2.2.6. Bioactivity Study

Bioactivity

The simulated body fluid (SBF) solution which is similar to the human physiological fluid was used for the in vitro bioactive study of the electrospun nanofibers. The procedure, which was suggested by Kokubo et al., 1990 [29] was used for the preparation of SBF solution. All chemicals were added together to 1 L of double distilled water, pH was adjusted to 7.35 ± 0.25, and stored in the refrigerator at −4 ◦C. For the analysis, 1 × 1 cm size of the electrospun nanofibrous mat (HPC/PLA and HAP-HPC/PLA) was immersed

in the SBF solution (60 mL) at 37 ◦C for a different time duration of 7, 15, and 30 days, respectively. The contamination was avoided by the replacement of SBF solution with fresh solution for every 24 h. Carbonated hydroxyapatite (HCA) was formed in the process of immersion in the SBF solution and hence resulting in the formation of apatite layer. SEM and EDAX analysis were used for the confirmation of apatite layer formation on the surface of the electrospun nanofibers.

### In Vitro Biodegradation Properties

Electrospun mat of dimensions 1 × 1 cm was immersed in SBF solution at a pH of 7.4 for different time intervals such as 7, 15, and 30 days, respectively. The samples were dried at room temperature after the respective time duration was completed [30]. The percentage degradation was calculated from the dried weight of the samples after degradation as follows:

$$\text{Percentage Depradation} = ((\text{Wi} - \text{Wd})) / \text{Wi} \times 100 \tag{2}$$

where Wi and Wd are initial and dried weight of the sample, respectively.

### 2.2.7. Cytotoxicity Analysis

Osteosarcoma (MG-63) cell lines were used for the cytotoxicity analysis of the fabricated mat. MG63 was passaged using Minimum Eagle's Medium (MEM), 10% fetal bovine serum, and 1% penicillin–streptomycin solution and maintained at 37 ◦C and 5% CO<sup>2</sup> concentration in a humidified incubator. Mats were sterilized by washing with 70% ethanol 2–3 times and exposing them to UV overnight (approximately for 12 h). The sterilized sample was soaked in cell culture medium for 2 h. The cells were harvested using 0.05% (*v/v*) trypsin–EDTA (ethylenediamine tetraacetic acid) and seeded on scaffolds followed by incubation at 37 ◦C in the presence of CO<sup>2</sup> environment. The medium was changed once in 2 days to supply the adequate amount of nutrients present in the culture plate. Further, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay was used to check the toxicity effects of samples. The media were washed off with 0.1 M PBS solution at the end of 24 and 48 h incubation. Further, 300 µL of fresh media and 60 µL of MTT solution were mixed together, added to PBS-washed mats, and incubated at 37 ◦C for 3 h. About 200 µL of the incubated mixture was filled in 96-well plate and absorbance was measured at 570 nm using a Vmax Microplate reader. Triplicates were used for calculating the average of two sets of the assay [31]. The % cell viability was calculated using the following formula:

$$(\% \text{cell viability ((A570 of treated cells))})/((570 \text{ of control cells})) \times 100\tag{3}$$

### 2.2.8. Statistical Analysis

Results were expressed as a mean and standard deviation. Comparative studies of means were performed using one-way analysis of variance (ANOVA). Significance was accepted with *p* < 0.05.

### 2.2.9. In Vivo Study

Sixteen 4–6 weeks-old Wistar albino rats weighing between 250 and 400 g were obtained from the Animal Center Laboratory of VIT University. All experimental rats were bred at the Animal Center Laboratory of VIT University, with a standard laboratory diet and environment. All animal experiments were approved and performed according to the regulations of the animal ethics committee of our university. VIT/IAEC/14/NOV5/47. The rats were anesthetized by intraperitoneal injection of pentobarbital (ketamine 0.2 mL and xylene 0.1 mL). Using sterile instruments and aseptic technique, a 1.0–1.5 cm sagittal incision was made on the scalp, and the calvarium was exposed by blunt dissection. A full-thickness defect (5 mm in diameter) was created in the central area of each parietal bone using a 5 mm electric trephine bur under constant irrigation with sterile 0.9% saline. The defects were implanted randomly with the HAP-HPC-PLA scaffolds (n 1/4 12). After 4 and 8 weeks of implantation, the calvaria of the rats was harvested and immediately immersed in a 10% tempered solution of formalin and further analysis was studied such as R-ray and histopathological.

### **3. Results and Discussion**

### *3.1. Preparation of HAP-HPC/PLA Nanofibers*

The solution of HPC/PLA was prepared by HPC and was added to boiling water. Since HPC has a solubility issue, PLA with high viscosity was added to HPC to make the solution suitable for electrospinning. The different amounts of HAP-HPC/PLA were considered to fabricate nanofibers of different compositions.

### *3.2. Viscosity, Electrical Conductivity, and Dielectric Constant Analysis*

HAP was blended with HPC/PLA polymer composite at different ratios such as 0:100 (HAP is absent here), 40:60, 50:50, 60:40, and 70:30 and these resulted in a viscosity of 387.6 2487, 1413, 1198, and 786.7 cP, respectively. Amongst the different composite solutions, 70:30 mixture had a viscosity of 786.7 cP, which is found to be the applicable viscosity, as it has to flow easily from the needle during electrospinning. Additionally, the viscosity parameter is significant, because the optimum viscosity can only yield a targeting diameter of nanofibers for various applications. The viscosity and conductivity of HAP-HPC/PLA composite solutions were measured and mentioned in Table 2.


**Table 2.** Optimizing different parameters and its blending properties.

Conductivity and dielectric constants depend on the input temperature, frequencies, and viscosity of the solution. The overall parameters and conductivity depend on the viscosity of the sample preparation. Voltage, RPM (rotations per minute), and conductivity are the main parameters to form thinner fiber diameters. Conductivity remains constant at low temperatures, and it increases only when the temperature is increased. The dielectric constant and conductivity study of the nanofiber scaffolds is shown in Figure 1. In this

5 50:50 1413 0.0024

6 60:40 1198 0.0036

7 70:30 786.7 0.0041

study, the conductivity (σ) and dielectric constants (ε) depend on the concentration of the HAP at different temperature and the frequency was studied. The conductivity (σ) and dielectric constant (ε) were found to be increasing with an increase in temperature (35 to 125 ◦C) and with an increase in HAP concentration. study, the conductivity (σ) and dielectric constants (ε) depend on the concentration of the HAP at different temperature and the frequency was studied. The conductivity (σ) and dielectric constant (ε) were found to be increasing with an increase in temperature (35 to 125 °C) and with an increase in HAP concentration.

1 1500 17 25

12 15 17

12 15 17

12 15 17 15 21 25

15 21 25

15 21 25

700 1000 1500

700 1000 1500

700 1000 1500

Conductivity and dielectric constants depend on the input temperature, frequencies, and viscosity of the solution. The overall parameters and conductivity depend on the viscosity of the sample preparation. Voltage, RPM (rotations per minute), and conductivity are the main parameters to form thinner fiber diameters. Conductivity remains constant at low temperatures, and it increases only when the temperature is increased. The dielectric constant and conductivity study of the nanofiber scaffolds is shown in Figure 1. In this

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0.5 0.7 1

0.5 0.7 1

0.5 0.7 1

**Figure 1.** (**a**) Conductivity of HAP-HPC/PLA (70:30); (**b**) different ratios of HAP-HPC/PLA; (**c**) dielectric constant at 30 ◦C of 0% HAP and HAP-HPC/PLA; (**d**) dielectric constant at 125 ◦C of 0% HAP and HAP-HPC/PLA.

The dielectric constant decreases with increasing frequency and we obtain the maximum value of the dielectric constant 96 K for a 70:30 ratio of the nanofibrous mat at 5 kHz at room temperature. This is because of the fact that at lower frequency dipole moment of hydroxyl ions in HAP follow the variation of the field while at higher frequency those ions do not follow the variation of the field and the dielectric permittivity value decreases. Figure 1b shows the frequency dependence of the dielectric constant for different compositions at room temperature and at 125 ◦C. The dielectric constant increases with increasing the HAP content and we obtain the value of dielectric constant 5329 K at 5 kHz at 125 ◦C and at 89 K for RT which is due to the interfacial flow rate of the viscous solution during electrospinning. Similarly, the conductivity has a much stronger concentration dependence with respect to frequency. Hence, from the results observed, the values of both conductivity and dielectric constant for the 70:30 ratio were found to be increased in HAP-HPC/PLA composition. The percolation threshold limit of 70:30 ratio of HAP-HPC/PLA is flexible and hence applicable for the fabrication of mat.

### *3.3. FT-IR (Fourier Transform Infrared) Analysis of HAP-HPC/PLA Nanofibers 3.3. FT-IR (Fourier Transform Infrared) Analysis of HAP-HPC/PLA Nanofibers*  The FT-IR spectra of the fabricated HPC/PLA nanofibrous mat and HAP with various

is flexible and hence applicable for the fabrication of mat.

*Polymers* **2022**, *14*, x FOR PEER REVIEW 8 of 22

and HAP-HPC/PLA.

The FT-IR spectra of the fabricated HPC/PLA nanofibrous mat and HAP with various composite ratios of 40:60, 50:50, 60:40, and 70:30 have been illustrated in Figure 2A. The absorption band at 3351 cm−<sup>1</sup> corresponds to a hydroxyl group in the pyranose unit of HPC. The absorption band at 2921 cm−<sup>1</sup> appears due to the CH2 and CH stretching vibration. The absorption peaks at 1717 and 1422 cm−<sup>1</sup> are attributed to the C=O stretch and –CH stretch, respectively. The absorption band appearing at 1079 cm−<sup>1</sup> is formed due to C-O stretching vibration. The results confirm the presence of HPC in the composite nanofibrous mat. composite ratios of 40:60, 50:50, 60:40, and 70:30 have been illustrated in Figure 2A. The absorption band at 3351 cm−1 corresponds to a hydroxyl group in the pyranose unit of HPC. The absorption band at 2921 cm−1 appears due to the CH2 and CH stretching vibration. The absorption peaks at 1717 and 1422 cm−1 are attributed to the C=O stretch and – CH stretch, respectively. The absorption band appearing at 1079 cm−1 is formed due to C-O stretching vibration. The results confirm the presence of HPC in the composite nanofibrous mat.

**Figure 1.** (**a**) Conductivity of HAP-HPC/PLA (70:30); (**b**) different ratios of HAP-HPC/PLA; (**c**) dielectric constant at 30 °C of 0% HAP and HAP-HPC/PLA; (**d**) dielectric constant at 125 °C of 0 % HAP

The dielectric constant decreases with increasing frequency and we obtain the maximum value of the dielectric constant 96 K for a 70:30 ratio of the nanofibrous mat at 5 kHz at room temperature. This is because of the fact that at lower frequency dipole moment of hydroxyl ions in HAP follow the variation of the field while at higher frequency those ions do not follow the variation of the field and the dielectric permittivity value decreases. Figure 1b shows the frequency dependence of the dielectric constant for different compositions at room temperature and at 125 °C. The dielectric constant increases with increasing the HAP content and we obtain the value of dielectric constant 5329 K at 5 kHz at 125 °C and at 89 K for RT which is due to the interfacial flow rate of the viscous solution during electrospinning. Similarly, the conductivity has a much stronger concentration dependence with respect to frequency. Hence, from the results observed, the values of both conductivity and dielectric constant for the 70:30 ratio were found to be increased in HAP-HPC/PLA composition. The percolation threshold limit of 70:30 ratio of HAP-HPC/PLA

**Figure 2.** (**A**) FTIR and 2 (**B**) XRD (X-ray diffraction) analysis of HAP-HPC/PLA nanofibers of different ratios. **Figure 2.** (**A**) FTIR and 2 (**B**) XRD (X-ray diffraction) analysis of HAP-HPC/PLA nanofibers of different ratios.

Figure 2A shows the broad peak at 3344 cm−1 corresponding to the OH stretch. Mainly, the prominent peaks appearing at 478 cm−1, 555 cm−1, 936 cm−1, 1021 cm−1, and 1081 cm−1 are characteristic bands assigned to PO43− of HAP. The distinguishable peak at 936 cm−1 appears due to the asymmetric P-O stretching vibration of PO43- bands. The sharp peaks at 478 cm−1 and 555 cm−1 correspond to the triply degenerate bending vibrations of PO43− in HAP and these results further confirm the presence of HAP. Additionally, the Figure 2A shows the broad peak at 3344 cm−<sup>1</sup> corresponding to the OH stretch. Mainly, the prominent peaks appearing at 478 cm−<sup>1</sup> , 555 cm−<sup>1</sup> , 936 cm−<sup>1</sup> , 1021 cm−<sup>1</sup> , and 1081 cm−<sup>1</sup> are characteristic bands assigned to PO<sup>4</sup> <sup>3</sup><sup>−</sup> of HAP. The distinguishable peak at 936 cm−<sup>1</sup> appears due to the asymmetric P-O stretching vibration of PO<sup>4</sup> 3- bands. The sharp peaks at 478 cm−<sup>1</sup> and 555 cm−<sup>1</sup> correspond to the triply degenerate bending vibrations of PO<sup>4</sup> <sup>3</sup><sup>−</sup> in HAP and these results further confirm the presence of HAP. Additionally, the characteristics peaks of HPC/PLA appear at 2931 cm−<sup>1</sup> and 1437 cm−<sup>1</sup> responsible for the asymmetric CH<sup>2</sup> and CH<sup>3</sup> stretch. The prominent peak at 1756 cm−<sup>1</sup> identifies the carbonyl group. Eventually, FTIR spectroscopy results of Figure 2A confirmed that fabricated nanofibers contain HAP and HAP-PLA in all composite nanofibrous mats.

### *3.4. XRD Analysis of HAP-HPC/PLA Nanofibers*

The phase analysis and purity of the fabricated HAP-HPC/PLA nanofibrous composite mats have been scanned using XRD studies which are shown in Figure 2B. The diffraction peaks at 19.8◦ and 30.48◦ are responsible for the HPC/PLA composite. Additionally, the diffraction peaks of HAP in HAP-HPC/PLA nanofibrous composite mats at 22.78◦ , 25.92◦ , 28.10◦ , 29.12◦ , 31.89◦ , 32.05◦ , 32.20◦ , 34.09◦ , 39.88◦ , 46.75◦ , 48.28◦ , 49.48◦ , 50.78◦ , and 51.23◦ are corresponding to (111), (002), (102), (210), (211), (112), (300), (202), (310), (222), (312), (213), (321), and (410) planes are confirmed. Hence both FTIR and XRD results confirmed the presence of pure HAP without any other secondary phases. Moreover, the triplet peak appearing at 31.89◦ , 32.05◦ , and 32.20◦ is due to an increase in the HAP concentration in the fabricated mats. The results suggest that the diffraction peak of HPC/PLA (Figure 2B) is slightly amorphous. Further, the intensity of the diffraction peaks is increasing with the addition of HAP to the HPC/PLA composite.

### *3.5. Mechanical Properties*

As explained above, the smaller diameter of fibers with a definite orientation of the polymer chains has been observed in this study. The extended amorphous structures of the HPC/PLA and HAP-HPC/PLA nanofibers yield results as shown in Figure 3. The obtained results suggest that 70:30 ratio shows moderate tensile strength compared to the other ratios (Figure 3a). The results demonstrated that tensile strength and Young's modulus are found to be increased due to the alignment of the polymer along the axes of fibers, although the percentage of fracture is found to be decreasing, respectively. HAP-HPC/PLA nanofibrous mat with a composition ratio of 70:30 resulted in the high tensile strength of 9.53 MPa. The other composite ratios of 0:100, 40:60, 50:50, and 60:40 gave a tensile strength of 3.7 MPa, 4.88 MPa, 8.21 MPa, and 8.91 MPa, respectively. Further, Table 3 shows an increase in tensile strength as the percentage of HAP increases; hence maximum tensile strength was observed for "70:30" composition of HAP-HPC/PLA nanofibrous mat. Additionally, the percentage of fracture has decreased, and Young's modulus has increased, exhibiting high stiffness, regular chain orientation, and elevated mechanical strength to the nanofibers. The composite mat without HAP shows less Young's modulus and tensile strength. All these results clearly state that the HAP-HPC/PLA nanofibrous mat of composite ratio 70:30 shows an optimal increase in tensile strength (9.53 MPa) for a decreased diameter (110 ± 66 nm). *Polymers* **2022**, *14*, x FOR PEER REVIEW 10 of 22 Structurally, all molecules of the nanofibers should be fully extended and aligned perfectly with the fiber axis. The secondary bonds of the molecular structure of the fibers help in the determination of the tensile strength. The high stiffness of the nanofibers can be obtained if the polymer chains are fully extended and oriented. Some polymer chains are defective due to the high molecular weight of the polymer leading to the high tenacity of the fiber. The mechanical strength of the nanofibrous mat is influenced by the size of the fiber diameter. This confinement effect is the main property for the improvement in the mechanical strength of the mat. Hence, the orientation and degree of alignment can be influenced by decreasing the diameter of the nanofiber [32,33].

**Figure 3.** Correlation of (**a**) mechanical property (**b**) tensile strength (**c**) Young modulus. **Figure 3.** Correlation of (**a**) mechanical property (**b**) tensile strength (**c**) Young modulus.



Structurally, all molecules of the nanofibers should be fully extended and aligned perfectly with the fiber axis. The secondary bonds of the molecular structure of the fibers help in the determination of the tensile strength. The high stiffness of the nanofibers can be obtained if the polymer chains are fully extended and oriented. Some polymer chains

are defective due to the high molecular weight of the polymer leading to the high tenacity of the fiber. The mechanical strength of the nanofibrous mat is influenced by the size of the fiber diameter. This confinement effect is the main property for the improvement in the mechanical strength of the mat. Hence, the orientation and degree of alignment can be influenced by decreasing the diameter of the nanofiber [32,33]. (**a**) (**b**) (**c**) **Figure 3.** Correlation of (**a**) mechanical property (**b**) tensile strength (**c**) Young modulus.

### *3.6. X-ray Photoelectron Spectroscopy (XPS) 3.6. X-Ray Photoelectron Spectroscopy (XPS)*  The results which are obtained from FT-IR, XRD and SEM (scanning electron micros-

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influenced by decreasing the diameter of the nanofiber [32,33].

Structurally, all molecules of the nanofibers should be fully extended and aligned perfectly with the fiber axis. The secondary bonds of the molecular structure of the fibers help in the determination of the tensile strength. The high stiffness of the nanofibers can be obtained if the polymer chains are fully extended and oriented. Some polymer chains are defective due to the high molecular weight of the polymer leading to the high tenacity of the fiber. The mechanical strength of the nanofibrous mat is influenced by the size of the fiber diameter. This confinement effect is the main property for the improvement in the mechanical strength of the mat. Hence, the orientation and degree of alignment can be

The results which are obtained from FT-IR, XRD and SEM (scanning electron microscopy) analysis suggest that HAP-HPC/PLA nanofibrous mat of composite ratio 70:30 is the desired and favorable composition, and the surface chemistry of the material was analyzed using XPS. The binding energy peaks which are available in the full survey spectrum confirm the presence of Ca2p (348.44), P2p (137.64), O1s (526.02) and C1s (287.22) in the fiber matrix (Figure 4). copy) analysis suggest that HAP-HPC/PLA nanofibrous mat of composite ratio 70:30 is the desired and favorable composition, and the surface chemistry of the material was analyzed using XPS. The binding energy peaks which are available in the full survey spectrum confirm the presence of Ca2p (348.44), P2p (137.64), O1s (526.02) and C1s (287.22) in the fiber matrix (Figure 4).

**Figure 4.** The XPS full survey for HAP-HPC/PLA nanofiber and core level spectrum of Ca 2p, P 2p, O 1s, and C1s **Figure 4.** The XPS full survey for HAP-HPC/PLA nanofiber and core level spectrum of Ca 2p, P 2p, O 1s, and C1s.

deconvoluted into three elements which are present as P-O (535.68 eV), OH (534.86 eV), and P-O-P (533.71 eV), respectively. Additionally, Table 4 (showed in Supplementary File) confirms the presence of C 1s of HPC and PLA appeared at 287.22 eV and its deconvoluted peak has four different elements at a different binding energy such as 288.50 eV (C-C=O), 286.73 eV (C-O), 286.16 eV (C-H), and 285.75 eV (C-CH3), respectively. Therefore, XPS analysis confirms the nanofiber matrix surface chemistry and the Ca/P (1.67) ratio, which

**Sample Ca 2P P 2p C1s O1s Ca/P** 

The morphology of the fabricated HAP-HPC/PLA electrospun nanofibrous mats was analyzed using SEM. Initially, the optimization study was performed with different parameters such as flow rate (0.5, 0.7, and 1 mL), collector rotation speed (700, 1000, and 1500 rpm), tip-to-collector distance (12, 15, and 17 cm), and voltage (15, 21, and 25 Kv (kilo volts)) as shown in Table 1. Accordingly, the samples were analyzed for the morphological

**2p3/2 2p1/2 2p3/2 2p1/2 Ratio** 

(a) 288.50-(C-C=O) (b) 286.73-(C-O) (c) 286.16-(C-H) (d) 285.75-(C-C/CH2)

(a) 535.68-OH (b) 534.86-P-O (c) 533.71-P-O-P

1.66

In HAP, the main core level of Ca (2P) appears at 348.44 eV region, and the two deconvoluted peaks such as Ca (2P1/2) and Ca (2P3/2) appear at 352.51 eV and 348.50 eV, respectively. Another core level of the P (2P) exists at 137.31.1 eV and 136.86 eV which are

**Table 4.** The binding energy of XPS spectra peak for HAP-HPC/PLA.

HPC/PLA 348.50 352.51 136.86 137.31

*3.7. SEM Morphology and EDAX* 

is specific to HAP [34].

HAP-

In HAP, the main core level of Ca (2P) appears at 348.44 eV region, and the two deconvoluted peaks such as Ca (2P1/2) and Ca (2P3/2) appear at 352.51 eV and 348.50 eV, respectively. Another core level of the P (2P) exists at 137.31.1 eV and 136.86 eV which are responsible for P (2P1/2) and 2(2P3/2), respectively [32,33]. The O1s appearing at 526.02 is deconvoluted into three elements which are present as P-O (535.68 eV), OH (534.86 eV), and P-O-P (533.71 eV), respectively. Additionally, Table 4 confirms the presence of C 1s of HPC and PLA appeared at 287.22 eV and its deconvoluted peak has four different elements at a different binding energy such as 288.50 eV (C-C=O), 286.73 eV (C-O), 286.16 eV (C-H), and 285.75 eV (C-CH3), respectively. Therefore, XPS analysis confirms the nanofiber matrix surface chemistry and the Ca/P (1.67) ratio, which is specific to HAP [34].


**Table 4.** The binding energy of XPS spectra peak for HAP-HPC/PLA.

### *3.7. SEM Morphology and EDAX*

The morphology of the fabricated HAP-HPC/PLA electrospun nanofibrous mats was analyzed using SEM. Initially, the optimization study was performed with different parameters such as flow rate (0.5, 0.7, and 1 mL), collector rotation speed (700, 1000, and 1500 rpm), tip-to-collector distance (12, 15, and 17 cm), and voltage (15, 21, and 25 Kv (kilo volts)) as shown in Table 1. Accordingly, the samples were analyzed for the morphological studies and pore size measurements using SEM and image J software, respectively (Figure 5A). *Polymers* **2022**, *14*, x FOR PEER REVIEW 12 of 22 studies and pore size measurements using SEM and image J software, respectively (Figure 5A).

(**A**)

**Figure 5.** *Cont*.

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**Figure 5.** (**A**) SEM Morphology of electrospun nanofibers at various magnifications and fiber diameter (Insert) of various compositions. (**B**) SEM Morphology of SBF immersed electrospun nanofibers for various time intervals by in vitro bioactivity study. Figure 5A represents the SEM micrograph for HAP-HPC/PLA composite nano-**Figure 5.** (**A**) SEM Morphology of electrospun nanofibers at various magnifications and fiber diameter (Insert) of various compositions. (**B**) SEM Morphology of SBF immersed electrospun nanofibers for various time intervals by in vitro bioactivity study.

fibrous mat of various compositions. The optimum parameters, i.e., a flow rate of 0.7 mL, a collector rotation speed of 1000 rpm, tip-to-collector distance of 15 cm, and voltage of 21 kV gave uniform nanofibers which were bead-free, porous, and non-woven amongst all the HAP-HPC/PLA blends. The other parameters resulted in the formation of improper HAP-HPC/PLA nanofibers. The Ca and P content in the HAP-HPC/PLA nanofibrous mat Figure 5A represents the SEM micrograph for HAP-HPC/PLA composite nanofibrous mat of various compositions. The optimum parameters, i.e., a flow rate of 0.7 mL, a collector rotation speed of 1000 rpm, tip-to-collector distance of 15 cm, and voltage of 21 kV gave uniform nanofibers which were bead-free, porous, and non-woven amongst all the HAP-HPC/PLA blends. The other parameters resulted in the formation of improper HAP-HPC/PLA nanofibers. The Ca and P content in the HAP-HPC/PLA nanofibrous mat were determined using EDAX (energy dispersive analysis X-ray) analysis featured in Figure 6 and Table 5 respectively. The stoichiometric value of HAP is 1.67, which is on par with the stoichiometric Ca/P (calcium/phosphate) ratio of HAP. HAP-HPC/PLA nanofibrous mat of all ratios showed the same stoichiometric ratio of Ca and P content.

The HAP-HPC/PLA mat showed different diameter sizes for each composition ratio of HAP-HPC/PLA fabrication. The higher HAP-containing mat showed a smaller fiber diameter, while the mat with no HAP showed a higher fiber diameter. From Table 6**,** it was observed that the HAP-HPC/PLA nanofibrous mat of the composite ratio 70:30 showed a diameter of 110 ± 66 nm, whereas the mat of composite ratio 40:60 showed a fiber diameter of 277 ± 11 nm. Additionally, as stated earlier, the fiber diameter decreases with an increase in the concentration of HAP. The viscoelastic property may be the cause for

the reduction in diameter size with an increase in HAP concentration since all parameters remained constant. fibrous mat of all ratios showed the same stoichiometric ratio of Ca and P content.

were determined using EDAX (energy dispersive analysis X-ray) analysis featured in Figure 6 and Table 5 respectively. The stoichiometric value of HAP is 1.67, which is on par with the stoichiometric Ca/P (calcium/phosphate) ratio of HAP. HAP-HPC/PLA nano-

**Figure 6.** EDAX analysis of (**a**) HPC/PLA (**b**) 40:60 (**c**) 50:50 (**d**) 60:40 (**e**) 70:30 of HAP-HPC/PLA. **Figure 6.** EDAX analysis of (**a**) HPC/PLA (**b**) 40:60 (**c**) 50:50 (**d**) 60:40 (**e**) 70:30 of HAP-HPC/PLA.


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of HAP-HPC/PLA fabrication. The higher HAP-containing mat showed a smaller fiber **Table 6.** The fiber pore size and diameter calculated from SEM images and solvent replacement method.

The HAP-HPC/PLA mat showed different diameter sizes for each composition ratio


### **Table 6.** The fiber pore size and diameter calculated from SEM images and solvent replacement *3.8. Pore Size and Porosity*

method. **S.no Sample Pore Size (µm) Pore Diameter (nm) Porosity (%)**  1 HPC/PLA 4.54 355 ± 10 59.56 2 40:60 HAP-HPC/PLA 7.21 277 ± 11 62.97 3 50:50 HAP-HPC/PLA 9.23 133 ± 33 75.23 4 60:40 HAP-HPC/PLA 11.21 122 ± 33 89.45 5 70:30 HAP-HPC/PLA 15.54 110 ± 66 98.11 *3.8. Pore Size and Porosity*  It has been observed that the pore size of HAP-HPC/PLA nanofibrous mats increases with increasing HAP concentration owing to a decrease in fiber diameter. The nanofibrous mat of composite ratios 0:100, 40:60, 50:50, 60:40, and 70:30 ratios yielded pore sizes of 4.54, 7.21, 9.23, 11.21, and 15.54 µm, respectively. Favoring cell proliferation and migration, pore size plays a major role in tissue engineering applications [35]. Factors such as cell growth, migration, and nutrient supply completely depend on the pore size and their interconnectivity. Cell migration becomes limited if the pore size is less, and also, decreases in the surface area may lead to limited cell adhesion if the pore size is very high. The interconnection of pores results in providing space for vasculature, required to promote new bone formation. Prior studies suggest that microporosity provides bone growth on scaffolds and it can increase the acting surface area of the mat for protein adsorption, it may also provide osteoblast attachment points during biocompatibility evaluation [36]. The pore size of the mat obtained from SEM images was processed using image J software and Porosity was measured by solvent replacement method. The results obtained from both studies are tabulated in Table 6.

### with increasing HAP concentration owing to a decrease in fiber diameter. The nanofibrous *3.9. Bioactivity*

mat of composite ratios 0:100, 40:60, 50:50, 60:40, and 70:30 ratios yielded pore sizes of The dissolution and precipitation process assists in the formation of an apatite layer on the surface of HAP which is essential for a good calcium-based biomaterial. HAP from the nanofibrous mat releases Ca2+ ions when it is immersed in the SBF solution, hence increasing apatite formation in the surrounding body fluid due to this ionic activity.

It has been observed that the pore size of HAP-HPC/PLA nanofibrous mats increases

The release of Ca2+ ions may lead to an increase in positive charge on the surface of hydroxyapatite in the nanofibrous mat [37]. Further, the calcium-rich surface interacts with the PO<sup>4</sup> 3- ions present in SBF. The calcium and phosphate ions migrate onto the surface and induce precipitation of apatite on the HAP surface of the nanofibrous mat. This formed apatite gets stabilized by the crystallization process and forms a bone analog. In addition, various ions play a key role in the formation of the apatite layer due to the ionic interaction taking place between the mat's surface and the ions present in SBF.

The apatite layer formation on the mat's surface was shown in Figure 5B. The apatite layer formation was obtained by immersing the Mat in SBF solution at different time intervals such as 7, 15, and 30 days. The SEM images exhibit the formation of apatite layers on the nanofibrous mat's surface as least after 7 days of immersion, and it was found that the quantity of apatite formation increases for 15 and 30 days of immersion. Further, the HAP-HPC/PLA nanofibrous mat with a composition ratio of 70:30 shows higher apatite layer formation after 30 days of immersion compared to the other composition ratios. The higher apatite layer formation is due to the high immersion time in SBF.

### *3.10. Degradation*

The biodegradation of polymers attacks the anhydride, ester, amide groups, or even enzymatically cleaves the structural or functional bonds. The molecular weight and structure, composition, crystallinity, and presence of cross-linking in a polymer affect the polymer degradation. In addition, the degradation of a polymer is influenced by the diffusion coefficient of water in the polymer matrix, the hydrolysis rate constant of the ester bond, the diffusion coefficient of the chain fragments within the polymeric matrix, and the solubility of the degradation product [38]. The results are demonstrated in Figure 7a. The fast degradation is due to a higher concentration of polymers (40:60, 50:50) with low molecular weight and the hydrolytic reaction is high in the early stage of degradation. By increasing the concentration of HAP, the ionic exchange (ca2+ and PO<sup>4</sup> <sup>3</sup>−) is very high between the stimulated body fluid and nanofibrous mat when compared with a low concentration of HAP. When the ions entered into the pores, the mineralization process also occurred predominantly. Once the mineralization process occurred, the interchange of ions might be less, and the degradation was reduced.

Figure 7b, the FT-IR analysis confirms by increasing the days of immersion exhibits the polymer decrease that shows the degradation rate. After 4 days of degradation, the nanofiber breakage was obtained and there is no morphology change was observed. In 7 days of degradation, the result confirms that the surface of pore fibers was covered due to the swelling effect. In 15 and 30 days of immersion shows that interconnected fiber shape was merged together and melted during the degradation process. Figure 7c represents the SEM analysis of the SBF-immersed scaffolds at various time intervals. After 4 days of immersion, the nanofibrous scaffold starts to degrade and there was a slight change in the morphology was observed. At 7 days of degradation, the results further confirmed that the pore in the fibers was covered due to the swelling effect. At 15 and 30 days of immersion, the complete coverage of apatite was clearly visible on the surface of the scaffold. From the figure, it was clearly confirmed the process of degradation rate with respect to various time intervals. Hence from the observation, the stability of the scaffold for 4 weeks is confirmed.

**Figure 7.** (**a**). Degradation rate of different composition ratios. (**b**). FTIR analysis of nanofibrous mat at different intervals of SBF immersion. (**c**). SEM analysis of nanofibrous mat at different intervals of SBF immersion **Figure 7.** (**a**). Degradation rate of different composition ratios. (**b**). FTIR analysis of nanofibrous mat at different intervals of SBF immersion. (**c**). SEM analysis of nanofibrous mat at different intervals of SBF immersion.

to the swelling effect. In 15 and 30 days of immersion shows that interconnected fiber shape was merged together and melted during the degradation process. Figure 7c represents the SEM analysis of the SBF-immersed scaffolds at various time intervals. After 4 days of immersion, the nanofibrous scaffold starts to degrade and there was a slight change in the morphology was observed. At 7 days of degradation, the results further confirmed that the pore in the fibers was covered due to the swelling effect. At 15 and 30 days of immersion, the complete coverage of apatite was clearly visible on the surface of the scaffold. From the figure, it was clearly confirmed the process of degradation rate with respect to various time intervals. Hence from the observation, the stability of the scaffold

### *3.11. Cytotoxic Analysis 3.11. Cytotoxic Analysis*

for 4 weeks is confirmed.

The microscopic images of the osteosarcoma (MG-63) cells seeded in HAP-HPC/PLA nanofibrous mat of different ratios (40:60, 50:50, 60:40, and 70:30) are shown in Figure 8a–e. Different patterns of cell proliferation have been observed within two days of culture. Cell proliferation differs at 24 h and 48 h, meaning cell viability is high even after 48 h when compared to 24 h. HPC/PLA nanofibrous mat shows moderate results in cell viability at 48 h (Figure 8a\*) than at 24 h (Figure 8a). Moreover, these microscopic images clearly conclude that complete cell proliferation was observed after 48 h for a 70:30 composition ratio (Figure 8a\*). This may be due to the infiltration of HAP with HPC/PLA composite, hence showing better performance in cell proliferation. The proliferation rate of cells in HAP-HPC/PLA nanofibrous mat is much higher at a 70:30 ratio compared to 40:60, 50:50, and 60:40 composition ratios for both 24 and 48 h cultures, respectively.

and 60:40 composition ratios for both 24 and 48 h cultures, respectively.

**Figure 8.** Cytotoxicity of electrospun nanofibrous (**a**–**e**) (24 h) and (**a\***–**e\***) (48 h) (**a**) HPC/PLA (**b**) 40:60 (**c**) 50:50 (**d**) 60:40 (**e**) 70:30 of HAP-HPC/PLA and its significant difference. **Figure 8.** Cytotoxicity of electrospun nanofibrous (**a–e**) (24 h) and (**a\***–**e\***) (48 h) (**a**) HPC/PLA (**b**) 40:60 (**c**) 50:50 (**d**) 60:40 (**e**) 70:30 of HAP-HPC/PLA and its significant difference.

The microscopic images of the osteosarcoma (MG-63) cells seeded in HAP-HPC/PLA nanofibrous mat of different ratios (40:60, 50:50, 60:40, and 70:30) are shown in Figure 8a– e. Different patterns of cell proliferation have been observed within two days of culture. Cell proliferation differs at 24 h and 48 h, meaning cell viability is high even after 48 h when compared to 24 h. HPC/PLA nanofibrous mat shows moderate results in cell viability at 48 h (Figure 8a\*) than at 24 h (Figure 8a). Moreover, these microscopic images clearly conclude that complete cell proliferation was observed after 48 h for a 70:30 composition ratio (Figure 8a\*). This may be due to the infiltration of HAP with HPC/PLA composite, hence showing better performance in cell proliferation. The proliferation rate of cells in HAP-HPC/PLA nanofibrous mat is much higher at a 70:30 ratio compared to 40:60, 50:50,

Hydrophobicity of the mat increases due to the cross-linking phenomenon and hence exhibiting cell adhesion as well as improved cellular functions. HPC and HAP usually show high biocompatibility and mechanical properties, therefore the cellulose derivatives are used as an important biomaterial for the fabrication of tissue engineering scaffolds. These biodegradable scaffolds are mainly used in tissue regeneration applications since the biodegradation rate of the scaffolds matches the biological process which takes place in tissue regeneration. Often, slow biodegradable scaffolds are preferred for regeneration applications due to minimal risks [39-41]. Interestingly, cellulose is an important biomaterial candidate for the design of tissue engineering scaffolds. The cells attach, grow and stimulate tissue growth depending on the stability of scaffolds in the body fluid; hence, scaffolds should be insoluble in water. Hydrophobicity of the mat increases due to the cross-linking phenomenon and hence exhibiting cell adhesion as well as improved cellular functions. HPC and HAP usually show high biocompatibility and mechanical properties, therefore the cellulose derivatives are used as an important biomaterial for the fabrication of tissue engineering scaffolds. These biodegradable scaffolds are mainly used in tissue regeneration applications since the biodegradation rate of the scaffolds matches the biological process which takes place in tissue regeneration. Often, slow biodegradable scaffolds are preferred for regeneration applications due to minimal risks [39–41]. Interestingly, cellulose is an important biomaterial candidate for the design of tissue engineering scaffolds. The cells attach, grow and stimulate tissue growth depending on the stability of scaffolds in the body fluid; hence, scaffolds should be insoluble in water.

Figure 8 shows the results of the MTT assay at 24 h and 48 h at various composition ratios of 40:60, 50:50, 60:40, and 70:30 of HAP-HPC/PLA nanofibrous mat. The mat of ratio 70:30 is found to have the highest cell proliferation rate compared to other ratios at both 24 and 48 h. The electrospun nanofiber at 70:30 ratios showed a significant difference with *p* < 0.01 (Figure 8) levels in the cell proliferation for 48 h, respectively. The obtained results confirm that the cells were attached and proliferated on HAP-HPC/PLA nanofibrous mat. This may be due to the presence of β-glucose linkages in HPC. The β-glucose linkage of carbohydrate derivatives plays a major role in cell metabolism, and it activates the formation of HAP. High cell proliferation was achieved at the highest concentration of HAP Figure 8 shows the results of the MTT assay at 24 h and 48 h at various composition ratios of 40:60, 50:50, 60:40, and 70:30 of HAP-HPC/PLA nanofibrous mat. The mat of ratio 70:30 is found to have the highest cell proliferation rate compared to other ratios at both 24 and 48 h. The electrospun nanofiber at 70:30 ratios showed a significant difference with *p* < 0.01 (Figure 8) levels in the cell proliferation for 48 h, respectively. The obtained results confirm that the cells were attached and proliferated on HAP-HPC/PLA nanofibrous mat. This may be due to the presence of β-glucose linkages in HPC. The β-glucose linkage of carbohydrate derivatives plays a major role in cell metabolism, and it activates the formation of HAP. High cell proliferation was achieved at the highest concentration of HAP in the HAP-HPC/PLA nanofibrous mat, hence proving the fact that this composition is non-toxic and significant material for regeneration and the rejuvenation of bone tissues.

### *3.12. In Vivo Study*

### 3.12.1. X-Ray Radiology Results

Figure 9 shows the X-ray examination of with and without implantation of the HAP-HPC-PLA nanofibrous composite. Instead of new bone formation, the hollow void space was observed in the defected area and is clearly visible in Figure 9A,C. Further, the results confirmed that there was no new bone formation at 4 and 8 weeks of implantation. Figure 9B displays, no osteogenesis, and bone formation were observed at 4 weeks of HAP-HPC-PLA implantation. The border of the unfilled void with no osteogenesis was observed at 8 weeks of implantation with HAP-HPC-PLA nanofibrous mat (Figure 9D). When compared with

4 weeks of implantation, 8 weeks of implantation confirmed the new bone formation. Further study is in progress to study the effect of new bone formation with respect to different ratios of HAP in the scaffolds. 8 weeks of implantation with HAP-HPC-PLA nanofibrous mat (Figure 9D). When compared with 4 weeks of implantation, 8 weeks of implantation confirmed the new bone formation. Further study is in progress to study the effect of new bone formation with respect to different ratios of HAP in the scaffolds.

in the HAP-HPC/PLA nanofibrous mat, hence proving the fact that this composition is non-toxic and significant material for regeneration and the rejuvenation of bone tissues.

Figure 9 shows the X-ray examination of with and without implantation of the HAP-HPC-PLA nanofibrous composite. Instead of new bone formation, the hollow void space was observed in the defected area and is clearly visible in Figure 9A,C**.** Further, the results confirmed that there was no new bone formation at 4 and 8 weeks of implantation. Figure 9B displays, no osteogenesis, and bone formation were observed at 4 weeks of HAP-HPC-PLA implantation. The border of the unfilled void with no osteogenesis was observed at

*Polymers* **2022**, *14*, x FOR PEER REVIEW 18 of 22

*3.12. In Vivo Study* 

3.12.1. X-Ray Radiology Results

**Figure 9.** X-ray images of (**A**) without implantation at 4 weeks (**B**) HAP-HPC-PLA at 4 weeks (**C**) without implantation at 8 weeks (**D**) HAP-HPC-PLA at 8 weeks. **Figure 9.** X-ray images of (**A**) without implantation at 4 weeks (**B**) HAP-HPC-PLA at 4 weeks (**C**) without implantation at 8 weeks (**D**) HAP-HPC-PLA at 8 weeks.

### 3.12.2. Histological Analysis 3.12.2. Histological Analysis

Figure 10 illustrates the highlights of the study of animal experiments. The histological section of in vivo studies with and without HAP-HPC-PLA polymer composite on rat skull defected area. The results demonstrated that the scaffolds promoted bone bonding activity at 4 and 8 weeks of implantation. The results of radiology and histology analysis indicated that this scaffold facilitated bone formation in the defects with excellent potential in bone defect repair. Figure 10 illustrates the highlights of the study of animal experiments. The histological section of in vivo studies with and without HAP-HPC-PLA polymer composite on rat skull defected area. The results demonstrated that the scaffolds promoted bone bonding activity at 4 and 8 weeks of implantation. The results of radiology and histology analysis indicated that this scaffold facilitated bone formation in the defects with excellent potential in bone defect repair.

Figure 10B shows the development of new cells and blood vessels at 4 weeks of HAP-HPC-PLA nanofiber implantation. When compared with 4 weeks intervals, 8 weeks of implantation of HAP-HPC-PLA nanofibers showed enhanced cell proliferation such as osteoblast, osteocyte with lacunae and osteoclast (Figure 10B1). The new bone formation was started to grow at 8 weeks of implantation. Architectural modification for scaffold such as pore size and fiber diameter also seems necessary for osteoblasts to favorably attach and grow on the hybrid scaffolds to substitute collagen sponge. Such scaffolds were reported to degrade in vivo after 2–6 months after their implantation [42]. Additionally, Pektok et al. discussed the use of scaffolds with better healing properties in vivo. They showed that faster extracellular matrix formation was achieved with the decomposition of nanofibers grafts [43]. So, these nanofibers with excellent healing properties can be applied for biomedical applications. Both in vitro and in vivo studies verified that these novel layered scaffolds can effectively deliver growth factors with better cell migration in a controlled manner for bone repair by promoting the healing process.

**Figure 10.** Histological images of (**A**) without implantation at 4 weeks (**B**) HAP-HPC-PLA at 4 weeks (**A1**) without implantation at 8 weeks (**B1**) HAP-HPC-PLA at 8 weeks. **Figure 10.** Histological images of (**A**) without implantation at 4 weeks (**B**) HAP-HPC-PLA at 4 weeks (**A1**) without implantation at 8 weeks (**B1**) HAP-HPC-PLA at 8 weeks.

### Figure 10B shows the development of new cells and blood vessels at 4 weeks of HAP-**4. Conclusions**

HPC-PLA nanofiber implantation. When compared with 4 weeks intervals, 8 weeks of implantation of HAP-HPC-PLA nanofibers showed enhanced cell proliferation such as osteoblast, osteocyte with lacunae and osteoclast (Figure 10B1). The new bone formation was started to grow at 8 weeks of implantation. Architectural modification for scaffold such as pore size and fiber diameter also seems necessary for osteoblasts to favorably attach and grow on the hybrid scaffolds to substitute collagen sponge. Such scaffolds were reported to degrade in vivo after 2–6 months after their implantation [42]. Additionally, Pektok et al. discussed the use of scaffolds with better healing properties in vivo. They showed that faster extracellular matrix formation was achieved with the decomposition of nanofibers grafts [43]. So, these nanofibers with excellent healing properties can be ap-Bone tissue engineering applications require fibrous mats, which have desired properties such as proper chemical integrity and crosslinking efficiency, and biodegradable properties to interact in the void space of the human native tissue without dissolving. Addressing porosity and optimum interconnectivity, optimum fiber diameter is required to ensure the necessary infiltration of cells and nutrients. The current study optimized the parameters required to produce the nanofibrous mat with the required porosity in terms of interconnectivity, mechanical property, bioactivity, and biocompatibility. Different concentrations of HAP were chosen to fabricate the nanofibrous mat resulting in the observation that the composite ratio of 70:30 exhibits all the desired properties required to be considered as an ideal biomaterial.

plied for biomedical applications. Both in vitro and in vivo studies verified that these novel layered scaffolds can effectively deliver growth factors with better cell migration in a controlled manner for bone repair by promoting the healing process. ✓ The XRD results confirm the existence of HAP in the presence of a polymeric network and it was found that the triplet peak at 31.89◦ , 32.05◦ , and 32.20◦ increases with an increase in the HAP concentration in the fibrous mat.

**4. Conclusions**  Bone tissue engineering applications require fibrous mats, which have desired properties such as proper chemical integrity and crosslinking efficiency, and biodegradable ✓ The mechanical property of 9.53 Mpa was obtained for the optimized composition with a high rate of HCA formation on SBF immersion, this may be due to the interconnected polymeric network and porosity of the sample which was confirmed favorable for cellular activity.

properties to interact in the void space of the human native tissue without dissolving. Addressing porosity and optimum interconnectivity, optimum fiber diameter is required to ensure the necessary infiltration of cells and nutrients. The current study optimized the parameters required to produce the nanofibrous mat with the required porosity in terms ✓ The retention of the Ca/P ratio of HAP in the polymeric network was analyzed by XPS analysis. Finally, the biocompatibility evaluation on the MG-63 osteoblast cell line was conducted for 24 h and 48 h which deemed the material fit for biomedical applications. All the compositions revealed enhanced cell proliferation at 48 h of duration.

of interconnectivity, mechanical property, bioactivity, and biocompatibility. Different concentrations of HAP were chosen to fabricate the nanofibrous mat resulting in the observation that the composite ratio of 70:30 exhibits all the desired properties required to ✓ In vivo animal study confirmed the effective bone formation at the 8th week of implantation of HAP-HPC-PLA grafted in the defective area with more cell differentiation when compared with the 4th week of implantation.

be considered as an ideal biomaterial.

Hence, from the above study, the fabricated nanofibrous mat of this composition ratio is found to be the desirable type for bone tissue engineering applications such as bone void filling, repair of bone damage, and in vitro and in vivo bone disease modeling.

**Author Contributions:** Conceptualization, methodology and writing.; U.V. and S.M.S.; formal analysis and data curation.; T.M.S. resources, R.R. and J.G. All authors have read and agreed to the published version of the manuscript.

**Funding:** APC was funded by Vellore Institute of Technology.

**Institutional Review Board Statement:** The animal study protocol was approved by the Institutional Animal Ethical Committee of Vellore Institute of Technology (VIT/IAEC/14/Nov5/47 dated November 5, 2017)." for studies involving animals.

**Acknowledgments:** The authors would like to thank the management of VIT, Vellore, Tamil Nadu for rendering the necessary laboratory and characterization facilities and financial support for this manuscript.

**Conflicts of Interest:** All the authors declare no conflict of interest.

## **References**


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