DIFFERENTIATION OF DIRECTLY CO-CULTURED BONE MARROW MESENCHYMAL STEM CELLS AND LIGAMENT FIBROBLASTS INTO LIGAMENT CELLS AFTER INDUCED BY TRANSFORMING GROWTH FACTORβ<sub>1</sub> AND BASIC FIBROBLAST GROWTH FACTOR 1_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

 LIUYi , ZHANGChenghao , FANQinghong , SUNPengpeng , WUShuhong

The First Department of Orthopaedics, the Affiliated Hospital of Zunyi Medical College, Zunyi Guizhou, 563003, P. R. China;

Corresponding author：

LIUYi, Email: 13308529536@163.com

Keywords：

Ligament fibroblasts; Bone marrow mesenchymal stem cells; Directly co-culture; Transforming growth factorβ₁; Basic fibroblast growth factor 1

DOI：

10.7507/1002-1892.20140304

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Objective To investigate the effect of transforming growth factorβ1 (TGF-β₁) and basic fibroblast growth factor 1 (bFGF-1) on the cellular activities, prol iferation, and expressions of ligament-specific mRNA and proteins in bone marrow mesenchymal stem cells (BMSCs) and ligament fibroblasts (LFs) after directly co-cultured. Methods BMSCs from 3-month-old Sprague Dawley rats were isolated and cultured using intensity gradient centrifugation. LFs were isolated using collagenase. The cells at passage 3 were divided into 6 groups: non-induced BMSCs group (group A), non-induced LFs group (group B), non-induced co-cultured BMSCs and LFs group (group C), induced BMSCs group (group D), induced LFs group (group E), and induced co-cultured BMSCs and LFs group (group F). The cellular activities and prol iferation were examined by inverted contrast microscope and MTT; the concentrations of collagen type Ⅰ and type Ⅲ were determined by ELISA; and mRNA expressions of collagen types I andⅢ, fibronectin, tenascin C, and matrix metalloproteinase 2 (MMP-2) were measured by real-time fluorescent quantitative PCR. Results A single cell layer formed in the co-cultured cells under inverted contrast microscope. Group F had fastest cell fusion ( > 90%). The MTT result indicated that group F showed the highest absorbance (A) value, followed by group D, and group B showed the lowest A value at 9 days after culture, showing significant difference (P < 0.05). Moreover, the result of ELISA showed that group F had the highest concentration of collagen type Ⅰ and type Ⅲ (P < 0.05); the concentration of collagen type Ⅲ in group E was significantly higher than that in group D (P < 0.05), but no significant difference was found in the concentration of collagen type Ⅰ between 2 groups (P > 0.05). The ratios of collagen type Ⅰ to type Ⅲ were 1.17, 1.19, 1.10, 1.25, 1.17, and 1.18 in groups A-F; group D was higher than the other groups. The real-time fluorescent quantitative PCR results revealed that the mRNA expressions of collagen type Ⅰ and type Ⅲ and fibronectin were highest in group F; the expression of tenascin C was highest in group D; the expression of MMP-2 was highest in group E; and all differencs were significant (P < 0.05). Conclusion Directly co-cultured BMSCs and LFs induced by TGF-β₁ and bFGF-1 have higher cellular activities, proliferation, and expressions of ligament-specific mRNA and protein, which can be used as a potential source for ligament tissue engineering.

Citation： LIUYi, ZHANGChenghao, FANQinghong, SUNPengpeng, WUShuhong. DIFFERENTIATION OF DIRECTLY CO-CULTURED BONE MARROW MESENCHYMAL STEM CELLS AND LIGAMENT FIBROBLASTS INTO LIGAMENT CELLS AFTER INDUCED BY TRANSFORMING GROWTH FACTORβ₁ AND BASIC FIBROBLAST GROWTH FACTOR 1. Chinese Journal of Reparative and Reconstructive Surgery, 2014, 28(11): 1406-1412. doi: 10.7507/1002-1892.20140304 Copy

1.	Jiang D, Xu B, Yang M, et al. Efficacy of tendon stem cells in fibroblastderived matrix for tendon tissue engineering. Cytotherapy, 2013, 16(5):662-673.
2.	Almarza AJ, Augustine SM, Woo SL. Changes in gene expression of matrix constituents with respect to passage of ligament and tendon fibroblasts. Ann Biomed Eng, 2008, 36(12):1927-1933.
3.	Segawa Y, Muneta T, Makino H, et al. Mesenchymal stem cells derived from synovium, meniscus, anterior cruciate ligament, and articular chondrocytes share similar gene expression profiles. J Orthop Res, 2009, 27(4):435-441.
4.	Cheng MT, Liu CL, Chen TH, et al. Comparison of potentials between stem cells isolated from human anterior cruciate ligament and bone marrow for ligament tissue engineering. Tissue Eng Part A, 2010, 16(7):2237-2253.
5.	张蕾, 陈槐卿, Wang Xiong, 等.与韧带成纤维细胞间接共培养对大鼠骨髓间充质干细胞胶原蛋白和韧粘素-C表达的影响.生物医学工程学杂志, 2007, 24(4):846-851.
6.	Mujaj S, Manton K, Upton Z, et al. Serum-free primary human fibroblast and keratinocyte coculture. Tissue Eng Part A, 2010, 16(4):1407-1420.
7.	Lee IC, Wang JH, Lee YT, et al. The differentiation of mesenchymal stem cells by mechanical stress or/and co-culture system. Biochem Biophys Res Commun, 2007, 352(1):147-152.
8.	Luo Q, Song G, Song Y, et al. Indirect co-culture with tenocytes promotes proliferation and mRNA expression of tendon/ligament related genes in rat bone marrow mesenchymal stem cells. Cytotechnology, 2009, 61(1-2):1-10.
9.	Ge Z, Goh JC, Lee EH. Selection of cell source for ligament tissue engineering. Cell Transplant, 2005, 14(8):573-583.
10.	Cooper JA Jr, Bailey LO, Carter JN, et al. Evaluation of the anterior cruciate ligament, medial collateral ligament, achilles tendon and patellar tendon as cell sources for tissue-engineered ligament. Biomaterials, 2006, 27(13):2747-2754.
11.	Nahai H, Terada K, Watanabe G, et al. Differentiation of liver epithel ial (stem-like) cells into hepatocytes induced by coculture with hepatics tellate cells. Biochem Biophys Res Commun, 2002, 293(5):1420-1425.
12.	Chen Y, Desautel M, Anderson A, et al. Collagen synthesis is not altered in women with stress urinary incongtinence, Neurourol Urodyn, 2004, 23(4):367-373.
13.	王春丽, 梅虎, 谢静, 等.共培养下前交叉韧带成纤维细胞中LOXs与MMPs的基因表达情况.生物医学工程杂志, 2013, 30(4):777-781.
14.	Pitiyage GN, Lim KP, Gemenitzidis E, et al. Increased secretion of tissue inhibitors of metalloproteinases 1 and 2(TIMPs-1 and-2) in fibroblasts are early indicators of oral sub-mucous fibrosis and ageing, J Oral Pathol Med, 2012, 41(6):454-462.
15.	Schumm MA, Cas tellanos DA, Frydel BR, et al. Direct cell-cell contact required for neurotrophic effect of chromaffin cells on neural progenitor cells. Brain Res Dev Brain Res, 2003, 146(1-2):1-13.
16.	Li H, Tang M, Liang H, et al. Coculture of embryonic ventricular myocytes and mouse embryonic stem cell enhance intercellular signaling by upregulation of connexin43. Cell Physiol Biochem, 2013, 32(1):53-63.
17.	Cheng MT, Liu CL, Chen TH, et al. Comparison of potentials between stem cells isolated from human anterior cruciate ligament and bone marrow for ligament tissue engineering. Tissue Eng Part A, 2010, 16(7):2237-2253.
18.	Saiga K, Furumatsu T, Yoshida A, et al. Combined use of bFGF and GDF-5 enhances the healing of medial collateral ligament injury. Biochem Biophys Res Commun, 2010, 402(2):329-334.
19.	Wwi XL, Lin L, Hou Y, et al. Construction of recombinant adenovirus co-expression vector carrying the human transforming growth factorbeta1 and vascular endothelial growth factor genes and its effect on anterior cruciate ligament fibroblasts. Chin Med J (Engl), 2008, 121(15): 1426-1432.
20.	Hagerty P, Lee A, Calve S, et al. The effect of growth factors on both collagen synthesis and tensile strength of engineered human ligaments. Biomaterials, 2012, 33(27):6355-6361.
21.	Bakhshayesh M, Soleimani M, Mehdizadeh M, et al. Effects of TGF-βand b-FGF on the potential of peri pheral blood-borne stem cells and bone marrow-derived stem cells in wound healing in a murine model. Inflammation, 2012, 35(1):138-142.
22.	Hasegawa T, Chosa N, Asakawa T, et al. Differential effects of TGF-β₁ and FGF-2 on SDF-1αexpression in human periodontal ligament cells derived from deciduous teeth in vitro. Int J Mol Med, 2012, 30(1):35-40.
23.	Xiao L, Du Y, Shen Y, et al. TGF-beta 1 induced fibroblast proliferation is mediated by the FGF-2/ERK pathway. Front Biosci (Landmark Ed), 2012, 17:2667-2674.

1. Jiang D, Xu B, Yang M, et al. Efficacy of tendon stem cells in fibroblastderived matrix for tendon tissue engineering. Cytotherapy, 2013, 16(5):662-673.
2. Almarza AJ, Augustine SM, Woo SL. Changes in gene expression of matrix constituents with respect to passage of ligament and tendon fibroblasts. Ann Biomed Eng, 2008, 36(12):1927-1933.
3. Segawa Y, Muneta T, Makino H, et al. Mesenchymal stem cells derived from synovium, meniscus, anterior cruciate ligament, and articular chondrocytes share similar gene expression profiles. J Orthop Res, 2009, 27(4):435-441.
4. Cheng MT, Liu CL, Chen TH, et al. Comparison of potentials between stem cells isolated from human anterior cruciate ligament and bone marrow for ligament tissue engineering. Tissue Eng Part A, 2010, 16(7):2237-2253.
5. 张蕾, 陈槐卿, Wang Xiong, 等.与韧带成纤维细胞间接共培养对大鼠骨髓间充质干细胞胶原蛋白和韧粘素-C表达的影响.生物医学工程学杂志, 2007, 24(4):846-851.
6. Mujaj S, Manton K, Upton Z, et al. Serum-free primary human fibroblast and keratinocyte coculture. Tissue Eng Part A, 2010, 16(4):1407-1420.
7. Lee IC, Wang JH, Lee YT, et al. The differentiation of mesenchymal stem cells by mechanical stress or/and co-culture system. Biochem Biophys Res Commun, 2007, 352(1):147-152.
8. Luo Q, Song G, Song Y, et al. Indirect co-culture with tenocytes promotes proliferation and mRNA expression of tendon/ligament related genes in rat bone marrow mesenchymal stem cells. Cytotechnology, 2009, 61(1-2):1-10.
9. Ge Z, Goh JC, Lee EH. Selection of cell source for ligament tissue engineering. Cell Transplant, 2005, 14(8):573-583.
10. Cooper JA Jr, Bailey LO, Carter JN, et al. Evaluation of the anterior cruciate ligament, medial collateral ligament, achilles tendon and patellar tendon as cell sources for tissue-engineered ligament. Biomaterials, 2006, 27(13):2747-2754.
11. Nahai H, Terada K, Watanabe G, et al. Differentiation of liver epithel ial (stem-like) cells into hepatocytes induced by coculture with hepatics tellate cells. Biochem Biophys Res Commun, 2002, 293(5):1420-1425.
12. Chen Y, Desautel M, Anderson A, et al. Collagen synthesis is not altered in women with stress urinary incongtinence, Neurourol Urodyn, 2004, 23(4):367-373.
13. 王春丽, 梅虎, 谢静, 等.共培养下前交叉韧带成纤维细胞中LOXs与MMPs的基因表达情况.生物医学工程杂志, 2013, 30(4):777-781.
14. Pitiyage GN, Lim KP, Gemenitzidis E, et al. Increased secretion of tissue inhibitors of metalloproteinases 1 and 2(TIMPs-1 and-2) in fibroblasts are early indicators of oral sub-mucous fibrosis and ageing, J Oral Pathol Med, 2012, 41(6):454-462.
15. Schumm MA, Cas tellanos DA, Frydel BR, et al. Direct cell-cell contact required for neurotrophic effect of chromaffin cells on neural progenitor cells. Brain Res Dev Brain Res, 2003, 146(1-2):1-13.
16. Li H, Tang M, Liang H, et al. Coculture of embryonic ventricular myocytes and mouse embryonic stem cell enhance intercellular signaling by upregulation of connexin43. Cell Physiol Biochem, 2013, 32(1):53-63.
17. Cheng MT, Liu CL, Chen TH, et al. Comparison of potentials between stem cells isolated from human anterior cruciate ligament and bone marrow for ligament tissue engineering. Tissue Eng Part A, 2010, 16(7):2237-2253.
18. Saiga K, Furumatsu T, Yoshida A, et al. Combined use of bFGF and GDF-5 enhances the healing of medial collateral ligament injury. Biochem Biophys Res Commun, 2010, 402(2):329-334.
19. Wwi XL, Lin L, Hou Y, et al. Construction of recombinant adenovirus co-expression vector carrying the human transforming growth factorbeta1 and vascular endothelial growth factor genes and its effect on anterior cruciate ligament fibroblasts. Chin Med J (Engl), 2008, 121(15): 1426-1432.
20. Hagerty P, Lee A, Calve S, et al. The effect of growth factors on both collagen synthesis and tensile strength of engineered human ligaments. Biomaterials, 2012, 33(27):6355-6361.
21. Bakhshayesh M, Soleimani M, Mehdizadeh M, et al. Effects of TGF-βand b-FGF on the potential of peri pheral blood-borne stem cells and bone marrow-derived stem cells in wound healing in a murine model. Inflammation, 2012, 35(1):138-142.
22. Hasegawa T, Chosa N, Asakawa T, et al. Differential effects of TGF-β₁ and FGF-2 on SDF-1αexpression in human periodontal ligament cells derived from deciduous teeth in vitro. Int J Mol Med, 2012, 30(1):35-40.
23. Xiao L, Du Y, Shen Y, et al. TGF-beta 1 induced fibroblast proliferation is mediated by the FGF-2/ERK pathway. Front Biosci (Landmark Ed), 2012, 17:2667-2674.

Chinese Journal of Reparative and Reconstructive Surgery

DIFFERENTIATION OF DIRECTLY CO-CULTURED BONE MARROW MESENCHYMAL STEM CELLS AND LIGAMENT FIBROBLASTS INTO LIGAMENT CELLS AFTER INDUCED BY TRANSFORMING GROWTH FACTORβ₁ AND BASIC FIBROBLAST GROWTH FACTOR 1

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DIFFERENTIATION OF DIRECTLY CO-CULTURED BONE MARROW MESENCHYMAL STEM CELLS AND LIGAMENT FIBROBLASTS INTO LIGAMENT CELLS AFTER INDUCED BY TRANSFORMING GROWTH FACTORβ1 AND BASIC FIBROBLAST GROWTH FACTOR 1

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DIFFERENTIATION OF DIRECTLY CO-CULTURED BONE MARROW MESENCHYMAL STEM CELLS AND LIGAMENT FIBROBLASTS INTO LIGAMENT CELLS AFTER INDUCED BY TRANSFORMING GROWTH FACTORβ₁ AND BASIC FIBROBLAST GROWTH FACTOR 1