纳米羟基磷灰石/壳聚糖复合VEGF转染BMSCs构建骨复合体修复骨缺损的实验研究
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摘要
研究背景
在生命活动中由外伤、感染、肿瘤及先天性疾病等原因造成的骨缺损十分常见,是骨科临床常见的难症之一。大的骨缺损难以自行愈合,需要植骨材料修复,但目前临床使用的骨修复材料各有利弊,不能达到修复骨缺损理想材料的要求。近年来随着组织工程技术的发展,研制寻找良好的骨替代材料,与种子细胞联合培养,同时采用缓释技术添加信号因子,构建有生命活性的组织工程骨复合体,无疑将会对临床修复骨缺损带来重大的科学意义和实用价值。羟基磷灰石(hydroxyapatite, HA)是人体骨组织的主要成分,自20世纪70年代末,羟基磷灰石作为新型生物材料问世以来,已在材料学科和医学界,广泛应用于整形外科、矫形外科、口腔科等医学相关领域各类骨缺损的修复。但普通的多孔型羟基磷灰石,质脆强度低,生物机械性能较差；致密型羟基磷灰石植入后不能被新骨完全替代,在局部形成占位。近些年来,利用纳米技术制备的纳米级组织工程骨材料,在纳米结构单元或纳米数量级下生产新型材料,其结构与天然骨相似,有助于人体细胞及大分子对其识别,从而可有效提高材料的生物活性、利用度和生物相容性,符合理想人工骨的性能要求。目前对纳米级羟基磷灰石人工制品的研究取得了长足的进展,并在临床上获得了广泛的应用,但是仍距离结构功能一体化的要求相差很远,还存在某些不可克服的缺点,如缺少连通的、均匀的孔结构,不利于骨组织的生长；其降解速度与天然骨组织生长替代速度不协调；力学强度与天然骨组织的力学性能不匹配等。而且国内外大多数学者认为单纯的羟基磷灰石只具有骨生长引导作用,但不具有骨诱导活性。天然骨是无机矿物质与生物大分子规则排列所组成的复合体,可被看成是一种有机／无机的纳米复合材料。因此以羟基磷灰石为基础构建与天然骨类似的复合骨替代材料已成为当前的研究热点。血管生成与血运重建是创面愈合、移植组织存活的关键。VEGF又称血管通透性因子(vascular permeability factor, VPF),是目前发现的作用最强的促血管生成细胞生长因子,VEGF在骨的形成及修复中也起到重要的作用,许多研究表明,VEGF是通过3个方面来实现的(1)通过促进内皮细胞增殖、血管生成参与骨发育形成。(2)作为旁分泌因子或直接参与骨形成与代谢：有研究发现,VEGF可促进血管内皮细胞分泌胰岛素样生长因子-1(IGF-1)及内皮素-1(ET-1),而这些细胞因子刺激人成骨细胞生长。(3)VEGF还可通过作用于人成骨细胞强表达的flt-1受体增加人成骨细胞移动和分化功能。VEGF本身也可以直接作用于成骨细胞,已有实验证明VEGF可以结合成骨细胞,增加其移动和分化功能,但不能促进其增殖。
研究目的
将无机纳米羟基磷灰石和有机羧甲基壳聚糖(CMCTS)制备成与天然松质骨的力学特性、几何形状和表面性质类似的三维孔洞网状结构,形成多孔状、孔隙率高、力学性能好的二级孔道的三维网络结构纳米人工骨基质材料,构建一种结构功能一体化的复合支架材料。同时,依照组织工程骨复合体三要素(支架材料、种子细胞、信号因子)的原则,利用基因转染技术将VEGF转染BMSCs植入复合材料内,使BMSCs在发挥成骨作用的同时,表达分泌VEGF,从而构建一种有生命活性的骨组织工程骨复合体,以期提高骨缺损修复的效果,为研制理想的骨缺损修复替代材料提供一定的实验依据和理论基础。研究方法
1nHA/CMCTS复合支架材料的制备
nHA粉体由本实验室采用化学沉淀法制备,京尼平(Genipin)做交联剂,通过粒子沥滤法、冷冻干燥法成功制备nHA/CMCTS多孔复合材料。
2nHA/CMCTS生物安全性测试
动物模型建立及分组后进行肝肾功能检测,实验组取移植nHA/CMCTS复合材料周围骨组织标本,对照组取缺损处骨组织标本进行组织学观察。体外生物安全性测试。利用细胞形态观测法检测其毒性。
3.构建pcDNA3.1-VEGF165重组载体
提取细胞总RNA,以提取的mRNA为模板,在逆转录酶的作用下反转录合成VEGF165cDNA序列,以VEGF165基因cDNA为模板,用引物特异性扩增。用Wizard PlusMinpreps DNA纯化系统抽提并纯化噬菌粒DH5α,进行3.5pMD18-T和VEGF165基因的双酶切,用凝胶回收试剂盒,自凝胶中分别回收双酶切后的pMD18-T片段和VEGF165基因,连接两个基因,制备大肠杆菌DH5α感受态细菌,构建重组载体并鉴定后,进行质粒的扩增与纯化。
4骨髓间充质干细胞培养
抽取骨髓,分离骨髓单个核细胞,反复洗涤后悬浮于含10%胎牛血清的DMEM培养基,37℃,5%C02及饱和湿度培养箱中培养并换液,观察细胞生长融合情况。胰蛋白酶消化传代。自第三代细胞条件培养基培养(体积分数10%胎牛血清、地塞米松10-8mol/L、β-甘油磷酸钠0.01mmol/L和维生素C0.05g/L)。对不同时期的骨髓间充质干细胞进行形态观察,在质粒转染之前对骨髓间充质干细胞进行了表面标志物鉴定。
5pcDNA3.1-VEGF165转染骨髓间充质干细胞(电转染法)
电转染前处理哺乳动物细胞,pcDNA3.1-VEGF165转染骨髓间充质干细胞复合纳米羟基磷灰石／羧甲基壳聚糖复合材料,即将制备的纳米羟基磷灰石／羧甲基壳聚糖浸泡于已转染pcDNA3.1-VEGF165的第三代骨髓间充质干细胞悬液内48小时,进行材料与细胞复合。
6动物模型制备
将实验兔前肢桡骨中段大约1.8cm的骨质连同骨膜完全切除。随机选择一侧骨缺损作为实验组,局部植入转染PCDNA3.1-VEGF165骨髓间充质干细胞的纳米羟基磷灰石／羧甲基壳聚糖复合材料(VEGF+n-Ha/CMCTs)或者单纯植入纳米羟基磷灰石／羧甲基壳聚糖复合材料(n-Ha/CMCTs);两组的对侧分别作为作为空白对照组1和空白对照2。
7一般情况观察
术后观察动物的一般情况、局部炎症反应、切口愈合时间
8.术后X线观察
术后4周,12周及24周,行常规X射线检查,观察骨缺损处断端及骨形成及修复情况。Lane-Sandhu法评分标准进行评分。
9.取材
分别于术后6周,12周,24周处死动物后沿原缝合切口切开达新生骨膜,仔细分离,观察骨缺损区愈合及修复情况,切取桡骨标本,取中段缺损处修复的组织,将所取标本以10%中性甲醛固定。
10.HE染色标本经脱钙,梯度脱水,透明、浸蜡,制备厚度为5μm切片,苏木素-伊红(H E)染色,观察术后复合材料降解及新生骨生长的情况,测量新生骨面积。
11.生物力学性能检测
分别于术后6周,12周,24周采用三点弯曲法对实验组及对照组新生骨进行生物力学性能测试,加载速率为2mm/min。
12.分子生物学检测
提取标本总蛋白,Western Blot法检测组织内血管内皮细胞生长因子(VEGF),血小板内皮细胞黏附分子1(CD-31),骨桥蛋白(OPN),胰岛素样生长因子1(IGF-1)的表达水平。
13.统计分析
采用SPSS13.0统计软件,定量数据均以均数±标准误表示,应用各组间单因素方差分析进行统计。P<0.05为有统计学意义。
研究结果
1采用粒子沥滤法制备了高孔隙率nHA/CMCTS多孔复合材料。复合材料的成分无变化,仍然为羟基磷灰石与壳聚糖,并且羧甲基以及胺基分别与羟基磷灰石的羟基或Ca2+发生了不同程度作用,形成了较为牢固的界面结合。该多孔材料最高孔隙率高达87%,孔形不一,以圆形为主,其尺寸分布大约从几微米到600微米,具有良好的贯通性,非常有利于组织在其中的长入与扩展。当复合材料中CMCTS含量为40%,复合材料／造孔剂的质量比为1：1时,多孔材料的孔隙率接近75%,其抗压强度可达21MPa以上,可以满足骨组织工程支架材料的要求。复合材料经SBF溶液浸泡之后的SEM分析,从中可以看到孔壁周围出现纳米级颗粒。分析认为具有骨生物活性的无机陶瓷材料在生理环境中通过与体液发生一系列离子交换和化学沉积反应,可在表面形成类骨碳酸羟基磷灰石晶体,与宿主骨组织发生化学键和,并具有诱导骨组织形成的作用
2复合材料的组织学及肝肾功能检测实验组3周可以见到急慢性炎细胞浸润,无异物巨细胞反应,未见骨坏死；对照组3周以炎性充血为主,并可见慢性炎症细胞浸润,实验组6周可见轻微炎症反应,骨组织未见坏死、基本正常；对照组6周可见少量炎细胞,骨组织基本正常,未发现引起骨组织明显的异物巨细胞反应及骨坏死,提示nHA/CMCTS具有良好的组织相容性。本实验对nHA/CMCTS进行了实验动物的肝肾毒性检测,各组动物血清ALT、AST、Cr和Urea的均值,经单因素方差分析,4组动物血清中ALT、AST、Cr和Urea的水平各组内组间比较及各组内组间两两比较均没有统计学意义,提示nHA/CMCTS复合材料未引起肝肾毒损害具有一定的安全性。小鼠胚胎成纤维细胞在复合材料培养不同时间后的细胞形态观察见细胞突充分伸展,可见多角形细胞,细胞逐渐铺满视野。根据细胞形态分析标准,复合材料无细胞毒性。
3.成功构建了构建pcDNA3.1-VEGF165重组载体采用VEGF165cDNA为模板,将VEGF165经大量扩增后,以1%琼脂糖凝胶电泳,在标准核酸分子量附近出现一条特异性扩增条带,片段大小约为237bp左右,图像分析表明产物纯度较高,无非特异性带干扰,适宜进行基因转化；分别以EcoR I和HindⅢ内切酶对二者酶切,结果前者在5.4kb左右有一条特异性条带,后者条带位于225bp左右；用PCR法对重组PcDNA3.1-VEGF165质粒进行鉴定,可见酶切产物为两个条带,分别在5.4kb、230bp左右片段,测序证实其序列与GENEBANK中兔VEGF序列相同。PcDNA3.1-VEGF165基因测序图峰值清晰,无双峰干扰。
4大体观察发现两组术后均无明显炎症反应,创面无感染,切口愈合顺利。
5X线评分结果不同时期X射线照片显示VEGF-nHA/CMCTS实验组较单纯nHA/CMCTS组及两个空白对照组骨愈合明显快,按Lane-Sandhu法评分标准评分,所得数据进行方差分析,Lane-Sandhu组织学评分差异均有统计学意义。
6HE染色观察所见术后6周VEGF-nHA/CMCTS实验组材料中间可见少量骨髓颗粒细胞,植入复合材料未见明显降解；术后12周VEGF-nHA/CMCTS实验组复合材料明显减少,骨髓颗粒细胞明显增多,并可见骨生长；术后24周VEGF-nHA/CMCTS实验组复合材料明显破碎减少,骨及纤维性骨痂生长良好。另将HE染色组织切片根据Lane-Sandhu组织学评分标准进行分析发现,各组组织学评分随着植入时间的延长而增加；在第6周和第12周,VEGF+n-Ha/CMC组评分不仅显著高于两个对照组,且高于n-Ha/CMC组,在第24周,VEGF+n-Ha/CMC与n-Ha/CMC相比无差异；在第6,12,24周,n-Ha/CMC组显著高于两个对照组；在各个时间点,空白对照组1与空白对照组2之间无差异。HE染色组织切片发现VEGF+n-Ha/CMCTS组较其他各组微小血管形成较多,有利于新骨的生长。
7生物力学性能检测实验组及对照组进行采用三点弯曲法对新生骨进行生物力学性能测试,加载速率为2mm/min。结果显示,抗弯强度随时间延长而增大,在VEGF+n-Ha/CMCTS组,从6周起强度突然增大,在12周时已接近达到最大值。而在Ha/CMCTS组,增大幅度则较为平缓,在24周时也接近最大值。在两组空白对照组,抗弯强度随时间延长增大不明显,统计结果显示,VEGF+n-Ha/CMCTS及n-Ha/CMCTS实验组抗弯强度差异性显著高于高于对照组,12周、24周显著性水平明显不同(p<0.05),但两个对照组之间无差异。
8分子生物学检测
术后6周,对四组组织内血管内皮细胞生长因子(VEGF),血小板内皮细胞黏附分子1(CD-31),骨桥蛋白(OPN),胰岛素样生长因子1(IGF-1)蛋白表达水平进行检测,发现VEGF+n-Ha/CMCTS组VEGF, CD-31, OPN, IGF-1的表达不仅显著高于两个对照组,且高于n-Ha/CMCTS组：n-Ha/CMCTS组的表达水平较两个对照组升高,而空白对照组1和空白对照组2之间,上述因子蛋白表达无差异。
结论
1该复合材料孔隙率、抗压强度,可以满足骨组织工程支架材料的要求。nHA/CMCTS植入大白兔骨缺损处未见引起骨组织明显的炎症反应及骨坏死,肝肾功能检测未发现有肝肾毒性,提示nHA/CMCTS具有良好的组织形容性及生物安全性。
2.纳米羟基磷灰石／壳聚糖复合VEGF转染BMSCs构建骨复合体植入骨缺损模型后,促进新生骨的生长,使骨折愈合加快,抗弯能力强,治疗效果明显。
3.纳米羟基磷灰石／壳聚糖复合VEGF转染BMSCs构建骨复合体植入骨缺损模型后,组织内VEGF, CD31, OPN, IGF-1表达水平升高,提示本模型植入后可能通过促进血管新生及直接促进成骨两个方面发挥作用。
4.将VEGF基因转入BMSCs后与复合材料结合,构建了一种新型的有生命活性的骨组织工程骨复合体,为研制理想的骨缺损修复替代材料提供一定的实验依据和理论基础,为提高组织修复与愈合质量与速度提供新的思路。
Background
Bone defect is a common challenge faced by orthopaedic surgeon resulting from various events such as trauma, infection, tumor or congenital disease. Massive bone defect requires graft implant due to limited self-restoration. Current bone repairing material has been imperfect. Recent development in tissue engineering introduced promising bioactive bone engineering material combining bone substitute with progenitor cells to which signal factors are added through controlled-release. Hydroxyapatite is the primary content of human bone. Since its advent in1970s, it has been widely used in orthoplastic, orthopaedic and stomatology to repair bone defect. Conventionally produced hydroxyapatite is crisp and weak; condensed hydroxyapatite halts osteogenesis, forming unwanted occupation. Fortunately, innovative bone substitute structurally similar to natural bone, whose building block is of nano level, has proved easily recognizable to human body cells and macromolecules, hence its bioactivity, practicability and biocompatibility. To date, artificial nanohydroxyapatite product has undergone marked progress, greatly enabling its clinical application. Notwithstanding that, integrated function and structure have yet to be reached. Identified pitfalls include unconnected inconsistent porosity, unsuitable degradation rate and lower mechanical strength than natural. The dominant concept is that bone directing hydroxyapatite is not osteoinductive. Natural bone is a sort of organic/inorganic compound in light of its combination of inorganic mineral and biomacromolecules. Therefore, focused research is now on hydroxyapatite-based scaffold to construct bonemimicing material. Angiogenesis and vascularization play pivotal role in defect heal and graft survival. VEGF, also termed as vascular permeability factor(VPF), is the most efficacious growth factor known, which counts in osteogenesis and defect repair. Multiple researches converged to conclude that VEGF works by:(1) promoting endothelial proliferation, and angiogenesis,(2) influencing bone turnover directly or as a paracrine factor—it's discorved that VEGF lifts insulin-like growth factor and endothelin secretion from endothelium,(3) exciting osteoblast migration and differetiation through impect on fit-1receptor expressed on the cell. VEGF also reacts directly with fibroblast. There are experiments proving its positive action on fibroblast migration and differention, although not proliferation.
Objective
To incorperate hydroxyapatite and conboxymethyl chitosan(CMCTS) constructing a three-dimensioned network similar to natural cancellous bone in mechanics, geometry, and surface properties, aiming at3D nanonetwork both highly porous and mechanically competent with two-graded channels as artificial bone matrix. The material were supposed to suffice in both structure and function. Meanwhile, in accordance with the three essentials of bone tissue engineering--scaffold, seedlings, signal factors--VEGF-transfected BMSCs were implanted into the composite so that VEGF was secreted along with osteogenesis, thus a bioactive bone engineering was established to improve defect repair, buttressing research for ideal bone substitute.
Method
1. Preparation of nHA/CMCT scaffold
Nanohydoxyapatite powder was produced through chemical deposition. Using genipin as cross-linking agent, porous nHA/CMCTS composite was successed through particle leaching and lyophilization.
2. Tests on the biological safety of nHA/CMCTS
After establishment and grouping, liver and kidney function of the animal models were tested. Bone samples were collected around nHA/CMCTS composite in experiment group and from the defect in control group. The toxicity was tested under cytomorphometry.
3. Preparation the Restructuring carrier of pcDNA3.1-VEGF165
The total RNA was extracted and the VEGF165DNA fragment was synthesized reverse transcriptionally from the primer designed. The gene was specifically amplified by the specific primer and template of cDNA to VEGF165.The bacteriphage DH5a was extracted and purified. The pMD18-T and VEGF165were double digeseed and the pMD18-T and VEGF165fragments were collected respectively on Gel Extraction Kit. pcDNA3.1and VEGF165were connected and competent E.Coli was prepared. At last, the combinant carrier was amplified and purified.
4. Culture of bone marrow stromal cells
Individual nucleus cell was seperated from bone marrow and cultured in DMEM with10%fetal calf serum and in the conditional medium with10%fetal calf serum,8-10mol/L dexamethasone,0.01mol/Lp-Glycerin sodium and0.05g/L VitC.
5. Transfection of BMSCs with pcDNA3.1-VEGF165plsmids (electrotransfection)
6. Establishment of animal models
1.8cm length of the middle part of the rabbits'both radii was removed. One foreleg was randomly selected to be implanted with pcDNA3.1-VEGF165transfected BMSCs on nHA/CMCTS scaffold or nHA/CMCTS simply, another foreleg as the empty control,respectively.
7. Postoperative observation.
Observation of general condition, regional inflammation, and time of heal.
8. Radiography examination
The fracture was inspected under radiography on osteogenesis and defect recovery at the end of4w,12w, and24w after operation.
9.Sample collection
The animals were sacrificed and collected from the defect through incision on the original operated sites. Slices5μm thick were aquired through decalcification, gradient dehydration, transparent and waxing.
10. HE staining Observation of material degradation, new bone formation and the new blood vessels with Haematoxylin and eosin (HE) staining.
11. Biomechanical testing
Biomechanical testing was carried out at the end of6w,12w, and24w after the operation with the three point bending method and the loading rate was2mm/min.
12. Melecular biochemestry testing
Melecular biochemestry testing was carried out at the end of6w,12w, and24w after the operation to test the expression of VEGF, CD31, OPN and IGF-1.
13. Statistical analysis
All analyses were performed using SPSS v13.0. Data were expressed as mean±SE. An independent-samples t-test was used to compare continuous data for between-group differences and comparisons among groups involved the use of ANOVA. P<0.05was considered statistically significant.
Result
1Porous nHA/CMCTS composite was prepared through particle-leeching. HA and CMCTS stayed unchanged. Carbonylmethyl group reacted with HA and Ca2+, forming rigid bond on the interface. The maixmal porosity can be as high as87%. The pores, mainly shaped spherical, ranging from a few nanometers to600nm in diameter, interconnected, are inductive to bone ingrowth and expansion.40%CMCTS content, composite/Porous agent ratio of1:1, and porosity of75%forms a ideal combination to reach a compressive strength up to21Mpa, satisfying as bone engineering scaffold. Nano level particles were detected on the pore wall under SEM analysis after immersion in the SBF solution. One theory is that osteoinductiv carbonated hydroxyapatite crystal results from chemical bond-forming reaction of ceramic material with body fluid.
2Histological and biochemical test of the composite. At week4, acute and chronic inflammatory cells infiltration, absent foreign body reaction and necrotic bone were detected in the experiment group, and inflammatory congestion with some chronic inflammatory cell infiltration in the control group. At week6, slight inflammatory reaction emerged in the experiment group and the bone was primarily intact; in the control group, no sign of abnormality showed except slight inflammation. The above suggested good biocompatibility of nHA/CMCTS. nHA/CMCTS was also tested for liver or kidney toxicity. Under analysis of variance, no statistically significant difference in ALT, AST, Cr and Urea was exhibited among the four groups, indicating biotical safety of the composite. Morphological observation of rat embryonic fibroblasts cultured in the composite further proved it non-cytotoxic.
3.The PcDNA-VEGF165recombinant vector was constructed successfully
VEGF165was amplified by template of its cDNA. In the subsequent electrophoresis, a distinct band of fragments of237bp appeared near the standard nucleic acid molecule weight. The product was pure and free of non-specific band disturbance, thus qualified for transfection use. pMD-18and VEGF165were double digested with EcoR I and Hind III endonucleases respectively. Specific band of the former forged around5.4kb, the latter around225kb. The sequence figure of PcDNA-VEGF165complied with established rabbit VEGF gene depicted clear peak free of double-peak disturbance.
4Neither group displayed significant inflammation in gross appearance.
5Radiography indicated better result in the experiment group. With the analyses of X-Way with lane-Sandhu score, we found the VEGF-nHA/CMCTS group seem to be healing faster than not only the two control groups,but also than the nHA/CMCTS, Lane-Sandhu score
6The observation of HE staining
The VEGF-nHA/CMCTS experiment group demonstrated some bone marrowgranular cells and hardly degraded implant at postoperative week4, markedly more bone marrow cells and bone growth at postoperative week12, and largely reduced implant and good newly formed bone and fibrous callus at postoperative week24. Graphical analysis of the slices revealed much higher Lane-Sandhu Histologic score in the VEGF-nHA/CMCTS experiment group than in the nHA/CMCTS group and two control groups (P<0.05). The new small vessel inductive to oseogenesis was also detected.
7The analyse of biomechanical test
The biomechanical property of the newly formed bone were tested with three point bending test, with loading rate of2mm/min. The compressive strength increased with time. In VEGF-nHA/CMCTS experiment group, it burst suddenly from week4, peaking at week12. In the nHA/CMCTS experiment group, it was raised gently and peaked at week24. No obvious increasement was observed in the two control groups.
8. The results of molecular biochemistry test
The expression level of VEGF, CD31, OPN and IGF was higher in VEGF-nHA/CMCTS group not only than the two control groups, but also than nHA/CMCTS group. The expression level of theses factors was higher in nHA/CMCTS group than the two control groups. There was no significant difference in the two control groups.
Conclusion
1The composite has adequate porosity and compressive strength to construct ideal bone engineering scaffold. nHA/CMCTS implanted on rabbit bone defect invoked no noted inflammation or bone necrosis. Its good compatibility and biotical safety were firmly validated..
2. The new Bioactive bone tissue engineering which was acquired by transporting pcDNA3.1-VEGF165-transfected BMSCs in the wound area could promote the new bone formation, the defect healing and the bending ability, so it should play promotative function in the fracture healing.
3.The new Bioactive bone tissue engineering with pcDNA3.1-VEGF165-transfected BMSCs shoul heal the defect by inducing osteogenesis and angiogenesis
4. We constructed a new bioactive bone engineering composite, which helps experimentally and theoretically to establish ideal bone substitute for defect repair. A new idea to promote tissue repair is come up with.
Background:We previously showed that nano-hydroxyapatite/carboxymethyl chitosan (n-Ha/CMCS) displayed excellent mechanical properties, good degradation rates and exceptional biocompatibility, with negligible toxicity. The aim of this study was to determine the effect of the same composite with vascular endothelial growth factor (VEGF)-transfected bone marrow stromal cells (BMSCs) in a rabbit radial defect model.
Methods:The nano-hydroxyapatite was produced through co-precipitation. The n-HA/CMCS scaffold was produced by particle filtration and lyophilization followed by genipin crosslinking. Total RNA from rabbit bone was reverse-transcribed to synthesize VEGF165-pcDNA3.1that was transfected into the BMSCs. The composite was implanted into a rabbit radial defect model, and the osteogenic activity examined by gross morphology, X-ray analysis and hematoxylin and eosin (HE) staining.
Results:The microstructure and mechanical property of the n-HA/CMCS scaffold resembled natural cancellous bone. Compared with glutaric dialdehyde crosslinked scaffolds, the genipin crosslinked scaffold is less toxic, and displays a higher capacity to promote cell adhesion and proliferation. Spontaneous fluorescence of the composite permitted visualization the composite-bone interface and the adhesion behavior of cells on the scaffold under laser scanning confocal microscopy. The scaffold with VEGF-transfected BMSC bridged the bony defect and promoted healing, with most of the implanted material being replaced by natural bone over time with little residual implant. Using X-ray, we noted obvious callus formation and recanalization of the bone marrow cavity. Furthermore, HE stained sections showed new cortical bone formation.
Conclusions:The n-HA/CMCS scaffold composite with VEGF-trasnfected BMSCs is biocompatible, nontoxic, promotes the infiltration and formation of the microcirculation, and stimulates bone defect repair. Furthermore, the degradation rate of the composite matched that of growing bone. Overall, this composite material is a potentially useful tool for bone defect repair.
在生命活动中由外伤、感染、肿瘤及先天性疾病等原因造成的骨缺损十分常见,是骨科临床常见的难症之一。大的骨缺损难以自行愈合,需要植骨材料修复,但目前临床使用的骨修复材料各有利弊,不能达到修复骨缺损理想材料的要求。近年来随着组织工程技术的发展,研制寻找良好的骨替代材料,与种子细胞联合培养,同时采用缓释技术添加信号因子,构建有生命活性的组织工程骨复合体,无疑将会对临床修复骨缺损带来重大的科学意义和实用价值。羟基磷灰石(hydroxyapatite, HA)是人体骨组织的主要成分,自20世纪70年代末,羟基磷灰石作为新型生物材料问世以来,已在材料学科和医学界,广泛应用于整形外科、矫形外科、口腔科等医学相关领域各类骨缺损的修复。但普通的多孔型羟基磷灰石,质脆强度低,生物机械性能较差；致密型羟基磷灰石植入后不能被新骨完全替代,在局部形成占位。近些年来,利用纳米技术制备的纳米级组织工程骨材料,在纳米结构单元或纳米数量级下生产新型材料,其结构与天然骨相似,有助于人体细胞及大分子对其识别,从而可有效提高材料的生物活性、利用度和生物相容性,符合理想人工骨的性能要求。目前对纳米级羟基磷灰石人工制品的研究取得了长足的进展,并在临床上获得了广泛的应用,但是仍距离结构功能一体化的要求相差很远,还存在某些不可克服的缺点,如缺少连通的、均匀的孔结构,不利于骨组织的生长；其降解速度与天然骨组织生长替代速度不协调；力学强度与天然骨组织的力学性能不匹配等。而且国内外大多数学者认为单纯的羟基磷灰石只具有骨生长引导作用,但不具有骨诱导活性。天然骨是无机矿物质与生物大分子规则排列所组成的复合体,可被看成是一种有机／无机的纳米复合材料。因此以羟基磷灰石为基础构建与天然骨类似的复合骨替代材料已成为当前的研究热点。血管生成与血运重建是创面愈合、移植组织存活的关键。VEGF又称血管通透性因子(vascular permeability factor, VPF),是目前发现的作用最强的促血管生成细胞生长因子,VEGF在骨的形成及修复中也起到重要的作用,许多研究表明,VEGF是通过3个方面来实现的(1)通过促进内皮细胞增殖、血管生成参与骨发育形成。(2)作为旁分泌因子或直接参与骨形成与代谢：有研究发现,VEGF可促进血管内皮细胞分泌胰岛素样生长因子-1(IGF-1)及内皮素-1(ET-1),而这些细胞因子刺激人成骨细胞生长。(3)VEGF还可通过作用于人成骨细胞强表达的flt-1受体增加人成骨细胞移动和分化功能。VEGF本身也可以直接作用于成骨细胞,已有实验证明VEGF可以结合成骨细胞,增加其移动和分化功能,但不能促进其增殖。
研究目的
将无机纳米羟基磷灰石和有机羧甲基壳聚糖(CMCTS)制备成与天然松质骨的力学特性、几何形状和表面性质类似的三维孔洞网状结构,形成多孔状、孔隙率高、力学性能好的二级孔道的三维网络结构纳米人工骨基质材料,构建一种结构功能一体化的复合支架材料。同时,依照组织工程骨复合体三要素(支架材料、种子细胞、信号因子)的原则,利用基因转染技术将VEGF转染BMSCs植入复合材料内,使BMSCs在发挥成骨作用的同时,表达分泌VEGF,从而构建一种有生命活性的骨组织工程骨复合体,以期提高骨缺损修复的效果,为研制理想的骨缺损修复替代材料提供一定的实验依据和理论基础。研究方法
1nHA/CMCTS复合支架材料的制备
nHA粉体由本实验室采用化学沉淀法制备,京尼平(Genipin)做交联剂,通过粒子沥滤法、冷冻干燥法成功制备nHA/CMCTS多孔复合材料。
2nHA/CMCTS生物安全性测试
动物模型建立及分组后进行肝肾功能检测,实验组取移植nHA/CMCTS复合材料周围骨组织标本,对照组取缺损处骨组织标本进行组织学观察。体外生物安全性测试。利用细胞形态观测法检测其毒性。
3.构建pcDNA3.1-VEGF165重组载体
提取细胞总RNA,以提取的mRNA为模板,在逆转录酶的作用下反转录合成VEGF165cDNA序列,以VEGF165基因cDNA为模板,用引物特异性扩增。用Wizard PlusMinpreps DNA纯化系统抽提并纯化噬菌粒DH5α,进行3.5pMD18-T和VEGF165基因的双酶切,用凝胶回收试剂盒,自凝胶中分别回收双酶切后的pMD18-T片段和VEGF165基因,连接两个基因,制备大肠杆菌DH5α感受态细菌,构建重组载体并鉴定后,进行质粒的扩增与纯化。
4骨髓间充质干细胞培养
抽取骨髓,分离骨髓单个核细胞,反复洗涤后悬浮于含10%胎牛血清的DMEM培养基,37℃,5%C02及饱和湿度培养箱中培养并换液,观察细胞生长融合情况。胰蛋白酶消化传代。自第三代细胞条件培养基培养(体积分数10%胎牛血清、地塞米松10-8mol/L、β-甘油磷酸钠0.01mmol/L和维生素C0.05g/L)。对不同时期的骨髓间充质干细胞进行形态观察,在质粒转染之前对骨髓间充质干细胞进行了表面标志物鉴定。
5pcDNA3.1-VEGF165转染骨髓间充质干细胞(电转染法)
电转染前处理哺乳动物细胞,pcDNA3.1-VEGF165转染骨髓间充质干细胞复合纳米羟基磷灰石／羧甲基壳聚糖复合材料,即将制备的纳米羟基磷灰石／羧甲基壳聚糖浸泡于已转染pcDNA3.1-VEGF165的第三代骨髓间充质干细胞悬液内48小时,进行材料与细胞复合。
6动物模型制备
将实验兔前肢桡骨中段大约1.8cm的骨质连同骨膜完全切除。随机选择一侧骨缺损作为实验组,局部植入转染PCDNA3.1-VEGF165骨髓间充质干细胞的纳米羟基磷灰石／羧甲基壳聚糖复合材料(VEGF+n-Ha/CMCTs)或者单纯植入纳米羟基磷灰石／羧甲基壳聚糖复合材料(n-Ha/CMCTs);两组的对侧分别作为作为空白对照组1和空白对照2。
7一般情况观察
术后观察动物的一般情况、局部炎症反应、切口愈合时间
8.术后X线观察
术后4周,12周及24周,行常规X射线检查,观察骨缺损处断端及骨形成及修复情况。Lane-Sandhu法评分标准进行评分。
9.取材
分别于术后6周,12周,24周处死动物后沿原缝合切口切开达新生骨膜,仔细分离,观察骨缺损区愈合及修复情况,切取桡骨标本,取中段缺损处修复的组织,将所取标本以10%中性甲醛固定。
10.HE染色标本经脱钙,梯度脱水,透明、浸蜡,制备厚度为5μm切片,苏木素-伊红(H E)染色,观察术后复合材料降解及新生骨生长的情况,测量新生骨面积。
11.生物力学性能检测
分别于术后6周,12周,24周采用三点弯曲法对实验组及对照组新生骨进行生物力学性能测试,加载速率为2mm/min。
12.分子生物学检测
提取标本总蛋白,Western Blot法检测组织内血管内皮细胞生长因子(VEGF),血小板内皮细胞黏附分子1(CD-31),骨桥蛋白(OPN),胰岛素样生长因子1(IGF-1)的表达水平。
13.统计分析
采用SPSS13.0统计软件,定量数据均以均数±标准误表示,应用各组间单因素方差分析进行统计。P<0.05为有统计学意义。
研究结果
1采用粒子沥滤法制备了高孔隙率nHA/CMCTS多孔复合材料。复合材料的成分无变化,仍然为羟基磷灰石与壳聚糖,并且羧甲基以及胺基分别与羟基磷灰石的羟基或Ca2+发生了不同程度作用,形成了较为牢固的界面结合。该多孔材料最高孔隙率高达87%,孔形不一,以圆形为主,其尺寸分布大约从几微米到600微米,具有良好的贯通性,非常有利于组织在其中的长入与扩展。当复合材料中CMCTS含量为40%,复合材料／造孔剂的质量比为1：1时,多孔材料的孔隙率接近75%,其抗压强度可达21MPa以上,可以满足骨组织工程支架材料的要求。复合材料经SBF溶液浸泡之后的SEM分析,从中可以看到孔壁周围出现纳米级颗粒。分析认为具有骨生物活性的无机陶瓷材料在生理环境中通过与体液发生一系列离子交换和化学沉积反应,可在表面形成类骨碳酸羟基磷灰石晶体,与宿主骨组织发生化学键和,并具有诱导骨组织形成的作用
2复合材料的组织学及肝肾功能检测实验组3周可以见到急慢性炎细胞浸润,无异物巨细胞反应,未见骨坏死；对照组3周以炎性充血为主,并可见慢性炎症细胞浸润,实验组6周可见轻微炎症反应,骨组织未见坏死、基本正常；对照组6周可见少量炎细胞,骨组织基本正常,未发现引起骨组织明显的异物巨细胞反应及骨坏死,提示nHA/CMCTS具有良好的组织相容性。本实验对nHA/CMCTS进行了实验动物的肝肾毒性检测,各组动物血清ALT、AST、Cr和Urea的均值,经单因素方差分析,4组动物血清中ALT、AST、Cr和Urea的水平各组内组间比较及各组内组间两两比较均没有统计学意义,提示nHA/CMCTS复合材料未引起肝肾毒损害具有一定的安全性。小鼠胚胎成纤维细胞在复合材料培养不同时间后的细胞形态观察见细胞突充分伸展,可见多角形细胞,细胞逐渐铺满视野。根据细胞形态分析标准,复合材料无细胞毒性。
3.成功构建了构建pcDNA3.1-VEGF165重组载体采用VEGF165cDNA为模板,将VEGF165经大量扩增后,以1%琼脂糖凝胶电泳,在标准核酸分子量附近出现一条特异性扩增条带,片段大小约为237bp左右,图像分析表明产物纯度较高,无非特异性带干扰,适宜进行基因转化；分别以EcoR I和HindⅢ内切酶对二者酶切,结果前者在5.4kb左右有一条特异性条带,后者条带位于225bp左右；用PCR法对重组PcDNA3.1-VEGF165质粒进行鉴定,可见酶切产物为两个条带,分别在5.4kb、230bp左右片段,测序证实其序列与GENEBANK中兔VEGF序列相同。PcDNA3.1-VEGF165基因测序图峰值清晰,无双峰干扰。
4大体观察发现两组术后均无明显炎症反应,创面无感染,切口愈合顺利。
5X线评分结果不同时期X射线照片显示VEGF-nHA/CMCTS实验组较单纯nHA/CMCTS组及两个空白对照组骨愈合明显快,按Lane-Sandhu法评分标准评分,所得数据进行方差分析,Lane-Sandhu组织学评分差异均有统计学意义。
6HE染色观察所见术后6周VEGF-nHA/CMCTS实验组材料中间可见少量骨髓颗粒细胞,植入复合材料未见明显降解；术后12周VEGF-nHA/CMCTS实验组复合材料明显减少,骨髓颗粒细胞明显增多,并可见骨生长；术后24周VEGF-nHA/CMCTS实验组复合材料明显破碎减少,骨及纤维性骨痂生长良好。另将HE染色组织切片根据Lane-Sandhu组织学评分标准进行分析发现,各组组织学评分随着植入时间的延长而增加；在第6周和第12周,VEGF+n-Ha/CMC组评分不仅显著高于两个对照组,且高于n-Ha/CMC组,在第24周,VEGF+n-Ha/CMC与n-Ha/CMC相比无差异；在第6,12,24周,n-Ha/CMC组显著高于两个对照组；在各个时间点,空白对照组1与空白对照组2之间无差异。HE染色组织切片发现VEGF+n-Ha/CMCTS组较其他各组微小血管形成较多,有利于新骨的生长。
7生物力学性能检测实验组及对照组进行采用三点弯曲法对新生骨进行生物力学性能测试,加载速率为2mm/min。结果显示,抗弯强度随时间延长而增大,在VEGF+n-Ha/CMCTS组,从6周起强度突然增大,在12周时已接近达到最大值。而在Ha/CMCTS组,增大幅度则较为平缓,在24周时也接近最大值。在两组空白对照组,抗弯强度随时间延长增大不明显,统计结果显示,VEGF+n-Ha/CMCTS及n-Ha/CMCTS实验组抗弯强度差异性显著高于高于对照组,12周、24周显著性水平明显不同(p<0.05),但两个对照组之间无差异。
8分子生物学检测
术后6周,对四组组织内血管内皮细胞生长因子(VEGF),血小板内皮细胞黏附分子1(CD-31),骨桥蛋白(OPN),胰岛素样生长因子1(IGF-1)蛋白表达水平进行检测,发现VEGF+n-Ha/CMCTS组VEGF, CD-31, OPN, IGF-1的表达不仅显著高于两个对照组,且高于n-Ha/CMCTS组：n-Ha/CMCTS组的表达水平较两个对照组升高,而空白对照组1和空白对照组2之间,上述因子蛋白表达无差异。
结论
1该复合材料孔隙率、抗压强度,可以满足骨组织工程支架材料的要求。nHA/CMCTS植入大白兔骨缺损处未见引起骨组织明显的炎症反应及骨坏死,肝肾功能检测未发现有肝肾毒性,提示nHA/CMCTS具有良好的组织形容性及生物安全性。
2.纳米羟基磷灰石／壳聚糖复合VEGF转染BMSCs构建骨复合体植入骨缺损模型后,促进新生骨的生长,使骨折愈合加快,抗弯能力强,治疗效果明显。
3.纳米羟基磷灰石／壳聚糖复合VEGF转染BMSCs构建骨复合体植入骨缺损模型后,组织内VEGF, CD31, OPN, IGF-1表达水平升高,提示本模型植入后可能通过促进血管新生及直接促进成骨两个方面发挥作用。
4.将VEGF基因转入BMSCs后与复合材料结合,构建了一种新型的有生命活性的骨组织工程骨复合体,为研制理想的骨缺损修复替代材料提供一定的实验依据和理论基础,为提高组织修复与愈合质量与速度提供新的思路。
Background
Bone defect is a common challenge faced by orthopaedic surgeon resulting from various events such as trauma, infection, tumor or congenital disease. Massive bone defect requires graft implant due to limited self-restoration. Current bone repairing material has been imperfect. Recent development in tissue engineering introduced promising bioactive bone engineering material combining bone substitute with progenitor cells to which signal factors are added through controlled-release. Hydroxyapatite is the primary content of human bone. Since its advent in1970s, it has been widely used in orthoplastic, orthopaedic and stomatology to repair bone defect. Conventionally produced hydroxyapatite is crisp and weak; condensed hydroxyapatite halts osteogenesis, forming unwanted occupation. Fortunately, innovative bone substitute structurally similar to natural bone, whose building block is of nano level, has proved easily recognizable to human body cells and macromolecules, hence its bioactivity, practicability and biocompatibility. To date, artificial nanohydroxyapatite product has undergone marked progress, greatly enabling its clinical application. Notwithstanding that, integrated function and structure have yet to be reached. Identified pitfalls include unconnected inconsistent porosity, unsuitable degradation rate and lower mechanical strength than natural. The dominant concept is that bone directing hydroxyapatite is not osteoinductive. Natural bone is a sort of organic/inorganic compound in light of its combination of inorganic mineral and biomacromolecules. Therefore, focused research is now on hydroxyapatite-based scaffold to construct bonemimicing material. Angiogenesis and vascularization play pivotal role in defect heal and graft survival. VEGF, also termed as vascular permeability factor(VPF), is the most efficacious growth factor known, which counts in osteogenesis and defect repair. Multiple researches converged to conclude that VEGF works by:(1) promoting endothelial proliferation, and angiogenesis,(2) influencing bone turnover directly or as a paracrine factor—it's discorved that VEGF lifts insulin-like growth factor and endothelin secretion from endothelium,(3) exciting osteoblast migration and differetiation through impect on fit-1receptor expressed on the cell. VEGF also reacts directly with fibroblast. There are experiments proving its positive action on fibroblast migration and differention, although not proliferation.
Objective
To incorperate hydroxyapatite and conboxymethyl chitosan(CMCTS) constructing a three-dimensioned network similar to natural cancellous bone in mechanics, geometry, and surface properties, aiming at3D nanonetwork both highly porous and mechanically competent with two-graded channels as artificial bone matrix. The material were supposed to suffice in both structure and function. Meanwhile, in accordance with the three essentials of bone tissue engineering--scaffold, seedlings, signal factors--VEGF-transfected BMSCs were implanted into the composite so that VEGF was secreted along with osteogenesis, thus a bioactive bone engineering was established to improve defect repair, buttressing research for ideal bone substitute.
Method
1. Preparation of nHA/CMCT scaffold
Nanohydoxyapatite powder was produced through chemical deposition. Using genipin as cross-linking agent, porous nHA/CMCTS composite was successed through particle leaching and lyophilization.
2. Tests on the biological safety of nHA/CMCTS
After establishment and grouping, liver and kidney function of the animal models were tested. Bone samples were collected around nHA/CMCTS composite in experiment group and from the defect in control group. The toxicity was tested under cytomorphometry.
3. Preparation the Restructuring carrier of pcDNA3.1-VEGF165
The total RNA was extracted and the VEGF165DNA fragment was synthesized reverse transcriptionally from the primer designed. The gene was specifically amplified by the specific primer and template of cDNA to VEGF165.The bacteriphage DH5a was extracted and purified. The pMD18-T and VEGF165were double digeseed and the pMD18-T and VEGF165fragments were collected respectively on Gel Extraction Kit. pcDNA3.1and VEGF165were connected and competent E.Coli was prepared. At last, the combinant carrier was amplified and purified.
4. Culture of bone marrow stromal cells
Individual nucleus cell was seperated from bone marrow and cultured in DMEM with10%fetal calf serum and in the conditional medium with10%fetal calf serum,8-10mol/L dexamethasone,0.01mol/Lp-Glycerin sodium and0.05g/L VitC.
5. Transfection of BMSCs with pcDNA3.1-VEGF165plsmids (electrotransfection)
6. Establishment of animal models
1.8cm length of the middle part of the rabbits'both radii was removed. One foreleg was randomly selected to be implanted with pcDNA3.1-VEGF165transfected BMSCs on nHA/CMCTS scaffold or nHA/CMCTS simply, another foreleg as the empty control,respectively.
7. Postoperative observation.
Observation of general condition, regional inflammation, and time of heal.
8. Radiography examination
The fracture was inspected under radiography on osteogenesis and defect recovery at the end of4w,12w, and24w after operation.
9.Sample collection
The animals were sacrificed and collected from the defect through incision on the original operated sites. Slices5μm thick were aquired through decalcification, gradient dehydration, transparent and waxing.
10. HE staining Observation of material degradation, new bone formation and the new blood vessels with Haematoxylin and eosin (HE) staining.
11. Biomechanical testing
Biomechanical testing was carried out at the end of6w,12w, and24w after the operation with the three point bending method and the loading rate was2mm/min.
12. Melecular biochemestry testing
Melecular biochemestry testing was carried out at the end of6w,12w, and24w after the operation to test the expression of VEGF, CD31, OPN and IGF-1.
13. Statistical analysis
All analyses were performed using SPSS v13.0. Data were expressed as mean±SE. An independent-samples t-test was used to compare continuous data for between-group differences and comparisons among groups involved the use of ANOVA. P<0.05was considered statistically significant.
Result
1Porous nHA/CMCTS composite was prepared through particle-leeching. HA and CMCTS stayed unchanged. Carbonylmethyl group reacted with HA and Ca2+, forming rigid bond on the interface. The maixmal porosity can be as high as87%. The pores, mainly shaped spherical, ranging from a few nanometers to600nm in diameter, interconnected, are inductive to bone ingrowth and expansion.40%CMCTS content, composite/Porous agent ratio of1:1, and porosity of75%forms a ideal combination to reach a compressive strength up to21Mpa, satisfying as bone engineering scaffold. Nano level particles were detected on the pore wall under SEM analysis after immersion in the SBF solution. One theory is that osteoinductiv carbonated hydroxyapatite crystal results from chemical bond-forming reaction of ceramic material with body fluid.
2Histological and biochemical test of the composite. At week4, acute and chronic inflammatory cells infiltration, absent foreign body reaction and necrotic bone were detected in the experiment group, and inflammatory congestion with some chronic inflammatory cell infiltration in the control group. At week6, slight inflammatory reaction emerged in the experiment group and the bone was primarily intact; in the control group, no sign of abnormality showed except slight inflammation. The above suggested good biocompatibility of nHA/CMCTS. nHA/CMCTS was also tested for liver or kidney toxicity. Under analysis of variance, no statistically significant difference in ALT, AST, Cr and Urea was exhibited among the four groups, indicating biotical safety of the composite. Morphological observation of rat embryonic fibroblasts cultured in the composite further proved it non-cytotoxic.
3.The PcDNA-VEGF165recombinant vector was constructed successfully
VEGF165was amplified by template of its cDNA. In the subsequent electrophoresis, a distinct band of fragments of237bp appeared near the standard nucleic acid molecule weight. The product was pure and free of non-specific band disturbance, thus qualified for transfection use. pMD-18and VEGF165were double digested with EcoR I and Hind III endonucleases respectively. Specific band of the former forged around5.4kb, the latter around225kb. The sequence figure of PcDNA-VEGF165complied with established rabbit VEGF gene depicted clear peak free of double-peak disturbance.
4Neither group displayed significant inflammation in gross appearance.
5Radiography indicated better result in the experiment group. With the analyses of X-Way with lane-Sandhu score, we found the VEGF-nHA/CMCTS group seem to be healing faster than not only the two control groups,but also than the nHA/CMCTS, Lane-Sandhu score
6The observation of HE staining
The VEGF-nHA/CMCTS experiment group demonstrated some bone marrowgranular cells and hardly degraded implant at postoperative week4, markedly more bone marrow cells and bone growth at postoperative week12, and largely reduced implant and good newly formed bone and fibrous callus at postoperative week24. Graphical analysis of the slices revealed much higher Lane-Sandhu Histologic score in the VEGF-nHA/CMCTS experiment group than in the nHA/CMCTS group and two control groups (P<0.05). The new small vessel inductive to oseogenesis was also detected.
7The analyse of biomechanical test
The biomechanical property of the newly formed bone were tested with three point bending test, with loading rate of2mm/min. The compressive strength increased with time. In VEGF-nHA/CMCTS experiment group, it burst suddenly from week4, peaking at week12. In the nHA/CMCTS experiment group, it was raised gently and peaked at week24. No obvious increasement was observed in the two control groups.
8. The results of molecular biochemistry test
The expression level of VEGF, CD31, OPN and IGF was higher in VEGF-nHA/CMCTS group not only than the two control groups, but also than nHA/CMCTS group. The expression level of theses factors was higher in nHA/CMCTS group than the two control groups. There was no significant difference in the two control groups.
Conclusion
1The composite has adequate porosity and compressive strength to construct ideal bone engineering scaffold. nHA/CMCTS implanted on rabbit bone defect invoked no noted inflammation or bone necrosis. Its good compatibility and biotical safety were firmly validated..
2. The new Bioactive bone tissue engineering which was acquired by transporting pcDNA3.1-VEGF165-transfected BMSCs in the wound area could promote the new bone formation, the defect healing and the bending ability, so it should play promotative function in the fracture healing.
3.The new Bioactive bone tissue engineering with pcDNA3.1-VEGF165-transfected BMSCs shoul heal the defect by inducing osteogenesis and angiogenesis
4. We constructed a new bioactive bone engineering composite, which helps experimentally and theoretically to establish ideal bone substitute for defect repair. A new idea to promote tissue repair is come up with.
Background:We previously showed that nano-hydroxyapatite/carboxymethyl chitosan (n-Ha/CMCS) displayed excellent mechanical properties, good degradation rates and exceptional biocompatibility, with negligible toxicity. The aim of this study was to determine the effect of the same composite with vascular endothelial growth factor (VEGF)-transfected bone marrow stromal cells (BMSCs) in a rabbit radial defect model.
Methods:The nano-hydroxyapatite was produced through co-precipitation. The n-HA/CMCS scaffold was produced by particle filtration and lyophilization followed by genipin crosslinking. Total RNA from rabbit bone was reverse-transcribed to synthesize VEGF165-pcDNA3.1that was transfected into the BMSCs. The composite was implanted into a rabbit radial defect model, and the osteogenic activity examined by gross morphology, X-ray analysis and hematoxylin and eosin (HE) staining.
Results:The microstructure and mechanical property of the n-HA/CMCS scaffold resembled natural cancellous bone. Compared with glutaric dialdehyde crosslinked scaffolds, the genipin crosslinked scaffold is less toxic, and displays a higher capacity to promote cell adhesion and proliferation. Spontaneous fluorescence of the composite permitted visualization the composite-bone interface and the adhesion behavior of cells on the scaffold under laser scanning confocal microscopy. The scaffold with VEGF-transfected BMSC bridged the bony defect and promoted healing, with most of the implanted material being replaced by natural bone over time with little residual implant. Using X-ray, we noted obvious callus formation and recanalization of the bone marrow cavity. Furthermore, HE stained sections showed new cortical bone formation.
Conclusions:The n-HA/CMCS scaffold composite with VEGF-trasnfected BMSCs is biocompatible, nontoxic, promotes the infiltration and formation of the microcirculation, and stimulates bone defect repair. Furthermore, the degradation rate of the composite matched that of growing bone. Overall, this composite material is a potentially useful tool for bone defect repair.
引文
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