纤维蛋白凝胶促进大鼠间充质干细胞的成骨分化

doi:10.3969/j.issn.2095-4344.2014.25.011

中国组织工程研究 ›› 2014, Vol. 18 ›› Issue (25): 3998-4003.doi: 10.3969/j.issn.2095-4344.2014.25.011

• 组织工程骨及软骨材料 tissue-engineered bone and cartilage materials • 上一篇下一篇

纤维蛋白凝胶促进大鼠间充质干细胞的成骨分化

买霞，李威，王英慧，扎拉嘎胡，陈小义

武装警察部队后勤学院临床医学系细胞生物学与医学遗传学教研室，天津市 300039

收稿日期:2014-05-28 出版日期:2014-06-18 发布日期:2014-06-18
通讯作者: 陈小义，硕士，教授，硕士生导师，武装警察部队后勤学院临床医学系细胞生物学与医学遗传学教研室，天津市 300039
作者简介:买霞，女，1972年生，山西省运城市人，回族，武警部队后勤学院在读硕士，实验师，主要从事干细胞定向分化研究。
基金资助:
中国人民武警部队资助重点项目(WKH2009Z04)，中国人民武警部队后勤学院资助项目(WHM201209)

Fibrin gel enhances osteogenic differentiation of rat mesenchymal stem cells

Mai Xia, Li Wei, Wang Ying-hui, Zha La Ga Hu, Chen Xiao-yi

Research Room of Cell Biology and Medical Genetics, Department of Clinical Medicine, Logistics College of Chinese People’s Armed Police Forces, Tianjin 300039, China

Received:2014-05-28 Online:2014-06-18 Published:2014-06-18
Contact: Chen Xiao-yi, Master, Professor, Master’s supervisor, Research Room of Cell Biology and Medical Genetics, Department of Clinical Medicine, Logistics College of Chinese People’s Armed Police Forces, Tianjin 300039, China
About author:Mai Xia, Studying for master’s degree, Experimentalist, Research Room of Cell Biology and Medical Genetics, Department of Clinical Medicine, Logistics College of Chinese People’s Armed Police Forces, Tianjin 300039, China
Supported by:
the Key Program of Chinese People’s Armed Police Forces, No. WKH2009Z04; the Program of Logistics College of Chinese People’s Armed Police Forces, No. WHM201209

摘要/Abstract

摘要：

背景：纤维蛋白是一种天然的可生物降解、组织相容性好的高分子材料，是一种能促进细胞和外源性生长因子释放的载体，其中血纤维蛋白稳定因子ⅩⅢ已证明有利于未分化的间充质干细胞在高度交联的凝胶支架内迁移，并且促进这些细胞的增殖与分化能力。
目的：观察大鼠间充质干细胞在纤维蛋白凝胶内的行为。
方法：无菌条件下分离大鼠胎肢细胞获得间充质干细胞，取第3代细胞分别接种于0，5，10，20 g/L纤维蛋白凝胶内，用倒置相差显微镜和激光扫描共聚焦显微镜分析细胞在凝胶内的形态学变化；酶标仪和Von Kossa染色分析碱性磷酸酶活性和钙盐沉积。
结果与结论：5 g/L低浓度纤维蛋白凝胶有利于细胞形态的发生，20 g/L高浓度凝胶有利于细胞的成骨分化。20 g/L纤维蛋白凝胶碱性磷酸酶活性高于对照组，10和20 g/L浓度纤维蛋白凝胶矿化结节出现在21至28 d，而对照组无矿化结节出现。提示大鼠间充质干细胞的形态与成骨分化依赖于纤维蛋白凝胶浓度，提示纤维蛋白凝胶有助于间充质干细胞的成骨分化。

中国组织工程研究杂志出版内容重点：生物材料；骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性；组织工程

全文链接：

关键词: 生物材料, 骨生物材料, 纤维蛋白凝胶, 间充质干细胞, 成骨分化, 细胞培养, 细胞形态, 碱性磷酸酶

Abstract:

BACKGROUND: Fibrin is a kind of high polymer materials with biodegradation and good histocompatibility, and is a vector that can promote cell and exogenous growth factor release. Fibrin stabilizing factor XIII has been verified to contribute to the migration of undifferentiated mesenchymal stem cells in gel scaffold with high crosslinking, and promote cell proliferation and differentiation.
OBJECTIVE: To observe rat mesenchymal stem cell behavior in a fibrin gel.
METHODS: The rat fetal limbs cells was separated under the aseptic condition. The passage 3 cells were seeded in 0, 5, 10 and 20 g/L fibrin gel. Cell morphology was observed by inverted phase microscope and laser scanning confocal microscopy. Alkaline phosphatase activity and calcium deposition were measured respectively using a microplate reader and von Kossa staining.
RESULTS AND CONCLUSION: 5 g/L fibrin gel contributed to cell morphological changes, and 20 g/L fibrin gel contributed to osteogenic differentiation. Compared with the control group, alkaline phosphatase activity was higher in the formulations containing a 20 g/L fibrinogen concentration. Small mineralization nodules were observed at 21 and 28 days in a formulation containing both 10 and 20 g/L fibrinogen concentration, but no mineralization was detected in the control group. These results indicate that morphology and osteogenic differentiation of rat mesenchymal stem cells depended on the fibrinogen concentration, suggesting that fibrin gel is conducive to osteogenic differentiation of mesenchymal stem cells.

中国组织工程研究杂志出版内容重点：生物材料；骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性；组织工程

全文链接：

Key words: fibrin, hydrogels, mesenchymal stem cells, factor XIII

中图分类号:

R318

买霞，李威，王英慧，扎拉嘎胡，陈小义. 纤维蛋白凝胶促进大鼠间充质干细胞的成骨分化[J]. 中国组织工程研究, 2014, 18(25): 3998-4003.

Mai Xia, Li Wei, Wang Ying-hui, Zha La Ga Hu, Chen Xiao-yi. Fibrin gel enhances osteogenic differentiation of rat mesenchymal stem cells[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(25): 3998-4003.

图/表（结果） 2

Fibrin gel effects on morphology of rat bone marrow mesenchymal stem cells

Microscopy image of a typical cell colony was seen at 7 days by inverted phase microscope. Laser scanning confocal microscopy results demonstrated that the majority of passage 3 cells stained by calcein dye in the gels were viable in all gel formulations and at all incubation time. Different cell morphologies were observed depending on the formulation of the fibrin gel. In gels containing 5 g/L of fibrinogen concentration cells started to elongate as early as day 3 and formed an interconnected network within the gels up to day 21. On the other hand, in gels containing 20 g/L of fibrinogen concentration cells tended to remain mostly round or ‘‘star-shaped’’ up to day 21 and formed some agglomeration, but not an interconnected network. Cells in no gels showed a spindle-shape or cuboidal morphology up to day 21, but no interconnected network was found during prolonged incubation time (Figure 1).

Fibrin gel affected the proliferation of rat mesenchymal stem cells

Alkaline phosphatase activity was relatively stable for all the formulations at 3 and 7 days. The alkaline phosphatase activity in cell-seeded fibrin gels containing a high (10, 20 g/L) fibrinogen concentration became significant at 14 days, and continued to increase up to 28 days, but that remained still lower in no gels and containing a low (5 g/L) fibrinogen concentration cultures of rat mesenchymal stem cells. In contrast to the proliferation results, alkaline phosphatase activity was higher in gels containing a high fibrinogen concentration (Table 1).

Von Kossa staining

Von Kossa staining in a formulation containing a high

(20 g/L) fibrinogen concentration showed the presence of mineralization nodules after 21 and 28 days of incubation. The nodules appeared in areas with cells, and mostly in the areas with holes in the fibrin gels. No nodules were observed on top of the gel, which was covered by a monolayer of cells (Figure 2).

参考文献

[1] Caplan AI. Mesenchymal stem cells. J Orthop Res. 1991; 9(5):641-650.
[2] Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells. Science. 1999;284(5411):143-147.
[3] Bianco P, Robey PG, Simmons PJ. Mesenchymal stem cells: revisiting history, concepts, and assays. Cell Stem Cell. 2008;2(4):313-319.
[4] Jaiswal RK, Jaiswal N, Bruder SP, et al. Adult human mesenchymal stem cell differentiation to the osteogenic or adipogenic lineage is regulated by mitogen-activated protein kinase. J Biol Chem. 2000;275(13):9645-9652.
[5] Wang X, Wang Y, Gou W, et al. Role of mesenchymal stem cells in bone regeneration and fracture repair: a review. Int Orthop. 2013;37(12):2491-2498.
[6] Cao L, Liu G, Gan Y, et al. The use of autologous enriched bone marrow MSCs to enhance osteoporotic bone defect repair in long-term estrogen deficient goats. Biomaterials. 2012;33(20):5076-5084.
[7] Guan M, Yao W, Liu R, et al. Directing mesenchymal stem cells to bone to augment bone formation and increase bone mass. Nat Med. 2012;18(3):456-462.
[8] Pino AM, Rosen CJ, Rodríguez JP. In osteoporosis, differentiation of mesenchymal stem cells (MSCs) improves bone marrow adipogenesis. Biol Res. 2012; 45(3):279-287.
[9] Dohan Ehrenfest DM, Doglioli P, de Peppo GM, et al. Choukroun's platelet-rich fibrin (PRF) stimulates in vitro proliferation and differentiation of human oral bone mesenchymal stem cell in a dose-dependent way. Arch Oral Biol. 2010;55(3):185-194.
[10] Wong C, Inman E, Spaethe R, et al. Fibrin-based biomaterials to deliver human growth factors. Thromb Haemost. 2003;89(3):573-582.
[11] Gorodetsky R, Clark RA, An J, et al. Fibrin microbeads (FMB) as biodegradable carriers for culturing cells and for accelerating wound healing. J Invest Dermatol. 1999; 112(6): 866-872.
[12] Horch RE, Bannasch H, Kopp J, et al. Single-cell suspensions of cultured human keratinocytes in fibrin-glue reconstitute the epidermis. Cell Transplant. 1998;7(3): 309-317.
[13] Catelas I, Dwyer JF, Helgerson S. Controlled release of bioactive transforming growth factor beta-1 from fibrin gels in vitro. Tissue Eng Part C Methods. 2008;14(2):119-128.
[14] Mosesson MW, Siebenlist KR, Meh DA. The structure and biological features of fibrinogen and fibrin. Ann N Y Acad Sci. 2001;936:11-30.
[15] Marktl W, Rudas B. The effect of factor XIII on wound granulation in the rat. Thromb Diath Haemorrh. 1974;32 (2-3):578-581.
[16] Lin B, Cai ZG, Yu GY, et al. Osteoblastoma of the maxilla and mandible: a report of 2 cases and literature review. Chin J Dent Res. 2012;15(2):153-158.
[17] Nöth U, Steinert AF, Tuan RS. Technology insight: adult mesenchymal stem cells for osteoarthritis therapy. Nat Clin Pract Rheumatol. 2008;4(7):371-380.
[18] Yu Y, Shao B, Shuai Y, et al. Role of bone marrow-derived mesenchymal stem cells in treating colitis through Fas/FasL-mediated immune regulation. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2013;29(10):1028-1031.
[19] Ishii M, Shibata R, Numaguchi Y, et al. Enhanced angiogenesis by transplantation of mesenchymal stem cell sheet created by a novel magnetic tissue engineering method. Arterioscler Thromb Vasc Biol. 2011;31(10): 2210-2215.
[20] Li X, van Blitterswijk CA, Feng Q, et al. The effect of calcium phosphate microstructure on bone-related cells in vitro. Biomaterials. 2008;29(23):3306-3316.
[21] Sun N, Yang L, Li Y, et al. Effect of advanced oxidation protein products on the proliferation and osteogenic differentiation of rat mesenchymal stem cells. Int J Mol Med. 2013;32(2):485-491.
[22] Tang X, Sheng L, Xie F, et al. Differentiation of bone marrow-derived mesenchymal stem cells into chondrocytes using chondrocyte extract. Mol Med Rep. 2012;6(4): 745-749.
[23] Scotti C, Pozzi A, Mangiavini L, et al. Healing of meniscal tissue by cellular fibrin glue: an in vivo study. Knee Surg Sports Traumatol Arthrosc. 2009;17(6):645-651.
[24] Chien CS, Ho HO, Liang YC, et al. Incorporation of exudates of human platelet-rich fibrin gel in biodegradable fibrin scaffolds for tissue engineering of cartilage. J Biomed Mater Res B Appl Biomater. 2012;100(4):948-955.
[25] Sha'ban M, Yoon SJ, Ko YK, et al. Fibrin promotes proliferation and matrix production of intervertebral disc cells cultured in three-dimensional poly(lactic-co-glycolic acid) scaffold. J Biomater Sci Polym Ed. 2008;19(9):1219- 1237.
[26] Even-Ram S. Fibrin gel model for assessment of cellular contractility. Methods Mol Biol. 2009;522:251-259.
[27] de Jonge N, Kanters FM, Baaijens FP, et al. Strain-induced collagen organization at the micro-level in fibrin-based engineered tissue constructs. Ann Biomed Eng. 2013;41(4): 763-774.
[28] Gerard C, Forest MA, Beauregard G, et al. Fibrin gel improves the survival of transplanted myoblasts. Cell Transplant. 2012;21(1):127-137.
[29] Lee F, Kurisawa M. Formation and stability of interpenetrating polymer network hydrogels consisting of fibrin and hyaluronic acid for tissue engineering. Acta Biomater. 2013;9(2):5143-5152.
[30] Allan P, Uitte de Willige S, Abou-Saleh RH, et al. Evidence that fibrinogen γ' directly interferes with protofibril growth: implications for fibrin structure and clot stiffness. J Thromb Haemost. 2012;10(6):1072-1080.
[31] Beck GR Jr. Inorganic phosphate as a signaling molecule in osteoblast differentiation. J Cell Biochem. 2003;90(2):234- 243.
[32] Simão AM, Beloti MM, Cezarino RM, et al. Membrane-bound alkaline phosphatase from ectopic mineralization and rat bone marrow cell culture. Comp Biochem Physiol A Mol Integr Physiol. 2007;146(4): 679-687.
[33] Kim A, Benning MM, OkLee S, et al. Divergence of chemical function in the alkaline phosphatase superfamily: structure and mechanism of the P-C bond cleaving enzyme phosphonoacetate hydrolase. Biochemistry. 2011;50(17): 3481-3494.
[34] Tobe BT, Hou J, Crain AM, et al. Phosphoproteomic analysis: an emerging role in deciphering cellular signaling in human embryonic stem cells and their differentiated derivatives. Stem Cell Rev. 2012;8(1):16-31.
[35] Brill LM, Xiong W, Lee KB, et al. Phosphoproteomic analysis of human embryonic stem cells. Cell Stem Cell. 2009;5(2):204-213.
[36] Tian XF, Heng BC, Ge Z, et al. Comparison of osteogenesis of human embryonic stem cells within 2D and 3D culture systems. Scand J Clin Lab Invest. 2008;68(1):58-67.
[37] Bensaïd W, Triffitt JT, Blanchat C, et al. A biodegradable fibrin scaffold for mesenchymal stem cell transplantation. Biomaterials. 2003;24(14):2497-2502.
[38] Ho W, Tawil B, Dunn JC, et al. The behavior of human mesenchymal stem cells in 3D fibrin clots: dependence on fibrinogen concentration and clot structure. Tissue Eng. 2006;12(6):1587-1595.
[39] Catelas I, Sese N, Wu BM, et al. Human mesenchymal stem cell proliferation and osteogenic differentiation in fibrin gels in vitro. Tissue Eng. 2006;12(8):2385-2396.
[40] Rose LC, Fitzsimmons R, Lee P, et al. Effect of basic fibroblast growth factor in mouse embryonic stem cell culture and osteogenic differentiation. J Tissue Eng Regen Med. 2013;7(5):371-382.
[41] Luan J, Cui Y, Zhang Y, et al. Effect of CXCR4 inhibitor AMD3100 on alkaline phosphatase activity and mineralization in osteoblastic MC3T3-E1 cells. Biosci Trends. 2012;6(2):63-69.
[42] Mauney JR, Blumberg J, Pirun M, et al. Osteogenic differentiation of human bone marrow stromal cells on partially demineralized bone scaffolds in vitro. Tissue Eng. 2004;10(1-2):81-92.
[43] Kitamura S, Ohgushi H, Hirose M, et al. Osteogenic differentiation of human bone marrow-derived mesenchymal cells cultured on alumina ceramics. Artif Organs. 2004;28(1):72-82.
[44] Lee SJ, Atala A. Scaffold technologies for controlling cell behavior in tissue engineering. Biomed Mater. 2013;8(1): 010201

引言

Mesenchymal cells appear early during embryogenesis and persist throughout the life span of mammalians in a body-wide distribution. They were originally thought to be connective tissue supportive cells (stroma). However, it was eventually found that single mesenchymal cells may exhibit multipotent differentiation and these were therefore termed mesenchymal stem cells^[1-2]. Careful examination of cultured mesenchymal cell populations revealed their heterogeneous nature^[3]. Mesenchymal stem cells are multipotent stromal cells that can differentiate into a variety of cell types such as osteoblasts, adipocytes, chondrocytes, myoblasts and neurons^[4]. Recent studies have demonstrated that mesenchymal stem cells could be used to treat bone fracture and osteoporotic bone defect^[5-6]. Furthermore, Guan et al ^[7] found that directing mesenchymal stem cells to bone could augment bone formation and increase bone mass. Therefore, enhancing osteogenesis of mesenchymal stem cells is thought to be a useful therapeutic strategy for bone diseases such as osteoporosis^[8].

Fibrin gels have been widely used as adhesives in plastic and reconstructive surgery^[9-10]. They consist of two human plasma-derived components: (1) a highly concentrated fibrinogen complex composed primarily of fibrinogen and fibronectin along with catalytic amounts of factor XIII and plasminogen and (2) a high-potency thrombin. They have been used for cell delivery^[11-12], but have also been shown to be a suitable delivery vehicle for exogenous growth factors that could be used to accelerate bone growth and vascularization^[13]. Fibrin-stabilizing factor XIII contained in fibrin sealant has been demonstrated to favor migration of undifferentiated mesenchymal stem cells on the highly cross-linked structure of the glue and enhance the proliferation of these cells^[14-16].

Some studies demonstrated the migration of marrow-derived stem cells to skeletal sites and their proliferation and differentiation at local

tissue sites^[17-19]. Mesenchymal stem cells isolated from a variety of species can be induced to differentiate into osteoblasts and have also been shown to form bone tissue when seeded into ceramic or calcium phosphate carriers and implanted in vivo ^[20-22]. The purpose of this study was to analyze the morphology and osteogenic differentiation of rat mesenchymal stem cells within a fibrin gel in vitro and to analyze the effects of variations in the fibrinogen concentrations on cell behavior. Our goal is to examine the potential of this fibrin gel as an injectable and biodegradable scaffold for mesenchymal stem cells for possible bone regeneration applications.

中国组织工程研究杂志出版内容重点：生物材料；骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性；组织工程
全文链接：

材料方法

Design

An animal experiment with cell observation.

Time and setting

The experiment was conducted at Department of Cell Biology and Medical Genetics, Logistics College of Chinese People’s Armed Police Forces from February 2009 to December 2011.

Materials

Animals

The specific pathogen free pregnant Sprague-Dawly rats were purchased from the Experimental Animal Center of Chinese People’s Liberation Army Academy of Military Medical Sciences (animal number SCXK-(Army) 2007-004). All rats were housed at about 25 ℃, humidity of 60%–70%, and handled in accordance with animal ethical standards.

Mean reagents and instrument used for culture of rat mesenchymal stem cells:
Reagent and instrument	Source
H-DMEM culture medium, trypsin	GIBCO Corporation, USA
Fibrinogen from cow, thrombin	Sigma Corporation, USA
Fetal bovine serum	Biochrom Corporation, Germany
Inverted phase contrast microscope, laser scanning confocal microscopy	Olympus Corporation, Japan

Methods

Culture of mesenchymal stem cells derived from rat bone marrow

Rat mesenchymal stem cells were isolated from fetal limbs, aspirated and cultured for 7 days at 37 ℃ in 5% CO₂. Expansion medium (Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and 80 U gentamycin/mL) was added twice a week, but spent medium was not removed until cells reached 60% confluence. Non-adherent hematopoietic cells were removed via PBS washes and the rat mesenchymal stem cells were cultured in expansion medium until passage. Passages 3–5 cells were used as subsequent experiments.

Fibrin gel preparation

According to a modification of the methods of Scotti et al ^[23], the thrombin component was reconstituted in

30 mmol/L calcium chloride into 2 U/mL (final concentrations in the gels). The fibrinogen component was reconstituted in serum-free medium. To analyze the effects of variations in the fibrinogen concentrations, three different formulations of fibrin gels were prepared using different fibrinogen concentrations, 5, 10, 20 g/L (final concentrations in the gels). At the time of experiment, rat mesenchymal stem cells were resuspended in the thrombin solution at a concentration of approximately

5.4×10⁵ cells/mL, and 150 μL of the diluted thrombin cell solution were added in each well of 24-well microplates (about 8 000 cells/well). Finally, 150 μL of fibrinogen solution were added to the wells and tapped lightly for thorough mixing of the two components to produce a uniform fibrin gel. The plates were covered and left undisturbed at room temperature for 2 hours to allow formation and stabilization of the gels, which were then washed twice with 37 ℃ serum-free medium, before addition of 1 mL of a serum containing medium. All culture plates were place at 37 ℃ in 5% CO₂.

Cell morphology

Morphology analysis of rat mesenchymal stem cells was conducted at each time point (1, 3, 5, 7, 14, 21, and 28 days). Cells were observed by inverted phase microscope and laser scanning confocal microscopy. Digital micrographs of cell morphology and distribution were taken at the top, middle, and bottom layers of the gels.

Alkaline phosphatase assay

Passage 3 cells were digested with 0.25% trypsin and then diluted to 5×10⁵ cell/mL according the method of the above. 50 μL fibrinogen and 50 μL cell suspension with thrombin were added to 96-wells plates. Every group included four duplication wells. At 3, 5, 7, 14 , 21 and 28 days, p-nitrophenyl phosphate staining was carried out according to a modification of the methods of reference^[16]. The absorbance of p-nitrophenol product formed at

405 nm, which is proportional to the alkaline phosphatase activity, was read according to the assay manufacturer’s protocol using a Micro Plate Reader (Tecan, Austria).

Von Kossa staining

At 21 and 28 days, the culture medium was removed from each well containing the gels. Gels were washed three times with PBS, and left in PBS for at least 2 hours for an extra wash. They were then fixed with 10% formalin at room temperature overnight and placed at 4 ℃for later staining. At the time of staining, gels were washed five times with distilled water. Next, 5% silver nitrate was added for 30 minutes under strong light. Gels were washed again five times with distilled water, and 5% thiosulfate was added for 5 minutes. Gels were washed more than three times with distilled water, and photographed with an inverted light microscope.

Main outcome measures

Fibrin-gel enhancement osteogenic differentiation in rat mesenchymal stem cells, the morphological structure, alkaline phosphatase activity and mineralization were measured in this study.

Statistical analysis

The data were statistically processed using SPSS 11.0 software and were expressed as mean ± SD. One-way analysis of variance and least significant difference were used. A value of P < 0.05 was considered statistically significant.

中国组织工程研究杂志出版内容重点：生物材料；骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性；组织工程

全文链接：

讨论

Fibrin gel is a natural bio-degradable polymer material with good histocompatibility, and is a carrier to promote the release of intracellular and exogenous growth factor, is fairly structure engineering bracket materials^[24-27]. Fibrin gel can provide a substrate for cells to proliferate and migrate^[28-30]. The present study demonstrated that rat mesenchymal stem cells were able to spread, and had the ability to differentiate along the osteogenic lineage as evidenced by cellular mineral deposition within the three-dimensional fibrin gel. However, cell behavior depended on the fibrinogen concentration. Alkaline phosphatase activity was higher in gels containing a high fibrinogen concentration. These results suggest that fibrinogen concentrations are necessary to optimize cell potential osteogenic differentiation. However, the small size of the mineralization nodules were deposited after 21 days of incubation suggest that rat mesenchymal stem cells did not undergo full osteogenic differentiation when seeded in the fibrin gels in vitro, and different cell morphologies were observed depending on the formulation of the fibrin gel.

Alkaline phosphatase is a glycosylated membrane-bound enzyme that catalyses the hydrolysis of phosphomonoester bonds and may also play a physiological role in the metabolism of phosphor-ethanolamine, inorganic pyrophosphate, and pyridoxal 5’-phosphate^[31-33]. Alkaline phosphatase is thought to play a primary role in mineralization and, because it is present early in osteoblast development, has been proposed to be a progression factor in osteoblast differentiation^[34]. In the present study, alkaline phosphatase assay results showed the highest levels in fibrin gels containing a high fibrinogen concentration

(20, 10 g/L) at 21 days as compared with the non-fibrin gel control, suggesting that fibrin gels containing a high fibrinogen concentration promoted a higher alkaline phosphatase activity and therefore potentially a higher osteogenic differentiation. These results were consistent with the observations reported in recent studies^[35-38], suggesting that specific fibrinogen concentrations are necessary to optimize cell differentiation. The increase of alkaline phosphatase activity in the gel formulations containing higher fibrinogen concentrations could be explained by a higher concentration of growth factors contained in the fibrinogen component, especially like tumor growth factor-ß1 and basic fibroblast growth factor that potentially induce osteogenesis in the fibrin gels^[39-40], as well as bone mineralization by von Kossa staining to further assess the osteogenic differentiation of rat mesenchymal stem cells in the fibrin gel.

Von Kossa staining in a gel containing a high fibrinogen concentration (10 or 20 g/L) demonstrated small nodules of calcium phosphate deposition, associated with the presence of cells and mostly in the regions of the gels depicting a partial dissolution. These nodules remained, however, only few in number and relatively small at 21 days. No nodule was observed on the top of the gels, where cells were present in larger numbers, and tended to form a monolayer. This suggests the influence of the gel structure and potentially the different chemical environment influencing cell-cell interactions on the formation of the bone nodules in the three-dimensional gel structure, where cells are entrapped and form clusters, compared to the uniform monolayer proliferation on top of the gels. The relatively low number of nodules observed in the gels could be attributable to the low number of cells initially seeded in the gel and the relatively large volume of growth medium used in the present experiment. It is therefore possible that increasing the initial cell seeding density and reducing the volume of growth medium in the present study would increase bone mineralization in the fibrin gel. It should be emphasized that standard growth medium was used in the present study for culturing mesenchymal stem cells in the fibrin gels, in contrast with most of the studies reported in the literature on stem cell differentiation in various scaffolds, using a supplement of dexamethasone and/or b-ascorbate, known to induce osteogenic differentiation^[41-44].

Overall, this study demonstrated that rat mesenchymal stem cell behavior in fibrin gels in vitro depended on the fibrinogen concentration. Fibrin scaffold supports the growth of rat mesenchymal stem cells, especially in formulations containing a low fibrinogen concentration, and offers a potential for their differentiation into osteoblasts, especially in formulations containing a high fibrinogen complex concentration. However, although rat mesenchymal stem cells appeared to undergo osteogenic differentiation in the gel formulations containing a high fibrinogen concentration, results also suggest that the cells did not fully differentiate.

Other future studies using different fibrin formulations in combination with synthetic materials and/or additional growth factors could therefore be conducted to allow a complete differentiation of the mesenchymal stem cells presence in this scaffold. Furthermore, other future work will explore animal models to extend the in vitro observations reported here to bone healing in vivo.

中国组织工程研究杂志出版内容重点：生物材料；骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性；组织工程
全文链接：

纤维蛋白凝胶促进大鼠间充质干细胞的成骨分化

Fibrin gel enhances osteogenic differentiation of rat mesenchymal stem cells

PDF

可视化

被引次数

摘要/Abstract

引用本文

使用本文

图/表（结果） 2

参考文献

相关文章 15

引言

材料方法

讨论

文章快阅

延伸阅读

编辑推荐

Metrics

本文评价

[1]	蒲锐, 陈子扬, 袁凌燕. 不同细胞来源外泌体保护心脏的特点与效应[J]. 中国组织工程研究, 2021, 25(在线): 1-.
[2]	林清凡, 解一新, 陈婉清, 叶振忠, 陈幼芳. 人胎盘源间充质干细胞条件培养液可上调缺氧状态下BeWo细胞活力和紧密连接因子的表达[J]. 中国组织工程研究, 2021, 25(在线): 4970-4975.
[3]	汪显耀, 关亚琳, 刘忠山. 提高间充质干细胞治疗难愈性创面的策略[J]. 中国组织工程研究, 2021, 25(7): 1081-1087.
[4]	万然, 史旭, 刘京松, 王岩松. 间充质干细胞分泌组治疗脊髓损伤的研究进展[J]. 中国组织工程研究, 2021, 25(7): 1088-1095.
[5]	王诗琦, 张金生. 中医药调控缺血缺氧微环境对骨髓间充质干细胞增殖、分化及衰老的影响[J]. 中国组织工程研究, 2021, 25(7): 1129-1134.
[6]	孔德胜, 何晶晶, 冯宝峰, 郭瑞云, Asiamah Ernest Amponsah, 吕飞, 张舒涵, 张晓琳, 马隽, 崔慧先. 间充质干细胞修复大动物模型脊髓损伤疗效评价的Meta分析[J]. 中国组织工程研究, 2021, 25(7): 1142-1148.
[7]	侯婧瑛, 于萌蕾, 郭天柱, 龙会宝, 吴浩. 缺氧预处理激活HIF-1α/MALAT1/VEGFA通路促进骨髓间充质干细胞生存和血管再生[J]. 中国组织工程研究, 2021, 25(7): 985-990.
[8]	史洋洋, 秦英飞, 吴福玲, 何潇, 张雪静. 胎盘间充质干细胞预处理预防小鼠毛细支气管炎[J]. 中国组织工程研究, 2021, 25(7): 991-995.
[9]	梁学奇, 郭黎姣, 陈贺捷, 武杰, 孙雅琪, 邢稚坤, 邹海亮, 陈雪玲, 吴向未. 泡状棘球绦虫原头蚴抑制骨髓间充质干细胞向成纤维细胞的分化[J]. 中国组织工程研究, 2021, 25(7): 996-1001.
[10]	樊全宝, 罗惠娜, 王丙云, 陈胜锋, 崔连旭, 江文康, 赵明明, 王静静, 罗冬章, 陈志胜, 白银山, 刘璨颖, 张晖. 低氧培养犬脂肪间充质干细胞的生物学特性[J]. 中国组织工程研究, 2021, 25(7): 1002-1007.
[11]	耿瑶, 尹志良, 李兴平, 肖东琴, 侯伟光. hsa-miRNA-223-3p调控人骨髓间充质干细胞成骨分化的作用[J]. 中国组织工程研究, 2021, 25(7): 1008-1013.
[12]	伦志刚, 金晶, 王添艳, 李爱民. 过氧化物还原酶6干预骨髓间充质干细胞增殖及体外向神经谱系诱导分化[J]. 中国组织工程研究, 2021, 25(7): 1014-1018.
[13]	朱雪芬, 黄成, 丁健, 戴永平, 刘元兵, 乐礼祥, 王亮亮, 杨建东. 胶质细胞神经营养因子诱导骨髓间充质干细胞向功能性神经元分化的机制[J]. 中国组织工程研究, 2021, 25(7): 1019-1025.
[14]	段丽芸, 曹晓沧. 人胎盘间充质干细胞来源细胞外囊泡调节肠炎小鼠肠黏膜胶原的沉积[J]. 中国组织工程研究, 2021, 25(7): 1026-1031.
[15]	裴丽丽, 孙贵才, 王弟. 丹酚酸B抑制骨髓间充质干细胞氧化损伤及促进分化为心肌样细胞[J]. 中国组织工程研究, 2021, 25(7): 1032-1036.