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\title{Fabrication and Characterization of Porous Nanohydroxyapatite/Chitosan-Cellulose Composite Scaffold for Biomedical Application}
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\begin{document}

             \author[1]{S  Nagalakshmi}

             \author[2]{G.M.  Pavithra}

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\date{\small \em Received: 14 June 2021 Accepted: 30 June 2021 Published: 15 July 2021}

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\begin{abstract}
        


Bones are stiff structures that upkeep and guard several body parts of the physique. A medical technique entitled bone grafting substitutes misplaced bone to overhaul bone fractures that are very intricate, Otherwise, that does not cure precisely.Methods: Several scaffold formulations are prepared (S1, S2, S3, and S4) using various polymers. The prepared scaffold was studied for their weight loss, swelling ability, X-Ray Diffraction (XRD), Scanning Electron Microscopy (SEM) Electron Dispersive X-Ray Analysis, Transmission electron microscopy (TEM), Fourier Transform Infrared Spectroscopy (FT-IR), optical microscopy, in vitro release studies, and in vitro antimicrobial studies.

\end{abstract}


\keywords{bone grafting, scaffolds, hydroxyapatite, ofloxacin, chitosan.}

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\let\tabcellsep& 	 	 		 
\section[{Introduction}]{Introduction}\par
n the past two decades, tissue engineering by bone regeneration has become an alternative method used to overcome the shortcomings of conventional bone defect treatments \hyperref[b0]{[1]}. Bones are upkeep and guard various organs of the body. Damage induces a significant decrease in the quality of our life. A medical technique called bone grafting substitutes lost bones to patchup bone fractures, which are very difficult, imparting substantial health hazards to a patient, or flop to cure appropriately. The grafts may be autologous, allograft, or synthetic. Many of the grafts get reabsorbed and substituted when the normal bone reconciles over some time. The doctrines in fruitful grafts include osteoconduction, osteoinduction, and osteogenesis \hyperref[b1]{[2]}.\par
The progress inthe medical discipline has upgraded biomaterials role in substituting injured tissue, organs and enhancing their functions. Bone tissue engineering is a novel treatable practice for bone grafting \hyperref[b2]{[3]}. The tissue engineering research is implemented mainly in two fields: osteo and dental applications. This technique implants scaffolds, which give mechanical strength in the crackzones. The scaffold remains as a momentary medium for cell multiplication until fresh tissue is entirely revived \hyperref[b3]{[4]}.\par
Hydroxyapatite (HAP) is one of the apatite materials that have a significant inorganic constituent of teeth and bone, which has high biocompatible and bioactive properties and hence employed in bone tissue engineering. Its flow property strength is very little than those required for bone tissue engineering materials and has a tend to migrate from implant sites. These limitations can be overwhelmed by combining hydroxyapatite with organic constituents, thus mimicking the ECM of bone \hyperref[b4]{[5]}.The contagions allied with the implantation recurrently minimize the usage of biomaterials in humans. Bacteria trigger the patient's immune system forming a protective film by sticking onto biomaterial exterior. To avoid these complications, ofloxacin which possesses antibacterial activity, has been incorporated in this biomaterial \hyperref[b5]{[6]}. Thus, the present research work was intended towards the formulation of nano biocomposite scaffold of hydroxyapatite-chitosan-cellulose. Five formulations namely, S1, S2, S3, S4, and S5, was developed. These five formulations are initially characterized for various properties. The optimized formulation, i.e.,. S5, was characterized by analytical techniques. 
\section[{II.}]{II.} 
\section[{Materials and Methods}]{Materials and Methods} 
\section[{a) Materials}]{a) Materials}\par
Hydroxyapatite, Chitosan, Sodium Carboxy Methyl Cellulose, Carboxy Methyl Cellulose, Hydroxy Propyl Methyl Cellulose, Ofloxacin and acetic acid were purchased from Sastha Scientific Services, Chennai. 
\section[{III.}]{III.} 
\section[{Methodology a) Preparation of Hydroxy Apatite Nanoparticles}]{Methodology a) Preparation of Hydroxy Apatite Nanoparticles}\par
The orthophosphoric acid solution was added drop by drop into calcium hydroxide solution under magnetic stirring at 70°C for 3 hours. The mixture is stirred until a clear and homogenous solution formed, and then sodium hydroxide solution was added to this solution until pH value was maintained at 10. The white precipitates were left for 4 hours. The obtained nanoparticles were parted, clarified with deionized water, and dried under ambient atmosphere. It was then 
\section[{b) Fabrication of Scaffold}]{b) Fabrication of Scaffold}\par
Hydroxy Propyl Methyl Cellulose(HPMC) and 100 mg of ofloxacin drug were dissolved in water using a mechanical stirrer until a homogenous solution was formed. Secondly, chitosan was solubilized in 2\% acetic acid, which was instilled dropwise into the HPMC mixture. It is then mixed at 500 rpm. These mixtures were added to the above-formed nanoparticles. The stirring is kept for 24hrs and, the gel formed was then transferred into the tissue culture dish and cooled at -24°C for 24 hrs and lyophilized to form scaffolds. These 
\section[{c) Preparation of Five Formulations of Scaffold}]{c) Preparation of Five Formulations of Scaffold}\par
By experimenting with different polymers, five formulations of the scaffold was prepared namely, S1, S2, S3, S4, and S5. 
\section[{IV.}]{IV.} 
\section[{Characterization Studies a) Calibration Curve of Ofloxacin}]{Characterization Studies a) Calibration Curve of Ofloxacin}\par
The calibration curve of ofloxacin was performed using various concentrations of ofloxacin, as given in Figure no.1 \hyperref[b10]{[11]}. 
\section[{b) Fabrication of the Nanocomposite Scaffold}]{b) Fabrication of the Nanocomposite Scaffold}\par
The scaffold was prepared as per the procedure described in Figure no.2. The quantities of the ingredients in each scaffold are described in  
\section[{d) Swelling Ability}]{d) Swelling Ability}\par
The parched mass of the scaffolds was represented as W i . Parched scaffolds were submerged in Phosphate Buffer solution at 37°C for 24 hours. Later, the scaffolds were removed from PBS solution, and its damp mass was denoted as W f . Swelling ability data was depicted in Table \hyperref[tab_0]{no}.3. Swelling Ability (\%) = [(W f -W i ) /W i ] x100 e) Porosity Measurement\par
W d was used to represent the dry weight of the scaffolds, while W l designated the mass of the scaffolds after immersing in ethyl alcohol for five minutes. After slight parching over the shallow area, W w was recorded. The porosity data is described in Table no.4 \hyperref[b11]{[12]}.Porosity (\%) = (W w -W d ) / (W w -W l ) x 100 f) FT-IR Analysis\par
The spectra of the Chitosan, HPMC, Ofloxacin, and the optimized F5 formulation were documented by means of potassium bromide pellet method in the FT-IR spectrophotometer (JASCO 4100 type A) within the range of 4000cm-1 to 400cm-1 \hyperref[b12]{[13]}.\par
V. 
\section[{Surface Analysis a) Scanning Electron Microscopy (SEM)}]{Surface Analysis a) Scanning Electron Microscopy (SEM)}\par
The powdered sample was taken and mounted on a double side carbon tape, which was fixed to sample specimen stub. The SEM (QUANTA FEG) instrument is used for analysis. The SEM images were described in Figure no.4 \hyperref[b13]{[14]}. 
\section[{b) Optical Microscopy}]{b) Optical Microscopy}\par
MOTIC digital microscope is used to image the scaffold at 10X and 40X, as given in Figure no.5. 
\section[{c) Transmission Electron Microscopy (TEM)}]{c) Transmission Electron Microscopy (TEM)}\par
TEM studies were useful in examining the morphological and crystalline arrangements of the scaffold. The principle employed to view the scaffolds is high-resolution transmission electron microscopy (HRTEM). The scaffold's (20 µl) solution was taken. On the carbon-coated side of the copper lattice, the mixture was dripped. At room temperature for few hours, the lattice was dehydrated. The grid was then placed in the sample holder and mounted in the instrument. The instrument TECHNAI T20 was used for the analysis. The TEM images were given in Figure no.6 \hyperref[b14]{[15]}. 
\section[{d) Electron Dispersive X-Ray Analysis}]{d) Electron Dispersive X-Ray Analysis}\par
The elements present in the scaffold were estimated using EDAX analysis. It is given in Figure no.7 \hyperref[b15]{[16]}. B heated in an electric furnace at 700°C to obtain pure nanoparticles \hyperref[b6]{[7,}\hyperref[b7]{8]}.© 2021 Global Journals\par
scaffolds were cross-linked with CaCl 2 solution for 30 minutes, followed by sopping in ethanol for 10 minutes. Finally, the scaffold was clarified with water and another timely ophilized \hyperref[b8]{[9,}\hyperref[b9]{10]}.  
\section[{VI.}]{VI.}\par
In-vitro Release Studies 100µg of the scaffold was pondered from each of the five formulations primed in different test tubes. To this, pH 7.4 phosphate buffer medium was added and placed in an orbital shaker. The quantity of ofloxacin expelled out from the scaffolds was assessed by amassing buffer medium from the test tubes and supplanting with fresh buffer at 30 minutes' intervals for 5 hours. The amount expelled out was recorded at 294 nm. The discharged amount was ascertained from the standard curve. From this percentage, drug release was calculated, and percentage drug release as plotted versus time. The in-vitro drug release graph was depicted in Figure no.9 \hyperref[b16]{[17]}.\par
VII. 
\section[{In-vitro Antibacterial activity a) Agar Disc Diffusion Method i. Preparation of Inoculum}]{In-vitro Antibacterial activity a) Agar Disc Diffusion Method i. Preparation of Inoculum}\par
On agar slant, cultures were conserved at 4°C. By relocating a coil of cells from the cultures to test tubes, lively cultures were developed. The anti-septic action was ascertained by the agar disc diffusion technique. 
\section[{ii. Antibacterial Activity}]{ii. Antibacterial Activity}\par
The antiseptic activity was ascertained by the well diffusion method on Muller Hinton agar (MHA) medium. MHA was solubilized in purified water, and the medium was sterilized after the addition of agar. Then, the media was transferred into disinfected Petri plates and solidified. By using disinfected swab saturated with the bacterial suspension, the inoculums were spread on the plates. To the wells made, 100,200, 400µg of (F5), 50 µl negative control (HCl), and positive control of streptomycin suspension were added on respective wells. These plates were gestated at 37ºC for a day. The area of inhibition was then recorded. The results were depicted in Table \hyperref[tab_0]{no}      
\section[{Results and Discussion}]{Results and Discussion} 
\section[{a) Calibration Curve of Ofloxacin}]{a) Calibration Curve of Ofloxacin} 
\section[{c) Weight Loss}]{c) Weight Loss}\par
From the above shown Fig. \hyperref[fig_6]{3} and Table \hyperref[tab_3]{3}, Scaffold S1 has a maximum weight loss of 8 \% during the study. The scaffold S3 showed less weight loss compared to S2. Scaffold S4 showed the minimum loss of weight (2.7 \%) in four weeks and had the less degradation \hyperref[b18]{[19]}.   The swelling was similar in all the scaffold formulations due to constant hydroxyapatite and chitosan concentrations, as given in table no.  {\ref 3 [20]}. Table \hyperref[tab_3]{3} shows the Parched mass (W i in g), damp mass (W f in g) and swelling ability (\%) of scaffold formulations. 
\section[{e) Porosity Measurement}]{e) Porosity Measurement}\par
The porosity of the scaffold formulations was similar to one another, as given in table no.4.  \hyperref[tab_4]{4} reveals the parched mass (W w in g), dry weight (W d in g), dipped mass (W l in g) and porosity (\%) of the scaffold formulations. 
\section[{f) FT-IR Analysis}]{f) FT-IR Analysis}\par
The results of the analysis showed various stretching, bending, and rocking vibrations based on the groups present. All the spectra indicated that there are no significant drug-excipient interactions. 
\section[{IX.}]{IX.} 
\section[{Surface Analysis a) Scanning Electron Microscopy (SEM)}]{Surface Analysis a) Scanning Electron Microscopy (SEM)}\par
The images exhibit that the scaffold has an elongated surface which is shown in figure no.4.    
\section[{d) Energy Dispersive X-Ray Analysis}]{d) Energy Dispersive X-Ray Analysis}\par
The scaffold contains oxygen(O), carbon(C), calcium(Ca), phosphorus(P), magnesium (Mg) and chlorine(Cl) at 50.80\%, 24.93\%, 24.64\%, 8.19\%, 0.50\% and 0.36\% respectively as shown in Figure no.7 \hyperref[b20]{[22]}. Fig.  {\ref 7}: EDAX analysis Fig.  {\ref 7} shows the presence of various elements and their composition of S4 scaffold. 
\section[{e) X-Ray Diffraction (XRD) Analysis}]{e) X-Ray Diffraction (XRD) Analysis}\par
The peaks were obtained at 2? level at positions 27.  {\ref 21, 29.15, 30.65, 34.35, 37.}  
\section[{In-vitro Release Studies}]{In-vitro Release Studies}\par
From the Figure no.9, the scaffold S4 showed an initial burst release succeeded by a persistent release and the release rate was found to be 100\% at the end of 8hours, whereas scaffolds S2 and S3 showed release of 62\%, and 82\% at the end of 8 hours study. However, S5 showed a sustained release profile over an extended period of study of upto 24 hours. Hence, the formulation S5 has been optimized for characterization \hyperref[b21]{[23]}.  From the report of the antibacterial activity of the formulated scaffold as shown in Table no.5 and Figure no.10, it was found that the scaffold with various concentrations 100µg, 200µg and 400µg when compared with standard positive and negative control, showed maximum zone of inhibition of 26mm, 32mm and 34mm respectively. Hence the prepared scaffold exhibits antibacterial activity \hyperref[b22]{[24]}.  
\section[{Conclusion}]{Conclusion}\par
The scaffold is a versatile bioactive product among wound dressing materials, whose production is flexible and economical. The present work was aimed towards fabricating a scaffold containing hydroxyapatite using various polymers like chitosan, carboxy methylcellulose (CMC), sodium carboxy methylcellulose (SCMC), and hydroxypropyl methylcellulose (HPMC) by freeze-drying technique by incorporating Ofloxacin as an anti-microbial agent. Five formulations, namely S1, S2, S3, S4, and S5, were prepared using various combinations of the polymers mentioned. The prepared scaffolds were studied for their characteristic properties like weight loss, swelling ability, porosity, and in-vitro drug release studies. The optimized formulation (S5) was characterized by SEM, optical microscopy, TEM, EDAX, XRD, FT-IR, and in-vitro antibacterial activity.\par
Due to the greater water acceptance, sufficient porosity, improved antibacterial activity, and extended drug release, the hydroxyapatite-chitosan-HPMCofloxacin scaffold would be a hopeful biomaterial for bone tissue engineering. From this research, it was concluded that the nano-composite scaffold is a viable alternative to existing conventional dosage forms, which lead to improved bioactivity and a promising biomaterial for bone tissue engineering in case of administration affords resulting in better patient compliance and costeffective therapy in the field of biomedical application.  
\section[{Conflicts of Interest}]{Conflicts of Interest}\begin{figure}[htbp]
\noindent\textbf{}\includegraphics[]{image-2.png}
\caption{\label{fig_0}Fabrication}\end{figure}
 \begin{figure}[htbp]
\noindent\textbf{}\includegraphics[]{image-3.png}
\caption{\label{fig_1}}\end{figure}
 \begin{figure}[htbp]
\noindent\textbf{}\includegraphics[]{image-4.png}
\caption{\label{fig_2}}\end{figure}
 \begin{figure}[htbp]
\noindent\textbf{1}\includegraphics[]{image-5.png}
\caption{\label{fig_3}Fig. 1 :}\end{figure}
 \begin{figure}[htbp]
\noindent\textbf{}\includegraphics[]{image-6.png}
\caption{\label{fig_4}Fabrication}\end{figure}
 \begin{figure}[htbp]
\noindent\textbf{2}\includegraphics[]{image-7.png}
\caption{\label{fig_5}Fig. 2 :}\end{figure}
 \begin{figure}[htbp]
\noindent\textbf{3}\includegraphics[]{image-8.png}
\caption{\label{fig_6}Fig. 3 :}\end{figure}
 \begin{figure}[htbp]
\noindent\textbf{4}\includegraphics[]{image-9.png}
\caption{\label{fig_8}Fig. 4 :}\end{figure}
 \begin{figure}[htbp]
\noindent\textbf{5}\includegraphics[]{image-10.png}
\caption{\label{fig_9}Fig. 5 :}\end{figure}
     \begin{figure}[htbp]
\noindent\textbf{no} \par 
\begin{longtable}{P{0.85\textwidth}}
.1.\\
c) Weight Loss\\
By imbibing the scaffolds in Simulated Body\\
Fluid (SBF), the weight losses of the five scaffold\\
formulations are conceded.\\
Weight Loss (\%) = [(W o -W t ) / W o ] x 100\end{longtable} \par
 
\caption{\label{tab_0}Table no}\end{figure}
 \begin{figure}[htbp]
\noindent\textbf{1} \par 
\begin{longtable}{P{0.35079365079365077\textwidth}P{0.12142857142857141\textwidth}P{0.1259259259259259\textwidth}P{0.1259259259259259\textwidth}P{0.1259259259259259\textwidth}}
Formulation Code\tabcellsep S1 (mg)\tabcellsep S2 (mg)\tabcellsep S3 (mg)\tabcellsep S4 (mg)\\
Hydroxyapatite\tabcellsep 100\tabcellsep 100\tabcellsep 100\tabcellsep 100\\
Chitosan\tabcellsep 30\tabcellsep 30\tabcellsep 30\tabcellsep 30\\
SCMC\tabcellsep -\tabcellsep 10\tabcellsep -\tabcellsep -\\
CMC\tabcellsep -\tabcellsep -\tabcellsep 10\tabcellsep -\\
HPMC\tabcellsep -\tabcellsep -\tabcellsep -\tabcellsep 10\\
Ofloxacin\tabcellsep 100\tabcellsep 100\tabcellsep 100\tabcellsep 100\\
2\% Acetic Acid\tabcellsep 50ml\tabcellsep 50ml\tabcellsep 50ml\tabcellsep 50ml\\
Water\tabcellsep 100ml\tabcellsep 100ml\tabcellsep 100ml\tabcellsep 100ml\end{longtable} \par
  {\small\itshape [Note: Note: Details the composition of S1, S2, S3, and S4 formulations.© 2021 Global Journals]} 
\caption{\label{tab_1}Table 1 :}\end{figure}
 \begin{figure}[htbp]
\noindent\textbf{2} \par 
\begin{longtable}{P{0.12350427350427351\textwidth}P{0.18162393162393162\textwidth}P{0.18162393162393162\textwidth}P{0.18162393162393162\textwidth}P{0.18162393162393162\textwidth}}
Time (d)\tabcellsep S1 (\%)\tabcellsep S2 (\%)\tabcellsep S3 (\%)\tabcellsep S4 (\%)\\
1\tabcellsep 1.5\tabcellsep 0.2\tabcellsep 0.1\tabcellsep 0.0\\
3\tabcellsep 2.7\tabcellsep 1.2\tabcellsep 0.9\tabcellsep 0.0\\
7\tabcellsep 3.9\tabcellsep 2.2\tabcellsep 1.7\tabcellsep 0.5\\
15\tabcellsep 5.4\tabcellsep 3.1\tabcellsep 2.4\tabcellsep 1.2\\
21\tabcellsep 6.9\tabcellsep 4.2\tabcellsep 3.3\tabcellsep 1.9\\
28\tabcellsep 8.0\tabcellsep 5.1\tabcellsep 4.0\tabcellsep 2.7\end{longtable} \par
  {\small\itshape [Note: Note: Represents the loss of weight in \% of scaffolds at predetermined time intervals for 28 days.]} 
\caption{\label{tab_2}Table 2 :}\end{figure}
 \begin{figure}[htbp]
\noindent\textbf{3} \par 
\begin{longtable}{P{0.19805825242718447\textwidth}P{0.18980582524271844\textwidth}P{0.18980582524271844\textwidth}P{0.27233009708737865\textwidth}}
Formulation Code\tabcellsep W i (g)\tabcellsep W f (g)\tabcellsep Swelling Ability (\%)\\
S1\tabcellsep 1.00\tabcellsep 2.40\tabcellsep 140\\
S2\tabcellsep 1.00\tabcellsep 2.60\tabcellsep 160\\
S3\tabcellsep 1.00\tabcellsep 2.50\tabcellsep 150\\
S4\tabcellsep 1.00\tabcellsep 2.90\tabcellsep 190\end{longtable} \par
 
\caption{\label{tab_3}Table 3 :}\end{figure}
 \begin{figure}[htbp]
\noindent\textbf{4} \par 
\begin{longtable}{P{0.1881679389312977\textwidth}P{0.14923664122137403\textwidth}P{0.14923664122137403\textwidth}P{0.14923664122137403\textwidth}P{0.21412213740458014\textwidth}}
Formulation Code\tabcellsep W w (g)\tabcellsep W d (g)\tabcellsep W l (g)\tabcellsep Porosity (\%)\\
S1\tabcellsep 0.58\tabcellsep 0.25\tabcellsep 1.15\tabcellsep 57.89\\
S2\tabcellsep 0.59\tabcellsep 0.25\tabcellsep 1.19\tabcellsep 56.66\\
S3\tabcellsep 0.62\tabcellsep 0.25\tabcellsep 1.20\tabcellsep 63.79\\
S4\tabcellsep 0.65\tabcellsep 0.25\tabcellsep 1.24\tabcellsep 67.79\\
Table\tabcellsep \tabcellsep \tabcellsep \tabcellsep \end{longtable} \par
 
\caption{\label{tab_4}Table 4 :}\end{figure}
 \begin{figure}[htbp]
\noindent\textbf{5} \par 
\begin{longtable}{P{0.03566433566433567\textwidth}P{0.1783216783216783\textwidth}P{0.04160839160839161\textwidth}P{0.04160839160839161\textwidth}P{0.1783216783216783\textwidth}P{0.14265734265734267\textwidth}P{0.23181818181818178\textwidth}}
\tabcellsep \tabcellsep \tabcellsep \tabcellsep \multicolumn{3}{l}{Zone of Inhibition (mm)}\\
S.No.\tabcellsep Microorganisms\tabcellsep 100µg\tabcellsep 200µg\tabcellsep 400µg\tabcellsep HCl (negative control)\tabcellsep Streptomycin 15 µg (positive control)\\
1\tabcellsep Escherichia coli\tabcellsep 26\tabcellsep 32\tabcellsep 34\tabcellsep 23\tabcellsep 16\end{longtable} \par
 
\caption{\label{tab_5}Table 5 :}\end{figure}
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\end{document}
