Biomolecule incorporated poly-ε-caprolactone nanofibrous scaffolds for enhanced human meniscal cell attachment and proliferation

Abstract

The present study investigates the impact of biomolecule (biotin and galactose) incorporated poly-ε-caprolactone (PCL) nanofibrous scaffolds on attachment and proliferation of human meniscal cells by three modes of biomolecule supplementation. Two different ratios of biomolecules like biotin and galactose were incorporated in nanofibers using a standardized electrospinning process. Surface morphologies of control and biomolecule incorporated nanofibers were analyzed by field emission scanning electron microscope (FESEM). The presence of the biomolecules in the nanofibers was confirmed by Fourier Transform Infrared (FTIR) Spectroscopy. The biodegradability of pure PCL and biomolecules incorporated nanofibers was determined. The biomolecule embedded inside PCL nanofibrous scaffolds were studied in terms of DNA content and extra cellular matrix (ECM) (glycosaminoglycans (GAG) and collagen) for meniscus cell attachment, growth and proliferation with and without addition of these biomolecules (in the scaffold and in the medium). FESEM and fluorescence microscopic studies were used to confirm cell proliferation on the surface of the scaffolds. In vitro human meniscal cell culture study revealed that galactose incorporation was more efficient when compared to biotin. Enhanced meniscal cell attachment and proliferation were achieved when half of the biomolecule was inside the nanofiber and the other half was in the medium. This approach to improve the cell attachment onto the scaffold is a promising strategy for meniscal tissue engineering.

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1. Introduction

Meniscus is a crescent shaped fibrocartilaginous tissue located on the tibial plateau. It serves as a shock absorber and transmits load between the femur and the tibia.¹ The semilunar medial and lateral components of the meniscus differ in their shape, size and mobility.² Meniscus is nourished by blood vessels at the periphery; however, the central region lacks vascularity, which makes it difficult to heal when injured.³ The injury is caused mainly due to trauma (in the case of athletes, younger patients) or degenerative processes (in the case of older patients).⁴ Conventionally, surgical and non-surgical methods are employed for meniscal tear treatment. The non-surgical methods are RICE (rest, ice, compression, elevation) and physiotherapy.⁵ The surgical methods include total and partial meniscectomy, which refer to the removal of the entire or damaged part of the meniscus, respectively.⁶ In order to overcome the shortcomings of these conventional methods, the idea of developing new meniscus with patients' own cells is being conceived using a tissue engineering approach.

In most of the meniscal tissue engineering approaches, various polymers are used in different forms to serve as a scaffold for the attachment of cells and to mimic the extracellular matrix of the tissue. Scientists have preferred synthetic polymers when compared to natural polymers because of various disadvantages of natural polymers, like complexity in processing and purification, and its immunogenicity response in the recipient.⁷ Among the various polymers, poly-ε-caprolactone (PCL) has been a material of choice due to its tunable properties, bioresorbability, excellent mechanical properties, slow degradation rate and FDA approval for in vivo studies.^8–10 One of the major problems faced by scientists in meniscal tissue engineering is the extended duration required for proper cell attachment and proliferation.¹¹ Improved cell attachment and proliferation have been obtained by adding various growth factors like recombinant human transforming growth factor-β1 (TGF-β1),¹² insulin-like growth factor (IGF), platelet derived growth factor (PDGF),¹³ fibroblast growth factor (FGF),¹⁴ hepatocyte growth factor,¹⁵ bone morphogenetic protein 2 (BMP-2),¹⁶ and human platelet lysate.¹⁷ However, these growth factors are very expensive and are less available for continuous usage. Hence, low cost alternative biomolecules like biotin and galactose are being investigated by many scientists for enhanced cell attachment and proliferation. Biotin plays an important role in cell proliferation, cell signalling and DNA repair.¹⁸ Apart from these functions, biotin also plays an active role in various metabolic pathways involved in cells.^19,20 Galactose embedded onto the PLGA surface induced efficient hepatocyte cell attachment.²¹ Blackburn and Schnaar²² reported the positive influence of galactose on hepatocyte cell adhesion onto a polymer surface. Selvakumar et al.²³ reported that co-electrospinning of biotin and galactose onto gelatin nanofibers increases the functionality of the nanofiber membrane and, subsequently, enhances cell attachment and proliferation. The electrospinning of polymeric nanofibers along with blending of biomolecules has been extensively used by several researchers to incorporate biomolecules into nanofibers or to functionalize nanofibers for tissue engineering applications.²⁴ However, so far researchers have mostly concentrated on one way of administration (either incorporated into scaffold or into the culture medium) of such biomolecules or growth factors in tissue engineering scaffolds.^24,25

In the present study, we have investigated the impact of the administration of biomolecules like biotin and galactose through PCL nanofibers as well as through the medium on primary human meniscal cell attachment, proliferation, and ECM secretion. As per our knowledge, the effect of biotin and galactose on human meniscal cells has not been studied so far. The control and biomolecule incorporated PCL scaffolds were characterized using suitable techniques before and after cell adhesion. The effectiveness of biomolecules for enhancing cell attachment and proliferation was studied in three different routes of administration: all inside the nanofibrous scaffold, all into growth medium and half inside nanofiber scaffold as well as half into growth medium.

2. Experimental

2.1. Materials

Poly-ε-caprolactone (Mw: 70 000–90 000), 1,9-dimethylmethylene blue (DMMB), sirius red dye, trypsin, Hoechst stain 33258, hexamethyldisilozane (HMDS), collagenase type II and D-galactose (Mw: 180.16) were purchased from Sigma-Aldrich, USA. N,N′-Dimethyl formamide (DMF), hydrochloric acid (HCl), glacial acetic acid and dimethyl sulfoxide (DMSO) were procured from Loba Chemie, India. D̲-Biotin (Mw: 244.31), Dulbecco's Modified Eagle's Medium (DMEM), fetal bovine serum (FBS), chondroitin sulphate A sodium salt, glucose, sodium chloride, sodium bicarbonate, magnesium sulfate heptahydrate, di-sodium hydrogen phosphate dihydrate, magnesium chloride hexahydrate, 3-(4,5-dimethyl thiazolyl-2)-2,5-diphenyl tetrazolium bromide (MTT) and methanol were obtained from Himedia, India. Calcium chloride, potassium chloride, potassium hydroxide and monosodium phosphate were procured from Merck, India. All the chemicals were used without further purification.

2.2. Preparation of nanofibrous scaffolds

Fig. 1 shows the schematic representation of the overall experiment. PCL (15% w/v) was dissolved in DMF at 45 °C for 5 h with constant stirring. Two different ratios of biomolecule (BM) were chosen and were added individually to the polymer solution such that the polymer biomolecule ratios were 15 : 1 and 15 : 0.5. The electrospinning set up includes a syringe pump in which polymer solutions were loaded in a 10 mL syringe and clamped.

Fig. 1 Schematic representation of the nanofibrous scaffold fabrication and methods used in this study.

The syringe was connected to the needle with the aid of a Teflon tube (15 cm) and a glass connector. The tip of the needle (19 gauge) was connected to the high voltage power supply (Glassman, USA) and encased within a transparent plastic chamber. A 5 × 5 cm aluminium sheet was placed on a flat grounded collector for collection of nanofibers. Nanofibers were prepared with optimized parameters of 0.2 mL per h flow rate, 35 kV voltage, 35 cm distance and 70% humidity. The electrospun nanofibers were kept in a vacuum oven for 48 h to remove the residual DMF from the fibrous mats and were stored in an airtight container at room temperature for further use.

The samples prepared were coded as given in Table 1.

Table 1 Coding of samples

S. no.	Sample description	Medium used	Sample code
1	Control meniscus cells	DMEM	C
2	PCL nanofibers	DMEM	P–C
3	PCL nanofibers	1 wt% biotin in DMEM	P + B
4	1 wt% biotin incorporated PCL nanofiber	DMEM	PB-1
5	0.5 wt% biotin incorporated PCL nanofiber	0.5 wt% biotin in DMEM	PB-0.5
6	PCL nanofibers	1 wt% galactose in DMEM	P + G
7	1 wt% galactose incorporated PCL nanofiber	DMEM	PG-1
8	0.5 wt% galactose incorporated PCL nanofiber	0.5 wt% galactose in DMEM	PG-0.5

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2.3. Characterization of scaffolds

2.3.1. Morphological characterization. The morphologies of the scaffolds were evaluated using a field emission scanning electron microscope (FESEM, Carl Zeiss, Germany). The images obtained from FESEM were fed into Image J software (NIH, USA), and the fiber diameters were measured at different places in the sample. The values were plotted as a histogram with frequency of occurrence.

2.3.2. Functional group analysis. Functional groups in nanofibers were analyzed using Fourier Transform Infrared (FTIR) Spectroscopy (Shimadzu (IRaffinity 1), Japan) to confirm the presence of the biomolecules in the nanofibers. The infrared spectrum of the D-Biotin, galactose, P–C and other biomolecule incorporated nanofiber mats were recorded in the spectral range of 4000–400 cm⁻¹.

2.3.3. In vitro degradation studies. The nanofiber scaffolds of ∼1 cm × 1 cm size were subjected to in vitro degradation studies in simulated body fluid (SBF)²⁶ at room temperature. The samples subjected to biodegradation were weighed periodically. The SBF was changed at regular time intervals in order to prevent fungal growth. The percentage of weight loss was calculated from the initial weight (W_i) and the weight obtained at a time t (W_t) using the following formula:²⁷

Weight loss (%) = [(W_i − W_t)/W_i] × 100 (1)

2.4. In vitro cell culture studies

2.4.1. Isolation of human meniscal cells and seeding onto scaffolds. The human meniscal cells for in vitro cell culture studies were isolated from surgical debris obtained from the donor with proper consent and information sheet, who underwent partial/complete meniscectomy. All procedures followed were in accordance with the ethical standards declared by the Institutional human ethical committee (IHEC), PSG Institute of Medical Sciences and Research, Coimbatore, India (Ethical committee approval number: 12/193 dated 24/01/2013).

Meniscal tissue obtained from hospital was washed with 70% ethanol followed by phosphate buffered saline (PBS) twice, dissected into small pieces and trypsinized for 1 h. The tissues were digested using 1% (w/v) collagenase enzyme for 3 h followed by PBS wash (twice) and re-suspended in culture medium containing DMEM, FBS (10% v/v), penicillin (100 units per mL), and streptomycin (100 mg mL⁻¹). The cells were incubated at 37 °C with 5% humidified CO₂ throughout the study and observed under inverted phase contrast epi-fluorescence microscope (Nikon Eclipse Ti-S series, Japan) for attachment onto the polystyrene petri plate surface. After one day, the unattached cells were removed and sufficient new medium was replaced. The cells were detached after the plates reached 100% confluency (after two weeks) using trypsin and serially sub-cultured. Meniscal cells of passage 2 (P2) were used to seed the scaffolds.

Scaffolds of known size (1 cm × 1 cm) were sterilized using the protocol suggested by Hosper et al.²⁸ In brief, the scaffolds were immersed in a series of ethanol (100, 90, 80, 70 and 60%) for 30 minutes followed by washing with sterile PBS. They were further washed repeatedly in sterile distilled water and exposed to ultraviolet (UV) radiation for 3 h.²⁹ The sterilized scaffolds were subjected to sterility tests by incubating the scaffolds for 48 h at 37 °C with 5% carbon dioxide (CO₂) in 1 mL DMEM medium and checked periodically for signs of contamination. In the control sample, the human meniscal cells were cultured in DMEM medium supplemented with FBS and incubated at 37 °C with 5% CO₂ (Eppendorf 170S, Germany) without a scaffold. Sterilized scaffolds were placed in 24 well plates and inoculated with primary human meniscal cells (cell density of 3.3 × 10⁷ cells per well) using modified/unmodified DMEM medium as given in Table 1. The attachment and morphology of the cells on the scaffolds were monitored at different time periods using FESEM and an inverted phase contrast epi-fluorescence microscope. For FESEM analysis, samples were prepared according to Moran and Coats³⁰ with some modifications. In brief, the scaffolds with and without cells were fixed with 4% glutaraldehyde for 8 h and rinsed thrice with fresh PBS. Followed by buffer wash, scaffolds were washed with a continuous series of ethanol concentrations (60%, 70%, 80%, 90% and 100%) and air dried. The washed scaffolds were transferred to a fume hood and fixed with 1 : 2 ratio of HMDS and ethanol for 20 min. After this step, the scaffolds were immersed again in 2 : 1 ratio of HMDS and ethanol for 20 min. Further, the scaffolds were again fixed with 100% HMDS alone without ethanol and air dried overnight. HMDS dried samples were gold sputtered and fixed onto a metal stub using double sided carbon tape.

Control samples (cells in polystyrene plates) and cell seeded scaffolds (without HMDS fixing) were stained with Hoechst stain and observed under inverted phase contrast epi-fluorescence microscope fitted with a filter having a wavelength of 460 to 490 nm. The images were captured using a Nikon CCD camera attached to the microscope.

2.4.2. MTT assay. The cytotoxicity of the nanofibrous scaffolds (both biomolecule incorporated and P–C) was estimated for 5 days using the standard MTT assay.³¹ MTT assay helps in evaluating the ability of mitochondrial dehydrogenase enzymes of viable cells present in the scaffolds/plate to reduce the yellow colour tetrazolium salt into soluble purple colour formazan crystals. The amount of purple colour precipitate/crystal formed was quantified using a spectrophotometer, which gives the number of metabolically viable cells present in the samples.³² Small pieces of nanofibrous scaffolds were placed in a 96 well plate and a known quantity of cells was seeded onto it along with the medium and incubated at 37 °C and 5% CO₂. 20 μL of MTT was added to each well and incubated for 3.5 h at 37 °C. The media was removed carefully from each well and 150 μL of DMSO was added followed by agitation in an orbital shaker for 15 min. The optical density of each well was read at 590 nm using a 96 well microplate reader (Thermo Scientific™ Multiskan™ GO Microplate Spectrophotometer). The cell viability was estimated by comparing the absorbance of the cells cultured on different scaffolds to that of the control.

2.4.3. DNA estimation using Hoechst stain. DNA binding Hoechst dye 33258 was used to determine the DNA content³³ of the cells detached from the nanofibrous scaffolds. The attached cells in scaffolds were removed periodically at definite time periods by trypsinization and cells were homogenised in PBS. The detached cells were mixed gently for 30 seconds and added with a solution containing a 1 : 10 ratio of Hoechst stain and PBS. The samples were read at 356 nm for excitation and 492 nm for fluorescence emission in a Fluoroskan Ascent™ Microplate Fluorometer (Thermo Scientific™). The corresponding absorbance readings were saved for further analysis. The DNA content was estimated by comparing the absorbance of the cells cultured on different scaffolds to that of the control on the 6^th day.

2.4.4. Estimation of extracellular matrix (ECM) production. The total content of ECM can be correlated with the amount of glycosaminoglycans (GAG) and collagen secreted in the medium.³⁴ In both control and cell seeded scaffolds, total GAG and collagen secreted by the cells were estimated at regular time intervals. These estimations enable us to quantify the effect of scaffolds on ECM secretion of the cells. The culture medium taken from the control scaffolds (without cells) was used as blanks for all the different estimations. DMMB assay was used to estimate the GAG content in the sample, spectrophotometrically. The aliquots of medium taken from different samples at various time periods were mixed with DMMB dye and its absorbance was measured at 525 nm using a Multiskan™ GO Microplate Spectrophotometer (Thermo Scientific, USA). The chondroitin sulphate A sodium salt was used as standard for GAG. The collagen content secreted in the medium was estimated using Sirius red dye as per the modified Hride Tullberg-Reinert method.³⁵ 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 seconds. The plates were incubated undisturbed for 30 min. The samples were centrifuged at 1500 rpm for 10 min and the pellet was washed with 0.01 N HCl to remove the unbound dye. The pellet was resuspended in 0.1 N KOH and the absorbance was measured at 540 nm using a microplate reader. Calf collagen was used as standard.

2.4.5. Cell adhesion studies. Adhesion of human meniscus cells to the scaffolds was monitored through fluorescence study.³⁶ Staining of control sample (cells on polystyrene plate) and meniscal cell seeded scaffolds was carried out at different time periods (6, 12, 18 and 24^th day). After the respective incubation time, DMEM medium was removed from the 24 well plate aseptically and washed with PBS to remove superficially attached cells. The scaffolds with cells adhered were treated with Carnoy's fixation solution for 10 min and this fixation step was repeated twice under the same conditions. After fixation, Hoechst stain was added to all the wells and incubated for 30 minutes followed by two times washing with water. The samples were allowed to air dry for 1 h in fume hood and observed under an inverted phase contrast epi-fluorescence microscope fitted with a filter having a wavelength range of 460 to 490 nm. The images were captured using a Nikon CCD camera attached to the microscope.

2.5. Statistical analysis

All the experiments were carried out in triplicate. Data are expressed as mean ± standard deviation. Statistical analysis was performed using one way ANOVA with Tukey's multiple comparison tests (p < 0.05) in Origin 8.0 software.

3. Results and discussion

3.1. Characterization of nanofibers

The control (P–C) and biomolecule incorporated PCL nanofibers were characterized using FESEM to determine the morphology and fiber diameter. All the nanofibers had a uniform morphology and were randomly aligned (Fig. 2). The overall fiber diameter in P–C and biomolecules incorporated nanofibers are given as a histogram (Fig. 2a1–c1).

Fig. 2 Representative FESEM micrographs showing morphology of electrospun and biomolecule incorporated PCL nanofibrous scaffold before seeding of cells. (a) P–C, (b) PB-1 and (c) PG-1; (a1), (b1) and (c1) show the histograms of fiber diameter for corresponding images.

All the nanofibrous mats were found to be bead free P–C scaffolds with an average diameter of 443 ± 122 nm. The fiber diameter was found to increase on electrospinning with biomolecules. The fiber diameter increased by 4.73% and 9% on nanofibers PB-1 and PG-1, respectively. The mean diameters of PB-1 and PG-1 were found to be 465 ± 126 nm and 572 ± 171 nm, respectively. The increase in fiber diameter may be attributed to the variation in total charge of the biomolecules and PCL mix when compared to the P–C.

The change in functional groups of nanofibers due to addition of biomolecules was characterized using FTIR (Fig. 3). The FTIR spectrum of P–C revealed the presence of two distinct characteristic peaks of PCL polymer at 2862 cm⁻¹ and 1728 cm⁻¹ corresponding to C–H and C=O stretching, respectively.³⁷ The FTIR spectrum of D-Biotin reveals the characteristic peak of D-Biotin in the range of 3400–3250 cm⁻¹ corresponding to N–H streching.³⁸ Similar N–H peaks were observed in PB-1 indicating the presence of biotin along with PCL peaks. The other characteristic peaks (1638 and 1707 cm⁻¹ correspond to C=O stretching) of biotin as mentioned by Li et al.³⁸ were overlapping with the high intensity P–C peaks of C=O stretching and thus not showing individual peaks. The FTIR spectrum of pure D-galactose (Fig. 3) shows the characteristic broad peak of D-galactose in the region of 3600–3200 cm⁻¹ due to the presence of OH bonds.³⁹ The FTIR spectrum of PG-1 (Fig. 3) showed similar broad peaks in the same region showing the presence of D-galactose along with the peaks of PCL. The characteristic peak of one axial OH group at C4 (1057 cm⁻¹)³⁹ was found in both galactose and PG-1 spectra, which also confirms the presence of galactose inside the nanofibers. The presence of an OH group indicates the more hydrophilic nature of PG-1 as compared to P–C.

Fig. 3 FTIR spectrum of P–C, PG-1, D-galactose, PB-1 and D-biotin.

These results clearly indicate the presence of biomolecules and corresponding changes in functional groups in the biomolecules incorporated PCL nanofibers. The intensities of the peaks corresponding to the biomolecules are lower in the nanofibers due to the low concentration of biomolecules added in the solution.

The biodegradation studies for different electrospun nanofibers were carried out in SBF. PCL pellets were taken as control and were subjected to biodegradation studies along with nanofibrous mats. Fig. 4 indicates the percentage weight loss of the samples with time, over a period of 50 days.

Fig. 4 Rate of biodegradation in SBF.

The degradation rate of PCL pellets was less (15.5% after 50 days in SBF) than the electrospun mats. The mean biodegradation rate of pure and biomolecules incorporated PCL nanofibrous scaffolds (i.e. P–C, PB-1, PB-0.5, PG-1, PG-0.5) were found to be 35%, 31%, 30.7%, 38% and 38.8%, respectively, up to 50 days. Biotin incorporated samples (PB-1 and PB-0.5) showed relatively slower degradation than other nanofibrous scaffolds. On further increase of test period to 110 days, PCL pellet and P–C showed weight loss of 26.5% and 75%, respectively (data not shown in figure). The biodegradation of PCL is reported as 2 to 4 years.^40,41 However, nanofibers showed increased degradation due to their high surface area to volume ratio as compared to PCL pellets.⁴² Bolgen et al.⁴³ had mentioned a similar biodegradation rate for electrospun PCL nanofibers. Electrospun poly(D,L-lactide) nanofibrous scaffolds also showed higher degradation rates.⁴⁴ Previous reports with blend electrospinning of biomolecules have shown promise in tissue engineering applications for sustained release.^24,25 The release of biomolecules from the polymer matrix is determined by multiple factors, such as erosion, surface and bulk diffusion. These factors are in turn dependent on various physical, chemical and processing steps involved during the fabrication of the polymer scaffold. However, the exact mechanisms behind these complicated processes are still unclear for many polymers and incorporated biomolecules due to differences in chemical moieties and structures. The first phase of the biomolecules' release may be due to random breaking of the polymer chain, which lacks significant weight loss. However, the second set of release is usually illustrated with significant weight loss in the polymer matrix. Thus, the incorporated molecules released in the medium may be controlled by the diffusivity of the molecules in the degrading polymer and the distribution of the molecules in the matrix.⁴⁵

3.2. In vitro cell culture studies

1 cm × 1 cm sterile control PCL nanofibers and co-electrospun nanofibers with biomolecules were seeded with primary human meniscus cells at an initial concentration of 3.3 × 10⁷ cells per well. The scaffolds were regularly monitored for cell attachment and proliferation. The random arrangement of nanofibers and the incorporation of biomolecules into the nanofiber and into the medium enhanced the cell attachment and proliferation. The cell adhesion and proliferation throughout the scaffold increased with increasing incubation time. The cells after attaching onto a favorable scaffold start expanding their cytoplasmic projections or cellular extensions to have contact with neighboring cells (to initiate cell–cell communication) which in turn contribute to form tissues.^46,47 After 6 days, all the scaffolds seeded with cells showed cellular extensions and proliferation (ESI Fig. 1†). These results clearly indicate that these nanofibrous scaffolds are non-toxic and favor cell attachment. Initially the cells' attachment was more favourable at the edges of the scaffold used and sparingly present in the centre of the scaffold. As the incubation time was increased, the cell proliferation was found to spread throughout the scaffolds. This phenomenon of cells attaching to the periphery may be attributed to the easy availability of nutrients from the medium as well as the curvature present at the fibrous scaffold.⁴⁸ Baker et al.⁴⁹ have also observed that the meniscus cells were attached to the periphery of PCL nanofibrous scaffolds during the initial days of culture. The possible reason for the initial preference for cell attachment to the periphery may be due to the availability of more oxygen and nutrients at the edge and easy removal of metabolic waste.⁵⁰ The same trend was seen in the case of control sample (C – without scaffold) as well (ESI Fig. 1a†). As the incubation period was increased to 12 days (figure not shown), the amount of cells attached to the scaffolds was found to vary. Biotin was found to have less influence on cell adhesion (ESI Fig. 1c–e†) and was comparable to the P–C scaffold. PG-0.5 and PG-1 showed higher cell density when compared to other scaffolds including P–C (ESI Fig. 2†). However, when galactose was added into the medium alone, the cell adhesion to the P–C scaffold was found to be less (ESI Fig. 2f†) when compared to galactose incorporated PCL nanofibers (both PG-0.5 and PG-1) (ESI Fig. 2g and h†). On the 18^th day of incubation, the cell density was found to be increased in all scaffolds (figure not shown).

After 24 days of cell seeding onto the scaffolds, meniscal cells were fully distributed over the surface of the scaffolds (Fig. 5). Among all scaffolds, PG-0.5 and PB-0.5 showed the highest cell attachment and colonization, followed by PG-1 and PB-1. The scaffolds supplemented with biomolecules from inside as well as from outside (i.e., PG-0.5 and PB-0.5) showed the highest cell attachment and proliferation when compared to the other scaffolds supplemented fully either via fiber (PG-1 and PB-1) or via medium (P + G and P + B).

Fig. 5 Fluorescence microscopic images of Hoechst stained meniscal cells on nanofibrous scaffolds (24^th day): (a) C, (b) P–C, (c) P + B, (d) PB-1, (e) PB-0.5, (f) P + G, (g) PG-1 and (h) PG-0.5.

These results indicated that in order to enhance meniscal cell attachment and proliferation, the supplements should be provided via both scaffold and medium rather than via a single route. In the case of PG-0.5 and PB-0.5, half the content of biomolecules was readily available in the medium, which helped the cells to grow faster, while the other half of the biomolecules incorporated inside nanofibrous scaffold might have helped in the attachment of cells by increasing the affinity of cells towards the scaffolds. But, in the case of biomolecules inside the nanofiber (PG-1 and PB-1), more time is required for the cells to grow in biomolecule free medium and get attached to the scaffold. This may be due to minimum exposure of embedded biomolecules to the cells during the initial period of culturing. Once the fibers start degrading, this trend may change, enabling the molecules to be accessed by the cells, directly, depending on the nature of the biomolecules and scaffold. Hence, more cells were attached to PG-0.5 and PB-0.5 scaffolds and higher proliferation was observed for the same as compared to PG-1 and PB-1. The scaffolds were further investigated using FESEM to visualize the human meniscal cell attachment and proliferation. The FESEM results (Fig. 6) were in correlation with the Hoechst stained images. The P–C scaffold showed a much lower number of individual cells with distinct cell morphology and less cellular extensions (Fig. 6a) when compared to the others. All scaffolds (Fig. 6b–e) seeded with human meniscal cells showed increased cell mass, cellular extensions and colonization. The cells were attached more onto the nanofiber surface. Few cells were found to grow in between the nanofibers below the surface (marked as arrow in Fig. 6).

Fig. 6 FESEM images of meniscal cells on nanofibrous scaffolds (24^th day): (a) P–C, (b) PB-1, (c) PB-0.5, (d) PG-1 and (e) PG-0.5.

The cell attachment below the nanofiber surface may be due to the porous structure of the nanofibrous scaffold. Similar penetrations of cells were observed in different nanofibrous scaffolds.^51,52 The cells formed tissue like organized structures over the scaffolds surface on 24^th day, especially in PG-0.5 (Fig. 6e).

The overall fibrous nature of the scaffold was fully masked by a dense mass of cells. This result shows that the scaffold PG-0.5 has the highest ability to favour meniscal cell attachment and proliferation. After 24 days of culture, PG-0.5 supported maximum cell attachment followed by PB-0.5, P + G, PG-1, P + B, PB-1 and P–C. Hence, the PG-0.5 scaffold was taken as a representative sample for showing the sequential morphological cell adhesion and proliferation on the nanofibrous scaffolds at different time periods. PG-0.5 scaffolds seeded with meniscus cells were monitored on the 6^th, 12^th, 18^th and 24^th days (Fig. 7). These days were chosen based on optimization studies carried out with the Hoechst staining method. The initial stage of cell attachment on the 6^th day showed a much lower number of cells (Fig. 7a). As the number of days of incubation increased, the cell number increased steadily on the surface of the scaffolds (Fig. 7b and c). The cell–cell interaction and proliferation of cells onto the nanofibrous scaffolds enabled them to form a thick sheet like cell mass over the scaffolds (Fig. 7d). This superior cell adhesion and proliferation on galactose based nanofibers may be due to carbohydrate specific adhesion between intact cells.^21,22

Fig. 7 FESEM images of meniscus cells seeded over PG-0.5 nanofibrous scaffolds. (a) 6^th day, (b) 12^th day, (c) 18^th day and (d) 24^th day.

3.3. Cell viability and proliferation studies

MTT assay was carried out to evaluate the viability of human meniscal cells on nanofibrous scaffolds (Fig. 8a). The viability of control cells were set at 100% on each day of estimation and the viability relative to the control is shown. Meniscus cells were found to be viable on all scaffolds. The cell viability was found to be less than the control. However, the percentage cell viability of all the scaffolds was found to be above 76%, which clearly indicates that electrospun PCL scaffolds are non-toxic to cells, as suggested by Jiang et al.,⁵³ and do not show any adverse effect on the growth of meniscus cells. Human cartilage cells respond slowly towards the scaffold and they usually take more time to adapt.⁵⁴ Thus, initial MTT assay results show lower cell viability on the scaffolds than control. The slight decrease in the viability may be attributed to the initial time taken by the cells to adapt to the new polymeric environment and the surface structure. The cell viability of P–C scaffolds showed significant variation till the 2^nd day. Similarly, PB-1 showed significant variation till the 3^rd day, beyond which the variations in cell viability were not significant. PG-1 did not show any statistically significant variation in cell viability throughout the study. Hence, these scaffolds can be positively considered for meniscus tissue engineering. Once cells get adapted to the new scaffold environment, they start dividing by taking nutrients from the substrate and the medium.

Fig. 8 (a) Cell viability study using MTT assay and (b) DNA estimation of cells grown on different nanofibrous scaffolds (*p < 0.05).

DNA estimation was carried out on cells extracted from different scaffolds up to the 24^th day to see the long term cell viability on the scaffolds (Fig. 8b). Initial absorbance of DNA content on day 6 of control sample was taken as 100%. The DNA content was found to be higher in galactose treated samples than those treated with biotin and it increased with increasing number of days. DNA content showed a significantly increasing trend with number of days till the 24^th day for PB-0.5 and for all galactose supplemented samples (P + G, PG-1 and PG-0.5) (Fig. 8b). The DNA content increased almost ∼1.6 fold in galactose based samples (P + G, PG-1 and PG-0.5) when compared to the control on day 24. However, statistical analysis between the galactose supplemented samples on the 24^th day (and also in the case of the 18^th day) showed no significant difference in the estimated DNA content. Among the biotin based samples, supplementation of biotin through the medium showed higher DNA content (∼1.3 fold in P + B and PB-0.5 on day 24). However, both samples showed significantly lower DNA content compared to the galactose supplemented samples on the 24^th day. This study clearly indicated that the DNA content of the cells was comparatively high when galactose or biotin were supplemented both in the scaffold as well as in the medium. These results are in correlation with the Hoechst stained and FESEM images discussed in the previous sections.

3.4. Estimation of GAG and collagen

The GAG and collagen contents were found to increase with increasing number of incubation days in all cases (Fig. 9). The GAG and collagen secretion into the medium increased up to the 24^th day. The P–C scaffolds showed less GAG and collagen secretion when compared to the biomolecules supplemented scaffolds. However, no appreciable differences in GAG secretion were observed till the 12^th day among all scaffolds (Fig. 9a and b). Beyond that, scaffolds having biomolecules inside, or in the medium, or both show significantly higher (p < 0.05) GAG secretion than the control and P–C. No statistically significant improvements were observed for collagen secretion in the case of biotin supplemented scaffolds (Fig. 9c). Biotin, a water soluble vitamin, regulates gene expression at the transcriptional and translational levels, which enhances glucose metabolism.⁵⁵ As biotin has no direct involvement in collagen secretion, this may be the reason for less significance in the data. Galactose supplemented scaffolds started showing significantly increased collagen secretion than P–C after the 6^th day (Fig. 9d). Considering both GAG and collagen secretion, PG-0.5 was found to be most effective for ECM formation. Galactose is the key component in the biosynthesis of GAG and collagen. Galactose is reported to be directly involved in heparan sulfate and chondroitin sulfate synthesis pathways, which are the major components of ECM.⁵⁶ Moreover, galactose enters the glycolytic pathway at the level of glucose 6 phosphate, whereas the glucose molecules come from the regular path.⁵⁷ This enables the galactose molecules to speed up the glycolysis pathway for more energy production. Also, the galactose molecule is directly involved in the post translational modification of procollagen⁵⁸ occurring during the synthesis of collagen in human cells. Hence, incorporation of galactose resulted in increased cell proliferation as compared to biotin.

Fig. 9 GAG production and total collagen content on cell seeded scaffolds on the 24^th day. (a) GAG concentration in PCL–biotin scaffolds, (b) GAG concentration in PCL–galactose scaffolds, (c) collagen concentration in PCL–biotin scaffolds and (d) collagen concentration in PCL–galactose scaffolds (*p < 0.05).

4. Conclusions

In meniscal tissue engineering, the major challenge is to create a favorable environment where meniscal cells take less time for cell adhesion onto the scaffolds with good overall distribution. Such a favorable environment increases the proliferation rate of these cells to replace the damaged tissue in a short time. To achieve this goal, functionalization of electrospun nanofibers with biomolecules (biotin and galactose) to enhance cell attachment and proliferation was carried out. This study showed similar biodegradation rate for these biomolecule incorporated nanofibers as compared to PCL nanofibers. The attachment and proliferation of human meniscus cell varied from one biomolecule to the other and also through the route of delivery. Galactose incorporated into the medium as well as in the scaffold promoted more meniscal cell adhesion and growth. The addition of galactose also increases DNA content, and GAG and collagen secretion. The biomolecule incorporated PCL based nanofibers not only provide physical support to the cells but also provide local release of biomolecules to influence regeneration of the surrounding meniscal tissue. The biomolecules embedded in the nanofibrous scaffolds provide nutrients in a sustained release manner, while the biomolecules added in medium will supply immediate nutrition towards the cells. Hence, the most efficient and potential route of administration for these biomolecules is via two routes simultaneously (i.e., 50% of the biomolecules are inside the fibers and 50% in the medium) to enhance the cell attachment, growth and proliferation in the PCL nanofibrous matrix. This is a promising strategy in meniscal tissue engineering to improve cell attachment onto scaffolds. This approach can be further extended for supplementing any potent growth factors, biomolecules, etc., and may be used in various other scaffolds for in vitro culturing of cells.

Acknowledgements

The authors sincerely acknowledge the financial support from Tamil Nadu State Council for Science and Technology (TNSCST) for this project [No.: TNSCST/S&T Projects/VR/MS/2013-2014]. The authors like to express their deep gratitude to the management of PSG Institutions for their financial and other shapes of 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, Head, Department of Orthopaedic Surgery, Ortho One Orthopaedic Speciality Centre and Dr S. Ramalingam, Principal, PSG IMS&R, Coimbatore.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra14315b

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