**1. Introduction**

Bone tissue engineering is one of the most important approaches to repairing damaged and diseased tissue and it facilitates the complete recovery of the tissue itself as well as its function [1]. Bone serves several functions such as body support, organ protection, and storage of nutrients. Due to the applicability of bone, it is considered a complex tissue in the human body [2]. Moreover, it has an extremely anisotropic nature due to a variety of mechanical properties extending in all directions. The design of scaffolds as an extracellular matrix (ECM) and the process of regeneration is the major goals in tissue engineering [3]. ECM should be nontoxic and biocompatible as well as show the desired degradation rate and porosity with excellent mechanical properties. Additionally, ECM should not be the cause of foreign body reactions [4].

Alternatively, scaffolds are used as supporting materials and the results have been better cell growth, increased proliferation rate, and healthier ECM production [5]. The fabricated artificial material will be useful for the repair of damaged tissue and the regeneration

**Citation:** Stella, S.M.; Sridhar, T.M.; Ramprasath, R.; Gimbun, J.; Vijayalakshmi, U. Physio-Chemical and Biological Characterization of Novel HPC

(Hydroxypropylcellulose):HAP (Hydroxyapatite):PLA (Poly Lactic Acid) Electrospun Nanofibers as Implantable Material for Bone Regenerative Application. *Polymers* **2023**, *15*, 155. https://doi.org/ 10.3390/polym15010155

Academic Editor: Subramanian Sundarrajan

Received: 5 September 2022 Revised: 27 October 2022 Accepted: 19 December 2022 Published: 29 December 2022

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

of new tissues by cell proliferation and growth of their own ECM. Generally, several types of material sources, be it natural, synthetic, semi-synthetic or even composites are used to synthesize scaffolds used in bone tissue regeneration.

Cellulose fiber is a good choice for bone tissue engineering due to its high strength and better mechanical properties. Subsequently, the fabrication of cellulose composite scaffolds is attracting many researchers due to its highly applicable mechanical properties [6]. The main advantages of cellulose-based scaffolds are biodegradability in chemical as well as biological environments and mechanical stress factors, good mechanical properties in terms of tensile strength, negligible foreign body and inflammatory response reactivity within in vitro and in vivo applications, and abundant presence of this naturally occurring polymer [7,8]. In tissue engineering, cellulose has been used as a permanently implantable scaffold establishing the fact that it is stable for a long-time application in vivo [9,10].

Among the other cellulose fibers, hydroxypropyl cellulose (HPC) has the abovementioned desired properties and it can be used for the fabrication of HAP-reinforced fibrous mat. Structurally, hydroxypropyl groups substitute the hydroxyl groups present in cellulose, hence yielding better mechanical properties than cellulose [11]. The material has adequate biomedical application due to its biodegradability and biocompatibility [12].

In scaffold fabrication, polymers play a major role as they are responsible to mimic the organic nature of human native bone. Poly(lactic acid) (PLA) [13], polyglycolic acid (PGA) [14], poly(lactic-co-glycolic acid) (PLGA) [15], poly(vinyl alcohol) (PVA) [16], poly(vinyl pyrrolidone) (PVP) [17], poly(methyl methacrylate) [18] and polycaprolactone (PCL) [19] are some of the important polymers that may be deployed in fabricating scaffolds for bone tissue engineering applications. Among the above polymers, PLA has advantages such as biocompatibility, biodegradability and good modulus with adequate strength [20].

Certain polymeric materials are unable to accomplish the desired scaffold to provide valuable inputs in biomedical applications. To overcome these issues, the composite of natural and synthetic polymers can be employed. HPC polymer has good biocompatibility and fine particle grades are most favorable for faster hydration, and uniform dissolution of particles during mixing [21]. On the other hand, it has some disadvantages such as hydrophobicity and low molecular weight. Additionally, for electrospinning to occur, polymer chain entanglement is one of the important criteria to form nanofibers. Due to these reasons, HPC as such is not spinnable and hence the polymers such as PLA are mixed to provide sufficient chain entanglement in the solution, whereby electro-spinnability is feasible [22].

Hydroxyapatite [Ca10(PO4)6(OH)2] is one of the important materials which offer significant applications in bone therapy and tooth reconstruction. It exhibits the properties and mechanisms to directly bond to the natural host tissue and results in regeneration [23]. The major challenge in recent research is producing an apatite layer similar to human tissues to be confirmed by morphological and physiochemical analysis. It is necessary to understand the mechanism of HAP bonding and the biomineralization process leading to cell growth [24]. In biomedical aspects, scaffolds have been fabricated to renew or repair damaged organs by fracture or defects caused by disease (osteoporosis, osteoarthritis, osteosarcoma) or surgeries (tumor removal) or congenital disabilities. To figure out, the risk and demands, artificial scaffolds have been fabricated with the properties of biocompatibility, biodegradability, mineralization, mechanical resistance, and cell proliferation.

Electrospinning is an emerging technique for the fabrication of nanofibrous scaffolds which is useful for the production of ECM better than other bone scaffold fabrication techniques [25]. As we know, other fabrication techniques do not offer any ability to control porosity, whereas the electrospinning technique offers continuous fiber production with a controlled diameter. The diameter and alignment of the fiber can be easily controlled by adjusting the properties of the HAP–polymer composite solution and parameters of the electrospinning instrument [26].

In the present study, the novelty of the work is divided into two categories: Firstly, HAP-HPC/PLA nanofibrous mat has been fabricated using electrospinning techniques. The composite of HAP-HPC/PLA prepared by 25% of HAP was gradually added into 5 wt% of HPC/PLA polymer solution. The different composition ratios such as 0:100, 40:60, 50:50, 60.40, and 70:30 of HAP-HPC/PLA have been used to optimize the composition in terms of mechanical strength, biodegradability, and bioactivity properties. Different physical, chemical, and mechanical characterization techniques were used for the analysis of composite nanofibers. Further, the optimized scaffold has been confirmed by in vitro biocompatibility study. In this study, the cytotoxicity analysis has been carried out using the MG63 (osteoblast) cell line to achieve the osteoconductive property in the form of new bone formation. Secondly, the optimized composite was further confirmed by in vivo performance using the calvarial defect on rats and qualitative analysis was carried out at 4 weeks and 8 weeks of implantation using radiological (X-Ray) evaluation and histological study. The objective of this work was to optimize composite scaffolds with respect to the different levels of cell growth (osteoblast and osteoclast etc.). Thus, the optimized study plays a favorable foundation for new bone formation both by in vitro and in vivo analysis.

### **2. Materials and Methods**

### *2.1. Materials*

PLA (molecular weight of 220 kDa), HPC (molecular weight of 100,000 kDa), aqueous ammonia, calcium nitrate tetrahydrate (Ca(NO3)4H2O) and ammonium dihydrogen orthophosphate (NH4H2PO4) were purchased from the SD-Fine chemicals, Mumbai, India.

MG63 cell lines were obtained from NCCS, Pune. DMEM (Dulbecco's Modified Eagle Medium), fetal bovine serum (FBS), trypsin and 1× antibiotic solution were purchased from Hi-Media Laboratories, Mumbai, India. Methyl thiazolyl diphenyl-tetrazolium bromide (MTT) and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich Chemicals Company, Mumbai, India. The cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% FBS, penicillin (10,000 U/mL), and streptomycin (10 mg/mL) in a humidified atmosphere of 50 µg/mL CO<sup>2</sup> at 37 ◦C.

### *2.2. Methods*

### 2.2.1. Preparation of Hydroxyapatite and Polymer Solution

1M solutions of calcium nitrate tetrahydrate and 0.6M of ammonium dihydrogen orthophosphate were prepared using double distilled water by adopting co-precipitation method. Both the solutions were mixed together under continuous stirring and pH of the solution was adjusted to 11 by adding aqueous ammonia [27]. Pure hydroxyapatite was obtained by aging the solution for 16 h, the raw powder was filtered and washed several times with distilled water followed by sintering the dry powder at 700 ◦C for 2 h. Nanofibrous scaffold was fabricated using 2.5 g of HAP powder dispersed in 10 mL of water under stirring conditions for 12 h. The solution of HPC/PLA was prepared by selecting 0.5 g of (5 wt %) HPC and 0.5 g of PLA in 10 mL of boiling water under stirring conditions. Finally, the resultant solution with the composition of HAP/HPC/PLA suspension was made for electrospinning.

The composite mixture was stirred for 3 h until a clear homogeneous solution was obtained. The homogenous solution is allowed to age for 12 h followed by ultra-sonication prior to electrospinning.

### 2.2.2. Viscosity and Electrical Conductivity Analysis

The obtained composite solutions were tested for their viscosity using Brookfield DV2 viscometer. The dielectric constant of the electrospun nanofibers was measured using a HIOKI 3532-50 LCR HITESTER meter at a temperature range of 35–125 ◦C at a frequency of 0–5 × 106 Hz.

## 2.2.3. Electrospinning

The nanofibrous mat was fabricated with different composite ratios (0:100, 40:60, 50:50, 60:40, and 70:30) of HAP and HPC/PLA. The composite mixture was stirred for 3 h until

a clear homogeneous solution is obtained. The homogenous solution is allowed to age for 12 h. The composite solution to be electrospun was taken in a plastic syringe (5 mL) with a hypodermic needle and a flat-filed tip, with an internal diameter of 0.8 mm. The electrospun nanofibers were collected on aluminum foils on a rotating drum collector at a controlled relative humidity (20–25%) environment. The obtained nanofibrous mat was dried along with aluminum foil in a desiccator to reduce the effect of humidity on the nanofibers [28]. The solution preparation is tabulated in Table 1.


**Table 1.** The composite mixture and the volume of the solution.

## 2.2.4. Physio Chemical Characterization

FT-IR analysis was used to distinguish species, and analyze the functional groups, vibration modes, chemical interaction of HAP with polymers. Evaluation by using FT-IR was accomplished using SHIMADZU CROP IRAFFINITY-1 flourier transform infrared spectrophotometer within the variety 400–4000. X-ray diffraction is used to identify the nanoparticles such as HAP in composites. This characterization was performed for dried and finely tailored nano composite samples on XRD machine (BRUKER Germany with Cu K radiation; =1.5405 Å). Mechanical study used to measure the tensile strength and elastic modulus. Tensile strength for electrospun nanofibers was performed using an ASTM standard (D695) on the Tinus Olsen H5K5 universal testing machine. Nanofibers were cut into a rectangular shape of width 12 mm and placed at the height of 6 cm between two clamps bearing a 500 N load cell with velocity of 1mm/min. The average of three trials of tensile modulus were calculated from stress–strain response. X-ray photoelectron spectroscopy (XPS, ESCALAB 250Xi, Richardson, TX, USA) is used to analyze materials surface chemistry. Specimens were analyzed using a monochromatic Al Kα source (10 mA, 15 kV). Scanning Electron Microscope (ZISS-EVO18, Horn, Austria) is used to investigate the morphology of composite at excessive magnification and resolution by using lively electron beam.

### 2.2.5. Porosity

The solvent replacement method was used for the measurement of porosity of the HPC/PLA and HAP-HPC/PLA nanofibrous mat. The initial weight (Wi) of the nanofibers was measured after drying in desiccator. The nanofibrous mat was immersed in absolute ethanol and dried off immediately. The porosity of the electrospun nanofibrous mat was determined as follows:

$$\text{Porosity} = (\text{(Ws} - \text{Wi)}) / \text{Ws} \times 100 \tag{1}$$

where Ws is the rehydrated nanofiber weight and Wi is the initial nanofiber weight.
