silk fibroin nanofibrous scaffolds for bone tissue engineering.

Journal of Biomaterials Applications http://jba.sagepub.com/

Precipitation of hydroxyapatite on electrospun polycaprolactone/aloe vera/silk fibroin nanofibrous scaffolds for bone tissue engineering Suganya Shanmugavel, Venugopal Jayarama Reddy, Seeram Ramakrishna, BS Lakshmi and VR Giri Dev J Biomater Appl published online 27 November 2013 DOI: 10.1177/0885328213513934 The online version of this article can be found at: http://jba.sagepub.com/content/early/2013/11/27/0885328213513934

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Original Article

Precipitation of hydroxyapatite on electrospun polycaprolactone/aloe vera/silk fibroin nanofibrous scaffolds for bone tissue engineering

Journal of Biomaterials Applications 0(0) 1–13 ! The Author(s) 2013 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0885328213513934 jba.sagepub.com

Suganya Shanmugavel1,2, Venugopal Jayarama Reddy3, Seeram Ramakrishna3, BS Lakshmi2 and VR Giri Dev1

Abstract Advances in electrospun nanofibres with bioactive materials have enhanced the scope of fabricating biomimetic scaffolds for tissue engineering. The present research focuses on fabrication of polycaprolactone/aloe vera/silk fibroin nanofibrous scaffolds by electrospinning followed by hydroxyapatite deposition by calcium-phosphate dipping method for bone tissue engineering. Morphology, composition, hydrophilicity and mechanical properties of polycaprolactone/aloe vera/silk fibroin–hydroxyapatite nanofibrous scaffolds along with controls polycaprolactone and polycaprolactone/aloe vera/silk fibroin nanofibrous scaffolds were examined by field emission scanning electron microscopy, Fourier transform infrared spectroscopy, contact angle and tensile tests, respectively. Adipose-derived stem cells cultured on polycaprolactone/aloe vera/silk fibroin–hydroxyapatite nanofibrous scaffolds displayed highest cell proliferation, increased osteogenic markers expression (alkaline phosphatase and osteocalcin), osteogenic differentiation and increased mineralization in comparison with polycaprolactone control. The obtained results indicate that polycaprolactone/aloe vera/silk fibroin–hydroxyapatite nanofibrous scaffolds have appropriate physico-chemical and biological properties to be used as biomimetic scaffolds for bone tissue regeneration. Keywords Aloe vera, silk fibroin, hydroxyapatite, electrospinning, bone tissue engineering

Introduction Electrospinning is a multipurpose technique that offers possible approach to produce continuous nano and micro-dimensional polymeric fibres with large surface areas making this technique very attractive for diverse applications. The major application of this technique in tissue regeneration is co-spinning of polymers with bioactive materials to create biomimetic scaffolds with suitable physical and biological environment to promote cell growth and new tissue formation.1–4 For bone tissue engineering, scaffold should be biocompatible, osteoconductive and osteoinductive and have appropriate structural and mechanical properties.5 Synthetic polymers such as poly(lactic acid), poly(glycolic acid), polycaprolactone (PCL) and natural polymers such as chitosan, gelatin, collagen, fibrin and hyaluronic acid have been enormously used for soft tissue regeneration and as drug-delivery systems,6

but their application in hard tissue surgery has raised several problems such as inflammatory reaction. This drawback can be significantly overcome by the incorporation of native biomaterials, such as hydroxyapatite (HA). HA is the major mineral constituent of the bone matrix and the most widely used therapeutic material. This bioceramic has showed excellent biocompatibility with bones and teeth because of its bioactivity and osteoconductivity and are widely used as bone

1

Department of Textile Technology, Anna University, Chennai, India Centre for Biotechnology, Anna University, Chennai, India 3 Nanoscience and Nanotechnology Initiative, National University of Singapore, Singapore 2

Corresponding author: VR Giri Dev, Department of Textile Technology, A.C. Tech, Anna University, Chennai 600025, Tamilnadu, India. Email: [email protected]



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substitutes and tissue engineering scaffolds.7 The major disadvantage with synthetic HA is its brittle nature, and this has limited its applications to non–load-bearing parts in the form of powders, granules or as a coating material.8 One promising strategy to overcome its brittleness is to introduce bioactive materials that fulfil the mechanical and biological properties that the tissues require. Silk fibroin (SF) is a natural protein isolated from silkworm (Bombyx mori) containing two main proteins: sericin (outer covering) and fibroin (central structure). Fibroin does not induce immune rejection unlike other bio-derived proteins and hence being focused for biomedical applications. Additionally, SF materials offer impressive mechanical properties, biodegradability, biocompatibility and versatility in processing for biomedical applications. Research findings have shown that fibroin-based scaffolds mimic the extracellular matrix and efficiently support cell attachment and proliferation.9–11 Besides possessing biocompatibility, osteoconductivity and appropriate structural and mechanical properties, the scaffolding materials for bone tissue engineering should also be osteoinductive, which is capable of initiating osteogenic differentiation. Aloe vera (AV) is one of the most commonly used medicinal herbs. The major component contained in AV is the polysaccharide acemannan. Recent studies demonstrated that acemannan has stimulating therapeutic action on both soft and hard tissue healing.12–14 Research finding showed that acemannan-treated rats significantly increased cell proliferation and differentiation of bone marrow stromal cells with increased alkaline phosphatase activity and mineralization as compared to untreated control rats.15 The result proves AV to be osteoinductive in bringing osteogenesis. Stem cells in addition to biomimetic scaffolds are required in bringing tissue regeneration. Adipose tissues are abundant source of adult multipotent stem cells capable of undergoing multilineage differentiation. Adipose-derived stem cells (ADSC) have similar surface immunophenotype and osteogenic differentiation like bone marrow-derived stem cells. Abundance, easy access and lower cytotoxicity make ADSC a better alternative to bone marrow stem cells, in conditions where large number of cells is required for regeneration.16–19 The current study focuses on the fabrication of nanofibrous scaffold using PCL, a biodegradable and excellent electrospinnable polymer,20 with incorporation of natural biomaterials AV for osteoinduction and SF for mechanical support with surfacemineralized HA to prevent immune response, and is investigated in terms of the initial cell adhesion, proliferation and further osteogenic differentiation and mineralization of ADSC cells for bone tissue engineering.

Materials and methods Materials ADSCs were obtained from Lonza, USA. Dulbecco’s modified Eagle’s medium (DMEM)/Nutrient Mixture F-12 (HAM), fetal bovine serum (FBS), antibiotics and trypsin–EDTA were purchased from GIBCO Invitrogen, USA. CellTiter 96Õ Aqueous one solution was purchased from Promega, Madison, WI, USA. PCL, 1,1,1,3,3,3-hexafluoro-2-propanol (HFP), CaCl2, Na2HPO4, Alizarin Red-S (ARS) and cetylpyridinium chloride were purchased from Sigma. Lyophilized AV and SF powder were purchased from Xia`n Yuensun Biological Technology Co., Ltd, China.

Fabrication of nanofibrous scaffolds PCL pellets were dissolved in HFP at 10% (w/v), and PCL–AV–SF solution was prepared at the ratio of 8:4:4 (w/w) at the concentration of 16% in HFP. The concentration was fixed based on various trials, and the concentration that produces bead-free fibres was used for the study. The solutions were magnetically stirred at room temperature overnight for better distribution and homogenization. The polymer solution was then loaded into a 3-ml standard syringe attached to 21G blunt needle using a syringe pump (KD 100 Scientific Inc., Holliston, MA, USA) at a constant flow rate of 1 ml/h with a voltage electric field of 17 kV (DC highvoltage power supply from Gamma high-voltage research, Florida, USA). The electrospun nanofibres were collected on aluminium foil wrapped on a flat collector plate kept at a distance of 10 cm between the tip of the spinneret and collector plate for physico-chemical analysis. Nanofibres were collected on 15-mm coverslips for cell-culture experiments. Fabricated electrospun nanofibrous scaffolds were consequently dried overnight under vacuum oven to eliminate residual solvents. Biomineralization procedure was then carried out on the PCL–AV–SF samples to precipitate HA by calcium-phosphate dipping method. The electrospun PCL–AV–SF nanofibres were initially immersed in 0.5 M CaCl2 solution for 10 min. The samples were then rinsed in DI water for 1 min. The samples were then immersed in 0.3 M of Na2HPO4 for 10 min and rinsed for 1 min in DI water. This entire procedure was considered as one cycle. The scaffolds were subjected to three cycles of the above treatment. The first cycle was for 10 min, and the subsequent cycles were for 5 min in each solution. After biomineralization, the scaffolds were freeze-dried overnight for further studies.



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Characterization of nanofibrous scaffolds The electrospun nanofibres were sputter-coated with gold (JEOL JFC-1200 Fine Coater, Japan) and visualized using a field emission scanning electron microscope (FESEM) (JEOL JSM 6700, Japan). Diameters of the electrospun fibres were analysed from the SEM images using image analysis software (Image J, National Institutes of Health, USA). Tensile properties of electrospun nanofibrous scaffolds were determined with a tabletop tensile tester (Instron 3345, USA) using load cell of 10 N capacities. Rectangular specimens of dimensions 10 mm 20 mm were used for testing, at a crosshead speed of 10 mm/min, and the data was recorded for every 50 ms. Tensile stress strain and elastic modulus were calculated based on the generated tensile stress–strain curve. Hydrophilic nature of the electrospun nanofibrous scaffolds was measured by sessile drop water contact angle measurement using a VCA Optima Surface Analysis system (AST products, Billerica, MA). Distilled water was used for drop formation. The measured contact angle value reflected the hydrophilicity of the scaffolds. FTIR spectroscopic analysis of electrospun nanofibrous scaffolds was performed on Avatar 380 (Thermo Nicolet, Waltham, MA, USA) over a range of 500–4000 cm1 at a resolution of 2 cm1.

Cell culture ADSCs were cultured in DMEM containing 10% FBS with 1% antibiotics in a 75-cm2 cell-culture flask. The ADSCs were incubated at 37 C humidified atmosphere containing 5% CO2 for 7 days, and the culture medium was changed once in every 3 days. The cultured cells (passage 4) were trypsinized by trypsin–EDTA and replated after counting with trypan blue using hemocytometre. The electrospun nanofibres that were collected on coverslips of 15 mm diameter were placed in a 24 well plate with a stainless steel ring to prevent lifting of nanofibres. The fibres were sterilized under UV light for 3 h, and the scaffolds were again sterilized with 70% ethanol for 30 min and washed thrice with phosphatebuffered saline (PBS) for 15 min each in order to remove any residual solvent and subsequently immersed in complete medium overnight before cell seeding. The ADSCs were seeded on the electrospun nanofibres at a cell density of 10,000 cells/well, and tissue culture polystyrene (TCP) was used as a control.

Cell proliferation The cell proliferation was determined using the colorimetric MTS assay (CellTiter 96 Aqueous one solution, Promega). The principle of this assay is that the

reduction of yellow tetrazolium salt [3-(4,5dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2 (4 sulfophenyl)-2H tetrazolium] in MTS to form purple formazan crystals by dehydrogenase enzymes is secreted by mitochondria of metabolically active cells. The formazan dye shows the absorbance at 490 nm, and the quantity of formazan crystals produced is directly proportional to the number of live cells. After culturing the cells for a period of 7, 14 and 21 days, the media were removed from the 24 well plates, and the scaffolds were washed with PBS to remove unattached cells. The scaffolds were then incubated in a 1:5 ratio mixture of MTS reagent in serum-free DMEM medium for 3 h at 37 C in 5% CO2 incubator. Thereafter, the samples were pipetted into 96 well plates, and the absorbance was measured at 490 nm using a microplate reader (Fluostar optima, BMG Lab Technologies, Germany).

Cell morphology The cell morphology was analysed using FESEM (Hitachi S4300, FESEM). After 21 days of seeding cells on the scaffolds, the media was removed from culture wells, the cell scaffolds were rinsed twice with PBS for 15 min to remove non-adherent cell, and then the samples were fixed with 3% glutaraldehyde in PBS for 3 h at room temperature. The scaffolds were again rinsed in distilled water for 15 min and then dehydrated with increasing concentrations of ethanol (30%, 50%, 70%, 90% and 100% v/v) for 10 min. Finally, the cell scaffolds were treated with hexamethyldisilazane (Sigma) solution and air-dried in fume hood overnight. Dried cellular constructs were coated with gold in an automatic sputter coater, and the cell morphology and biomineralization were observed under FESEM at an accelerating voltage of 10 kV.

Alkaline phosphatase activity Alkaline phosphatase (ALP) activity of ADSCs differentiation into osteogenic lineages was measured using Alkaline Phosphate Yellow Liquid substrate system for ELISA (Sigma Life Sciences, USA). In this reaction, ALP catalyses the hydrolysis of a colourless organic phosphate ester substrate, p-nitrophenylphosphate (pNPP), to a yellow product, p-nitrophenol and phosphate. After seeding the cells for a period of 7, 14 and 21 days, the media was removed from 24-well plates, and the nanofibrous scaffolds with osteogenic cells were washed twice with PBS, and 400 mL pNPP in liquid form was added to cells on the scaffolds and incubated for 30 min until the colour of solution became yellow. The reaction was stopped by the addition of 100 mL of 2 N NaOH solution following which the yellow colour



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product was aliquoted in 96 well plates and read in spectrophotometric plate reader at 405 nm.

Mineralization of osteogenic cells ARS is a dye that binds selectively calcium salts and is widely used for mineral staining. On day 14, the scaffolds seeded with ADSCs were washed three times in PBS and fixed in 70% ice cold ethanol for 1 h. These constructs were washed three times with distilled water and stained with ARS (40 mM) for 20 min. After several washes with distilled water, the scaffolds were observed under inverted optical microscope, and images were taken using Leica FW 4000 (version V 1.0.2). The stain was eluted by incubating the scaffolds with 10% cetylpyridinium chloride for 1 h. The dye was collected and absorbance read at 540 nm in spectrophotometer (Thermo Spectronics, Waltham, MA, USA).

Immunofluorescent staining for osteocalcin Osteogenic differentiation of ADSCs was confirmed using immunofluorescent staining by employing both ADSCs-specific marker protein CD 105 and osteoblast-specific marker protein osteocalcin (OCN). The cells were primarily fixed with 100% ice-cold methanol for 15 min. After fixing, the scaffolds were washed with PBS once for 15 min and incubated in 0.5% Triton X-100 solution in PBS for 5 min to permeabilize the cell membrane. Non-specific sites were blocked by incubating the cells in 3% bovine serum albumin for 1 h. Primary antibody ADSCs-specific marker protein CD 105 (Abcam, USA) was added in the dilution (1:100) for 90 min at room temperature. This was followed by adding secondary antibody Alexa Fluor 594 (Invitrogen-Red) in the dilution 1:250 for 60 min. The scaffolds were washed thrice with PBS and then incubated with osteoblast-specific protein OCN (Sigma) in the dilution 1:100 for 90 min. Further secondary antibody Alexa Fluor 488 (Invitrogen-Green) was added in the dilution 1:250 for 60 min. The scaffolds were washed thrice with PBS for 15 min to remove the excess staining. Finally, the cells were incubated with 4,6,diamino-2 phenyl indole (Invitrogen Corp, Carlsbad, CA) in the dilution 1:5000 for 30 min. The samples were removed from 24 well plates and mounted over glass slide using vectashield mounting medium and examined under the fluorescence microscope (Olympus FV 1000).

Statistical analysis The data presented are expressed as mean standard deviation. Statistical analysis was done using Student’s t-test, and the significance level of the data was

obtained. The p-value 0.05 was considered to be statistically significant.

Results and discussion Characterization of nanofibrous scaffolds Nanostructured scaffolds are favourable platforms to recapitulate the organization of natural extracellular matrix for functional bone tissues formation, because nanotopography provides a closer approximation to native bone architecture.21 Polymeric fibres developed should be in nano dimensions for providing favourable structural support in the formation of functional tissues. Figure 1(a) to (c) shows the FESEM micrographs of PCL, PCL–AV–SF and PCL–AV–SF–HA nanofibrous scaffolds. The average fibre diameters of PCL and PCL–AV–SF nanofibres calculated using ImageJ analysis software were in the range of 515 19 and 133 28 nm. PCL–AV–SF–HA nanofibrous scaffolds showed densely deposited HA nanoparticles on their surface with a mean fibre diameter of 133 28 nm imparting a rough texture to the scaffolds. Research finding reported that the initial osteoblast attachment and mineralization are favourably enhanced by the rough surface provided by HA.22 The water contact angle parameter of PCL, PCL–AV–SF and PCL–AV– SF–HA nanofibrous scaffolds was analysed and was found that PCL nanofibres were hydrophobic having a contact angle of 133.00 . The contact angles obtained for PCL–AV–SF and PCL–AV–SF–HA were 51.00 and 26.90 , respectively. The wettability nature of HA and water absorption property of AV has imparted hydrophilicity to the hydrophobic polymeric scaffold, thereby improving their cell-adhesion property.23,24 Figure 2 shows the nonlinear stress strain graph of PCL, PCL–AV–SF and PCL–AV–SF–HA nanofibrous scaffolds. Tensile stress for PCL, PCL–AV–SF, and PCL–AV–SF–HA nanofibrous scaffolds was 1.54 MPa, 4.80 MPa and 10.89 MPa and can bear a strain of 71%, 67%, and 70%, respectively. The Young’s modulus value for PCL, PCL–AV–SF, and PCL–AV–SF–HA nanofibrous scaffolds was 13.67 MPa, 14.60 MPa and 23.49 MPa. Previous studies showed that beta structure of SF provides outstanding mechanical property, which, in combination with HA, gives excellent compressive modulus.25 The obtained result with increased tensile stress and tensile modulus values for PCL–AV–SF–HA shows synergistic effect of SF and HA, which in turn increases the rigidity of the scaffolds, which is very important for clinically useful scaffolds that retain their shape during implantation and ideally bear mechanical loads after implantation especially for bone tissue engineering.26 The values obtained for nanofibres diameter, contact angle and



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Figure 1. FESEM micrographs of (a) PCL, (b) PCL–AV–SF and (c) PCL–AV–SF–HA nanofibrous scaffolds. PCL–AV–SF–HA nanofibrous scaffolds show densely deposited HA on their surface.

tensile stress, strain and modulus for PCL, PCL–AV– SF and PCL–AV–SF–HA scaffolds were tabulated in Table 1. The FTIR spectra of PCL, PCL–AV–SF and PCL– AV–SF–HA scaffolds are shown in Figure 3(a) to (c). The characteristic peaks of PCL namely C ¼ O vibration at 1728 cm1, CH2 band vibrations at 1362, 1407 and 1465 cm1, esteric COO vibrations at 1181 and 1238 cm1, O-C vibrations at 1099, 1047 and 961 cm1 and CH2 rocking vibration occurring at 729 cm1 are clearly seen in the spectrum (Figure 3(a)). The FTIR spectrum of PCL–AV–SF scaffolds is shown in Figure 3(b). The characteristic peak of functional groups of components present in AV namely O-acetyl esters, glucan units, pyranoside ring and mannose is at 1736 cm1, 1032 cm1, 879 cm1 and 811 cm1, respectively. Similarly, the secondary structure of SF consists of the major conformations including random coils and b-sheet. Peaks for random coil of Amide I, Amide II and Amide III are seen at around 1650 cm1, 1540 cm1 and 1230 cm1, respectively. Peaks for b-sheet for Amide I, Amide II and Amide III are seen at around 1620 cm1, 1520 cm1

and 1270 cm1, respectively. These characteristic peaks of AV and SF are seen in Figure 3(b) but slightly broadened due to blending confirming the successful incorporation of AV and SF to the polymeric base. The spectrum (Figure 3(c)) of PCL–AV–SF–HA shows the features of spectrum (Figure 3(b)) and additionally characteristic peaks of HA namely stretching vibration for PO43 at 1053 cm1, P-O stretch coupled P-O bend peaks at 573 cm1 and CO32 group at 1400 cm1 were obtained similar to bone composition. Thus, the physicochemical properties of PCL–AV–SF– HA show desirable properties in terms of morphology, chemical, hydrophilicity and tensile strength, and therefore expected to have better cell adhesion, proliferation and mineralization on this scaffolds for bone tissue engineering.

Interaction of cells and scaffolds Initial interactions between cells and scaffolds are an important phenomenon in bringing enhanced proliferations, which mainly depend on the physical and chemical composition of the scaffolds.27 The ability for




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PCL PCL-AV-SF

10 PCL-AV-SF-HA

Tensile stress (MPa)

8

6

4

2

0 0

15

30

45

60

75

Tensile strain (%)

Figure 2. Tensile stress–strain curves of PCL, PCL–AV–SF and PCL–AV–SF–HA nanofibrous scaffolds.

Table 1. Characterization of electrospun nanofibres for fibre diameter, water contact angle and mechanical properties. Nanofibre construct

Fibre diameter (nm)

Water contact angle ( )

Tensile strength (MPa)

Tensile break (%)

Young’s modulus (MPa)

PCL PCL–AV–SF PCL–AV–SF–HA

515 19 133 28 133 28

133.00 51.00 26.90

1.54 4.80 10.89

71 67 70

13.67 14.60 23.49

nanofibrous scaffolds to support adhesion and proliferation of ADSCs on TCP, PCL, PCL–AV–SF and PCL–AV–SF–HA nanofibrous scaffolds on days 7, 14 and 21 was determined by MTS assay as shown in Figure 4. The proliferation rate of ADSCs on PCL– AV–SF–HA nanofibrous scaffolds increased by 26.58% (p 0.001) on day 14 and about 59.19% (p 0.001) on day 21 compared to PCL nanofibrous scaffolds. This is because of the presence of bioactive AV, SF, and HA in PCL–AV–SF–HA nanofibrous scaffolds, which impart favourable factors for efficient cell adhesion. SF provides integrin recognition sequence on their surface favourable for osteoblast cell adhesion and proliferation.28 The hydrophilic

nature imparted by AV and rough surface topography provided by HA favoured initial cell adhesion29,30 and stimulated increased proliferation in PCL–AV–SF–HA nanofibrous scaffolds compared to PCL scaffold. Similarly, PCL–AV–SF–HA nanofibrous scaffolds favoured cell proliferation, which almost increased linearly by 10.46% (p 0.01) and 12.01% (p 0.001) compared to PCL–AV–SF nanofibrous scaffolds on days 7 and 14 due to the presence of precipitated HA on the surface of PCL–AV–SF–HA nanofibrous scaffold, which acts as osteoconductive medium and favours ingrowth of cells, thereby inducing cell migration, adhesion and growth. This result demonstrates that the unique physical and chemical composition of




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7

(a)

120

Transmittance (%)

100

729 cm–1

(b) 80 1736 cm–1 1032 cm–1

60 (c)

879 cm–1

811 cm–1

40

1053 cm–1

20 1650 cm–1

573 cm–1

1540 cm–1

0 4000

3500

3500

2500

2000

1500

1000

500

Wavenumber cm–1

Figure 3. FTIR spectroscopic analysis of (a) PCL, (b) PCL–AV–SF and (c) PCL–AV–SF–HA nanofibrous scaffolds showing characteristic peaks of PCL, AV, SF and HA.

2500 TCP

PCL

PCL-AV-SF

**

PCL-AV-SF-HA

** **

2000

Absorbance Index

* 1500

1000

500

0 7

14 Days

21

Figure 4. Cell proliferation study of ADSCs on TCP, PCL, PCL–AV–SF and PCL–AV–SF–HA nanofibrous scaffolds on days 7, 14 and 21. * indicates significant difference of p 0.01; ** indicates significant difference of p 0.001.



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Figure 5. FESEM images showing the cell-scaffold interactions on day 21 on (a) TCP, (b) PCL, (c) PCL–AV–SF and (d) PCLAV–SF–HA nanofibrous scaffolds at 5000 magnification. PCL–AV–SF–HA nanofibrous scaffolds show densely synthesized mineral on their surface.

PCL–AV–SF–HA nanofibrous scaffolds favoured the cell adhesion and proliferation compared to other scaffolds. The FESEM images of ADSCs (Figure 5(a) to (d)) showed the cell morphology on TCP, PCL, PCL–AV– SF and PCL–AV–SF–HA nanofibrous scaffolds with the secretion of minerals on the surface of cells on day 21. The initial adhesion, migration and proliferation of cells on the nanofibrous scaffolds were consequently followed by differentiation and mineralization. The trigger for osteogenic differentiation of ADSCs followed by mineralization is the bony environment.31 It was seen that cells grown on PCL–AV–SF–HA nanofibrous scaffolds showed densely synthesized mineral on their surface, which is due to the presence of HA, which provided bone-like environment for the mineralization of osteoblasts for bone regeneration. The obtained result clearly proves the ADSC differentiation followed by mineralization process. Precipitated HA is responsible for forming dense layer of minerals on the surface of PCL–AV–SF–HA nanofibrous scaffolds as clearly seen in Figure 5(d) compared to other scaffolds proving it to be a potential scaffold for conditions where there is

reduction in mineralization process like patients with osteoporosis and inactive osteoblast precursors.

Mineralization of differentiated osteogenic cell and OCN expression Pluripotent cells, which are undergoing differentiation, have a very high-elevated level of ALP activity.32 The osteogenic differentiation of ADSC on different scaffolds can be confirmed by determining the activity of the osteogenic marker ALP. Figure 6 shows the ALP activity of cells on TCP, PCL, PCL–AV–SF and PCL– AV–SF–HA nanofibrous scaffolds on days 7, 14, and 21. The ALP activity of cells on PCL–AV–SF–HA was significantly higher (p 0.001) by 35.85% compared to PCL–AV–SF nanofibrous scaffolds on day 7. Research finding showed that HA in the presence of pluripotent cells induces osteogenesis;33,34 similarly, in another study, acemannan present in AV showed induction of osteogenesis.13 PCL–AV–SF–HA showed increased rate of cell ALP activity of about 2.86%, 198.13% (p 0.01) and 60.03% (p 0.01) compared to PCL on days 7, 14 and 21. From the data, it is clear that the



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1600 TCP

PCL-AV-SF

PCL

PCL-AV-SF-HA

* *

1400

*

Absorbance Index

1200

**

1000

800

600

400

200

0 7

14 Days

21

Figure 6. Alkaline phosphatase activity showing the osteogenic differentiation of ADSCs on TCP, PCL, PCL–AV–SF and PCL–AV– SF–HA nanofibrous scaffolds on days 7, 14 and 21. * indicates significant difference of p 0.01; ** indicates significant difference of p 0.001.

ALP activity of cells grown on PCL–AV–SF–HA nanofibrous scaffolds was very high compared to PCL scaffold on all three days confirming the enhanced differentiation of ADSC cells into osteogenic lineage. Elevated levels of ALP activity were noticed on PCL– AV–SF–HA nanofibrous scaffolds on day 14 compared to day 21. This down-regulation of ALP at day 21 is considered to be related to the cellular process switching onto further steps, such as mineralization. Mineralization following osteogenic differentiation can be determined quantitatively (Figure 7) and qualitatively (Figures 8 and 9) using Alizarin Red Staining methodology. PCL–AV–SF–HA scaffolds (Figure 7) showed significantly about 18.83%, 107.97% (p 0.01) and 183.60% higher mineral deposition compared to PCL scaffolds on days 7, 14 and 21, respectively, and about 37.42%, 93.30% and 57.16% compared to PCL–AV–SF scaffolds on days 7, 14 and 21. The measured absorbance from PCL–AV–SF–HA scaffolds was increased linearly on days 7, 14 and 21 compared to all other scaffolds. These data show more ADSCs cells on PCL–AV–SF–HA scaffolds and have differentiated leading to mineralization phase to secrete more ECM. Presence of HA is the major factor responsible for high-mineral secretion. Optical microscopic images in Figures 8 and 9 of scaffolds with ARS staining after 14 and 21 days of cell culture supported the

above quantitative analysis data where high intensity of red coloured stain confirming the presence of calcium deposition was observed on PCL–AV–SF–HA scaffolds. OCN is an osteoblast-secreted protein playing major role in bone mineralization. OCN level is generally correlated with bone mineral density. The expression of OCN can be used as a marker for determining osteoblast activity in bone mineralization.35,36 For the present analysis, both ADSC-specific marker protein and OCN-specific marker protein were used to discriminate the undifferentiated ADSC cells and differentiated osteoblast cells. Figure 10(a) to (d) showed that the cells express ADSCs-specific marker protein CD 105 (red colour) on TCP, PCL, PCL–AV–SF and PCL– AV–SF–HA nanofibrous scaffolds, respectively. Similarly, Figure 10(e) to (h) showed differentiated osteogenic cells expressing OCN (green colour) marker protein OCN on TCP, PCL, PCL–AV–SF and PCL– AV–SF–HA nanofibrous scaffolds. Dual expression of both CD 105 and OCN in Figure 10(i) to (l) confirmed the osteogenic differentiation of ADSCs on TCP, PCL, PCL–AV–SF and PCL–AV–SF–HA nanofibrous scaffolds. Among all the scaffolds, namely TCP (a, e, i), PCL (b, f, j), PCL–AV–SF (c, g, k) and PCL–AV– SF–HA (d, h, l), nanofibrous scaffolds ADSCs cultured on PCL–AV–SF–HA (Figure 10(d), 10(h), and 10(l))



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PCL

PCL-AV-SF

PCL-AV-SF-HA

*

Absorbance Index

0.8

0.6

0.4

0.2

0

7

14 Days

21

Figure 7. Quantitative analysis of the mineralization by differentiated ADSCs on TCP, PCL, PCL–AV–SF and PCL–AV–SFHA nanofibrous scaffolds on days 7, 14 and 21. * indicates significant difference of p 0.01.

Figure 8. Optical microscope images showing the secretion of extracellular matrix by ADSCs using Alizarin red staining on day 14 on (a) TCP, (b) PCL, (c) PCL–AV–SF and (d) PCL–AV–SF–HA nanofibrous scaffolds at 10 magnification.



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Figure 9. Optical microscope images showing the secretion of extracellular matrix by ADSCs using Alizarin red staining on day 21 on (a) TCP, (b) PCL, (c) PCL–AV–SF and (d) PCL–AV–SF–HA nanofibrous scaffolds at 10 magnification.

Figure 10. Confocal microscopy images showing the osteogenic differentiation of ADSCs using ADSC-specific marker protein CD 105 (a, b, c, d), osteoblasts specific marker protein osteocalcin (e, f, g, h) and dual expression of CD 105 and osteocalcin (i, j, k, l) on TCP, PCL, PCL–AV–SF and PCL–AV–SF–HA nanofibrous scaffolds at 60 magnification.



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scaffolds exhibit the characteristic osteoblastic phenotype and high-OCN expression representing complete osteogenic differentiation compared to all other nanofibrous scaffolds. From the observed results, we have demonstrated that ADSCs cells on PCL–AV–SF–HA nanofibrous scaffolds undergo a well-defined pathway of cell adhesion, proliferation and differentiation followed by ECM secretion, making them excellent scaffolds for bone tissue engineering.

Conclusion The significance of this study is the incorporation of natural biomaterials like SF and AV on polymeric electrospun nanofibrous scaffolds with surface precipitation of HA to regulate and improve specific biological functions like adhesion, proliferation and differentiation of ADSC cells for bone tissue engineering. ADSC cells are desirable cell therapeutics for bone repair and regeneration due to their abundance and accessibility, which showed osteogenic differentiation, in the absence of an induction medium, with the presence of osteoconductive HA and osteoinductive AV with SF providing the necessary mechanical support. This demonstrates the therapeutic potential of ADSCs-loaded PCL–AV–SF–HA scaffolds that may benefit patients with reduced numbers of native osteoblast precursors in vivo in conditions like osteoporosis.

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