Experimental study on transplantation of microencapsulated transgenic bone marrow mesenchymal stem cells for early steroid-induced osteonecrosis of femoral head in rabbits_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

YOU Wulin ¹ , HUANG Guicheng ² ,  WANG Jianwei ¹

1. Department of Joint Orthopedics, Wuxi Affilliated Hospital of Nanjing University of Chinese Medicine, Wuxi Jiangsu, 214071, P.R.China;
2. Department of Orthopedics and Traumatology, Nanjing University of Chinese Medicine, Nanjing Jiangsu, 210023, P.R.China;

Corresponding author：

WANG Jianwei, Email: osteoWjW@163.com

Keywords：

Microencapsulation; bone marrow mesenchymal stem cells; transgene; cell transplantation; osteonecrosis of femoral head; rabbit

DOI：

10.7507/1002-1892.202003021

Video：

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Objective To investigate the effect of microencapsulated transgenic bone marrow mesenchymal stem cells (BMSCs) transplantation on early steroid induced osteonecrosis of femoral head (SONFH) in rabbits.Methods Alginate poly-L-lysine-sodium alginate (APA) microencapsulated transgenic BMSCs with high expression of Foxc2 were prepared by high-voltage electrostatic method. Part of the cells were cultured in osteoblasts and observed by alizarin red staining at 2 and 3 weeks. Forty New Zealand white rabbits were used to prepare SONFH models by using hormone and endotoxin. Thirty two rabbits who were successful modeling were screened out by MRI and randomly divided into 4 groups (groups A, B, C and D, n=8); another 6 normal rabbits were taken as normal control (group E). The rabbits in group A did not receive any treatment; and in groups B, C, and D were injected with normal saline, allogeneic BMSCs, and APA microencapsulated transgenic BMSCs respectively after core decompression. At 6 and 12 weeks after operation, specimens of femoral head were taken for HE staining to observe bone ingrowth; the expressions of osteocalcin (OCN), peroxisome proliferative activated receptor γ 2 (PPARγ-2), and vascular endothelial growth factor (VEGF) proteins were observed by immunohistochemistry staining. At 12 weeks after operation, the bone microstructure was observed by transmission electron microscope, and the maximum compressive strength and average elastic modulus of cancellous bone and subchondral bone were measured by biomechanics.Results After 2 and 3 weeks of induction culture, alizarin red staining showed the formation of calcium nodules, and the number of calcium nodules increased at 3 weeks when compared with 2 weeks. The rabbits in each group survived until the experiment was completed. Compared with groups A, B, and C, the trabeculae of group D were more orderly, the empty bone lacunae were less, there were abundant functional organelles, and obvious osteogenesis was observed, and the necrotic area was completely repaired at 12 weeks. Immunohistochemical staining showed that, at 6 and 12 weeks after operation, the expressions of OCN and VEGF in groups A, B, and C were significantly lower than those in groups D and E, while those in groups B and C were significantly higher than those in group A, and in group E than in group D (P<0.05). The expression of PPARγ-2 was significantly higher in groups A, B, and C than in groups D and E, and in group A than in groups B and C, and in group D than in group E (P<0.05). At 12 weeks after operation, biomechanical test showed that the average elastic modulus and maximum compressive strength of cancellous bone and subchondral bone in groups D and E were significantly higher than those in groups A, B, and C (P<0.05); there was no significant difference between groups A, B, and C and between groups D and E (P>0.05).Conclusion In vivo transplantation of microencapsulated transgenic BMSCs can repair early SONFH in rabbits.

Citation： YOU Wulin, HUANG Guicheng, WANG Jianwei. Experimental study on transplantation of microencapsulated transgenic bone marrow mesenchymal stem cells for early steroid-induced osteonecrosis of femoral head in rabbits. Chinese Journal of Reparative and Reconstructive Surgery, 2020, 34(11): 1446-1453. doi: 10.7507/1002-1892.202003021 Copy

1.	Kaushik AP, Das A, Cui Q. Osteonecrosis of the femoral head: an update in year 2012. World J Orthop, 2012, 3(5): 49-57.
2.	Huang Z, Cheng C, Cao B, et al. Icariin protects against glucocorticoid-induced osteonecrosis of the femoral head in rats. Cell Physiol Biochem, 2018, 47(2): 694-706.
3.	傅维民, 刘保一, 王本杰, 等. 激素性股骨头坏死的保髋治疗策略及疗效. 中华骨科杂志, 2019, 39(23): 1424-1431.
4.	Li J, Li ZL, Zhang H, et al. Long-term outcome of multiple small-diameter drilling decompression combined with hip arthroscopy versus drilling alone for early avascular necrosis of the femoral head. Chin Med J (Engl), 2017, 130(12): 1435-1440.
5.	Luo P, Gao F, Han J, et al. The role of autophagy in steroid necrosis of the femoral head: A comprehensive research review. Int Orthop, 2018, 42(7): 1747-1753.
6.	Kuroda Y, Matsuda S, Akiyama H. Joint-preserving regenerative therapy for patients with early-stage osteonecrosis of the femoral head. Inflamm Regen, 2016, 36: 4.
7.	Fu Q, Tang NN, Zhang Q, et al. Preclinical study of cell therapy for osteonecrosis of the femoral head with allogenic peripheral blood-derived mesenchymal stem cells. Yonsei Med J, 2016, 57(4): 1006-1015.
8.	Zhang Y, Wang W, Xie Y, et al. Optimization of microencapsulated recombinant cho cell growth, endostatin production, and stability of microcapsule in vivo. J Biomed Mater Res B Appl Biomater, 2008, 84(1): 79-88.
9.	Ding HF, Liu R, Li BG, et al. Biologic effect and immunoisolating behavior of bmp-2 gene-transfected bone marrow-derived mesenchymal stem cells in apa microcapsules. Biochem Biophys Res Commun, 2007, 362(4): 923-927.
10.	Paek HJ, Campaner AB, Kim JL, et al. Microencapsulated cells genetically modified to overexpress human transforming growth factor-beta1: viability and functionality in allogeneic and xenogeneic implant models. Tissue Eng, 2006, 12(7): 1733-1739.
11.	You W, Fan L, Duan D, et al. Foxc2 over-expression in bone marrow mesenchymal stem cells stimulates osteogenic differentiation and inhibits adipogenic differentiation. Mol Cell Biochem, 2014, 386(1-2): 125-134.
12.	Leslie SK, Kinney RC, Schwartz Z, et al. Microencapsulation of stem cells for therapy. Methods Mol Biol, 2017, 1479: 251-259.
13.	Asada T, Kushida T, Umeda M, et al. Prevention of corticosteroid-induced osteonecrosis in rabbits by intra-bone marrow injection of autologous bone marrow cells. Rheumatology (Oxford), 2008, 47(5): 591-596.
14.	Caldarelli I, Speranza MC, Bencivenga D, et al. Resveratrol mimics insulin activity in the adipogenic commitment of human bone marrow mesenchymal stromal cells. Int J Biochem Cell Biol, 2015, 60: 60-72.
15.	Gangji V, De Maertelaer V, Hauzeur JP. Autologous bone marrow cell implantation in the treatment of non-traumatic osteonecrosis of the femoral head: five year follow-up of a prospective controlled study. Bone, 2011, 49(5): 1005-1009.
16.	Zhao D, Cui D, Wang B, et al. Treatment of early stage osteonecrosis of the femoral head with autologous implantation of bone marrow-derived and cultured mesenchymal stem cells. Bone, 2012, 50(1): 325-330.
17.	Hernigou P, Flouzat-Lachaniette CH, Delambre J, et al. Osteonecrosis repair with bone marrow cell therapies: state of the clinical art. Bone, 2015, 70: 102-109.
18.	Wada Y, Kataoka H, Yokose S, et al. Changes in osteoblast phenotype during differentiation of enzymatically isolated rat calvaria cells. Bone, 1998, 22(5): 479-485.
19.	Babister JC, Tare RS, Green DW, et al. Genetic manipulation of human mesenchymal progenitors to promote chondrogenesis using “bead-in-bead” polysaccharide capsules. Biomaterials, 2008, 29(1): 58-65.
20.	Davis KE, Moldes M, Farmer SR. The forkhead transcription factor FoxC2 inhibits white adipocyte differentiation. J Biol Chem, 2004, 279(41): 42453-42461.
21.	Hernández RM, Orive G, Murua A, et al. Microcapsules and microcarriers for in situ cell delivery. Adv Drug Deliv Rev, 2010, 62(7-8): 711-730.
22.	Sobajima S, Vadala G, Shimer A, et al. Feasibility of a stem cell therapy for intervertebral disc degeneration. Spine J, 2008, 8(6): 888-896.

1. Kaushik AP, Das A, Cui Q. Osteonecrosis of the femoral head: an update in year 2012. World J Orthop, 2012, 3(5): 49-57.
2. Huang Z, Cheng C, Cao B, et al. Icariin protects against glucocorticoid-induced osteonecrosis of the femoral head in rats. Cell Physiol Biochem, 2018, 47(2): 694-706.
3. 傅维民, 刘保一, 王本杰, 等. 激素性股骨头坏死的保髋治疗策略及疗效. 中华骨科杂志, 2019, 39(23): 1424-1431.
4. Li J, Li ZL, Zhang H, et al. Long-term outcome of multiple small-diameter drilling decompression combined with hip arthroscopy versus drilling alone for early avascular necrosis of the femoral head. Chin Med J (Engl), 2017, 130(12): 1435-1440.
5. Luo P, Gao F, Han J, et al. The role of autophagy in steroid necrosis of the femoral head: A comprehensive research review. Int Orthop, 2018, 42(7): 1747-1753.
6. Kuroda Y, Matsuda S, Akiyama H. Joint-preserving regenerative therapy for patients with early-stage osteonecrosis of the femoral head. Inflamm Regen, 2016, 36: 4.
7. Fu Q, Tang NN, Zhang Q, et al. Preclinical study of cell therapy for osteonecrosis of the femoral head with allogenic peripheral blood-derived mesenchymal stem cells. Yonsei Med J, 2016, 57(4): 1006-1015.
8. Zhang Y, Wang W, Xie Y, et al. Optimization of microencapsulated recombinant cho cell growth, endostatin production, and stability of microcapsule in vivo. J Biomed Mater Res B Appl Biomater, 2008, 84(1): 79-88.
9. Ding HF, Liu R, Li BG, et al. Biologic effect and immunoisolating behavior of bmp-2 gene-transfected bone marrow-derived mesenchymal stem cells in apa microcapsules. Biochem Biophys Res Commun, 2007, 362(4): 923-927.
10. Paek HJ, Campaner AB, Kim JL, et al. Microencapsulated cells genetically modified to overexpress human transforming growth factor-beta1: viability and functionality in allogeneic and xenogeneic implant models. Tissue Eng, 2006, 12(7): 1733-1739.
11. You W, Fan L, Duan D, et al. Foxc2 over-expression in bone marrow mesenchymal stem cells stimulates osteogenic differentiation and inhibits adipogenic differentiation. Mol Cell Biochem, 2014, 386(1-2): 125-134.
12. Leslie SK, Kinney RC, Schwartz Z, et al. Microencapsulation of stem cells for therapy. Methods Mol Biol, 2017, 1479: 251-259.
13. Asada T, Kushida T, Umeda M, et al. Prevention of corticosteroid-induced osteonecrosis in rabbits by intra-bone marrow injection of autologous bone marrow cells. Rheumatology (Oxford), 2008, 47(5): 591-596.
14. Caldarelli I, Speranza MC, Bencivenga D, et al. Resveratrol mimics insulin activity in the adipogenic commitment of human bone marrow mesenchymal stromal cells. Int J Biochem Cell Biol, 2015, 60: 60-72.
15. Gangji V, De Maertelaer V, Hauzeur JP. Autologous bone marrow cell implantation in the treatment of non-traumatic osteonecrosis of the femoral head: five year follow-up of a prospective controlled study. Bone, 2011, 49(5): 1005-1009.
16. Zhao D, Cui D, Wang B, et al. Treatment of early stage osteonecrosis of the femoral head with autologous implantation of bone marrow-derived and cultured mesenchymal stem cells. Bone, 2012, 50(1): 325-330.
17. Hernigou P, Flouzat-Lachaniette CH, Delambre J, et al. Osteonecrosis repair with bone marrow cell therapies: state of the clinical art. Bone, 2015, 70: 102-109.
18. Wada Y, Kataoka H, Yokose S, et al. Changes in osteoblast phenotype during differentiation of enzymatically isolated rat calvaria cells. Bone, 1998, 22(5): 479-485.
19. Babister JC, Tare RS, Green DW, et al. Genetic manipulation of human mesenchymal progenitors to promote chondrogenesis using “bead-in-bead” polysaccharide capsules. Biomaterials, 2008, 29(1): 58-65.
20. Davis KE, Moldes M, Farmer SR. The forkhead transcription factor FoxC2 inhibits white adipocyte differentiation. J Biol Chem, 2004, 279(41): 42453-42461.
21. Hernández RM, Orive G, Murua A, et al. Microcapsules and microcarriers for in situ cell delivery. Adv Drug Deliv Rev, 2010, 62(7-8): 711-730.
22. Sobajima S, Vadala G, Shimer A, et al. Feasibility of a stem cell therapy for intervertebral disc degeneration. Spine J, 2008, 8(6): 888-896.

Chinese Journal of Reparative and Reconstructive Surgery

Experimental study on transplantation of microencapsulated transgenic bone marrow mesenchymal stem cells for early steroid-induced osteonecrosis of femoral head in rabbits

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