Defatting effect of gradient alcohol treatment on cancellous bone allograft and the biomechanical properties of defatted cancellous bone allograft

doi:10.3969/j.issn.2095-4344.2322

Abstract

Abstract:

BACKGROUND: Defatting is considered to be the primary procedure for obtaining bone allograft. However, the current defatting method cannot avoid the problem of residual organic solvents.

OBJECTIVE: To investigate the defatting effect of gradient alcohol treatment and whether this defatting method can reduce the mechanical properties of bone.

METHODS: Sixty pieces of fresh cancellous bone mass were obtained from human femoral condyles. They were divided into three groups according to different defatting treatments: gradient alcohol group, acetone group, and fresh group (control group). The general shape of the bone mass and the morphology and color of the cancellous bone allograft were observed with the naked eye. Soxhlet extraction was used to determine the residual lipid content of the bone mass after defatting. Infrared spectroscopy was used to observe the changes of various components in the bone mass after different defatting treatments. The maximum stress and elastic modulus were used to evaluate the effect of defatting treatment on the biomechanical properties of bone mass.

RESULTS AND CONCLUSION: (1) Gross observation revealed that fresh bone mass was yellow with a large amount of fat in the pores, and the defated bone mass was white with a clear porous network structure. (2) There was no statistical difference in residual lipid content between the gradient alcohol and the acetone groups (P=0.385). (3) Infrared spectroscopy showed that the defatting effects of the two defatting treatments were similar and had no effect on other components in the bone, such as PO₄^3-, carbonate, and hydroxyapatite. (4) Biomechanical tests showed that there were no significant differences in the maximum stress and elastic modulus between groups. (5) These results suggest that the defatting effect of the gradient alcohol treatment is similar to that of the traditional acetone solvent extraction, and gradient alcohol treatment cannot reduce the biomechanical properties of bone

Key words: materials">, , allograft bone">, , alcohol">, , defatting">, , biomechanics">, , experiment

CLC Number:

Hua Kunchi, Hu Yongcheng. Defatting effect of gradient alcohol treatment on cancellous bone allograft and the biomechanical properties of defatted cancellous bone allograft[J]. Chinese Journal of Tissue Engineering Research, 2020, 24(34): 5502-5507.

Figures/Tables 5

2.3 红外光谱仪的测定结果在新鲜组骨块的红外光谱(图4A)中：559 cm-1和605 cm-1处吸收峰是由PO43-中的O-P-O键产生的；1 037 cm-1处吸收峰是由PO43-中的P-O键产生的；1 464 cm-1和1 561 cm-1处的吸收峰是由骨中碳酸盐成分的CO32-产生的；1 637 cm-1出现的是骨中水分的羟基产生的；3 417 cm-1处吸收峰是由骨中羟基磷灰石成分的羟基产生的；2 853 cm-1和2 924 cm-1处的吸收峰，主要是由骨中饱和脂肪酸成分的C-H键产生的。在梯度乙醇组骨块的红外光谱(图4B)中：559 cm-1处的吸收峰是由PO43-中的O-P-O键产生的；1 038 cm-1处的吸收峰是PO43-中的P-O键产生的；1 384 cm-1处的吸收峰是由骨中碳酸盐成分的CO32-产生的；1 654 cm-1处的吸收峰是由骨中水分的羟基产生的；3 434 cm-1处的吸收峰是由骨中羟基磷灰石成分的羟基产生的；2 853 cm-1和2 923 cm-1处的吸收峰，主要是由骨中饱和脂肪酸成分的C-H产生的。在丙酮组骨块的红外光谱(图4C)中：605 cm-1处的吸收峰是由PO43-中的O-P-O键产生的；1 036 cm-1处的吸收峰是PO43-中的P-O键产生的；1 407 cm-1处的吸收峰是由骨中碳酸盐成分的CO32-产生的；1 638 cm-1的吸收峰是由骨中水分的羟基产生的；3 415 cm-1处的吸收峰是由骨中羟基磷灰石成分的羟基产生的；2 853 cm-1和2 924 cm-1处的吸收峰，主要是由骨中饱和脂肪酸成分的C-H键产生的。观察3组红外光谱图可见，由骨中饱和脂肪酸成分的C-H键所产生的吸收峰高度存在明显差异，2组脱脂骨块在此处的吸收峰高度相近，且明显低于新鲜组骨块，证明这2种脱脂方法的脱脂效果相近，且均能有效去除骨中脂质。而其他成分，如PO43-、碳酸盐及羟基磷灰石等，在3组图中不能观察到明显的差异，证明这2种脱脂处理不会破坏骨的天然结构和无机物成分。 "

References

[1] DEVRIES JG, SCHARER B. Comparison and Use of Allograft Bone Morphogenetic Protein Versus Other Materials in Ankle and Hindfoot Fusions.J Foot Ankle Surg.2018;57(4):707-711.

[2] 张看,赵彦涛,白玉龙,等.不同脱脂方法对异种松质骨脱脂效果及免疫原性的影响[J].中国骨与关节损伤杂志,2019,34(6): 585-587.

[3] 田志超,蔡启卿,王鑫,等.四肢骨肿瘤大段同种异体骨移植术后骨愈合的分析[J].中华解剖与临床杂志,2019,24(1):11-16.

[4] CZITROM AA. The Immune Response: The Afferent Arm.Clin Orthop Relat Res.1996; 326(326):11-24.

[5] HOROWITZ MC, FRIEDLAENDER GE, QIAN HY. The Immune Response: The Efferent Arm.Clin Orthop Relat Res. 1996;(326):25-34.

[6] URIST MR, MIKULSKI A, BOYD SD. A chemosterilized antigen-extracted autodigested alloimplant for bone banks. Arch Surg.1975;110(4):416-428.

[7] CHAPPARD D, FRESSONNET C, GENTY C, et al. Fat in bone xenografts: importance of the purification procedures on cleanliness,wettability and biocompatibility.Biomaterials.1993; 14(7):507-512.

[8] THORÉN K, ASPENBERG P, THORNGREN KG. Lipid extraction decreases the specific immunologic response to bone allografts in rabbits.Acta Orthop Scand. 1993;64(1): 44-46.

[9] NGUYEN H, MORGAN DAF, FORWOOD MR. Sterilization of allograft bone: effects of gamma irradiation on allograft biology and biomechanics.Cell Tissue Bank. 2007;8(2): 93-105.

[10] FRAYSSINET P, ROUQUET N, MATHON D, et al. Histological integration of allogeneic cancellous bone tissue treated by supercritical CO2 implanted in sheep bones. Biomaterials. 1998;19(24):2247-2253.

[11] PARHAMI F, MORROW AD, BALUCAN J, et al. Lipid oxidation products have opposite effects on calcifying vascular cell and bone cell differentiation. A possible explanation for the paradox of arterial calcification in osteoporotic patients.Arterioscler Thromb Vasc Biol. 1997; 17(4):680-687.

[12] FAGES J, MARTY A, DELGA C, et al. Use of supercritical CO2 for bone delipidation.Biomaterials.1994;15(9):650-656.

[13] TADIC D, EPPLE M. A thorough physicochemical characterisation of 14 calcium phosphate-based bone substitution materials in comparison to natural bone. Biomaterials.2004;25(6):987-994.

[14] GARDIN C, RICCI S, FERRONI L, et al. Decellularization and Delipidation Protocols of Bovine Bone and Pericardium for Bone Grafting and Guided Bone Regeneration Procedures. PLoS One.2015; 10(7):e0132344.

[15] 中华人民共和国卫生健康委员会.人体捐献器官获取与分配管理规定[S]. 2019-01-28.

[16] 黄爱军,彭建强,陈丽萍,等.不同程度脱钙对皮质骨生物力学的影响[J].广东医学,2018,39(suppl):16-19.

[17] IORGI ND, ROSOL M, MITTELMAN SD, et al. Reciprocal Relation between Marrow Adiposity and the Amount of Bone in the Axial and Appendicular Skeleton of Young Adults.J Clin Endocrinol Metab.2008;93(6):2281-2286.

[18] FRIEDLAENDER GE. Immune responses to osteochondral allografts. Current knowledge and future directions.Clin Orthop Relat Res.1983;174(174):58-68.

[19] HOROWITZ MC, FRIEDLAENDER GE. Induction of specific T-cell responsiveness to allogeneic bone.J Bone Joint Surg Am.1991;73(8):1157-1168.

[20] MUSCOLO DL, CALETTI E, SCHAJOWICZ F, et al. Tissue-typing in human massive allografts of frozen bone. J Bone Joint Surg Am.1987;69(4):583-595.

[21] MÖLLER E. Advances in and future of tissue typing. Transplant Proc. 1991;23(1 Pt 1):63-66.

[22] ECKELS DD. Alloreactivity: Allogeneic presentation of endogenous peptide or direct recognition of MHC polymorphism? A review.Tissue Antigens.1990;35(2):49-55.

[23] MUSCOLO DL, AYERZA MA, CALABRESE ME, et al. Human Leukocyte Antigen Matching, Radiographic Score, and Histologic Findings in Massive Frozen Bone Allografts.Clin Orthop Relat Res.1996;326(326):115-126.

[24] THORÉN K, ASPENBERG P, THORNGREN KG. Lipid extracted bank bone. Bone conductive and mechanical properties.Clin Orthop Relat Res.1995;311(311):232-246.

[25] SCHWARZ N, REDL H, SCHIESSER A, et al. Irradiation-sterilization of rat bone matrix gelatin.Acta Orthop Scand.1988;59(2):165-167.

[26] OSTROWSKI K, DZIEDZIC-GOCLAWSKA A, STACHOWICZ W, et al. Radiation-induced paramagnetic centers in research on bone physiopathology.Clin Orthop Relat Res. 1991;(272): 21-29.

[27] 王娴,崔龙,董威杰,等.电子束和γ射线对油料氧化及霉菌的影响[J].包装工程,2019,40(3):30-37.

[28] AKKUS O, BELANEY RM, DAS P. Free radical scavenging alleviates the biomechanical impairment of gamma radiation sterilized bone tissue.J Orthop Res.2005;23(4):838-845.

[29] BURGESS HW, MACKRELL J, TOMS D, et al. Response of Bone Subjected to Optimized High Dose Irradiation. J Biomater Appl.2010;24(5):387-400.

[30] MOREAU MF, GALLOIS Y, BASLE MF, et al. Gamma irradiation of human bone allografts alters medullary lipids and releases toxic compounds for osteoblast-like cells. Biomaterials.2000;21(4):369-376.

[31] KALUS U, HELGA MÜLLER, BAUDISCH H, et al. A method for the determination of the residual chloroform in defatted cancellous bone transplants.Cell Tissue Bank. 2005;6(1): 71-75.