Abstract
BACKGROUND:Stem cell differentiation potential is strongly correlated with culture condition. The alteration in scaffold material surface function, three dimensional (3D) structure, and addition of growth factors can control stem cell proliferation and differentiation.
OBJECTIVE: To develop 3D macroporous scaffolds with optimal porosity and porous structure to provide a microenvironment that promotes the growth of multi-potent stem cells.
DESIGN: Repetitive measurement.
SETTING: Department of Anatomy, College of Basic Medicine, Guangzhou University of Chinese Medicine.
MATERIALS: Healthy adult SD rats were provided by the Experimental Animal Center in Guangzhou University of Chinese Medicine. Chitosan and basic fibroblast growth factor (bFGF) were purchased from Sigma Corporation (St. Louis, MO).
METHODS: The experiment was performed at the Department of Anatomy, College of Basic Medicine, Guangzhou University of Chinese Medicine from March 2003 to December 2006. Using a freeze-drying method, 3D macroporous scaffolds made of different ratios of chitosan-gelatin with bFGF were fabricated that could release bFGF with controlled porosity and porous structure. Bone marrow was obtained from the femur and tibia of SD rats, and mesenchymal stem cells (MSCs) were isolated, cultured and seeded on the scaffolds with bFGF. MSCs seeded on scaffolds with no bFGF served as control. The procedure during experiment was accorded with animal ethical requirements.
MAIN OUTCOME MEASURES: 3D structure and release performance of the scaffolds were observed by ELISA and scanning electron microscope; the effect of 3D macroporous scaffolds that released bFGF on MSC growth and viability were observed by HE staining, MTT, cell counting and SEM.
RESULTS: There was no significant difference in pore size between scaffolds with and without bFGF (P > 0.05). Scaffolds with bFGF significantly improved MSC survival rate, promoted cell adhesion, proliferation, and viability compared with scaffolds without bFGF (P < 0.05).
CONCLUSION: The results suggest that 3D macroporous scaffolds with bFGF release improve MSC survival on scaffolds, and lay a foundation for its application in tissue engineering.




INTRODUCTION

Mesenchymal stem cells (MSCs) in adult bone marrow are capable of self-renewal and differentiation into all mesodermal cell types and neuro-ectodermal cells, such as osteoblast, chondrocyte, myoblast, stromal cell, adipocytes, neuron, astrocytes and so on[1-5]. These abilities make MSC an excellent seed cell of cell transplantation, tissue engineering and gene therapy. Tissue engineering is a promising strategy for the treatment of neuro-traumatic injuries, stroke, neurodegenerative and muscle degenerative diseases. Many kinds of cells, including embryonic stem cells and tissue stem cells, have been considered as candidates for transplantation therapy. MSCs have great potential as therapeutic agents since they are easy to be isolated and can be expanded from patients without serious ethical or technical problems.
However, the response of MSCs to the culture environment and the mechanisms of their proliferation and differentiation are still not clear. It is very important to control or regulate the behaviors of MSCs during cell culturing in order to induce them to perform desired functions or to differentiate into specific cell lineage. Tissue development requires proper control of cell proliferation and differentiation in three dimensions (3D). In tissue engineering, this is often achieved by variable biomaterial properties and culture conditions. The examples include material surface function [6], addition of growth factors, alternation of growth conditions [7], and delivery of tissue-inductive proteins from polymer matrices[8].
Chitosan is a naturally derived polysaccharide. It has gained much attention as a biomaterial in diverse tissue engineering applications due to its low cost, large-scale availability, anti-micro-bioactivity, and biocompatibility[9]. Chitosan scaffolds with various geometries, pore sizes, and pore orientation can be obtained by using controlled-rate freezing[10]. Chitosan marginally supports biological activity of diverse cell types[11]. To improve the mechanical or biological properties of chitosan over a broad range, its blending with other polymers is widely investigated. Gelatin is blended with chitosan to improve the biological activity since gelatin contains Arg-Gly-Asp (RGD)-like sequence that promotes cell adhesion and migration, and then a polyelectrolyte complex is formed. Gelatin-chitosan scaffolds have been formed without or with cross-linkers such as glutaraldehyde[12] or enzymes [13] and been tested in
regenerating various tissues including skin[14], cartilage [15], and bones[16]. But the amount of MSCs in chitosan-gelatin scaffolds is small and the ability of proliferation is weak; this limited their clinical application. Therefore, in order to meet the demand of further clinical therapy usage, MSC system needs to be highly standardized and proliferated. Current researches in this field focus particularly on the role of growth factors towards MSC proliferation. Furthermore, fibroblast growth factor (FGF) is the most effective in promoting growth of these cells in vitro[17].
It is known that MSC proliferation in vitro is greatly influenced by basic fibroblast growth factor (bFGF), while these studies show the beneficial effects of bFGF on proliferation of MSCs in 2D culture, little is known on the role of bFGF as component of bio-mimetic microenvironment on MSC adhesion and subsequent proliferation. Therefore, in this study, to develop three-dimensional (3D) macroporous scaffolds with optimal porosity and porous structure to provide a microenvironment that promotes the growth of multi-potent stem cells, both 3D porous chitosan-gelatin scaffolds with or without bFGF were fabricated, and then MSCs were seeded onto both scaffolds and their growth condition was investigated.

MATERIALS AND METHODS

Materials
The experiment was performed at the Department of Anatomy, Guangzhou University of Chinese Medicine from March 2003 to December 2006. Healthy 4-week-old SD rats, half male and half female, were supplied by Experimental Animals Center in Guangzhou University of Chinese Medicine [certificated number: SYXK(yue)2003-0001]. Chitosan, bFGF, N, N-(3-dimethylaminopropyl)- N'-ethyl-carbodiimide (EDC), N-hydroxysuccinimide (NHS) and 2-morpholinoethane sulphonic acid (MES) were purchased from Sigma Corporation (St. Louis, MO); DMEM and Penicillin/Streptomycin from Life Technologies (Rockville, MD); Fetal bovine serum (FBS) from Invitrogen Corporation (Gibco, USA).

Methods
Scaffold fabrication and bFGF release
Scaffolds were prepared by solid-liquid phase separation and subsequent sublimation of solvent. Pulverized chitosan 500 mg was mixed with 50 mL acetic acid (2 mol/L) followed by dissolved incubation at 40 ℃. Different concentrations of bFGF was accurately weighed and added into deionized distilled water, and then mixed with 10% (w/v) 50 mL chitosan-acetic acid solution. After stirring, the chitosan-acetic acid solution was remained overnight. Then 5 mL gelatin (1%) was added to the mixture and placed in water bath at 40 ℃ for 2 hours, and 50 mmol/L MES buffer containing 100 μL EDC (30 mmol/L) and 8 mmol/L NHS was added to crosslink after stirring. Before the hydrogel formed, the mixture was poured into several polyethylene pipes (1 cm diameter), immediately frozen with liquid nitrogen, incubated at 37 ℃ and frozen with liquid nitrogen repeatedly. Temperature and time were controlled to regulate pores and keep liquid-full state in scaffolds. The sample was finally dried under vacuum condition and prepared in foam approximately 5 mm thickness, and then it was cut into 1 cm diameter disks and stored for following usage.
Prior to cell seeding, the scaffolds were treated with 50 mL NaOH solution (10%) in 50 mL ethanol to neutralize the acetic acid followed by repeatedly washing with deionized water. The samples were then immersed into 70% ethanol for 24 hours for sterilization, and the ethanol was then removed by soaking for 72 hours with three changes of phosphate-buffered saline (PBS), and then for 3 hours with two changes of complete DMEM medium containing 10% FBS and 1% Penicillin/Streptomycin.
An in vitro release study was performed, and the concentration of bFGF released from the scaffolds was measured by ELISA. The scaffolds containing different concentrations of bFGF were plated in DMEM and centrifuged at 10 000×g for 20 minutes at 4 ℃, and then the supernatant was collected. ELISA for bFGF in the supernatant was performed by using a commercially available kit specific for rat bFGF according to the manufacture's instructions (Shanghai Shenergy Biocolor BioScience and Technology Co, Ltd, China).

Culture of MSCs [18]
Bone marrow was obtained from the femur and tibia of the rats (all experimental procedures were approved by the Care of Experimental Animals Committee of Guangzhou University of Chinese Medicine). The marrow samples were diluted with DMEM containing 10% FBS. MSCs were prepared by gradient centrifugation at 900×g for 30 minutes on Percoll at a density of 1.073 g/mL. The cells were washed, counted and plated at 1×106 cm-2 on Petri dishes in DMEM-LG supplemented with 10% FBS. Medium was replaced and the unattached cells were removed every 3 days. MSCs formed as confluent layers were detached by treatment with 0.25% trypsin and were passaged into cultural flasks at 1×104 cm-2.

Morphological analysis
Morphology was monitored using an inverted microscope outfitted with a CCD camera. Digital
micrographs were captured from different locations to observe cell spreading area, pore sizes, and shape factors.

HE staining
MSCs cultured on scaffolds were fixed and then stained with hematoxylin/eosin.

Scanning electron microscope (SEM) analysis
The morphology of the scaffolds and MSCs were analyzed by using SEM (S-4500, Hitachi, Japan) at an accelerating voltage of 15 kV. Samples were dried by using a series of increasing concentrations of ethanol followed by a brief vacuum drying. They were coated with gold at 40 mA prior to observation under SEM.

Cell proliferation by MTT assay
Analysis of MTT was made on the third passage cultures of MSCs. MSC suspensions (at a cell concentration of 1× 108 L-1) were seeded scaffolds at 500 cells per well in 96-well plates and incubated in 5% CO2, at 37 ℃ for 24 hours, then the culture medium was replaced. The wells were washed with PBS twice. MSCs were divided into different groups for a dose-response experiment in the presence or absence of bFGF scaffolds, respectively. They were incubated for 72 hours. Each sample was repeated in five independent wells. After incubation, 20 μL MTT (5 g/L) was added and incubated for another 4 hours. The culture medium was discarded and replaced with 150 μL DMSO. The absorbance at 490 nm was measured by Bio-kinetics reader (PE-1420, USA).

Effect of scaffolds on numbers of MSCs
MSC suspensions (at a cell concentration of 1×108 L-1) were seeded on scaffolds at 1.2×104 cells per well in 24-well plates, and then the culture medium was added (1 mL per well). The wells of scaffolds without bFGF served as control group, and those of scaffolds with bFGF at 1μg/mL as experimental groups. At each indicated time points after culture (1, 3, and 7 days), cells were trypsinized from scaffolds and the absolute cell numbers were determined by a cell counter.

Statistical analysis
All data were expressed by Mean±SD. One-way analysis of variance was carried out using SPSS 10.0 for windows software. Effects were considered to be significant at P value less than 0.05.

RESULTS

Micro-architecture of chitosan-gelatin without and with bFGF scaffolds
To investigate the influence of bFGF on micro-architecture of scaffolds, scaffolds were analyzed via SEM. An open pore microstructure with a high degree of interconnectivity was generated in porous scaffolds with or without bFGF by SEM (Figure 1). Images were used to determine the pore structure of the scaffolds. The mean pore sizes of both scaffolds were (105±18) and (102±16) μm respectively, with no statistically significant differences. These results showed no significant changes in the porous structure in chitosan scaffolds with bFGF or without bFGF.

Release of bFGF from porous scaffolds
We quantified the amount of bFGF released from 3D scaffolds in the culture medium, and fabricated porous scaffolds that contained a different dose of bFGF. The fabricated scaffolds released bFGF on day 7 (Figure 2). The release of bFGF followed almost in dose dependent way, which was an ideal release pattern for maintaining a constant concentration of a drug.

Morphological analysis
Morphological differences were rather evident in the third passage of MSCs between scaffolds with bFGF or without bFGF under the inverted microscope. The original fibroblast-like phenotype of MSCs was gradually lost in the scaffolds without bFGF and changed into a more rounded cell phenotype; whereas, it maintained well when bFGF was present in the scaffolds. HE staining clearly showed that to scaffolds without bFGF, the round or round-flat-like cellular body in MSC was big; nucleolus was predominant and the ratio of nucleus to cytoplasm was high. Generally, MSCs exhibited characteristics of primitive un-differentiation. Meanwhile, cells on scaffolds with bFGF appeared as fibroblast-like phenotype of MSCs. It suggested that bFGF can promote the proliferation of MSC on scaffolds.

SEM analysis
Cell adhesion on scaffold surface
Morphological differences were significant in MSC cultures between scaffolds with bFGF and scaffolds without bFGF (Figures 4 a-f) under the SEM. The original fibroblast-like phenotype of MSCs was gradually lost in the scaffolds without bFGF and changed into a more rounded cell phenotype with increasing culture time (Figures 4 a-c); whereas, it maintained well exhibiting a highly flattened shape with an elongated cell body and intercellular connection when bFGF was present in the scaffolds (Figures 4 d-f).

Cell growth in scaffold pores
SEM micrographs showed distinct differences in growth between MSCs in the scaffold pores with bFGF and without bFGF. Some cells grew around pores in the scaffolds without bFGF (Figure 5a), while some cells grew in bridges between adjacent pores in the scaffolds without bFGF (Figure 5b); however, semi-spread cells and rounded cells were still observed, particularly around a pore in the scaffolds without bFGF, on which the majority of cells exhibited rounded shape (Figures 5 c and d). In contrast, fibroblast-like phenotype of MSCs still appeared, particularly cell aggregates in the pore of scaffolds with bFGF. Long spindle cells were observed at (Figure 5 e) low or (Figure 5 f) high magnification, and a large cell aggregate was also found in the pore of the scaffolds with bFGF. Long spindle cells formed intercellular connections, and then multi-shape cell body and long spindle cell process were measured (Figures 5 g and h). The micrographs depicted typical cell-material interactions on both scaffolds with bFGF and scaffolds without bFGF and suggested that the presence of bFGF played a significant role in regulating cell adhesion and proliferation on scaffolds.

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MSCs activity on scaffolds
To understand the importance of bFGF in scaffolds, MSCs were seeded onto scaffolds with or without bFGF. Cell viability results showed (Figure 6a) a significant increase in the viability on scaffolds with bFGF by day 3. Cell viability on scaffolds with bFGF concentration showed a significant increase in the absorbance indicating the presence of more cells in the scaffolds.
To understand the difference in the cell number from scaffolds with or without bFGF further, MSCs were also seeded onto scaffolds with or without bFGF. After 1 day, there was no difference in the cell number between scaffolds with or without bFGF. However, after 3 days, MSCs on scaffolds with bFGF exhibited significant increase in number of cells. After 7 days, MSCs on scaffolds with bFGF also maintained a high number of cells (Figure 6b). The increased cell adhesion and proliferation were probably due to the presence of bFGF. These results suggest that the presence of bFGF plays a significant role in regulating cell adhesion and proliferation on scaffolds.

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DISCUSSION

Current challenge in tissue engineering is functional tissue engineering involving the use of stem cells seeded with specific growth factors in biomaterial scaffolds. Ideally, the scaffold should be a functional and structural bio-mimetic of the native extra-cellular matrix and can support cell seeded growth. Chitosan is a positively charged specific polysaccharide, which stimulates cell growth and protein adsorption. It has already been reported that, besides its good biocompatibility, chitosan has an excellent nerve cell affinity. Extra-cellular molecules such as laminin, fibronectin, and other serum proteins have a good affinity to chitosan[18]. Although there are some differences in cellular adhesion and proliferation rate according to the degree of deacetylation[19], chitosan has good cellular proliferation and cell attachment properties in chondrogenic hMSCs. However, it lacks bioactivity and is mechanically weak[20-21]. For these reasons, more than two different characterized materials have to be used to achieve ideal scaffolds. The combination of biomaterials provides an alternative to integrate the best properties of each material, meanwhile it overcame many of their shortcomings when used as homogenous materials. Recently, chitosan-gelatin scaffolds have gained much attention in various tissue engineering applications[22]. But the amount of MSCs in chitosan-gelatin scaffolds is small and the ability of the proliferation is weak[23], which have limited their clinical application. In order to meet the demand for further clinical therapy, MSC system needs to be highly standardized and proliferated. Current research in this field focuses particularly on the role of growth factors towards MSC proliferation. The introduction of bFGF into the chitosan-gelatin scaffolds has also modified the scaffold biological activity. It is known that bFGF is the most effective factor in promoting growth of the cells in vitro [17]. Therefore, we hypothesized that the biological response to our composite scaffolds may be due to the strong biological activity of bFGF.
Consistent with our initial hypothesis, cell number, distinct cell density, and MTT activity were observed depending on the type of scaffolds on which the cells were grown. Striking results were obtained from scaffolds with bFGF, where the cell number and MTT activity were remarkably higher than those obtained from scaffolds without bFGF. Moreover, the scaffold with bFGF was the only material on which the cells maintained a high fibroblast-like phenotype of MSCs throughout the study. Also, SEM observation tended to indicate that the scaffolds with bFGF offered surface and pore conditions allowing the fastest cell attachment and the highest degree of cell spreading among the biomaterials used in the present study. All these observations are in agreement with composite of chitosan, gelatin and bFGF, especially those having a high bFGF content. They are also in concord with the amount of bFGF releasing from scaffolds, suggesting that the scaffolds with bFGF facilitate cell proliferation.
In order to achieve effective bFGF in scaffolds, the regulation of the amount of bFGF in scaffolds is also important. The amount of bFGF on the scaffolds showed a high correlation with the concentrations of bFGF solution in a range of 1-10 mg/L. In this study, we evaluated short-term bFGF releasing from scaffolds set into culture environments. These results were consistent with a sustained release of biologically active bFGF from the chitosan fleeces[24]. Cell viability results reported here show the effective dose of bFGF in scaffolds is between 1 and 10 mg/L; however, scaffolds with 20 μg/L bFGF which promoted MSCs activity on 2-D membranes showed viability similar to 3D scaffolds without bFGF. Our findings are consistent with the usefulness of growth factors for tissue engineering, which has been investigated by many researchers [25-28]. Tanihara et al [29] showed that 1 mg of bFGF was effective for angiogenesis in the dorsal area of rat, and Lu et al [30]showed that 1 μg/L of transforming growth factor (TGF)-β1 was effective for the proliferation and differentiation of marrow stromal cells.
Further support for the hypothesis that the presence of bFGF plays a significant role in regulating cell adhesion and proliferation on scaffolds is obtained by examining the micro-architecture of scaffolds. The spatial cell organization in a 3D environment is affected by the pore size of fibrous matrix and is an important factor in regulating long-term cellular events that are essential to tissue development and function [31]. Thus, optimization of the spatial properties of the fibrous matrix as a tissue scaffold is critical to tissue engineering. A 3D porous scaffold has been developed, using a phase separation method for bone tissue engineering [32]. The scaffold has an inter-connective structure and controllable porosity, and benefits osteoblast proliferation, bone-like tissue formation and mineralization. In this study, our porous scaffolds were fabricated by using the same method to detect the effects of bFGF on the development of 3D MSC tissue development. The pore sizes of the scaffolds with or without bFGF were carefully controlled within the same range to eliminate the structural influence of the materials, which has shown the influence on cellular activity [33]. Our results analyzed via SEM show no significant changes in the porous structure in chitosan scaffolds with bFGF or without bFGF; however, there are significant changes in scaffold biological activity. Therefore, results reported here suggest that scaffold based on composite of chitosan, gelatin and bFGF can be used as attractive scaffolds for developing new strategies for tissue engineering.
The advantage of using this strategy was obvious. If we can regulate the combined amount of growth factors on scaffolds, cell proliferation and differentiation would be controlled, that would be the first step toward the development of composite scaffolds model system for tissue engineering.
Our future work will focus on culturing MSC population committed to multiple differentiation lineage in this scaffold architecturally designed to generate a multi-phasic tissue, when provided with the appropriate inductive agents. MSCs are generally considered a highly promising cell source for tissue engineering application because of their multi-differentiation capabilities and their expandability. Structural and functional tissue engineering requires that cells be seeded within biomaterials scaffolds that permit specific cellular differentiation and expression of specific cellular phenotypes. Therefore, it is important that future experiments examine the possible role of 3D porous scaffolds in mediating differentiation of MSCs in vivo, and further animal study will be needed before this treatment can be used clinically.
In conclusion, the presence of bFGF in chitosan-gelatin composite promotes initial MSCs adhesion and supports growth in porous scaffolds. MSCs also maintain higher proliferative potentials in scaffolds. The significantly improved bioactivity of the scaffolds is attributed to the increased bFGF from scaffolds.

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壳聚糖-明胶-碱性成纤维细胞生长因子
三维大孔支架对骨髓间充质干细胞
增殖的影响**☆

黎 晖1，陈东风1，刘金元2，周健洪1，杜少辉3，李伊为1，邓汝东1，张赛霞4，曾和平5
1广州中医药大学基础医学院解剖教研室，广东省广州市 510006；2广州中医药大学电镜中心, 广东省广州市 510006；3广州中医药大学附属深圳市中医院内科，广东省深圳市 518033；4广州中医药大学基础医学院组织与胚胎学教研室, 广东省广州市 510006；5华南理工大学化学学院有机化学系，广东省广州市 510641
黎 晖☆，男，1972年生，湖北省通城县人，汉族，中山大学在读博士，副教授，主要从事中医药调控干细胞的机制与应用研究。
通讯作者：陈东风，广州中医药大学（基础医学院解剖教研室），广东省广州市 510006
国家自然科学基金资助项目（30371837*;30472272）*

摘要
背景：干细胞的分化潜能与培养条件有密切关系，改变支架材料的表面特性，三维结构，增加生长因子均可实现对干细胞增殖分化的控制。
目的：制备适合骨髓间充质干细胞附着生长的、具有最佳孔隙率和孔隙结构的药物缓释组织工程支架——三维大孔支架，提供能促进多能干细胞生长的微环境。
设计：重复测量设计。
单位：广州中医药大学基础医学院解剖教研室。
材料：实验所用健康成年SD大鼠由广州中医药大学实验动物中心提供。壳聚糖、碱性成纤维细胞生长因子购自Sigma公司。
方法：实验于2003-03/2006-12主要在广州中医药大学基础医学院解剖教研室完成。采用冷冻干燥的方法，用不同比例的壳聚糖-碱性成纤维细胞生长因子-明胶依次混匀，通过控制冷冻、复温和干燥时间处理使其具有最佳孔隙率和孔结构，制备具缓释碱性成纤维细胞生长因子功能的三维大孔支架。取SD大鼠股骨和胫骨骨髓，分离、培养骨髓间充质干细胞并移植于缓释碱性成纤维细胞生长因子的三维大孔支架上进行三维培养，与无碱性成纤维细胞生长因子的支架对照。实验过程中对动物的处置符合动物伦理学标准。
主要观察指标：用ELISA和扫描电镜观察支架的三维结构和缓释性能，用苏木精-伊红染色、MTT、细胞计数及扫描电镜方法观察缓释碱性成纤维细胞生长因子的三维大孔支架对骨髓间充质干细胞生长状态和细胞活力的影响。
结果：含有碱性成纤维细胞生长因子的三维大孔支架有碱性成纤维细胞生长因子缓释性能，孔隙尺寸与不含碱性成纤维细胞生长因子的支架三维结构相比，差异无显著性意义（P > 0.05）。含有碱性成纤维细胞生长因子的三维大孔支架能提高在支架上立体培养的骨髓间充质干细胞存活率，促进骨髓间充质干细胞黏附、增殖和活力，与不含碱性成纤维细胞生长因子的支架相比，差异有显著性意义（P < 0.05）。
结论：含有碱性成纤维细胞生长因子的三维大孔支架能缓释碱性成纤维细胞生长因子，有利于在支架上立体培养的骨髓间充质干细胞存活，为其在组织工程中的应用打下基础。
关键词：壳聚糖-明胶-碱性纤维生长因子支架；骨髓间充质干细胞；增殖；体外；干细胞组织工程
中图分类号: R318.08 文献标识码: A 文章编号: 1673-8225(2008)10-01943-07
黎晖，陈东风，刘金元，周健洪，杜少辉，李伊为，邓汝东，张赛霞，曾和平.壳聚糖-明胶-碱性成纤维细胞生长因子三维大孔支架对骨髓间充质干细胞增殖的影响[J].中国组织工程研究与临床康复，2008，12(10):1943-1949
[www.zglckf.com/zglckf/ejournal/upfiles/12-10/10k-1943(ps).pdf]
(Edited by Amir Al-Munajjed/Su LL/Wang L)