Egg shell membrane – a potential natural scaffold for human meniscal tissue engineering: an in vitro study

Abstract

In the present study, natural egg shell membrane (ESM), harvested from locally available single comb white leghorn hen eggs was investigated for its ability to support adhesion and proliferation of human meniscal cells. The harvested ESM was subjected to moist heat (autoclaving) and compared with raw egg shell membrane (RESM) for meniscal cell growth. RESM and autoclaved egg shell membrane (AESM) were characterized using suitable techniques like field emission scanning electron microscopy (FESEM), solid surface zeta potential, Fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA) and biodegradability in trypsin and phosphate buffered saline (PBS). From the characterization studies, it was evident that autoclaving resulted in surface modification of RESM. RESM was cationic in nature and was covered with a mucilaginous coating on the surface. However, AESM showed almost neutral surface charge without any mucilaginous coating. The AESM showed increased resistance to biodegradation when compared to RESM. The primary human meniscal cells, seeded onto the fibrous side of both ESM showed increased cell adhesion and proliferation in AESM than RESM. Extracellular matrix (collagen and glycosaminoglycan) secretion into the external medium by the adhered cells was higher with AESM than RESM. Cell attachment, DNA content on the scaffold, cell proliferation index, cytotoxicity and biodegradation studies also confirmed that AESM scaffolds supports better cell attachment and growth of meniscal cells when compared to RESM. Hence, AESM can be a potential and interesting natural scaffold matrix for meniscal tissue engineering.

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Introduction

Meniscus tearing has been reported to occur commonly in sports persons, elderly people and persons undergoing excessive physical activities.¹ These tears are difficult to self-heal due to avascularity in the periphery of the meniscus and hence may require continuous treatment.² Depending on the level of injury, the patients are usually advised to rest with medication or go for surgery which involves partial or complete meniscectomy. Such meniscectomy always leads to osteoarthritis and knee instability problems in the long run.³ These after effects of meniscectomy, warrants the need for meniscus tissue engineering,⁴ where synthetic or natural scaffolds are used to grow the cells to facilitate a possible replacement of meniscus cells. Various synthetic scaffolds like polyurethane,⁵ polycaprolactone (PCL),⁶ poly(glycolic acid) (PGA),⁷ and natural scaffolds like collagen,⁸ silk fibroin,⁹ bacterial cellulose¹⁰ etc. have been investigated by many scientists for meniscus cell growth. When compared to synthetic scaffold, natural scaffold are more preferable due to various reasons like higher biocompatibility, reduced toxicity, closer mimic of the natural damaged tissue etc.¹¹

Egg shell membrane (ESM) is one such naturally occurring double layered membrane present inside the egg shell. The ESM is rich in amino acids like proline, glutamic acid and glycine along with uronic acid, sialic acid and nitrogen.^12,13 The fibers of ESM are also composed of type I, V, X collagen, glycosaminoglycans (dermatan sulfate and chondroitin sulfate), glycoproteins and hyaluronic acids.¹² This composition of ESM naturally mimics the extracellular matrix composition of human meniscus and hence can be a suitable matrix for cartilage based tissue engineering.¹⁴ Similar scaffolds with closure mimic to the natural human meniscal tissue have not been reported so far. ESM also has high structural strength, high porosity, large surface area, antibacterial and anti-inflammatory activity and permits the diffusion of gas and water molecules which help cell attachment and proliferation.¹⁴ Thus, ESM is a potential candidate for meniscal tissue engineering research. However, the major limitations of using these natural ESM for tissue engineering are its size and available quantity. These limitations were overcome by dissolving it in organic solvents like 3-mercaptoropionic acid and acetic acid.¹⁵ The solubilized ESM have been further electrospun/casted/blended with polymers to obtain different structure, size and morphology.¹⁵ However, such soluble ESM lacks the innate antimicrobial activity which makes it less preferable matrix for tissue engineering.¹⁶ The natural architecture of ESM (double layered) is lost when we solubilize and hence we could not compare the structure of natural ESM with the blended, solubilized ESM based matrices.¹⁷ Although various cell adhesion studies using polymer blended soluble ESM have been studied with cell lines like L929 fibroblasts cells,¹⁸ NIH3T3 ¹⁷ fibroblast cells, there is no study on the impact of human primary meniscus cells on natural ESM to our knowledge. In this study, we have tried to understand the influence of natural ESM matrix (with and without autoclaving) for meniscal cell growth and proliferation. The ESM before and after autoclaving have been characterized for its functional and structural properties and have studied for their use in meniscal tissue engineering.

Experimental

Collection of egg shell membrane

The commercially available single comb white leghorn hen egg shell after cleaning was used to collect the ESM. The ESM was washed in isopropanol for a few minutes, followed by repeated rinsing with phosphate buffer saline (PBS). The ESM was blot dried and UV sterilized for 20 min. The UV sterilized ESM was considered as raw ESM (RESM). Similarly, the stripped ESM was further treated with moist heat by autoclaving at 121 °C, 15 lbs pressure for 20 min. The autoclaved ESM (AESM) and RESM were used for further studies.

Characterization of RESM and AESM

The RESM and AESM were characterized for surface morphology, surface charge, functional groups and thermal properties using appropriate techniques. The surface morphology and elemental analysis of RESM and AESM were characterized using field emission scanning electron microscopy (FESEM) (Carl Zeiss, Germany) attached with energy dispersive spectroscopy (EDS). 1 cm × 1 cm of RESM and AESM were cut and placed on to a FESEM stub containing a double sided carbon tape and pressed gently to fix it. The samples were sputter coated with gold under standard coating conditions. The images were taken at an operating voltage of 5–10 kV. Zeta potential of RESM and AESM were measured using Delsa™ Nano C (Beckman Coulter, USA) with a special solid cell assembly accessory. Reference colloidal suspension was made up of polystyrene latex particles dispersed in 0.1 mM KCl (pH 6.7, supplied by manufacturer). 30 mV DC was used across two platinum electrodes throughout the analysis. Measurements were taken using 658 nm laser at five different planes spacing at equal distance from the central line. The tests were repeated twice for both sides of the samples. Fourier transform infrared spectroscopy (FTIR) (Shimadzhu, Japan) was used to examine functional groups on the surface of RESM and AESM. The spectrum was measured and recorded at 600–4000 cm⁻¹ on a spectrometer with a resolution of 2.0 cm⁻¹. Thermogravimetric analysis (TGA) was performed using NETZSCH STA 449F3, Bavaria, in static nitrogen atmosphere from room temperature to 600 °C at a heating rate of 5 °C min⁻¹.

In vitro degradation study

The dried samples (RESM and AESM) were cut into 2.5 × 2.5 cm² pieces and were placed in a test tube containing 10 ml of PBS (pH 7) and in 0.25% trypsin–EDTA solution separately, for in vitro degradation studies.¹⁹ The tubes were kept at 37 °C in a sterile environment and were observed for total period of 30 days. Samples were withdrawn at different time intervals, washed with distilled water and vacuum dried at room temperature. The percentage degradation was calculated based on initial and final weight of the scaffold before and after exposure to trypsin–EDTA solution and PBS.

Cell culture studies

Meniscus sample collection. Human meniscus samples were collected from the patients undergoing partial or complete meniscectomy. The samples were collected from patients with proper informed consent. All procedures followed were in accordance with the ethical standards declared by the Institutional Human Ethics Committee (IHEC), PSG Institute of Medical Sciences and Research, Coimbatore, India (Ethical committee approval number: 12/193).

Isolation and expansion of human meniscus cells. Meniscal cells were released from surgical debris of human meniscus through sequential enzymatic digestion using a known volume of 0.25% (w/v) trypsin and 0.25% (w/v) collagenase type II. Isolated meniscal cells were resuspended in DMEM medium supplemented with 10% fetal bovine serum (FBS) and 0.1% penicillin, streptomycin and amphotericin for 2 weeks. After reaching 100% confluency, meniscal cells were subsequently detached using 0.25% trypsin/1 mM ethylene diamine tetra acetic acid (EDTA) and subcultured as first passage cells (Passage 1, P1) until it reached confluency. Throughout the study, all the plates were maintained at 37 °C at 5% CO₂ inside incubator under humid conditions. Culture medium was changed every 3–4 days in all studies.

Preparation of RESM and AESM for cell culture. The fibrous side of RESM and AESM were used for cell culture studies. Before seeding the isolated human meniscus primary cells, the ESM were screened for sterility by monitoring RESM and AESM in DMEM medium for 1 week at 37 °C with 5% CO₂. Only the sterile ESM samples were used for the cell seeding.

Cell adhesion and proliferation studies. Confluent Passage 2 (P2) meniscus cells were trypsinised, washed with sterile DMEM medium and used to seed the scaffolds. Meniscus cells were seeded (10³ cells per well) on to fibrous surface of RESM and AESM placed in a 24 well plate. Appropriate control lacking scaffold were maintained in parallel. The cells were maintained under optimum conditions and monitored regularly for cell adhesion and proliferation on to the scaffold and to the plate. The scaffolds were removed and washed with sterile medium and PBS to remove superficially attached cells. The cell adhesion and proliferation was monitored using FESEM. The scaffolds with and without cells were fixed with 4% glutaraldehyde for 8 h and rinsed thrice with fresh PBS buffer. Followed by buffer wash, scaffolds were washed with series of ethanol concentration (60%, 70%, 80%, 90% and 100%) and air dried. The washed scaffolds were transferred to fume hood and fixed with 1 : 2 ratio of hexamethyldisilazane (HMDS) and ethanol (80%) for 20 min. The scaffolds were then immersed in 2 : 1 ratio of HMDS and ethanol (80%) for 20 min. The fixed scaffolds were further treated with 100% HMDS alone without ethanol.²⁰ The scaffolds were air dried in fume hood overnight. The dried samples were fixed onto a metal stub using double sided carbon tape, gold sputtered and imaged in FESEM.

The washed scaffolds were also subjected to Hoechst staining to visualize the live adhered cells. Hoechst-33258 stain (Sigma) was used for cell nuclei visualization by staining the chromatin. Cells were prewashed with PBS solution and incubated for 10 min in room temperature after adding Carnoy's fixative (acetic acid : methanol, 1 : 3). The cells were treated with Hoechst-33258 stain and incubated for 30 min. Cells were monitored for its fluorescence using inverted phase contrast epifluorescence microscope (Nikon, Japan) under 460–490 nm filter.²¹ Images were captured using Nikon CCD camera attached to the microscope.

Cytotoxicity assay. The cytotoxicity of the AESM and RESM scaffolds were estimated using standard 3-(4,5-dimethyl thiazolyl-2)-2,5-diphenyl tetrazolium bromide (MTT) assay. MTT assay helps in evaluating the ability of mitochondrial dehydrogenase enzymes of viable cells present in the scaffolds/plate to reduce the yellow colored tetrazolium salt into in soluble purple colored formazan crystals. The amount of formation of purple colored precipitate/crystal was quantified using spectrophotometer, which gives the number of metabolically viable cells present on the samples.²² Small pieces of scaffolds were placed in 96 well plates and a known quantity of cells were seeded on to it along with the DMEM medium. 20 μl of MTT was added to each well and incubated for 3.5 h at 37 °C in laminar air flow chamber. The medium was removed carefully from each well and 150 μl of dimethyl sulfoxide (DMSO) was added followed by agitating in an orbital shaker for 15 min. Optical density of each well was taken at different time intervals, at 570 nm using 96 well microplate reader (Thermo Scientific, USA).

DNA estimation in cells attached to scaffolds. The DNA content of the meniscal cells attached to the RESM and AESM scaffold were estimated by staining with DAPI (4′,6-diamidino-2-phenylindole) stain.²³ The attached cells in scaffolds were completely removed by trypsinization and homogenised in PBS. DAPI stain (0.8 μg ml⁻¹) was added to the homogenised filtrate and incubated for 30 s at room temperature in dark. The fluorescence intensity of the samples were read at an excitation and emission wavelength of 358 nm and 461 nm respectively using Fluoroskan Ascent Microplate Fluorimeter (Thermo Scientific). The DNA content was estimated after cells were cultured on RESM and AESM scaffolds for 2nd to 14th day and compared to that of control (without scaffold).

Cell proliferation index. Cell proliferation index (%) was determined as the percentage of cells adhered to the scaffolds when compared to control after day 14.²⁴ Image J software was used to count cells from a unit area of Hoechst stained images at 20× magnification. The cell count in RESM and AESM were compared with control images.

Estimation of extracellular glycosaminoglycan (GAG) and collagen. The total extra cellular matrix (ECM) secreted by cells can be correlated with the quantity of collagen and GAG content present in the medium. The ECM collagen content secreted in the medium was estimated using modified sirius red dye method.²⁵ A known concentration of sirius red dye prepared in 0.5 M acetic acid solution was added to a known quantity of medium from the cell culture plates and mixed well for 5 s. The plates were incubated undisturbed for 30 min, centrifuged at 1500 rpm for 10 min and the pellet was washed with 0.01 N hydrochloric acid (HCl) to remove unbound dye. The pellet was re-suspended in 0.1 N potassium hydroxide (KOH) and absorbance was measured at 540 nm using a microplate reader. Calf collagen was used as a standard. 1,9-Dimethylmethylene blue (DMMB) assay was used to estimate the GAG content in the sample, spectrophotometrically.²⁶ The aliquots of ECM taken from cell seeded scaffolds at various time intervals were mixed with DMMB dye and its absorbance was measured at 525 nm in Multiskan™ GO Microplate Spectrophotometer (Thermo Scientific, USA). The chondroitin sulphate A sodium salt was used as standard for GAG.

Statistical analysis

Image J software (NIH) was used for analyzing the images. All data were expressed in mean ± s.d. One way ANOVA was carried out using Origin Pro8 for hypothesis testing, with p < 0.05.

Results and discussion

Characterization of RESM and AESM

The morphological and structural variation between RESM and AESM was observed under FESEM (Fig. 1). The ESM was found to be double layered which has an inner (side which contacts with the egg white and consists of limiting membrane as shown in Fig. 1a and c) and an outer membrane (side which is sticking to the shell as shown in Fig. 1b and d) with distinct structural morphologies. In the inner membrane of both RESM and AESM (Fig. 1a and c), the fibers were arranged randomly throughout the matrix with random distribution of pores on the surface. Such pores help in gas permeation and nutrient diffusion during cell culture.²⁶ The fiber morphology was rough throughout the matrix, where spherical shaped single or grouped particles with a size range of ∼1 μm were seen. The particles on surface of fibers did not have any particular arrangement or pattern. The diameter of the fibers was found to be 2.2 ± 0.5 μm with a membrane thickness of 14.4 ± 3.3 μm. Fig. 1b and d show the compact particulate outer membrane of ESM which is densely packed with spherical shaped particles (∼1 μm) and has no visible pores on the surface.

Fig. 1 FESEM images of RESM and AESM; inner surface (a) and outer surface (b) of RESM, inner surface (c) and outer surface (d) of AESM (respective EDS spectra as insert).

Similar observations have been reported by Balaz et al.¹³ with RESM. However, the RESM showed variation in the structure once it was autoclaved. A mucilaginous coating between the fibers were observed in RESM which fused the adjacent fibers (Fig. 1a). On autoclaving, such mucilaginous materials were not visible and individual fibers could be observed (Fig. 1c). The outer surface of RESM also shows similar mucilaginous cover (Fig. 1b). Similar kind of coating has been reported by Yi et al.²⁷ for RESM. After autoclaving, the particle boundaries appeared more distinct (Fig. 1d). The EDS spectra around the spherical particles on both RESM and AESM fibers indicated the presence of calcite type of minerals. The Ca²⁺ ions in calcite can act as a secondary messenger and helps in proliferation of cartilage cells.^28,29 The RESM and AESM showed significant variation in the composition (Fig. 1a and c insert). Sodium and calcium content was found to be more pronounced in AESM when compared to RESM while the sulphur content reduced considerably.

Surface charge of scaffold plays an important role in cell adhesion.³⁰ The zeta potential of RESM inner and outer surfaces were found to be 32.29 ± 2.73 mV and 35.31 ± 0.34 mV, respectively which showed the cationic nature of membrane surface. This result was in line with previous reports which confirms RESM surface is positively charged.²⁷ But in AESM, the zeta potential value of both inner and outer surfaces were observed to be almost neutral i.e., 2.94 ± 1.98 mV and −1.30 ± 0.02 mV, respectively. All these observations clearly indicate the presence of some coating onto the surface of the ESM which get removed on autoclaving. The material which is getting degraded or removed by autoclaving may be predicted as a cationic substance. Yi et al.²⁷ has reported the existence of some toxic substance on the ESM which gets removed during autoclaving or treatment with mild acids like 3-mercaptopropionic acid. Yeung et al.³¹ reported heat sterilisation (autoclaving) induces the degradation of proteins and free amino acids. Similar to the above reported study, the degradation of proteins/amino acids present in the mucilaginous coating on the ESM may be possible during autoclaving which affects the surface zeta.

The surface functional groups of the RESM and AESM were analysed using FTIR (Fig. 2). Both the ESM showed similar N–H stretching of primary and secondary amines at 3410 and 3309 cm⁻¹ respectively. However the N–H stretching peaks was masked by broad OH stretching peak in case of AESM. The peak at 2962 cm⁻¹ indicated the presence of S–H bonding vibrations which confirms the presence of sulphur compounds. The peak at 1592 cm⁻¹ corresponds to N–H bending. The peaks at 2070 and 1635 cm⁻¹ exhibit the presence of amines and amides respectively in ESM.^13,31–33

Fig. 2 FTIR spectra of RESM and AESM.

Thermal properties of RESM and AESM were investigated using TGA (Fig. 3). The TGA graph clearly indicates two stage degradation processes for RESM while AESM has only one stage degradation. Torres et al.,³⁴ also reported similar multiple decomposition phases for RESM using TGA. An initial mass loss was observed in case of RESM which may be attributed to the water loss and the thermal degradation of collagen and other volatile components.³⁴ After 90 °C, there was no mass loss till 220 °C for both the ESM. Beyond 220 °C, the second phase of degradation starts which extends up to 400 °C. The higher thermal stability of AESM may be due to the introduction of some cross-linking³⁵ and removal of amorphous materials from ESM.

Fig. 3 Thermal degradation profile of RESM and AESM.

Biodegradation studies

RESM and AESM were subjected to biodegradation in trypsin and PBS separately under standard conditions (pH 7.4; laboratory temperature (∼30 °C)). The percentage weight retained is shown in Fig. 4. RESM degradation was faster in presence of trypsin as compared to AESM till 30th day. On 10th day, RESM retained only 42% weight in trypsin treated samples while AESM retained more than 68%. There was not much difference in the percentage weight loss after 20th day in both ESM samples. On 30th day, RSEM retained only 25% weight when compared to AESM (45%) in trypsin solution (Fig. 4a). In PBS, both the scaffolds showed similar degradation till 20th day (Fig. 4b). However, faster degradation was observed in the case of RESM (∼30% weight retained on 30th day) as compared to AESM (∼46% weight retained on 30th day). Among two different solutions used, trypsin induced higher degradation rate compared to PBS solution because of its enzymatic ability to denature proteins present in ESM. Over all, after 30 days of biodegradation study, the RESM scaffolds showed higher degradation rate compared to AESM in both trypsin and PBS. This lower degradation rate with AESM may be correlated with the TGA results which show more thermal stability. Moreover, the possibility of cross-linking³⁵ may influence lower degradation through less exposure of cleavage sites for enzymatic action.

Fig. 4 Biodegradation profile of RESM and AESM in (a) trypsin and (b) PBS.

Meniscus cell culture studies

In order to evaluate the biocompatibility of RESM and AESM, human meniscus primary cells were cultured on inner fibrous surface of both scaffolds and compared with control. The cytotoxicity of RESM and AESM on human meniscus cells in vitro was measured using MTT assay and the data were plotted (Fig. 5a). The viability percentage of cells attached on AESM was higher than that of the RESM scaffold (p < 0.05). The cell viability was less with RESM than control till 6th day. Initial toxicity with RESM may be attributed to the presence of some toxic mucilaginous coating as discussed in FESEM section and as reported by Yi et al.²⁷ The viability of RESM increased gradually with time and became nontoxic that can be due to the removal of the mucilaginous content during routine medium change. DNA estimation data were also in line with MTT assay results (Fig. 5b).

Fig. 5 (a) Cell viability (b) DNA content and (c) cell proliferation index on 14th day; all data were compared with control (cells seeded on tissue culture plate) taken as 100% (p < 0.05).

DNA content of cells adhered on to the RESM and AESM scaffolds was estimated and compared with control. The total DNA content of AESM was two fold higher than RESM. Cell proliferation index (%) for RESM and AESM was calculated from Hoechst stained images on 14th day and compared with control. The AESM and RESM showed an average cell proliferation index (%) of 173 ± 19 and 60 ± 32, respectively, when compared to control (Fig. 5c). These results clearly indicate that the cell proliferation was higher in AESM when compared to RESM.

As discussed in the earlier sections, autoclaving removes mucilaginous coating from the ESM and renders it cytocompatible for meniscal cell attachment and proliferation. Fig. 6a and b shows the FESEM images of the meniscus cells attached onto the surfaces of the RESM and AESM after 14 days of incubation. The cell attachment and proliferation of meniscal cells was found to be higher in AESM when compared to RESM. The cells in AESM showed more filopodial projections and covered the entire surface as continuous monolayer. However, in RESM, such filopodial projections and distribution of cells were found to be very less on the surface. The cell proliferation and adhesion in both ESM were confirmed through Hoechst staining (Fig. 6c and d). The AESM and RESM showed an average cell count of 410 ± 19 and 99 ± 32 cells per scaffold respectively (data as average of cells in 5 scaffold) and was in line with FESEM data. These results were also in correlation with the ECM secretion. For enhanced cell adhesion and proliferation, mimicking the ECM environment in scaffolds is a widely used approach in cartilage tissue engineering.³⁶ ESM major components includes collagen and glycosaminoglycan like CS and hyaluronic acid (HA) which can mimic the ECM of meniscus cartilage.¹⁴ Collagen present in the ESM gives mechanical strength to the membrane and also helps to mimic the natural ECM of meniscus³⁷ thereby enhancing cell adhesion and proliferation. HA also imparts major role in cell attachment and proliferation.³⁸ Other than that, HA plays critical role in matrix metalloproteinases (MMPS) synthesis which helps in tissue regeneration and repair.³⁸ CS enhances cellular metabolism, cell proliferation and helps in HA synthesis.³⁹ These biomimetic environments present in ESM can be a possible reason for enhanced cell adhesion and proliferation.

Fig. 6 FESEM images of attached meniscus cells on RESM (a) and AESM (b); Hoechst images of attached meniscus cells on RESM (c) and AESM (d).

Extracellular matrix (ECM) analysis

ESM is rich in proteins and amino acids, and provides suitable environment for human meniscal cell attachment and proliferation.²⁵ The extracellular collagen and GAG secretion was found to be higher in AESM than RESM and control (Fig. 7). The total amount of ECM secretion into the medium was statistically insignificant till 7th day between the samples (RESM, AESM and control). However after 7th day, the ECM secretion significantly varied between samples (p < 0.5). The cells seeded on to AESM secreted more collagen and GAG when compared to control. RESM showed the lowest ECM secretion. Collagen content in the medium was estimated to be 13.4 μg ml⁻¹ and 7.4 μg ml⁻¹ with AESM and RESM, respectively on 15th day while control cells showed 10.3 μg ml⁻¹ of collagen synthesis (Fig. 7a). The GAG secreted into the medium also showed similar trend (Fig. 7b). GAG secretion was high with AESM (12 μg ml⁻¹) on 15th day when compared to RESM (5.2 μg ml⁻¹) and control (8.6 μg ml⁻¹). The scaffold composition also plays an important role in extracellular matrix production. As discussed earlier, components of ESM like collagen³⁷ and GAGs such as HA³⁸ and CS³⁹ are reported to enhance the synthesis of ECM. Hence natural biomimetic scaffolds like AESM can be a better option for meniscal cartilage tissue engineering.

Fig. 7 Extracellular matrix secreted into medium with RESEM and AESM, (a) Collagen and (b) GAG.

Conclusions

The natural egg shell membrane is a potential candidate for use in cartilage tissue engineering due to its structural strength, high collagen content and antimicrobial activity. In this study, we have observed that autoclaving of RESM results in modification of various morphological and biological properties making it more suitable for meniscal tissue engineering. The surface properties of RESM changes a lot due to autoclaving process which results in improved meniscal cell attachment and proliferation and induces excess ECM secretion. The cytocompatibility of RESM improved considerably on autoclaving. We have also observed that, this process is comparable and much easier method than solubilising egg shell membrane for preparation of scaffolds. Hence, autoclaved egg shell membrane represents an interesting scaffold for tissue engineering.

Acknowledgements

The authors like to express their deep gratitude to the management of PSG Institutions, Tamil Nadu, India and Tamil Nadu State Council for Science and Technology (TNSCST) for their support to carry out this work. We appreciate the support, guidance and contribution from Dr P. Radhakrishnan, Director and Dr T. Lazar Mathew, Advisor, PSG Institute of Advanced Studies, Dr David V. Rajan, Ortho One Orthopaedic Specialty Centre and Dr S. Ramalingam, PSG Institute of Medical Sciences and Research, Coimbatore. The authors acknowledge the help of COE-Medical textiles, SITRA, Coimbatore, India for helping us in FESEM analysis.

Notes and references

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