【作者】 孙大炜；

【导师】 金大地； 白晓春；

【作者基本信息】 南方医科大学 ， 骨外科学（专业学位）， 2014， 博士

【摘要】 一、研究背景全世界目前每年已经有超过220万例骨移植手术,手术领域包括骨科、神经外科和牙科。由于自体骨移植具有良好的成骨诱导作用,自体骨移植被认为是骨移植手术的“金标准”。然而,自体骨移植供区可产生相关并发症,如供区血肿形成、软组织损伤、疼痛及恢复期延长等,此外,自体骨移植是严重骨质疏松症患者的禁忌症。因此,研发出完全合成的、易得的,且具有成骨诱导作用的人工合成材料作为自体骨移植的辅助材料是目前的研究热点,理想的人工材料研发成功也将会是临床领域的里程碑事件。在合成骨生物材料领域,合成材料已经从使用永久性的、内置金属材料向模拟天然骨成分的组织工程、生物可降解材料转化。生物可降解材料方面,最初合成的骨生物材料是生物陶瓷,即磷酸钙(calcium phosphates；CaPs)和β-磷酸三钙(beta tricalcium phosphate；TCP),这两种材料具有模拟骨组织矿化结构的特点。尽管这两种人工合成材料仿生及成骨方面与人体骨骼的多空结构相差甚远,而且这两种材料本身材料的脆性以及极其缓慢的降解率,均限制了它们在临床的应用。为了改进上述生物陶瓷的缺点,人们已经在生物陶瓷中参杂可降解的多聚体,以增加其机械性能及生物活性,使人工材料最终能达到临床应用。虽然如此,目前合成材料仍有许多显著的不足之处,诸如机械强度不足,无诱导骨再生作用,骨整合作用弱,并且不能模仿天然骨的理化结构,尤其是天然骨胶原基质中含有60-65%质量的羟基磷灰石。为了改善人工骨材料的缺陷,我们的实验室关注于研制基于柠檬酸人工合成材料与生物陶瓷合成材料,用于骨组织工程中。近年来研究发现,柠檬酸作为三羧酸循环中的中间产物可能在骨发育和骨生物材料发展中起非常重要的作用。天然骨组织中含有大量的柠檬酸,并且柠檬酸在骨代谢中具有重要的作用,因此在骨生物材料及支架的设计中应当考虑到柠檬酸的应用。柠檬酸不仅仅是骨钙的增溶剂,而且近来发现柠檬酸在骨纳米结构的组成中调节羟基磷灰石纳米晶体的形成。尽管柠檬酸在骨形成作用方面的研究只是一个开始,不过我们近期关于外源性柠檬酸的研究取得了鼓舞人心的结果,我们在基于柠檬酸的移植物的体内研究中或把柠檬酸加入培养基中的体外研究中发现,柠檬酸均能增加碱性磷酸酶(ALP)基因和成骨细胞特异基因osterix (OSX)的表达,成骨细胞表形增加以及出现基于柠檬酸的移植物骨诱导及骨整合作用。如上所述,基于柠檬酸多聚体/羟基磷灰石复合物(CABP-HAs)是一种新型的骨生物活性材料,这种材料在骨组织工程应用中具有明显的优势。第一个被报道的基于柠檬酸的复合材料是poly(1,8-octanediol)-HA (POC-HA), POC是一种人工合成、成本低、无毒性的单体,通过自由羧基键的钙螯合能力展现出与HA更强的整合能力。POC具有大量的羧基化学键,能提供65%重量HA的结合,达到天然骨的HA含量,这种特性是之前所有人工材料都达不到的。从POC-HA研制开始,我们实验室一直从事于基于柠檬酸可降解多聚体的研究,如力学性能强的crosslinked urethane-doped polyesters (CUPEs), biodegradable photoluminescent polymers (BPLPs), dual-crosslinkable poly (alkylene maleate citrate)(PAMC),以及clickable POC-based elastomers (POC-Click),这些基于柠檬酸的生物可降解材料用于多种医学用途,用于骨骼、血管、神经组织工程,肿瘤影像学和药物输送中。为了进一步改善POC-HA的机械性能,我们通过加入聚氨酯键得至CUPE-HA复合物,使得CUPE-HA复合物达到突破性的抗压强度116.23±65.37MPa,虽然这种抗压强度仍逊于人体皮质骨的抗压强度(100-230MPa)。尽管CUPE-HA在兔股骨外侧髁缺损模型中,移植缺损处表现出极微的慢性炎症反应和很强的骨诱导性,但是这种材料参杂聚氨酯键的设计丢失了有用的羧基侧化学键,进而也失去了螯合羟基磷灰石的能力,进而使得材料的生物力学强度下降。为了改善上述缺点,我们近期研发了clickable biodegradable elastomer,即poly (octanediol citrate)-click (POC-Click),这种新材料增加了叠氮-炔环加成反应(click化学反应),新增的交联机理增加了材料的机械强度又保持了结合羟基磷灰石(HA)螯合钙的羧基侧化学键。以上叙述了基于柠檬酸的生物活性材料特性可以满足骨生物降解材料的设计机械强度,骨传导性(osteocondutive)和骨诱导性(osteoinductive),但是这种新型骨生物材料目前仍局限于固态非孔性移植物的研究,也没有修复颅骨骨缺损的相关研究。本研究中,我们合成CUPE-HA和POC-Click-HA多空支架,用于修复大鼠颅骨极限骨缺损(critical sized cranial defects)。二、材料与方法1.基于柠檬酸生物可降解支架材料的制作1.1POC预聚物和CUPE预聚物的合成1.2合成多孔的POC-CLICK-HA和CUPE-HA基于柠檬酸生物可降解盘形支架2.扫描电镜观察POC-CLICK-HA和CUPE-HA支架结构3、Sprague Dawley (SD)大鼠自体骨髓间充质干细胞(BMSCs)与基于柠檬酸的生物可降解支架共培养BMSCs的分离采用密度梯度离心法。经过Co60消毒盘形的基于柠檬酸的生物可降解支架使用前浸泡于MEM/10%FBS3小时,使得材料孔隙水化、材料孔隙表面充分吸收粘附蛋白,以利于接种的BMSCs在材料表面粘附生长。将上述处理的支架移入培养皿中,用移液器在材料上加入密度为1×106的第三代BMSC混悬液,将所接种BMSCs的支架在37℃、5%CO2的培养箱内饱和湿度条件下培养5天。扫描电镜观察BMSCs在支架上的生物相容性。4、大鼠颅骨骨缺损动物模型的制作及分组8周龄SPF级Sprague Dawley雄性大鼠74只(其中2只用于SD大鼠自体BMSCs取材,72只用于体内动物实验),购于南方医科大学实验动物中心。我们用不添加任何生长因子或细胞成分的CABP-HA盘型支架(65%质量HA含量；70%孔隙率)去修复大鼠颅骨骨缺损(n=72,每组18只),随机分为四组：骨缺损未修复组(对照组)、CUPE-HA支架治疗组(CUPE-HA组)、POC-Click-HA支架治疗组(POC-CLICK-HA组),以及自体骨移植组(AB组)。颅骨骨缺损造模术后1月、3月及6月取标本。5.将4%多聚甲醛固定好的大鼠颅骨标本,送至广东中科恺盛医疗科技有限公司,使用该公司显微CT成像系统进行断层扫描成像,扫描软件三维重建、拍摄照片,骨分析软件计算颅骨缺损处与正常颅骨交界区环形骨定量分析。6.将标本脱钙切片,行骨组织学染色分析及免疫组化染色分析,并行形态计量学分析骨缺损处的血管计数。7.统计学处理：统计学分析采用IBM SPSS Statistics20统计软件分析处理。计量资料统计描述用均数、标准差(mean、SD)表示。不同组间术后3个时间点的比较采用Two-Way ANOVA检验。单独效应分析采用One-Way ANOVA检验,方差不齐时使用Welch检验结果。两两比较方差齐时采用LSD法,方差不齐时使用Games-Howell法。显著性标准为a=0.05。三、结果1、CABP-HA支架扫描电镜评价本实验中合成的CUPE-HA和POC-Click-HA盘形支架扫描电镜可见多空的支架平均孔径大小为200-400μm的立方体结构,两者结构相似,50倍扫描电镜下,CUPE-HA支架结构较POC-CLICK-HA支架结构略微疏松。体外实验中大鼠BMSCs种植于CUPE-HA和POC-Click-HA盘形支架,共培养5天后,可见BMSCs健康贴于支架上生长。2.颅骨骨缺损新骨形成分析造模术后1月,3月和6月,3D micro-CT观察颅骨骨缺损处新骨形成。影像学可见非材料移植组(CON组和AB组)与CABP-HA材料移植组(CUPE-HA组和POC-Click-HA组)有明显的不同。在未治疗组(CON组)中,术后各个时间点均没有完全骨形成,只有缺损处边缘有少量的骨形成,这也证明4mm的大鼠颅骨骨缺损模型符合极限骨缺损模型(CSD)模型。新骨形成最明显的仍然是自体骨移植组(AB组),术后6月,达到临床骨愈合的标准。在两个基于柠檬算骨生物材料组中均可见不透X线的组织广泛填充于颅骨缺损处,这暗示了新骨形成,其中CUPE-HA组较POC-Click-HA组新骨形成更加明显。进一步micro-CT骨形成定量分析,我们选取颅骨缺损的环形边缘为新生矿化颅骨定量分析的关注区域(VOI the volume of interest)。根据统计结果分析,AB组中新骨形成显著高于其它各组,与非治疗组(CON组)相比较,支架修复的两组展现出良好的修复效果,不过这种修复效果仍不如自体骨移植组。术后1月,3月和6月,AB组的BMD值均显著高于其它各组(F=24.526,P=0.016),而且术后各个时间点CUPE-HA组和POC-Click-HA组这两个支架修复组的BMD均高于CON组。所测得的Tb.Th值结果与BMD值结果也表现出同样的趋势。CUPE-HA组和POC-Click-HA组两种支架组之间,BMD值与Tb.Th值各个时间点均没有统计学差异。(p>0.05)3.组织学分析从缺损处组织学的结果来看,CUPE-HA组和POC-Click-HA组两个材料组与自体骨移植治疗组(AB组)相似。特别是两个材料组治疗的颅骨缺损边缘可见纤维间质增生和反应性成骨。纤维间质围绕支架处增生,表现出相对血管形成增多；未治疗组(CON组)纤维间质增生少于与这两个材料组,也少于自体骨移植组。造模1月,3月和6月后,VEGF-b免疫组化染色结果示,未治疗组几乎没有成纤维细胞和炎性细胞,与其他各治疗组相比较,VEGF-b免疫组化染色低表达。相反的是,在CUPE-HA组和POC-Click-HA组中,可见VEGF-b免疫组化高表达,并且与AB组的免疫组化染色表达相似,尤其是术后1月组的VEGF-b免疫组化结果。造模1月,3月和6月后,各组缺损处的血管计数结果形态计量学分析示,两种基于柠檬酸的生物可降解支架治疗组有较多的成血管数量,其中术后1月时间点,CUPE-HA组的成血管数量显著高于AB组和POC-CLICK-HA组。(P=0.049)术后3月和6月时间点,各组血管血管数均无著性不同。(P=0.261；P=0.187)造模1月,3月和6月后,碱性磷酸酶(ALP)特染和骨钙素(OCN)免疫组化染色结果示,在CUPE-HA组和POC-Click-HA组材料中,可见ALP阳性细胞表达,以及OCN免疫组化阳性细胞表达,并且与AB组的免疫组化染色表达相似。四、结论我们认为,大鼠极限骨缺损模型适合验证高孔隙率支架的成骨效果；基于柠檬酸多聚体羟基磷灰石复合物盘状支架适合于修复大鼠颅骨极限骨缺损；无需复合生物生长因子或成骨细胞, POC-Click-HA和CUPE-HA支架均能通过诱导颅骨缺损处骨形成和血管形成而表现出理想的宿主反应和潜在成骨作用。因此,基于柠檬酸多聚体羟基磷灰石复合物支架是一种有应用前景的即用移植材料,该材料可做为骨缺损的填充修复材料。骨性关节炎(OA)是世界范围内最为常见的退行性骨科疾病,发病率极高。伴随着人口老龄化,骨性关节炎的发病率也逐年上升。近年,据经济学评估,关节炎发病这一单一因素可造成国内生产总值(GDP)0.5-1%的下滑,如何有效的治疗,预防骨性关节炎一直以来是医学研究的难题之一。过去,在OA的发病过程中,主要的研究热点集中于关节软骨血管增生,滑膜入侵,软骨细胞内的线粒体自噬。然而,血管增生,滑膜入侵,线粒体自噬发生时已出现软骨丢失。因此,针对这三点的研究,并不能达到提前保护软骨的目的。软骨下骨是介于软骨下与骨基质区之间的一层松质骨,较早便有报道在骨性关节炎病患的尸检病理切片中,发现患者膝关节的软骨下骨排列异常。而在骨性关节炎患者的MRI检查时,也发现一定病例出现软骨下骨的异常增生。目前认为骨性关节炎早期,由于应力改变,软骨下骨会出现病理性增生,而这一改变的来源,并未被清楚的阐述。哺乳动物雷帕霉素特异性靶标(mTOR)信号通路自发现以来,被认为是调控哺乳动物生长发育,能量代谢的重要信号通路。mTOR信号通路中,有两种关键的蛋白复合体,mtorc1与mtorc2。mtorc1是雷帕霉素敏感复合物,体内可被诸多生长因子激活,调控能量代谢。本研究将重点讨论软骨下骨mTOR信号通路的活性与骨性关节炎发病病理的重要关系。探讨mTOR信号通路可能在骨性关节炎中的扮演的重要调控角色。以mTOR信号通路为基础为骨性关节炎寻找全新的治疗靶点。我们选取选取成年C57雄性小鼠20只,体重16-18g,行前交叉韧带离断模型做为骨性关节炎疾病模型,右膝关节为骨性关节炎组(OA group),左膝关节作为假手术(sham group)。造模后2周及1个月时间点处死老鼠。其中一个月时间点取材后脱钙切片,进行苏木素-伊红染色及甲苯胺蓝染色确定模型建立的成败,并评估大致病程。mTOR信号通路特异性靶蛋白磷酸化S6免疫组织化学染色评估早期(2周)骨性关节炎软骨下骨中mTOR信号通路的活性。碱性磷酸酶特异性染色评估骨性关节炎中软骨下骨成骨细胞活性与骨性增生。血管内皮生长因子免疫组织化学染色评估骨性关节炎中软骨下骨及软骨血管增生情况。术后1月骨性关节炎造模发现,出现了明显的软骨丢失。所有HE染色结果和甲苯胺蓝染色均一稳定。ACLT造模可被认为是一种稳定的骨性关节炎造模方法。早期骨性关节炎可用ACLT一周结果进行模式替代。与假手术组比较,骨性关节炎组软骨下骨在早期出现了显著的PS6活性增高,说明mTOR信号通路在软骨下骨的活化可能在骨性关节炎病程中扮演关键作用。同时,骨性关节炎组的软骨下骨出现了碱性磷酸酶活性与血管内皮生长因子的增强。暗示mTOR信号通路可能通过影响软骨下骨的成骨与血管再生过程影响骨性关节炎的发病过程。更多还原

【Abstract】 IntroductionOver2.2million bone transplantation procedures are performed annually worldwide in a variety of fields including orthopedics, neurosurgery, and dentistry. Autologous bone grafts are considered the gold standard for bone grafting procedures due to their superior osteogenic potential. However, autologous grafts are associated with various complications such as hematoma, soft tissue breakdown, pain, and prolonged recovery times. Moreover, the use of bone autografts is contraindicated in osteoporotic populations due to a significant reduction in bone quality and quantity. Thus, the development of a fully synthetic, readily available, and osteogenic bone substitute as an adjunct to autologous tissue grafts is strongly encouraged and considered as a great milestone in the clinical field.For synthetic orthopedic biomaterials, research in the field has witnessed a shift from the use of permanent, inert metals towards tissue-engineered biodegradable composites designed to mimic the native composition of bone. Initially, the majority of synthetic orthopedic materials were based off calcium phosphates (CaPs), such as hydroxyapatite (HA) and beta tricalcium phosphate (TCP), because of their ability to replicate the native mineral constituent of bone tissue. Although biomimetic and osteogenic, their applications are severely limited when fabricated into porous structures due to the inherent brittleness and very slow degradation rates. To improve the utility bioceramics, the hybridization of bioceramics and biodegradable polymers has been widely used to improve the mechanical properties and bioactivity of the resulting materials for orthopedic applications. Nevertheless, the current composite materials still suffer from several significant problems such as unsatisfactory mechanical strength, inefficient bone regeneration, poor bone integration, and the inability to mimic the native bone chemical composition, which is composed of60-65wt.-%hydroxyapatite embedded in a collagen matrix.To address these limitations, our lab has been focused on the development of citric acid-based materials to composite with bioceramics for orthopedic tissue engineering. Recent research has suggested that citrate, a naturally occurring Kreb’s cycle product, may play a significant role in bone development and orthopedic biomaterial development. The natural abundance of citrate in native bone tissue and its importance in skeletal metabolism hints that citrate should be incorporated in orthopedic biomaterial and scaffold design. Citrate is not only a dissolved calcium-solubilizing agent, but has recently been found to be an integral part of the bone nanocomposite playing roles in regulating apatite nanocrystal formation and thickness. Although the role of citrate in bone formation is still largely unknown, our recent exciting results showed that the exogenous citrate, whether presented on a biomaterial or supplemented into culture media, can enhance alkaline phosphatase (ALP) and osterix (OSX) gene expression, osteoblast phenotype progression, implant osteoinductivity, and osteointegration both in vitro and in vivo. The natural abundance of citrate in bone tissue, its importance in bone physiology, and our recent findings on stem cell culture hints that citrate may have significant impacts on bone development and orthopedic biomaterial development.As previously mentioned, citric acid-based polymer/hydroxyapatite composites (CABP-HAs) are a novel class of orthopedic biomaterial, which offer distinct advantages for bone tissue engineering applications. The first reported citric acid-based composite, poly (1,8-octanediol)-HA (POC-HA), is synthesized with non-toxic monomers using simple and cost-effective procedures, and displayed enhanced HA integration through the calcium chelating ability of free carboxylic chemistry. The abundant carboxyl chemistry allowed for the incorporation of up to65wt.-%of HA in the composites to match the native mineral content of bone tissue and is a feature that is not possible with previous materials. Since the development of POC-HA, our laboratory has long been working on a series of citrate-based biodegradable polymers such as mechanically strong crosslinked urethane-doped polyesters (CUPEs), biodegradable photoluminescent polymers (BPLPs), dual-crosslinkable poly (alkylene maleate citrate)(PAMC), and clickable POC-based elastomers (POC-Click) or various biomedical applications such as bone, vascular, and neural tissue engineering, cancer imaging, and drug delivery. To improve upon the mechanical properties of POC-HA through urethane chemistry, we have shown that CUPE-HA composites display impressive compressive strengths of116.23±65.37MPa, which falls within the range of native human cortical bone (100-230MPa). Although, CUPE-HA showed minimal chronic inflammation and full osteointegration when implanted in a rabbit lateral femoral condyle defect model, the urethane-doping strategy sacrificed valuable pendant carboxyl chemistry to chelate with hydroxyapatite limiting the mechanical potential of the material. To address this situation, we have recently developed a clickable biodegradable elastomer, poly (octanediol citrate)-click (POC-Click), which employs azide-alkyne cycloaddition (click chemistry) as an additional crosslinking mechanism to improve the mechanical strength of the bulk material without sacrificing valuable pendant citric acid carboxyl chemistry for hydroxyapatite (HA) calcium chelation.Although we have previously shown that citrate-based materials can address the challenges in designing mechanically strong, osteoconductive, and osteoinductive orthopedic biomaterials, this new class of orthopedic biomaterials has only been studied as solid non-porous implants, and their use as a scaffold for cranial bone defect repair has not been studied yet. In this study, CUPE-HA and POC-Click-HA porous scaffolds were fabricated and compared for their potential to repair critical sized cranial defects in rats.Materials and MethodsScaffold fabricationCUPE pre-polymers and POC-Click pre-polymers were synthesized according to previously published methods. To fabricate porous CUPE-HA disc shaped scaffolds, CUPE pre-polymer was first dissolved in1,4-dioxane and mixed with hydroxyapatite (65wt.-%). Next, sodium chloride salt with an average size in the range of200-400μm was added to the mixture (85wt.-%) and stirred in a Teflon dish until a homogenous viscous paste was formed. To fabricate disc-shaped scaffolds, the viscous paste was inserted into Teflon tubes (4×2mm; inner diameter x height) purchased from McMaster-Carr. Following solvent evaporation, the scaffolds were post-polymerized in an oven maintained at100℃for3days. Salt was leached out from the scaffolds by immersion in deionized water under vacuum for72hours with water changes every12hours. Finally, the scaffolds were dried using lyophilization to obtain the final disc-shaped scaffolds (4×2mm; diameter x height). POC-Click-HA disc shaped scaffolds were also fabricated as described above except with POC-Click pre-polymers.Rat bone mesenchymal stem cells (BMSCs) isolation and scaffold seedingTo evaluate material cytocompatibility, rat BMSCs were seeded onto CABP-HA scaffolds in vitro. BMSCs were harvested from both tibias under general anesthesia from ketamine (90mg/kg) and xylazine (10mg/kg). Briefly, blood was collected from the tibial bone marrow and BMSCs were then isolated by centrifugation and suspended in a minimum essential medium supplemented with 10%fetal bovine serum and1%penicillin-streptomycin solution. The cells were incubated at37℃in a humidified atmosphere of95%air and5%CO2with the medium changed twice per week. Cells from the third passage were used for scaffold cytocompatibility evaluation. Disc-shaped scaffolds were sterilized by soaking in70%ethanol followed by washing with phosphate buffered saline (PBS). The sterilized scaffolds were soaked in MEM/10%FBS for3h to ensure sufficient adsorption of adhesion proteins onto the accessible scaffold surface and to fully hydrate the interior scaffold surface pores. For scaffold cell seeding, rat BMSCs were counted using a hemocytometer and seeded directly by pipetting the BMSCs suspension onto scaffolds at a density of1×106cells/graft. Scaffolds were then incubated for5days at37℃.Surgical procedureSeventy-two male Sprague-Dawley rats (200-220g) were used for the animal experiments. The objective of this study was to compare the efficacy of two different CABP-HAs, poly (1,8-octanediol citrate)-click-HA (POC Click-HA) and crosslinked urethane-doped polyester-HA (CUPE-HA), as scaffold materials for the repair of critical sized calvarial defects. CABP-HA disc-shaped scaffolds (65wt.-%HA with70%porosity) were used as bare implants without the addition of growth factors or cells to renovate4mm diameter rat calvarial defects (n=72, n=18per group). Animals were ultimately anesthetized and sacrificed by xylazine injection at1,3,6months post-surgery.Scanning electron microscopeBare CABP-HA scaffolds, rat BMSCs seeded scaffolds, and explanted scaffolds isolated6months after surgery were fixed with2.5%glutaraldehyde for12h and characterized using scanning electron microscopy (SEM). After tension-free drying, the samples were coated in gold and analyzed with a S-3000N SEM under high-vacuum conditions,20kV voltage, and18mm working distance.Microcomputed tomography (micro CT)Micro CT analysis was used to quantify the volume of bone formation within the defect. The tomography of fixed rat calvarial bone specimens was performed using a microtomographic imaging system with a70kV scanning voltage,30W power, and429μA current.3D-MED3.0software was used for image capture and three-dimensional reconstruction. To calculate the bone mineral density (BMD) and trabecular thickness (Tb.Th), a hollow cylindrical volume of interest (VOI)4mm in external diameter,2.8mm in inner diameter, and1.5mm in height was selected for scanning and corrected for the CT value.Histological assessmentFor histological assessment, the defect sites were fixed in4%paraformaldehyde for48h, decalcified with0.5M ethylenediaminetetraacetic acid (EDTA) at pH7.4for3weeks, and then embedded in paraffin. Tissues were longitudinally sectioned with a4-μm thickness, deparaffinated with xylene, gradually hydrated, and stained with hematoxylin and eosin (H&E) for light microscopic analysis. Images were captured at200x magnification using an Olympus bx51microscope with a20×objective lens and a digital camera. Five random images were obtained for each rat along the length of the defect. Overall, a total of25to30images were acquired from five to six rats in each group. The number of blood vessels was determined by counting luminal structures lined by vascular endothelium and partially filled with red blood cells.Immunohistochemical analysisTo stain for Vascular Endothelial Growth Factor-b (VEGF-b), tissue sections were treated with proteinase K for antigen recovery, washed with PBS, and blocked with5%bovine serum albumin at room temperature for30min, and exposed to antibodies for VEGF-b overnight at4℃. Peroxidase activity was detected using the enzyme substrate3-amino-9-ethyl carbazole. For negative controls, sections were treated in an identical manner, except they were incubated in PBS-buffered saline without primary antibodies. Vascular numbers were determined by three single blind pathologists and calculated according to the average number of vessels in five random areas of an image at200×magnification.ResultsSEM analysis of CABP-HA scaffoldsSEM images of the disc-shaped CUPE-HA and POC-Click-HA scaffolds fabricated in this study show the presence of a porous structure with an average pore size in the range of200-400μm. The SEM images show that the seeded BMSCs were healthy with a normal morphology and adhered tightly to the surface of the scaffolds. Explanted samples after6months were also viewed under SEM, and no obvious differences in the bone defect areas were seen between the CUPE-HA and POC-Click-HA groups.Radiographic assessment of new bone formationTo observe new bone formation within the defects,3D images were reconstructed using micro-CT at1,3, and6months post-surgery. Radiographic evidence of new bone formation was highly variable between non-scaffold groups (CON and AB groups) and CABP-HA scaffold groups (CUPE-HA and POC-Click-HA groups). In the untreated defects, no bone formation was observed after6months of surgery. In contrast, new bone formation was evident in autologous bone treated animals. In the scaffold groups, radiopaque tissue was found spread over the entire defect site indicating new bone formation and was especially evident in the CUPE-HA group.Next, micro-CT analysis was conducted to quantify the mineralized skeletal bone formation at the edge of the defect site. The most apparent difference observed was the greater amount of newly formed bone in the AB group compared to other groups. Compared to the negative control (CON), the two scaffolds-treatment groups exhibited a greater repair effect, but less of an effect compared to the AB group. The BMD of the AB group at1,3, and6months after surgery were significantly higher than that of the other groups (p<0.05). Furthermore, the BMD of the CUPE-HA and POC-Click-HA groups was higher than the CON group. The Tb.Th of each group showed a similar trend with no significant differences observed between the CUPE-HA group and the POC-Click-HA groups (p>0.05).Histological assessmentHistological findings for defect sites in the CUPE-HA and POC-Click-HA groups were similar to findings in the autologous bone treated group (AB). Specifically, the edge of the defect site was composed of fibrous stroma and reactive bone. The fibrous stroma appeared loose around the scaffolds and exhibited a relatively high level of angiogenesis. In contrast, the defect sites in the negative control group (CON) did not exhibited reactive bone and displayed less fibrous stroma compared to the AB and both scaffolds groups. After1,3, and6months of implantation, immunohistochemical staining for VEGF-b was performed. In the negative control group (without any material implantation), very few fibroblasts and inflammatory cells were concentrated in the defect space, and lower expression levels for VEGF-b were also observed when compared to the same area of other groups. In contrast, CUPE-HA and POC-Click-HA groups showed higher level of VEGF-b staining similar to that of the AB group, especially at1month post-surgery. Quantification of the vessel numbers found in the defect sites after1,3, and6months for all experimental groups are shown in Figure7. Greater vessel numbers in scaffolds-treated rats were confirmed by morphometric analysis of the vessels. The vascular numbers of CUPE-HA groups were significantly higher than the AB or POC-CLICK-HA group at1month post-surgery (p<0.05). In addition, there were no significant differences in vascular numbers between each group at3and6months post-surgery (p<0.05).ConclusionsIn conclusion, highly porous disc-shaped citric acid-based polymer hydroxyapatite composite scaffolds were fabricated to repair critical sized rat calvarial defects. Without incorporating biological growth factors or osteoblastic cells, both POC-Click-HA and CUPE-HA scaffolds displayed satisfactory host responses and osteogenic potential through the stimulation of proximal bone formation and angiogenesis in the repair of calvarial defects. Therefore, citric acid-based polymer hydroxyapatite scaffolds could serve as a promising off-the-shelf implant for the regeneration of critical size bone defects. Osteoarthritis (OA) is one of the most common types of degenerated diseases which is associated with significant morbidity in modern life, of which prevalence is growing rapidly with ageing social population. Recently, an economic estimation revealed that the burden on health economics of OA has previously approached to0.5-1%on gross domestic product. In past two decades, investigator focused their power of view on angiogenesis, synovial invasion and mitochondrial autophagy of cartilage. However, the cartilage protective effects which can attenuate OA process in OA was considered to be presented before cartilage degradation. Therefore, an effective therapy contributed to cartilage degradation in earlier period is expected to be approved.Subchondral bone was described as the cancellous bone located between articular cartilage and bone matrix. In autopsy of OA patients, subchondral bone was cluttered compared with common people. Magnetic Resonance Imaging (MRI) scans reported that a dysplasia of subchondral bone was observed in OA patients. Nevertheless, the pathological mechanism of the dysplasia of subchondral bone presence in OA was not clear now.1994, mammalian target of rapamycin (mTOR) was first posed as an energy metabolism signal pathway which associated with mammalian growth. There are two key complexs in mTOR signal pathway:mtorc1and mtorc2. mtorc1is a rapamycin sensitive complex plays a significant role in energy metabolism that can be activated by growth factors and energy deficit.In our research, we view to investigate the regulatory role of mTOR signal pathway in OA processes. To investigate a novel notion of OA therapy target, we reveal positional target of mTOR activating in subchondral bone for the primary cartilage protective purpose.20mature C57male mice (weight16-18g) were chosen in this research. Anterior cruciate ligament (ACLT) transaction was treated as an OA animal model operated on right knee joint of mice as well as the left knee joint in sham.1week and4weeks post ACLT were considered as the OA process period to confirm this animal model. Knee joint was resected intactly and cut into5um slices respectively. Hematoxylin-Eosin stain and T-blue stain were posed to reveal the OA process post ACLT. Phosphorylated s6, the target protein of mTOR activating was estimated by immunohistochemical stain in subchondralbone post ACLT1week (earlier period of OA process). Alkaline phosphatase (ALP) specific stain was aprose to reveal the osteogenesis in subchondral bone. The immunohistochemical stain of Vascular Epithelial Growth Factor (VEGF) was determined to estimate the angiogenesis in subchondral bone and cartilage in OA mice.In1month post ACLT, HE stain and T-blue stain claim that the cartilage degradation presence in4weeks time prolong and cannot be observed in1week time prolong. It hints that mice live1week post ACLT is a reasonable replacement of earlier OA process. Meanwhile, ACLT is a notable animal model of OA motivation is proved by our results. In1week post ACLT, compared with sham group, Ps6, VEGF IHC stain located in subchondral bone in OA group are significantly activating, ALP stain poses an enhancement of osteogenesis. These results suggest that activating mTOR signal pathway promotes osteogenesis and angiogenesis in subchondral bone is associated with OA process. The possibility of mTOR activating in subchondral bone play a regulatory role in OA pathology would be more sufficient identified in our further studies.更多还原