VIABILITY OF ADIPOSE-DERIVED STEM CELLS (ASCs) ON POROUS CHITOSAN SCAFFOLD

VIABILITY OF ADIPOSE-DERIVED STEM CELLS (ASCs) ON POROUS CHITOSAN SCAFFOLD

M. Mohamed ¹, S. Gomathysankar ¹, AZ Mat Saad ¹, Kartini Noorsal ² and AS Halim ^*3

Reconstructive Sciences Unit ¹, Dean and Senior Consultant Plastic and Reconstructive Surgeon ³,  School of Medical Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia.

Advanced Materials Research Centre (AMREC) ², SIRIM Berhad, Lot 34, Jalan Hi Tech 2/5, Kulim Hi Tech Park, 09000 Kulim, Kedah, Malaysia

ABSTRACT: Background: Stem cells have great potential in regenerative medicine due to their multipotentiality. Treatments involving cell sheets for tissue reconstruction require a large number of cells to repair the tissue damage, and the cells are fragile. Combined with a porous chitosan scaffold (PCS), adipose-derived stem cells (ASCs), which are ubiquitous, can be delivered to a target tissue in vivo. This approach provides mechanical support to the skin until the normal tissue is regenerated. In the present study, ASCs were characterized and tested for their viability on a PCS in vitro prior to in vivo application. Results: The ASCs expressed CD29, CD73, CD90 and CD105 but did not express CD34, as demonstrated by immunocytochemistry. Most of the ASCs were found to be viable on the PCS at 24 and 72 hours of culture. These results suggest that ASCs seeded on a PCS have high viability, allowing ASCs to be enriched for therapeutic applications. Conclusions: This qualitative study has proven ASCs were able to survive on PCS as indicated by the number of live cells exceeded the number of dead cells as demonstrated by live/dead assay

Adipose derived stem cells,

Porous chitosan scaffold, Immunocytochemistry, Viability assay

INTRODUCTION: Autologous transplantation of bone marrow-derived stem cells has been used to treat various abnormalities and skin lesions. In vitro studies and animal models have demonstrated that these bone marrow-derived stem cells have the potential to reconstitute tissue vascularization and to restore normal function ^{1, 2, 3}. Adipose-derived stem cells (ASCs) have similar biological properties as bone marrow-derived cells and can be induced to differentiate into different cell lineages. Adipose tissue is a potential source of adult and somatic stem cells and has a wide range of clinical and therapeutic applications

In addition, adipose tissue is an abundant source of various growth factors and cytokines that stimulate the growth, proliferation and differentiation of ASCs ^{4, 5}. Recent studies have proven that ASCs play a significant role in tissue regeneration. To evaluate the possibility of improving tissue regeneration, the ASCs have been characterized in this study.

Reconstruction of skin after injury involves different types of wound healing, depending on the wound’s classification. Over several decades, studies have been performed on different reconstructs seeded with ASCs. Skin reconstructs create the optimal conditions for accelerating wound healing, are easily handled for transplantation and may replace the use of skin grafts ⁶. Most of the biomaterials that are available on the market lack these properties, and therefore, the demand for perfect skin reconstructs is high. The remarkable properties of chitosan offer unique opportunities for the development of biomedical applications. However, to date, no study has examined the viability of human ASCs on a porous chitosan scaffold (PCS). The present study reveals the greater viability of ASCs on a PCS, given that the chitosan scaffold allows greater cell attachment and proliferation when the cells are seeded on it.

MATERIALS AND METHODS:

Materials:

Adipose tissue:

Adipose tissue was obtained from individuals who had provided informed consents and who had undergone elective surgery at Hospital Universiti Sains Malaysia, Kelantan, as approved by the Human Research Ethics Committee of Universiti Sains Malaysia (USMKK/PPP/JEPeM (236.3[16])).

Porous chitosan scaffold (PCS):

Chitosan scaffolds were obtained from Advanced Materials Research Centre, Standards and Industrial Research Institute of Malaysia (SIRIM) Berhad in Malaysia ⁷. The thickness of the PCS was 2 mm, and the diameter was 5 mm. These scaffolds were sterilized with ethylene oxide (EO) gas and stored at room temperature for one month in a dark and dry place prior to use.

Methods:

Derivation and culture of ASCs:

The tissue obtained was washed with Dulbecco’s phosphate-buffered saline (DPBS) (Gibco®, USA) with 1% antibiotic-antimycotic. The tissue was then digested with collagenase type I (200 U/mL) (Worthington, NJ) overnight at 37°C. The cells were passed through a 70 µm cell strainer, washed with DPBS and centrifuged at 805g for 5 minutes. The cells were then cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco®, Life Technologies, USA) 10% mesenchymal stem cell (MSC)-qualified fetal bovine serum (Gibco®, Mexico) and 1% antibiotic-antimycotic. The cells were seeded at a density of 4x10⁴/cm² in T25 culture flasks and incubated at 37°C with 5% CO₂.

Verification of adipose-derived stem cell (ASC) phenotype by immunocytochemistry: This experiment was performed using an IHC Select® HRP/DAB Kit (DAB150, Chemicon) according to the instructions given by the manufacturer, with certain modifications. The staining was performed in a 24-well plate.

The culture medium was discarded, and the cells were washed with PBS and fixed in cold methanol for 15 minutes at 4°C. Blocking reagent was added and incubated at room temperature for 30 minutes. The cells were incubated with optimized dilutions of mouse anti-human IgG1 monoclonal antibodies against CD29 (Abcam, clone number 12G10) (1:1000), CD34 (Abcam, clone number BI-03C5) (1:1000), CD105 (Abcam, clone number SN6) (1:1000), CD73 (BD Pharmingen™, clone AD2, 550256) (1:1000), and CD90 (BD Pharmingen™, clone 5E10, 555593) at 4°C for one hour.

After washing with PBS, the cells were incubated with biotinylated goat anti-mouse IgG at 4°C for 15 minutes. Streptavidin-horseradish peroxidase was added to the wells, incubated for 10 minutes and washed away with PBS. The cells were incubated in diluted Chromogen A+B (1:25) for 10 minutes and, after washing, counterstained with hematoxylin for 5 minutes. The staining was observed under an inverted phase-contrast microscope.

Seeding of ASCs on PCS:

Cells at passage three were detached by incubation in TrypLE Express for 10 minutes and centrifugation at 805g for 5 minutes. A cell suspension with a seeding density of 2.5x10⁴ cells was seeded onto PCS within the wells of a 96-well plate and incubated at 4°C for 30 minutes. Next, 50 µL of media was added to each well. The constructs were incubated in low-glucose DMEM, GlutaMAX-I and 10% MSC-qualified fetal bovine serum at 37°C and 5% CO₂ incubator. The growth media were changed daily.

Viability of ASCs, assessed using LIVE/DEAD® Viability/Cytotoxicity Kit (Molecular Probes):

The constructs were washed in DPBS for 5 minutes. The constructs were then incubated in 100 µL of LIVE/DEAD reagent for 30 minutes at room temperature. The constructs were observed and image were captured using a confocal laser scanning microscope (CLSM) (Leica MicrosystemsHeide GmbH) with an excitation wavelength of 488 nm and an emission wavelength of 534 nm.

RESULTS:

Immunocytochemical analysis of cultured ASC:

FIG.1: SELECTED IMAGES OF IMMUNOCYTOCHEMICAL STAINING OF CULTURED ASCs POSITIVE FOR (b) CD29, (c) CD105, (d) CD73 AND (e) CD90, WHEREAS ASCs DID NOT EXPRESSED CD34 (a) AS OBSERVED UNDER A PHASE-CONTRAST MICROSCOPE. MAGNIFICATION (10x). THE POSITIVE EXPRESSION OF MARKERS IS INDICATED BY BROWN COLOR AT PROTEIN MEMBRANE OF ASCs. THE NEGATIVE EXPRESSION OF MARKERS IS INDICATED BY AN ABSENCE OF BROWN COLOR AT PROTEIN MEMBRANE OF ASCs

Viability of ASCs

Table 1: Selected CLSM images of ASCs at 24 and 72 hours post-seeding (magnification: 10x). The images were taken at four different parts of a PCS. The green dots represent live cells, whereas the red dots are dead cells as shown by white and blue arrows respectively. Numerous live ASCs can be observed lining the interconnected pores of the PCS (yellow color) compared with dead ASCs.

TABLE 1: SELECTED CLSM IMAGES OF ASCS AT 24 AND 72 HOURS POST-SEEDING (MAGNIFICATION: 10X)

DISCUSSION: Tissue engineering involves the combination of cells with a scaffold to enhance the basic functions of the cells, such as attachment, viability, proliferation, migration and differentiation ⁸. Human ASCs have been shown to exhibit great proliferative potential and the ability to differentiate into different cell lineages ⁹. Although these ASCs have various preclinical and clinical applications, their use in wound repair and regeneration is more promising¹⁰. Chitosan, a partially deacetylated form of chitin composed of glucosamine and N-acetylglucosamine, has been tested for its effects on the growth of various types of cells, such as bone-marrow-derived stem cells ¹¹, embryonic stem cells ¹², and fibroblasts and keratinocytes ^{13, 14}. Chitosan was chosen due to its interesting biological properties, such as wound healing acceleration ¹⁵, drug delivery potential ¹⁶ and antibacterial activities ¹⁷. In addition, chitosan has been proven to be nontoxic, biodegradable into a harmless product and biocompatible¹⁸ . The glycosaminoglycans present in chitosan form a structure analogous to the extracellular matrix, to which cells can adhere while proliferating to generate a functional tissue or organ.

Immunocytochemistry is a technique in which antibodies are used to detect the presence of antigens in cells ¹⁹. The interpretation of immunocytochemistry is qualitative, usually based on observation by personnel, and this technique indicates whether a marker is expressed. In the current study, ASCs were characterized based on their expression of the MSC markers CD73, CD90, and CD105; the adhesion molecule CD29; and the hematopoietic stem cell marker CD34. ASCs were found to be positive for CD29, CD73, CD90 and CD105, as demonstrated by the precipitation of brown deposits in the cells as shown in Fig. 1(b), (c), (d), (e).

This phenomenon was due to the reaction of the horseradish peroxidase with the DAB substrate, which resulted in the precipitation of brown-to-black deposits at the antigenic sites (membrane) where antigen-antibody complexes formed. Our findings showed an absence of brown color at the membrane of cells for CD34, indicating a lack of expression this marker (Figure 1a). The findings were in accordance with previous studies, which suggested expression of CD73, CD90 and CD105 ^{20, 21, 22}, but not expression of CD34 ²⁰.

Recent studies have shown that ASCs seeded on a silk fibroin-chitosan scaffold enhance wound healing ²³. Studies have been performed on ASCs seeded on chitosan particle-agglomerated scaffolds for cartilage and osteochondral tissue engineering ²⁴. ASCs derived from rats and seeded on chitosan film differentiated into osteogenic cell lineages ¹¹. However, studies based on human ASCs seeded on a PCS have not been well established. Therefore, in the present study, ASCs were seeded on a PCS, and the viability of the ASCs was tested. This viability analysis enhances understanding of the growth and proliferation of ASCs.

The viability of cells and their proliferation are essential to produce a functional tissue. The viability of cells within a scaffold reflects the cells’ enzymatic activity. The calcein-AM in live/dead assay kits enters live cells, which have intact membranes and converting the calcein-AM into green fluorescence. The ethidium homodimer binds to DNA following the membrane rupture of dead cells, thus resulting in red fluorescence. This in vitro test was performed to evaluate the viability of ASCs on a PCS 24 and 72 hours post-seeding.

Table 1 shows images of the green fluorescence and red fluorescence as dots on the chitosan. Post-seeding, after 24 and 72 hours, rounded green dots indicated that the live cells were scattered, lining the interconnected pores of the chitosan. The number of live ASCs within the chitosan was higher compared with the number of dead cells.

This study proved that ASCs are able to grow on a PCS for 72 hours. The cells either can be differentiated into desired cell lineages or can deliver growth factors upon transplantation into damaged tissue. Therefore, this finding is relevant to engineering ASCs on PCS.

CONCLUSIONS: This qualitative study has proven ASCs were able to survive on PCS as indicated by the number of live cells exceeded the number of dead cells as demonstrated by live/dead assay. The high potential of viable ASC growth on PCS could be applied for tissue engineering purposes. This study only highlighted the viability of human ASCs seeded on a PCS. Further investigations on the proliferation rate and differentiation of ASCs into desired lineages on PCS should be explored.

COMPETING INTERESTS: The authors declare that they have no competing of interest.

ACKNOWLEDGEMENTS: The work related to this paper was supported by Research University Grant (1001/PPSP/813058) Universiti Sains Malaysia.

REFERENCES:

1. Ojeh NO, Navsaria HA: An in vitro skin model to study the effect of mesenchymal stem cells in wound healing and epidermal regeneration. J Biomed Mater Res A 2014; 102:2785-2792.
2. Qian D, Gong J, He Z, Hua J, Lin S, Xu C, et al.: Bone Marrow-Derived Mesenchymal Stem Cells Repair Necrotic Pancreatic Tissue and Promote Angiogenesis by Secreting Cellular Growth Factors Involved in the SDF-1 alpha /CXCR4 Axis in Rats. Stem Cells Int 2015; 2015:306836.
3. Sun J, Wei ZZ, Gu X, Zhang JY, Zhang Y, Li J, et al.: Intranasal delivery of hypoxia-preconditioned bone marrow-derived mesenchymal stem cells enhanced regenerative effects after intracerebral hemorrhagic stroke in mice. Experimental Neurology 2015.
4. Peroni D, Scambi I, Pasini A, Lisi V, Bifari F, Krampera M, et al.: Stem molecular signature of adipose-derived stromal cells. Experimental Cell Research 2008; 314:603-615.
5. Batra A, Pietsch J, Fedke I, Glauben R, Okur B, Stroh T, et al.: Leptin-dependent toll-like receptor expression and responsiveness in preadipocytes and adipocytes. American Journal of Pathology 2007; 170:1931-1941.
6. Mohd Hilmi AB, Halim AS, Jaafar H, Asiah AB, Hassan A: Chitosan dermal substitute and chitosan skin substitute contribute to accelerated full-thickness wound healing in irradiated rats. BioMed Research International 2013; 2013:795458.
7. Zainol I, Halim A, Ghani S, Deraman M, Saliman M, Mohamed M, et al.: Preparation and characterisation of bilayer chitosan membrane as skin regenerating template. Malaysian Journal of Microscopy 2008; 4:24-29.
8. Mieszawska AJ, Kaplan DL: Smart biomaterials - regulating cell behavior through signaling molecules. BMC Biology 2010; 8:1-3..
9. Fraser JK, Zhu M, Wulur I, Alfonso Z:Mesenchymal Stem Cells.Springer,2008: 59-67.
10. Kim WS, Park BS, Sung JH, Yang JM, Park SB, Kwak SJ, et al.: Wound healing effect of adipose-derived stem cells: a critical role of secretory factors on human dermal fibroblasts. Journal of Dermatological Science 2007; 48:15-24.
11. Ratanavaraporn J, Kanokpanont S, Tabata Y, Damrongsakkul S: Growth and osteogenic differentiation of adipose-derived and bone marrow-derived stem cells on chitosan and chitooligosaccharide films. Carbohydrate Polymers 2009; 78:873-878.
12. Lin HR, Chen KS, Chen SC, Lee CH, Chiou SH, Chang TL, et al.: Attachment of stem cells on porous chitosan scaffold crosslinked by Na5P3O10. Materials Science and Engineering C 2007; 27:280-284.
13. Neamnark A, Sanchavanakit N, Pavasant P, Rujiravanit R, Supaphol P: In vitro biocompatibility of electrospun hexanoyl chitosan fibrous scaffolds towards human keratinocytes and fibroblasts. European Polymer Journal 2008; 44:2060-2067.
14. Lim CK, Yaacob NS, Ismail Z, Halim AS: In vitro biocompatibility of chitosan porous skin regenerating templates (PSRTs) using primary human skin keratinocytes. Toxicology in Vitro 2010; 24:721–727.
15. Caetano GF, Frade MA, Andrade TA, Leite MN, Bueno CZ, Moraes AM, et al.: Chitosan-alginate membranes accelerate wound healing. J Biomed Mater Res B ApplBiomater 2014;.
16. Tajmir-Riahi HA, Nafisi S, Sanyakamdhorn S, Agudelo D, Chanphai P: Applications of chitosan nanoparticles in drug delivery. Methods in Molecular Biology 2014; 1141:165-184.
17. Li Z, Yang F, Yang R: Synthesis and characterization of chitosan derivatives with dual-antibacterial functional groups. International Journal of Biological Macromolecules 2015; 75:378-387.
18. Dai T, Tanaka M, Huang YY, Hamblin MR: Chitosan preparations for wounds and burns: antimicrobial and wound-healing effects. Expert Review of Anti-Infective Therapy 2011; 9:857-879.
19. Burry RW: Controls for immunocytochemistry: an update. Journal of Histochemistry and Cytochemistry 2011; 59:6-12.
20. B Blasi A, Martino C, Balducci L, Saldarelli M, Soleti A, Navone SE, et al.: Dermal fibroblasts display similar phenotypic and differentiation capacity to fat-derived mesenchymal stem cells, but differ in anti-inflammatory and angiogenic potential. Vascular Cell 2011; 3:1-14.
21. Yang XF, He X, He J, Zhang LH, Su XJ, Dong ZY, et al.: High efficient isolation and systematic identification of human adipose-derived mesenchymal stem cells. Journal of Biomedical Science 2011; 18:2-9.
22. Pachón-Peña G, Yu G, Tucker A, Wu X, Vendrell J, Bunnell BA, et al.: Stromal stem cells from adipose tissue and bone marrow of age-matched female donors display distinct immunophenotypic profiles. Journal of Cellular Physiology 2011; 226:843-851.
23. Altman AM, Yan Y, Matthias N, Bai X, Rios C, Mathur AB, et al.: IFATS collection: Human adipose-derived stem cells seeded on a silk fibroin-chitosan scaffold enhance wound repair in a murine soft tissue injury model. Stem Cells 2009; 27:250-258.
24. PP BM, Pedro AJ, Peterbauer A, Gabriel C, Redl H, and Reis RL: Chitosan particles agglomerated scaffolds for cartilage and osteochondral tissue engineering approaches with adipose tissue derived stem cells. Journal of Materials Science: Materials in Medicine 2005; 16:1077-1085.
    

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    How to cite this article:

    Mohamed M, Gomathysankar S, Mat Saad AZ and Halim AS: Viability of Adipose-Derived Stem Cells (ASCs) on Porous Chitosan Scaffold. Int J Pharm Sci Res 2015; 6(9): 3781-87.doi: 10.13040/IJPSR.0975-8232.6(9).3781-87.

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