丝素蛋白/羟基磷灰石复合材料修复骨缺损的微循环研究

了解丝素蛋白(silk fibroin,SF)表面修饰的羟基磷灰石(hydroxyapatite,HA)修复骨缺损过程中,实验动物血液流变学和骨缺损修复区血流量的变化。选择20只新西兰白兔,制作15 mm长的桡骨节段性骨缺损模型,根据植入不同移植材料分为实验组和对照组,实验组于动物左侧桡骨缺损区植入MSCs复合SF表面修饰的HA培养制备的组织工程骨,对照组植入MSCs复合HA培养制备的组织工程骨,观察各组动物术后7、14 d血液流变学和术后14 d骨缺损修复区血流量的变化。实验组与对照组比较,血液流变学指标和骨缺损修复区血流量差异显著,实验动物全身血液粘度降低,骨缺损修复区的局部血流量增加。SF表面修饰对以HA为支架材料组织工程骨的修复作用有明显优化作用。     

组织工程骨修复兔桡骨节段性缺损血液流变性及局部血流量的变化

目的 观察组织工程骨修复骨缺损过程中，实验动物血液流变性和骨缺损修复区血流量的变化。方法 选择30只新西兰白兔，制作15mm长的桡骨节段性骨缺损模型，根据植入不同移植材料分为实验组和对照组，实验组于动物左侧桡骨缺损区植入组织工程骨，对照组植入部分脱蛋白骨，观察各组动物术后1h、7h、14d血液流变性和术后14d骨缺损修复区血流量的变化。结果 实验组与对照组比较，血液流变学指标和骨缺损修复区血流量差异显著。组织工程骨修复骨缺损，实验动物全身血液黏度降低，骨缺损修复区局部血流量增加。结论 与单纯材料比较，组织工程骨可促进血液流变学及局部血流量的改善。     

表面修饰羟基磷灰石修复骨缺损骨形态发生蛋白-2的表达

目的了解精氨酸-甘氨酸-天冬氨酸多肽表面修饰的羟基磷灰石（hydroxyapatite,HA）修复节段性骨缺损局部骨形态发生蛋白-2（bone morphogenefic protein-2，BMP-）的表达。方法以骨髓基质干细胞（marrow stromal cels，MSCs）复合Arg-Gly-Asp（RGD）多肽表面修饰的HA或单纯材料培养制备组织工程骨，选择60只新西兰白兔。制作15mm长的桡骨节段性骨缺损模型，根据植入不同的材料分为A、B、C、D组。A组：骨缺损区植入MSCs复合RGD多肽表面修饰的HA培养制备的组织工程骨；B组：骨缺损区植入MSCs复合HA培养制备的组织工程骨；C组：骨缺损区植入RGD多肽表面修饰的HA；D组：骨缺损区植入HA。术后4周取材，行修复区局部BMP-2免疫组化分析。结果术后4周各组骨缺损区均有新骨生成，修复区局部BMP-2表达水平依次为：A〉B〉C〉D（P〈0．05）。结论RGD多肽表面修饰对以HA为支架材料组织工程骨的修复作用有明显优化作用。     

骨髓基质干细胞复合纳米材料修复骨缺损的微循环研究

目的:了解骨髓基质干细胞(MSCs)复合纳米羟基磷灰石/聚乳酸(n-HA/PLA)构建组织工程骨修复骨缺损过程中,实验动物血液流变学和骨缺损修复区血流量的变化。方法:选择20只新西兰白兔,制作15mm长的桡骨节段性骨缺损模型,根据植入不同移植材料分为实验组和对照组,实验组于动物左侧桡骨缺损区植入组织工程骨,对照组植入n-HA/PLA,观察各组动物术后1h、7天、14天血液流变学指标和术后14天骨缺损修复区血流量的变化。结果:实验组与对照组比较,血液流变学指标和骨缺损修复区血流量差异显著。组织工程骨修复骨缺损,实验动物血液粘度降低,骨缺损修复区局部血流量增加。结论:与单纯n-HA/PLA材料比较,组织工程骨可促进骨缺损的修复。     

表面修饰对羟基磷灰石异位成骨的影响

目的 了解精氨酸-甘氨酸-天冬氨酸(Arg-Gly-Asp,RGD)多肽表面修饰对羟基磷灰石(hydroxyapatite,HA)异位成骨的影响.方法 以骨髓基质干细胞(marrow stromal cells,MSCs)复合RGD多肽表面修饰的HA或单纯材料培养制备组织工程骨,将材料植入新西兰白兔脊柱旁的肌肉内,根据植入不同材料分为A、B、C和D组.A组植入MSCs复合RGD多肽表面修饰的HA培养制备的组织工程骨;B组植入MSCs复合HA培养制备的组织工程骨;C组植入RGD多肽表面修饰的HA;D组植入HA.术后4、8周取材,行组织学观察和计算机图像分析.结果 术后4、8周,各组异位成骨组织学评估,A＞B(P＜0.05),C和D组无异位成骨.结论 RGD多肽表面修饰对以HA为支架材料组织工程骨的异位成骨有明显优化作用.     

复合同种异体成骨细胞的β-磷酸三钙人工骨修复桡骨缺损

背景：作为骨支架材料,β-磷酸三钙具有良好的生物相容性、骨诱导性及生物力学性能。 目的：探讨β-磷酸三钙复合同种异体成骨细胞修复兔桡骨节段性骨缺损的效果。 方法：建立45只兔单侧桡骨骨缺损模型,随机分为3组,实验组缺损区植入复合同种异体成骨细胞的β-磷酸三钙,对照组缺损区植入β-磷酸三钙,空白对照组缺损区不植入任何材料。植入后4,8,16周观察新骨生成情况,通过形态学、X射线等观察指标客观评价各组成骨及骨缺损修复能力。 结果与结论：随着修复时间的延长,实验组骨缺损逐渐修复,至16周时X射线见支架与宿主骨之间的骨痂已完全骨化,骨缺损完全修复;组织学见缺损区皮质骨连续,髓腔再通,并且不同时间点实验组修复效果明显优于对照组与空白对照组(P < 0.01)。表明复合同种异体成骨细胞的β-磷酸三钙人工骨具有良好的成骨能力,有引导组织再生、防止骨不连的作用。     

表面修饰对羟基磷灰石细胞相容性的影响

探讨精氨酸-甘氨酸-天冬氨酸(Arg-Gly-Asp,RGD)多肽表面修饰对羟基磷灰石(hydroxyapatite,HA)细胞相容性的影响。以骨髓基质干细胞(marrow stromal stem cells,MSCs)复合精氨酸-甘氨酸-天冬氨酸多肽表面修饰的羟基磷灰石或单纯材料培养制备组织工程骨,观察骨髓基质干细胞的粘附和生长情况,检测细胞活力和碱性磷酸酶(alkaline phosphatase,ALP)活性,流式细胞仪分析细胞周期。结果表明:骨髓基质干细胞在材料表面和孔隙内均可粘附和生长,粘附于RGD多肽修饰羟基磷灰石的细胞活力和碱性磷酸酶活性明显高于未经RGD多肽修饰组(P<0.01,P<0.05)。各组细胞周期未见明显变化,未见异倍体细胞。说明RGD多肽表面修饰对HA材料的细胞相容性有明显的优化作用。     

自体骨髓间充质干细胞载体复合物修复骨缺损的组织学观察

背景：在骨缺损修复过程中,从修复质量、免疫排斥和疾病传播等多方面来衡量,自体骨都是最佳的选择,但来源有限且取骨区可能产生并发症,给骨缺损的修补及自体骨移植临床应用带来了很大局限。 目的：以含自体骨髓间充质干细胞脱钙骨载体复合支架材料植入骨缺损的同时,向植入处微环境内添加碱性成纤维细胞生长因子等因素,从而达到增强骨修复能力,改进修复效果的目的。 方法：选择3月龄新西兰大耳白兔45只,建立双侧前臂桡骨中下段骨-骨膜缺损模型,然后将实验兔等分为3组：实验组、对照组和空白组,均于左侧髂骨和股骨转子处抽取骨髓,分离培养扩增骨髓间充质干细胞后,与不同材料体外复合,植入兔桡骨干10 mm缺损处。实验组兔缺损处植入骨髓间充质干细胞、脱钙骨、藻酸钙、碱性成纤维细胞生长因子、维生素C;对照组兔缺损处植入骨髓间充质干细胞、脱钙骨、藻酸钙;空白组兔双侧缺损处均不植入任何材料,自然愈合。 结果与结论：植入后30,60,90 d各组之间组织学检查新骨生成速度、生成量差异均有显著性意义。实验组兔缺损修复部位骨痂和移植物化骨及材料降解明显快于对照组和空白组;但对照组和空白组兔缺损修复部位残存物明显多于实验组。实验组兔骨缺损以多点方式直接成骨,对照组和空白组则从两端以“爬行替代”方式成骨。空白组兔自然愈合后90 d骨缺损均无愈合。说明植入体外培养移植物的同时向该植入微环境添加碱性成纤维细胞生长因子和维生素C等有利骨修复,提高骨损伤的愈合。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程全文链接：     

硅胶膜管联合BMP／HA修复大块骨缺损实验研究

目的：探讨硅胶膜（SGM）管与骨形态发生蛋白（BMP）和羟基磷灰石（HA）复合的修复兔长骨缺损的效果。方法：制备兔桡骨中段1．2cm缺损模型,实验组缺损区外围包绕硅胶膜,其内分别填充自体骨,BMP／HA,HA三种材料：对照组仅填充HA;空白组骨缺损区未填充,通过X线摄片,光学显微镜,立体计量学分析和生物力学评价对骨缺损区愈合效果进行.分析。结果：术后1个月,SGM＋BMP／HA组骨缺损区见有大量的类骨质,明显优于其他组,第2个月,SGM＋自体骨组与SGM＋BMP／HA组植入区有大片新骨形成,术后3个月,SGM＋自体骨级瑟SGM＋BMP／HA组骨缺损基本修复并出现骨组织改建现象,此时,SGM＋HA组与对照组骨缺损区也有少量新骨形成,空白缺损区为纤维组织充填,结论：硅胶膜管与自体骨或BMP／HA联合用于修复骨缺损成骨效果更佳,因为硅胶膜的屏障作用可使骨缺损区受到引导成骨和放导成骨的双重作用。     

天然牡蛎壳纳米体复合型骨材料修复骨缺损

背景：研究发现,牡蛎壳等很多海洋生物外壳等形成矿物质盐的过程与人体实际情况较为接近。 目的：观察天然牡蛎壳纳米体复合型骨材料修复骨缺损的效果。 方法：取30只成年大耳白兔,制作双侧桡骨骨缺损模型,随机均分为两组,实验组于骨缺损处植入天然牡蛎壳纳米体复合型骨材料,对照组于骨缺损处植入医用硫酸钙可注射型植骨材料,植入后2,8,12周进行X射线检查,了解植入骨材料周围组织生长结合情况;于第12周末获得双侧桡骨,利用生物力学测试系统检测桡骨抗弯曲强度,并利用彩色图像分析仪定量分析成骨情况。 结果与结论：植入后2周,两组骨材料密度较周边正常骨组织呈偏低,缺损与材料间边界清晰,未发现明显骨修复现象;植入后8周,两组骨材料均被较厚软组织全部包裹,实验组血管成分显著减少;植入后12周,两组骨材料紧密结合相邻组织,包裹骨材料的组织质地较韧,二者之间无界线,实验组桡骨表面形态已恢复至正常水平,在形态和质地结构方面与正常组织无明显区别,对照组仍存在明显投射分界影像。实验组桡骨抗弯曲强度和成骨量均显著大于对照组(P < 0.05)。表明天然牡蛎壳纳米体复合型骨材料修复骨缺损可以获得更好的桡骨抗弯曲强度,并促进新骨形成。     

The effect of adsorbed serum proteins, RGD and proteoglycan-binding peptides on the adhesion of mesenchymal stem cells to hydroxyapatite

Prior studies from our laboratory have shown that RGD peptides increase the attachment of mesenchymal stem cells (MSCs) to hydroxyapatite (HA), however, RGD does not induce cell spreading when coupled to this type of biomaterial. In an effort to improve MSC spreading, and possibly cell attachment, proteoglycan-binding peptides (KRSR or FHRRIKA) were combined with RGD in the current study. It was found that the peptide combinations did not enhance MSC attachment relative to RGD alone, although a slight amount of spreading was elicited by both KRSR and FHRRIKA. Similar results were obtained with proteoglycan-binding peptides modified with a heptaglutamate domain, a motif that improves peptide tethering to HA. To determine whether differentiation status affected cell responses, MSCs were in vitro differentiated into osteoblasts, and evaluated as before. These experiments revealed that, like MSCs, osteoblasts did not adhere in greater numbers to the peptide combinations. Finally, none of the peptides or peptide combinations were able to stimulate the robust amount of cell adhesion and spreading elicited by serum-coated HA surfaces (of note, five different species of serum were tested). Given the propensity of HA to adsorb proadhesive proteins from blood/serum, we question the utility of functionalizing HA with RGD and/or proteoglycan-binding peptides.     

The effect of the addition of a polyglutamate motif to RGD on peptide tethering to hydroxyapatite and the promotion of mesenchymal stem cell adhesion

Mimicking endogenous bone-binding proteins, RGD peptides have been synthesized with polyacidic amino acid domains in order to ionically tether the peptides to bone-like synthetic biomaterials, including hydroxyapatite (HA). However, a direct comparison of unmodified RGD with polyacidic-conjugated RGD has not been performed, and thus a benefit for the acidic domain has not been established. We evaluated the peptide/HA bond of RGD peptides with and without an attached polyglutamate sequence (E(7)), as well as examined mesenchymal stem cell (MSC) adhesion and morphology as they were affected by the conjugated peptide. We found that significantly more E(7)RGD was bound to HA than RGD at all coating concentrations tested, and moreover, more E(7)RGD was retained on the HA surface even after extended washing in serum-free media. Consistent with in vitro results, higher levels of E(7)RGD than RGD remained on HA that had been implanted in vivo for 24 h, indicating that the polyacidic domain improved peptide-binding efficiency. At several peptide concentrations, E(7)RGD increased cell adhesion compared to RGD surfaces, establishing a biological benefit for the E(7) modification. In addition, HA pre-coated sequentially with low-density E(7)RGD (1-10 microg/ml) and serum (FBS) stimulated cell adhesion and spreading, compared to either coating alone, suggesting that an ionic linkage allows for the potential adsorption of serum proteins to unoccupied sites, which may be important for bone formation in vivo. Collectively, these results suggest that tethering peptides to HA via a polyglutamate domain is an effective method for improving the peptide/HA bond, as well as for enhancing MSC adhesion.     

The effect of RGD peptides on osseointegration of hydroxyapatite biomaterials

Given that hydroxyapatite (HA) biomaterials are highly efficient at adsorbing proadhesive proteins, we questioned whether functionalizing HA with RGD peptides would have any benefit. In this study, we implanted uncoated or RGD-coated HA disks into rat tibiae for 30 min to allow endogenous protein adsorption, and then evaluated mesenchymal stem cell (MSC) interactions with the retrieved disks. These experiments revealed that RGD, when presented in combination with adsorbed tibial proteins (including fibronectin, vitronectin and fibrinogen), has a markedly detrimental effect on MSC adhesion and survival. Moreover, analyses of HA disks implanted for 5 days showed that RGD significantly inhibits total bone formation as well as the amount of new bone directly contacting the implant perimeter. Thus, RGD, which is widely believed to promote cell/biomaterial interactions, has a negative effect on HA implant performance. Collectively these results suggest that, for biomaterials that are highly interactive with the tissue microenvironment, the ultimate effects of RGD will depend upon how signaling from this peptide integrates with endogenous processes such as protein adsorption.     

RGD多肽修饰的改性PLGA仿生支架材料对骨髓间充质干细胞粘附、增殖及分化影响的研究

目的：探索RGD多肽修饰的改性PLGA支架材料上骨髓基质细胞的增殖、粘附及分化情况。方法用异型双功能交联剂Sulfo-LC-SPDP将GRGDSPC多肽共价结合到改性PLGA支架材料上，以未接多肽的改性PLGA材料做对照，取第三代MSC接种到材料上，培养1d、2d、3d、4d后比较材料上的细胞密度来反映细胞的增殖程度；取第三代MSC接种到材料上，培养4h、12h后沉淀法定量检测粘附的细胞数，培养24h后摄光镜图像比较粘附细胞的数量和形态，并用FITC连接的鬼笔环肽对细胞骨架染色，在荧光显微镜下观察细胞骨架的组织情况；取第三代MSC接种到材料上，用成骨性培养基培养7d、14d、21d，检测细胞中ALP活性来了解MSC分化情况。结果：培养1d、2d、3d、4d后细胞的增殖程度无显著性差异；培养4h、12h后实验组细胞粘附率均显著高于对照组，且24h后细胞的粘附质量、细胞骨架的组织情况也较对照组为好；培养14d后实验组细胞表达显著高的ALP活性。结论：RGD多肽修饰对细胞增殖无明显促进作用，但能提高改性PLGA支架材料对骨髓基质细胞的粘附性，对MSC向成骨细胞分化有显著促进作用。     

Collagen-infiltrated porous hydroxyapatite coating and its osteogenic properties: in vitro and in vivo study

Bone-implant interface is critical for the early fixation of orthopedic implants. In this study, porous hydroxyapatite (HA) coatings were prepared through a liquid precursor plasma spraying process and were infiltrated with the collagen, alone and with the additional incorporation of recombinant human bone morphogenetic protein-2 (rhBMP-2) and RGD peptide (RGD). The results showed significantly improved mesenchymal stem cell (MSC) adhesion, proliferation, and differentiation on collagen-modified HA coatings, partially benefited from the formation of a fibrous network due to the self-reconstitution of collagen on the HA surface. Further enhancements on MSC proliferation and differentiation were generally observed through the additional incorporation of bone morphogenetic protein (BMP) and RGD. The osteoinductive and osteoconductive properties of the collagen/BMP-modified HA coatings were studied in vivo. Clear ectopic bone formation and significantly accelerated bone growth rate (29% increase, p < 0.05) have been observed after 1-month implantation of HA-collagen/rhBMP-2-coated Ti alloy samples into the rabbit muscle and dog femora, respectively. Overall, our results suggest that collagen-modified HA coating surface is a far superior substrate for cell attachment, proliferation, and differentiation, and collagen can be used an efficient carrier for BMP in vivo. Therefore, modification of HA coating with collagen is a simple but effective biomimetic approach to enhancing the osteointegration and early fixation of bone-implant interface.     

The effect of collagen I mimetic peptides on mesenchymal stem cell adhesion and differentiation,and on bone formation at hydroxyapatite surfaces

Integrin-binding peptides increase cell adhesion to naive hydroxyapatite (HA), however, in the body, HA becomes rapidly modified by protein adsorption. Previously we reported that, when combined with an adsorbed protein layer, RGD peptides interfered with cell adhesion to HA. In the current study we evaluated mesenchymal stem cell (MSC) interactions with HA disks coated with the collagen-mimetic peptides, DGEA, P15 and GFOGER. MSCs adhered equally well to disks coated with DGEA, P15, or collagen I, and all three substrates, but not GFOGER, supported greater cell adhesion than uncoated HA. When peptide-coated disks were overcoated with proteins from serum or the tibial microenvironment, collagen mimetics did not inhibit MSC adhesion, as was observed with RGD, however neither did they enhance adhesion. Given that activation of collagen-selective integrins stimulates osteoblastic differentiation, we monitored osteocalcin secretion and alkaline phosphatase activity from MSCs adherent to DGEA or P15-coated disks. Both of these osteoblastic markers were upregulated by DGEA and P15, in the presence and absence of differentiation-inducing media. Finally, bone formation on HA tibial implants was increased by the collagen mimetics. Collectively these results suggest that collagen-mimetic peptides improve osseointegration of HA, most probably by stimulating osteoblastic differentiation, rather than adhesion, of MSCs.     

Regulation of mesenchymal stem cell attachment and spreading on hydroxyapatite by RGD peptides and adsorbed serum proteins

The successful development of biomaterials must take into consideration how those surfaces will interact with in vivo processes such as adsorption of endogenous proteins. In this study, we examined whether modifying highly adsorbent materials like hydroxyapatite (HA) with RGD peptides would improve mesenchymal stem cell (MSC) adhesion. We found that RGD, alone, was not sufficient to promote full cell spreading. However, given that RGD-modified HA will likely adsorb osteogenic serum proteins in vivo, we evaluated MSC behavior on HA pre-coated with RGD, then over-coated with serum (RGD/FBS). Interestingly, RGD/FBS coatings additively stimulated MSC attachment and spreading compared to either coating alone, but only at low RGD coating concentrations. High RGD concentrations inhibited cell attachment, and completely eliminated cell spreading on RGD/FBS surfaces. To better understand the mechanism by which RGD and adsorbed serum proteins interactively regulate cell behavior, we monitored the deposition of fibronectin (FN) from serum onto HA pre-coated with increasing RGD concentrations. These studies showed that high RGD concentrations did not inhibit FN adsorption, therefore cell spreading is attenuated by mechanisms other than lack of FN availability. Collectively, our results suggest a potential therapeutic benefit for functionalizing HA with RGD, however such a benefit will likely depend upon the RGD density.     

Bone regeneration using an alpha 2 beta 1 integrin-specific hydrogel as a BMP-2 delivery vehicle

Non-healing bone defects present tremendous socioeconomic costs. Although successful in some clinical settings, bone morphogenetic protein (BMP) therapies require supraphysiological dose delivery for bone repair, raising treatment costs and risks of complications. We engineered a protease-degradable poly(ethylene glycol) (PEG) synthetic hydrogel functionalized with a triple helical, α2β1 integrin-specific peptide (GFOGER) as a BMP-2 delivery vehicle. GFOGER-functionalized hydrogels lacking BMP-2 directed human stem cell differentiation and produced significant enhancements in bone repair within a critical-sized bone defect compared to RGD hydrogels or empty defects. GFOGER functionalization was crucial to the BMP-2-dependent healing response. Importantly, these engineered hydrogels outperformed the current clinical carrier in repairing non-healing bone defects at low BMP-2 doses. GFOGER hydrogels provided sustained in vivo release of encapsulated BMP-2, increased osteoprogenitor localization in the defect site, enhanced bone formation and induced defect bridging and mechanically robust healing at low BMP-2 doses which stimulated almost no bone regeneration when delivered from collagen sponges. These findings demonstrate that GFOGER hydrogels promote bone regeneration in challenging defects with low delivered BMP-2 doses and represent an effective delivery vehicle for protein therapeutics with translational potential.