摘要
第一部分BMP-2相关多肽促进三维培养的骨髓基质干细胞成骨分化及矿化的实验研究
目的评价BMP-2相关多肽诱导体外三维培养的骨髓基质干细胞(bone marrowstromal cells，BMSCs)成骨分化及矿化的能力。
方法原代培养大鼠BMSCs，细胞传至第3代时，将细胞消化收集，与Cytodex~(TM) 3微载体三维培养体系复合培养，完全培养基培养1d后，将培养基换为成骨诱导培养基，实验组培养基加入终浓度为200μg／ml的BMP-2相关多肽，对照组不加多肽。复合培养7天和14天后，进行死／活细胞染色，观察微载体三维培养体系中BMSCs的存活情况，通过检测细胞内钙含量和碱性磷酸酶(alkaline phosphatase，ALP)染色评价BMSCs成骨分化情况，并进行钙黄绿素染色评价细胞矿化情况。
结果细胞培养7天和14天后，BMSCs在实验组和对照组微载体三维培养体系中均存活良好，差异无统计学意义(P＞0.05)；实验组钙含量明显高于对照组，差异具有统计学意义(P＜0.05)，实验组ALP阳性细胞明显多于对照组，钙黄绿素染色结果表明实验组与对照组相比细胞矿化明显。
结论BMP-2相关多肽能够有效促进体外三维培养的BMSCs成骨分化及矿化。
第二部分纳米羟基磷灰石／胶原／聚乳酸／BMP-2相关多肽复合材料的制备、特征及其对骨髓基质干细胞生物学行为的影响
目的制备纳米羟基磷灰石／胶原／聚乳酸／BMP-2相关多肽复合材料，观察其特征并评价其对大鼠骨髓基质干细胞粘附、增殖及分化等生物学行为的影响。
方法用生物仿生自组装的方法制备纳米羟基磷灰石／胶原复合材料(nano-Hydroxyapatite／collagen，nHAC)，在此基础上联合聚乳酸(poly lacticacid，PLA)制备胶原矿化骨仿生骨基质材料——纳米羟基磷灰石／胶原／聚乳酸(nano-Hydroxyapatite／collagen／poly lactic acid，nHAC／PLA)，用X射线衍射仪分析其晶体结构，扫描电镜观察其表面微观形貌，将纳米羟基磷灰石／胶原／聚乳酸与BMP-2活性多肽复合，构建纳米羟基磷灰石／胶原／聚乳酸／BMP-2相关多肽复合材料。取第三代BMSCs接种到材料上，以未结合多肽的纳米羟基磷灰石／胶原／聚乳酸作为对照，采用MTT法检测BMSCs在材料表面的增殖；沉淀法检测BMSCs在材料表面的粘附率；扫描电镜观察比较BMSCs在材料表面的生长形态；通过检测细胞中的碱性磷酸酶活性及钙离子浓度，观察BMSCs在材料表面的分化情况。
结果X射线衍射分析结果显示，纳米羟基磷灰石／胶原／聚乳酸复合材料的主要成分是羟基磷灰石(HA)；扫描电镜结果显示：该复合材料是一种疏松多孔结构，孔径从几十微米至300μm不等，并且孔与孔之间有连通。细胞实验结果表明：纳米羟基磷灰石／胶原／聚乳酸／BMP-2相关多肽复合材料与单纯纳米羟基磷狄石／胶原／聚乳酸相比，能够促进BMSCs的粘附和向成骨细胞方向，差异具有统计学意义(P＜0.05)，而对BMSCs的增殖能力没有影响(P＞0.05)。
结论BMP-2活性多肽可以显著改善纳米羟基磷灰石／胶原／聚乳酸复合材料的细胞相容性和生物活性，负载BMP-2活性多肽的纳米羟基磷灰石／胶原／聚乳酸复合材料是一种理想的骨组织工程支架材料。
第三部分纳米羟基磷灰石／胶原／聚乳酸／BMP-2相关多肽仿生骨基质材料异位成骨的实验研究
目的评价纳米羟基磷灰石／胶原／聚乳酸／BMP-2相关多肽仿生骨基质材料的异位成骨能力。
方法制备纳米羟基磷灰石／胶原／聚乳酸／BMP-2相关多肽复合材料，采用高效液相色谱仪检测不同时间点BMP-2相关多肽的释放量，观察BMP-2相关多肽的体外释放规律。将48只成年雄性SD大鼠随机分为四组，在A组、B组、C组大鼠背部肌肉内分别植入3mg、2mg、1mg的BMP-2相关多肽与纳米羟基磷灰石／胶原／聚乳酸复合材料；在D组大鼠背部肌肉内植入单纯纳米羟基磷灰石／胶原／聚乳酸，于4周和8周两个时间点将大鼠分批处死，经CT三维重建、组织学观察及钙含量测定检测植入材料的异位成骨情况。
结果体外释放实验结果显示BMP-2相关多肽释放规律为：第1天表现为爆发性释放，释放量为29.57％，以后缓慢持续释放，至21d累积释放量达65.98％。A、B、C组，在各时间点的CT三维重建、组织学观察及钙含量检测结果均表明其异位成骨能力优于D组，差异有统计学意义(P＜0.01)，其中A、B两组的异位成骨能力差异无统计学意义(P＞0.05)，但两组均优于C组，差异有统计学意义(P＜0.01)。
结论BMP-2相关多肽以纳米羟基磷灰石／胶原／聚乳酸支架材料为载体能够缓慢释放。BMP-2相关多肽可以显著增强纳米羟基磷灰石／胶原／聚乳酸的骨诱导活性，这种骨诱导性存在一定的剂量效应关系。纳米羟基磷灰石／胶原／聚乳酸／BMP-2相关多肽复合材料能够诱导异位骨形成，是一种具有良好骨诱导性的仿生骨修复材料。
第四部分纳米羟基磷灰石／胶原／聚乳酸／BMP-2相关多肽仿生骨基质材料促进大鼠颅骨缺损修复的实验研究
目的探讨纳米羟基磷灰石／胶原／聚乳酸／BMP-2相关多肽仿生骨基质材料促进大鼠颅骨缺损修复的可行性和有效性。
方法成年SD大鼠24只，在大鼠顶骨左、右两侧分别制备直径5mm的颅骨缺损，左侧设为实验组，植入纳米羟基磷灰石／胶原／聚乳酸／BMP-2相关多肽复合材料，对侧设为对照组，植入纳米羟基磷灰石／胶原／聚乳酸，于4和12周时间点将大鼠分批处死，进行大体观察、CT三维重建、组织学观察、扫描电镜观察和透射电镜观察，取术后12周的标本进行Masson三染、免疫组化染色和生物力学检测，比较各组材料促进骨再生修复骨缺损的能力。
结果CT三维重建结果显示：实验组4周时可见片状高密度影，12周时可见骨缺损完全愈合。对照组4周时仅骨缺损边缘可见轻微的片状致密影，12周时高密度影有所增强，面积增大，接近缺损面积的1／2，未能完全修复骨缺损。组织学观察结果显示术后4周，实验组骨缺损处可见大量新骨生成，而对照组仅有少量新骨生成；术后12周，实验组可见大部分支架材料降解，新生骨与宿主骨之间达到完全骨性结合，对照组支架材料部分降解，残余支架被新生骨组织包围。在各时间点，实验组新骨形成面积百分比均大于对照组，差异有统计学意义(P＜0.05)。扫描电镜结果显示：4周时两组材料部分降解，可见成骨细胞黏附和类骨质形成，实验组材料与正常骨组织有部分键合，对照组材料与正常骨组织间存在明显间隙；12周时实验组材料大部分降解并由板层骨取代，骨缺损完全修复，对照组骨缺损部分修复。透射电镜结果显示：4周时实验组材料内部可见功能活跃，代谢旺盛的成骨细胞，分泌的胶原排列无序，12周时可见大量成骨细胞，在骨小梁表面成层排列，部分成骨细胞演变为骨细胞，胶原排列有序紧密。对照组在材料周围仅见少量成骨细胞，其分泌胶原功能不活跃。术后12周时，Masson三染结果显示实验组骨缺损处存在大量红色的成熟钙化组织，对照组仅见少量绿色的新生骨样组织。骨钙素和骨桥蛋白免疫组化染色结果进一步印证了HE染色和Masson三染的发现，实验组可见大量骨钙素阳性和骨桥蛋白阳性的新生骨组织，明显多于对照组骨钙素阳性和骨桥蛋白阳性的骨组织。生物力学检测结果显示：实验组最大压力载荷和载荷／应变比值均明显高于对照组差异有统计学意义(P＜0.01)。
结论纳米羟基磷灰石／胶原／聚乳酸／BMP-2相关多肽复合材料促进骨缺损修复能力明显强于单纯纳米羟基磷灰石／胶原／聚乳酸，纳米羟基磷灰石／胶原／聚乳酸／BMP-2相关多肽复合材料是一种优良的具有骨诱导活性的骨缺损修复材料。
PartⅠEffect of BMP-2-related peptide on osteogenic differentiation andmineralization of BMSCs cultured in three-dimensional system
Objective To evaluate the effect of BMP-2-related peptide on osteogenic differentiationand mineralization of BMSCs cultured in three-dimensional system
Methods BMSCs were harvested from 4-week-old SD rats and cultured with normalmedium containing low-glucose Dulbecco's modified Eagle's medium (DMEM), 10% fetalbovine serum (FBS) and 1% antibiotics. The 3rd generation BMSCs were dissociated.collected and cultured with Cytodex~(TM) 3 collagen microcarrier in three-dimensional system.After one day, the medium was changed to osteogenetic differentiation medium whichconsisted of the culture media previously described, supplemented with 10mMβ-glycerophosphate and 50 mg/ml ascorbic acid. In the experimental group, the medium wasadded to 200μg/ml BMP-2-related compared with the control group. After 7d and 14d. thecell survival of BMSCs was determined by a fluorescent live/dead assay. The calciumcontents and alkaline phosphatase(ALP) staining were measured to assess the differentiationof BMSCs towards osteoblasts. The mineralization of BMSCs was determined by calceinstaining.
Results BMSCs cultured with Cytodex~(TM) 3 collagen microcarrier grew well in bothexperimental group and control group. There was no statistical significance between the twogroups (P＞0.05).The result of calcium contents and ALP staining suggested theexperimental group was significant higher than the control group(P＜0.05). The result ofcalcein staining showed that the mineralization level of BMSCs in the experimental groupwas significant higher than that in the control group.
Conclusion BMP-2-derived peptide can promote osteogenic differentiation andmineralization of BMSCs cultured in three-dimensional system effectively.
PartⅡPreparation and characteristic of scaffold based onmineralized collagen loaded with synthetic BMP-2-related peptide andits effect on biological behavior of bone marrow stromal cells
Objective In this study, a new biomimetic bone tissue engineering scaffold material, thescaffold based mineralized collagen loaded with BMP-2-related peptide was prepared. Theeffect of BMP-2-related peptide on the adhesion, proliferation and differentiation of BMSCswere investigated.
Methods The scaffold material nano-hydroxyapatite/collagen/poly(lactic acid)(nHAC/PLA) was developed by biomimetic synthesis.The hierarchical microstructure of thescaffold material was analyzed by X-ray diffraction. The microcosmic appearance of thescaffold was observed by scanning electron microscope. BMP-2-related peptide wasintroduced into the porous nHAC/PLA scaffold. The third generation of BMSCs was seededonto the scaffolds and the scaffold without BMP-2-related peptide as a control. The adhesionof BMSCs was assessed by precipitation method. The proliferative ability of BMSCs weremeasured by MTT assay and scanning electron microscope. The alkaline phosphatase activities and calcium contents were measured to assess the differentiation of BMSCstowards osteoblasts.
Results The result of X-ray diffraction suggested that the inorganic phase in themineralized collagen scaffold was determined as HA. The results of scanning electronmicroscope showed that the surface of the scaffold was porous and the pores wereinterconnected. The pore sizes were from several 10μm to about 300μm. The abilities ofadhesion and differentiation to osteoblast of the BMSCs in the experimental group weresignificantly greater than those in the control group (P＜0.05), but with regard to the abilitiesof proliferation of the BMSCs. there is no significant difference between the experimentalgroup and the control group(P＞0.05).
Conclusion The synthetic BMP-2-related peptide could improve biocompatibility andbioactivity of the nHAC/PLA scaffold material. The nHAC/PLA scaffold loaded withBMP-2-related peptide is a kind of ideal scaffold material for bone tissue engineering.
PartⅢEctopic osteogenesis of scaffold based on mineralizedcollagen loaded with synthetic BMP-2-related peptide
Objective To evaluated the ectopic osteogenetic capacity of the scaffold based onmineralized collagen loaded with synthetic BMP-2-related peptide.
Methods The scaffold material nano-hydroxyapatite/collagen/poly(lactic acid)(nHAC/PLA) was prepared. BMP-2-related peptide was introduced into the porousnHAC/PLA scaffold. For observing the releasing character of BMP-2-related peptide.thereleased peptide content from the scaffold was detected using high performance liquidchromatography at different set times. 48 SD rats were allocated into four groups randomly.Four kinds of scaffold materials were respectively implanted subcutaneously into rats. Group A: the nHAC/PLA scaffold loaded with 3 mg BMP-2-related peptide, Group B: thenHAC/PLA scaffold loaded with 2 mg BMP-2-related peptide, Groups C: the nHAC/PLAscaffold loaded with 1 mg BMP-2-related peptide, Groups D: the nHAC/PLA scaffold alone.At the 4th and 8th weeks after implantation, the rats were sacrificed in batch and the sampleswere harvested. Their osteogenic capability was detected by CT scan and histologicalobservation.
Results The releasing character of BMP-2-related was that an initial burst releasing(29.57%) was observed over the first day, followed by a sustained release and reached 65.98% after 21 days. The results of CT scan and histological observation demonstrated that theosteogenic capability of 3 mg, 2 mg and 1 mg of the BMP-2-related peptide was superior tothe implants without the BMP-2-related peptide(P＜0.05). There was no significant differencebetween implants with 3 mg and 2 mg BMP-2-related peptide(P＞0 05), but the osteogeniccapability of the two dosage groups was significantly better than that of the 1 mggroup(P＜0.05).
Conclusion BMP-2-related peptide can increase the osteoinduction of nHAC/PLAscaffold and the BMP-2-related peptide induced ectopic bone formation in a dose-dependentmanner. The nHAC/PLA scaffold loaded with the BMP-2-related peptide is a kind of idealscaffold material with good osteoinductivity.
PartⅣRepair of cranial bone defects with scaffold based onmineralized collagen loaded with synthetic BMP-2-related peptide
Objective To investigate the osteogenetic capacity of nHAC/PLA loaded with aBMP-2-related peptide biomimetic scaffold materials.
Methods Rat cranial defects were created on both sides of the parietal bone of 24 adult Sprague-Dawley rats. BMP-2-related peptide combined with nHAC/PLA scaffold wereimplanted on the left side as experimental group, leaving the other side implanted withnHAC/PLA scaffold alone as control group. Up to the 4~(th) and 12~(th) weeks, the rats were killedin batch respectively. Macroscopic observation, three-dimensional reconstruction of computedtomography, histological observation and scanning electron microscope observation wereperformed on these samples. Biomechanics detection, Masson trichome staining, andimmunohistochemical analysis were performed on the 12~(th) week's sample additionally.
Results Three-dimensional reconstruction of computed tomography indicated thatthere were radiodense areas in the left dorsal regions of each rat. but the density of theradiodense areas of experimental group were obviously denser than the density of controlgroup at the 4~(th) week. The defects completety healed at the 12~(th) week in the experimentalgroup that was treated with BMP-2-related peptide loaded nHAC/PLA, while the completebone healing rate in control group was just 1/2. Histological observation showed a largeamount of osteoid tissue and new bone had been observed in the experimental groups at the4th week. While the control group showed only minimal new bone formation at the defectmargins. At the 12th week, in the experimental group, the bony-union between new bone andhost bone was observed. Meanwhile. the composite was almost completely degraded. In thecontrol group, there were still slight amounts of new bone, the scaffolds were only partlydegraded and the residual materials were surrounded by areas of new bone formation. Thepercentage of bone formation areas in the experimental group was superior to the controlgroup at 4~(th) and 12~(th) weeks, the differences were statistically significant (P＜0.05). Scanningelectron microscope demonstrated that the bone defects were repaired completely in theexperimental group, where most composite was degraded and replaced by lamellar bone,compared with partly repaired in the control group. Masson trichome staining at the 12~(th) weekshowed that massive calcified bones stained red color were observed in the experimentalgroup. However, in the control group there was only slight amounts of fresh-formed bonestained blue color. The results of immunohistochemical staining for osteocalcin (OCN) and osteopontin (OPN) further supported the H&E and Masson trichrome staining findings.Positive OCN and OPN staining were found throughout the overall region of the regeneratedbone in the experimental group. In contrast, the regenerated tissue from the control groupshowed much lower OCN and OPN immunoreactivity than the experimental group.Biomechanics detection indicated the maximum compression loading and loading/strain ratioon repairing tissue of defects in the experimental group was obviously superior to those in thecontrol group. The differences were also statistically significant (P＜0.01)
Conclusion The osteogenic capability of BMP-2-related peptide loaded nHAC/PLA isobviously superior to nHAC/PLA alone. BMP-2-related peptide loaded nHAC/PLAbiomimetic scaffold material can be a potential repairing material for bone defects.
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