Bone marrow mesenchymal stem cells combined with calcium phosphate cement to repair articular cartilage defects in rabbits

doi:10.3969/j.issn.2095-4344.2015.08.009

Abstract

Abstract:

BACKGROUND: In previous experiments, bone marrow mesenchymal cells have been successfully seeded onto the calcium phosphate cement scaffold, which is confirmed to have good mechanical strength and biocompatibility.

OBJECTIVE: To observe the feasibility of rabbit bone marrow mesenchymal stem cells combined with calcium phosphate cements embedded in articular cartilage defects.

METHODS: Eighteen New Zealand rabbits were used to create 5 mm bone-cartilage defects on both sides of the mandible, and then, 15 rabbits were randomly selected and implanted with simple calcium phosphate cements into the left bone defects as control group and with bone marrow mesenchymal stem cells combined with calcium phosphate cement into the right bone defects as experimental group. Another three rabbits were implanted with nothing as blank group. After transplantation 4, 8, 16 weeks, rabbits from each group were sacrificed. Clinical observation, X-ray imaging, histological observation were performed at different periods.

RESULTS AND CONCLUSION: After 4,8,16 weeks, bone tissue regeneration in the experimental group was better than the other two groups in the bone formation speed and quantity. The maturity of new bone tissue, scaffold degradation, bone maturation in the experimental group was better than that in the other two groups. After 16 weeks, the maximum load, maximum stress and damage energy of the bone specimens in the experimental group were significantly higher than those in the control group. These findings indicate that bone marrow mesenchymal stem cells combined with calcium phosphate cement can promote bone regeneration, restore bone stiffness and strength, which is expected to act as a new artificial bone material in repairing bone defects.

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: Bone Marrow, Mesenchymal Stem Cells, Calcium Phosphates

CLC Number:

R318

Zhou Chang-yan, Zhou Qing-huan, Bian Jing, Chen Ke, Chen Wen. Bone marrow mesenchymal stem cells combined with calcium phosphate cement to repair articular cartilage defects in rabbits [J]. Chinese Journal of Tissue Engineering Research, 2015, 19(8): 1195-1199.

Figures/Tables 1

2.1 实验动物数量分析 18只新西兰兔标本均进入结果分析，在实验过程中，未发生动物意外死亡事件。 2.2 兔骨髓间充质干细胞的形态采用全骨髓培养法分离出兔骨髓间充质干细胞，传代细胞形态同原代细胞，呈集落样螺旋状生长，经过多次传代后椭圆形形态的细胞可变为纺锤形、长梭形等形态，生长进入相对平台期，见图2。 2.3 X射线拍片观察结果术后4周， X线射片可见空白组骨缺损区出现低密度影，对照组骨缺损区可见磷酸钙骨水泥材料阻射影，实验组骨缺损区也可见材料阻射影，但较对照组减轻。术后8周，各组骨缺损低密度阴影均减小，术后16周，空白组的骨缺损区可见轻微骨凹陷，对照组和实验组骨缺损区材料阻射影消失(图3)。 2.4 大体形态观察和功能评价所有动物均在术后4 h左右苏醒，创口均愈合良好，未发生创口红肿、炎症、化脓及裂开现象。术后4周，空白组骨组织凹陷裂隙较深，未有新骨组织生成，仅见软组织，对照组骨缺损区凹陷不明显，磷酸钙骨水泥两端有少量新骨形成，并可见纤维骨样组织形成，实验组骨缺损区凹陷不明显，植入物两端可见较多骨痂形成。术后8周，空白组仍未有新骨组织生成，骨组织凹陷裂隙较深，对照组磷酸钙骨水泥两端形成的新骨较4周明显增多，实验组植入物两端可见骨痂，与骨端联系较紧密。术后16周，空白组同前，无新生骨变化。对照组磷酸钙骨水泥部分被新骨代替，骨痂未完全包绕，塑性不完整，实验组植入物两端骨痂形成，完全包绕植入物，缺损区骨端皮质连续，塑性完整。 2.5 各组骨缺损区力学性能比较骨髓间充质干细胞与磷酸钙骨水泥复合培养6 h后，电镜下可见磷酸钙骨水泥支架材料表面和内部有兔骨髓间充质干细胞紧密黏附于支架材料，但是支架的空间结构未发生明显改变。术后第16周实验组骨标本抗弯曲能力的最大负荷、最大应力和破坏能量的数值均明显高于对照组，两组间差异有显著性意义(P < 0.05，表1)，实验组和对照组各项数值均明显高于空白组，差异有显著性意义(P < 0.05)。 2.6 各组兔骨缺损修复标本骨密度比较术后4，8，16周实验组兔缺损区骨标本密度大于对照组，差异有显著性意义(P < 0.05，表2) ，实验组和对照组各项数值均明显高于空白组，差异有显著性意义(P < 0.05)。 2.7 组织学观察结果术后4周，空白组仅见软组织，未有新骨组织生成，对照组骨缺损处有少量软骨化骨，实验组两侧新骨继续生长，可见不同时期的软骨细胞及成骨细胞，骨缺损部位无软组织长入。术后8周，空白组骨组织凹陷裂隙较深，可见少量新骨形成，有少许成骨细胞，对照组缺损处大部分由纤维组织填充，有少许的软骨细胞增生，散在的骨小梁、骨基质形成，实验组骨缺损部位被新生骨填充，可见成熟的骨小梁，软骨细胞更成熟，可见少量的成骨细胞、破骨细胞。术后16周，空白组同前，无新生骨变化。对照组可见成熟的骨小梁，可见少量的成骨细胞、破骨细胞，实验组新生骨较前更致密和连续。"

References

[1] Zhao L, Burguera EF, Xu HH, et al. Fatigue and human umbilical cord stem cell seeding characteristics of calcium phosphate-chitosan-biodegradable fiber scaffolds. Biomaterials. 2010;31(5):840-847.
[2] Zhao L, Weir MD, Xu HH. Human umbilical cord stem cell encapsulation in calcium phosphate scaffolds for bone engineering. Biomaterials. 2010;31(14):3848-3857.
[3] Fayaz HC, Giannoudis PV, Vrahas MS, et al. The role of stem cells in fracture healing and nonunion. Int Orthop. 2011; 35(11):1587-1597.
[4] Janicki P, Boeuf S, Steck E, et al. Prediction of in vivo bone forming potency of bone marrow-derived human mesenchymal stem cells. Eur Cell Mater. 2011;21: 488-507.
[5] Kagami H, Agata H, Tojo A. Bone marrow stromal cells (bone marrow-derived multipotent mesenchymal stromal cells) for bone tissue engineering: basic science to clinical translation. Int J Biochem Cell Biol. 2011;43(3):286-289.
[6] Sommeling CE, Heyneman A, Hoeksema H, et al. The use of platelet-rich plasma in plastic surgery: a systematic review. J Plast Reconstr Aesthet Surg. 2013;66(3):301-311.
[7] Namazi H. Enhancing bone healing during distraction osteogenesis with platelet-rich plasma: a novel molecular mechanism. Injury. 2012;43(7):1225-1226.
[8] Latalski M, Elbatrawy YA, Thabet AM, et al. Enhancing bone healing during distraction osteogenesis with platelet-rich plasma. Injury. 2011;42(8):821-824.
[9] Kanthan SR, Kavitha G, Addi S, et al. Platelet-rich plasma (PRP) enhances bone healing in non-united critical-sized defects: a preliminary study involving rabbit models. Injury. 2011;42(8):782-789.
[10] Gustavsson J, Ginebra MP, Engel E, et al. Ion reactivity of calcium-deficient hydroxyapatite in standard cell culture media. Acta Biomater. 2011;7(12):4242-4252.
[11] Weir MD, Xu HH. Culture human mesenchymal stem cells with calcium phosphate cement scaffolds for bone repair. J Biomed Mater Res B Appl Biomater. 2010;93(1):93-105.
[12] Yuan J, Zhang WJ, Liu G, et al. Repair of canine mandibular bone defects with bone marrow stromal cells and coral. Tissue Eng Part A. 2010;16(4):1385-1394.
[13] Zhou H, Weir MD, Xu HH. Effect of cell seeding density on proliferation and osteodifferentiation of umbilical cord stem cells on calcium phosphate cement-fiber scaffold. Tissue Eng Part A. 2011;17(21-22):2603-2613.
[14] Chen W, Zhou H, Weir MD, et al. Human embryonic stem cell-derived mesenchymal stem cell seeding on calcium phosphate cement-chitosan-RGD scaffold for bone repair. Tissue Eng Part A. 2013;19(7-8):915-927.
[15] Yasuda H, Yano K, Wakitani S, et al. Repair of critical long bone defects using frozen bone allografts coated with an rhBMP-2-retaining paste. J Orthop Sci. 2012;17(3):299-307.
[16] Ginebra MP, Espanol M, Montufar EB, et al. New processing approaches in calcium phosphate cements and their applications in regenerative medicine. Acta Biomater. 2010; 6(8):2863-2873.
[17] Xu HH, Zhao L, Weir MD. Stem cell-calcium phosphate constructs for bone engineering. J Dent Res. 2010; 89(12): 1482-1488.
[18] Zhao L, Weir MD, Xu HH. Human umbilical cord stem cell encapsulation in calcium phosphate scaffolds for bone engineering. Biomaterials. 2010;31(14):3848-3857.
[19] Zhao L, Weir MD, Xu HH. An injectable calcium phosphate- alginate hydrogel-umbilical cord mesenchymal stem cell paste for bone tissue engineering. Biomaterials. 2010; 31(25): 6502-6510.
[20] Habraken WJ, Liao HB, Zhang Z, et al. In vivo degradation of calcium phosphate cement incorporated into biodegradable microspheres. Acta Biomater. 2010;6(6):2200-2211.
[21] Zou D, Guo L, Lu J, et al. Engineering of bone using porous calcium phosphate cement and bone marrow stromal cells for maxillary sinus augmentation with simultaneous implant placement in goats. Tissue Eng Part A. 2012;18(13-14): 1464-1478.
[22] Bonewald LF. The amazing osteocyte. J Bone Miner Res. 2011;26(2):229-238.
[23] Sell SA, Wolfe PS, Ericksen JJ, et al. Incorporating platelet-rich plasma into electrospun scaffolds for tissue engineering applications. Tissue Eng Part A. 2011;17(21-22): 2723-2737.
[24] Riaz R, Ravindran C, Ramkumar, et al. Efficacy of platelet rich plasma in sinus lift augmentation. J Maxillofac Oral Surg. 2010; 9(3):225-230.
[25] Franco D, Franco T, Schettino AM, et al. Protocol for obtaining platelet-rich plasma (PRP), platelet-poor plasma (PPP), and thrombin for autologous use. Aesthetic Plast Surg. 2012;36(5): 1254-1259.
[26] Aberg J, Brisby H, Henriksson HB, et al. Premixed acidic calcium phosphate cement: characterization of strength and microstructure. J Biomed Mater Res B Appl Biomater. 2010; 93(2):436-441.
[27] Giocondi JL, El-Dasher BS, Nancollas GH, et al. Molecular mechanisms of crystallization impacting calcium phosphate cements. Philos Trans A Math Phys Eng Sci. 2010;368(1917): 1937-1961.
[28] Pina S, Vieira SI, Torres PM, et al. In vitro performance assessment of new brushite-forming Zn- and ZnSr-substituted beta-TCP bone cements. J Biomed Mater Res B Appl Biomater. 2010;94(2):414-420.
[29] Vazquez D, Takagi S, Frukhtbeyn S, et al. Effects of Addition of Mannitol Crystals on the Porosity and Dissolution Rates of a Calcium Phosphate Cement. J Res Natl Inst Stand Technol. 2010;115(4):225-232.
[30] Silva TS, Primo BT, Silva Júnior AN, et al. Use of calcium phosphate cement scaffolds for bone tissue engineering: in vitro study. Acta Cir Bras. 2011;26(1):7-11.
[31] van de Watering FC, van den Beucken JJ, Walboomers XF, et al. Calcium phosphate/poly(D,L-lactic-co-glycolic acid) composite bone substitute materials: evaluation of temporal degradation and bone ingrowth in a rat critical-sized cranial defect. Clin Oral Implants Res. 2012;23(2):151-159.
[32] Lee GS, Park JH, Won JE, et al. Alginate combined calcium phosphate cements: mechanical properties and in vitro rat bone marrow stromal cell responses. J Mater Sci Mater Med. 2011;22(5):1257-1268.
[33] Lee GS, Park JH, Shin US, et al. Direct deposited porous scaffolds of calcium phosphate cement with alginate for drug delivery and bone tissue engineering. Acta Biomater. 2011; 7(8):3178-3186.
[34] Zhao L, Tang M, Weir MD, et al. Osteogenic media and rhBMP-2-induced differentiation of umbilical cord mesenchymal stem cells encapsulated in alginate microbeads and integrated in an injectable calcium phosphate-chitosan fibrous scaffold.Tissue Eng Part A. 2011;17(7-8):969-979.