hydroxyapatite as wound dressings: Morphology, cell adhesion, and antibacterial activity

Journal Pre-proofs Polycaprolactone based electrospun matrices loaded with Ag/hydroxyapatite as wound dressings: Morphology, cell adhesion and antibacterial activity Abeer A. Hassan, Hyam A. Radwan, Said A. Abdelaal, Najlaa S. Al-Radadi, M.K. Ahmed, Kamel Shoueir, Mayssa Abdel Hady PII: DOI: Reference:

S0378-5173(20)31128-5 https://doi.org/10.1016/j.ijpharm.2020.120143 IJP 120143

To appear in:

International Journal of Pharmaceutics

Received Date: Revised Date: Accepted Date:

3 July 2020 25 November 2020 28 November 2020

Please cite this article as: A.A. Hassan, H.A. Radwan, S.A. Abdelaal, N.S. Al-Radadi, M.K. Ahmed, K. Shoueir, M. Abdel Hady, Polycaprolactone based electrospun matrices loaded with Ag/hydroxyapatite as wound dressings: Morphology, cell adhesion and antibacterial activity, International Journal of Pharmaceutics (2020), doi: https://doi.org/10.1016/j.ijpharm.2020.120143

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Polycaprolactone based electrospun matrices loaded with Ag/hydroxyapatite as wound dressings: Morphology, cell adhesion and antibacterial activity Abeer A. Hassana,b, Hyam A. Radwana,b, Said A. Abdelaalc, Najlaa S. Al-Radadid, M. K. Ahmede, f,*, Kamel Shoueirg, Mayssa Abdel Hady h

a

Department of Chemistry, College of Science, King Khalid University, Abha, Saudi Arabia

b

Department of Chemistry, Faculty of Women for Arts, Science and Education, Ain Shams University, Cairo, Egypt

c Department d Chemistry

of Chemistry, Faculty of Science, Jazan University, Saudi Arabia,

Department, Faculty of Science, Taibah University, P.O. Box 30002, Al-Madinah

Monawara 14177, Saudi Arabia e f

g

Department of Physics, Faculty of Science, Suez University, Suez, 43518, Egypt Egypt Nanotechnology Center (EGNC), Cairo University, El‑Sheikh Zayed 12588, Egypt Institute of Nanoscience & Nanotechnology, Kafrelsheikh University, 33516, Kafrelsheikh, Egypt

h

Department of Pharmaceutical Technology, National Research Centre, Dokki, Cairo, Egypt

*Corresponding

author: *M. K. Ahmed: e-mail: [email protected] Abstract The development of a scaffold matrix that can inhibit bacterial infection and promote wound healing simultaneously is an essential demand to improve the health care system. Hydroxyapatite (HAP) doped with different concentrations of silver ions (Ag+) were incorporated into electrospun nanofibrous scaffolds of polycaprolactone (PCL) using the electrospinning technique. The formed phase was identified using XRD, while the morphological and roughness behavior were investigated using FESEM. It was shown that scaffolds were configured in randomly distributed nanofibers with diameters around of 0.19-0.40, 0.31-0.54, 1.36, 0.122-0.429 μm for 0.0AgHAP@PCL, 0.2Ag-HAP@PCL, 0.6Ag-HAP@PCL, and 0.8Ag-HAP@PCL, respectively. Moreover, the maximum roughness peak height increased significantly from 179 to 284 nm, with 1

the lowest and highest contributions of Ag. The mechanical properties were examined and displayed that the tensile strength increased from 3.110.21 MPa to its highest value at 3.570.31 MPa for 0.4Ag-HAP@PCL. On the other hand, the cell viability also was enhanced with the addition of Ag and improved from 97.14.6 % to be around 102.33.1 % at the highest contribution of Ag. The antibacterial activity was determined, and the highest imbibition zones were achieved at the highest Ag dopant to be (12.51.1 mm and 11.41.5 mm against E. coli and S. aureus). The in vitro cell proliferation was observed through human fibroblasts cell lone (HFB4) and illustrated that cells were able to grow and spread not only on the fibers’ surface but also, they were spreading and adhered through the deep pores. Keywords: Wound healing; electrospun nanofibers; hydroxyapatite; silver. 1. Introduction Restoring skin functionality is a serious concern to keep its role as a protective barrier and immunoreactive defender [1, 2]. However, this repair could be accompanied by obstacles, especially with burns or chronic injuries, as well as diabetic ulcers. It could be stated that around 425 million diabetic patients are suffering from chronic wounds [2]. Wound healing is a complicated process; however, it could be summarized into three main stages; inflammation, proliferation, and finally, remodeling [2]. The inflammation starts after the stopping of blood flowing and contains intricate interplay through the wound milieu [2-4]. The proliferation stage commences after 3 days of the former step approximately. It contains the formation of a new extracellular matrix (ECM) based on the proliferation and migration of fibroblasts [5-7]. Moreover, fibroblasts are provoked in this stage to be differentiated into myofibroblasts to close the wound [8, 9]. The last process (remodeling) may start after 2-3 weeks, which could be done via increasing the rate of crosslinking and hence the formation of normal tissue [10-12]. The former stages represent the normal pathway of wound healing; however, the deviation in these processes may lead to chronicity behavior. Such deviation is accompanied by a delay owing to chronic disease [2]. Furthermore, in this scope, a bacterial infection is prospected to be grown, and thus may delay the skin regeneration or even lead to a failure of healing [13-16]. An intensive effort has been made to introduce a scaffold membrane with multifunctional behaviors, including antibacterial and anti-fungi. Nanofibrous scaffolds based on polymeric 2

blending is a facile strategy to combine versatile advantages such as; high surface area to volume, high porosity, and high permeability, besides offering delivery of drugs to inhibit bacterial growth [17-19]. Nanofibrous membranes could be produced via electrospinning technique, while their properties, including fibers’ diameters, porosity ratio, homogeneity, and orientation, can be controlled via preparation conditions. Furthermore, electrospun nanofibrous scaffolds could stimulate the structure of ECM, which may introduce an appropriate structure for wound dressing usage [20-22]. -Polycaprolactone (PCL) is one of the most polymeric substances that have been examined for biomedical applications including wound healing, drug delivery, and bone regeneration [23-25]. It is a semi-crystalline polymer, while it shows good mechanical properties, biocompatibility, slow biodegradability, and high capacity. However, it possesses high hydrophobicity, which may put an obstacle against enlarging its real utilization. This could be done by a modification of the produced nanofibers using a new substance to provoke a high roughness, and hence a high tendency to be adhered to over the ambient environment. Different additional materials could be added to PCL nano-membranes, including bioceramic substances. Hydroxyapatite (HAP) is one of the most bioceramics that have been used for clinical applications due to its similarity to the mineral constituent of bone tissue [26-28]. Bone tissue consists of two main parts; collagen which is formed in the nanofibrous scaffold, whereas HAP crystals are embedded through these oriented fibers. This hierarchical design offers high resistance to both compression and tensile strengths, besides the high porosity which ensures permeability and low density. However, it should be mentioned that biological HAP is not a pure one; rather, it contains versatile dopants including carbonate ions, Mg, F, Fe ...[29-31]. Therefore, stimulation of this design includes HAP dopant to be carried in a polymeric nanofiber. The overuse of antibiotics provokes a formation of bacterial resistance; therefore, the suggestion of inorganic nanoparticles with antibacterial effectiveness may circumvent this obstacle [32-34]. These nanoparticles such as Ag, ZnO, and CuO, however, the compositional singularity introduces a simple technique for controlling behavior; therefore, an antibacterial agent that is suggested should include Ag during the ionic substitution within the HAP structure rather than disparate nanoparticles [35, 36]. It could be stated that Ag ions were used intensively as an antibacterial agent; for instance, S. F. Mansour

3

studied the antibacterial efficiency of Mg/Ag co-dopant into HAP, and the results displayed that increasing of Ag ions has inhibited the bacterial growth with efficiency reaching to 75 % [37]. Combining the former ideas, this work aims to introduce nanofibrous scaffolds of PCL containing HAP doped with different concentrations of Ag ions. Thus, structural and morphological behavior could be investigated, whereas the antibacterial and the biological response towards the scaffolds will be investigated. 2. Materials and methods 2.1. Synthesis of Ag-HAP@PCL nanofibrous Calcium

chloride

dihydrate

[CaCl2.2H2O],

diammonium

hydrogen

phosphate

[(NH4)2HPO4], silver nitrate [AgNO3], chloroform (99.5 %), and methanol (99 %) were purchased from LOBA, India, and they were utilized without further purifications. PCL (Mw = 80,000 g/mol) was purchased from Sigma-Aldrich. Firstly, 8 g of PCL (pellets) was dissolved through 100 ml of the solvent (66.7 ml of chloroform + 33.3 ml of methanol) to obtain a PCL solution (8 %). Then HAP was synthesized using the co-precipitation method. To obtain Ag-free HAP; 0.5 M of [CaCl2.2H2O] was dissolved in 50 mL of distilled water, and 0.3 M of [(NH4)2HPO4] in 50 mL. Next, phosphorous (P) solution was added drop wisely with a rate of 1 mL/s into the Ca solution, whereas the pH of the (Ca+P) solution was fixed at 11.00  0.05. Ag-HAP samples were synthesized with addition of 0.01, 0.02, 0.03, or 0.04 M of AgNO3 on account of Ca content, while Ca concentrations could be 0.49, 0.48, 0.47, and 0.46 M upon the equation: 𝑥𝐴𝑔𝑁𝑂3 + 5(𝑁𝐻4)2𝐻𝑃𝑂4 + (10 ― 0.5𝑥)𝐶𝑎𝐶𝑙2.2𝐻2𝑂 𝐴𝑔𝑥𝐶𝑎(10 ― 0.5𝑥)(𝑃𝑂4)6(𝑂𝐻)2 + 3( 𝑁𝐻4)(𝑁𝑂3) + 6(𝑁𝐻4)𝐶𝑙 (1) Where 0.0 x 0.8, with a step of 0.2. After the addition of (Ca+Ag) into P solution, the stirring is kept at 1200 rpm for 1 h, and the solution was aged for 24 h. Then the precipitate was filtered and washed via double distilled water several times and dried at 50-60 oC before use. The nanofibers solutions were prepared via the addition of 110 mg of each powder sample of (Ag-HAP) to 10 ml of PCL (8 %) in a sealed bottle to be suspended using an ultrasonic probe device for 15 min. Then the obtained viscous gel was introduced into a syringe pump to be 4

produced via electrospinning at fixed processing parameters. The operating parameters of the electrospinning technique are; high voltage is around 180.1 kV, injection rate of 1 mL/h, distance gap between the electrode and the wall is 15 cm, while the needle of the syringe is 22φ. The flow diagram of the synthesis process is illustrated in Fig. 1. 2.2.

Measurements

2.2.1. XRD analysis X-ray diffraction (XRD) patterns were recorded on a (Pertpro, Cu kα1 radiation, λ=1.5404 Å, 45 kV, 40 mA, USA) diffractometer. The taken range at 2 =5-60o and the average crystalline size of the PCL nanofibrous containing Ag-HAP at different contents was calculated using the well-known Debye–Scherrer equation [37]. 2.2.2.

FTIR spectra Fourier transformed infrared (FTIR) spectral analysis was recorded using (Perkin-Elmer

2000) in the range 4000-400 cm-1, 4cm-1 resolution. 2.2.3.

FESEM The morphology and roughness were discussed by a field emission scanning electron

microscope (FESEM) to investigate the nanofibrous scaffold’s morphology and to scan the surface roughness using an operating voltage around 20-30 kV (model: QUANTA-FEG250, Netherlands). Micrographs of nanofiber scaffolds were obtained using FESEM before cultivation with cell lines and were treated using Gwyddion 2.45 software to scan their roughness behavior [26]. Firstly, the 3D micrographs were processed for each composition whereas the resolution was kept at 1450×950 pixels. The edges of micrographs were removed to avoid excess boundaries. Then, the dependency of roughness parameters on the variation of compositions was estimated via the same software in (nm). 2.2.4. Porosity measurement The samples were processed in rectangular pieces of (23) cm for each sample to be ready for the analysis. It was then introduced into a sealed cell, while the helium gas has flowed within the crucible, and then real density was obtained and derived from three independent results on each 5

sample. The measured porosity and density have been repeated three times to obtain the standard deviation. 2.2.5. Mechanical properties Mechanical testing was applied onto the prepared samples, the samples measured in a uniform shape of strips with dimensions of (80200.1) mm. Then the stress-strain examination was done by pulling the scaffold with a rate of 5 mm/min reaching the fracture point upon the standard code of ASTM D882. Furthermore, the mechanical test has been repeated three times to obtain the statical investigation. 2.3.

In vitro cell viability tests The human osteoblast cell line HFB4 was grown in Dulbecco's modified Eagle medium

(DMEM, Gibco) at 37 °C and 5% CO2 to investigate the viability of cells sown on microfibers. Cells seeded at a density of 5 × 103 cells / cm2 were grown on fibers in 12-well plates. After three days of incubation, the medium was removed and MTT (3- (4,5-dimethylthiazol-2-yl) -2,5diphenyltetrazolium bromide) was introduced into each well, after which its cell viability was determined using an optical analyzer was determined. It could be reported the cell viability has been repeated 3 times to get calculate the standard deviation. The cell viability is the percentage of viable cells in the total number of cells [29]: Viability (%) = 2.4.

Mean optical density of test samples Mean optical density of the control

100

(2)

Antibacterial activity The activity of antibacterial was investigated against two types of bacteria: Gram-positive

(Staphylococcus aureus) and Gram-negative (Escherichia coli) under the same conditions. The starting concentration of the nanofibrous scaffolds was introduced as 50 mg/ml. The antibacterial activity was then scanned after an incubation time of 3 days at 37 oC. The same experiment has been repeated three times to obtain the standard deviation. 2.5.

Cells growth on the scaffold FESEM has been used to monitor the behavior of human HFB4 osteoblast cells seeded on

nanofibers. Each sample was cut into two parts ranging in size from 0.5 to 0.5 cm, which were then exposed to a 12-well plate UV lamp for 30 minutes for sterilization. Followed by adding 1.5 6

mL of HFB4 cells (5 x 105 cells) to each well. Finally, the plate was covered and incubated at 37 °C for several days. The fibers were washed with phosphate-buffered saline (PBS). To allow the cells to attach to the surface of the nanofibers, the scaffolds were immersed in a glutaraldehyde solution (2.5% concentration) for two hours. They were then dehydrated with air for 30 minutes. Finally, they were sprayed with gold for 1 minute to be ready for FESEM imaging. 3. Results and discussion 3.1. Phase investigation The XRD patterns are shown in Fig. 2 indicates the formation of PCL containing HAP. The high peaks about 2 = 21.45 o and 23.81o could be attributed to the semi-crystalline PCL. The low peak which is detected around 2 = 31.81 o, 32.15 o, 34.15 o, 39.77 o, and 1.94 o refer to the Miller indices of (211), (112), (202), (130), and (222) of HAP. It could be shown that HAP was crystallized in a hexagonal symmetry upon the ICDD card no. 01-073-0293. The observed low peaks of HAP could be assigned to the lower contribution of HAP compared with PCL, besides it could be also because HAP is an encapsulated interior of PCL nanofibers. Table 1. The dependency of the measured density () and porosity ratio of Ag-HAP@PCL nanofibrous scaffolds upon Ag content

Composition

 (g/cm3)

0.0Ag-HAP@PCL

0.1120.02

91.95.3

0.2Ag-HAP@PCL

0.1240.05

91.12.4

0.6Ag-HAP@PCL

0.2030.06

85.44.5

0.6Ag-HAP@PCL

0.1340.03

90.43.8

0.8Ag-HAP@PCL

0.1750.04

87.44.6

Porosity (%)

The real density of the nanofibrous scaffolds is reported in Table 1, and it was 0.1120.02 g/cm3 and increased to its highest value at 0.2030.06 g/cm3, and reduced to be around 0.1750.04 g/cm3 for 0.0Ag-HAP@PCL, 0.6Ag-HAP@PCL, and 0.8Ag-HAP@PCL respectively. The calculated porosity was reached its lowest value of 85.44.5 %, and its highest one of 91.12.4 % 7

for 0.6Ag-HAP@PCL and 0.0Ag-HAP@PCL respectively. It could be stated that the porosity ratio has a pivotal effect on both mechanical properties and cells growth. Even though porosity may cause deterioration of tensile strength, it may facilitate cell growth by acting as a nutrient transporter. Therefore, controlling porosity is an important parameter to develop a proper nanofibrous matrix. 3.2. FTIR spectra FTIR spectral investigation is shown in Fig. 3. The bands around 569.5 and 599.2 cm-1 denotes the formation of the stretching mode of (4) belong to the vibration of PO34 ― [38-40]. The band of 960.2 cm-1 is assigned to the band of (1) PO34 ― [29, 38], while the bonding modes of CO and C-C are detected around 1291.4 cm-1. The bands of 1415.8, 1463.4 cm-1 are assigned to the presence of CO33 ― group, which is suggested to substitute PO34 ― position (B-type) or (OH)- position (A-type) or both of them simultaneously (AB type). This affinity of carbonated structural substitution comes from the susceptibility of HAP lattice to absorb CO2 from the ambient air during the synthesis stage. However, the carbonated substitution introduces a beneficial behavior because it may enhance the biodegradation rate of HAP, and thus increase its bioactivity towards the physiological environment. Furthermore, the bands around 1727.5 and 2937.4 cm-1 could be attributed to the presence of C=O bonding mode and stretching mode of C–H system, while the range starting from 3068.5 up to 3436.6 cm-1 refers to the vibrational mode of O–H [30, 39, 41]. The characteristics bands of FTIR are reported in Table 2. Table 2 FTIR characteristic bands of Ag-HAP@PCL nanofibrous scaffolds. 0.0Ag-

0.2Ag-

HAP@PCL

HAP@PCL HAP@PCL HAP@PCL HAP@PCL

454.2

453.3

569.5

---

599.2

0.4Ag-

0.6Ag-

454.2

0.8Ag-

Assignment

Ref.

(4) of O–P–

[26]

454.2

453.0

569.1

571.5

571.2

597.1

---

---

598.2

730.2

729.3

731.1

730.3

731.1

Se-O

960.2

959.3

960.5

960.6

961.7

(1) PO34 ―

8

O (4)PO34 ―

[38-40]

[41, 42]

(3) PO34 ―

[43]

stretching of

[44]

1042.2

1043.5

1041.2

1043.6

1044.4

1103.3

1103.5

1102.2

1101.3

1101.3

1181.1

1177.3

1178.5

1181.6

1181.6

Vibration of

1241.1

1239.4

1238.5

1240.8

1238.8

C–O–C

1291.4

1293.3

1291.4

1292.5

1290.5

1415.8

1415.4

1416.6

1418.6

1416.7

P–O

C-O and C-

[41, 45] [42, 45]

C (3) CO33 ―

[46, 47]

(B-type) CO33 ― (B-

[44, 46]

1463.4

1461.5

1463.6

1462.5

1461.6

1727.5

1725.2

1724.5

1725.8

1726.6

2862.5

2861.5

2858.5

2860.6

2861.5

2937.4

2931.5

2933.6

2936.6

2935.7

C–H

---

3068.5

---

---

---

Vibration of

[30,

---

---

---

3210.6

O–H

41]

3224.4

---

---

---

---

---

---

3255.5

3259.6

---

3435.4

---

---

3436.6

---

type) C=O

[42, 45]

Stretching of [42, 45]

3.3. Morphological investigation The morphological behavior upon the variation of Ag into HAP through nanofibrous scaffolds of PCL is shown in Fig. 4(a-d). It could be noticed that at no addition of Ag, the nanofibrous was formed in a non-oriented network with diameters in the range of 0.19-0.40 μm. A high ratio of interconnected porosity was found, while the fibers tend to be with low roughness. The nanofibers are branched, which indicates their good coherence. The composition of 0.2AgHAP@PCL is depicted in Fig. 4(b), it was configured as a network with diameters around 0.310.54 μm. The interconnected porosity ratio and surface roughness seem to be higher than the former one. The higher composition (0.6Ag-HAP@PCL) is illustrated in Fig. 4(c) was formed as networked fibers in two classes of diameters; 25-51 nm and 1.36 μm. It could be seen that the thin 9

39,

class is branched from the larger one, which may indicate their good tensile resistance. Furthermore, small spherical shapes tend to be scattered through the nanofibers with approximate diameters of about 15.4-25.6 nm. These spheres may refer to the presence of AgNPs rather than the ionic Ag that was hypothesized to be substituted into HAP lattice. It could be stated that Ag+ prefers to substitute Ca2+ sites partially reaching a crystallographic limit; in which the distortion of HAP crystal does not accept a more ionic replacement. At this time, there is a tendency to extrude the additional ions to be deposited on the fibers’ surface. The highest contribution of Ag (0.8Ag-HAP@PCL) was illustrated in Fig. 4d and is shown to be randomly distributed nanofibers with diameters starting from 0.122 μm and reaching to 0.429 μm. The dispersed spherical spots that may refer to the formation of AgNPs display grain diameters around 12.2-30.7 nm. The behavior of surface roughness is shown in Fig. 5(a-d). It could be noticed from Table 3, that the roughness average (Ra) starts from 32 nm at 0.0Ag-HAP@PCL, then it decreases to be 26.1 nm at 0.2Ag-HAP@PCL. The addition of Ag into the composition induces an increase of Ra to be around 30 nm for both 0.6Ag-HAP@PCL and 0.8Ag-HAP@PCL. It could be seen that the root mean square roughness (Rq) follows the trend of Ra. On the other hand, the maximum height of the roughness (Rt) begins with 321 nm at no contribution of Ag and then increases significantly to be 489.7 nm at 0.2Ag-HAP@PCL. The high concentrations of Ag provoke a slight decrease of Rt to be around 454 nm. Moreover, both the maximum roughness valley depth (Rv) and the maximum roughness peak height (Rp) possess a similar trend. In other words, Rp reaches 179 nm at no additional Ag, increases to 277, 250 nm, and 284 nm for 0.2Ag-HAP@PCL, 0.6AgHAP@PCL, and 0.8Ag-HAP@PCL respectively. This noticeable trend for both Rv and Rp about Ra and Rq refers to the high divergence among peaks and notches. In detail, while Ra indicates the average values of both peaks and notches, Rv refers to notches solely and Rp represents peaks only. The high difference between those two properties (peaks and notches) comes from versatile sources, including crystallographic defects, compositional disorder, besides the preparation conditions consequences. It could be benefited from this trend in two types of adhesion mechanisms [48-50]. The integration between the nanofibrous scaffold and the host tissue requires physical adhesion which could be done via the interlocking procedure and chemical cohesion that is facilitated by crystallographic defect sites [51-53]. The higher surface roughness is the lower contact angle, which may induce higher 10

bioactivity towards the host environment; and hence good correlation is expected to be done via these compositional nanofibrous scaffolds. Table. 3 Surface roughness parameters of the Ag-HAP@PCL fibers upon the Ag content including roughness average (Ra), root mean square roughness (Rq), the maximum height of the roughness (Rt), maximum roughness valley depth (Rv), maximum roughness peak height (Rp), the average maximum height of the roughness (Rtm).

Composition 0.0Ag-HAP@PCL 0.2Ag-HAP@PCL 0.6Ag-HAP@PCL 0.8Ag-HAP@PCL

Ra (nm) 32 26.1 30 30

Rq (nm) 41 38.7 40 42

Rt (nm) 321 489.7 454 454

Rv (nm) 142 212.6 204 169

Rp (nm) 179 277 250 284

Rtm (nm) 253 259.9 280 321

3.4. Mechanical properties The resistance of nano scaffolds towards mechanical loading is a pivotal factor in estimating the effectiveness of the implanted biomaterial. Mechanical properties of the nanofibers of PCL containing Ag-HAP tend to be varied considerably upon the change of compositional contributions. As illustrated in Fig. 6, the tensile strength started from 3.110.21 MPa at no contribution of Ag ions, and changed to be around 2.350.25 MPa at 0.2Ag-HAP@PCL, then it achieved its highest value at 3.570.31 MPa for 0.4Ag-HAP@PCL, and then deteriorated to be 2.330.34 MPa at the highest contribution of Ag. The strain at break is an important factor because it denotes the ability to be stretched. It was around 60.24.3 % at no additional Ag, and grew to 107.85.6 % at 0.2Ag-HAP@PCL, then deteriorated to reach its lowest value of 82.93.6 % at 0.4Ag-HAP@PCL. The total energy that could be absorbed through the scaffold before the break is defined as the roughness, which begun with 1.130.11 MJ/m3 at no Ag addition, and enhanced up to 1.720.19 MJ/m3 at 0.4Ag-HAP@PCL, then it plunged slightly, as reported in Table 4. Despite the low additional concentrations of Ag ions into HAP structure, the variation of mechanical properties was represented in an exponential proportional. This may refer to the ability of ionic dopant to provoke high disorder within the crystalline structure, and thus may induce the formation of crystallographic defects including dislocations and volumetric defects such as pores [54, 55]. The break occurs when the dislocation is slipped; however, the presence of a secondary 11

phase such as AgNPs through the composition may act as an inhibitor for dislocation motion and thus may enlarge the mechanical resistance towards applied stresses. Table 4 Mechanical properties of Ag-HAP@PCL nanofibrous scaffolds at different contributions of Ag dopant, including tensile strength, max strain before the break, and toughness, with their deviation values.

Composition 0.0Ag-HAP@PCL 0.2Ag-HAP@PCL 0.4Ag-HAP@PCL 0.6Ag-HAP@PCL 0.8Ag-HAP@PCL

Tensile strength (MPa) 3.110.21 2.350.25 3.570.31 2.520.26 2.330.34

Max strain before the break (%) 60.24.3 107.85.6 82.93.6 85.33.5 96.85.6

Toughness (MJ/m3) 1.130.11 1.680.21 1.720.19 1.380.13 1.560.17

3.5. Cell viability The cytotoxicity response against the nanofibrous scaffolds whereas they are exposed to human fibroblasts cell line in vitro is an important parameter to estimate the feasibility of these implants. The cell viability ratio was estimated in vitro after 3 days of cultivation and as depicted in Fig. 7, it could be noticed that the ratio of the viable cells was improved with the addition of Ag into the composition. It started from 97.14.6 % at no Ag contribution, then it reached its lowest value of 96.13.8 %, and then improved significantly to achieve around 102.33.1 % at the highest contribution of Ag. The low mortality ratio and the relatively high viability indicate the high biocompatibility of the nanofibrous scaffolds. The released species due to the presence of Ag ions and AgNPs does not show a toxic interaction owing to their low contribution [56-58]. 3.6. Antibacterial effect It could be mentioned that one of the most causes of wound repairing failure is a bacterial invasion. A bacterial infection is often accompanied by an imbalance of physical behavior among tissue degradation and reformation through the wound. The antibacterial activity of the AgHAP@PCL nanofibrous scaffolds was investigated against E. coli and S. aureus as shown in Fig. 8. It was illustrated that no inhibition activity was detected for scaffolds with no contribution of Ag. However, it was increased at the 0.2Ag-HAP@PCL composition to be 5.60.5 mm and 4.50.9 mm against E. coli and S. aureus respectively. Furthermore, the antibacterial activity 12

increased significantly with the addition of Ag ions and achieved its highest value of 12.51.1 mm and 11.41.5 mm against E. coli and S. aureus respectively for the highest Ag contribution. It could be postulated that the antibacterial activity was established through the biological milieu due to the release of Ag ions [36]. These ions possess a chemical activity towards bacterial membranes and thus may degenerate the bacterial wall, which leads to bacteria mortality. The Ag+ ions were suggested to be incorporated into the HAP structure up to a specific limit, and the over concentrations were agglomerated to be formed in AgNPs [59-61]. Even though these AgNPs seem to have lower activity than ionic ones, their high contributions might compensate for their potency, and thus additional Ag could be expected to exhibit good antibacterial activity. Therefore, inhibition zones were grown exponentially at 0.6Ag-HAP@PCL and 0.8Ag-HAP@PCL compared with the lower compositions, and thus matches well with FESEM results. 3.7. Cells attachment in vitro The performance of nanofibrous scaffolds of PCL containing Ag-HAP to reconstruct the wounded area is suggested to be examined by the seeding of the human fibroblasts cell line in vitro and let them be grown within this scaffold as obvious in Fig. 9(a-e). It is illustrated that at no contribution of Ag, the cells have adhered to the nanofibers and the mineralization process tends to be triggered through the porous surface of the scaffold. The deposition of mineral components on the scaffold’s surface is provoked via the ionic release of HAP including Ca and P ions. The composition with higher Ag, as Fig. 9b, depicts that cells were proliferated and spread through the scaffold. Furthermore, the filopodia of cells seem to follow the curvature of fibers, while cells are more cohesive than the former composition. Fig. 9c shows the development of cell integration towards the nanofibrous scaffold of (0.4Ag-HAP@PCL) and it is obvious that cells are growing deeply through the scaffold, while the upper composition is illustrated in Fig. 9d shows a higher spreading area. Furthermore, cells seem to be mature with a rough surface than the former, which may refer to their potency. The highest contribution of Ag as Fig. 9e, cells tend to spread to cover the whole surface of the scaffold. Moreover, the cells are not only growing on the scaffold’s surface, but they are also proliferating from the bottom of pores, and thus offers good integration and adhesion between both implant and host tissue. The health cells could be triggered to differentiate to myofibroblasts, which may close the wound. The considerable variation of cell activity towards the different compositions of the nanofibrous scaffold may refer to the high 13

sensitivity of cells upon the conditions of the milieu. The ionic release containing Ca, P, and AgNPs (for the 0.6, 0.8Ag-HAP@PCL) may introduce essential elements that are required for the reconstruction of tissue. Besides, the co-presence of AgNPs may offer an additional characteristic via inhibiting bacterial growth and thus may accelerate differentiation and spreading of health one (fibroblast cells) [62-64]. Moreover, at the higher contribution of AgNPs, higher surface roughness was provoked, and that may induce higher cell adhesion than the lower one. Therefore, combining these factors may introduce a facile strategy to develop fibroblast cells through a nanofibrous scaffold matrix. 4. Conclusion Nanofibrous scaffolds of polycaprolactone containing hydroxyapatite doped with different concentrations of Ag were fabricated. The morphological investigation of the manipulated scaffolds indicated that they were formed in a nanofibers network in non-orientation distribution with diameters in the range of 0.19-0.40, 0.31-0.54, 1.36, 0.122-0.429 μm for 0.0Ag-HAP@PCL, 0.2Ag-HAP@PCL, 0.6Ag-HAP@PCL, and 0.8Ag-HAP@PCL, respectively. Furthermore, the tensile strength increased from 3.110.21 MPa to its highest value at 3.570.31 MPa for 0.4AgHAP@PCL, and then deteriorated. The antibacterial efficiency was examined and showed that the inhibition zone increased upon the incorporation of Ag, and achieved around 12.51.1 mm and 11.41.5 mm against E. coli and S. aureus respectively at the highest Ag contribution. The cells' attachment test was carried out in vitro through the HFB4 cell line. Spreading and proliferation of cells were observed to be enhanced considerably upon the addition of Ag. Therefore, tailoring of a new scaffold with appropriate characteristics for wound healing utilizations could be developed via the gathering of biocompatible nanoparticles to be incorporated into nanofibers. Acknowledgment The authors extend their appreciation to the Deanship of Scientific Research at King Khalid University, Saudi Arabia for funding this work through the Research Groups Program under grant number R.G.P.1/171/41. Conflict of interest: Not present

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Fig. 1 Flow chart of Ag-HAP@PCL nanofibrous scaffold fabrication.

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Fig. 2 XRD patterns of nanofibrous scaffolds of PCL containing Ag-HAP at different contents of Ag; (*: HAP).

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Fig. 3 FTIR spectra of nanofibrous scaffolds of PCL containing Ag-HAP at different concentrations of Ag ionic dopant.

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(a)

(b)

(c)

(d)

Fig. 4 FESEM micrographs of nanofibrous matrix Ag-HAP@PCL at different contributions of Ag ionic dopant; (a) 0.0Ag-HAP@PCL, (b) 0.2Ag-HAP@PCL, (c) 0.6Ag-HAP@PCL and (d) 0.8AgHAP@PCL.

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(b)

(a)

(c)

(d)

Fig. 5 Surface roughness behaviors of Ag-HAP@PCL nanofibrous scaffolds upon the variation of Ag content; (a) 0.0Ag-HAP@PCL, (b) 0.2Ag-HAP@PCL, (c) 0.6Ag-HAP@PCL, and (d) 0.8AgHAP@PCL.

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Fig. 6 Mechanical properties (tensile test) of the Ag-HAP@PCL nanofibrous matrix upon the additional concentration of Ag.

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Fig. 7 Cell viability of nanofibrous scaffolds of Ag-HAP@PCL upon Ag contribution after cultivation through HFB4 cell line for 3 days in vitro.

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Fig. 8 Antibacterial activity of Ag-HAP@PCL nanofibrous scaffolds upon the contribution of Ag against E. coli and S. aureus.

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(a)

(b)

(c)

(d)

(e)

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Fig.

9 The attachment behavior of HFB4 cell line towards nanofibrous scaffolds of Ag-

HAP@PCL at different contributions of Ag after 3 days of cultivation in vitro; (a) 0.0AgHAP@PCL, (b) 0.2Ag-HAP@PCL, (c) 0.6Ag-HAP@PCL and (d) 0.8Ag-HAP@PCL.

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Graphical Abstract

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Abeer A. Hassan: Conceptualization, Methodology, Hyam A. Radwan: Data curation, Writing, Mayssa Abdel Hady: Original draft preparation. Said A. Abdelaal: Visualization, Investigation. Najlaa S. Al-Radadi: Supervision: Kamel Shoueir: Software, Validation.: M. K. Ahmed: Writing- Reviewing and Editing,

30