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. 2021 Jun 16;18(5):787–795. doi: 10.1007/s13770-021-00341-4

Nigella Sativa-Coated Hydroxyapatite Scaffolds: Synergetic Cues to Stimulate Myoblasts Differentiation and Offset Infections

Touseef Amna 1,✉, Abdullah A A Alghamdi 1, Ke Shang 2, M Shamshi Hassan 3,✉
PMCID: PMC8440700  PMID: 34132986

Abstract

Background:

At present osteoporosis has come into view as a major health concern. Skeletal diseases typified by weak and fragile bones have imposed threats of fissure. Hydroxyapatite (HAP) is known to induce osteoblast like differentiation and provide mechanical strength, hence, used in bone tissue engineering; whereas, Nigella sativa has also demonstrated potential to treat bone and muscle diseases. This study was aimed to develop potential orthopedic scaffold exploiting natural resources of Saudi Arabia which can be used as prospective tissue engineering implant.

Methods:

The bone scaffold was developed by grafting biogenic HAP with N. sativa essential oil. N. sativa was applied for boosting osteogenesis and to stimulate antimicrobial potential. Antimicrobial potential was investigated utilizing S. aureus bacteria. Spectroscopic and surface characters of N. sativa grafted HAP scaffolds were analyzed using Fourier-transform infrared spectroscopy, X-ray crystallography and Scanning electron microscopy. To ensure biocompatibility of scaffolds; we selected C2C12 cell-lines; best model to study mechanistic pathways related to osteoblasts and myoblasts differentiation.

Results:

Grafting of HAP with N. sativa did not affect typical spherical silhouette of nanoparticles. Characteristically; protein loaded polynucleated myotubes are result of in vitro myogenesis of C2C12 myoblasts in squat serum environment.

Conclusion:

It is first study of unique combination of N. sativa and HAP scaffold as a possible candidate of implantation for skeletal muscles regeneration. Outcome of this finding revealed N. sativa grafted HAP enhance differentiation significantly over that of HAP. The proposed scaffold will be an economical natural material for hard and soft tissue engineering and will aid in curing skeletal muscle diseases. Our findings have implications for treatment of muscular/bone diseases.

Graphic Abstract

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Keywords: N. sativa, Differentiation, Hydroxyapatite, Myoblasts, Antibacterial

Introduction

In old age, especially women suffer from major bone illness called osteoporosis, (a skeletal sickness described by low bone density and micro-architectural weakening of bones). The people suffering from osteoporosis possess fragile bones and are vulnerable to fractures. In females, the key cause of osteoporosis has been recognized as menopause; typically absence or less production of estrogen hormone [1]. In recent times, ever growing authentication of postmenopausal osteoporosis and linked fractures have become international fitness concern [2]. Nevertheless, hormone proxy remedy has established as an efficient method in curing bone ailments, however, it produces undesirable side effects to women [3]. From ancient times, the natural products are being employed as suitable replacement to allopathic treatment for cure of many illnesses. N. sativa belongs to family Ranunculaceae; commonly acknowledged as black cumin or habatus sauda. It has been used from decades for treatment of various persistent and severe diseases. The seeds are resource of active constituents of N. sativa [4] viz; linoleic and oleic which are assumed to be beneficial for humans [5]. Interestingly, accessible literature have reported plethora of curative aspects of N. sativa for instance; anticancer, antioxidant, antibacterial, antifungal, antiparasitic and anti-asthmatic [6] etc. Additionally, N. sativa particularly thymoquinone have illustrated advantageous impact on bone and joint ailments [6]. Integrated effect of N. sativa and parathyroid hormone was found beneficial in reversing osteoporosis and ameliorate bone vigor [7]. N. sativa has also been recognized to improve diabetic impairment of bones [8] by rehabilitating bone metabolism and blood glucose [9]; therefore, recommended as effectual anti-diabetic treatment [7]. In recent times, organic–inorganic hybrids; ceramic biomaterials are being used for bone tissue engineering. Hydroxyapatite, Ca10(PO4)6(OH)2, classified in calcium phosphate group possess stupendous characters and has shown biocompatibility with human tissues [10, 11]. Therefore, it is clinically being used as remedy in osseous flaws, grafting material, filler for bone cavities as well as scaffold in prosthesis surgical treatment [12]. It has also been utilized as functionalization material to dental and orthopedic implants in order to induce propagation of osteoblasts as well as biofixation between tissues and implant [13].

However, microbial infections of implant are all the time a major distress. More specifically, prevalence of bacterial drug confrontation is ever escalating. Accordingly, effective options to counteract these resistant contagions are in high demand. Surface amendments and coatings techniques are the most suitable possible ways to fight these infections [14]. In this course; essential oils of aromatic foliage which are overloaded with antimicrobial activities are of immense significance [15]. In the reported literature, it is mentioned that N. sativa essential oil contain thymoquinone, dithymoquinone, thymohydroquinone, thymol as main bioactive components. The quinine constituent is found as most potent and pharmacologically active compound in N. sativa which depicted useful influence on infected bones and joints [16]. Within light of literature, it has been proved that N. sativa possess great potential to treat bone and muscle diseases and also have power to cease microbial growth. To our knowledge, hitherto, no study about grafting of biogenic HAP with N. sativa to prepare a novel scaffold for differentiation of myoblasts has been instituted. Thus, current research work premeditated to assess effectiveness of N. sativa in extenuating the differentiation of myoblasts on N. sativa coated HAP scaffolds as the regenerative medicine to treat skeletal muscle diseases.

Materials and methods

Grafting of biogenic HAP nanoparticles with N. sativa oil

Biogenic cow bones were collected from local Albaha butcher house and diced in small parts and washed enough to eliminate any impurity. The pristine bones were immersed in acetone and ethanol for 30 min to remove collagen, fat and superfluous contaminations. The samples were transferred to dry oven for 24 h at 160 °C. Furthermore, the crushed sample was kept in a crucible and shifted to the muffle furnace (Dubuque, IA, USA) at 600 °C for 5 h at scan rate of 10 °C /min. The obtained calcined samples were once again compressed into fine powder using mortar and pestle. The capping of synthesized HAP with N. sativa oil was done by physical mixing of HAP powder with natural N. sativa oil. First, 1 g of HAP powder was consistently muddled up in 1 ml of ethanol. Then, 0.25 ml of N. sativa oil was spread to polymerize at 37 °C for 48 h to obtain a coating and preserved in the dark till further use.

Characterization techniques

The biogenic HAP and N. sativa grafted HAP scaffolds were characterized using modern physicochemical techniques. The scaffolds were characterized for crystallinity and phase content by X-Ray Diffraction (XRD, Rigaku, Japan) with Cu Kα radiation over Bragg angle ranging from 20° to 60°. Exterior silhouette of N. sativa grafted HAP was affirmed by Scanning Electron Microscopy (Blk 192 Pandan Loop, Pantech Industrial Complex, Singapore). Comprehensible morphological details of scaffolds were analyzed by Transmission Electron Microscopy (North Ryde, NSW, Australia). Energy dispersive X-ray (CBlk 192 Pandan Loop, Pantech Industrial Complex, Singapore) Spectroscopy has utilized to check elements. Diverse functional moieties in biogenic HAP and N. sativa grafted HAP scaffolds was analyzed by Fourier Transform Infrared Spectroscopy (Billerica, MA, USA) from wavenumber 400 to 4000 cm−1.

Cytocompatibility test

C2C12 myoblasts have developed in Dulbecco’s modified Eagle’s (DMEM, pH 7.4) medium complemented with suitable antibiotics (1%) and fetal bovine serum (FBS–10%) [17]. The cultured myoblasts were placed at 37 °C with 5% CO2 in a humidified atmosphere in the incubator. Recently revived culture of myoblasts (1 × 104 cells/well) was inoculated in 96-well plates for proper attachment and confluence (70–80%). Customary CCK-8 assessment system has applied to supervise viability of adherent cells. After attaining confluence, myoblasts were cultivated on biogenic HAP and N. sativa grafted HAP scaffolds for different (0, 1, 2, 3) days. 10 μl of water soluble tetrazolium-8 was cast to each cultured well and incubated for 4 h at 37 °C as per regulations given in protocol and absorbance was noted at 450 nm with the help of microplate spectrophotometer (model 680; Bio-Rad Laboratories, Hercules, CA, USA). Viability of cultures was premeditated as percentage of unprocessed standard [18]. The morphologic alterations were reviewed with the help of phase contrast microscope (Shinjuku-ku, Tokyo, Japan) [19].

Immunofluorescence microscopy

The assenting influence of N. sativa grafted HAP scaffolds on myoblasts was also substantiated by immunofluorescence labeling. The live/dead staining kit contains two distinct fluorescent stains that tag living and dead culture. Integral cells are marked green whereas compromised plasma membranes tag red.

Differentiation

For C2C12 differentiation, 5 × 104 cells were pre-seeded in six-well plates filled with DMEM growth media to obtain ~ 70–80% confluence. Eventually; obtaining desired cell count; spent media was substituted with differentiation medium which comprise of DMEM complemented with 5% (v/v) horse serum, 2 mM glutamine, penicillin (100 U/ml), and streptomycin (100 lg/ml). The cultured myoblasts were maintained in differentiation medium till termination of analysis (0–7 days). The establishment and organization of myotube was regularly tracked by means of an inverted microscope. Briefly, myoblasts cultured on N. sativa grafted HAP scaffolds. The culture was rinsed by phosphate buffered saline, immobilized with methanol (100%) for 2–5 min and air dried (10 min). In brief, Jenner staining solution (BDH, Poole UK) was attenuated 1:3 in 1 mM sodium phosphate buffer (pH 5.6), and 1 ml was introduced into cultured wells and kept for incubation for 5 min. The cells were rinsed by DW. Subsequently, culture was stained with 1 ml Giemsa stain attenuated 1:20 in 1 mM sodium phosphate buffer and incubated for 10 min at room temperature. Culture was again rinsed with DW. Notably, cultured myoblasts were dyed concurrently by Jenner and Giemsa stain. Finally, cultured wells were snapped at random spots by means of a digital camera fixed with an inverted microscope. In case cells are not to be stained immediately, the immobilized cells have been preserved at room temperature till further study. The development of myotubes can be detected by dark purple shade whereas nuclei retain pinkish color. For every domain, nuclei counts in myotubes as well as aggregated nuclei were labeled. The fusion index has been computed as percent of sum total of all embedded nuclei in myotubes.

Antibacterial activity

A standard Staphylococcus aureus strain obtained from American Type Culture Collection (ATCC strain no.29213) was utilized for antibacterial activity. S. aureus colonies were developed on nutrient agar (Merck, Darmstadt, Germany); virgin colonies were sealed in cryo-vials and stocked at − 80 °C until future exploitation. N. sativa oil grafted HAP scaffolds have been tested [20] for biofilm inhibition. The bacterial strain was cultured (106 CFU) with different concentration of samples. Twofold concentration technique was used to find minimum inhibitory concentration (MIC) of N. sativa grafted HAP scaffolds. Dilutions employed in present investigation were 0, 25, 50 and 100 mg/tube correspondingly. Expansion pattern was observed at every 4 h through OD inspection by spectrophotometer to validate MIC. Required growth parameters such as incubation temperature (37 °C), rpm (150) were maintained in shaker. Complete time duration to supervise inhibition by HAP and N. sativa grafted HAP scaffolds was 16 h.

Results

Since ancient times, N. sativa oil was most extensively being consumed for cure of various health issues. This study involves successful fabrication and characterization of N. sativa grafted biogenic HAP scaffolds in order to analyze myoblasts proliferation and differentiation. We used XRD to define crystal structure of synthesized scaffolds. Figure 1 demonstrates XRD spectra of pure HAP particles. All observed XRD peaks of pure HAP can be assigned to solitary hexagonal organization of HAP (JCPDS No. 82–1943) [21]. Pointed crests have been noted in calcined sample (at 600 °C for 5 h) signifying high crystalline phase owing to disintegration of organic constituents of cow bones at elevated temperature (Fig. 1). No impurities like hydroxide and phosphates of calcium were noticed, which indicates formation of pure HAP. Figure 2 illustrated micro-structural study of N. Sativa oil grafted HAP scaffolds, which are spherical in shape, highly dispersed and size was less than 1 μm (Fig. 2A). To further confirm size of particles, we performed TEM characterization. Figure 2B shows a typical TEM image of N. sativa oil grafted HAP composite particles. It again confirmed that particles are having spherical morphology along with some elongated shape. The average size of particles was ~ 500 nm. The chemical composition of N. sativa oil coated HAP particles was analyzed by EDX spectra (Fig. 3). As shown in spectra (Fig. 3), it comprised of calcium (Ca), phosphorous (P), oxygen (O) and a prominent peak of carbon (C) only. FT-IR studies were done to know molecular structure or to detect functional groups present in HAP particles. Figure 4A depicts FTIR spectra of HAP samples synthesized at 600 °C. The assimilation group at 3570 cm−1 was assigned to characteristic hydroxyl groups (–OH) stretching vibration in crystallized pattern of HAP. Furthermore, weak peaks at about 1405 and 1460 cm−1 were assigned to absorption bands of CO32−, indicating carbonate ions formation. The major peaks at 559, 605, 962.0 and 1027 cm−1 could be assigned to (PO4 group) PO43−. Figure 4B depicts FT-IR spectra of pure N. sativa oil. The pure N. sativa oil displays distinctive peaks at 720, 1185 and 1464 cm−1 that could be assigned to peaks of alkene (=C–H bending), ester (C–O Stretch) and alkanes (C–H bending) groups, respectively [22]. N. sativa oil has spiky peaks at 1744 cm−1. The ridges have accredited to carbonyl (C=O) stretching vibration [23]. Notable characteristic peaks for N. sativa oil were at 2854 cm−1 (C–H in-CH2), 2923 cm−1 (C–H in-CH2), and 3009 cm−1 (C–H in HC=CH) owing to domination of carbon shackles of fatty acids [24, 25]. Figure 4C depicts N. sativa oil coated HAP scaffolds. In case of N. sativa coated HAP scaffolds, all characteristic peaks of HAP and N. sativa oil are present in the scaffolds (Fig. 4C). It suggested that the identity of HAP and N. sativa oil is maintained and they coexist in N. sativa coated HAP scaffolds [26, 27].

Fig. 1.

Fig. 1

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XRD spectrum of biogenic HAP scaffolds

Fig. 2.

Fig. 2

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A SEM, B TEM micro-structure of N. sativa oil grafted HAP scaffolds

Fig. 3.

Fig. 3

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The chemical composition spectra of N. sativa oil grafted HAP scaffolds

Fig. 4.

Fig. 4

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FT-IR spectra of pristine biogenic HAP, N. sativa oil and N. sativa oil grafted HAP scaffolds

The biocompatibility of N. sativa grafted HAP scaffolds were tested in vitro using myoblasts. The survival of cultured C2C12 was monitored by CCK-8 calorimetric assay and no negative impact has been perceived; rather, cells were found exponentially growing on N. sativa grafted HAP scaffolds (Fig. 5A). Healthy and integral cells were marked green whereas compromised plasma membranes tag red. The magnitude of viable myoblasts on N. sativa grafted HAP scaffolds was best possible. Contrariwise; the quantity of compromised cells was meager (Fig. 5B, C). The outcomes of immunofluorescence labeling and CCK-8 test are in fine correlation. The N. sativa coated HAP scaffolds demonstrated antibacterial effect against S. aureus strain. The logarithmic stage was recognized from 4 to 8 h in pristine cultures (Fig. 5D). The perceptible changes in kinetics were observed from 4 to 8 h point time. The N. sativa coated HAP scaffolds were found excellent to counteract the biofilm formation.

Fig. 5.

Fig. 5

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In vitroA CCk-8 cytotoxicity assay, B immunofluoresence staining at low. Arrows indicate the mature myotubes with visible nuclei and C high magnification. Arrows indicate the mature myotubes with visible nuclei, D A bar diagram representation of optical density (600 nm) of supernatants of S. aureus cultured with different concentrations of HAP and N. sativa oil grafted HAP scaffolds (100% growth represents S. aureus in nutrient medium without N. sativa and HAP)

Moreover; a facile system to gauge myotube organization has been established. Calculations of darkly stained spots were utilized to compute myotube density (Fig. 6). Estimation of myotube compactness mirrored blending index all through C2C12 differentiation (Fig. 7). Figure 7 shows histograms drawn on basis of data obtained from randomly taken images after applying ImageJ software.

Fig. 6.

Fig. 6

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Jenner–Giemsa stain exposes dark myotube formation at point in C2C12 differentiation. C2C12 cells differentiated in lesser amount of serum with 70–80% growth (day 0). Descriptions are model pictures of cultures mounted at days 0, 1, 3, 5, and 7. Deeply tainted multinucleated myotubes appeared at day 3 with faint textured myoblasts. Myotube numerals and dimension increased at days 5 and 7 respectively. Yellow dashed arrows indicate the formation of myotubes with increased size in differentiating myogenic cell cultures

Fig. 7.

Fig. 7

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Variations in nuclei digit, fusion index, and myotube mass throughout C2C12 differentiation. Snaps of C2C12 cells for days 0, 1, 3, 5, and 7 post-confluence A Total nuclei number B Fusion index calculated as fraction of total nuclei present inside myotubes C Myotube density estimated as summation of pixels endorsed to tones 0–75 D Average image histograms during C2C12 differentiation. All photos were evaluated with ImageJ to get histogram. The x-axis describes array of grayish tones from 0 (black) to 255 (white), whereas y-axis depicts numeral of pixels credited to each tone. Lines symbolize typical pixel count for each tone, and error bars correspond to SD of three replicates Tissue Eng Regen Med

Discussions

The C2C12 cell line is an excellent replica to investigate myogenic process, differentiation of myoblasts as well as osteoblasts, owing to its quick growth and maturation into skeletal muscle cells which possess great capacity to contract and produce vigor [28]. In addition; frequency of muscle generation in C2C12 could be adjusted through introducing loss of functions of crucial genes responsible for union of myoblasts as well as myogenesis [29]. Nevertheless; C2C12 cells have also been applied to scan cell cycle allocated to its rapid multiplication. More to the point, C2C12 has also demonstrated outstanding ability to articulate a variety of proteins and has facilitated in mechanistic studies [30, 31]. Considering, aforementioned, properties of C2C12, in present study we utilized this cell line to check differentiation on N.sativa coated HAP scaffolds which can be used as a possible candidate of implantation.

Rheumatoid arthritis is categorized by degradation of collagen, persistent pain in joints as well as atrophy of skeleton muscles. Earlier studies have evidenced health-giving implications of N. sativa on joint and bone ailments. It is reported in literature that N. sativa reverses osteoporosis is possibly due to presence of ample amount of unsaturated fatty acids and its antioxidant and anti-inflammatory qualities. In folk medicine the N. sativa seeds have been applied for diverse infections and poor health conditions probably due to presence of thymoquinone. A research investigation has indicated positive influence of N. sativa oil in curing the skeletal muscle disease along with many other clinical illnesses [32]. Another study has indicated that thymoquinone reduces muscular oxidative anxiety and also shields skeletal muscles from ischemic injury [33]. Therefore, this study was carried out to throw out light on preventive impact of N. sativa grafted HAP with respect to differentiation of myoblasts into myotubes. It has been observed that cultured C2C12 cells depicted optimum growth on N. sativa coated HAP scaffolds. As a matter of fact the essential oils are also known to smash up the cell membrane, clot cytosol, hamper ATP generation [34] as well.

The potential reason for complete impedance of bacteria could be the hydrophobicity of essential oil (N. sativa seed oil) which ostensibly destroys bacterial wall. The distorted cell wall enhances permeableness, so, interrupt cellular operations (i.e. energy equilibrium, solute movement as well as metabolism).

Furthermore; C2C12 myoblasts experienced in vitro myogenesis in order to configure poly-nucleated protein loaded myotubes. Conventionally, magnitude of differentiation is calculated by quantity of all nuclei embodied in to myotubes. However; this method was laborious and vulnerable to operator bias. Therefore; Jenner–Giemsa staining technique [35] has been applied. It has been reported [36] that polyploidy is natural phenomenon accompanied by differentiation. Insufficient information is available, even though in general, it is acknowledged that it can participate in gene augmentation, cell survival, genome realignment as well as chromosome interplays [37].

To conclude, our data demonstrate efficacy of differentiation arrangement that gradually convert undifferentiated fusiform myoblasts into elongated multinucleated myotubes demonstrating contractile muscle cell characteristics. The biological tests indicated that N. sativa grafted HAP scaffolds could be novel biogenic material for myogenic differentiation. This will be first report on unique combination of N. sativa grafted HAP scaffolds for implant use. Remarkably, N. sativa grafted HAP has indicated incredible capacity as effectual antiosteoporotic scaffolds, inheriting antioxidant and anti-inflammatory features; so, can be utilized as upcoming bone and muscle tissue engineering implants.

Compliance with ethical standards

Conflict of interest

The authors declare no conflicts of interest.

Ethical statement

There is no use of animal models for the experiments. All authors approve the submission.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Contributor Information

Touseef Amna, Email: touseefamna@gmail.com.

M. Shamshi Hassan, Email: mshasan@bu.edu.sa.

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