Preliminary studies of PVA/PVP blends incorporated with HAp and β -TCP bone ceramic as template for hard tissue engineering

Abstract

Polyvinylalcohol (PVA) and Polyvinylpyrrolidone (PVP) blend incorporated with hydroxyapatite (HAp) and β-tricalcium phosphate (β-TCP) is electrospun as nanofibrous composite scaffolds to act as suitable template for bone tissue engineering. Microscopic and spectroscopic characterizations confirm uniform integration of the crystalline calcium phosphate ceramics in the scaffolds. PVA-PVP blends are usually amorphous in nature and addition of phosphate ceramic particles specifically HAp elevates its crystalline behavior which is substantiated by XRD details. Further incorporation of ceramics is confirmed using FT-IR as characteristic ${PO}_{4}^{3 -}$ groups for HAp and β-TCP were observed in the distinguished composites. Single glass transition temperature is observed for pure and composite blends indicating the formation of highly miscible blends. Also, addition of these ceramics augments the thermal stability of the blend scaffolds. Biocompatibility of the prepared (PVA: PVP)-HAp and (PVA: PVP)-TCP scaffolds is assessed using MG-63 Osteoblast cell lines in the time interval of 1st, 4th and 7th day. The cell viability percentage for (PVA: PVP)-HAp is high compared to β-TCP added blend composites, reinforcing the fact that scaffolds with good mechanical strength and enhanced porosity supports better cell adhesion.

Keywords

Electrospinning polymer-ceramic composites structural characterization biomaterial

1. Introduction

Extensive investigation has been carried out on calcium phosphate based ceramics due to their applicability in a wide range of biomedical applications like bone tissue engineering and drug delivery systems. Hydroxyapatite (HAp, ${Ca}_{10} {({PO}_{4})}_{6} {(OH)}_{2}$ ) and β-tricalcium phosphate (β-TCP, ${Ca}_{3} {({PO}_{4})}_{2}$ ) are the most prominently used biomaterials in the treatment of orthopedic defects. The chemical composition of these biomaterials resembles the mineral phase of natural bone to a large extent. These chemically anisotropic biomaterials are classified mainly as polycrystalline ceramics [1,2]. They are highly biocompatible and have excellent osteoconductive properties, as numerous in-vitro studies have demonstrated good attachment, migration, growth, and differentiation of osteogenic cells on these ceramic surfaces [3–5]. The major drawback of these biomaterials are their inferior mechanical properties which makes them unsuitable to be used alone as bone scaffolds [6]. To reinforce the inherent mechanical properties and suitable biocompatibility they are incorporated in natural or synthetic polymer membranes [7]. Apart from enhancing the mechanical properties of the material they not only control the degradation rate of composite matrix but also modify the surface features of the matrix along with the medium surrounding the cell [8].

Polyvinylpyrrolidone (PVP) is a hydrogel having a distinctive combination of properties; like good solubility in both water and wide range of organic solvents, non-toxicity, high viscoelastic strength resulting in enhanced mechanical properties of PVP composites and excellent biocompatibility [9–11]. PVP has been in use in the biomedical field [12] for a very long time, for example it was used as a colloidal plasma substitute in World War II, and in the cosmetic and food industrial sectors for decades. It has already been substantiated that the presence of graft PVP chains upon the surface of a substrate enhances the biocompatibility and haemocompatibility of that substrate when implanted into a biological system [13].

Another synthetic polymer hydrogel having good membrane forming abilities with low toxicity in physiological conditions and excellent biocompatibility is polyvinylalcohol (PVA). However a major setback factor for semi crystalline PVA is that it suffers suspension under physiological conditions. Blends of PVA and PVP may overcome this limitation as they have interchain hydrogen bonding which improves their stability. These hydrogel blends are highly stable under physiological conditions due to the presence of intermolecular hydrogen bonding between PVA and PVP and intramolecular hydrogen bonds existing within PVA crystals. Both PVA and PVP have excellent biocompatibility and biodegradability [14–16].

HAp/polymer blends have been successfully developed as bone cements, dental implants and as bone tissue engineering matrix [17]. In our present study we report the synthesis of pure HAp, β-TCP and individually HAp and β-TCP embedded PVP/PVA blend nanofibrous composite scaffolds for bone tissue engineering applications. The prepared scaffolds were extensively characterized for their physical, chemical and thermal behavior using SEM, XRD, EDAX, FTIR and DSC. Biocompatibilities of the prepared scaffolds were investigated and compared using MG-63 cell lines.

2. Materials and methods

2.1. Materials

Poly (vinyl alcohol) (PVA, $M_{W} 1$ , 15,000, 98% hydrolyzed), Polyvinylpyrrolidone (PVP, molecular weight, 40,000 g/mol) and Poly(ethylene glycol) (PEG) were obtained from Loba Chemie, India. Ethanol ( $C_{2} H_{5} OH$ ), Calcium chloride dehydrate ( ${CaCl}_{2} \cdot 2 H_{2} O$ ), Trisodium phosphate ( ${Na}_{3} {HPO}_{4}$ ), Calcium hydroxide $Ca {(OH)}_{2}$ and Phosphoric acid ( $H_{3} {PO}_{4}$ ) were obtained from Qualigens Fine Chemicals, India.

2.2. Synthesis of beta tricalcium phosphate (β-TCP) and hydroxyapatite (HAp)

${CaCl}_{2}$ and PEG were dissolved in deionized water using constant stirring to form 0.100M ${CaCl}_{2}$ solution with a PEG/Ca molar ratio of 0:4. 0.14M ${Na}_{3} {PO}_{4}$ solution is prepared in deionized water and gradually admitted into ${CaCl}_{2}$ solution maintaining the Ca/P molar ratio at 1:5. The reaction is carried out at a pH value of 9 and temperature of 5°C [18]. The precipitates were washed several times to remove ${Cl}^{-}$ and ${Na}^{+}$ ions, and then dried in a freezing-dryer for 48 h. After calcining the precipitates at 900°C for 3 h, β-TCP is obtained.

Hydroxyapatite (HAp) is synthesized using wet precipitation route with calcium hydroxide [ $Ca {(OH)}_{2}$ ] and orthophosphoric acid [ $H_{3} {PO}_{4}$ ] as mentioned in our previous studies [19].

2.3. Electrospinning of PVA/PVP, (PVA/PVP)-HAp and (PVA/PVP)-β-TCP composites

PVA solutions of 8 wt% is prepared in distilled water at 70°C with constant stirring using a magnetic stirrer for 1 h and cooled before electrospinning. Ethanol is used as the solvent to spin PVP nanofibers. 30 wt% of PVP solution is prepared in ethanol with constant stirring for 3 h. 8 wt% of PVA solution and 30 wt% of PVP solution were mixed in ratio of 50/50, with intense stirring to prepare homogenous blends for electrospinning. 0.5 g of HAp and β-TCP were added separately in 10 ml of (50/50) PVA: PVP blend solution with constant stirring and sonication. The electrospinning set up used in the present study is indigenously fabricated which consists of a syringe pump (NE-300, New Era Pump Systems, Inc.). Electrospinning parameters were optimized at 10 cm tip-target distance, 12 kV applied voltage and $0.5 μ l / \min$ flow rate for all the scaffolds.

2.4. Structural characterization

FTIR spectroscopic analysis of electrospun nanofibrous scaffolds is performed on Bruker Tensor 27 FTIR, Germany, over a range of 500–4000 cm⁻¹ at a resolution of 2 cm⁻¹. XRD patterns of prepared PVA-PVP ceramic composites were recorded using a Schimadzu X-ray diffractometer (model Lab XRD-600) with Cu K_ß radiation ( $λ = 1.5418$ Å) at 40 kV and 30 mA in the $2 θ$ range of 0° to 90° with scan speed of 10°/min. The percent crystallinity is determined via XRD, i.e. the crystallinity is calculated by dividing the total area of crystalline peaks by the total area under the diffraction curve (crystalline plus amorphous peaks). % Crystallinity = (total area of crystalline peaks) / (total area of all peaks). Image J software is used to calculate the crystallinity percentage of samples. The surface morphology of electrospun nanofibrous scaffolds is studied under field emission scanning electron microscopy (FEI-QUANTA 200 F, Netherlands) at an accelerating voltage of 10 kV, after sputter coating with gold (JEOL JFC-1200 fine coater, Japan). The average fiber diameter is then calculated along with SD using image analysis software (Image J, National Institutes of Health, US). The composition and Ca/P ratio of HAp is found out using Energy Dispersive X-ray analysis (EDAX). Differential scanning calorimetry (DSC) analysis of the nanocomposites is performed from 0 to 350°C at 10°C/min using DSC 200F3 Maia instruments. The porosity of electrospinning nanofibrous scaffold is calculated using the following equation given below [20]. $\begin{array}{l} (1) & Porosity = [\frac{Fiber mat apparent density}{Bulk density of scafflod}] \times 100 \\ (2) & Fiber apparent density = [\frac{Fiber mat mass}{Fiber mat thickness \times Fiber mat area}] . \end{array}$

2.5. Measurement of hydrophilicity of prepared scaffolds

The water retention capacity of scaffold is determined by calculating the swelling ratio. Firstly, the samples were cut into uniform rectangular shapes and immersed in deionized water for 12 h. Further the specimens were removed from water and extra water is removed using filter paper and the specimen is weighed in wet condition. The swelling ratio is calculated according to the following equation: $\begin{array}{l} (3) & Swelling ratio % = {[W - W_{0}] / W_{0}} \times 100 %, \end{array}$ where $W_{0}$ is the initial weight and W is the wet weight of sample. Each experiment is repeated for three times [20].

2.6. Biocompatibility activity on MG-63 cell lines

The MG-63 cells (The National centre for cell science, Pune, India) were grown in a 96-well plate in Dulbecco’s Minimum Eagle’s Medium (DMEM) (Hi Media) supplemented with 10% fetal bovine serum (Gibco, Laboratories) and antibiotics (streptomycin, penicillin-G, kanamycin, amphotericin-B). About $25 μ L$ cell suspension ( $5 \times 10^{3}$ cells/well) is seeded in each 96 well and incubated at 37°C for 48 hour in 5% ${CO}_{2}$ for the formation of confluent monolayer. The monolayer of cells in the plate is exposed to various time intervals of scaffold material. The cell viability is measured using MTT assay with MTT (5 mg/mL) and 10% DMSO. This tetrazolium salt is metabolically reduced by viable cells to yield a blue insoluble Formozan product measured at 570 nm spectro-photometerically. Controls were maintained throughout the experiment (untreated wells as cell control and diluents treated wells as diluents control). The assay is performed in triplicate for each of the scaffolds. The mean of the cell viability values is compared to the control to determine the effect of the scaffold on cells and % cell viability is plotted against different scaffolds.

2.7. Cell morphology

The cell morphology on the scaffold is investigated using scanning electron microscopy {JEOL, JSM-6390} operating at an accelerating voltage of 5–20 kV. The cell-seeded scaffolds were rinsed with phosphate buffer saline (PBS) and fixed using 10% neutral formalin buffer for 5 h at 4°C. Scaffolds were dehydrated by graded ethanol (10–100%) treatment, dried overnight, (to prevent shrinkage of structures) and to remove moisture content. The samples were coated with platinum using a JEOL JFC-1600 auto fine coater at 10 mA for 60 seconds before SEM analysis. Samples were carefully analyzed at lower and higher magnifications to assess the morphology of adhered cells.

3. Results and discussions

3.1. FT-IR studies

Figure 1 shows FT-IR absorption spectra for pure HAp, β-TCP and corresponding PVA: PVP nanocomposites recorded at room temperature in the region 4000–500 cm⁻¹. The fairly visible absorption bands at 1041 cm⁻¹ and 1994 cm⁻¹ detected in the spectra are attributed to the ${PO}_{4}^{3 -}$ ion and [ ${PO}_{4}$ ] $γ_{3}$ , $γ_{1}$ modes. The broad peak around 3568 cm⁻¹ for the sample corresponds to the adsorbed water [21,22]. A very feeble characteristic band corresponding to HAp is visible at around 630 cm⁻¹, which represents the OH end-group with lower steric impediment of the structure [23]. Similarly, IR spectra for β-TCP reveals characteristic broad absorption bands at 3408 cm⁻¹ and 1629 cm⁻¹ which is attributed to adsorbed water and the peak at 3545 cm⁻¹ appears due to the stretching vibration of the lattice ${OH}^{-}$ ions. The bands at 900–1200 cm⁻¹ are due to the stretching mode of the ${PO}_{4}^{3 -}$ group. The characteristic bands due to vibration of ${PO}_{4}^{3 -}$ group appeared at 574 and 1020 cm⁻¹ [24].

Fig. 1.

FTIR spectra for pure HAp, β-TCP and corresponding PVA: PVP Nanocomposites.

The characteristic peaks of (PVA: PVP) blend observed in composite scaffolds are interpreted as follows. The observed broad and strong band around 3385 cm⁻¹ is attributed to -OH stretching vibrations of (PVA: PVP) blend. An overlapped and weak band at 2930–2933 cm⁻¹ is assigned to- ${CH}_{2}$ asymmetric stretching of (PVA: PVP) blend. Another strong band at 1658 cm⁻¹ observed in (PVA: PVP)-β-TCP composite is attributed to the stretching vibration of $C = O$ of PVP. $C = C$ stretching vibration of the blend occurred for the composite scaffolds around 1439–1437 cm⁻¹. Characteristic C-N stretching vibrations of PVP are observed in both composite scaffolds at 1282–1260 cm⁻¹ [25,26]. The prominent peaks corresponding to β-TCP are ascribed to 580, 925, 1094 cm⁻¹ ( ${PO}_{4}^{3 -}$ groups) in the respective composite scaffold. (PVA: PVP)-HAp exhibited characteristic groups pertaining to ${PO}_{4}^{3 -}$ groups of hydroxyapatite around 600, 960, and 1050 cm⁻¹ [27].

Fig. 2.

XRD patterns for pure HAp, β-TCP andcorresponding PVA: PVP nanocomposites.

3.2. XRD analysis

To investigate structural and crystalline forms of the prepared samples X-ray diffraction scans were studied. Figure 2 represents the XRD patterns for pure HAp, β-TCP and corresponding PVA: PVP nanocomposites. The XRD spectra for pure PVA-PVP shows short broadened and highly diffused peaks around $2 θ \sim 19.19$ °–20.70° which reveals that the pure blend contains highly amorphous structure. This peak corresponds to (110) reflection, which are consistent with the values in the standard card (JCPDS file No. 41-1049). XRD analysis of HAp powder revealed peaks at 25.68°, 31.55°, 31.98°, 32.68°, 39.58°, 46.50° and 49.30° having (hkl values) as (201), (217), (211), (300), (310), (222) and (213) which are characteristic of hydroxyapatite. The observed diffraction peaks are identified by standard JCPDS (File no. 09-0432) file and indicates highly crystalline form with hexagonal crystal structure of HAp [28]. The following characteristic peaks of HAp are observed in the (PVA-PVP)-HAp blend composite at $2 θ \sim 31.95$ °, 32.40°, 33.05°, 39.98°, 46.88°, 49.69° corresponding to (211), (300), (306), (1016), (222) and (213). The values finely corroborated with values in standard card (JCPDS file No. 09-0432). The HAp peaks retained their crystalline behavior and phase in composite scaffolds to some extent. The percentage crystallinity of the HAp incorporated blend membrane is found to be around 65%. XRD pattern substantiated that the obtained precipitate calcined at 900°C had a pure β-TCP phase with high crystallinity and rhombohedral crystal structure according to JCPDS 70-2065 and 09-0169 cards. Prominent peaks ascribed to β-TCP (JCPDS file No. 09-0169) in the blend composite scaffolds are 31.77°, 32.40°, 33.80°, 37.96°, 44.25° relating to (211), (128), (1112), (315) and (400). The intensity of peaks corresponding to pure blend in both (PVA-PVP)-HAp and (PVA-PVP)-TCP composite is drastically reduced due to the stronger diffraction of ceramic crystals or probably due to overlapping of diffraction peaks of HAp and β-TCP. Reduced percentage crystallinity is observed for β-TCP incorporated blend composite at 42%. Small peaks with low intensity at $2 θ \sim 10.96$ °–11.04° and 21.91°–22.27° are observed for pure PVP in both the composites, as reported in literature [29,30]. Detailed explanations of all the peaks for composite scaffolds with respective (hkl) values are given in Table 1.

Table 1
XRD results for pure PVA: PVP, (PVA: PVP)-HAp and (PVA: PVP)-TCP scaffolds

S. no. particular 2 Theta (deg) d (A) FWHM (deg) (hkl) values Crystallinity (%)

PVA-PVP

1 11.72 7.54 5.61 – –

20.65 4.29 5.75 –

(PVA-PVA)-HAp

1 11.66 7.58 0.32 – 65

2 19.90 4.45 0.90 –

3 31.95 2.79 0.33 (2 1 1)

4 33.05 2.70 0.31 (3 0 0)

(PVA-PVP)-TCP

1 11.00 8.00 0.37 – 42

2 20.70 4.28 1.05 –

3 31.77 2.81 0.43 (2 1 1)

4 32.80 2.72 0.38 (3 0 0)

S. no. particular	2 Theta (deg)	d (A)	FWHM (deg)	(hkl) values	Crystallinity (%)
PVA-PVP
1	11.72	7.54	5.61	–	–
	20.65	4.29	5.75	–
(PVA-PVA)-HAp
1	11.66	7.58	0.32	–	65
2	19.90	4.45	0.90	–
3	31.95	2.79	0.33	(2 1 1)
4	33.05	2.70	0.31	(3 0 0)
(PVA-PVP)-TCP
1	11.00	8.00	0.37	–	42
2	20.70	4.28	1.05	–
3	31.77	2.81	0.43	(2 1 1)
4	32.80	2.72	0.38	(3 0 0)

3.3. SEM and EDAX analysis

To investigate the morphology of pure HAp, β-TCP and the composite nanofibers, the prepared scaffolds were studied by scanning electron microscope. Also the chemical compositions and Ca/P ratio of ceramics were analyzed using EDAX. Figure 3(A)–(D) shows the SEM images of pure HAp and β-TCP with their respective EDAX graphs.

Fig. 3.

(A) and (B) SEM images of HAp particles & EDX result of Hap particles (C) and (D) SEM image of β-TCP particles & EDX result of β-TCP particles.

The SEM results of synthesized HAp ceramic demonstrated that grain size ranged from 150 to 300 nm as shown in (Fig. 3(A)). EDX results showed that the HAp particles had a Ca/P ratio of 1.62 (Fig. 3(B)), very close to natural bone. The chemically prepared granules represent hexagonal crystal structure typical for Hydroxyapatite. Similarly the grain size of β-TCP is found in the range of 100–180 nm with spherical shape. Presence of calcium and phosphate is observed in the samples and the concentration of calcium is found to be more than phosphate as the peak of the calcium dominated the phosphate one with ratio of Ca/P as 1.49 which confirms the formation of β-tricalcium phosphate [31,32].

Figure 4(A)–(F) shows SEM images of pure blend and composite nanofibers. A pure blend nanofiber (A&B) represents ultrafine and uniform nonwoven architecture characteristic of electrospun materials with AFD of 189 nm. With the incorporation of both HAp and β-TCP, particles the fiber diameter increased to a greater extent. It is observed that all the prepared composite nanofibers are homogeneous having random surface morphology and interconnected porosity, revealing a fine dispersion of calcium phosphate ceramic in the blend scaffolds. The average fiber diameter (AFD) of (PVA: PVP)-HAp and (PVA: PVP)-TCP composite were found to be 261 nm and 408 nm.

Fig. 4.

(A) and (B) SEM images of pure PVA: PVP blend (C) and (D) (PVA: PVP) $- β$ TCP nanofibers in different magnification (E) and (F) SEM images of (PVA: PVP)-Hap nanofibers in different magnification.

3.4. DSC study

To determine the thermal properties and miscibility of polymeric blend composites DSC measurements were carried out. DSC thermograms were obtained for pure and composite blends and glass transition temperature ( $T_{g}$ ) is analyzed. All samples were heated from room temperature to 200°C with a heating rate of 10°C min⁻¹ under nitrogen atmosphere. The glass transition temperature of the semicrystalline PVA polymer is 70°C and PVP is 170°C. The $T_{g}$ values of the PVA–PVP blend may lie between 70 and 170°C [33,34]. Single glass transition temperature is observed for pure and composite blends at 147°C for (PVA:PVP), 144°C and 142°C for (PVA:PVP)-HAp and (PVA:PVP)-TCP scaffolds (Fig. 5), indicating the hydrogen bonding formation between hydroxyl groups of PVA and carbonyl groups of PVP resulting in highly miscible blends. $T_{g}$ of HAp blended composite is found slightly higher compared to β-TCP blend attributing to enhanced thermal stability with addition of Hydroxyapatite granules [35]. Sharp endothermic peaks were observed for all the scaffolds in the range of 50–70°C.

Fig. 5.

DSC thermograms for pure PVA: PVP blend and HAp, β-TCP incorporated blend Scaffolds.

3.5. Percent porosity and pore diameter measurement

The interconnected pore structure is important to bone cell growth, tissue regeneration and interface support. Furthermore, the micro-porous structure is beneficial to capillary growth, nutrient transport and biological properties of the implant [36]. Porosity of both composite scaffolds is in the range of (60–70)%. Slightly enhanced porosity for composite scaffolds can be explained on the basis of addition of HAp or β-TCP filler particles. It may be attributed to the fact that addition of ceramic particle results in more dense and thicker pore walls with lower porosity. Small pore diameters of 471 nm and 461 nm were observed for ultrafine and dense nanofibrous network of PVA-PVP and HAp loaded blend scaffolds as seen from SEM images. The pore structure of (PVA-PVP)-HAp composite scaffold seems to be uniform, with a lot of highly interconnected pores in the network. β-TCP incorporated structure demonstrates larger pore size of 691 nm as coarse and non uniform morphology is observed from SEM images.

3.6. Hydrophilicity

The (PVA: PVP)-HAp showed improved water absorption ability with the introduction of HAp in the composite scaffolds, while the pure PVA: PVP blend shows poor water absorption ability. The enhanced water absorption ability observed with the introduction of HAp might be due to the interaction between scaffolds and water because of the P-OH groups present on the surface of HAp powders. Also the porous nature of nanofibrous scaffolds enhances the water uptake by retaining more amount of water within in the fibers and pores. (PVA: PVP)-TCP demonstrated reasonably good hydrophilicity compared to pure blend as the water molecules tends to concentrate at the particle matrix interface improving the volume of liquid absorbed. Unfilled blend films have no interfaces between filler particles and matrix through which water can diffuse. Also percentage increase of the filler particle has an impact on the amount of volume absorbed by the composite scaffold [37–41].

3.7. Biocompatibility of polymer nanocomposite with osteoblast MG-63 cell lines

In this study, the scaffolds made of (PVA: PVP)-HAp and (PVA: PVP)-TCP along with control were investigated for the application as substrates for MG-63 cells. The changes in cell viability percentage at 1, 4, and 7 days after loading MG-63 cells to the scaffolds were monitored as shown in Fig. 6 to verify whether these cells could adhere to and proliferate on the scaffolds or not. The scaffolds had porosity of 65%, 67% and pore sizes of 466 nm and 691 nm. The initial cell seeding density is $5.00 \times 10^{3}$ cells/sample, and the viability percentage of (PVA: PVP)-HAp (PVA: PVP)-TCP scaffolds is 110% and 92%, respectively after 7 day in culture, as shown in Fig. 6. It is observed that viability percentage increases after each interval for all the scaffolds. The blend composites demonstrate less viability percentage on 1st day compared to control, the reason for this observation being that some cells may have infiltrated into cell culture medium after initial seeding due to porous nature of nanofibrous scaffolds. The percentage of cell viability enhances fairly for the case of (PVA: PVP)-HAp scaffold and particularly steep rise is seen on 7th day. This supports the fact of good adherence of remaining cells and faster proliferation and growth of cells on the scaffold.

Fig. 6.

The % cell viability studies of osteoblast cell (MG-63) cultured on PVA: PVP-Hap and PVA:PVP-TCP bilayer nanocomposites scaffolds for the time intervals of 1st, 4th and 7th day.

Biocompatibility of HAp and increased overall surface area of nanofibrous membrane definitely provides suitable ambience of extra cellular matrix (ECM) to the seeded cells proving it as a suitable matrix for MG-63 cell lines. (PVA: PVP)-TCP scaffold also shows increasing % cell viability after each time interval but the values are lot inferior compared to other scaffold and the control. As the water swelling ratio of this scaffold is high, the scaffold might have lost some mechanical strength to retain cells for adherence and their growth. Larger pore diameter observed for this scaffold also contributes to the effect of cells passing through the membrane to the culture media resulting in the decreased number of cells on the scaffold.

Figure 7 shows the SEM images of MG-63osteoblast cell lines cultured on (PVA: PVP)-HAp scaffolds on time intervals of 1st, 4th and 7th day respectively at low and high magnifications. All the low magnification images clearly illustrate the spread of osteoblast cells on the nanofibers matrix. The spread increases with every interval and 7th day matrix seems to be fully covered with highly spread cells proving the excellent biocompatibility of composite matrix. High magnification images give idea about the morphology of the adhered cells. Densely proliferated cells with thin elongated spindle like morphology are observed, which is also characteristic for MG-63 cell lines. Cells have beautifully covered nanofibers matrix exemplifying the importance of resemblance of nanofibers membranes with ECM.

Fig. 7.

SEM micrograph of osteoblast MG-63 cells seeded on electrospun (PVA: PVP-HAp) fibers after 1st (A) and (D) 4th (B) and (E) and 7th (C) and (F) day at different magnifications.

Figure 8 represents the SEM micrographs of MG-63 osteoblast cultured on (PVA: PVP)-TCP scaffolds. Average spread of cells is observed on 7th day of seeding and normal morphology of cells is observed in high magnification images. Still the nature of proliferation is same as seen in (PVA: PVP)-HAp scaffolds i.e., densely populated and cells covering the non woven fibers are seen. Though the number of cells retained on these scaffolds might be less but they have grown well and have definitely had a nice grip on nanofibers matrix to show good adherence.

Fig. 8.

SEM micrograph of osteoblast MG-63 cells seeded on electrospun (PVA: PVP TCP) fibers after 1st (A) and (B) and 7th (C) and (D) day at different magnifications.

Fig. 9.

Scheme: (PVA-PVP)-HAp blend nanofibers with interchain hydrogen bonding seeded with MG-63 cell lines.

4. Conclusions

In Conclusion PVA/PVP blend composites incorporated with HAp and β-TCP was prepared using electrospinning technique. Characteristic XRD peak revealed the presence of added ceramics and defines the overall semicrystalline behaviour of the composite scaffolds. SEM results show that homogenous smooth nanofibrous morphology was obtained with AFD in the range of 250 nm. EDAX analysis confirms the formation of appropriate composition of ceramics hydroxyapatite and β-TCP. DSC analysis confirmed the formation of highly miscible blend due to the presence of a single glass transition temperature. The thermal properties of the polymer ceramic composite scaffold were almost similar to pure blend scaffolds which already have high thermal stability. Excellent cell adherence with normal morphology of cells on (PVA: PVP)-HAp scaffolds was observed. Hence, PVA/PVP polymer blend filled with HAp is a very promising biomaterial for bone graft application.

Footnotes

Acknowledgements

The authors would like to thank Central Research Facilities Lab, Karunya University for supporting characterization of samples. Also, we are thankful to Pondicherry Centre for Biological Sciences for helping us with cell culture studies.

Conflict of interest

The authors have no conflict of interest to report.

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