Effect of injectable composites of calcium sulfate and hyaluronate in enhancing osteogenesis_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

HUANG Zhifeng ¹ , LI Bo ¹ , LI Qiang ¹ , HUANG Zhenfei ¹ , YIN Bo ² , MA Pei ³ ,  WU Zhihong ^4,5 , QIU Guixing ^1,4 , XU Derong ¹

1. Department of Orthopaedic Surgery, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, 100730, P.R.China;
2. Department of Maxillofacial Surgery, Plastic Surgery Hospital, Peking Union Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, 100144, P.R.China;
3. Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, 100050, P.R.China;
4. Beijing Key Laboratory for Genetic Research of Bone and Joint Diseases, Beijing, 100730, P.R.China;
5. Central Laboratory, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, 100730, P.R.China;

Corresponding author：

WU Zhihong, Email: pumch@163.com

Keywords：

Bone substitute; injectable; hyaluronic acid; cross-linking; calcium sulfate

DOI：

10.7507/1002-1892.201612145

Video：

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ObjectiveTo fabricate an injectable composite bone substitute with hyaluronic acid (HA) and calcium sulfate and to evaluate the biocompatibility and effect of the composite on cell proliferation, osteogenic differentiation in vitro and osteogenic capability in vivo. MethodsCalcium sulfate powder was mixed with HA solution, cross-linked HA solution, and phosphate buffer solution (PBS) in a ratio of 2∶1 (W/V) to get composites of CA+HA, CA+HAC, and CA. The standard extracts from above 3 materials were prepared according to ISO10993-5, and were used to culture mouse MC3T3-E1 cells. The composite biocompatibility and cell proliferation in different concentrations of extract were tested with cell counting kit-8 (CCK-8). The cells were cultured with standard medium as a control. The optimal concentration was selected for osteogenic differentiation test, and ELISA Kit was used to determine the alkaline phosphatase (ALP), collagen type I (COL-I), and osteocalcin (OCN). The femoral condylar bone defect was made on New Zealand white rabbits and repaired with CA+HA, CA+HAC, and CA. Micro-CT was done to evaluate new bone formation with bone volume/tissue volume (BV/TV) ratio at 6 and 12 weeks. HE staining was used to observe bone formation. ResultsCA+HA and CA+HAC were better in injectability and stability in PBS than CA. The biocompatibility test showed that absorbance (A) value of CA group was significantly lower than that of control group (P<0.05) at 6, 12, and 24 hours after culture, but no significant difference was found inA values between CA+HA group or CA+HAC group and control group (P>0.05). The proliferation test showed 25% and 50% extract of all 3 materials had significantly higherA value than control group (P<0.05). For 75% and 100% extract, only CA+HA group had significantly higherA value than control group (P<0.05). And 50% extract was selected for osteogenic differentiation test. At 14 and 21 days, ALP, COL-I and OCN concentrations of CA+HA group and CA+HAC group were significantly higher than those of CA group and control group (P<0.05). Micro-CT results showed higher BV/TV in CA+HA group and CA+HAC group than CA group at 6 and 12 weeks (P<0.05), but no significant difference was found between CA+HA group and CA+HAC group (P>0.05). HE staining revealed that a little bone tissue was seen in CA+HA group and CA+HAC group, but there was no bone formation in CA group at 6 weeks; more streak bone tissue in CA+HA group and CA+HAC group than CA group at 12 weeks. ConclusionComposites prepared with calcium sulfate and HA or with cross-linked HA are stable, injectable, and biocompatible. The materials have excellent effect on proliferation and differentiation of mouse MC3T3-E1 cells. They also show good osteogenic capability in vivo. So it is a potential bone substitutes for bone defective diseases.

Citation： HUANG Zhifeng, LI Bo, LI Qiang, HUANG Zhenfei, YIN Bo, MA Pei, WU Zhihong, QIU Guixing, XU Derong. Effect of injectable composites of calcium sulfate and hyaluronate in enhancing osteogenesis. Chinese Journal of Reparative and Reconstructive Surgery, 2017, 31(6): 730-737. doi: 10.7507/1002-1892.201612145 Copy

1.	Fillingham Y, Jacobs J. Bone grafts and their substitutes. Bone Joint J, 2016, 98-B (1 Suppl A): 6-9.
2.	Fillingham YA, Cvetanovich GL, Haughom BD, et al. Bioceramic bone graft substitute for treatment of unicameral bone cysts. J Orthop Surg (Hong Kong), 2016, 24(2): 222-227.
3.	Nguyen TB, Lee BT. A combination of biphasic calcium phosphate scaffold with hyaluronic acid-gelatin hydrogel as a new tool for bone regeneration. Tissue Eng Part A, 2014, 20(13): 1993-2004.
4.	Orellanaa BR, Thomasb MV, Dziublac TD, et al. Bioerodible calcium sulfate/poly (β-amino ester) hydrogel composites. J Mech Behav Biomed Mater, 2013, 26(10): 43-53.
5.	Sawatjui N, Damrongrungruang T, Leeanansaksiri W, et al. Silk fibroin/gelatin-chondroitin sulfate-hyaluronic acid effectively enhances in vitro chondrogenesis of bone marrow mesenchymal stem cells. Mater Sci Eng C Mater Biol Appl, 2015, 52: 90-96.
6.	Solis MA, Chen YH, Wong TY, et al. Hyaluronan regulates cell behavior: a potential niche matrix for stem cells. Biochem Res Int, 2012, (2012): 346972.
7.	Wu AT, Aoki T, Sakoda M, et al. Enhancing osteogenic differentiation of MC3T3-E1 cells by immobilizing inorganic polyphosphate onto hyaluronic acid hydrogel. Biomacromolecules, 2015, 16(11): 166-173.
8.	Kumar CY, K B N, Menon J, et al. Calcium sulfate as bone graft substitute in the treatment of osseous bone defects, a prospective study. J Clin Diagn Res, 2013, 7(12): 2926-2928.
9.	Zhang C, Li Z, Li Q, et al. Properties and Osteogenicity of Two Calcium Sulfate Materials with Micro or Nano Morphology. J Nanosci Nanotechnology, 2016, 16(3): 2277-2282.
10.	Fillingham YA, Cvetanovich GL, Haughom BD, et al. Bioceramic bone graft substitute for treatment of unicameral bone cysts. J Orthop Surg (Hong Kong), 2016, 24(2): 222-227.
11.	Mao K, Zhou F, Cui F, et al. Preparation and properties of α-calcium sulphate hemihydrate and β-tricalcium phosphate bone substitute. Biomed Mater Eng, 2013, 23(3): 197-210.
12.	Brown ME, Zou Y, Dziubla TD, et al. Effects of composition and setting environment on mechanical properties of a composite bone filler. J Biomed Mater Res Part A, 2013, 101(4): 973-980.
13.	Cheng P, Han P, Zhao C, et al. High-purity magnesium interference screws promote fibrocartilaginous entheses regeneration in the anterior cruciate ligament reconstruction rabbit model via accumulation of BMP-2 and VEGF. Biomaterials, 2016, 81: 14-26.
14.	李阳, 雷伟, 王征, 等. 生物玻璃和壳聚糖改性的多孔活性骨水泥体内实验研究. 中国修复重建外科杂志, 2013, 27(3): 320-325.
15.	Zhao N, Wang X, Qin L, et al. Effect of hyaluronic acid in bone formation and its applications in dentistry. J Biomed Mater Res A, 2016, 104(6): 1560-1569.
16.	Hulsart-Billström G, Yuen PK, Marsell R, et al. Bisphosphonate-linked hyaluronic acid hydrogel sequesters and enzymatically releases active bone morphogenetic protein-2 for induction of osteogenic differentiation. Biomacromolecules, 2013, 14(9): 3055-3363.
17.	Bae MS, Kim JE, Lee JB, et al. ZrO ₂ surface chemically coated with hyaluronic acid hydrogel loading GDF-5 for osteogenesis in dentistry. Carbohydr Polym, 2013, 92(1): 167-175.
18.	Qi X, Li H, Qiao B, et al. Development and characterization of an injectable cement of nano calcium-deficient hydroxyapatite/multi (amino acid) copolymer/calcium sulfate hemihydrate for bone repair. Int J Nanomedicine, 2013, 8: 4441-4452.
19.	Kim YK, Lee JY, Kim SG, et al. Guided bone regeneration using demineralized allogenic bone matrix with calcium sulfate: case series. J Adv Prosthodont, 2013, 5(2): 167-171.
20.	Yang X, Li Y, Huang Q, et al. Evaluation of a biodegradable graft substitute in rabbit bone defect model. Indian J Orthop, 2012, 46(3): 266-273.
21.	Cao L, Weng W, Chen X, et al. Promotion of in vivo degradability, vascularization and osteogenesis of calcium sulfate-based bone cements containing nanoporous lithium doping magnesium silicate. Int J Nanomedicine, 2017, 12: 1341-1352.
22.	Komorowicz E, Balázs N, Varga Z, et al. Hyaluronic acid decreases the mechanical stability, but increases the lytic resistance of fibrin matrices. Matrix Biol, 2016. [Epub ahead of print].
23.	Gómez-Aristizábal A, Kim KP, Viswanathan S. A Systematic Study of the Effect of Different Molecular Weights of Hyaluronic Acid on Mesenchymal Stromal Cell-Mediated Immunomodulation. PLoS One, 2016, 11(1): e0147868.
24.	Kwon TR, Seok J, Jang JH, et al. Needle-free jet injection of hyaluronic acid improves skin remodeling in a mouse model. Eur J Pharm Biopharm, 2016, 105(8): 69-74.
25.	Wu PT, Kuo LC, Su FC, et al. High-molecular-weight hyaluronic acid attenuated matrix metalloproteinase-1 and -3 expression via CD44 in tendinopathy. Sci Rep, 2017, 7(1): 40840.
26.	Lin WJ, Lee WC, Shieh MJ. Hyaluronic acid conjugated micelles possessing CD44 targeting potential for gene delivery. Carbohydr Polym, 2017, 155(1): 101-108.
27.	Esguerra KV, Tolg C, Akentieva N, et al. Identification, design and synthesis of tubulin-derived peptides as novel hyaluronan mimetic ligands for the receptor for hyaluronan-mediated motility (RHAMM/HMMR). Integr Biol (Camb), 2015, 7(12): 1547-1560.
28.	Savage JR, Pulsipher A, Rao NV, et al. A Modified Glycosaminoglycan, GM-0111, Inhibits Molecular Signaling Involved in Periodontitis. PLoS One, 2016, 11(6): e0157310.

1. Fillingham Y, Jacobs J. Bone grafts and their substitutes. Bone Joint J, 2016, 98-B (1 Suppl A): 6-9.
2. Fillingham YA, Cvetanovich GL, Haughom BD, et al. Bioceramic bone graft substitute for treatment of unicameral bone cysts. J Orthop Surg (Hong Kong), 2016, 24(2): 222-227.
3. Nguyen TB, Lee BT. A combination of biphasic calcium phosphate scaffold with hyaluronic acid-gelatin hydrogel as a new tool for bone regeneration. Tissue Eng Part A, 2014, 20(13): 1993-2004.
4. Orellanaa BR, Thomasb MV, Dziublac TD, et al. Bioerodible calcium sulfate/poly (β-amino ester) hydrogel composites. J Mech Behav Biomed Mater, 2013, 26(10): 43-53.
5. Sawatjui N, Damrongrungruang T, Leeanansaksiri W, et al. Silk fibroin/gelatin-chondroitin sulfate-hyaluronic acid effectively enhances in vitro chondrogenesis of bone marrow mesenchymal stem cells. Mater Sci Eng C Mater Biol Appl, 2015, 52: 90-96.
6. Solis MA, Chen YH, Wong TY, et al. Hyaluronan regulates cell behavior: a potential niche matrix for stem cells. Biochem Res Int, 2012, (2012): 346972.
7. Wu AT, Aoki T, Sakoda M, et al. Enhancing osteogenic differentiation of MC3T3-E1 cells by immobilizing inorganic polyphosphate onto hyaluronic acid hydrogel. Biomacromolecules, 2015, 16(11): 166-173.
8. Kumar CY, K B N, Menon J, et al. Calcium sulfate as bone graft substitute in the treatment of osseous bone defects, a prospective study. J Clin Diagn Res, 2013, 7(12): 2926-2928.
9. Zhang C, Li Z, Li Q, et al. Properties and Osteogenicity of Two Calcium Sulfate Materials with Micro or Nano Morphology. J Nanosci Nanotechnology, 2016, 16(3): 2277-2282.
10. Fillingham YA, Cvetanovich GL, Haughom BD, et al. Bioceramic bone graft substitute for treatment of unicameral bone cysts. J Orthop Surg (Hong Kong), 2016, 24(2): 222-227.
11. Mao K, Zhou F, Cui F, et al. Preparation and properties of α-calcium sulphate hemihydrate and β-tricalcium phosphate bone substitute. Biomed Mater Eng, 2013, 23(3): 197-210.
12. Brown ME, Zou Y, Dziubla TD, et al. Effects of composition and setting environment on mechanical properties of a composite bone filler. J Biomed Mater Res Part A, 2013, 101(4): 973-980.
13. Cheng P, Han P, Zhao C, et al. High-purity magnesium interference screws promote fibrocartilaginous entheses regeneration in the anterior cruciate ligament reconstruction rabbit model via accumulation of BMP-2 and VEGF. Biomaterials, 2016, 81: 14-26.
14. 李阳, 雷伟, 王征, 等. 生物玻璃和壳聚糖改性的多孔活性骨水泥体内实验研究. 中国修复重建外科杂志, 2013, 27(3): 320-325.
15. Zhao N, Wang X, Qin L, et al. Effect of hyaluronic acid in bone formation and its applications in dentistry. J Biomed Mater Res A, 2016, 104(6): 1560-1569.
16. Hulsart-Billström G, Yuen PK, Marsell R, et al. Bisphosphonate-linked hyaluronic acid hydrogel sequesters and enzymatically releases active bone morphogenetic protein-2 for induction of osteogenic differentiation. Biomacromolecules, 2013, 14(9): 3055-3363.
17. Bae MS, Kim JE, Lee JB, et al. ZrO ₂ surface chemically coated with hyaluronic acid hydrogel loading GDF-5 for osteogenesis in dentistry. Carbohydr Polym, 2013, 92(1): 167-175.
18. Qi X, Li H, Qiao B, et al. Development and characterization of an injectable cement of nano calcium-deficient hydroxyapatite/multi (amino acid) copolymer/calcium sulfate hemihydrate for bone repair. Int J Nanomedicine, 2013, 8: 4441-4452.
19. Kim YK, Lee JY, Kim SG, et al. Guided bone regeneration using demineralized allogenic bone matrix with calcium sulfate: case series. J Adv Prosthodont, 2013, 5(2): 167-171.
20. Yang X, Li Y, Huang Q, et al. Evaluation of a biodegradable graft substitute in rabbit bone defect model. Indian J Orthop, 2012, 46(3): 266-273.
21. Cao L, Weng W, Chen X, et al. Promotion of in vivo degradability, vascularization and osteogenesis of calcium sulfate-based bone cements containing nanoporous lithium doping magnesium silicate. Int J Nanomedicine, 2017, 12: 1341-1352.
22. Komorowicz E, Balázs N, Varga Z, et al. Hyaluronic acid decreases the mechanical stability, but increases the lytic resistance of fibrin matrices. Matrix Biol, 2016. [Epub ahead of print].
23. Gómez-Aristizábal A, Kim KP, Viswanathan S. A Systematic Study of the Effect of Different Molecular Weights of Hyaluronic Acid on Mesenchymal Stromal Cell-Mediated Immunomodulation. PLoS One, 2016, 11(1): e0147868.
24. Kwon TR, Seok J, Jang JH, et al. Needle-free jet injection of hyaluronic acid improves skin remodeling in a mouse model. Eur J Pharm Biopharm, 2016, 105(8): 69-74.
25. Wu PT, Kuo LC, Su FC, et al. High-molecular-weight hyaluronic acid attenuated matrix metalloproteinase-1 and -3 expression via CD44 in tendinopathy. Sci Rep, 2017, 7(1): 40840.
26. Lin WJ, Lee WC, Shieh MJ. Hyaluronic acid conjugated micelles possessing CD44 targeting potential for gene delivery. Carbohydr Polym, 2017, 155(1): 101-108.
27. Esguerra KV, Tolg C, Akentieva N, et al. Identification, design and synthesis of tubulin-derived peptides as novel hyaluronan mimetic ligands for the receptor for hyaluronan-mediated motility (RHAMM/HMMR). Integr Biol (Camb), 2015, 7(12): 1547-1560.
28. Savage JR, Pulsipher A, Rao NV, et al. A Modified Glycosaminoglycan, GM-0111, Inhibits Molecular Signaling Involved in Periodontitis. PLoS One, 2016, 11(6): e0157310.

Chinese Journal of Reparative and Reconstructive Surgery

Effect of injectable composites of calcium sulfate and hyaluronate in enhancing osteogenesis

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