含重组碱性成纤维细胞生长因子培养基中3T3成纤维细胞在血纤维蛋白凝块上生长的可能性
作者：王一飞 戴云 刘杰生 林剑 崔蕴霞
单位：王一飞 戴云 刘杰生 林剑(暨南大学生物工程研究所，广东 广州 510632)；崔蕴霞(中山医科大学分子医学研究中心， 广东 广州 510089)
关键词：成纤维细胞；凝集；成纤维细胞生长因子，碱性
中国病理生理杂志000201
[摘 要] 目的：重组碱性成纤维细胞生长因子(rhbFGF)对培养条件下3T3成纤维细胞在血纤维蛋白凝块上生长的可能性研究。方法：采用Giemsa染色和MTT检测法，经过电镜扫描，分段对比观察，研究3T3细胞在血纤维蛋白凝块上的生长情况。 结果：rhbFGF促进细胞生长并维持细胞存活的最佳浓度是100 ng/mL，在含有100 ng/mL的低血清培养基中细胞可在血纤维蛋白凝块上生长。经48h培养以后仍有大量的细胞存活。 结论：3T3成纤维细胞可在含有rhbFGF的低血清培养基上生长并存活，rhbFGF与血纤维蛋白凝块共存可以促进创伤愈合。
, 百拇医药
[中图分类号]Q 813 [文献标识码] A
[Article ID] 1000-4718(2000)02-0097-05
Possibility of 3T3 fibroblast growth on blood fibrin clot in culture medium with recombinant human basic fibroblast growth factor
WANG Yi-fei, DAI Yun, LIU Jie-shen, LIN Jian
(Bioengineering Institute of Jinan University, Guangzhou 510632, China)
CUI Yun-xia
, 百拇医药
(The Research Center of Molecular Medicine, Sun Yet-Sen University of Medical Sciences,Guangzhou 510089, China)
[Abstract] AIM:
To investigate the possibility of 3T3 fibroblast growth on blood fibrin clot in culture medium with recombinant human basic fibroblast growth factor (rhbFGF). METHOD: Growth of the cells on blood fibrin clot was studied by phase-contrast, scanning and transmission electron microscopy and by Giemsa stain and MTT assay. RESULTS: The optimal concentration of rhbFGF for proliferation and survival of the cells was 100 ng/mL. The cells also grew on blood fibrin clot scaffold in the low-serum medium containing 100 ng/mL rhbFGF, and a greater number of the cells survived after 48 hours incubation compared to that after 24 hours. The elongated filopodia appeared to bridge the gaps among the fibroblasts after 24 hours incubation. Further incubation to 72 hours, a greater number of platycytes were found to be joined together by lamellopodia. CONCLUSION: 3T3 fibroblasts could grow and survive on blood fibrin clot in the low-serum medium containing rhbFGF, and a combination of blood fibrin clot and rhbFGF may have over proportional effects on wound healing.
, 百拇医药
[MeSH] Fibroblasts;Agglutination;Fibroblast growth factor, basic
[CLC number] Q813 [Document code] A
INTRODUCTION
Fibrin clot obtained from fresh blood is a kind of biomaterial with low immunogenicity or no antigenicity, usually acting as a physiological scaffold in wound healing[1～4]. rhbFGF is an active peptide growth factor capable of facilitating the proliferation and differentiation of the majority of cells derived from the mesoderm and neuroectoderm as well as promting processes in wound healing[5～8]. A series of investigation concerning the effect of rhbFGF on the formation of three-dimensional tissue model has been made and we here report the possibility of 3T3 fibroblasts on blood fibrin clot immersed in a low-serum medium supplemented with rhbFGF.
, 百拇医药
MATERIALS AND METHODS
Experimental animals and reagents BALB/C mice aged 8～12 weeks were used. Trypsin, penicillin, streptomycin, fetal bovine serum and Dulbecco's Modified Eagle Medium (DMEM) were purchased from Giebco Ltd., Paisley, UK; MTT [3-(4, 5-dimethylthiazal-2yl)2,5-diphenyltetrazolium bromide] was a product of Sigma Chemical Co., St. Louis, MO; rhbFGF (No. 199806) was a purified gene recombinant product of Bioengineering Institute of Jinan University.
, 百拇医药
Preparation of blood fibrin clot The eyeballs of six BALB/C mice were ablated with an eye-tweezers under a relatively aseptic condition, and the blood collected was let to stand for 30 minutes in a centrifuge tube. The serum was then removed after centrifugation by 1 500 r/min for 10 minutes. The blood fibrin clot thus obtained was washed repeatedly with double-distilled water until it became free of red color, and then twice with Hanks solution containing penicillin (50 U/mL) and streptomycin(50 μL/mL). The blood fibrin clot was subsequently cut into a number of pieces which were put separately into the centre of a series of culture flasks to be lyophilized in a cryodesiccator (HETOTR CT-60-e) for 2 hours.
, 百拇医药
Cell culture 3T3 fibroblasts were cultured in DMEM containing 10% (vol/vol) fetal bovine serum and penicillin (50 U/mL)/streptomycin ( 50 μL/mL). When the bottom of the flasks was nearly packed with confluent cells, 0.1% (wt/vol) trypsin was used to detached the fibroblasts at 37 ℃ for 3 minutes. The dissociated cells were then washed and resuspended in DMEM with 10% (vol/vol) fetal bovine serum. Concentration of the cell suspension was calculated and adjusted to 5×105/mL.
, http://www.100md.com
MTT assay[9,10] Cell suspensions (5×103/mL) containing 10%(vol/vol) fetal bovine serum in DMEM were seed onto 96-well flat-bottomed plates (100 μL/well) and incubated 24 hours ,the culture medium was removed , the cells were then washed with phosphate buffered saline three times and re-fed with 100 μL/well of 0.4% (vol/vol) fetal bovine serum in DMEM containing a series of different dilutions of rhbFGF for further incubation. Baseline control (100 μL/well fetal bovine serum in DMEM) parallel to the sample for each plate. 48 hours after the further incubation , 10 μL MTT solution (5 mg/mL) was added immediately to each well of the assay and the plates were incubated at 37 ℃ for another 4 hours. Acid-isopropanol (100 μL of 0.04 mol/L in isopropanol) was added to each well and the contents of which were mixed thoroughly to dissolve the dark blue crystals. The plates were let to stand for a few minutes at room temperature to ensure complete dissolution of the crystals . The OD vaule of each well of the 3T3 fibroblast suspensions incubated with different concentrations of rhbFGF was read out on an EL311 autoplate reader (BIO-TEK Instruments) with a test wavelength of 570nm and a reference wavelength of 630nm, so that the optimal concentration of rhbFGF for stimulation of the proliferation of 3T3 fibroblasts could be determined.
, 百拇医药
Phase contrast microscopic observation 5 mL of cell suspension (1.5×105/mL) in DMEM with 10%(vol/vol) fetal bovine serum were added separately to each of the 25 cm×25 cm culture flasks with and without blood fibrin clot specimen. Incubated 24 hours, the medium containing 10%(vol/vol) fetal bovine serum was removed out from the culture flasks and the behavior of the cells plated on the blood fibrin clots or glasses was monitored and photographed under a phase contrast microscope. Subsequently, 5 mL each of fresh DMEM containing 10% (vol/vol) fetal bovine serum were added separately into half of the flasks while another half of the flasks were added 5 mL each of fresh DMEM containing 0.4%(vol/vol) fetal bovine serum supplemented with rhbFGF in an optimal concentration as shown in the MTT assay. The growth behaviour of 3T3 fibroblasts plated on blood fibrin clot in each of the flasks was monitored and photographed under the phase contrast microscope after 24 hours as well as 48 hours of incubation, respectively, so as to compare the behaviour of the cells grown in the two different culture media.
, http://www.100md.com
Giemsa stain After 72 hours of incubation, the culture medium in each of the flasks was quikly removed out and the cells were washed three times with 0.1 mol/L PBS, fixed in cold methanol:glacial acetic acid (3∶1) for 30 minutes, washed twice in 0.1 mol/L PBS again and then observed under the phase contrast microscope after Giemsa staining. 3T3 fibroblasts were stained blue in Giemsa staining, whereas blood fibrin clots, light red in color, thus rendering the latter to be readily distinguished from the former . With the increase in the number of the fibroblasts, the colour of the cells turned from light to deep blue.
, http://www.100md.com
Scanning electron microscopic observation 3T3 fibroblasts with both blood fibrin clot and glass control as substratum were fixed successively in 2% glutaraldehyde in 0.1 mol/L PBS and osmium cacodylate in 0.1 mol/L PBS. The specimens were then dehydrated through a grade series of acetone and water, and dried with CO2 and gold sputter-coated for examination under a scanning electron microscope (JEOL JSM-7300).
Transmission electron microscopic observation 3T3 cell specimens with blood fibrin clot as substratum were fixed successively with 2% glutaraldehyde in 0.1 mol/L PBS at 4℃ and 1% osmium tetraoxide for 2 hours. Ultrathin sections were stained with 2% uranyl acetate and lead citrate and were inspected under a transmission electron microscope (JEOL 100×II/T).
, http://www.100md.com
RESULTS AND DISCUSSION
Effect of rhbFGF on 3T3 fibroblast proliferation and survival To evaluate the effect of rhbFGF on 3T3 fibroblast proliferation and survival, it is necessary to observe the impact of rhbFGF in different concentrations on the cells.It was shown that rhbFGF in concentrations higher than 25 ng/mL displayed a promotive effect on the proliferation of the cells incubated in DMEM with 0.4% (vol/vol) fetal bovine serum, and this effect was gradually augmented with increasing concentrations of rhbFGF. rhbFGF in still higher concentration, ie 800 ng/mL, however, depressed the proliferation of the cells. The optimal concentration of rhbFGF for the promotion of cell proliferation was found to be 100 ng/mL (P<0.01). The results also showed that 3T3 fibroblast adhered and grew well on blood fibrin clot bathed in DMEM containing 0.4% (vol/vol) fetal bovine serum supplemented with 100 ng/mL rhbFGF, indicating that rhbFGF can be used as an important component of a novel low-serum or serum-free medium for the growth of fibroblasts (Tab 1).
, 百拇医药
Tab 1 OD of 3T3 fibroblasts in culture media with different concentrations of rhbFGF in MTT assay(
±s, n=12) rhbFGF concentration (ng/mL)
Control
800
400
100
25
6.25
3.125
1.562
, 百拇医药
0.103±0.002*
0.138±0.005△△
0.152±0.005△
0.140±0.008△△
0.130±0.008*
0.129±0.007*
0.130±0.008*
0.115±0.011
*P>0.05, △P<0.05, △△P<0.01, vs control
, 百拇医药
3T3 fibroblast growth on blood fibrin clot 3T3 fibroblasts grew well on blood fibrin clot in DMEM supplemented 10% (vol/vol) fetal bovine serum, as shown under the phase-contrast microscope (Fig 1). The cells also can grew on blood fibrin clot scaffold in the low-serum containing 100 ng/mL of rhbFGF (Fig 2), and a significantly greater number of 3T3 fibroblasts were seen to grow on blood fibrin clot after 48 hours of incubation as compared with these after 24 hours of culture in the low-serum DMEM(Fig 3). These results suggest that the blood fibrin clot may serve not only as a support system, but also a carrier of rhbFGF in wound healing.
, 百拇医药
3T3 fibroblast phenotypes on blood fibrin clot A number of high resolution images of the key behavioural phenotypes of 3T3 fibroblasts grown on blood fibrin clot or glass at different intervals of incubation were shown by scanning electron microscopy. Elongated filopodia were seen to bridge the gaps among 3T3 fibroblasts that had been incubated for 24 hours on blood fibrin clots bathed in DMEM containing 0.4% (vol/vol) fetal bovine serum supplemented with 100 ng/mL of rhbFGF (Fig 4). After 72 hours of incubation, a great number of platycytes were found to be joined together by lamellopodia (Fig 5). Besides, 3T3 fibroblassts could grow and spead well on both blood fibrin clot and glass (Fig 4, Fig 6).
, 百拇医药
Fig 1 3T3 fibroblast culture on blood fibrin clot bathed in 10% fetal bovine serum for 24 hours ×150
Fig 2 3T3 fibroblast culture on blood fibrin clot bathed in 0.4% fetal bovine serum DMEM supplemented 100 ng/mL rhbFGF for 24 hours ×150
Fig 3 3T3 fibroblast culture on blood fibrin clot in 0.4% fetal bovine serum DMEM supplemented 100 ng/mL rhbFGF for 48 hours ×150
, 百拇医药
Fig 4 3T3 fibroblasts growth on blood fibrin clot bathed in DMEM containing 0.4% fetal bovine serum supplemented with 100 ng/mL of rhbFGF for 24 hours
The structure of 3T3 fibroblasts and blood fibrin clot Transmission electron microscopic examination revealed that 3T3 fibroblasts could well adhere to and on the surface of blood fibrin clot. Besides, blood fibrin clot was found to possess a large number of minute pores through which adequate nutrients can be readily supplied to the cells (Fig 7).
, 百拇医药
In conclusion, as a non-antigenic derived from fresh blood, fibrin clot may serve as a physiological support system and carrier of cellular growth factor rhbFGF to promote wound healing. We envisage that combined application of blood fibrin clot and rhbFGF should have a prospect in clinical medicine, notably in the treatment of traumas and wounds. The combination of blood fibrin and rhbFGF may also serve as a useful tool in the study of three-dimensional tissue culture, and the biological activity of rhbFGF should also be utilized to develop an economical and feasible low-serum or free-serum three dimentional tissue culture medium.
, 百拇医药
Fig 5 3T3 fibroblasts growth on blood fibrin clot bathed in DMEM containing 0.4% fetal bovine serum supplemented with 100 ng/mL of rhbFGF for 72 hours
Fig 6 3T3 fibroblasts growth on glass bathed in DMEM containing 0.4% fetal bovine serum supplemented with 100 ng/mL of rhbFGF for 24 hours
Fig 7 The structure of 3T3 fibroblast growth on blood fibrin clot for 24 hours ×4031
, 百拇医药
[Foundation item] Supported by Grant (No. 96-C02-01-03) from the Key Scientific Item in National “9.5” of China
[REFERENCES]
[1] Henning CE, Lynch MA, Yearout KM, et al. Arthroscopic meniscal repair using an exogenous fibrin clot[J]. Clin Orthop, 1990, 252: 64～69.
[2] Roddecker K, Munnich U, Jochims J, et al. Measurement of the biomechanical stability of the healing menisci in animal: fibrin gluing, an alternative to traditional therapy methods[J]. Z Orthop, 1991, 129(4): 350～355.
, http://www.100md.com
[3] Ishimura M. Arthoscopic meniscal repair with fibrin glue[J]. Arthroscopy, 1991, 7(2): 177～182.
[4] Arnoczky SP. Meniscal repair using an exogenous fibrin clot[J]. J Bone Joint Surg, 1988, 70-A: 1209～1213.
[5] Gospodarowicz D. Fibroblast growth factor[J]. Clin Orthop, 1990, 257: 231～235.
[6]Martin, P. Wound healing: Aiming for perfect skin regeneration[J]. Science, 1997, 276: 75～82.
, 百拇医药
[7] Michalopoulos GK, DeFrances MC. Liver regeneration[J]. Science, 1997, 276: 60～68.
[8] Baird A. The regulation of basic fibroblast growth factor (FGF-2) through limited bioavailability[M]. In: Ziegler, TR, Pierce GF, Herndon DN. eds. Growth factor and wound healing, basic science and potential clinical application. New York: Springer, 1997. 27～31.
[9] Mosmann T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays[J]. J Immunol Methods, 1983, 65: 55～60.
[10] James C, William GD, Adi FG, et al. Evaluation of a tetrazolium-based semiautomated colorimetric assay: assessment of chemosensitivity testing. Cancer Res, 1987, 47(4): 936～943.
Received]1999-04-01 [Accepted]1999-08-26, http://www.100md.com
单位：王一飞 戴云 刘杰生 林剑(暨南大学生物工程研究所，广东 广州 510632)；崔蕴霞(中山医科大学分子医学研究中心， 广东 广州 510089)
关键词：成纤维细胞；凝集；成纤维细胞生长因子，碱性
中国病理生理杂志000201
[摘 要] 目的：重组碱性成纤维细胞生长因子(rhbFGF)对培养条件下3T3成纤维细胞在血纤维蛋白凝块上生长的可能性研究。方法：采用Giemsa染色和MTT检测法，经过电镜扫描，分段对比观察，研究3T3细胞在血纤维蛋白凝块上的生长情况。 结果：rhbFGF促进细胞生长并维持细胞存活的最佳浓度是100 ng/mL，在含有100 ng/mL的低血清培养基中细胞可在血纤维蛋白凝块上生长。经48h培养以后仍有大量的细胞存活。 结论：3T3成纤维细胞可在含有rhbFGF的低血清培养基上生长并存活，rhbFGF与血纤维蛋白凝块共存可以促进创伤愈合。
, 百拇医药
[中图分类号]Q 813 [文献标识码] A
[Article ID] 1000-4718(2000)02-0097-05
Possibility of 3T3 fibroblast growth on blood fibrin clot in culture medium with recombinant human basic fibroblast growth factor
WANG Yi-fei, DAI Yun, LIU Jie-shen, LIN Jian
(Bioengineering Institute of Jinan University, Guangzhou 510632, China)
CUI Yun-xia
, 百拇医药
(The Research Center of Molecular Medicine, Sun Yet-Sen University of Medical Sciences,Guangzhou 510089, China)
[Abstract] AIM:
To investigate the possibility of 3T3 fibroblast growth on blood fibrin clot in culture medium with recombinant human basic fibroblast growth factor (rhbFGF). METHOD: Growth of the cells on blood fibrin clot was studied by phase-contrast, scanning and transmission electron microscopy and by Giemsa stain and MTT assay. RESULTS: The optimal concentration of rhbFGF for proliferation and survival of the cells was 100 ng/mL. The cells also grew on blood fibrin clot scaffold in the low-serum medium containing 100 ng/mL rhbFGF, and a greater number of the cells survived after 48 hours incubation compared to that after 24 hours. The elongated filopodia appeared to bridge the gaps among the fibroblasts after 24 hours incubation. Further incubation to 72 hours, a greater number of platycytes were found to be joined together by lamellopodia. CONCLUSION: 3T3 fibroblasts could grow and survive on blood fibrin clot in the low-serum medium containing rhbFGF, and a combination of blood fibrin clot and rhbFGF may have over proportional effects on wound healing.
, 百拇医药
[MeSH] Fibroblasts;Agglutination;Fibroblast growth factor, basic
[CLC number] Q813 [Document code] A
INTRODUCTION
Fibrin clot obtained from fresh blood is a kind of biomaterial with low immunogenicity or no antigenicity, usually acting as a physiological scaffold in wound healing[1～4]. rhbFGF is an active peptide growth factor capable of facilitating the proliferation and differentiation of the majority of cells derived from the mesoderm and neuroectoderm as well as promting processes in wound healing[5～8]. A series of investigation concerning the effect of rhbFGF on the formation of three-dimensional tissue model has been made and we here report the possibility of 3T3 fibroblasts on blood fibrin clot immersed in a low-serum medium supplemented with rhbFGF.
, 百拇医药
MATERIALS AND METHODS
Experimental animals and reagents BALB/C mice aged 8～12 weeks were used. Trypsin, penicillin, streptomycin, fetal bovine serum and Dulbecco's Modified Eagle Medium (DMEM) were purchased from Giebco Ltd., Paisley, UK; MTT [3-(4, 5-dimethylthiazal-2yl)2,5-diphenyltetrazolium bromide] was a product of Sigma Chemical Co., St. Louis, MO; rhbFGF (No. 199806) was a purified gene recombinant product of Bioengineering Institute of Jinan University.
, 百拇医药
Preparation of blood fibrin clot The eyeballs of six BALB/C mice were ablated with an eye-tweezers under a relatively aseptic condition, and the blood collected was let to stand for 30 minutes in a centrifuge tube. The serum was then removed after centrifugation by 1 500 r/min for 10 minutes. The blood fibrin clot thus obtained was washed repeatedly with double-distilled water until it became free of red color, and then twice with Hanks solution containing penicillin (50 U/mL) and streptomycin(50 μL/mL). The blood fibrin clot was subsequently cut into a number of pieces which were put separately into the centre of a series of culture flasks to be lyophilized in a cryodesiccator (HETOTR CT-60-e) for 2 hours.
, 百拇医药
Cell culture 3T3 fibroblasts were cultured in DMEM containing 10% (vol/vol) fetal bovine serum and penicillin (50 U/mL)/streptomycin ( 50 μL/mL). When the bottom of the flasks was nearly packed with confluent cells, 0.1% (wt/vol) trypsin was used to detached the fibroblasts at 37 ℃ for 3 minutes. The dissociated cells were then washed and resuspended in DMEM with 10% (vol/vol) fetal bovine serum. Concentration of the cell suspension was calculated and adjusted to 5×105/mL.
, http://www.100md.com
MTT assay[9,10] Cell suspensions (5×103/mL) containing 10%(vol/vol) fetal bovine serum in DMEM were seed onto 96-well flat-bottomed plates (100 μL/well) and incubated 24 hours ,the culture medium was removed , the cells were then washed with phosphate buffered saline three times and re-fed with 100 μL/well of 0.4% (vol/vol) fetal bovine serum in DMEM containing a series of different dilutions of rhbFGF for further incubation. Baseline control (100 μL/well fetal bovine serum in DMEM) parallel to the sample for each plate. 48 hours after the further incubation , 10 μL MTT solution (5 mg/mL) was added immediately to each well of the assay and the plates were incubated at 37 ℃ for another 4 hours. Acid-isopropanol (100 μL of 0.04 mol/L in isopropanol) was added to each well and the contents of which were mixed thoroughly to dissolve the dark blue crystals. The plates were let to stand for a few minutes at room temperature to ensure complete dissolution of the crystals . The OD vaule of each well of the 3T3 fibroblast suspensions incubated with different concentrations of rhbFGF was read out on an EL311 autoplate reader (BIO-TEK Instruments) with a test wavelength of 570nm and a reference wavelength of 630nm, so that the optimal concentration of rhbFGF for stimulation of the proliferation of 3T3 fibroblasts could be determined.
, 百拇医药
Phase contrast microscopic observation 5 mL of cell suspension (1.5×105/mL) in DMEM with 10%(vol/vol) fetal bovine serum were added separately to each of the 25 cm×25 cm culture flasks with and without blood fibrin clot specimen. Incubated 24 hours, the medium containing 10%(vol/vol) fetal bovine serum was removed out from the culture flasks and the behavior of the cells plated on the blood fibrin clots or glasses was monitored and photographed under a phase contrast microscope. Subsequently, 5 mL each of fresh DMEM containing 10% (vol/vol) fetal bovine serum were added separately into half of the flasks while another half of the flasks were added 5 mL each of fresh DMEM containing 0.4%(vol/vol) fetal bovine serum supplemented with rhbFGF in an optimal concentration as shown in the MTT assay. The growth behaviour of 3T3 fibroblasts plated on blood fibrin clot in each of the flasks was monitored and photographed under the phase contrast microscope after 24 hours as well as 48 hours of incubation, respectively, so as to compare the behaviour of the cells grown in the two different culture media.
, http://www.100md.com
Giemsa stain After 72 hours of incubation, the culture medium in each of the flasks was quikly removed out and the cells were washed three times with 0.1 mol/L PBS, fixed in cold methanol:glacial acetic acid (3∶1) for 30 minutes, washed twice in 0.1 mol/L PBS again and then observed under the phase contrast microscope after Giemsa staining. 3T3 fibroblasts were stained blue in Giemsa staining, whereas blood fibrin clots, light red in color, thus rendering the latter to be readily distinguished from the former . With the increase in the number of the fibroblasts, the colour of the cells turned from light to deep blue.
, http://www.100md.com
Scanning electron microscopic observation 3T3 fibroblasts with both blood fibrin clot and glass control as substratum were fixed successively in 2% glutaraldehyde in 0.1 mol/L PBS and osmium cacodylate in 0.1 mol/L PBS. The specimens were then dehydrated through a grade series of acetone and water, and dried with CO2 and gold sputter-coated for examination under a scanning electron microscope (JEOL JSM-7300).
Transmission electron microscopic observation 3T3 cell specimens with blood fibrin clot as substratum were fixed successively with 2% glutaraldehyde in 0.1 mol/L PBS at 4℃ and 1% osmium tetraoxide for 2 hours. Ultrathin sections were stained with 2% uranyl acetate and lead citrate and were inspected under a transmission electron microscope (JEOL 100×II/T).
, http://www.100md.com
RESULTS AND DISCUSSION
Effect of rhbFGF on 3T3 fibroblast proliferation and survival To evaluate the effect of rhbFGF on 3T3 fibroblast proliferation and survival, it is necessary to observe the impact of rhbFGF in different concentrations on the cells.It was shown that rhbFGF in concentrations higher than 25 ng/mL displayed a promotive effect on the proliferation of the cells incubated in DMEM with 0.4% (vol/vol) fetal bovine serum, and this effect was gradually augmented with increasing concentrations of rhbFGF. rhbFGF in still higher concentration, ie 800 ng/mL, however, depressed the proliferation of the cells. The optimal concentration of rhbFGF for the promotion of cell proliferation was found to be 100 ng/mL (P<0.01). The results also showed that 3T3 fibroblast adhered and grew well on blood fibrin clot bathed in DMEM containing 0.4% (vol/vol) fetal bovine serum supplemented with 100 ng/mL rhbFGF, indicating that rhbFGF can be used as an important component of a novel low-serum or serum-free medium for the growth of fibroblasts (Tab 1).
, 百拇医药
Tab 1 OD of 3T3 fibroblasts in culture media with different concentrations of rhbFGF in MTT assay(
±s, n=12) rhbFGF concentration (ng/mL)Control
800
400
100
25
6.25
3.125
1.562
, 百拇医药
0.103±0.002*
0.138±0.005△△
0.152±0.005△
0.140±0.008△△
0.130±0.008*
0.129±0.007*
0.130±0.008*
0.115±0.011
*P>0.05, △P<0.05, △△P<0.01, vs control
, 百拇医药
3T3 fibroblast growth on blood fibrin clot 3T3 fibroblasts grew well on blood fibrin clot in DMEM supplemented 10% (vol/vol) fetal bovine serum, as shown under the phase-contrast microscope (Fig 1). The cells also can grew on blood fibrin clot scaffold in the low-serum containing 100 ng/mL of rhbFGF (Fig 2), and a significantly greater number of 3T3 fibroblasts were seen to grow on blood fibrin clot after 48 hours of incubation as compared with these after 24 hours of culture in the low-serum DMEM(Fig 3). These results suggest that the blood fibrin clot may serve not only as a support system, but also a carrier of rhbFGF in wound healing.
, 百拇医药
3T3 fibroblast phenotypes on blood fibrin clot A number of high resolution images of the key behavioural phenotypes of 3T3 fibroblasts grown on blood fibrin clot or glass at different intervals of incubation were shown by scanning electron microscopy. Elongated filopodia were seen to bridge the gaps among 3T3 fibroblasts that had been incubated for 24 hours on blood fibrin clots bathed in DMEM containing 0.4% (vol/vol) fetal bovine serum supplemented with 100 ng/mL of rhbFGF (Fig 4). After 72 hours of incubation, a great number of platycytes were found to be joined together by lamellopodia (Fig 5). Besides, 3T3 fibroblassts could grow and spead well on both blood fibrin clot and glass (Fig 4, Fig 6).
, 百拇医药
Fig 1 3T3 fibroblast culture on blood fibrin clot bathed in 10% fetal bovine serum for 24 hours ×150
Fig 2 3T3 fibroblast culture on blood fibrin clot bathed in 0.4% fetal bovine serum DMEM supplemented 100 ng/mL rhbFGF for 24 hours ×150
Fig 3 3T3 fibroblast culture on blood fibrin clot in 0.4% fetal bovine serum DMEM supplemented 100 ng/mL rhbFGF for 48 hours ×150
, 百拇医药
Fig 4 3T3 fibroblasts growth on blood fibrin clot bathed in DMEM containing 0.4% fetal bovine serum supplemented with 100 ng/mL of rhbFGF for 24 hours
The structure of 3T3 fibroblasts and blood fibrin clot Transmission electron microscopic examination revealed that 3T3 fibroblasts could well adhere to and on the surface of blood fibrin clot. Besides, blood fibrin clot was found to possess a large number of minute pores through which adequate nutrients can be readily supplied to the cells (Fig 7).
, 百拇医药
In conclusion, as a non-antigenic derived from fresh blood, fibrin clot may serve as a physiological support system and carrier of cellular growth factor rhbFGF to promote wound healing. We envisage that combined application of blood fibrin clot and rhbFGF should have a prospect in clinical medicine, notably in the treatment of traumas and wounds. The combination of blood fibrin and rhbFGF may also serve as a useful tool in the study of three-dimensional tissue culture, and the biological activity of rhbFGF should also be utilized to develop an economical and feasible low-serum or free-serum three dimentional tissue culture medium.
, 百拇医药
Fig 5 3T3 fibroblasts growth on blood fibrin clot bathed in DMEM containing 0.4% fetal bovine serum supplemented with 100 ng/mL of rhbFGF for 72 hours
Fig 6 3T3 fibroblasts growth on glass bathed in DMEM containing 0.4% fetal bovine serum supplemented with 100 ng/mL of rhbFGF for 24 hours
Fig 7 The structure of 3T3 fibroblast growth on blood fibrin clot for 24 hours ×4031
, 百拇医药
[Foundation item] Supported by Grant (No. 96-C02-01-03) from the Key Scientific Item in National “9.5” of China
[REFERENCES]
[1] Henning CE, Lynch MA, Yearout KM, et al. Arthroscopic meniscal repair using an exogenous fibrin clot[J]. Clin Orthop, 1990, 252: 64～69.
[2] Roddecker K, Munnich U, Jochims J, et al. Measurement of the biomechanical stability of the healing menisci in animal: fibrin gluing, an alternative to traditional therapy methods[J]. Z Orthop, 1991, 129(4): 350～355.
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[3] Ishimura M. Arthoscopic meniscal repair with fibrin glue[J]. Arthroscopy, 1991, 7(2): 177～182.
[4] Arnoczky SP. Meniscal repair using an exogenous fibrin clot[J]. J Bone Joint Surg, 1988, 70-A: 1209～1213.
[5] Gospodarowicz D. Fibroblast growth factor[J]. Clin Orthop, 1990, 257: 231～235.
[6]Martin, P. Wound healing: Aiming for perfect skin regeneration[J]. Science, 1997, 276: 75～82.
, 百拇医药
[7] Michalopoulos GK, DeFrances MC. Liver regeneration[J]. Science, 1997, 276: 60～68.
[8] Baird A. The regulation of basic fibroblast growth factor (FGF-2) through limited bioavailability[M]. In: Ziegler, TR, Pierce GF, Herndon DN. eds. Growth factor and wound healing, basic science and potential clinical application. New York: Springer, 1997. 27～31.
[9] Mosmann T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays[J]. J Immunol Methods, 1983, 65: 55～60.
[10] James C, William GD, Adi FG, et al. Evaluation of a tetrazolium-based semiautomated colorimetric assay: assessment of chemosensitivity testing. Cancer Res, 1987, 47(4): 936～943.
Received]1999-04-01 [Accepted]1999-08-26, http://www.100md.com