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Evaluation and Assessment of Differentiation Potential of Human Adipose-derived Stem Cells on Chitosan Hydrogel.

Introduction

Biomaterial provides scaffold and mimic extracellular matrix (ECM). It affords great potential in regenerating large tissue injuries and focal defects. Hence, it is imperative to understand the local niche of the tissue injury to be able to repair them with biocompatible tissue constructs. Langer and Vacanti have paved the way to utilize these cell laden biocompatible materials in regenerating the tissues which mimic natural organs [1]. Since then, modulation of matrix properties for tissue engineering applications has been of great interest. The study therefore addresses the chitosan based hydrogel, optimized for its role in modulating the site of cellular niche.

Stem cell-based therapies to treat soft tissue defects due to trauma, tumor resection, aging, and congenital abnormalities significantly depend on the availability of organs. Paucity of the available organs has been the driving force to develop alternate strategies that utilize the technological advances made in the areas of tissue engineering. Adipose tissue source is a major source of attention which has least ethical implications and increased donation. Current research is focused on utilizing adipose tissue to overcome the limitations, and combine the functionalized biomaterial for the intended purpose. Further, adipose tissue has demonstrated trilineage differentiation potential to osteocytes, chondrocytes and adipocytes representing an ideal source for autologous cells [2]. Adipose tissue obtained from omentum, is comparable to bone marrow and sub cutaneous fat in terms of proliferation and differentiation potential. hADSCs make upto 7% of the cells in a lipoaspirate.

For tissue engineering applications, biomaterials often serve as scaffold for a specific cell type. Furthermore, the biomaterial must integrate mechanically and physiologically with the repair tissue, or should be degraded without leaving gaps or fissures in the tissue that encapsulates the stem cells [3].

Synthetic materials that are commonly used for tissue engineering applications include poly- lactic acid, poly-glycolic acid, or a combination of the two. These are fibrous, non-toxic, and biodegradable molecules, and can be easily manipulated without encouraging cell adhesion. In the present study, chitosan hydrogel, an amino polysaccharide copolymer of 1, 4-D- glucosamines and N-acetyl glucosamines derived from chitin by alkaline or enzymatic deacetylation, was characterized. A chitosan hydrogel scaffold is hydrated and has been shown to provide a supporting matrix for human adipose-derived stem cells (hADSCs) [4]. In contrast, unmodified chitosan can only be dissolved in acidic solutions due to its strong intermolecular hydrogen bonds, and this limits its applications. The crosslinking reaction is mainly influenced by the size and type of crosslinker agent and the functional groups of chitosan. The smaller the molecular size of the cross linker, the faster the cross linking reaction. This enables easy diffusion. Depending on the nature of the cross-linker interactions, a network of covalent or ionic bonds is formed. Glutaraldehyde is an effective bi-functional crosslinking agent that is water soluble, highly efficient, and economical. Glutaraldehyde can be optimized to subliminal toxicity and can serve as an effective cross linker to natural polymers such as chitosan-gelatin [5]. The current study is focused on chitosan derivatives gelled via glutaraldehyde crosslinking.

Herein, we hypothesize that an alternate tissue source such as adipose tissue would provide an ideal cell source for tissue engineering applications. In the present study, a preliminary characterization of glutaraldehyde-crosslinked chitosan hydrogel is presented with an objective to evaluate its use in adipose derived stem cell culture models in vitro, and its potential differentiation capabilities of the ectoderm, mesoderm and endoderm lineages.

Results

Characterization by infrared spectroscopy and SEM

The chemical structure of inter penetrating network 3 (IPN3) was investigated using Fourier transform infrared spectroscopy (FTIR). Briefly, a pellet of IPN3 was prepared by adding dried hydrogel powder to solid potassium bromide (KBr) and grinding and pressing the mixture into discs. An analysis was then performed using a Nicolet 170SX FTIR spectrometer (Madison, WI, USA) in the range of 4,000-500 [cm.sup.-1] (Fig. 1a).

SEM micrographs illustrated that the surface morphology of hydrogel was interconnected and rough. Whereas, chitosan based hydrogel with glutaraldehyde cross linking confirmed pores of 110im in size with a smooth surface. (Fig.lb).

Isolation and characterization of mesenchymal stem cells from adipose tissue

An average yield of 1 x [10.sup.6] cells/mL was obtained from 3-5 g adipose tissue. Morphologically, the hADSCs exhibited spindle shape and a fibroblast-like appearance after 14 days of culturing. A homogenous hADSCs population that resembled an MSC origin was also observed during passage #3, and these MSCs were positive for CD90 and CD73, and were negative for CD45, CD34, and HLA DR as characterised by immunophenotyping.

Distribution and biocompatibility of cells on the hydrogel hADSCs grown on 10im hydrogel sections were found to be stable throughout the entire culture period, and the cells did not undergo any contractions. It was noted the hADSCs were spindle-like shape on the hydrogel on 10th day (Fig. 2a). In SEM images, adiposederived MSCs were found to form continuous sheets of cells and to fill the pores of the hydrogel. In addition, small, round-shaped cells were found to be embedded within the pores of the chitosan hydrogel (Fig. 2b).

Annexin V-FITC assays were performed to check cytotoxicity thus assessing the biocompatibility with the hydrogel scaffold. After hADSCs were cultured on the hydrogel for 7 d, no signs of phosphatidyl serine translocation were detected compared with cells induced with 20iM [H.sub.2][O.sub.2]. These results indicate that the hydrogel tested does not induce a toxic effect on the hADSCs that were cultured with it (Fig. 3).

Differentiation potential of hADSCs

Differentiation of fibroblastic cells obtained from adipose tissue could be directed towards adipogenic, chondrogenic and osteogenic lineages using appropriate differentiation media and supplements. For these studies, un-induced fibroblasts served as negative controls. For the chondrogenic cultures, spherical micro-mass cultures were observed, and deposits of acid mucopolysaccharides were confirmed with Alcian blue staining as shown in Fig. 4a. The cells in each group were separated by extensive regions of a diffuse extracellular matrix that had high collagen content. Similarly, under osteogenic induction conditions, a dark ECM material was detected after the induction period. Deposition of a calcified matrix was confirmed with Von Kossa staining (Fig. 4b). Adherent hADSCs which underwent adipogenic differentiation were characterized by an accumulation of cytoplasmic triglycerides that were represented as lipid droplets by Oil Red O staining (Fig. 4c).

Discussion

Successful tissue engineering requires three key factors: (a) a unique niche with a defined medium, (b) a biocompatible scaffold with favorable structural features for cell adherence, and (c) the ability to aid in cell metabolic activities [23]. The FITR results of this study is consistent with many previous studies [24], which have demonstrated that there was a difference in FITR profile between chitosan and chitosan cross linked with glutaraldehyde. Manynew peaks appeared at 3206, 1647, and 1562 cm-1 and these may be due to amide NH stretchingafter cross linking. However, Oyrton et al., [6] revealed that the increase of glutaraldehyde in chitosan causes an increase of ethylene bond frequency at 1562 cm"1. The SEM results indicated a difference in the surface morphology between chitosan and cross linked hydrogels. SEM images in the present study revealed smooth and porous morphology of glutaraldehyde cross linked hydrogel, which is consistent with the result of previous studies. Covalently crosslinked hydrogels present the crosslinking degree as the main parameter influencing important properties such as mechanical strength, swelling and drug release. Such gels generally exhibit pH-sensitive swelling and drug release by diffusion through their porous structure. Therefore, hydrogels based on covalently and ionically cross linked chitosan can be considered as good candidates for the cell delivery. The structural organization of chitosan also serves as a matrix for retaining the cells at a specific site and initiating appropriate cell-to-cell interactions.

A chitosan-based hydrogel was created and characterized to explore various strategies for its effective use. Chitosan acts a substrate for cell attachment, proliferation by mimicking the glycosaminoglycan of the extracellular matrix. In particular, the use of different crosslinkers can obtain distinct architectures. Variable pH levels were also evaluated in order to obtain a stable cell/gel formulation. The goal was to provide hydration of the scaffold, yet provide a favorable niche for cells to easily adhere, survive, and proliferate into the desired cell lineage based on the growth factors provided.

Human adipose-derived MSCs share an identical phenotype with bone marrow-derived MSCs and represent a feasible, minimally invasive, and alternate tissue source for exploring the potential lineage specific differentiation of these cells into cartilage or bone. In the present study, hADSCs exhibited a similar phenotype to bone marrow cells and were also positive for MSC markers (i.e., CD90, CD73), which is consistent with previously published results. For example, in a direct comparison by Zuk et al., adipose- and marrow-derived MSCs were found to share more than 90% similarity in cell surface expression markers.

Traditional chondrogenic inducers such as TGF-a, insulin, and dexamethasone have been reported to support chondrogenic differentiation. In contrast, isobutyl xanthine and beta glycerophosphate in the presence of BMP-2 have been found to promote adipogenic and osteogenic differentiation, respectively. In the present study, hADSCs were able to differentiate into specific cell lineages while being grown on a hydrogel matrix. Similarly, a recent study demonstrated that a hydrogel could induce mesenchymal cells to adopt a long striated, or spindle-shaped, morphology. Formation of an adherent mesh of cells on a hydrogel also substantiates the suitability of this matrix to accommodate hADSCs as demonstrated by Song et al. [7].

However, hADSCs undergo replicate senescence as indicated by the altered morphology that was observed for passage 6 cells and beyond, when they were maintained on the hydrogel matrix. Initial cell seeding density was found to be an important parameter for controlling proliferation within the matrix under normal metabolic conditions and growth kinetics as compared with monolayer cell culturing.

A hydrogel matrix has been shown to successfully support cell viability as well as nutrient and protein transport. Annexin V FITC staining further demonstrated that cells were viable throughout the entire thickness of the matrix. Therefore, the growth and metabolic activity of hADSCs appears to be influenced by the cross-linking of chitosan, which provides appropriate and favorable cell-cell contacts within the gel and facilitates the efficient transport of oxygen and nutrients. Our results are consistent with Cheburu et al. concluding that chitosan hydrogels have no inhibitory effect on cell growth [8]. The latter may be attributed to the microsphere surfaces that exist in a hydrogel, based on the results of a recent study that demonstrated micro-cavities on scaffold surfaces improving the cell adhesion and eliciting differential cellular responses compared with smooth surfaces. Compared with other studies [9, 10] the glutaraldehyde cross linked chitosan hydrogel evaluated in the present study was found to provide a good substrate for cell attachment and controlled proliferation. Furthermore, over time, 90% of the hADSCs have differentiated into chondrocytes and osteocytes, as evidenced by the presence of acid mucopolysaccharides and calcium deposits, respectively. The finding suggests that the crosslinking affects the cell-matrix interactions and plays a critical role in supporting differentiation. Thus, this present study demonstrates chitosan hydrogel as a scaffold for hADSCs, which is biocompatible and has the propensity to incorporate the chemical cues for differentiation to three distinct lineages. It has further exhibited an innate hydrated structure. Due to these improved functionalities, it can be further tested as a macro-carrier for various cell types in the treatment of large tissue defects.

References

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[3.] Takeshi I, Kahori I, Kouhei Y, Hidetaka I, Yoshizawa Y, K Yanagiguchi K, et al. Fabrication and characteristics of chitosan sponge as a tissue engineering scaffold. Bio Med Res Int. 2014; Article ID 786892:

[4.] Gunatillake PA, Adhikari R. Biodegradable synthetic polymers for tissue engineering. Eur Cells and Mat. 2003; 5: 1-16.

[5.] Distantina S, Rochmadi R, Fahrurrozi M, Wiratni W. Preparation and characterization of glutaraldehyde-crosslinked kappa carrageenan hydrogel. Eng J. 2013; 17:57-66.

[6.] Oyrton AC, Monteiro J, Claudio A. Some studies of crosslinking chitosan-glutaraldehyde interaction in a homogeneous system. Int. J. Biol. Macromol. 1999; 26:119-128.

[7.] Song K, Qiao M, Liu T, Jiang B, Macedo HM, Ma X, et al. Preparation, fabrication and biocompatibility of novel injectable temperature-sensitive chitosan/glycerophospahte/ collagen hydrogels. J Mater Sci Mater Med. 2010; 21:2835-42.

[8.] Cheburu CN, Stoica B, Neampu A, Vasile C. Biocompatibility testing of chitosan hydrogels. Rev Med Chir Soc Med Nat Iasi. 2011; 115:864-70.

[9.] Yun MK, Bit NL, Jae HK, Gyeong HK, Kkot NK, Kim DY, et al. In vivo biocompatibility study of electrospun chitosan microfiber for tissue engineering. Int J Mol Sci. 2010; 11: 4140-4148.

[10.] 43. Kim KS, Lee JY, Kang YM, Kim ES, Lee B, Chun HJ, et al. Electrostatic cross linked in situ-forming in vivo scaffold for rat bone marrow mesenchymal stem cells. Tissue Eng. Part A. 2009; 15: 3201-9.

Tanya Debnath (1), Lakshmi Kiran Chelluri (2), Lakshmi K.Kona (3), K.S. Ratnakar (1)

(1) Global Medical Education and Research Foundation (GMERF), Lakdikapool, Hyderabad, India

(2) Transplant Biology & Stem Cell Unit, Gleneagles Global Hospitals, Lakdikapool, Hyderabad, India

(3) Department of Bariatric Surgery, Gleneagles Global Hospitals, Lakdikapool, Hyderabad, India

Caption: Figure 1: FTIR spectra and SEM micrograph of IPN3

Caption: Figure 2: Biocompatibility of cells on the hydrogel

Caption: Figure 3: Annexin V-FITC assay for cytotoxicity

Caption: Figure 4: Differentiation potential of hADSCs on hydrogel
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Title Annotation:Original Article
Author:Debnath, Tanya; Chelluri, Lakshmi Kiran; K. Kona, Lakshmi; Ratnakar, K.S.
Publication:Trends in Biomaterials and Artificial Organs
Date:Jan 1, 2018
Words:2231
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