Comparison of biological characteristics between bone marrow mesenchymal stem cells and anterior cruciate ligament derived mesenchymal stem cells in rats_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

HAO Zichen ¹ , WU Hao ¹ , LI Yang ¹ , WANG Shanzheng ² ,  LU Jun ²

1. Medical School of Southeast University, Nanjing Jiangsu, 210009, P.R.China;
2. Department of Orthopaedics, Zhongda Hospital, Medical School of Southeast University, Nanjing Jiangsu, 210009, P.R.China;

Corresponding author：

LU Jun, Email: ljseueducn@126.com

Keywords：

Bone marrow mesenchymal stem cells; anterior cruciate ligament derived mesenchymal stem cells; proliferation and differentiation; rat

DOI：

10.7507/1002-1892.201611021

Video：

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Objective To compare the biological characteristics of bone marrow mesenchymal stem cells (BMSCs) and anterior cruciate ligament derived mesenchymal stem cells (ACL-MSCs) from ratsin vitro. Methods Ten male SPF-level BN rats, weighing 200-220 g, were selected to obtain anterior cruciate ligaments and bone marrows, and ACL-MSCs and BMSCs were isolated for passage culture respectively under sterile condition. The cell morphology was observed, and the cells at passage 3 were used to detect the surface markers of CD34, CD45, CD90, and CD29 by flow cytometry, the ability of cell proliferation by cell counting kit 8 (CCK-8), and colony formation ability by clone forming test. The mRNA levels of differentiation related genes [alkaline phosphatas (ALP), bone gamma-carboxyglutamate protein, runt related transcription factor 2, bone morphogenetic protein 2 (BMP-2), secreted phosphoprotein 1 (Spp1), collagen type II α1 (Col2α1), Aggrecan (Acan), Sox9, peroxisome proliferator activated receptor γ2 (PPARγ2), and CCAAT-enhancer-binding protein-α] were also determined by real-time fluorescent quantitative PCR. Results BMSCs and ACL-MSCs had similar morphology, adherent cells displaying long fusiform. The immunoprofile of ACL-MSCs and BMSCs at passage 3 was positive for CD29 and CD90 and was negative for CD45 and CD34. The absorbance (A) value of ACL-MSCs (1.11±0.08) was significantly higher than that of BMSCs (0.78±0.05) (t=3.599,P=0.023); the number of colonies of ACL-MSCs [(53.00±5.51)/hole] was significantly more than that of BMSCs [(30.67±4.84)/hole] (t=3.045,P=0.038). The results of toluidine blue staining, alizarin red staining, and oil red O staining were positive in BMSCs and ACL-MSCs at 21 days after osteogenic, chondrogenic, and adipogenic induction. The mRNA expressions of BMP-2, Spp1, Col2α1, Acan, Sox9, and PPARγ2 in ACL-MSCs were significantly higher than those in BMSCs (P<0.01). Conclusion The proliferation potential of ACL-MSCs is greater than that of BMSCs, and the former is apt to differentiate into chondrocytes. ACL-MSCs are promising cells to promote tendon-bone healing.

Citation： HAO Zichen, WU Hao, LI Yang, WANG Shanzheng, LU Jun. Comparison of biological characteristics between bone marrow mesenchymal stem cells and anterior cruciate ligament derived mesenchymal stem cells in rats. Chinese Journal of Reparative and Reconstructive Surgery, 2017, 31(4): 473-480. doi: 10.7507/1002-1892.201611021 Copy

1.	Lu HH, Thomopoulos S. Functional attachment of soft tissues to bone: development, healing, and tissue engineering. Annu Rev Biomed Eng, 2013, 15: 201-226.
2.	Hoshino Y, Fu FH, Irrgang JJ, et al. Can joint contact dynamics be restored by anterior cruciate ligament reconstruction. Clin Orthop Relat Res, 2013, 471(9): 2924-2931.
3.	Bissell L, Tibrewal S, Sahni V, et al. Growth factors and platelet rich plasma in anterior cruciate ligament reconstruction. Curr Stem Cell Res Ther, 2015, 10(1): 19-25.
4.	Zhu Z, Yu A, Hou M, et al. Effects of Sox9 gene therapy on the healing of bone-tendon junction:An experimental study. Indian J Orthop, 2014, 48(1): 88-95.
5.	Soon MY, Hassan A, Hui JH, et al. An analysis of soft tissue allograft anterior cruciateligament reconstruction in a rabbit model: a short-term study of the use ofmesenchymal stemcells to enhance tendon osteointegration. Am J Sports Med, 2007, 35(6): 962-971.
6.	Kanazawa T, Soejima T, Noguchi K, et al. Tendon-to-bone healing using autologous bone marrow-derived mesenchymal stem cells in ACL reconstruction without a tibial bone tunnel-A histological study-. Muscles Ligaments Tendons J, 2014, 4(2): 201-206.
7.	Figueroa D, Espinosa M, Calvo R, et al. Anterior cruciate ligament regenerationusing mesenchymal stem cells and collagen type I scaffold in a rabbit model. Knee Surg Sports Traumatol Arthrosc, 2014, 22(5): 1196-1202.
8.	Mifune Y, Matsumoto T, Ota S, et al. Therapeutic potential of anterior cruciate ligament-derived stem cells for anterior cruciate ligament reconstruction. CellTransplant, 2012, 21(8): 1651-1665.
9.	Matsumoto T, Kubo S, Sasaki K, et al. Acceleration of tendon-bone healing of anterior cruciate ligament graft using autologous ruptured tissue. Am J Sports Med, 2012, 40(6): 1296-1302.
10.	Mifune Y, Matsumoto T, Takayama K, et al. Tendon graft revitalization using adult anterior cruciate ligament (ACL)-derived CD34+ cell sheets for ACLreconstruction. Biomaterials, 2013, 34(22): 5476-5487.
11.	Jiang Y, Jahagirdar BN, Reinhardt RL, et al. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature, 2002, 418(6893): 41-49.
12.	Jones E, McGonagle D. Human bone marrow mesenchymal stem cells in vivo. Rheumatology (Oxford), 2008, 47(2): 126-131.
13.	Caplan AI. Adult mesenchymal stem cells for tissue engineering versus regenerative medicine. J Cell Physiol, 2007, 213(2): 341-347.
14.	Muir P, Hans EC, Racette M, et al. Autologous Bone Marrow-Derived Mesenchymal Stem Cells Modulate Molecular Markers of Inflammation in Dogs with Cruciate Ligament Rupture. PLoS One, 2016, 11(8): e0159095.
15.	Sakaguchi Y, Sekiya I, Yagishita K, et al. Comparison of human stem cells derived from various mesenchymal tissues: superiority of synovium as a cell source. Arthritis Rheum, 2005, 52(8): 2521-2529.
16.	Shirasawa S, Sekiya I, Sakaguchi Y, et al.In vitro chondrogenesis of human synovium-derived mesenchymal stem cells: optimal condition and comparison with bone marrow-derived cells. J Cell Biochem, 2006, 97(1): 84-97.
17.	Fu WL, Zhou CY, Yu JK. A new source of mesenchymal stem cells for articular cartilage repair: MSCs derived from mobilized peripheral blood share similar biological characteristics in vitro and chondrogenesis in vivo as MSCs from bone marrow in a rabbit model. Am J Sports Med, 2014, 42(3): 592-601.
18.	Fu WL, Zhang JY, Fu X, et al. Comparative study of the biological characteristics of mesenchymal stem cells from bone marrow and peripheral blood of rats. Tissue Eng Part A, 2012, 18(17-18): 1793-1803.
19.	Huang TF, Chen YT, Yang TH, et al. Isolation and characterization of mesenchymal stromal cells from human anterior cruciate ligament. Cytotherapy, 2008, 10(8): 806-814.
20.	Hao ZC, Wang SZ, Zhang XJ, et al. Stem cell therapy: a promising biological strategy for tendon-bone healing after anteriorcruciate ligament reconstruction. Cell Prolif, 2016, 49(2): 154-162.
21.	Steinert AF, Kunz M, Prager P, et al. Mesenchymal stem cell characteristics of human anterior cruciate ligament outgrowth cells. Tissue Eng Part A, 2011, 17(9-10): 1375-1388.
22.	Cheng MT, Yang HW, Chen TH, et al. Isolation and characterization of multipotent stem cells from human cruciate ligaments. Cell Prolif, 2009, 42(4): 448-460.
23.	Ghebes CA, Kelder C, Schot T, et al. Anterior cruciate ligament- and hamstring tendon-derived cells:in vitro differential properties of cells involved in ACL reconstruction. J Tissue Eng Regen Med, 2015. [Epub ahead of print].
24.	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.
25.	Fu W, Li Q, Tang X, et al. Mesenchymal stem cells reside in anterior cruciate ligament remnants in situ. Int Orthop, 2016, 40(7): 1523-1530.
26.	Dominici M, Le BK, Mueller I, et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy, 2006, 8(4): 315-317.
27.	Harichandan A, Bühring HJ. Prospective isolation of human MSC. Best Pract Res Clin Haematol, 2011, 24(1): 25-36.
28.	Nery AA, Nascimento IC, Glaser T, et al. Human mesenchymal stem cells: from immunophenotyping by flow cytometry to clinical applications. Cytometry A, 2013, 83(1): 48-61.
29.	Mifune Y, Matsumoto T, Ota S, et al. Therapeutic potential of anterior cruciate ligament-derived stem cells for anterior cruciate ligament reconstruction. Cell Transplant, 2012, 21(8): 1651-1665.
30.	Matsumoto T, Ingham SM, Mifune Y, et al. Isolation and characterization of human anterior cruciate ligament-derived vascular stem cells. Stem Cells Dev, 2012, 21(6): 859-872.
31.	Lee DH, Ng J, Chung JW, et al. Impact of chronicity of injury on the proportion of mesenchymal stromal cells derived from anterior cruciate ligaments. Cytotherapy, 2014, 16(5): 586-598.
32.	Al-Nbaheen M, Vishnubalaji R, Ali D, et al. Human stromal (mesenchymal) stem cells from bone marrow, adipose tissue and skin exhibit differences in molecular phenotype and differentiation potential. Stem Cell Rev, 2013, 9(1): 32-43.
33.	Bianco P, Cao X, Frenette PS, et al. The meaning, the sense and the significance: translating the science of mesenchymal stem cells into medicine. Nat Med, 2013, 19(1): 35-42.
34.	Lee DH, Ng J, Kim SB, et al. Effect of donor age on the proportion of mesenchymal stem cells derived from anterior cruciate ligaments. PLoS One, 2015, 10(3): e0117224.

1. Lu HH, Thomopoulos S. Functional attachment of soft tissues to bone: development, healing, and tissue engineering. Annu Rev Biomed Eng, 2013, 15: 201-226.
2. Hoshino Y, Fu FH, Irrgang JJ, et al. Can joint contact dynamics be restored by anterior cruciate ligament reconstruction. Clin Orthop Relat Res, 2013, 471(9): 2924-2931.
3. Bissell L, Tibrewal S, Sahni V, et al. Growth factors and platelet rich plasma in anterior cruciate ligament reconstruction. Curr Stem Cell Res Ther, 2015, 10(1): 19-25.
4. Zhu Z, Yu A, Hou M, et al. Effects of Sox9 gene therapy on the healing of bone-tendon junction:An experimental study. Indian J Orthop, 2014, 48(1): 88-95.
5. Soon MY, Hassan A, Hui JH, et al. An analysis of soft tissue allograft anterior cruciateligament reconstruction in a rabbit model: a short-term study of the use ofmesenchymal stemcells to enhance tendon osteointegration. Am J Sports Med, 2007, 35(6): 962-971.
6. Kanazawa T, Soejima T, Noguchi K, et al. Tendon-to-bone healing using autologous bone marrow-derived mesenchymal stem cells in ACL reconstruction without a tibial bone tunnel-A histological study-. Muscles Ligaments Tendons J, 2014, 4(2): 201-206.
7. Figueroa D, Espinosa M, Calvo R, et al. Anterior cruciate ligament regenerationusing mesenchymal stem cells and collagen type I scaffold in a rabbit model. Knee Surg Sports Traumatol Arthrosc, 2014, 22(5): 1196-1202.
8. Mifune Y, Matsumoto T, Ota S, et al. Therapeutic potential of anterior cruciate ligament-derived stem cells for anterior cruciate ligament reconstruction. CellTransplant, 2012, 21(8): 1651-1665.
9. Matsumoto T, Kubo S, Sasaki K, et al. Acceleration of tendon-bone healing of anterior cruciate ligament graft using autologous ruptured tissue. Am J Sports Med, 2012, 40(6): 1296-1302.
10. Mifune Y, Matsumoto T, Takayama K, et al. Tendon graft revitalization using adult anterior cruciate ligament (ACL)-derived CD34+ cell sheets for ACLreconstruction. Biomaterials, 2013, 34(22): 5476-5487.
11. Jiang Y, Jahagirdar BN, Reinhardt RL, et al. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature, 2002, 418(6893): 41-49.
12. Jones E, McGonagle D. Human bone marrow mesenchymal stem cells in vivo. Rheumatology (Oxford), 2008, 47(2): 126-131.
13. Caplan AI. Adult mesenchymal stem cells for tissue engineering versus regenerative medicine. J Cell Physiol, 2007, 213(2): 341-347.
14. Muir P, Hans EC, Racette M, et al. Autologous Bone Marrow-Derived Mesenchymal Stem Cells Modulate Molecular Markers of Inflammation in Dogs with Cruciate Ligament Rupture. PLoS One, 2016, 11(8): e0159095.
15. Sakaguchi Y, Sekiya I, Yagishita K, et al. Comparison of human stem cells derived from various mesenchymal tissues: superiority of synovium as a cell source. Arthritis Rheum, 2005, 52(8): 2521-2529.
16. Shirasawa S, Sekiya I, Sakaguchi Y, et al.In vitro chondrogenesis of human synovium-derived mesenchymal stem cells: optimal condition and comparison with bone marrow-derived cells. J Cell Biochem, 2006, 97(1): 84-97.
17. Fu WL, Zhou CY, Yu JK. A new source of mesenchymal stem cells for articular cartilage repair: MSCs derived from mobilized peripheral blood share similar biological characteristics in vitro and chondrogenesis in vivo as MSCs from bone marrow in a rabbit model. Am J Sports Med, 2014, 42(3): 592-601.
18. Fu WL, Zhang JY, Fu X, et al. Comparative study of the biological characteristics of mesenchymal stem cells from bone marrow and peripheral blood of rats. Tissue Eng Part A, 2012, 18(17-18): 1793-1803.
19. Huang TF, Chen YT, Yang TH, et al. Isolation and characterization of mesenchymal stromal cells from human anterior cruciate ligament. Cytotherapy, 2008, 10(8): 806-814.
20. Hao ZC, Wang SZ, Zhang XJ, et al. Stem cell therapy: a promising biological strategy for tendon-bone healing after anteriorcruciate ligament reconstruction. Cell Prolif, 2016, 49(2): 154-162.
21. Steinert AF, Kunz M, Prager P, et al. Mesenchymal stem cell characteristics of human anterior cruciate ligament outgrowth cells. Tissue Eng Part A, 2011, 17(9-10): 1375-1388.
22. Cheng MT, Yang HW, Chen TH, et al. Isolation and characterization of multipotent stem cells from human cruciate ligaments. Cell Prolif, 2009, 42(4): 448-460.
23. Ghebes CA, Kelder C, Schot T, et al. Anterior cruciate ligament- and hamstring tendon-derived cells:in vitro differential properties of cells involved in ACL reconstruction. J Tissue Eng Regen Med, 2015. [Epub ahead of print].
24. 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.
25. Fu W, Li Q, Tang X, et al. Mesenchymal stem cells reside in anterior cruciate ligament remnants in situ. Int Orthop, 2016, 40(7): 1523-1530.
26. Dominici M, Le BK, Mueller I, et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy, 2006, 8(4): 315-317.
27. Harichandan A, Bühring HJ. Prospective isolation of human MSC. Best Pract Res Clin Haematol, 2011, 24(1): 25-36.
28. Nery AA, Nascimento IC, Glaser T, et al. Human mesenchymal stem cells: from immunophenotyping by flow cytometry to clinical applications. Cytometry A, 2013, 83(1): 48-61.
29. Mifune Y, Matsumoto T, Ota S, et al. Therapeutic potential of anterior cruciate ligament-derived stem cells for anterior cruciate ligament reconstruction. Cell Transplant, 2012, 21(8): 1651-1665.
30. Matsumoto T, Ingham SM, Mifune Y, et al. Isolation and characterization of human anterior cruciate ligament-derived vascular stem cells. Stem Cells Dev, 2012, 21(6): 859-872.
31. Lee DH, Ng J, Chung JW, et al. Impact of chronicity of injury on the proportion of mesenchymal stromal cells derived from anterior cruciate ligaments. Cytotherapy, 2014, 16(5): 586-598.
32. Al-Nbaheen M, Vishnubalaji R, Ali D, et al. Human stromal (mesenchymal) stem cells from bone marrow, adipose tissue and skin exhibit differences in molecular phenotype and differentiation potential. Stem Cell Rev, 2013, 9(1): 32-43.
33. Bianco P, Cao X, Frenette PS, et al. The meaning, the sense and the significance: translating the science of mesenchymal stem cells into medicine. Nat Med, 2013, 19(1): 35-42.
34. Lee DH, Ng J, Kim SB, et al. Effect of donor age on the proportion of mesenchymal stem cells derived from anterior cruciate ligaments. PLoS One, 2015, 10(3): e0117224.

Chinese Journal of Reparative and Reconstructive Surgery

Comparison of biological characteristics between bone marrow mesenchymal stem cells and anterior cruciate ligament derived mesenchymal stem cells in rats

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