Evaluation of the ability of natural and synthetic scaffolds in providing an appropriate environment for growth and chondrogenic differentiation of adipose-derived mesenchymal stem cells.

BACKGROUND: Although progenitor cells have been observed in articular cartilage, this part has a limited ability to repair due to a lack of blood supply. Formerly, tissue engineering was mainly based on collecting chondrocytes from the joint surface, culturing them on resorbable scaffolds such as poly D, L-lactic glycolic acid (PLGA) and then autologous transplantation. In recent times, due to difficulties in collecting chondrocytes, most of the researchers are focused on stem cells for producing these cells. Among the important factors in this approach, is using appropriate scaffolds with good mechanical and biological properties to provide optimal environment for growth and development of stem cells. In this study, we evaluated the potential of fibrin glue, PLGA and alginate scaffolds in providing a suitable environment for growth and chondrogenic differentiation of mesenchymal stem cells (MSCs) in the presence of transforming growth factor-β3.
MATERIALS AND METHODS: Fibrin glue, PLGA and alginate scaffolds were prepared and MSCs were isolated from human adipose tissue. Cells were cultured separately on the scaffolds and 2 weeks after differentiation, chondrogenic genes, cell proliferation ability and morphology in each scaffold were evaluated using real time-polymerase chain reaction, MTT chondrogenic assay and histological examination, respectively.
RESULTS: Proliferation of differentiated adipose tissue derived mesenchymal stem cells (AD-MSCs) to chondrogenic cells in Fibrin glue were significantly higher than in other scaffolds. Also, Fibrin glue caused the highest expression of chondrogenic genes compared to the other scaffolds. Histological examination revealed that the pores of the Fibrin glue scaffolds were filled with cells uniformly distributed.
CONCLUSION: According to the results of the study, it can be concluded that natural scaffolds such as fibrin can be used as an appropriate environment for cartilage differentiation.

INTRODUCTION

Repairing the injuries and damage to the cartilage is still one of the most important problems in the orthopedics.1 Recently, tissue engineering techniques have shown acceptable potential to solve this problem. Tissue engineering is the science of design and production of new tissues to repair the damaged organs and replace missing parts due to various causes.2

Polymers used in preparation of cell scaffolds are classified into two groups: Natural and synthetic polymers.34 Each scaffold should actually be able to show certain biological and mechanical properties to improve and alter cell behavior so that each scaffold can be designed on the basis of the characteristics of its target tissue.5 Furthermore, Some of the other characteristics of cellular scaffolds are nontoxicity, biocompatibility, biodegradability, nonimmunogenicity, easy fabrication, appropriate physical and mechanical properties and good stability.6

One of the most important parts of tissue engineering is to select an appropriate scaffold to replace the damaged tissue.7 Scaffolds allow cell migration and transferring of biochemical factors.

Also, the great success in tissue engineering application can be achieved through coordinated application of suitable scaffolds with proper cell types.89 Recently, there is a growing interest in using hydrogels as scaffolds.1011 One of the most widely used hydrogels in medicine is fibrin.1112 Fibrin glue is commonly used in abdominal surgery, orthopedics, neurosurgery and oral surgery. Combination of fibrinogen clot and thrombin can induce binding, migration and growth of various types of cells such as chondrocytes, fibroblasts, smooth muscle cells and keratinocytes.13

During the past two decades, poly D, L-lactic glycolic acid (PLGA) biopolymer has been used as one of the most interesting candidates for fabrication of scaffolds needed in tissue engineering research.14 PLGA is a biodegradable and biocompatible polymer with optimal mechanical properties and is approved by the U.S. Food and Drug Administration.15 However, it has inferior hydrophilic properties and low water absorbency and is slowly degraded.16

Alginate is a polysaccharide derived from brown algae and has unique properties that enable it to switch to gel form in the presence of bivalent cations. Stable cellular growth of some types of cells such as chondrocytes has been observed in alginate.17 Alginate can be used as a scaffold due to its favorable properties.181920

Chondrogenic differentiation of mesenchymal stem cell (MSC) is affected by different intrinsic and extrinsic factors. Growth factors play a key role in this process. Growth factors are biologically active polypeptides synthesized by the body, which can induce cell proliferation and differentiation. One of the most known growth factor from the transforming growth factor-β (TGF-β) superfamily with respect to cartilage tissue engineering is TGF-β3.2122 In presence of TGF-β3 in medium culture, cartilaginous extracellular matrix (ECM) synthesis can be increased. This event is importantly ameliorated by supplementation of bone morphogenetic protein Type II.21

Mesenchymal stem cells are of great attention in medical and scientific communities due to their special capabilities.23 They are pluripotent progenitor cells that are capable of growth, reproduction and regeneration in media.2425 They are applied in tissue engineering much more than embryonic stem cells, the use of which is restricted due to ethical issues.26 They can differentiate to several cell lines such as chondrocytes, osteoblasts, adipocytes, endothelial cells, neurocytes, cardiac myocytes, hepatocytes and pancreatic cells.2728 Due to their special capabilities in tissue engineering and regenerative medicine, these cells are used in the repair of damaged tissue such as joints lesions, immunological diseases, bone and cartilage defect.30313233343536373839404142434445 Adipose-derived mesenchymal stem cells (AD-MSCs) have shown great capability in chondrogenic differentiation in the field of tissue engineering.264647484950 In this study, we evaluated the capability of three different scaffolds in growth and differentiation of AD-MSCs to chondrocytes in the presence of TGF-β3. Also, we hypothesized that hAD-MSCs could be suitable for chondrogenic differentiation using natural scaffolds specially Fibrin glue.

MATERIALS AND METHODS

In this study, poly D, L-lactic glycolic acid 50/50 (PLGA) (sigma) and Dulbecco's Modified Eagles Medium (DMEM) (sigma) and penicillin-streptomycin (sigma) and fetal bovine serum (FBS) (Gibco) were used.

Isolation and passage of adipose-derived mesenchymal stem cells

Human liposuction aspirates were obtained from three patients undergoing nephrectomy after informed consent was obtained from each patient. Adipose tissue was transferred to the laboratory in saline containing 1% antibiotic (penicillin-streptomycin) at 4°C and was divided into small pieces after being washed several times with phosphate buffered saline (PBS) and saline. Next it was digested with Type I collagenase (1.5 mg/1 g adipose tissue) for 45-60 min at 37°C and centrifuged at 1800 rpm for 10 min. Cell sediment in Dulbecco's Modified Eagles Medium containing penicillin-streptomycin and 10% FBS, was transferred to cell culture flasks and incubated at 37°C with 5% CO2 and 95% humidity. After 24 h, cells which were not attached to the bottom of the flask were removed by changing the medium. Cell culture medium was changed every 3 days. After trypsinization, cells were transferred to passage 3 and were ready to use.51

Preparation of fibrin glue

Preparation of fibrinogen and thrombin

Fresh-frozen plasma and fibrinogen were obtained from Qom blood transfusion organization. Fresh-frozen plasma was added to calcium gluconate at 5:3 ratio, incubated for 1 h at 37°C and centrifuged at 220 rpm for 10 min. After centrifugation, the supernatant was discarded and thrombin was separated. Fibrinogen and thrombin were then prepared for cell culture. For this purpose, AD-MSCs were converted to a soluble form at a concentration of 5 × 106 cells in 1cc of thrombin and fibrinogen was added to the solution. The solution was incubated at 37°C/5% CO2/99% humidity in chondrogenic medium (CM; DMEM-high glucose [HG] supplemented with 50 mg/ml bovine serum albumin, 5 µg/ml ascorbate-2-phosphate, 1% insulin-transferrin-selenium, 1 nM dexamethasone, 5 µg/ml linoleic acid, 1% penicillin-streptomycin and 10 ng/ml TGF-β3) for 14 days.1251

Preparation of poly D, L-lactic glycolic acid scaffold

Initially, 5% PLGA particles with dimensions of 5 mm × 3 mm were dissolved in methylene chloride and then NaCl was added. The solution was poured into a plastic container with 3 mm height and 6 mm diameter and was frozen at −20°C. Frozen polymer scaffolds were washed in distilled water to remove the salt particles and then samples were washed with 70% alcohol and were sterilized using ultraviolet. Human MSCs were trypsinizated at passage 3 and centrifuged and after transmission to differentiation medium, they were distributed on PLGA scaffold by the density of 1 × 106 cell/ml. Finally, the scaffold was incubated at 37°C/5% CO2/99% humidity in differentiation environment.1532

Preparation of alginate scaffold

Initially, 1.2% alginate powder was suspended in 0.15 M/L sodium chloride and sterilized using a filter. AD-MSCs of passage 3 were trypsinizated and centrifuged at 1400 rpm for 8 min and were suspended in alginate solution. The samples were added to 105 mM calcium chloride solution and kept in room temperature for 15 min. After washing by calcium chloride and Dulbecco's Modified Eagles Medium, alginate beads were incubated at 37°C/5% CO2 in chondrogenic differentiation medium for 14 days.1851

Chondrogenic differentiation of adipose-derived mesenchymal stem cells

Mesenchymal stem cells cultured in passage 3 were separated from the bottom of the culture plate by trypsinization and after resuspension, were incubated at 37°C/5% CO2/99% humidity in chondrogenic medium (DMEM-HG supplemented with 50 mg/ml bovine serum albumin, 5 µg/ml ascorbate-2-phosphate, 1% insulin-transferrin-selenium, 1 nM dexamethasone, 5 µg/ml linoleic acid, 1% penicillin-streptomycin and 10 ng/ml TGF-β3) for 14 days.51 The culture medium was changed every 2-3 days.51

Cell proliferation-MTT assay

The human adipose-derived mesenchymal stem cells (hAD-MSCs) proliferates with the existence of scaffolds, hAD-MSCs cultured in chondrogenic medium were seeded on the scaffold into 24-well plate and incubated at 37°C in 5% CO2 for 14 days. In order to prepare the cells and the scaffolds for MTT assay, first we removed the medium culture from the scaffolds on which the cells had been cultured after than the medium washed with sterile PBS. Later, 40 μL of the solution MTT (0.05 mg/ml) and 400 μL of fresh DMEM medium were added and incubated for 4 h at 37°C. The reaction was terminated by the addition of 400 μL dimethyl sulfoxide. Finally, the absorbance of the optic density was read at a 570 nm wavelength using an enzyme-linked immunosorbent assay plate reader (Biokit, EL × 800 Reader, Spain).

Real time-polymerase chain reaction for evaluating the expression of chondrogenic genes

Fourteen days after chondrogenic differentiation, RNA samples were obtained from each scaffold, separately. The scaffolds were digested in fluid nitrogen. Extraction of RNA and reverse transcription of RNA to cDNA was performed using AccuZol™ Total RNA Extraction Solution (bioNEER, Daedeok-gu, Daejeon, Korea) and AccuPower® CycleScript RT Premix (bioNEER, Daedeok-gu, Daejeon, Korea), respectively, as per manufacturer's instructions. Briefly, samples were washed twice with phosphate buffered saline. Then, 1 mL of AccuZol™ was added and samples were harvested into 1.5 mL centrifuge tube using a cell scraper. After adding 200 µL of chloroform and shaking vigorously for 15 seconds, the mixture was left in ice for five minutes. The mixture was next centrifuged for 15 minutes at 12,000 rpm at 4°C. After the supernatant was transferred to a 1.5 mL tube, an equal amount of isopropyl alcohol was added, and the mixture was left at −20°C for 10 minutes. At 4°C the mixture was centrifuged again for 15 minutes at 12,000 rpm. The supernatant was removed and washed with 1 mL of 80% ethanol. The solution was centrifuged once again for 10 minutes in 12,000 rpm at 4°C and the supernatant was removed. The ribonucleic acid (RNA) pellet was dried at room temperature for 10 to 20 minutes. It was dissolved in DEPC water and left for 5 minutes at 55°C. The total RNA that had been separated was treated with DNase. RNA 1 ug was added to AccuPower® CycleScript RT Premix (dT20, bioNEER, Daedeok-gu, Daejeon, Korea) and cDNA synthesis carried out [Table 1].

Table 1

The temperature, time and cycle required for the synthesis of cDNA

Real time-polymerase chain reaction (PCR) performance using AccuPower® 2X Greenstar qPCR Master Mix (bioNEER, Korea) and primers of each gene were designed as follows utilizing primer 3 program [Table 2].

Table 2

Real time PCR primer sequences

The reaction was initiated by heating to 95°C for 15 min., followed by 40 cycles of elongation at 59°C for 30 s and denaturation at 95°C for 15 s.

The mRNA expression level of Target gene was normalized based on GAPDH reference gene. The level of expression of each target gene was calculated using 2−ΔΔCt.51

Histological evaluation

28 days after chondrogenic differentiation, scaffolds and cartilage tissue as control, were analyzed histologically: After fixation with 10% neutral-buffered formalin for at least 24 h, specimens (N = 4) were embedded within paraffin and sectioned at 5 μm thickness. For histological evaluation, samples were stained with hematoxylin/eosin counterstaining.

Statistical analysis

For the real time-PCR analysis, significant differences in expression levels of four genes in 3 scaffolds were identified by the least significant difference test. Real time-PCR for every sample was repeated 4 times. Statistical analysis was performed using SPSS (Statistical Package for the Social Sciences) software (version 17) and data were considered significant with a P 0.05.

RESULTS

In primary cultures, growth of fibroblast-like or spindle-shaped AD-MSCs with distinct nuclei was observed by phase-contrast microscope [Figure 1]. In MTT assay, optical density values of Fibrin Glue were significantly higher than those of PLGA, Alginate Scaffolds and control after 2 weeks [Figure 2].

Figure 1

The image produced by phase-contrast microscope of living mesenchymal stem cells isolated from human adipose tissue which spindle cells in the third passage are visible

Figure 2

The MTT assay of chondrogenic differentiated adipose-derived mesenchymal stem cells in poly D, L-lactic glycolic acid, Fibrin glue and alginate in comparison with control group

Differentiation of stem cells to chondrocytes was confirmed by real time-PCR. Expression of mRNA of collagen Types I and II, sox9 and aggrecan in differentiated cells was evaluated after 14 days [Figure 3]. The highest expression of differentiation genes was observed in the fibrin glue scaffold. There was a significant difference in expression of aggrecan and collagen Type II genes on fibrin glue and PLGA scaffolds and their expression in AD-MSCs without scaffold (P < 0.05). Collagen Type II and aggrecan genes showed the highest expression in fibrin glue compared with other scaffolds and the lowest expression was observed in AD-MSCs without scaffold (P < 0.05). Expression level of collagen Type II in fibrin glue and AD-MSCs was 0.515 ± 0.45 and 0.02 ± 0.003, respectively. Expression level of aggrecan gene in fibrin glue and AD-MSCs was 135.83 ± 5716.52 and 519.12 ± 31.74, respectively. The highest and lowest level of expression of sox9 gene was observed in fibrin glue and PLGA, respectively (396.995 ± 27.39 vs. 3.834 ± 28.815).

Figure 3

The mRNA expression of collagen Type I, collagen Type II, Sox9 and Aggrecan in the differentiated adipose-derived mesenchymal stem cells on different Scaffolds (a-d respectively) *statically significant (P < 0.05)

The accumulation and presence of chondrocytes were examined by hematoxylin/eosin staining at 4th week [Figure 4]. Histological examination revealed that pores were filled with chondrocytes and the cells were uniformly distributed in the cell Fibrin glue constructs at 4 weeks [Figure 4a]. The histological evaluations for PLGA and alginate scaffolds showed week results.

Figure 4

Histological evaluation of cells cultured on Fibrin glue, poly D, L-lactic glycolic acid (PLGA) and Alginate scaffolds at 4th week. (a) Fibrin glue scaffold, (b) PLGA scaffold and (c) alginate scaffold with ×10 magnification. Staining of native cartilage used as control with hematoxylin/eosin is demonstrated in (d) (×10 magnification)

DISCUSSION

As the cartilage does not have blood vessels and has limited ability for self-healing, repairing a damaged cartilage requires efficient methods.1525354 Tissue engineering techniques based on the utilization of stem cells are useful techniques with great potential in treatment of such injuries. One of the most important factors in successful tissue engineering is selecting an appropriate scaffold to facilitate cell growth and differentiation. As a result, in cartilage tissue engineering, an ideal scaffold seems necessary to maintain the chondrocyte phenotype in the differentiation process.54555657 Most of the tissue engineering studies that are based on using stem cells utilize chemical factors (such as growth factors) or signals for chondrogenic differentiation.2958 Thus, less attention has been paid to the importance of scaffolds as influential factors in regulation of tissue growth and differentiation.18 In this study, it was shown that differentiation capacity of MSCs varies in different biological scaffolds and scaffold selection is an important factor in the stem cell differentiation process. This study confirmed the results of previous studies in the field of differentiation ability of AD-MSCs under certain conditions.425960

Hydrogels show characteristics similar to soft tissue and thus are able to provide a supportive matrix for chondrocyte activity and secretion of cartilage ECM. High efficiency of encapsulation and uniform distribution of cells within the hydrogels are some of their benefits and can be effective in the quality of the formed tissue.195861 Hydrogel scaffolds perform the uniform proliferation of the cells and easy integration of growth factors, while synthetic polymers provide the required load-bearing capacity.4562 Hydrogels are easily prepared and are surrounded by chondrocytes, thus they protect chondrocyte phenotype and morphology.636465 They also contain natural biomaterial based on carbohydrates (such as alginate) and protein (such as fibrin glue).5866 Results of several studies suggested that different hydrogels are suitable in order to be used in tissue engineering in vitro.18586367 Fibrin, as a scaffold, is utilized in cartilage tissue engineering accompanied with MSCs.545868 Based on previous reports, not only fibrin is not less efficient than other hydrogels in cartilage tissue engineering, but fibrin can also support chondrogenesis of different tissues derived- MSCs.5458

It has been shown that softer biomaterials can support chondrocyte phenotype in a better way.1920 Therefore, based on the results of our study, it seems that less stiffness of fibrin can lead to a better chondrogenesis. Different studies showed contradictory results. Results of a previous study have shown that fibrin gel scaffold provides an opportunity for the growth of AD-MSCs.69 Unlike synthetic hydrogels, fibrin is not only a cell supplier matrix, but its biological activity makes it suitable for differentiation of stem cells and tissue engineering.70 Integrin binding sequence of arginine-glycine-aspartic acid existing in fibrin is a stimulating factor for cell binding and growing. Some studies reported that in cartilage tissue engineering, fibrin stimulates collagen Type II, while it prevents digestion of collagen Type I by stem cell derived chondrocytes.70 Some of the researches have shown that purified fibrin scaffolds cause less expression of collagen Type II and aggreacan compared with alginate.131754 In another study, alginate showed better stimulation of chondrogenesis in bone marrow derived MSCs when compared to fibrin.58 Evidence show that chondrogenesis is recognized by increase in expression and accumulation of collagen Type II and aggreacan genes compared to collagen Type I and sox9. In this study, we verified that the relative expression amount of collagen Type I gene in fibrin in comparison to MSCs is 2:9, respectively, while relative expression amount of collagen Type II gene in fibrin is 25:1 in comparison with MSCs. These results indicate that the ratio of chondrogenic related genes expression is increased several times. Consequently, fibrin glue scaffold had a lower expression of collagen Type I and higher expression of collagen Type II and aggrecan, when compared to MSCs. Results of our study showed that fibrin glue scaffold can be considered as an appropriate scaffold for differentiation of AD-MSCs due to higher expression of the genes involved in chondrocyte differentiation compared to PLGA and alginate scaffolds.

Furthermore, according to our results it is suggested that the proliferation of differentiated AD-MSCs to chondrogenic cells could be enhanced by the application of fibrin glue scaffold as a suitable environment.

CONCLUSION

It seems that natural scaffolds such as fibrin glue can be used as appropriate scaffold for chondrogenic differentiation of human MSCs and to develop new efficient strategies in tissue engineering and regenerative medicine.