细胞外基质成份涂层椎弓根螺钉在负荷与非负荷状态下诱导、传导骨生成的研究
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摘要
目的:观测有机和无机细胞外基质成份涂层椎弓根螺钉,在幼羊体内负荷和非负荷状态下是否能有效的传导、诱导骨形成。指导涂层椎弓根螺钉在骨骼不断发育的脊柱侧凸患者中的应用。方法:随机将胶原纤维Ⅰ/硫酸软骨素/HA涂层、胶原纤维Ⅰ/硫酸软骨素涂层、HA涂层以及没有涂层的四组钛金属椎弓根螺钉植入幼羊双侧椎弓根内。一侧非负荷实验,另一侧加入链接棒固定,进行负荷实验。3个月后进行显微CT、组织形态学和生物力学观测。结果:1.组织形态学观测。无负荷下涂层椎弓根螺钉表面周围均有骨形成,与周围骨组织牢固结合。负荷状态下各组椎弓根螺钉的螺纹间和螺纹周围有大量的结缔组织形成,未见新骨的长入。2.无负荷状态下各组椎弓根螺钉涂层的显微CT观测。涂层椎弓根螺钉周围ROI的BMC、BMD大于没有涂层的,具有显著性意义(P﹤0.01或P﹤0.05)。胶原/硫酸软骨素/HA涂层椎弓根螺钉周围ROI的BVF、骨连接密度大于没有涂层的,且具有显著性意义(P﹤0.05)。3.各组椎弓根螺钉的生物力学观测。非负荷情况下,胶原/硫酸软骨素/HA涂层钛金属椎弓根螺钉的拔出力最大,胶原/硫酸软骨素涂层和HA涂层螺钉的拔出力次之,无涂层螺钉的拔出力最小(P﹤0.01)。负荷状态下,各组椎弓根螺钉的最大拔出力是202.8333±70.6638N,小于无负荷状态各组螺钉的最大拔出力(P﹤0.01),且组间未见明显差异(P﹥0.05)。结论:在没有负荷的情况下,钛合金椎弓根螺钉的有机和无机细胞外基质成份涂层均可传导或诱导螺钉周围的骨形成,而胶原/硫酸软骨素/HA涂层较其它涂层诱导骨生成的能力更强。在负荷状态下,各组螺钉周围均形成大量的结缔组织。本课题组在国内外首次成功建立了涂层椎弓根螺钉在幼羊体内负荷和非负荷情况的力学动物模型,模拟涂层椎弓根螺钉在正处于生长发育过程中的青少年脊椎内所受力的情况。本课题组也首次采用显微CT,进行各组椎弓根螺钉周围ROI骨密度和形态学参数的组间比较。
In pathology, spinal scoliosis is a spinal deformity of three-dimensional structure, involving the anterior, middle and posterior columns of the spine. Therefore, scoliosis surgery is a three-dimensional orthopedic surgery, which has been the difficulty in orthopedic surgery. The orthopedic fixation system with hook (e.g., pedicle hooks, facet hook, and laminar hooks) as the main structure only fixes in the middle and posterior column of the spine, which lacks of a real three-dimensional correction of spinal forces. In recent years, in scoliosis surgery, the pedicle screw is more and more used. Clinical Practice shows that the main advantage of pedicle screws is their directly effect on the three columns of spine to make the three-dimensional correction of scoliosis more effective: fixing solidly, uneasily moving, fewer intraoperative complications without effect on the spinal canal. In spite of these advantages, its complications are reported a lot, such as loosing, pulling nails, breaking, etc. Youth is characterized as those who require orthopedic surgery of scoliosis, with the spine at the stage of rapid growth, big curvature angle, and three-dimensional spinal deformity. Therefore, force in the vertebral body is very complex and varies with age, which requires pedicle screws in the vertebral body firmly integrating with the vertebrae, having good biomechanical stability. Many studies reported a variety of plants can increase the compatibility of bone and bony tissue through surface coatings. However, pedicle screw coating is very little studied and the study of the pedicle coating with the organic extracellular matrix components is not reported at home and abroad.
In recent years, the coating technology develops a lot with the theory of conducting and inducting bone formation around the surface of internal fixation, contributing to the progress of the compatibility and biomechanical stability of internal fixation in the host body. Nowadays, the most commonly used coating material is HA, collagenⅠa nd chondroitin sulfate (CS).
Hydroxyapatite (HA), a biologically active substance, can conduct early bone formation, compared to the peptide alloys plant. Vitro experiments proved that HA improves adhesion and differentiation of not only osteoblasts, but also bone marrow stromal cells. HA particles have only a little effect on inducing osteoclast differentiation and can promote collagen formation in extracellular matrix. HA can strongly adsorb RGD peptide sequence, with biologically active molecules inducing bone cell adhesion, differentiation and expansion. In the HA surface, RGD sequence with the help of the glycoprotein (saliva protein) can regulate adhesion, proliferation and differentiation of osteoblast more effectively, which shows that HA can increase and induce reactivity of the RGD sequence and glycoprotein formatting bone.
CollagenⅠi s a kind of three-peptide micro fiber protein (its diameter is 30-300nm) with 15-25 non-helical amino terminals. It is the basic subunit of collagen fibers and is the main organic ingredients of bone. As a stent and the plant coating, CollagenⅠguides bone regeneration. The titanium metal coating rods with CollagenⅠimplanted in tibia of rats can increase the bone reconstruction around the rods. By implanting plant with CollagenⅠcoating in rabbit's femur, dog mandible, goat and the femur of other animal models, bone reconstruction and the new bone ingrowths can be successfully observed on the of coating surface.
CollagenⅠ, as a scaffold of mineral deposition, contains RGD sequence (arginine-工团glycineaspartic acid). This kind of protein can be identified by integrin [97-102]. Integrin is a cell surface specific receptor and a heterodimer transmembrane receptors to regulate binding of cell and extracellular matrix components (such as collagen, fibronectin, laminin, fibronectin glass, glass adhesion protein). CollagenⅠmainly integrates with integrinα2β1 andα1β1, to further affect the adsorption and osteoblast differentiation. Culturing osteoblasts in fetal rat's calvaria on the plate coated with collagenⅠ, CollagenⅠpromotes the maturation of osteoblasts, decreases expression of acid-soluble collagen, and promotes the high expression of alkaline phosphatase, osteonectin, osteopontin, osteogenic gene. By culturing rat's calvarial osteoblasts on titanium plate coated with CollagenⅠ, we find that CollagenⅠpromotes the adsorption and differentiation of osteoblasts. CollagenⅠinteracts with integrins of osteoblast, not only because it contains RGD sequence, but also because DGEA motif of CollagenⅠa nd integrinβ1 plays a very important role on recognition of CollagenⅠconformation. This shows that the CollagenⅠcan induce adsorption, mature and differentiate of osteoblasts, not only because it contains protein motifs, but its conformation plays a key role as well.
In vivi or vitro, CollagenⅠcan take fibrillogenesis and cross-linking. Cellular response is affected by different chemical stability of collagen through fibrillogenesis and cross-linked. Unassembled collagen coating is not favorable to cell amplification and absorption, which is more obvious in a high density of collagen coating. However, after collagen fibrillogenesis, the collagen coating on the titanium surface can effectively enhance the response of osteoblast activity. As to the effect on collagen cross-linking, cell response is better than unassembled collagen coating. However, after collagen fibrosis, cell reactivity is not changed by more cross-linking. Although chemical stability of the fibrotic collagen is improved by cross-link, the response activity of osteoblast was not affected by cross-linking with collagen fiber. In short, collagen fibril cross-linking coating is more conducive to cell reactivity,compared with unassembled collagen fibril coating; but it does not affect the assembled collagen fibers. Assembly of collagen fibrils will not only improve the chemical stability of the collagen coating on the titanium surface, but also improve the induction of cell reactivity. A major weak point of cross-linking is that in collagen cross-linking agent, coating residue is caused by toxicity, and may change the natural conformation of the collagen fibers, and cross-linking with physical methods will also make collagen partially degenerated. Therefore, in the experiment, we did not carry out collagen cross-linking, and directly increase collagen chemical stability and biological activity using fibrillogenesis.
Chondroitin sulfate is a glucosamine chitosan polymer composed by disaccharide units. In recent years, chondroitin sulfate has been widely used in tissue engineering experiments and clinical studies. As additives of calcium phosphate compounds, chondroitin sulfate successfully restores the andibular bone defect. Using scanning electron microscopy, it showed that the proteoglycan coating on the titanium surface is a necessary condition for cell adsorption amplification. And condroitin sulfate can promote cell adsorption, differentiation and expansion. In the process of collagen fibrosis, adding chondroitin sulfate can improve the formation of collagen fibers, and make them more stable. Plant coating similar to the natural body structure, improves the induction of osteoblast activity. We designed four groups of titanium pedicle screws coated with the CollⅠ/ CS/HA, the CollⅠ/CS, HA and uncoated four groups. We observed whether four coated screws can conduct and induce osteogeny under loaded and non-loaded condition using young goats as experimental samples, in order to guide coated pedicle screws used in young patients whose bones are under developing. Methods: The four groups of coated titanium pedicle screws were randomly implanted into the bilateral pedicles (L2～L5) using young goats with 2.5 ~3 months old. On one side, the non-loaded test was taken; on the other side, the loaded test was taken by adding the connection rod to fix the L2-L3, L4-L5 segment. We observed the loaded situation of coated pedicle screws in young goats with the process of spine growth, and the combination of the screw surface and the surrounding bone organizations with age increasing. After three months, using micro CT, histological and biomechanical observations were taken.
Results: 1. Histomorphological observations: osteogeny around non-loaded coated pedicle screws was observed. Bone deposition was formed a lot on the surface of the pedicle screws coated with CollⅠ/CS/HA, which combined with surrounding both new and old bone tissues firmly. The surfaces of titanium pedicle screws coated with CollⅠ/CS and HA were rough, which indicated mineral deposition on the surfaces and new bone formation on the surfaces. However, compared with the screw surfaces coated with CollⅠ/CS/HA, the amount of bone formed was much less than that on the surfaces coated with CollⅠ/CS and HA. There was no significant difference between the two groups coated with CollⅠ/CS and HA. We also observed the process of the coated screw surface combined with the surrounding bone. First of all, on the screw surface, cell adhesion and mineral deposition formed, and gradually new bone formed. Bone tissues around the screw progressively formed new bone at the direction of the coated surface. When two directions of bone tissues joined, the bone remodeled, the surface of the pedicle screw coated was solidly combined with the surrounding bones. Less new bone formed on the surface of titanium uncoated pedicle screw, but much connective tissue appeared. The screw surface was smooth, no mineral deposited, and gap existed between partial surface of uncoated screw and the surrounding bone tissues. After removing the connecting rod, for each loaded screw group, screws were loose. When fixing sections, the screws in the bone were broken off. A large amount of connective tissue formed around screw threads and among screw threads of each loaded screw group without new bone formation. Mineral salt deposition was found in connective tissues.
2. Micro-CT observation. As to the morphological parameters (BMC, BMD, TMC, TMD, BVF, BS / TV and the connectivity density of ROI), those of the pedicle screws coated with CollⅠ/ CS/HA were the largest, followed by the screws coated with CollⅠ/CS and HA, and those of uncoated scews were the smallest. It has remarkable significance (P> 0.01 or P> 0.05) that BMC, BMD of ROI around coated pedicle screws are larger than those of uncoated ones.
3.Biomechanical observations: as to pullout strength, under non-load condition, that of pedicle screws coated with CollⅠ/CS/HA was the most, followed by the pedicle screws coated with CollⅠ/ CS and HA and that of uncoated pedicle screws was the smallest ( P<0.01). There was no remarkable significance for the pullout strength of the screws coated with Coll I/CS and the screws coated with HA is. Under non-loaded condition, the maximum pullout strength was 202.8333±70.6638N, no significant difference among the groups (P﹥0.05). Under non-loaded condition, after maximum pullout strength was reached, the mechanical curve reached the peak, which was the only peak in the image, and gradually dropped to the lowest value. However, under loaded condition, the mechanical curve gradually decreased after peak value reached, and then increased again, in which case, many peaks appeared in the image.
Conclusion: under non-loaded condition, organic and inorganic components of extracellular matrix coated on titanium pedicle screws can conduct or induct the bone formation around the screw, increasing the biomechanical stability of pedicel screws within the vertebral body. CollⅠ/CS/HA coating has stronger capacity to induce bone formation. Under loaded condition, a lot of connective tissues form around the screws, no significant difference among the screw groups. We have successfully established the mechanical animal model of loaded and non-loaded pedicle screws using young goats, whose lumbar is in process of growth, the first time at home and abroad, to simulate the situation of pedicle screws implanted in the spine during growth process of young people. It is the first experiment using micro CT to help compare density and morphometric parameters among various pedicle screw groups.
In pathology, spinal scoliosis is a spinal deformity of three-dimensional structure, involving the anterior, middle and posterior columns of the spine. Therefore, scoliosis surgery is a three-dimensional orthopedic surgery, which has been the difficulty in orthopedic surgery. The orthopedic fixation system with hook (e.g., pedicle hooks, facet hook, and laminar hooks) as the main structure only fixes in the middle and posterior column of the spine, which lacks of a real three-dimensional correction of spinal forces. In recent years, in scoliosis surgery, the pedicle screw is more and more used. Clinical Practice shows that the main advantage of pedicle screws is their directly effect on the three columns of spine to make the three-dimensional correction of scoliosis more effective: fixing solidly, uneasily moving, fewer intraoperative complications without effect on the spinal canal. In spite of these advantages, its complications are reported a lot, such as loosing, pulling nails, breaking, etc. Youth is characterized as those who require orthopedic surgery of scoliosis, with the spine at the stage of rapid growth, big curvature angle, and three-dimensional spinal deformity. Therefore, force in the vertebral body is very complex and varies with age, which requires pedicle screws in the vertebral body firmly integrating with the vertebrae, having good biomechanical stability. Many studies reported a variety of plants can increase the compatibility of bone and bony tissue through surface coatings. However, pedicle screw coating is very little studied and the study of the pedicle coating with the organic extracellular matrix components is not reported at home and abroad.
In recent years, the coating technology develops a lot with the theory of conducting and inducting bone formation around the surface of internal fixation, contributing to the progress of the compatibility and biomechanical stability of internal fixation in the host body. Nowadays, the most commonly used coating material is HA, collagenⅠa nd chondroitin sulfate (CS).
Hydroxyapatite (HA), a biologically active substance, can conduct early bone formation, compared to the peptide alloys plant. Vitro experiments proved that HA improves adhesion and differentiation of not only osteoblasts, but also bone marrow stromal cells. HA particles have only a little effect on inducing osteoclast differentiation and can promote collagen formation in extracellular matrix. HA can strongly adsorb RGD peptide sequence, with biologically active molecules inducing bone cell adhesion, differentiation and expansion. In the HA surface, RGD sequence with the help of the glycoprotein (saliva protein) can regulate adhesion, proliferation and differentiation of osteoblast more effectively, which shows that HA can increase and induce reactivity of the RGD sequence and glycoprotein formatting bone.
CollagenⅠi s a kind of three-peptide micro fiber protein (its diameter is 30-300nm) with 15-25 non-helical amino terminals. It is the basic subunit of collagen fibers and is the main organic ingredients of bone. As a stent and the plant coating, CollagenⅠguides bone regeneration. The titanium metal coating rods with CollagenⅠimplanted in tibia of rats can increase the bone reconstruction around the rods. By implanting plant with CollagenⅠcoating in rabbit's femur, dog mandible, goat and the femur of other animal models, bone reconstruction and the new bone ingrowths can be successfully observed on the of coating surface.
CollagenⅠ, as a scaffold of mineral deposition, contains RGD sequence (arginine-工团glycineaspartic acid). This kind of protein can be identified by integrin [97-102]. Integrin is a cell surface specific receptor and a heterodimer transmembrane receptors to regulate binding of cell and extracellular matrix components (such as collagen, fibronectin, laminin, fibronectin glass, glass adhesion protein). CollagenⅠmainly integrates with integrinα2β1 andα1β1, to further affect the adsorption and osteoblast differentiation. Culturing osteoblasts in fetal rat's calvaria on the plate coated with collagenⅠ, CollagenⅠpromotes the maturation of osteoblasts, decreases expression of acid-soluble collagen, and promotes the high expression of alkaline phosphatase, osteonectin, osteopontin, osteogenic gene. By culturing rat's calvarial osteoblasts on titanium plate coated with CollagenⅠ, we find that CollagenⅠpromotes the adsorption and differentiation of osteoblasts. CollagenⅠinteracts with integrins of osteoblast, not only because it contains RGD sequence, but also because DGEA motif of CollagenⅠa nd integrinβ1 plays a very important role on recognition of CollagenⅠconformation. This shows that the CollagenⅠcan induce adsorption, mature and differentiate of osteoblasts, not only because it contains protein motifs, but its conformation plays a key role as well.
In vivi or vitro, CollagenⅠcan take fibrillogenesis and cross-linking. Cellular response is affected by different chemical stability of collagen through fibrillogenesis and cross-linked. Unassembled collagen coating is not favorable to cell amplification and absorption, which is more obvious in a high density of collagen coating. However, after collagen fibrillogenesis, the collagen coating on the titanium surface can effectively enhance the response of osteoblast activity. As to the effect on collagen cross-linking, cell response is better than unassembled collagen coating. However, after collagen fibrosis, cell reactivity is not changed by more cross-linking. Although chemical stability of the fibrotic collagen is improved by cross-link, the response activity of osteoblast was not affected by cross-linking with collagen fiber. In short, collagen fibril cross-linking coating is more conducive to cell reactivity,compared with unassembled collagen fibril coating; but it does not affect the assembled collagen fibers. Assembly of collagen fibrils will not only improve the chemical stability of the collagen coating on the titanium surface, but also improve the induction of cell reactivity. A major weak point of cross-linking is that in collagen cross-linking agent, coating residue is caused by toxicity, and may change the natural conformation of the collagen fibers, and cross-linking with physical methods will also make collagen partially degenerated. Therefore, in the experiment, we did not carry out collagen cross-linking, and directly increase collagen chemical stability and biological activity using fibrillogenesis.
Chondroitin sulfate is a glucosamine chitosan polymer composed by disaccharide units. In recent years, chondroitin sulfate has been widely used in tissue engineering experiments and clinical studies. As additives of calcium phosphate compounds, chondroitin sulfate successfully restores the andibular bone defect. Using scanning electron microscopy, it showed that the proteoglycan coating on the titanium surface is a necessary condition for cell adsorption amplification. And condroitin sulfate can promote cell adsorption, differentiation and expansion. In the process of collagen fibrosis, adding chondroitin sulfate can improve the formation of collagen fibers, and make them more stable. Plant coating similar to the natural body structure, improves the induction of osteoblast activity. We designed four groups of titanium pedicle screws coated with the CollⅠ/ CS/HA, the CollⅠ/CS, HA and uncoated four groups. We observed whether four coated screws can conduct and induce osteogeny under loaded and non-loaded condition using young goats as experimental samples, in order to guide coated pedicle screws used in young patients whose bones are under developing. Methods: The four groups of coated titanium pedicle screws were randomly implanted into the bilateral pedicles (L2～L5) using young goats with 2.5 ~3 months old. On one side, the non-loaded test was taken; on the other side, the loaded test was taken by adding the connection rod to fix the L2-L3, L4-L5 segment. We observed the loaded situation of coated pedicle screws in young goats with the process of spine growth, and the combination of the screw surface and the surrounding bone organizations with age increasing. After three months, using micro CT, histological and biomechanical observations were taken.
Results: 1. Histomorphological observations: osteogeny around non-loaded coated pedicle screws was observed. Bone deposition was formed a lot on the surface of the pedicle screws coated with CollⅠ/CS/HA, which combined with surrounding both new and old bone tissues firmly. The surfaces of titanium pedicle screws coated with CollⅠ/CS and HA were rough, which indicated mineral deposition on the surfaces and new bone formation on the surfaces. However, compared with the screw surfaces coated with CollⅠ/CS/HA, the amount of bone formed was much less than that on the surfaces coated with CollⅠ/CS and HA. There was no significant difference between the two groups coated with CollⅠ/CS and HA. We also observed the process of the coated screw surface combined with the surrounding bone. First of all, on the screw surface, cell adhesion and mineral deposition formed, and gradually new bone formed. Bone tissues around the screw progressively formed new bone at the direction of the coated surface. When two directions of bone tissues joined, the bone remodeled, the surface of the pedicle screw coated was solidly combined with the surrounding bones. Less new bone formed on the surface of titanium uncoated pedicle screw, but much connective tissue appeared. The screw surface was smooth, no mineral deposited, and gap existed between partial surface of uncoated screw and the surrounding bone tissues. After removing the connecting rod, for each loaded screw group, screws were loose. When fixing sections, the screws in the bone were broken off. A large amount of connective tissue formed around screw threads and among screw threads of each loaded screw group without new bone formation. Mineral salt deposition was found in connective tissues.
2. Micro-CT observation. As to the morphological parameters (BMC, BMD, TMC, TMD, BVF, BS / TV and the connectivity density of ROI), those of the pedicle screws coated with CollⅠ/ CS/HA were the largest, followed by the screws coated with CollⅠ/CS and HA, and those of uncoated scews were the smallest. It has remarkable significance (P> 0.01 or P> 0.05) that BMC, BMD of ROI around coated pedicle screws are larger than those of uncoated ones.
3.Biomechanical observations: as to pullout strength, under non-load condition, that of pedicle screws coated with CollⅠ/CS/HA was the most, followed by the pedicle screws coated with CollⅠ/ CS and HA and that of uncoated pedicle screws was the smallest ( P<0.01). There was no remarkable significance for the pullout strength of the screws coated with Coll I/CS and the screws coated with HA is. Under non-loaded condition, the maximum pullout strength was 202.8333±70.6638N, no significant difference among the groups (P﹥0.05). Under non-loaded condition, after maximum pullout strength was reached, the mechanical curve reached the peak, which was the only peak in the image, and gradually dropped to the lowest value. However, under loaded condition, the mechanical curve gradually decreased after peak value reached, and then increased again, in which case, many peaks appeared in the image.
Conclusion: under non-loaded condition, organic and inorganic components of extracellular matrix coated on titanium pedicle screws can conduct or induct the bone formation around the screw, increasing the biomechanical stability of pedicel screws within the vertebral body. CollⅠ/CS/HA coating has stronger capacity to induce bone formation. Under loaded condition, a lot of connective tissues form around the screws, no significant difference among the screw groups. We have successfully established the mechanical animal model of loaded and non-loaded pedicle screws using young goats, whose lumbar is in process of growth, the first time at home and abroad, to simulate the situation of pedicle screws implanted in the spine during growth process of young people. It is the first experiment using micro CT to help compare density and morphometric parameters among various pedicle screw groups.
引文
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