Journal of Biomimetics, Biomaterials and Biomedical Engineering ...

Journal of Biomimetics, Biomaterials and Biomedical Engineering ISSN: 2296-9845, Vol. 30, pp 45-60 doi:10.4028/www.scientific.net/JBBBE.30.45 © 2017 Trans Tech Publications, Switzerland

Submitted: 2016-09-14 Revised: 2016-11-02 Accepted: 2016-11-03 Online: 2017-01-25

Novel Nanocrystal Clay Materials with Potential Bone Cells Growth Enhancement or Inhibition Characteristics In Vitro Elvis K. Tiburu1,a*, Benjamin W. Kankpeyeng2,b, Samuel N. Nkumbaan2,c, Ali A. Salifu3,d, and Jianqin Zhuang4,e 1

Department of Biomedical Engineering, University of Ghana;

2

Department of Archaeology and Heritage Studies, University of Ghana;

3

Department of Mechanical Engineering Sciences, University of Surrey, UK; 4

Department of Chemistry, College of Staten Island, NY, USA..

a

[email protected],[email protected],[email protected], [email protected] and [email protected]

Keywords: Archaeology, Ethnographic Clay, XRD, FTIR, EDX, Osteoblasts

Abstract. The application of clay nanocrystals in healing has gained popularity in recent years. The objective of this work was to investigate whether healing clays obtained from different geographical locations could influence differential cell growth. X-ray diffraction analyses of both nanocrystal materials revealed orthorhombic chamosite structure with lattice parameters: a =15 Å, b= 8 Å and c=7 Å whereas energy dispersive x-ray results showed the presence of Al, Si, Fe and O in both materials. However the porosity measurements of the two materials revealed different pore structures. Both materials were tested on human fetal osteoblast cells and the results showed differential cell growth in vitro. The results underscore the significance of pore structures in cell response as against the chemical composition or the structure of the material. Future mechanistic evaluation would be conducted to better understand the pathways leading to the increased/decrease osteoblast adhesion and proliferation by these materials and possible modification of the clay materials for biomedical applications. Abbreviations: BET, Brunauer, Emmett and Teller; XRD, X-ray diffraction; FTIR, Fourier transform infrared spectroscopy; SEM, Scanning electron microscopy; EDX, Energy dispersive x-Ray spectroscopy; AAS, Atomic absorption spectroscopy; ANOVA, Analysis of variance; TGA, Thermogravimetric analysis Introduction Medical practices in prehistoric times used magico-spiritual procedures in combination with materials, such as clay, soil and plant parts to cure various ailments including chronic and infectious diseases [1-4]. To date, clay minerals have been identified in biomedical applications including human treatment and healthcare delivery [5-7]. The ingestion of a clay preparation has been reported to serve as a rich source of essential elements such as iron, copper, calcium, zinc, and manganese for pharmaceutical and cosmetic applications [8]. Additionally, the ability of clay to detoxify and also for treating gastrointestinal diseases like diarrhea has been reported [6, 9-11]. Clay minerals also possess antibacterial properties, which is depended on the concentrations of metal ions like Cu2+, Co2+, and Zn2+ among others in the clay [12]. In recent times, nanoparticles from clay have been used to reinforce polymeric materials in the preparation of supporting structures for tissue engineering constructs [13-15]. According to some researchers, the incorporation of clay nanoparticles into bacterial cellulose for tissue engineering constructs, improved the mechanical and thermal properties of the constructs [16]. Along these lines, there is an interest in recent times to explore past human practices including the use of clay to cure certain disease states [17]. Archaeology, which is the study of material remains of past human life and activities, is therefore gaining prominence in modern medical research [18-20]. Archaeological All rights reserved. No part of contents of this paper may be reproduced or transmitted in any form or by any means without the written permission of Trans Tech Publications, www.ttp.net. (#73053498-16/01/17,20:57:51)

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Journal of Biomimetics, Biomaterials and Biomedical Engineering Vol. 30

surveys and excavations in Komaland in the Northern Region of Ghana from 2007 to the present have recovered material remains with medicinal practical indications [21, 22]. A pot containing clay materials was recovered from a feature excavated during the January 2011 Field Season from a mound radiocarbon dated to the ninth and twelfth century. The feature consisted of local ceramics with special assemblage with clay figurines, quartz querns, and biotite muscovite schist grinders [23]. An iron razor blade was recovered with the clay suggesting a causal linkage with some medical implications since the use of natural clay for cell growth and treatment of diseases has been extensively documented [24-28]. There are ethnographic parallels to the use of the clay in treating related ailments including fractured bones in Yikpabongo in the Northern Region of Ghana. The ethnographic clay is consumed in large quantities to treat various diseases especially in pregnant women. Contemporary societies within the region still use clay for such medicinal or health reasons, and therefore we seek to compare the nature and morphology of the ethnographic medical clay from the area to that of the archaeological clay material utilizing biophysical techniques such as X-ray diffraction (XRD), Scanning Electron Microscope (SEM), Fourier Transform Infrared spectroscopy (FTIR), and nitrogen adsorption desorption (BET) studies. We also analyzed the mineral composition of the clays using Energy Dispersive X-Ray (EDX) analysis. The study was intended to also investigate the effects on bone growth which is crucial for fetal development in the mother’s womb. Both clays were tested on bone cells (human fetal osteoblast cells) adhesion and growth. The systematic analyses and testing of both clays will allow us to draw conclusions that can support their past and present usage as medications to treat diseases or as nutritional supplements to the indigenous people. The main objective of this work was to analyze the clays and provide experimental evidence of their safety for treating diseases, and use them as model systems to conduct mechanistic studies on broad range of cells lines. The results reported in this paper indicate the enhancement of human fetal osteoblast cells growth in vitro in the presence of the archaeological clay but decrease in bone cell growth with the ethnographic medical clay. These findings highlight the health risk associated with the use of the ethnographic (geophagy) clay to treat medical ailments especially among pregnant women. The studies also reveal the potential use of these clays to enhance bone tissue rejuvenation or to treat bone diseases. Materials and Methods Clay Materials The archaeological clay was obtained from a depth of about 60 and 70 centimeters at Yikpabongo in the Northern Region of Ghana in West Africa. The ethnographic clay was obtained from the locals living in the same communities. The following materials were used for tissue cultures: human fetal osteoblast cells (hFOB 1.19) (LGC/ATCC, Teddington, UK), alamar blue reagent (Life Technologies Ltd, Paisley, UK), 1:1 mixture of Dulbecco’s Modified Eagle Medium and Ham’s F12 Medium (DMEM/F12, 1:1) (Life Technologies Ltd, Paisley, UK), fetal bovine serum (Life Technologies Ltd, Paisley, UK), and geneticin® selective antibiotic (G418) (Life Technologies Ltd, Paisley, UK). The clay was sieved with a 106 µm cut-off sieve, heated at 600 °C and used for this study. XRD, SEM, AAS and EDX Sample Preparation A gap was left in the middle after two strips of a 50 µm thick blue tape were stuck on either edge of an aluminium stub. With the aid of a toothpick, colloidal graphite (Agar Scientific Ltd, Stansted, UK) was deposited in the gap and spread along the stub with a blade that was touching both tape strips at the edges. The strips were subsequently removed leaving approximately 50 µm of thick colloidal graphite. Each powdered sample was then deposited on the sticky colloidal graphite and excess particles were removed by tapping the stub sideways followed by blowing with an aerosol duster (air duster).


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Atomic Absorption Spectroscopy Atomic absorption spectrometer (PinAAcle 900T) with a combined flame/longitudinal Zeeman furnace system and a solid-state detector was used for the elemental analysis. XRD Experiments XRD was performed with a PanAnalytical diffractometer using CuKα radiation at 2θ, scanning from 5 to 80 degrees in steps of 0.05 degrees, with a tube voltage of 45 kV and a current of 40 mA. FTIR Experiments The FTIR spectrum was recorded with a Nicolet Instrument Co. MAGNA-IR 750 Fourier transform infrared spectrometer. Nitrogen BET Adsorption Nitrogen adsorption measurements were performed at −196 °C on a Micrometrics ASAP 2020 volumetric adsorption analyzer. Before the analysis, samples were degassed under vacuum at 200 °C for 2 h in the port of the adsorption analyzer. SEM Experiments Each sample was then removed from the FE-SEM machine after the EDX analysis and sputtercoated with 3 nm of gold. SEM micrographs were obtained at an accelerating voltage of 10 kV using the JEOL JSM-7100F Field Emission Scanning Electron Microscope. EDX or EDS Experiments EDX spectra and elemental composition of each sample were obtained at accelerating voltages of 5, 10 and 15 kV using a JEOL JSM-7100F Field Emission Scanning Electron Microscope equipped with a Thermo Scientific UltraDry EDX detector. TGA Experiments Thermogravimetric analysis (TGA) was used to examine the thermal stability of the clay samples during the pretreatment at a heating rate of 10oC min-1 under nitrogen atmosphere of 40 mL min-1 from 25 – 1000oC. Tissue Culture The complete growth medium for the hFOB 1.19 cells consisted of the DMEM/F12 (1:1) basal medium supplemented with 10% fetal bovine serum and 0.3 mg/ml G418 antibiotic. Alamar blue reagent was diluted 1 in 10 with the complete cell culture medium. Thereafter, the clay material was added to the diluted alamar blue solution to produce working solutions with final concentrations of 2 mg/ml, 5 mg/ml and 10 mg/ml. The hFOB 1.19 cells were seeded in T75 flasks at a seeding density of 4 x 103 cells/cm2 (i.e. 3 x 105 cells seeded in total per T75 flask) after which 6 ml of each of the 2, 5 and 10 mg/ml working solutions containing the test samples were added to separate flasks. A separate T75 flask containing only the cells and alamar blue solution was used as a control. The hFOB 1.19 cells and the samples were incubated at 37 °C and 5% CO2 for 72 hours and 100 µl aliquots were transferred to Nunc 96-well plates (Fisher Scientific, Loughborough, UK) after 3, 24, 48 and 72 hours for absorbance measurements in a FLUOstar Omega microplate reader (BMG Labtech GmbH, Offenburg, Germany) at 570 nm. The absorbance values were normalized against the 3 hour values and used for analysis. Statistical Analysis All data were expressed as the mean with standard deviation for n=3 repeats using an in-house MATLAB code for the analysis of variance (ANOVA). We used one way analysis of variance (ANOVA) following multiple comparisons with a Fisher’s least significant difference (LSD)

48


procedure at the 95.0% confidence level to determine statistical significance between groups. We assumed that all cell populations involved follow a normal distribution and had the same variance (or standard deviation). The data values were also randomly selected and were independent of one another. Results EDX, XRD and FTIR Analysis Biomedical applications of the archaeological clay and ethnographic clays will depend on their dispersion properties associated with their mineral content that underpin well-defined structural features to support biological activity. The mineral composition of the clay materials were investigated with EDX. Five sampling sites were used to estimate the elemental composition using EDX analysis (Fig. 1). The analyses indicated that both samples contained Si, Al, Fe, O and C (Fig. 1). A

B

Fig. 1: The EDX spectra showing the major peaks originating from Fe, O, Al, Si and C from (A) archaeological clay material and (B) ethnographic medical clay all confirming the chemical composition the major constituents within each material. However, the ethnographic medical clay sample also contained K. Based on the elemental composition, the weight percentage ratio of the ions Si/Al≈1.5 and Si/Al≈5 for the archaeological and the ethnographic samples respectively were obtained. Detailed elemental analysis was conducted to estimate the minerals including the sodium composition within the materials, as high concentrations of this metal ion has been linked to the imbalance of electrochemical forces between the particles leading to clay dispersion (Table 1). The low concentration of Na is an indicative of clay materials which are aggregated with well-defined crystal lattice structures.


49

Table 1: Chemical analysis of the archaeological and the ethnographic clays Table 1: The elemental analysis of the archaeological and the ethnographic clays. The amounts are expressed in mg/kg. Ni Pb Cr Zn Ca Na Mg Fe Arch.

155.40

105.90

28.74

7.09

128.74

31.45

Ethno.

134.32

189.32

25.56

9.15

119.82

43.19

484.20 391.91

3282.08 3067.26

This was confirmed by the X-ray diffraction (XRD) studies with characteristic signature peaks of d001 and d100 basal spacing at 7.1 Å and 12.6 Å respectively (Fig. 2A). Additional signature peaks at positions 21 and 27 Å at angles were assigned and are all indicative of a chamosite clay based on matching data from the XRD database (Fig. 2A). However, majority of the components in the ethnographic clay were quartz (Fig. 2B). A 401

6000

5000

310

2000

001

3000

100

Counts

4000

1000

0 20

40

2 -Theta

B 1200

1000

Count

800

600

400

200

0 20

40

2-Theta

Fig. 2: The XRD spectra of the (A) archaeological clay material and (B) ethnographic medical clay all exhibiting characteristics peaks at 2-theta angles of 7.1, 12.6, 21 and 27 A°.

50


The FTIR was used to study the molecular interactions that inform the surface chemistry. The bands located in the range from 650–745 cm-1 were assigned to symmetric T–O–T vibrations of the clay framework (Fig. 3). There is also a small shoulder at 930 cm-1 which is a characteristic band for SiO-Si and Si-O-Al asymmetric stretching vibrations exhibited in most aluminosilicate materials. Another major dip at 1000 cm-1 is due to Si-O-Al symmetric stretching (Fig. 3). There is a band at 1600 cm-1 due to Si-O modes in the silica matrix. A broad peak spanning from 3000 to 3500 cm-1 are OH species interacting through H-bonding (Fig. 3). The most characteristic peaks exhibited by the clays are 3620 cm-1 and 3700 cm-1 due to NH2 stretching. There is another peak at cm-1 due to C=O in both spectra. The only unique difference between the archaeological and ethnographic clays is the appearance of an additional band at 1700 cm-1 due to C-O stretching modes in the ethnographic clay sample (Fig. 3B). A

1.00

OH C=O NH2

Si-O-Si and Si-O-Al Asymmetric Stretches

% Transmittance

0.95

0.90

0.85

0.80

0.75

4000

3500

3000

2500

2000

Wavenumber(cm-1)

1500

1000


51

B 105 100

C-O C=O

OH

90

Si-O-Si and Si-O-Al Asymmetric Stretches

% Transmittance

95

85 80 75

NH2

70 65 3500

3000

2500

2000

1500

1000

500

Wavenumber (cm-1)

Fig. 3: FTIR spectrum of the (A) archaeological clay material and (B) ethnographic medical clay both showing characteristics signature bands for various vibrational modes for T-O-T, Si-O-Si and Si-O-Al. The XRD and FTIR results provided key structural information of the clays that can inform whether such materials can support the mechanics and biological behavior of cells. SEM and BET Analysis Information regarding the particle size, pore structures and pore volume of the archaeological clay material was determined by the Brunauer, Emmett and Teller (BET) adsorption technique (Fig 4). The experimental curves (black, Fig. 4A)) represent the adsorption-desorption profile typical of a type IV isotherm where the presence of the knee indicates strong adsorbate-adsorbent interaction. The average pore width after BET Nitrogen desorption experiments was 5.6 nm and the pore diameter was 29 nm, putting the clay material beyond microporous and into a mesoporous material (Fig. 4B). The linear plot from the BET measurements was used to estimate the surface area of the zeolite (Fig. 4C). The experimental values (solid dots) fit very well with the theoretical line with an R-square value of 0.998 (Fig. 4C). The slope was determined from the graph and used to calculate the BET surface area. The BET surface area of the zeolite pore at P/Po = 0.199926141 was 14.7817 m²/g with a pore volume of 0.020763 cm³/g. There was no such structural arrangement observed in the ethnographic clays (data not shown).

52


A

B 0.0006

30

arch106

0.0005

25

Pore Volume (cm-3/gnm)

Volume Absorbed cm3/STP/g

Type IV

20

15

10

0.0004 0.0003 0.0002 0.0001

5

0.0000 0 0.0

0.2

0.4

0.6

0.8

0

1.0

20

40

60

80

100

120

Pore Diameter(nm)

P/Po

C

experimentat fit theoretical fit

0.06

B (Unit 2)

0.05

0.04

0.03 Equation

y = a + b*x

Weight

No Weighting

Residual Sum of Squares

0.02

3.55037E-6 0.99945

Pearson's r

0.99884

A dj. R-Square

Value ?$OP:A =1

0.01 0.04

0.08

0.12

Intercept

Standard Error

6.72487E-4

2.55797E-4

0.29296

0.00243

Slope

0.16

0.20

A (ASAP 2020 V3.04 E)

Fig. 4: BET measurements of the archaeological clay (A) Nitrogen adsorption-desorption studies of the clay material. (B) The pore distribution within the clay material. (C) The BET linear plot to estimate the surface area of the clay material. The solid squares represent the experimental points and the straight line represents the theoretical fit. The SEM image of the clays revealed the layered arrangement of the particles and their size distribution before pretreatment (Fig. 5A & B)) and after pretreated at elevated temperature to remove all contaminants and to kill microorganisms (Fig. 5C & D).


B

A

C

53

D

Fig. 5: Scanning electron image of the archaeological clay material: (A) the archaeology raw material excavated from a depth of seventy centimeters beneath the ground and (B) after sieving the archaeological clay sample with a 106 µm cut-off sieve and pretreated at 650 oC. : (C) ethnographic medical clay (D) after sieving the ethnographic clay sample with a 106 µm cut-off sieve and pretreated at 650 oC. All monographs were taken at 10,000x magnification. Scale bar = 1 micron. The TGA profiles of the archaeological clay showed three step decomposition patterns corresponding to 0.7%, 1% and 3% at temperature ranges 0-50 °C, 50-200 °C, 200-600 °C respectively (Fig. 6A) with the first step revealing the decomposition of the organic materials and the evaporation of water from the surface at about 50 °C to 200 °C. At 600 °C, the water within the pore structures begin to evaporate followed by rearrangement of the metal ions within the material framework. Similar three step decomposition was exhibited by the ethnogarphic medical clay except that the decomposition percentages were 1.5%, 1.8% and 1.0% at temperature ranges 0-50 °C, 50-200 °C, 200-600 °C respectively (Fig 6B).

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A 102

Weight Loss (%)

100

98

94 0

200

400

600

800

600

800

Temperature (oC) B

Weight Loss (%)

102

100

98

94 0

200

400

Temperature (oC) Fig. 6: TGA analysis of the clay material indicating a three step decomposition of the samples. (A) archaeological clay and (B) ethnographic clay. Alamar Blue Cell Viability Assay First the choice of using human fetal osteoblast cells over adult bone cell is because the former proliferates rapidly to produce homogenous mineralized nodules and is therefore a better model to study certain stages of human osteoblast differentiation. The fetal bone cells are also a good choice because the clays are used extensively by pregnant women and therefore health risk to fetus can only be examined by the use of this cell line. The principle of operation of the Alamar Blue assay is based on the reduction of resazurin into a highly fluorescent red-colored product known as resorufin


55

within the cytosols of metabolically active viable (live) cells. When live cells are exposed to the Alamar blue reagent, the resazurin enters into the cells and gets converted into resorufin which then diffuses out of the cells into the surrounding culture medium. An increase in the population of viable cells leads to an increase in the amount of red color/fluorescence generated in the culture medium as resazurin is continuously converted into resorufin. Thus, cell viability can be quantified by measuring the absorbance of resorufin at 570 nm or measuring its fluorescence. Resazurin cannot be converted into resorufin by non-viable (dead) cells due to the rapid loss of their metabolic abilities making dead cells unable to produce any red color or fluorescence after exposure to the Alamar Blue reagent. The normalized cell growth at 570 nm after 3 hours of cell growth for the control, 2 mg, 5 mg and 10 mg in the presence of the archaeological clay material were 0.216 ± 0.0015, 0.227 ± 0.0096, 0.510 ± 0.0180, and 1.036 ± 0.0744, respectively, with similar results obtained from the ethnographic clay. A comparative adhesive behavior of the cells with 10 µg/µL concentrations of the clays using the cell adhesive test after 4 hours were also determined (data not shown). We converted the absolute absorbance values in the presence of the clay materials into cell count using a standard curves to construct a response curve as a function of 0, 24, 48 and 72 hours of cell growth (Fig. 7). General trends indicated progressive enhancement of cell viability from 24 hours to 72 hours as compared to the control in the presence of 2, 5 and 10 µg/µL of the archaeological clay material (Fig. 7A). We noticed a modest decrease in cell viability when 2, 5 and 10 µg/µL of the clay material was introduced into the cells within the 24-hour time frame and this could be due to the adaptive behavior of the cells to the foreign material (Fig. 7A). However, up to 48 hours, we saw elevated levels of cell proliferation at 5 and 10 µg/µL concentrations of the material (Fig 7A). At 72 hours, with the exception of the 10 µg/µL clay treated cells, lower concentrations of the material showed slight decreases in cell growth. This trend suggests a more favorable osteoblast environment created by the clay material. The ethnographic sample was also studied under similar cell culture conditions and the results clearly indicated that the archaeological clay enhances bone cell growth, whereas the ethnographic medical clay rather inhibited the growth of bone cells (Fig. 7B). A 2000000

Control 2mg 5mg 10mg

1800000 1600000

Cell count

1400000 1200000 1000000 800000 600000 400000 200000 0

24

48

Time (hours)

72

56


B 1600000

0 mg/ml 2 mg/ml 5 mg/ml 10 mg/ml

1400000

Cell Count

1200000 1000000 800000 600000 400000 200000 0

24

48

72

Time (hours)

Fig. 7: (A) Effects of varying concentrations of the archaeological clay material on the growth of human fetal osteoblast cells (hFOB 1.19). Error bars represent standard deviation (n = 3). (b) The effect of varying concentrations of the ethnographic medical clay on the growth of human fetal osteoblast cells (hFOB 1.19). Error bars represent standard deviation (n = 3). A

Control 2 µg/µL 5 µg/µL 10 µg/µL

2000000

Control

1500000

1000000

500000

0 0

20

40

Time (hours)

60

80


57

B *

1600000

Control 2 µg/µL 5 µg/µL 10 µg/µL

1400000 1200000

*

* Cell count

1000000

*

800000 600000

*

400000 200000 0 0

24

48

72

Time (hours)

Fig. 8: (A) A comparison of the cell numbers as a function of time in the presence of 0, 2, 5 and 10 µg/µL of the archaeological clay material. (B) Similar comparison with the ethnographic medical clay. Error bars represent standard deviation (n = 3). *Based on the computed p-value of 0.05 and 0.003 for the archaeological clay material and the ethnographic medical clay respectively as against the untreated cells (which is almost equal to the significance level testing at 5%), we conclude that the means are statistically significant at higher concentrations. We compared the inhibitory effects of the control with those in the presence of 2, 5 and 10 µg/µL at 24, 48 and 72 hours (Fig. 8). The patterns clearly indicated a tremendous influence of the archaeological clay material after 72 hours of cell growth. There was an increase in cell count from 1.5 E6 to 1.8 E6 after 72 hours of cell growth upon the addition of the archaeological clay material but a drastic drop form 1.5 E6 to 0.6 E6 in the presence of the ethnographic clay at the same time frame resulting in 2.5 fold drop in cell concentrations when the former was used. These results clearly reveal the health risk regarding the continuous use of the ethnographic clay material in these contemporary societies within the region especially for women who used these clay materials for various health reasons. Discussion The major groups of clay minerals include kaolinites, smectite (bentonite), chlorites, spiolite and illites [6]. Porous chitosan-organically modified montmorillonite, halloysite, clinoptilolite clays have been investigated to understand their intercalation and exfoliation capabilities which are key for use of these organoclays as scaffold for tissue growth and also in drug delivery applications as well as for anti-inflammatory, anti-bacterial, and cytotoxicity effects [29-31]. It is widely used for medical applications in Africa and other parts of the world, Geophagy (eating clay) is common among children and adults with average daily consumption rate in Africa ranging from 10-30 grams [32, 33]. The most geophagical customs are that related to pregnancy and lactation where large amounts of clay is consumed by pregnant women to provide the fetus with essential minerals such as calcium, magnesium and iron [34, 35]. For example the clay deposits in Ghana especially in the Tongo Hills of northern Ghana (Gbankil, Kusanaab, and Yaane medicinal clays) and Yikpabongo where these clays are obtained are mainly composed of quartz, feldspar, albite and kaolinites and are known for their medicinal efficacy [22, 36, 37]. However their characteristics, mechanics and biological behavior on disease control is yet to be understood.

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In this work we characterized and investigated a clay sample which was excavated in context from the ancient city of Yikpabongo in Northern Ghana and compared this material with an ethnographic medical clay samples from the same geographical location. Through biophysical characterization of the archaeological as well as the ethnographic medical clay samples, we were able to identify one as a chamosite and the other as quartz. The chamosite has an ideal structure, Fe6(Al,Si)4O10(OH)8. This type of clay is very rare and possessed elevated levels of Fe by substituting Al and Si in kaolinite as revealed in our elemental analysis and by other researchers [38-41]. The extensive use of the ethnographic clay by the contemporary societies and potential benefits that may be derived for biomedical applications lead to the detailed analysis of the chemical compositions in the two clays to estimate and quantify the elements especially the sodium ion concentration which is a key ingredient that have influence on the intercalating or exfoliating properties of clay samples. The level of sodium was very negligible suggesting these clays exhibit nondispersive properties which can be modified to support biological activity. However, the two clays revealed elevated amounts of Fe which studies have indicated is an important element to maintain bone health and regeneration. However, when the two clays were tested on bone cell growth, the chamosite clay revealed cell growth enhancement whereas the ethnographic clay rather has inhibitory effect on bone cell growth in vitro (suggesting Fe is not the key for bone growth in this case). The studies therefore highlight key findings regarding the use of the ethnographic clay in contemporary societies within the Yikpabongo region to treat diseases among pregnant women because excessive consumption of the material by pregnant women may affect fetal development. To the best of our knowledge, this is the first time chamosite clays have been tested for biological activity in addition to what is already known about their catalytic efficiency demonstrated previously as versatile for various organic synthesis reactions [39, 42]. While, extensive studies have been conducted on medical clays, such as montmorillonite, halloysite and bentonites, to our knowledge, there are no studies on the influence of chamosite clays on biological activity (especially, bone cells) [31, 43]. The uniqueness of this clay may be a potential material in developing three-dimensional ceramic or composite porous or fibrous scaffolds that could be used in bone tissue engineering applications [44, 45]. The addition of this clay to what is already discovered about the natural occurring medical clays will enhance the efforts being directed towards nanotechnology methods for preparing nanocomposites with novel physical, chemical and biological properties using clays [46-48]. For example, numerous studies have now demonstrated that nanoscale surface features (which these clays possess) have greater surface energy to promote the adsorption of select proteins that increase osteoblast functions [38] the same events may be happening here. The novelty in this work is that by developing in vitro testing of the effect of different healing clays on a broad spectrum of cell lines including human bacterium pathogens, we can understand the healing process of these clays. By understanding the mechanism of cell-clay material interaction, we can identify easy and readily available materials for scaffold synthesis to aid in studies of biological systems including tissue growth. Conclusion The evidence demonstrated in this work that the growth of human fetal osteoblast could be enhanced on a chamosite clay material excavated from the Northern Region of Ghana has the potential to stir interest in the scientific community for the search for new materials from ancient material practices for today’s biological applications [49]. Further work will be undertaken to develop strategies to purify the clay and develop nanocomposite with enhance properties involving their dispersion, exfoliation ability and controlling their intercalation and their overall impact on mineralization as well as cellular response of stem cells differentiation. Acknowledgements We will like to thank the Office of Research, Innovation and Development for supporting this work with Grant #: UGRF/9/LMG-011(EKT).


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