Cartilage tissue engineering (CTE) applications are focused towards the use of implantable biohybrids consisting of biodegradable scaffolds combined with
Regenerative medicine is a multidisciplinary field of research which involves the use of biomaterials, growth factors, and stem cells in order to repair, replace, or regenerate tissues and organs damaged by injury or disease [
Cartilage tissue engineering (CTE) has been increasingly explored in the recent years [
Novel scaffolds which facilitate the differentiation of stem cells into cartilaginous phenotype concomitant with their assembly into 3D tissue [
To successfully mimic the cartilage tissue’s environment, the fundamental structure of the designed biomaterial should be a tridimensional system [
Among the most beneficial prochondrogenic factors used in the design of new biomaterials for CTE are hyaluronic acid (HA) and chondroitin sulfate (CS). HA is a natural molecule component of the ECM from many tissues, including cartilage, with multiple physical and biological functions. HA plays a vital role in the development of cartilage, the maintenance of the synovial fluid, and the regeneration of tendons [
CS is one of the natural glycosaminoglycans (GAG) found in the structure of the aggrecan molecule of the cartilage. Among other properties, CS is responsible for the water retention of cartilage, due to the negative charge ensured by its structure [
Regarding the cellular component of the implantable biohybrids, stem cells are ideal candidates for regenerative medicine due to their ability to commit to multiple cell lineages and to self-renew [
Several sources of stem cells are likely to meet these requirements, yet human adipose-derived stem cells (ASCs) have multiple benefits [
ASCs can be reproducibly isolated from liposuction aspirates through a procedure involving collagenase digestion, differential centrifugation, and expansion in culture [
ASCs have the potential to differentiate into bone, cartilage and muscle as well as adipose and neural tissue [
In this context, our aim was not only to build novel 3D porous scaffolds based on sericin and collagen, improved with prochondrogenic factors such as chondroitin sulfate or hyaluronic acid for CTE, but also to evaluate their
Human adipose-derived stem cells (ASCs) provided by Invitrogen (Life Technologies, Foster City, CA, USA) were used for this study. Cells were isolated from human lipoaspirate tissue, then they were expanded for one passage in MesenPRO RS Medium (Invitrogen, Life Technologies, Foster City, CA, USA), a low (2%) serum concentration medium that reduces ASCs doubling times and finally they were cryopreserved from primary cultures. According to the manufacturer, each lot of ASCs originates from a single donor of human subcutaneous adipose tissue and the cells can be expanded for 4-5 passages before losing their ability to grow or differentiate into all potential phenotypes, including adipocytes, osteoblasts, and chondrocytes. These ASCs express the following cell-surface markers profile: CD29+, CD44+, CD73+, CD90+, CD105+, CD166+ and CD14-, CD31−, CD45−, and Lin1−.
The culture was propagated for two passages and split 1 : 4 after achieving 75–80% confluence in order to achieve the cell number required for 3D cultures assessment. Therefore, cells plated in T75 culture flasks (Nunc, Thermo Scientific, Waltham, MA, USA) were incubated in MesenPRO RS Medium, at 37°C in a humidified atmosphere of 5% CO2 and 95% air, with growth media changed every 48 h.
Cell morphology was observed by phase contrast microscopy (Nikon Eclipse TS 100, Nikon Instruments Europe, Amsterdam, Netherlands) every day.
Type I collagen gel (Coll) with an initial concentration of 2.11% and pH 2.8 was extracted from calf hide using the technology previously described [
The cartilage scaffolds based on collagen, sericin, and glycosaminoglycans were prepared in a similar manner as previously described by Dinescu et al. [
The novelty of these scaffolds resides not only in the original combination of collagen, chondroitin sulfate or hyaluronic acid with sericin but also from the 3D porous nature of the scaffolds.
ASCs in the 3rd passage were seeded on top of Coll-SS, Coll-SS : HA 10%, Coll-SS : HA 5%, Coll-SS : CS 10% and Coll-SS : CS 5% biomatrices at an initial density of 2 × 105 cells/cm2. The cell suspension was allowed to diffuse through the hydrogels in order for the cells to adhere to the biomaterial. After 1 hour, the resulting 3D bioconstructs were incubated in standard conditions of cultivation in MesenPRO RS Medium. In our experiments, we defined as bioconstructs the porous 3D hybrids resulting after Coll-SS-based hydrogels were put in contact with ASCs.
For further simplicity, the following abridgements will be introduced to designate the studied 3D scaffolds: Coll-SS = control; Coll-SS : HA 10% = sample A; Coll-SS : HA 5% = sample B; Coll-SS : CS 10% = sample C; Coll-SS : and CS 5% = sample D.
The CTE-designed scaffolds were characterized by Fourier transform infrared (FTIR), thermal analysis, scanning electron microscopy (SEM) and water uptake.
Thermal properties of the obtained matrices were determined by differential thermal calorimetry (DSC) using a Netzsch DSC 204 F1 Phoenix equipment. Samples of about 2 mg were heated from 20 to 300°C under a constant nitrogen flow rate (20 mL/min). A heating rate of 10°C/min was applied.
Scanning electron microscopy (SEM) was used to determine the morphological structure of the scaffolds. The analysis was performed using a QUANTA 200 SEM device.
Biocompatibility was evaluated in terms of the viability and the proliferative activity of ASCs in contact with the control and samples A, B, C, and D using qualitative Live/Dead Assay and quantitative MTT test. The cytotoxic potential of the biomaterials on ASCs was evaluated by spectrophotometric quantification of the LDH released in culture medium.
Briefly, at 2, 4, and 7 days after seeding, the control, A, B, C, and D bioconstructs were incubated with a staining solution prepared according to manufacturer’s instructions for 15 minutes at dark. Next, the stained 3D cultures were analyzed by fluorescence microscopy using an Olympus IX71 inverted microscope and images were captured with Cell F Imaging Software (Olympus, Hamburg, Germany, 2008).
In addition, confocal 3D images were acquired with a Carl Zeiss LSM710 laser-scanning confocal microscopy system using Zeiss 20x 0.5NA objective. Carl Zeiss Zen 2010 software version 6.0 was used for image acquisition and analysis. The 488 and 543 nm laser lines were used for excitation and fluorescence emission was detected at 520–550 nm for calcein and 600–680 nm for ethidium bromide. The confocal aperture used corresponded to a backprojected size of 1 Airy unit. Images were acquired as z-stacks using depth brightness correction, and a maximal projection algorithm was used for 3D reconstruction.
A combination approach, consisting of MTT and lactate dehydrogenase (LDH) assays, was used to provide information about cell viability and possible cytotoxic effects of the analyzed materials.
ASCs capacity to proliferate into Coll-SS, Coll-SS : HA 10%, Coll-SS : HA 5%, Coll-SS : CS 10%, and Coll-SS : CS 5% biomatrices was quantitatively determined using MTT spectrophotometric assay at 2, 4, and 7 days afte seeding. In this context, all cell-scaffold bioconstructs studied were incubated in 1 mg/mL MTT (thiazolyl blue tetrazolium bromide) solution (Sigma Aldrich Co., Steinheim, Germany) and after 4 h the formazan crystals were solubilized in isopropanol for 1 h. The absorbance of the resulting solution was measured by spectrophotometry at 550 nm (Appliskan Thermo Scientific, Waltham, MA, USA).
The environmental cytotoxic potential of the Coll-SS, Coll-SS : HA 10%, Coll-SS : HA 5%, Coll-SS : CS 10% and Coll-SS : CS 5% materials on the ASCs was evaluated using “
The spectrophotometrical data were statistically analyzed using GraphPad Prism 3.03 Software, one-way ANOVA, Bonferroni test. Data are presented as the average of three replicates (mean ± standard deviation).
Freeze drying of collagen-sericin-glycosaminoglycan gel compositions led to porous 3D sponges, which resembled the ECM of cartilage tissue. The properties of these spongious polymeric samples were evaluated by various tests.
FTIR spectra of Coll-SS, Coll-SS : HA, and Coll-SS : CS (with 5% glycosaminoglycan) are shown in Figure
FTIR spectra of Coll-SS-based scaffolds.
The spectra of Coll-SS scaffolds are characterized by typical protein absorption bands. As revealed by FTIR analysis, the amide A band appeared at 3296 cm−1, amide B at 3075 cm−1, amide I at 1645 cm−1, amide II at 1545 cm−1 and amide III at 1241 cm−1 in IR spectra. The band of 1452 cm−1 is typical for the pyrolidonic ring of hydroxyproline and gives information about the denaturation degree of the collagen triple helix. The ratio between intensity of Amide III and 1452 cm−1 was higher than the one for all the studied samples, which indicated that no alterations or significant changes took place. The spectra of the samples are very similarls due to the small content of HA or CS which do not influence the FT-IR spectroscopy.
The differential thermal calorimetry (DSC) thermograms showed thermal denaturation caused by the breaking of hydrogen bonds which stabilize the collagen native helical structure. The results of thermal analysis are presented in Table
Thermal properties of Coll-SS-based scaffolds, during the heating process.
Samples | Denaturation temperature (°C) | Melting temperature (°C) | Thermooxidation temperature (°C) |
---|---|---|---|
Control | 58 | 219 | 310 |
Sample A | 66 | 222 | 309 |
Sample B | 63 | 216 | 310 |
Sample C | 62 | 219 | 310 |
Sample D | 56 | 219 | 310 |
Collagen is denaturated when heating between 56 and 66°C. The differences in the denaturation temperatures between samples are most probably due to the glycosaminoglycans interaction with Coll-SS and also due to their different ratio. The DSC results showed a strong increase of denaturation temperature for samples containing 10% HA and a decrease for the sample with CS. The melting temperatures showed insignificant differences between samples, and the most ordered structure was noticed for the sample A. Thermooxidation temperatures for all the samples were found to be at about 309°C.
Similarities between structures could also be seen in the SEM images presented in Figure
SEM images of (a) Coll-SS, (b) sample A, (c) sample B, (d) sample C and, (e) sample D.
The porous structures with inner pores interconnected by collagen fibrils were visible in all studied samples, as shown in Figure
The morphology of the samples was also investigated by water uptake and the results are presented in Figure
Water uptake of Coll-SS-based scaffolds after 1 h.
According to our results, the scaffolds became stable in terms of water uptake after 1 h of immersion. Although the scaffolds showed similar values of water uptake with variations between 42.80 and 43.15 g/g, sample A and sample B displayed higher hydrophilic character, probably due to their HA content.
Cell behavior in terms of viability, proliferation, morphology, and distribution, was qualitatively investigated after 2, 4, and 7 days of culture in standard conditions by fluorescence and confocal microscopy, based on the simultaneous staining of live (green labeled) and dead (red labeled) cells.
High cellular viability was revealed by Live/Dead assay (Figure
Fluorescence microscopy assessment of living (green-labeled) and dead (red-labeled) ASCs at 2, 4, and 7 days post-seeding on samples A, B, C, D and the control.
Additionally, cell distribution on the surface of the hydrogels was observed to be homogenous, suggesting an uniform and ordered material structure which was able to allow cells to adhere. Furthermore, cell phenotype on the surface of sample A and, to a lower extent, sample D, resembles to that of cells attached to a substrate. However, clear distinction could be made after 7 days of culture between ASCs phenotype in the presence of HA versus CS, since cells in samples C and D appear to be smaller and more rounded in shape than those in samples A and B, which display fibroblast-like phenotype.
Cell distribution inside samples A and D was investigated at day 7 by laser scanning confocal microscopy. Three dimensional reconstructions of the scanned volumes (Figures
Three dimensional reconstructions of the z-stacks obtained by confocal microscopy in (a) ASCs-Coll-SS : HA 10% (sample A) and (b) ASCs-Coll-SS : CS 5% (sample D) Live/Dead labeled systems 7 days after seeding.
The highly positive ratio between live (green-labeled) and dead (red-labeled) cells in both systems was confirmed once more through confocal microscopy (Figure
To validate the viability and the proliferation rate, MTT assay was employed as a more accurate approach. In this context, the Coll-SS scaffold as well as the samples A, B, C, and D were seeded with ASCs and subjected to MTT spectrophotometric assay at 2, 4, and 7 days of culture (Figure
ASCs viability and proliferation profile evaluation after 2, 4, and 7 days of culture using spectrophotometric MTT assay
Regarding the cellular viability, our results show that at 2, 4, and 7 days of culture the amount of metabolically active cells in sample A was found to be significantly increased (
As shown in Figure
The cytotoxic potential of samples A, B, C, and D was evaluated by spectrophotometric quantification of the LDH enzyme release in the culture media by the embedded ASCS. A collagen-sericin hydrogel, unimproved with HA or CS, preseeded with the same number of ASCs, was used as reference (Figure
Collagen-based scaffolds cytotoxic potential evaluation by spectrophotometric LDH assay after 2, 4, and 7 days of culture (
At 2 days of culture only sample C displayed significant cytotoxic potential on ASCs as compared to control (
Although sample B displayed a constant ratio of cytotoxicity as compared to the control during the experiment, sample A showed significant lower values as compared to it. In addition, at 7 days of culture, the cytotoxic potential of sample A was found to be lower (
Regarding samples C and D, starting with the 4th day of culture, both displayed a significant cytotoxic effect on ASCs (
Correlating the results obtained via quantitative and microscopy assays, we can generally conclude that the ratio between live and dead cells is strongly positive for all the studied bioconstructs, reflecting good biocompatibility of these materials. However, cells inside sample C were proved to have lower than 80% of viability, which makes it not eligible for further studies. Conversely, significant higher cell viability and lower cytotoxic potential were displayed by samples A and D, when compared to the other samples and to the control system. This observation suggests that not only the ratio between live and dead cells inside the scaffold, but also the material’s formulation effect on cell viability is critical for cell behavior analysis in contact with novel materials proposed for tissue engineering.
The scaffolds prepared in this study show similar physical properties, having a porous structure with pore sizes between 20–150
The results obtained using Live/Dead assay confirmed the quantitative determinations performed during MTT and LDH tests, suggesting that all samples are biocompatible, but the most equilibrated formulas in terms of cell behavior were collagen-sericin hydrogels improved with 10% HA and 5% CS (samples A and D). Furthermore, the scaffold’s structure and pore distribution in both samples allow cell viability and proliferation, but sample A pore interconnectivity and composition favor ASCs distribution in deeper layers of the hydrogel than in sample D, simultaneous with maintaining the appropriate conditions for cell survival in the depth of the material.
MTT and LDH assays revealed that sample A exerts no cytotoxicity on ASCs and allows their proper proliferation, while regarding the CS compositions, sample D could be used for further studies of chondrogenesis but taking care of maintaining its cytotoxic potential at a level that does not interfere with cell proliferation or differentiation. Sample C was found to be cytotoxic and was excluded from further studies.
Since scaffold physical characterization revealed no significant differences between the samples, ASCs behavior in contact with each sample composition served as 3D system selection criteria for further
The authors declare no conflict of interests.
Sorina Dinescu and Bianca Gălăţeanu contributed equally to this work.
This research was supported by Romanian CNCS-UEFISCDI, Complex Exploratory Research Project (Grant no. PCCE248/2010). We thank Prof. Dr. Dana Iordăchescu (University of Bucharest, Department of Biochemistry and Molecular Biology) for the project idea.