Early weight bearing of porous HA/TCP (60/40) ceramics in vivo: a longitudinal study in a segmental bone defect model of rabbit

Porous interconnected hydroxyapatite (HA) and HA/tricalcium phosphate (TCP) (60/40) ceramics are promising materials for hard tissue repair. However, the mechanical properties of these materials have not been accurately determined under weight-bearing conditions. In this study, newly developed HA and HA/TCP (60/40) ceramics were used with intramedullary fixation in segmental bone defects of rabbits. Early radiological, histological, densitometric and biomechanical changes were evaluated. The mean radiological grade of healing and bonding to bone was higher in HA/TCP (60/40) ceramics than that of pure HA ceramics but the difference was not statistically significant. The densities of both implanted ceramics improved with time, supported by the histological evaluation of bone matrix ingrowth into ceramic pores, whereas the densities at the bone–ceramic interface decreased gradually. Flexural resonant frequencies and three-point bending strength increased, revealing an increase in mechanical stability during this early critical time interval where implant and/or bone–implant interface failures occur frequently. It can be concluded that both HA and HA/TCP (60/40) ceramics have a limited application in the treatment of load-bearing segmental bone defects but did not fail at the early stages of implantation.     

Control of in vivo mineral bone cement degradation

The current study aimed to prevent the formation of hydroxyapatite reprecipitates in brushite-forming biocements by minimizing the availability of free Ca²⁺ ions in the cement matrix. This was achieved by both maximizing the degree of cement setting to avoid unreacted, calcium-rich cement raw materials which can deliver Ca²⁺ directly to the cement matrix after dissolution, and by a reduction in porosity to reduce Ca²⁺ diffusion into the set cement matrix. In addition, a biocement based on the formation of the magnesium phosphate mineral struvite (MgNH₄PO₄·6H₂O) was tested, which should prevent the formation of low-solubility hydroxyapatite reprecipitates due to the high magnesium content. Different porosity levels were fabricated by altering the powder-to-liquid ratio at which the cements were mixed and the materials were implanted into mechanically unloaded femoral defects in sheep for up to 10 months. While the higher-porosity brushite cement quantitatively transformed into crystalline octacalcium phosphate after 10 months, slowing down cement resorption, a lower-porosity brushite cement modification was found to be chemically stable with the absence of reprecipitate formation and minor cement resorption from the implant surface. In contrast, struvite-forming cements were much more degradable due to the absence of mineral reprecipitates and a nearly quantitative cement degradation was found after 10 months of implantation.     

磷酸钙强化椎弓根螺钉的动态生物力学研究

目的 研究磷酸钙骨水泥强化椎弓根螺钉后生物力学强度的体内动态变化。方法 成年雌性绵羊12只,年龄4～6岁;体重33～45Kg。经每只羊的L1～L6椎体双侧椎弓根,分直接植入、CPC强化和PMMA强化三种植钉方法植入椎弓螺钉,每只动物体内植钉方法按平衡不完全随机进行分配,每种方法各植4枚。植钉后动物被随机分配至术后1d,4w,12w和24w四个观察时间点,每点3只。在观察点获取植钉腰椎标本,测定螺钉的轴向拔出力和拔出能量吸收值,进行相同时间点各植钉方法之间和同一植钉方法各时间点之间的指标对比分析。结果 四个观察时间点动物的骨密度按序依次是,0.923±0.082g/cm2,0.910±0.098 g/cm2,0.952±0.123 g/cm2和0.912±0.126 g/cm2,组间均未见显著差异（P﹥0.05）。在术后1d,4w,12w和24w四个观察时间点CPC强化组的最大轴向拔出力（Fmax）依次是,893.13±63.91N,916.50±109.88N,1022.04±73.85N和1104.91±112.33N。拔出能量吸收值（EAV）与相应的Fmax呈正相关（P﹤0.001）。同一方法各时间点内的Fmax 和EAV均无显著性差异（P﹥0.28）。相同时间点,CPC强化组的Fmax和EAV均显著高于对照组（P﹤0.001）。另外,除24周的EAV二者无差异（P﹥0.18）外,PMMA强化组两项指标均高于CPC强化组（P﹤0.003）。结论 结果表明用CPC强化能显著提高椎弓根螺钉的固定强度,且强化作用在体内是动态稳定的。在椎弓根螺钉强化方面,CPC是PMMA理想的替代材料。     

SiO2 and ZnO dopants in three-dimensionally printed tricalcium phosphate bone tissue engineering scaffolds enhance osteogenesis and angiogenesis in vivo

Calcium phosphate (CaP) scaffolds with three-dimensionally-interconnected pores play an important role in mechanical interlocking and biological fixation in bone implant applications. CaPs alone, however, are only osteoconductive (able to guide bone growth). Much attention has been given to the incorporation of biologics and pharmacologics to add osteoinductive (able to cause new bone growth) properties to CaP materials. Because biologics and pharmacologics are generally delicate compounds and also subject to increased regulatory scrutiny, there is a need to investigate alternative methods to introduce osteoinductivity to CaP materials. In this study silica (SiO₂) and zinc oxide (ZnO) have been incorporated into three-dimensional printed β-tricalcium phosphate (β-TCP) scaffolds to investigate their potential to trigger osteoinduction in vivo. Silicon and zinc are trace elements that are common in bone and have also been shown to have many beneficial properties, from increased bone regeneration to angiogenesis. Implants were placed in bicortical femur defects introduced to a murine model for up to 16 weeks. The addition of dopants into TCP increased the capacity for new early bone formation by modulating collagen I production and osteocalcin production. Neovascularization was found to be up to three times more than the pure TCP control group. The findings from this study indicate that the combination of SiO₂ and ZnO dopants in TCP may be a viable alternative to introducing osteoinductive properties to CaPs.     

Preparation and characterization of a novel strontium-containing calcium phosphate cement with the two-step hydration process

A novel Sr-containing calcium phosphate cement (CPC) with excellent compressive strength, good radiopacity and suitable setting time was developed in this work. The two-step hydration reaction resulted in a high compressive strength, with a maximum of up to 74.9 MPa. Sr was doped into the calcium-deficient hydroxyapatite as a hydrated product during the hydration reaction of the CPC. Because of the existence of Sr element and the compact microstructure after hydration, the Sr-containing CPC shows good radiopacity. It is expected to be used in orthopedic and maxillofacial surgery for bone defects repairing.     

Synthesis of a novel b-tricalcium phosphate/hydroxyapatite biphasic calcium phosphate containing niobium ions and evaluation of its osteogenic properties

To promote the osteogenic properties of osteoblasts, we synthesized a hydroxyapatite (HAp) with β-tricalcium phosphate (β-TCP) biphasic calcium phosphate containing Nb ions (NbTCP/HAp). NbTCP/HAp was prepared by annealing precipitates obtained by coprecipitation of an aqueous solution of Ca(NO₃)₂ and a mixture of (NH₄)₂HPO₄ and aqueous Nb solution. The precipitates can be regarded as a calcium-deficient HAp, the PO₄ sites of which are partly occupied by Nb ions. NbTCP/HAp was successfully synthesized by thermal decomposition of the precipitates. NbTCP/HAp enhanced the calcification of normal human osteoblasts (NHOst), and the amount of calcified tissue increased in proportion to the Nb ion concentration in the NbTCP/HAp. The alkaline phosphatase (ALP) activity of NHOst was also enhanced by NbTCP/HAp. Because Nb ions significantly enhance the ALP activity of NHOst, calcification by NbTCP/HAp is considered to be due to enhancement of ALP activity induced by Nb ions dissolved from NbTCP/HAp. These results indicate that NbTCP/HAp can be an effective bone repair material.     

PLGA/O-CMC载药纳米微粒的体外降解及释药行为研究

本研究以聚乳酸-乙醇酸共聚物（PLGA）和自行制备的O-羧甲基壳聚糖（O-CMC）为原料，以5-氟尿嘧啶（5-FU）为抗癌药物模型，采用自身设计的改良复乳法制备了载药纳米微粒。微粒平均粒径为98.5nm，粒径分布指数为0.192，粒子表面∈电位为61．48eV，载药率高达18.9％。然后用SEM动态监测载药纳米粒子降解过程中表面形貌的变化，并连续追踪粒子降解过程中的质量损失和降解介质的pH变化。载药纳米粒子在PBS中的释药行为研究表明，（1）前12h的释药动力学符合Huguchi方程，具有一级释放特性；（2）在20d内的释药动力学符合零级释放特性。细胞凋亡实验结果表明载药纳米粒子对TJ905脑胶质瘤细胞增殖有明显的抑制作用。     

微创可注射双相骨水泥的制备及性能表征和复合DBM成骨能力研究

目的以硫酸钙、磷酸钙及羟丙甲纤维素(hypromellose,HPMC)为固相原料,磷酸盐溶液为液相,制备一种双相骨水泥(CS/TCP),可作为脱钙骨基质(decalcified bone matrix,DBM)微创注射的载体,同时与DBM协同作用,提高骨愈合能力。方法固相以球磨工艺制备,与液相混合成泥状,在模具中固化成型,然后放入到37℃生理盐水中,静置,分别于3 d、5 d、14 d、28 d、56 d取出干燥,研究其体外降解性能和力学性能及微观结构变化;将骨水泥单独或与40wt%DBM混合后注入到生理盐水中,观察注射性能和溃散性能;将骨水泥与40wt%DBM混合,植入到8周大鼠颅骨,分别于术后6周、8周和15周取材,HE染色后观察成骨性能。结果该骨水泥在生理盐水中逐渐降解,56 d材料降解了21.6%,形成了多孔结构,孔径为0～150 m;力学强度第5 d最高,为(21.4±3.1) MPa,56 d后下降为(9.7±2.2) MPa;水泥单独或者复合DBM注射流畅,均能保持1 d以上不溃散;6周切片显示明显成骨,15周骨缺损基本愈合。结论该骨水泥具有良好的注射性能、抗溃散性能和降解性能,复合DBM后具有良好的成骨性能。     

Synergistic effects of bisphosphonate and calcium phosphate nanoparticles on peri-implant bone responses in osteoporotic rats

The prevalence of osteoporosis will increase within the next decades due to the aging world population, which can affect the bone healing response to dental and orthopedic implants. Consequently, local drug targeting of peri-implant bone has been proposed as a strategy for the enhancement of bone-implant integration in osteoporotic conditions. In the present study, an established in-vivo femoral condyle implantation model in osteoporotic and healthy bone is used to analyze the osteogenic capacity of titanium implants coated with bisphosphonate (BP)-loaded calcium phosphate nanoparticles (nCaP) under compromised medical conditions. After 4 weeks of implantation, peri-implant bone volume (%BV; by μCT) and bone area (%BA; by histomorphometry) were significantly increased within a distance of 500 μm from implant surfaces functionalized with BP compared to control implants in osteoporotic and healthy conditions. Interestingly, the deposition of nCaP/BP coatings onto implant surfaces increased both peri-implant bone contact (%BIC) and volume (%BV) compared to the deposition of nCaP or BP coatings individually, in osteoporotic and healthy conditions. The results of real-time PCR revealed similar osteogenic gene expression levels to all implant surfaces at 4-weeks post-implantation. In conclusion, simultaneous targeting of bone formation (by nCaP) and bone resorption (by BP) using nCaP/BP surface coatings represents an effective strategy for synergistically improvement of bone-implant integration, especially in osteoporotic conditions.     

硫酸钙人工骨在良性骨肿瘤刮除术后骨缺损填充中的愈合情况及其重建作用研究

目的观察硫酸钙人工骨在良性骨肿瘤刮除术后骨缺损填充中的愈合情况,探讨其在骨肿瘤患者中的重建作用。方法回顾性分析在2010年1月~2013年9月期间,我院骨外科收治的临床随访资料完整的良性骨肿瘤患者,根据植骨材料的不同,分别采用硫酸钙人工骨、同种异体骨以及自体骨修复肿瘤刮除术后骨缺损。每组各选择随机样本40例。根据术后随访的X线结果和相关临床资料。评估不同移植骨在良性骨肿瘤缺损填充中的降解、吸收和愈合情况。结果 120例患者获得3~48个月随访,平均随访时间14.7个月。随访期自体骨组、硫酸钙人工骨组和同种异体骨组均未发生深部感染、植骨不愈合、伤口不愈合、植骨部位骨折及肿瘤复发等相应的并发症,临床获益率达100%。三组病例在骨愈合时间无统计学意义,愈合率也无显著性差异(0.05)。结论用硫酸钙人工骨材料填充良性骨肿瘤刮除后的骨缺损,愈合效果与自体骨和同种异体骨相近。且硫酸钙人工骨生物相容性好,并发症少,值得在临床上推广。     

Cytological Evaluation of the Tissue-Implant Reaction Associated with S/C and I/P Implantation of ALCAP and HA Bioceramics in vivo

It is well documented that several ceramic materials are highly compatible and non-immunogenic with host tissues. Recent studies have demonstrated the need for further investigation of these devices in vivo to further elucidate the possible mechanisms involved in biocompatibility. The purpose of this investigation was to study the morphological characteristics of the fibrous tissue capsule resulting from the implantation of aluminum calcium phosphate (ALCAP) and hydroxyapetite (HA) bioceramics. Implants of ALCAP and HA were implanted into 10 adult male rats subcutaneously (S/C) and intraperitoneally (I/P). At 90 days post-implantation, the animals were euthanized, and the ceramic devices, the fibrous tissue, and vital organs were harvested. Evaluation of routine stained sections (5 m, hematoxylin & eosin) of the fibrous tissue capsule surrounding the HA and ALCAP ceramics revealed the following: 1) all the ceramic devices had fibrous connective tissue capsules of slightly varying degrees of thickness at the time of sacrifice, depending on the site of implantation and type of material, and 2) there were statistically significant differences (p < 0.05) in the numbers and types of cellular components with respect to implantation site. The number of macrophages, neutrophils, fibroblasts, degree of vascularity, and thickness of the fibrous tissue matrix was found to be statistically different between the S/C implanted ceramic groups. The number of macrophages, neutrophils, fibroblasts, and collagen content comparing the fibrous tissue surrounding the ALCAP and HA ceramics (I/P), was found to be statistically different.     

磷酸钙活性人工骨与自体骨颗粒环抱式植骨治疗四肢骨不连#br#

目的　探讨固化型磷酸钙活性人工骨(CPC/rhBMP-2)与自体骨颗粒环抱式植骨治疗四肢非感染性萎缩型骨不连的临床疗效。 方法　对2013年9月至2017年9月年间治疗的16例四肢非感染性萎缩型骨不连患者进行回顾性分析,其中男12例,女4例;年龄（37.06±16.31）岁（16~67岁）。骨不连部位：下肢11例（胫骨6例、股骨3例、跟骨1例、腓骨1例）,上肢5例（桡骨3例、锁骨1例、手舟骨1例）,所有患者均行断端新鲜化,骨缺损中央植入自体骨颗粒,外周植入CPC/rhBMP-2形成环抱式植骨。术后复查X线片观察骨折愈合情况,记录并发症发生情况,随访患者直到骨折愈合。采用上肢功能障碍评定量表（DASH）、下肢功能量表(LEFS)和SF-12生活质量量表对治疗前、后进行疗效评定。 结果　本组16例均获随访,随访时间（16.93±4.06）月（12~26月）。X片显示全部病例均获得骨性愈合,愈合时间（5.43±2.25）月（3~10月）;术后DASH、LEFS和SF-12评分与术前比较均有统计学差异(P<0.05)。 结论　固化型磷酸钙活性人工骨(CPC/rhBMP-2)和自体骨颗粒环抱式植骨治疗四肢萎缩型骨不连的愈合率及安全性高,疗效可靠。     

Induction of strong immune response against a multicomponent antigen of Mycobacterium tuberculosis in BALB/c mice using PLGA and DOTAP adjuvant

A promising strategy for preventing illness and death caused by Mycobacterium tuberculosis (Mtb) is vaccination. In this study, we aimed to evaluate the capacity of a multicomponent vaccine comprising HspX/EsxS‐fused protein, PLGA (poly (lactide‐co‐glycolide)) and DOTAP (1, 2‐dioleoyl‐3‐trimethylammonium propane) in eliciting immune responses against Mtb in BALB/c mice. A preparation of PLGA nanoparticles (NPs) containing fused protein and DOTAP adjuvant was made using double emulsion solvent evaporation (w/o/w) and lipid film hydration methods, respectively. After three subcutaneous immunization of BALB/c mice with various formulations, ELISA technique was used to measure interferon‐γ (IFN‐γ) and interleukin‐4 (IL‐4) cytokines levels in splenocytes as well as serum anti‐HspX/EsxS IgG1 and IgG2a titers. The results of the current study showed that PLGA/HspX/EsxS/DOTAP formulation was able to induce higher levels of FN‐γ, IgG1, and IgG2a responses compared with BCG as the positive control, HspX/EsxS, HspX/EsxS/DOTAP and PLGA/HspX/EsxS formulations. Our results suggest that PLGA NPs, as delivery system, and DOTAP, as adjuvant, have a good potential to enhance immune responses against HspX/EsxS antigen after subcutaneous immunization of BALB/c mice.     

硫酸钙/庆大霉素、硫酸钙/聚乳酸/庆大霉素释药系统的制备与特性分析

目的构建新型骨植入型抗生素释放系统,硫酸钙／庆大霉素、硫酸钙／聚乳酸／庆大霉素释药系统,探讨抗生素释放系统的制备可行性。方法以自行研制的硫酸钙或硫酸钙与聚乳酸的复合物作为抗生素载体,构建抗生素释放系统,对其进行电镜扫描,晶体衍射分析和体外降解实验以分析其构成特点、理化特性和降解特点。结果所构建的抗生素释药系统主要成分为二水硫酸钙,具有初始的生物力学抗压缩强度,具有一定的孔隙率,硫酸钙／聚乳酸／庆大霉素中,聚乳酸对硫酸钙形成包裹。体外可降解,硫酸钙／聚乳酸／庆大霉素降解时间长于硫酸钙／庆大霉素。结论以硫酸钙和硫酸钙聚乳酸复合物作为载体构建抗生素释放系统能够满足药物释放和骨内植入的要求,具有制备的可行性。     

rhBMP-2／PLGA缓释支架的制备及对MSCs细胞成骨活性的体外研究

目的探讨骨形态发生蛋白（rhBMP-2）的聚乳酸聚乙醇酸共聚物（PLGA）体外缓释生物支架对人骨髓间充质干细胞（MSCs）细胞的影响。方法采用粒子沥滤-冷冻干燥复合工艺制备了附载rhBMP-2的PLGA生物支架,并检测了在PLGA的降解过程中rhBMP-2的释药规律;同时分离培养人骨髓间充质干细胞,体外培养后分别接种于附载和未附载rhBMP-2的PLGA支架上。扫描电镜观察不同时间段MSC在支架上的生长情况;MTT法测定细胞增殖情况。结果rhBMP-2能被包裹进PLGA支架中,而且可以在PLGA支架降解过程中持续释放出来并诱导骨发生。结论骨形态发生蛋白的PLGA复合载体是一种较为理想的新型生物支架。     

In vivo 31P MRS detection of an alkaline inorganic phosphate pool with short T1 in human resting skeletal muscle

Non‐invasive determination of mitochondrial content is an important objective in clinical and sports medicine. ³¹P MRS approaches to obtain information on this parameter at low field strength typically require in‐magnet exercise. Direct observation of the intra‐mitochondrial inorganic phosphate (Pi) pool in resting muscle would constitute an alternative, simpler method. In this study, we exploited the higher spectral resolution and signal‐to‐noise at 7T to investigate the MR visibility of this metabolite pool. ³¹P in vivo MR spectra of the resting soleus (SOL) muscle were obtained with ¹H MR image‐guided surface coil localization (six volunteers) and of the SOL and tibialis anterior (TA) muscle using 2D CSI (five volunteers). A resonance at a frequency 0.38 ppm downfield from the cytosolic Pi resonance (Pi₁; pH 7.0 ± 0.04) was reproducibly detected in the SOL muscle in all subjects and conditionally attributed to the intra‐mitochondrial Pi pool (Pi₂; pH 7.3 ± 0.07). In the SOL muscle, the Pi₂/Pi₁ ratio was 1.6 times higher compared to the TA muscle in the same individual. Localized 3D CSI results showed that the Pi₂ peak was present in voxels well away from blood vessels. Determination of the T1 of the two Pi pools in a single individual using adiabatic excitation of the spectral region around 5 ppm yielded estimates of 4.3 ± 0.4 s vs 1.4 ± 0.5 s for Pi₁ and Pi₂, respectively. Together, these results suggest that the intra‐mitochondrial Pi pool in resting human skeletal muscle may be visible with ³¹P MRS at high field. Copyright © 2010 John Wiley & Sons, Ltd.     

胶原/羟基磷灰石复合材料的制备及用于骨缺损修复的研究现状

通过模拟天然骨的结构,制备胶原/羟基磷灰石复合材料,与天然骨具有相似的组成、结构和性能,并具有良好的生物活性和生物降解性。本文就胶原/羟基磷灰石复合材料的制备方法、仿生形成机制、表征手段及骨缺损修复的应用等进行综述,并展望其未来发展方向。     

托吡酯对儿童骨代谢的影响及其相关机制的研究

目的：探讨抗癫痫药（AED）托吡酯（TPM）对儿童骨代谢的影响及其发生机制,企为临床制定防治措施提供一个理论基础。方法：收集43例无肝、肾损害及其他特殊疾病,近期未应用糖皮质激素和治疗剂量维生素D的癫痫患儿的血清和尿液,并以其中服药前的癫痫患儿13例（作为对照组）和TPM组30例,测定其血清碱性磷酸酶（ALP）和尿脱氧吡啶啉／尿肌酐（DPD／Cr）,同时,对服用TPM的患儿体内血Ca2＋、HCO-水平进行常规实验室检查。采用SPSS10．0统计软件进行统计学分析。结果：TPM组血清AI。PF（20．13±4．44）U／L]与对照组[（14．75±2．39）U／L]之间差异有非常显著意义（P〈o．01）,TPM组尿DPD／Cr与对照组之间差异有显著意义（P〈0．05）,两组中的血清ALP和尿DPD／Cr之间的相关性（相关系数r分别为-0．083、-0．396）均无统计学意义（P〉0．05）。TMP组患儿血Ca2＋、HCO-的结果分别为：（1．10±0．08）mmol／L,（16．28±1．09）mmol／L,均低于正常值范围2．12～2．75mmol／L和22～28mmol／L。服用TPM的癫痫患儿血Ca2＋水平与其ALP和DPD／Cr水平无显著相关性（r=-0．183,-0．288P〉0．05）,该组患儿血HCO。水平与其ALP和DPD／Cr水平无显著相关性（r=0．321,0．285P〉0．05）,血Ca2＋与HCO一之间无显著相关性（相关系数r=0．064,P〉0．05）。结论：TPM对儿童骨代谢的影响包括骨形成和骨吸收两个方面,服用TPM的癫痫患儿不仅需要补充钙剂和维生素D,还应定期检测血浆HCO3-水平。     

Adenovirus-mediated transfer of VEGF into marrow stromal cells combined with PLGA/TCP scaffold increases vascularization and promotes bone repair in vivo

Introduction

Large osseous defect remains a serious clinical problem due to the lack of sufficient blood supply and it has been proposed that this situation can be relieved by accelerating the formation of new vessels in the process of bone defect repair. The aim of this study was to develop a new type of artificial bone by transferring the VEGF gene into marrow stromal cells (MSCs) and seeding them into a porous scaffold.

Material and methods

An adenovirus vector was employed to transfer the VEGF gene into MSCs and expression of the exogenous gene was confirmed by ELISA. Next the transduced cells were seeded into a collagen I modified PLGA/TCP scaffold. The constructed new complex artificial bone was then assessed for biocompatibility in vitro and blood vessel formation and bone formation in vivo.

Results

We found that adenovirus mediated VEGF gene transfer into MSCs sustained VEGF expression in MSCs for 3 weeks. Porous scaffold PLGA/TCP made by rapid prototyping technology exhibited improved biocompatibility resulting from crosslinking with collagen I. Furthermore, the in vivo study showed that large amounts of blood vessels were detected histologically 1 week after artificial bone implantation, and significant bone formation was detected 8 weeks after implantation.

Conclusions

Our findings suggest that gene transfer of VEGF into MSCs combined with PLGA/TCP scaffold enhances bone repair in vivo by promoting vascularization.