaluated by AFM (Rq)corresponded to 77 nm (Figure 2). This increased surfaceroughness of the braids due to the interconnected porousstructures aids in the formation of neo-bone tissue, allowingadequate integration of host tissue/cells and channels fornutrient/metabolic waste diffusion.20 Moreover, it is well-known that increasing roughness and stiffness of scaffolds leadsto superior differentiation of osteogenic progenitors.21
Particularly, human bone marrow stem cells differentiatinginto mature osteoblasts on stiffer matrices have beenextensively researched.3 Also, the fact that the braid wasfabricated directly from native silk fibers without the usualdissolution process further contributed toward the increasedstability of the protein chains and resultant rigidity of thebraided construct (Table 2).
3.2. Cell Behavior on Braids. 3.2.1. Initial Cell Attach-ment. Cell counting data revealed that approximately 59000 +23 out of the total 2 × 105 hFOBs seeded were found floating/dead in culture wells on day 1, resulting in 70.5 + 6.7% of cellattachment (Table 3).
3.2.2. Cell Growth Kinetics. Surprisingly, at the 33.5 °Cproliferation temperature of the cell line,13 no significantincrease in metabolic activity of hFOB-laden braids wasobserved (p < 0.05), both with and without differentiationfactors (Figure 3). However, when the temperature was raisedto 39 °C (differentiation temperature),13 the metabolic activityof hFOBs declined by 2.4-fold (p < 0.05) in the absence ofosteogenic differentiation factors, while the value increasedsignificantly following treatment with osteogenic differentiationfactors. This increase in hFOB activity at 39 °C, which has notbeen witnessed earlier, emphasizes the interaction of theosteogenic differentiation factors with the hFOB-seeded A.mylitta braids.
3.2.3. Morphological Analysis. By day 1, a conspicuoussheath of cells along with extracellular matrix components(which starts synthesizing as early as 4 h post-cell seeding)22
covered the majority of the braided surface, encapsulating theunderlying pores and interyarn spaces (Figure 4A), as cellattachment was significantly high (Table 3). On highermagnification (2500×), the individual cells adhered onto thebraids (Figure 4B; white arrow) and attained an elongatedmorphology along the long axis of the fibers (Figure 4C). Thiswas possible as the diameter of the silk fibers was similar to thatof hFOBs and hence ensured proper wrapping of the cellsacross the surface of the fibers in a 3D orientation.
Table 1. Characterization of A. mylitta Two Ply Yarn
After 7 days of incubation at 33.5 °C without the presence ofany differentiation factors (Figure 4D−F), a confluent sheath ofcellular matrix was evident (Figure 4D). In regions where the
cells were directly in contact with the braid, cell morphologyappeared more rounded (Figure 4E, boxed regions), out ofwhich few assumed long dendritic processes resemblingosteocyte-like morphology (Figure 4E, inset) suggesting therole of the A. mylitta surface in accelerating differentiation. Onthe other hand, hFOBs associated with the cell-synthesizedmatrix were more flattened (Figure 4F). In the presence ofosteogenic factors, the cellular matrix was more prominent(Figure 4G). Because of this extensively spread sheath on thebraided surface, most of the cells were entrapped within thematrix region spread across yarns and pores of the braids(Figure 4H and I).
Figure 2. Surface roughness of silk yarn (n = 3) by atomic force microscopy displaying the height sensor, phase, and 3D micrograph.
Table 3. Summary of Quantitation Data from Cell Adhesion
matrixhFOBsseeded
no. offloatingcells
%floatingcells
no. ofattachedcells
%attachment
braid(3D)
2 × 105 5.9 × 104 29.5 1.41 × 105 70.5
Petri dish(2D)
2 × 105 1.6 × 104 8 1.84 × 105 92
Figure 3. MTT of hFOB-laden A. mylitta braids measured at different temperatures. * represents statistically significant data where p < 0.05 and n =3/group.
When cultured at 39 °C, a confluent cell sheath, observed inall other groups, was not evident in hFOB-laden braids culturedin the absence of differentiation factors (Figure 4J), also evidentfrom lower MTT values (Figure 3). Cell morphology washighly irregular and distorted (Figure 4K, asterisk) with tracesof debri/fragments (Figure 4L, arrowheads), which werepossibly an outcome of cellular degradation due to apoptosis.On the contrary, in the presence of differentiation factors, acontinuous sheath of cellular matrix (data not shown) invadedby distinct cellular morphologies later indicated different stagesof osteogenic differentiation (Figure 4M−O): (i) a roundedhFOB with high contrast particle deposition on the surface(asterisk *) indicative of mineral-like deposition (Figure 4M)and (ii) typical osteocyte-like morphology with prominent
dendritic protrusions (Figure 4N and O) spread across multiplefibers (Figure 4O). This coexistence of osteoblasts andosteocytes on A. mylitta matrix may facilitate a bidirectionalcommunication between the two cell types and generateimportant signals which may influence the differentiation ofosteoblasts toward osteocytes;23 however, this is onlyspeculation at this stage and would require a controlled seriesof experimentation to be validated.
3.2.4. Effect of Varying Differentiation Conditions onOsteogenic Gene Expression. The expression of osteogenic-specific markers for hFOBs cultured either on braids (Figure 5)or 2D monolayers (Figure S1) with and without differentiationfactors at 39 °C only. hFOBs cultured at 33.5 °C (which is theproliferative temperature), demonstrated nominal expression of
Figure 4. SEM micrographs of hFOB-laden A. mylitta braids imaged at (A−C) Day 1 and (D−O) Day 7 post-cell seeding. Morphological differencesin cellular morphology and extracellular matrix components were visualized at day 7 in cell-laden braids cultured at 33.5 °C with (G−I) and without(D−F) differentiation factors; 39 °C with (M−O) and without (J−L) differentiation factors. Abbreviations: w/o factors, without osteogenicdifferentiation factors; with factors, osteogenic differentiation factors. Scale bar for inset E = 10 μm.
all differentiation markers throughout the culture period (datanot shown).3.2.4.1. Osteoblast Markers. The expression of ALP in
hFOB-laden braids was increased with time in all theconditions, corresponding to 1.1-fold upregulation with differ-entiation factors and 30.6-fold upregulation without factorsfrom 7 to 14 days (Figure 5). The addition of differentiationfactors rapidly upregulated the expression by 6.4-fold as early asday 1 (p < 0.05). Addition of TGF-β1, drastically down-regulated (p < 0.05) the ALP transcript levels at all time pointsin hFOB-laden braids (Figure 5) as well as the 2D monolayer(Figure S1). Moreover, as expected, the 2D monolayerdemonstrated 1.8-fold upregulation of ALP in the presence ofdifferentiation factors over standard media conditions by day 7;however, the expression was significantly lower (p < 0.05) thanthat of cell-laden 3D braids (Figure S1).The expression of COL1A1, an important extracellular
matrix (ECM) protein of the bone, was significantlyupregulated in hFOB-laden braids by day 7 (p < 0.05) anddeclined by 14 days for both conditions (Figure 5); indicating a
temporal pattern of osteogenic differentiation.24 Negligibleexpression of COL1A1 (≤1 fold change) was detected in thepresence of TGF-β1, both with and without differentiationfactors. For 2D monolayer control (Figure S1), COL1A1transcript levels were nominal for all conditions tested.Similarly, the expression of Runx2, the master gene for bone
differentiation, demonstrated drastic upregulation by day 7 inhFOB-laden braids cultured only in the presence of differ-entiation factors (p < 0.05) and subsequently declined by 62.2-fold by day 14, typically following the pattern of osteogenicdifferentiation.24 A similar pattern of expression was alsoobserved for the 2D monolayer cultured with differentiationfactors (Figure S1); however, the expression levels weresignificantly lower than those in braids. In comparison,hFOB-laden braids cultured without differentiation factorsdepicted significantly low expression of RUNX2 at all timepoints (p < 0.05). Upon addition of TGF-β1, the Runx2transcript levels were found to be significantly downregulatedby day 14 (p < 0.05) for both conditions as compared to groupswhere no TGF-β1 was added.
Figure 5. Gene expression analysis of hFOB-laden A. mylitta braids (n = 3/group) cultured at 39 °C in different experimental conditions up to day14. Abbreviations: w/o, without osteogenic factors/standard media conditions; w/o + TGF, without osteogenic factors, with 10 ng/mL TGF-β1;with, with osteogenic factors; with + TGF, with osteogenic factors, with 10 ng/mL TGF-β1.
The expression of SPARC, a midstage ECM marker, inhFOB-laden braids depicted an expression pattern similar tothat of Runx2, wherein drastic upregulation was observed byday 7 only in the presence of differentiation factors, whichsignificantly declined by day 14 (52.4-fold decrease). Similarly,in the presence of TGF-β1, nominal expression of SPARC wasobserved in both experimental groups in braids (Figure 5) aswell as monolayer (Figure S1) cultures.For SPP1 or osteopontin, a reverse trend could be identified
compared to that of the above groups (Figure 5) withsignificant upregulation of SPP1 expression in hFOB-ladenbraids without differentiation factors at day 7 (3.8 fold) and day14 (4.6 fold) compared to that in differentiation culture media.Moreover, the transcript levels were drastically upregulated (p< 0.05) in the presence of TGF-β1 by day 14. This pattern ofdifferentiation was also observed for monolayer cultures(Figure S1).The expression of BGLAP, indicative of later stages of
osteoblast differentiation involving the deposition of calcifiedmatrix, was also analyzed. While the expression was significantlyupregulated (p < 0.05) in the presence of differentiated factorsby day 7, the transcript levels subsequently declined by day 14.An interesting trend that was observed in this particular genewas the drastically upregulated activity of BGLAP in thepresence of TGF-β1 in both groups; however, the expressionwas maximal in the presence of differentiation factors by day 14(p < 0.05). Similarly, in 2D monolayer culture, the expressionof BGLAP was maximal in the case of differentiation factors(26-fold) and minimal (2-fold) by day 7. The expression was
either similar (differentiation conditions) or upregulated(standard media conditions) in the presence of TGF-β1.
3.2.4.2. Osteocytic Markers. Podoplanin, an early osteocyticmarker, was drastically upregulated in hFOB-laden braidscultured both with (826-fold) and without differentiationfactors (333.1-fold) by day 7, and the subsequent expressiondiffered significantly between the two groups (Figure 5). Whilethe expression significantly declined to 129.8-fold in thepresence of differentiation factors, an upregulation by 5.1times was noticed without differentiation factors by day 14. Theaddition of TGF-β1 triggered the podoplanin expression by 1.5times, albeit only in the hFOB-laden braids cultured withdifferentiation media. In comparison, the 2D monolayer culturedemonstrated negligible expression of podoplanin by day 7suggesting no evidence of transformation to the osteocyticphenotype in monolayer conditions.DMP1, an important regulator of matrix mineralization,
demonstrated maximal expression in the case of hFOB-ladenbraids cultured in the presence of TGF-β1 with (123.6-fold)and without (98.4-fold) differentiation factors as compared totheir non-TGF-β1 counterparts by day 14. hFOB-laden braidscultured in standard culture media without differentiationfactors demonstrated nominal expression throughout theculture period. As DMP1 expresses in the later osteocyticstages of osteoblast to osteocyte transition, its expression wasnot evident in the early days of osteoblast differentiation (i.e.,up to day 7) in all groups. Similar to podoplanin (Figure 5), theexpression of DMP1 was negligible in the 2D monolayerculture.
Figure 6. Gene expression analysis of β-catenin (key regulator of Wnt signaling) demonstrating; (A) dose dependent response of TGF-β1 on cell-laden braids; (B,C) β-catenin expression monitored at the selected concentration of TGF-β1 (10 ng/mL) with respect to braids (B) and 2Dmonolayer (C) cultures. The reactions were set up in triplicate.
SOST, suggesting the terminal osteocytic phenotype, showeda trend similar to that of DMP1 (Figure 5) transcript levels.The hFOB-laden braids cultured without differentiation factorsshowed negligible expression throughout the culture period;however, the expression got significantly (p < 0.05) upregulatedin the presence of TGF-β1. However, in the presence ofdifferentiation factors, the expression levels of SOST were more
or less comparable, indicating no significant contribution of the
presence of the TGF-β1 monolayer culture (Figure S1) showed
negligible expression of this osteocytic marker.That fact that osteocytic expression was only evident in
hFOB-laden braids and not the monolayer culture emphasizes
on the role of the 3D environment and silk surface chemistry
Figure 7. Confocal micrographs of hFOB-laden braids after 7 days: (A) actin (red) and (B) osteopontin (red). Blue represents nuclear staining. Thesilk fibers visible in blue are due to autofluorescence. Scale bars = 50 μm.
for providing essential biomechanical, topographical, andchemical cues for silk matrix-mediated cell signaling.3.2.5. Dose Dependent Response of TGF-β1. A biphasic
response of TGF-β1 was observed on hFOB-laden A. mylittabraids (Figure 6A), as reported earlier.25 The expression of β-catenin showed a steep increase at day 7, albeit only in TGF-β1concentrations ranging from 2 to 6 ng/mL. By day 14, β-catenin expression declined in these groups showing signifi-cantly reduced expression (p < 0.05). However, in concen-trations >6 ng/mL, a slightly different trend was observed. Agradual increase in β-catenin expression was noticed demon-strating 22-fold and 59.7-fold upregulation at 8 ng/mL (Figure6A) and 10 ng/mL (Figure 6B), respectively, after 14 days. Onthe basis of these results, the desired concentration of 10 ng/mL was selected. Controls, consisting of hFOB-laden braidscultured in standard culture media without additional differ-entiation factors or TGF-β1, demonstrated a pattern similar tothat of groups containing lower dosages of TGF-β1 (<6 ng/mL), albeit with lower expression levels (maximal 5.8-foldincrease by day 7) (Figure 6C). As expected, the 2D monolayerculture depicted nominal expression of β-catenin after 7 days(Figure 6C).3.2.5.1. hFOB-Laden A. mylitta Braids Regulate Osteo-
genesis through Wnt/β-Catenin Signaling. As reportedearlier,25 we found that TGF-β1 demonstrated a dosedependent response on the expression of β-catenin (Figure6A; section 3.2.5), hence validating its involvement inregulating Wnt/β-catenin signaling on hFOB-laden braids. Asevident in later stages of differentiation, β-catenin demonstratedmaximal upregulation in TGF-β1 supplemented groups (59.7-and 100.4-fold with and without differentiation factors,respectively) by day 14 (p < 0.05) which would in turn dictatethe dependency of hFOBs to follow osteogenic signaling viaWnt/β-catenin signaling in the presence of TGF-β1. Although,a drastic increase in β-catenin expression was also noted by day7 (121.7-fold) even without TGF-β1 addition, the expressiondecreased by day 14, suggesting the requirement of additionalfactors in promoting the end-term differentiation of cells on A.mylitta braids. In contrast, negligible expression of β-cateninwas observed in the 2D monolayer culture after 14 days (Figure6C).3.2.6. Fluorescence Microscopy. In the absence of differ-
entiation factors, cell morphology was more rounded (Figure7A), and actin stress fibers were not very evident by day 7, asobserved in SEM micrographs (Figure 4J−L). On the contrary,hFOBs cultured in the presence of differentiation factorsdemonstrated extensively elongated stress fibers (Figure 7B,yellow arrows) resembling dendritic processes associated withtransition to the osteocytic phenotype, as also evident in SEM(Figure 4M−O) and RT-PCR (Figure 5). Further, thesynthesis of osteopontin was localized to a majority ofhFOBs with discrete patches of staining evident within thehFOB-laden braids in both differentiation conditions, albeitexpression was relatively higher in the absence of differentiationfactors by day 7 (Figure 7C) as also evident in the geneexpression analysis (Figure 5; SPP1). On the contrary, thesynthesis of osteonectin by day 7 was mostly localized tohFOB-laden braids cultured in the presence of differentiationfactors (Figure 7E and F) similar to gene expression data(Figure 5; SPARC). However, one limitation with theimmunofluorescence of silk is the inherent autofluorescencearising from the aromatic amino acids (tyrosine andtryptophan),26 as seen by the blue staining of silk fibers.
4. DISCUSSION
To our knowledge, this is the first report on the development ofa braided hierarchy, constituted of native (nonregenerated)nonmulberry silk fibers, as a potential scaffold for bone tissueengineering. The braids fabricated in this study mitigate thelack of adequate osteoconductivity associated with regeneratedsilk scaffolds while avoiding the fabrication challengesassociated with most tissue engineered scaffolds, as thedissolution of this variety of silk is a difficult task to achieve.2
These native fibers of nonmulberry silk would be moreosteogenic compared to regenerated silk scaffolds, as theprotein assembly has not been disrupted during manufactur-ing.4 Other reported studies on A. mylitta scaffolds, such as withconventional freeze-drying,2 have reconstituted dissolved silkfibers that unfortunately result in decreased apatite depositiondue to the disrupted protein chains as a result of processingparameters.4,5 Moreover, the method of textile braiding isadvantageous due to its ease of fabrication, customizability, andcost-effectiveness.We extensively investigated the mechanics of this braided
architecture of A. mylitta silk fibers, as mechanical propertiescan act as a potential limiting factor in the context of boneengineering scaffolds, especially because bone is a load-bearingtissue. The suitability of A. mylitta as a potential scaffold forbone partially arises from its exceptional mechanical properties(stiffness of 1.41 MPa) in the range of cancellous bone (0.5−14.6 MPa).27 As reported in our previous study, the rate ofdegradation of A. mylitta silk is incomparably higher than thatof the mulberry varieties (B. mori) both in vitro and in vivo.2
Moreover, the size of the pores, a critical feature ofosteoconductive materials, is comparable to that of a humanosteon (223 μm),19 while still retaining appropriate moduli.The tailored braid angle, which was in the range of 35−40°,also aided in sufficient compaction of the braided structure,hence adding to the mechanical properties. Taken together,these data clearly indicate that the said mechanical properties ofthe braid used here are favorable for implantation studies.Subsequently, we examined important aspects of cell-matrix
interactions between human preosteoblasts and the A. mylittabraids taking into consideration cell adhesion and distributionthat influence subsequent cell behavior, metabolic activity, cellmorphology, gene expression analysis, and protein synthesis.The SEM and AFM results on pore size and surface roughness,respectively, indicated the topographical relevance of the A.mylitta fibers that could enhance cell adhesion, orientation, andinfiltration at different orders of magnitude; fiber assembly isshown at the micrometer level by SEM and surface roughnessas demonstrated by AFM in the order of nanometers. Afteranalyzing the cellular distribution profile on a braided surface,we found that osteoblasts were localized both within the porespaces as well as also being oriented parallel to the longitudinalaxis of the fibers. Overall, from the cell distribution data, it islogical to speculate these two important aspects of boneformation: bone formation occurring from the infiltratedosteoblasts within pores which can regenerate bone from thescaffold’s interior, as well as cellular alignment along the fibersas they orient along the periphery of fibers forming bone fromoutside. However, this hypothesis warrants in vivo investigationsto provide a clearer picture on the mechanism of boneformation as a function of braided geometry.Moreover, the data demonstrate relatively high cell adhesion
(lower than that in the 2D Petri dish) and orientation of seeded
osteoblasts in compliance with the surface chemistry and braidmorphology. However, the values depicted for cell attachment(Table 3) include both initial adhesion of hFOBs on braids aswell as proliferation up to 1 day; nevertheless, the fact thatnative silk fibroin consisting of a specific sequence of aminoacids leads to enhanced cellular responses, along with otherfactors including surface roughness, rigidity, surface chemistry,or all of these combined, remains true. Another interestingrevelation made by a recent study was that mechanical stiffnessplays a crucial role in determining the fate of osteoprogeni-tors.23 While softer matrices (0.58 kPa) facilitated osteocyticdifferentiation of seeded MC3T3-E1 progenitors on gelatin-based 3D hydrogels, stiffer matrices (1.47 kPa), on thecontrary, retained the osteoblastic phenotype until 56 days inculture. However, it will be difficult to compare results as thematerial and cell line used are entirely different.The gene expression data clearly demonstrate that A. mylitta
braids are innately osteoconductive and enable rapid differ-entiation of cultured preosteoblasts to mature osteocytes within2 weeks. This was also evident from the extensively elongateddentritic cellular extensions as shown by SEM and confocalimaging data, a characteristic feature of the mature osteocyticphenotype. The Ct (copy number) values reported arenormalized to the expression in the 2D monolayer, culturedunder similar conditions. As expected, gene expression of cell-laden braids cultured in the presence of pro-osteogenic factorsincreased, compared to that in A. mylitta braids alone or in the2D monolayer. Interestingly, transient expression of osteocyticdifferentiation was evident in cell-laden A. mylitta braids alonedespite the absence of pro-osteogenic differentiation con-ditions. The observation that A. mylitta structures correlateclosely with previously reported in vivo studies where 3Dporous scaffolds of A. mylitta successfully regenerated criticalsized defects of rat calvariae,2 an effect which might be moreenhanced in the natively spun braids, demonstrates theosteogenic ability of the material in directing cellular differ-entiation since no differentiation was evident in 2D monolayercultures. Moreover, significant changes in the gene expressionprofile of cell-laden A. mylitta braids following supplementationwith TGF-β1 further produced a series of questions about therole of underlying signaling mechanisms involved. Note that tobe able to identify the exact signaling pathway involved, the cellbehavior on braids was extensively analyzed under variedexperimental parameters including temperature, pro-osteogenicfactors, and the presence or absence of nonmulberry braids (2Dvs 3D), which is discussed in detail below.4.1. Wnt/β-Catenin Signaling in Regulating Mineral-
ized Bone Differentiation on A. mylitta Braids. Aninteresting observation was the rapid transition of hFOBs onA. mylitta braids into the osteocytic phenotype within 2 weeksof culture, a phenomenon not yet reported on silk-basedmatrices. While the A. mylitta braids alone (without osteogenicdifferentiation factors) facilitated the transition of hFOBstoward osteocytic morphology, this was only transient. Clearly,in the absence of osteogenic factors, while early to midosteoblast differentiation in hFOB-laden braids (ALP, COLA1,and SPP1) increased with time, the transition towardmineralized osteoblasts (osteocalcin) was highly suppressedor suboptimal. While by passing this critical stage ofmineralization responsible for calcified bone formation in situ,the cultured hFOBs expressed drastically upregulated levels ofosteocytic markers within 7−14 days of culture suggesting rapidtransition of hFOBs into osteocytic cells, a phenomenon not
yet reported for hFOBs even under extended culture periods.14
While the early osteocyte gene continued to increase(podoplanin), we found a nominal expression of Dmp1(which participates in the process of matrix mineralization)28
at all time points studied. A possible explanation could be thereduced production of associated cytokines/growth factorsinvolved in mineralization, which was also validated from thenominal osteocalcin expression. However, within the commer-cial setup, this rapid transition of preosteoblasts to transitoryosteocytes may not serve as an ideal model of bonedifferentiation. Therefore, standard osteogenic reagents (dexa-methasone, ascorbic acid, and β-glycerophosphate) were addedto improve the extent of osteogenesis in cell-laden braids. Theaddition of factors convincingly upregulated the expression ofosteogenic markers over A. mylitta braids alone.Another major reason for the suppressed activity of
mineralized bone apatite could be the upregulated expressionof Sost gene. It is known that sclerostin, secreted by matureosteocytes, binds to Lrp5 to antagonize the action of Wnthence inhibiting Wnt/β-catenin signaling.29,30 Literaturesuggests that deletion of the Sost gene in mice resulted inincreased bone mass and strength,31 whereas overexpression ofSOST demonstrated lower bone mass.32 Similarly, loss ofSOST expression in humans is often associated with bonedisorders associated with high bone mass such as Van Buchem’sdisease33 and sclerosteosis.34 Hence, it was logical to believethat osteocyte secreted SOST caused the inhibition ofmineralized bone tissue by blocking Wnt/Lrp signaling inosteoblasts.28,29 This mechanism resulted in the lack of apatitedeposition on nonmulberry silk braids.Under physiological conditions of bone differentiation,
accumulation of β-catenin starts in the cytoplasm subsequentlytranslocating into the cell nucleus wherein it interacts with theTcf/Lef family of transcription factors. This interaction furtherregulates the activation of several important genes associatedwith proliferation, differentiation, and apoptosis of osteogeniccells.35 Therefore, to examine if this arrest of osteoblastdifferentiation observed at the later stages is possibly due tocompromised Wnt/β-catenin signaling,36,37 we supplementedthe cultures with TGF-β1. It is well-known that activation ofthe Wnt/β-catenin signaling pathway results in a decrease ofapoptosis in osteogenic cells.35 In this context, the firstindicator for the enhanced activity of Wnt/β-catenin signalingin the case of TGF-β1 supplemented braids was the increasedmetabolic activity found in hFOB-laden braids even whencultured at 39 °C, a restrictive temperature of the cell line.hFOB-laden braids cultured in TGF-β1 were found to restorethe antagonizing action of SOST, thus validating the directinvolvement of Wnt/β-catenin signaling in hFOBs cultured onnonmulberry silk braids. The expression of both mineralizationassociated genes, BGLAP and DMP1, was found to be restoredsuggesting a natural process of bone differentiation. Moreover,the significant relationship between SOST and β-catenin wasestablished by the reciprocal levels of expression observed inhFOB-laden braids cultured without any differentiation factors.With a 8.5-fold decline in the SOST expression level, thecorresponding increase in β-catenin was roughly 23-fold after14 days in culture. However, the addition of TGF-β1successfully upregulated the expression of β-catenin, hencetriggering Wnt/β-catenin signaling in regulating osteogenesis inhFOB-laden A. mylitta braids in vitro, with increased SOSTexpression suggesting a more stable phenotypic transition.
Furthermore, it is often contemplated that a cross-talk withthe prostaglandin pathway activates the Wnt/β-catenin pathwayin osteocytes under mechanical stimulus subsequently resultingin the downregulation of Sost.35 Since no mechanical load wasapplied to the cell-laden braids in our case, the SOST levelswere constantly upregulated while simultaneously inhibitingmineralized bone differentiation. The compelling evidenceindicates that supplementation of TGF-β1 increases mineralbone in hFOB-laden A. mylitta braids in vitro whilesimultaneously upregulating SOST to ensure the transition ofpreosteoblasts into phenotypically stable osteocytes within 2weeks of the culture period.Though beyond the scope of this study, extensive research
may be needed in determining how osteocytes perceive andenable signal transduction in response to such fibroushierarchies. Moreover, mediation of the physical attributes ofthe textile braid such as the number of fibers per yarn, thicknessof yarns, pore size, braid angle, rigidity as well as surfacechemistry (comparison of different silk species) and theirimpact on cellular differentiation may provide a morecomprehensive overview for controlling osteocyte differ-entiation for long-term sustainable cultures. Also, applicationof a mechanical stimulus and/or a bioreactor system forfacilitating proper nutrient diffusion and cellular infiltrationunder load bearing conditions will aid in providing abiequivalent model replicating the in vivo microenvironmentmore closely. Furthermore, the coexistence of osteoblasts andosteocytes, as observed in A. mylitta braids cultured withosteogenic factors, their relevance and cross-talk in facilitatingcell−cell communication for osteogenic differentiation needs tobe established. As evident, targeting the Wnt/β-cateninpathway because of its critical role in controlling osteocyte-related functions, i.e., regulation of bone mass strength, holdsdefinitive potential for developing new paradigms andtherapeutic applications for treating bone diseases in the nearfuture.
5. CONCLUSION
Recently, much attention is being given to osteocyte-relatedbiology as its significance in regulating the structural andfunctional mechanisms of bone hierarchy is being realized.Having said that, there still remain several obstacles which needto be addressed to fully untap the potential of this field ofresearch. In conclusion, hFOB-laden A. mylitta braids display allof the characteristics of an in vitro 3D osteocytic modelincluding the extensive dendritic processes, lacunocanalicularstructure, distinct genetic expression, and responses to growthfactors (TGF-β1), all indicating the development of amineralized bone-like microenvironment. These cells on A.mylitta braids alone rapidly induce the differentiation ofpreosteoblasts into osteocytes, albeit this transition was onlytransient. The additional of osteogenic differentiation factorssignificantly improved the expression of osteogenic-relatedmarkers marking a more discrete process of bone differ-entiation. However, the combinatorial effect of osteogenicfactors with TGF-β1 and the nonmulberry braided matrixresulted in completely establishing the process of osteogenicdifferentiation from preosteoblasts to terminal osteocytesexpressing functional sclerostin regulated via Wnt/β-cateninsignaling. This makes A. mylitta braided constructs an idealmodel system for studying in vitro bone differentiation.
■ ASSOCIATED CONTENT*S Supporting InformationThe Supporting Information is available free of charge on theACS Publications website at DOI: 10.1021/acsbiomater-ials.7b00006.
ORCIDSourabh Ghosh: 0000-0002-1091-9614NotesThe authors declare no competing financial interest.
■ ACKNOWLEDGMENTSThis study was funded by Department of Science andTechnology, Science and Engineering Board, India (YSS/2014/000472) and Department of Biotechnology, India (BT/PR8038/MED/32/303/2013).
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