Liu Zuo-qiang1, Huang Hai2, Huang Jian2, Lin Tian-xin2, Xu Ke-wei2, Guo Zheng-hui2, Jiang Chun2, Han Jin-li2
|
1Department of
Liu Zuo-qiang★, Master, Department of
huanghai257@126.
com
Correspondence to: Huang Jian, Professor, Tutor of doctor, Department of
Supported by: the Special Program for Scientific Research of Public Welfare of Health Department, No. ＃03104*; the Tackle Key Programs in Science and Technology of Guangzhou City, No. 2005Z2-E0121*; the Clinical Key Program of Hospital of Health Department (5010 Program), No. 2007018*
Received: 2008-01-24 Accepted: 2008-02-12 (08-50-1-96/GW)
Liu ZQ, Huang H, Huang J, Lin TX, Xu KW, Guo ZH, Jiang C, Han JL.Culture and differentiation of bone marrow mesenchymal stem cells on bladder acellular matrix.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(6): 1193-1195(China)
[www.zglckf.com/zglckf/ejournal/upfiles/ 12-6/6k-1193
(ps).pdf]
|
BACKGROUND: Smooth muscle cells and transitional epithelial cells were traditionally used to construct tissue-engineered bladder and to perform double-sided implantation of scaffold. However, double-sided implantation is difficult to perform, because smooth muscle cells are difficult to isolate or culture in vitro and passage is limited.
OBJECTIVE: To verify the feasibility of tissue-engineered bladder reconstruction with bone marrow mesenchymal stem cells (BMSCs) and bladder acellular matrix (BAM).
DESIGN: A basic empirical study.
SETTING: Linbaixin Medical Research Center, Second Affiliated Hospital, Sun Yat-sen University.
MATERIALS: Experiments were performed at the Linbaixin Medical Research Center, Second Affiliated Hospital, Sun Yat-sen University from March 2006 to May 2007. The laboratory was the Opening Laboratory of Hospital Affiliated to Health Department of China. One-month old SD rats of either sex, weighting 80-100 g were provided by Animal Experimental Center of Sun Yat-sen University. Fresh porcine bladders were offered by Animal Experimental Center of Southern Medical University. The protocol was in accordance with animal ethical standard.
METHODS: Whole bone marrow culture and successive adherence method was used to culture rat BMSCs in vitro. Flow cytometry was employed to detect surface antigen. Eradicator washing method was applied to prepare porcine BAM and measure its purity and characteristics. Third passage of BMSCs were inoculated in BAM and cultured in a medium containing vascular endothelial growth factor (VEGF165) (25 ng/L) in vivo and in vitro to test compatibility. Cells cultured alone were considered to be controls for in vivo trial, and materials non-implanted with cells were considered to be controls for in vitro trial. Suitable microenvironment was simulated to induce the differentiation of BMSCs. Four weeks and eight weeks later, compound materials were respectively removed to perform tissue section test. Simultaneously, immunohistochemistry keratin staining was conducted to examine regeneration of epithelial cells.
MAIN OUTCOME MEASURE: Biocompatibility of BMSCs and BAM.
RESULTS: ①BMSCs were cultured by whole bone marrow method. Flow cytometry demonstrated that third passage of cells were positive for CD29 (99.43%). ②BAM had good biological characteristics. Homogen matrix and byssoid collagen appeared under a microscope. Compatibility trials showed good compatibility of BMSCs and BAM and well-growth cells. ③Four weeks later, histological section test confirmed inflammatory cell infiltration, closely-arranged collagen and elastic fiber. Immunohistochemistry keratin staining showed lamellar and discontinuous simple epithelium. Eight weeks later, no inflammatory cell infiltration was found, and closely-arranged collagen and elastic fiber were detected. Immunohistochemistry keratin staining showed lamellar and continuous multiple epitheliums.
CONCLUSION: With good compatibility, BMSCs and BAM appear to be an ideal material for bladder tissue engineering.
INTRODUCTION
Bladder tumor is a common tumor appeared in urinary system. How to replace the bladder is a difficult problem following cystectomy. Traditional bladder repair materials affect quality of life of patients after surgery such as injuries to the body and various complications. Presently, someone believes that tissue-engineered materials can be used to construct new bladders. Seed cells, scaffold and suitable microenvironment are key factors in bladder tissue engineering. This study aimed to in vitro culture bone marrow mesenchymal stem cells (BMSCs) as seed cells, to prepare porcine bladder acellular matrix as scaffolds, and then to perform in vivo and in vitro compound culture for detecting their compatibility, to simulate a suitable microenvironment for differentiation, and to primarily investigate the feasibility of constructing tissue-engineered bladders.
MATERIALS AND METHODS
Materials
Experiments were performed at the Linbaixin Medical Research Center, Second Affiliated Hospital, Sun Yat-sen University between March 2006 and May 2007. The laboratory was the Opening Laboratory of Hospital Affiliated to Health Department of China. One-month old SD rats of either sex, weighting 80-100 g were provided by Animal Experimental Center of Sun Yat-sen University. After general anesthesia with pentobarbital sodium, the experiment was conducted and carcass was cremated in a concentrated way. The protocol was in accordance with animal ethical standards. Fresh porcine bladders were offered by Animal Experimental Center of Southern Medical University. DMEM-LG, fetal bovine serum (FBS), 0.25% trypase (containing 0.02% EDTA), heparin sodium, phosphate buffered saline (PBS), Triton
X-100, 1% bromogeramine solution, 10% gentamicin sulfate solution, swing bed, cell counting chamber, recombinant human VEGF165, dimethyl benzene, dehydrated alcohol, Mouse-anti-Pan Cytokeratin, sp immunohistochemistry kit, DAB developer, hematoxylin, pentobarbital sodium and surgical instruments were used in this study.
Methods
Culture of rat BMSCs
In vitro isolation and culture of BMSCs: Whole bone marrow culture method[1] was used to culture rat BMSCs in vitro. One-month rats were sacrificed by cervical dislocation, and sterilized with alcohol of 0.75 volume fraction for 10 minutes. The femur and tibia were removed and osteoepiphysis was sheared off. After bone marrow was rinsed with PBS solution containing heparin sodium, DMEM-LG solution of the same volume was added. Their combination were transferred into centrifuge tube, blown and centrifuged at 1 000 r/min for 5 minutes. Supernatant and adipose layer were discarded when 5 mL of DMEM containing 10% FBS was added, misce bene. Cells at the density of 1×109L-1 were inoculated in 25 cm2 culture flask at 37 ℃ in the 5% CO2 incubator at saturated humidity. Half liquor was changed at hour 48, and non-adherent cells and erythrocytes were discarded. After that, liquor was changed every 3-4 days. Morphous and growth of cells were observed under an inverted microscope day by day. Cells were digested with 0.25% trypsin containing 0.02% EDTA when cells were near confluence (80%-90%), and then passaged at 1:2-1:3.
Determination of BMSCs: Third passage of BMSCs were collected, and made into monoplast suspension (3×109 L-1). Cell surface antigen was examined by flow cytometry (FACScan).
Preparation of porcine bladder acellular matrix (BAM)
Fresh porcine bladder was washed with purified water, and soaked with 1% bromogeramine solution for 30 minutes (step 1). The bladder was rinsed with purified water three times to wash away bromogeramine. Seromuscular layer was discarded with a dissecting scissor under direct vision to obtain 3 cm×3 cm tissue sections, which were laid in a big beaker at room temperature and treated with 0.1％ trypsin containing 0.01％EDTA for 24 hours (step 2). After washing with PBS, tissue sections were inoculated in 1% Triton X-100 solution, and then treated with water bath shaking for 120 hours at 26-28℃ (step 3). After immersing with 1% bromogeramine for 30 minutes, BAM was stored in 10% gentamicin sulfate solution at 4 ℃. BAM after random sampling was measured by histological test to detect the acellular presence. If necessary, step 3 could be repeated to determine the presence of acellular matrix [1].
Compatibility of cells and scaffolds
Disposal of seed cells: Third passage of rat BMSCs was inoculated in DMEM containing 20% FBS and vascular endothelial growth factor (VEGF165) (final concentration of 25 ng/L) in an incubator for 5 days, and digested into cell suspension.
In vitro test of compatibility of cell materials: Cells were sliced into blocks, stored in a 24-well plate, and preweted with DMEM for 48 hours. Cell slices at the density of 108 L-1 were inoculated in a 24-well plate in an incubator. Cells cultured alone were considered to be controls. Two days after culture, cells were counted. Cells in two wells from each group were digested and centrifuged every day for counting, successively for 12 days, and cell growth curve was drawn for paired comparison between two groups.
In vivo test of compatibility of cell materials: Cells were cut into blocks, prepared in a 6-well plate, and preweted with DMEM for 48 hours. Cell slices at the density of 108 L-1 were inoculated in a 6-well plate in an incubator for 14 days. A 1.0 cm incision was made on the back of four-weeks old rats. Skin and subcutaneous tissues were incised to obtain two blind pouches at both sides by blunt separation. Cell material compounds cultured for 14 days were implanted into the left side, whereas materials non-implanted with cells were implanted into the right side and considered to be controls. Four weeks and eight weeks later, histological test was conducted respectively. After general observation, samples were fixed with 10% paraformaldehyde, imbedded with paraffin, made into 5 μm slices, and stained with hematoxylin-eosin and immunohistochemistry keratin.
RESULTS
Culture of rat BMSCs
Morphology of BMSCs
After inoculation, BMSCs were round, various in size and suspended in the culture solution (Figure 1). Twenty-four hours later, a small fraction of BMSCs began to adhere to the wall, presenting round or polygon and scattered on the bottom of the culture flask. At forty-eight hours, culture solution was changed. Two to three days later, fusiform attached cells with plenty cytoplasm and big nucleus were detected. Seven to ten days later, cells rapidly proliferated; Attached cells parallelly arrayed, appeared whirlpool shape, mesh shape, radialized, and the cell boundary was unclear. Attached cells were near confluence (80%-90%) 2-3 weeks later. Passaged cells completely attached to the wall from 12-24 hours, and became spindle cells; Cells after passage rapidly grew and proliferated, near confluence (80%-90%) from 3-4 days (Figure 2).
|
Figure 1 Primary bone marrow mesenchymal stem cells （×200）
|
Identification of BMSC surface antigen
Third passage of cells were made into monoplast suspension (3×109 L-1). Flow cytometry for cell surface antigen demonstrated that 99.43% cells were positive for CD29 and 4.53% cells were positive for CD45 (Figure 3). This indicated that these cells were purified mesenchymal stem cells.
|
Figure 2 Third passage of bone marrow mesenchymal stem cells（×200）
|
|
Figure 3 Flow cytometry for third passage of cell surface antigen demonstrated that 99.43% cells were positive for CD29 and 4.53% cells were positive for CD45
|
Preparation of porcine bladder acellular matrix (BAM)
About 0.3 cm white semi-transparent smooth BAM with elasticity and tenacity were obtained. Hematoxylin-eosin staining showed homogeneous matrix, byssoid collagen fibers, and no cell residual (Figure 4).
|
Figure 4 Hematoxylin-eosin staining of pathological sections of bladder acellular matrix（×200）
|
Coculture of cells and scaffold materials
Inverted microscope
Seven days after static culture, a fraction of cells on the scaffold materials melted with the scaffold materials; Fourteen days later, cells mixed with the scaffold materials, well grew.
In vitro compatibility of cells and scaffold materials
After combined culture, growth curve of cells and scaffold materials were similar to the control group (Figure 5).
vivo compatibility of cells and scaffold materials
Four and eight weeks after implantation of cells and scaffold materials, rats were sacrificed by cervical dislocation. Subsequently, an incision was made by opening the original incision on the back. Implanted compounds were observed. At week 4, general observation demonstrated significant inflammatory reaction around materials, severe adhesion, massive bleeding. Hematoxylin-eosin staining showed inflammatory cell infiltration, closely collagen and elastic fiber arrangement. Immunohistochemical staining demonstrated lamellar discontinuous simple epithelium (Figure 6). Epithelial cells were positive for keratin antibody in three cases (positive rate of 50%) in the experimental group, and in one case (positive rate of 16.7%) in the control group. Eight weeks later, general observation confirmed attenuated inflammatory reaction, degradation of a large fraction of composites, which mixed with surrounding tissues, but the texture was slightly fragile and easy to bleed. Hematoxylin-eosin staining showed no significant inflammatory cell infiltration, closely arranged collagen and elastic fibers. Immunohistochemical staining demonstrated lamellar continuous multilayer epithelium (Figure 7). Epithelial cells were positive for keratin antibody in five cases (positive rate of 83.3%) in the experimental group, and in one case (positive rate of 16.7%) in the control group.
Adverse reaction
One rat died of overdose of anesthesia and three rats died of severe infection, and they were compensated.
Analysis of influential factors
With limited technique, this study could not simulate a optimal microenvironment for normal bladder cells. This deserves further studies.
CUSSION
Smooth muscle cells and transitional epithelial cells were traditionally used to construct tissue-engineered bladder and to perform double-sided implantation of scaffold [3-4]. However, double-sided implantation is difficult to perform, because smooth muscle cells are difficult to isolate or culture in vitro and passage is limited. In this study, BMSCs with the properties of typical stem cells have strong reproductive activity and can differentiate into epithelial cells, chondrocytes, adipocyte, neural cells and myoblasts [5-10]. BMSCs can widely isolate and operate, and have a crucial effect on the treatment of defected tissue or organ disease, degenerative disease, some hereditary disease, malignant tumor, gene therapy and tissue engineering [7-11]. In this study, we collected simple BMSCs by direct adherent screening method. Cells appeared spindle or polygon under an inverted microscope. Cell surface antigen test confirmed a purified BMSCs. Seventh to eighth passage of BMSCs does no greatly change in morphous. This confirmed that BMSCs have good reproductive potential and are an ideal source of seed cells.
BAM as a bio-scaffold was obtained by physically or chemically removing cell components containing antigen in the bladder, only by retaining collagen protein, proteoglycan and glycoprotein and some cytokines, which could promote the regeneration of epithelium of the bladder mucous membrane, smooth muscle, blood vessel and nerves [11-12]. We used white semi-transparent smooth BAM with elasticity and tenacity extracted from triton X-100. After hematoxylin-eosin staining, BAM showed homogeneous matrix, byssoid collagen fibers, and no cell residual, so BAM could be used as a scaffold material for tissue-engineered bladder.
Cells and scaffold materials should have good biocompatibility. Present biocompatibility consists of two principles. One is "biological safety"; i.e. toxic properties and destructiveness of biomaterials on human organs should be avoided. Another is "biological function"; i.e. the materials should keep and stimulate the normal function of cells and hosts under a special application [13].
This study evaluated the biocompatibility of BMSCs and BAM by coculture and heterotopic transplantation of cells and scaffold materials. In vitro culture demonstrated good adhesion and proliferation of BMSCs on the surface of materials with the time prolonged. Materials were sliced into small blocks and implanted into 24-well plate. Growth curve of implanted cells was similar to that in the control group. This study confirmed that cells cocultured with small blocks of materials had a rapid proliferative speed compared to the cells as a single agent. This indicated that BAM contained some cytokines, which could promote cell proliferation. It is reported that these cytokines could enhance vascular and neural regeneration [14]. In vivo culture showed that significant inflammatory reaction, severe adhesion and massive bleeding around the materials at week 4 by general observation. Hematoxylin-eosin staining showed inflammatory cell infiltration and scattered collocation of collagen and elastic fiber. Immunohistochemistry demonstrated lamellar discontinuous simple epithelium. At week 8, general observation showed weak inflammatory reaction, a large fraction of compound materials grew together with surrounding tissues, but the material was fragile and easy to bleed. Hematoxylin-eosin staining showed no significant inflammatory cell infiltration, but closely arranged collagen and elastic fibers. Immunohistochemical staining demonstrated lamellar continuous multilayer epithelium. With good compatibility, BMSCs and BAM appear to be an ideal material for constructing tissue-engineered bladder.
A suitable microenvironment is another crucial factor for bladder tissue engineering and the direction of BMSC differentiation. In vivo, generation and function of stem cells were associated with transcription factor of gene encoding and extracellular signal transduction [15]. However, directional differentiated mechanism of in vitro stem cells was unclear. Basic nutrition, cell density, space constitution, mechanical force and growth factor greatly affect BMSC differentiation [16]. Studies had reported that vascular endothelial growth factor (VEGF) induced BMSCs could differentiate into endothelial cells, smooth muscle cells and vascular endothelial cells [5,17]. VEGF as a ligand of protein tyrosine kinase receptor Flk-1/KDR and Flt and a secretory glycoprotein can increase the permeability of small vessels and regulate vascularization, including lysis, migration, hyperplasy of endothelial extracellular matrix and lumen formation [18-20].
In this study, cells and materials were inoculated in DMEM containing 20% fetal bovine serum (FBS) and VEGF (25 ng/L of final concentration) in an incubator with 5% CO2 saturated humidity at 37 ℃. The microenvironment is fit for the survival of cells and materials and for the differentiation into a normal bladder. Under this microenvironment, VEGF is important to induce BMSC differentiation into endothelial cells. LG-DMEM containing 20% FBS (Ferric ion, other microelements, protein, factor and so on) can cause well growth of BMSCs and differentiated endothelial cells. The incubator with 5% CO2 saturated humidity at 37 ℃ is the basis for cell growth. In addition, first to second passage of cells could be obtained two weeks after in vitro coculture of cells and materials, which were simultaneously implanted into the body. The present study suggested that cells could well grow on the materials, and then were implanted into the body two weeks later. At week eight, multilamellar epithelium was detected. This indicated that the microenvironment (DMEM containing high concentration of FBS and 25 ng/L VEGF, an incubator with 5% CO2 saturated humidity at 37 ℃) is suitable to construct a tissue-engineered bladder.
It is presumed that we could construct a tissue-engineered bladder with normal structure and function by using BAM as scaffold materials, by inoculating BMSCs on scaffold materials in vitro for a short time, by differentiating from BMSCs into endothelial cells, smooth muscle cells and vascular endothelial cells induced by a microenvironment.
