Properties and in vitro biological evaluation of nano-hydroxyapatite/chitosan membranes for bone guided regeneration

Nano-hydroxyapatite(n-HA)/chitosan(CS) composite membranes were prepared by solvent casting and evaporation methods for the function of guided bone regeneration (GBR). The effect of n-HA content and solvent evaporation temperature on the properties of the composite membranes was studied. The n-HA/CS membranes were analyzed by scanning electron microscopy, Fourier transformed infrared spectroscopy, X-ray diffraction, swelling measurement, mechanical test, cell culture and MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenylte-2H-tetrazolium bromide) assay. The results show that the surface roughness and micropores of the composite membranes increase with the rise of n-HA content, suitable for adhesion, crawl and growth of cells. The hydroxyapatite holds nano size and distributes uniformly in the composite membranes. Chemical bond interaction exists between Ca ions and –OH groups of n-HA and –NH₂ or –OH groups of CS. The n-HA content and solvent evaporation temperature have obvious influence on the swelling ratio, tensile strength and elongation rate of the composite membranes. Cell culture and MTT assays show that n-HA and its content can affect the proliferation of cells. The n-HA/CS composite membranes have no negative effect on the cell morphology, viability and proliferation and possess good biocompatibility. This study makes the n-HA/CS composite membrane be a prospective biodegradable GBR membrane for future applications.     

Preparation of electrospun siloxane-poly(lactic acid)-vaterite hybrid fibrous membranes for guided bone regeneration

A nonwoven fabric scaffold for guided bone regeneration (GBR) consisting of siloxane-poly(lactic acid) (PLA)-vaterite hybrid material (SiPVH) was prepared by an electrospinning method. To improve the cellular compatibility of the fabric, the fibers were completely coated with hydroxyapatite (HA) by soaking in simulated body fluid. The HA-coated SiPVH nonwoven fabric contained large-sized spaces and showed the ability of releasing soluble silica and calcium species, which have previously been reported to stimulate osteogenic cells at the genetic level. A new type of GBR membrane was prepared by bonding SiPVH fabric with PLA nonwoven fabric with small-sized pores for preventing the intrusion of soft tissue. The resultant bi-layered membrane was expected to be effective not only for having an open structure for bone formation and a barrier to soft tissue, but also for enhancing bone growth by the release of ionic species.     

Comparison of ready-made and self-setting alginate membranes used as a barrier membrane for guided bone regeneration

In order to understand the requirements of guided bone regeneration (GBR) involving alginate base self-setting barrier membranes, GBR was performed in the case of bicortical bone defects formed at the tibiae of experimental animals employing self-setting and ready-made alginate membranes. Connective tissue ingress into the bone defects at the skin side of the tibia was observed when GBR was generated utilizing ready-made alginate membrane. In contrast, bone defects were reconstructed with bone tissue when GBR was generated with self-setting alginate membrane formed from aqueous 3% sodium alginate and 3% CaCl₂ solutions. The unreacted aqueous sodium alginate solution inherent to self-setting alginate membrane did not inhibit bone tissue regeneration. Rather, callus bone was formed using sodium alginate as the nucleus. However, when GBR was effected with self-setting alginate membrane formed from aqueous 10% CaCl₂ solution, membrane was too thick and thus regeneration of bone tissue in the bone cavity was prevented. Therefore, we concluded that self-setting alginate membrane is very useful as a barrier membrane for GBR upon appropriate adjustment of conditions with respect to preparation of alginate membrane.     

纳米羟基磷灰石/壳聚糖复合膜的制备和表征

以壳聚糖(chitosan,CS)为高分子相,纳米羟基磷灰石(nano-hydroxyapatite,n-HA)为无机相,采用溶液共混和真空下溶剂挥发的方法制备了n-HA/CS复合膜,通过SEM、XRD、FTIR、接触角及力学性能等测试对此复合膜进行分析和表征.结果表明,复合膜具有非对称结构,上表面组分主要是CS,下表面组分是n-HA和CS复合体,并在底部形成了一层致密层,中间是疏松层;复合膜中CS与HA之间存在一定的化学键合并复合均匀,没有明显的相分离,且复合膜中的HA为类似于自然骨矿物相的弱结晶结构.复合膜的非对称结构对其接触角也有一定的影响,反映了膜表面亲、疏水性的不同,为细胞的粘附和旺长提供了一定的微环境.复合膜干态下的拉伸强度和断裂伸长率较纯CS膜的低,而在湿态时却较纯CS膜高.     

Comparison of the performance of natural latex membranes prepared with different procedures and PTFE membrane in guided bone regeneration (GBR) in rabbits

This work assessed the performance of membranes made of natural latex extracted from Hevea brasiliensis prepared with three different methods: polymerized immediately after collection without the use of ammonia (L1); polymerized after preservation in ammonia solution (L2); and polymerized after storage in ammonia, followed by Soxhlet technique for the extraction of substances (L3). Polytetrafluoroethylene (PTFE) membrane was used as control. Two 10-mm diameter bone defects were surgically made in the calvaria of thirty adult male New Zealand rabbits. Defects (total n = 60) were treated with guided bone regeneration (GBR) using L1, L2, L3 or PTFE membranes (n = 15 for each membrane). Ten animals were euthanized after 7, 20 and 60 days postoperatively so that five samples (n = 5) of each treatment were collected at each time, and bone regeneration was assessed microscopically. The microscopic analysis revealed defects filled with blood clot and new bone formation at the margins of the defect in all 7-day samples, while 20-day defects were mainly filled with fibrous connective tissue. After 60 days defects covered with L1 membranes showed a significantly larger bone formation area in comparison to the other groups (P < 0.05, ANOVA, Tukey). Additionally, bone tissue hypersensitization for L1 and PTFE membranes was also investigated in six additional rabbits. The animals were subjected to the same surgical procedure for the confection of one 10-mm diameter bone defect that was treated with L1 (n = 3) or PTFE (n = 3). Fifty-three days later, a second surgery was performed to make a second defect, which was treated with the same type of membrane used in the first surgery. Seven days later, the animals were euthanized and samples analyzed. No differences among L1 and PTFE samples collected from sensitized and non-sensitized animals were found (P > 0.05, Kruskal–Wallis). Therefore, the results demonstrated that latex membranes presented performance comparable to PTFE membranes, and that L1 membranes induced higher bone formation. L1 and PTFE membranes produced no hypersensitization in the bone tissue.     

Human amniotic membrane for guided bone regeneration of calvarial defects in mice

Due to its biological properties, human amniotic membrane (hAM) is widely studied in the field of tissue engineering and regenerative medicine. hAM is already very attractive for wound healing and it may be helpful as a support for bone regeneration. However, few studies assessed its potential for guided bone regeneration (GBR). The purpose of the present study was to assess the potential of the hAM as a membrane for GBR. In vitro, cell viability in fresh and cryopreserved hAM was assessed. In vivo, we evaluated the impact of fresh versus cryopreserved hAM, using both the epithelial or the mesenchymal layer facing the defect, on bone regeneration in a critical calvarial bone defect in mice. Then, the efficacy of cryopreserved hAM associated with a bone substitute was compared to a collagen membrane currently used for GBR. In vitro, no statistical difference was observed between the conditions concerning cell viability. Without graft material, cryopreserved hAM induced more bone formation when the mesenchymal layer covered the defect compared to the defect left empty. When associated with a bone substitute, such improved bone repair was not observed. These preliminary results suggest that cryopreserved hAM has a limited potential for GBR.

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Analysis of bone formation and membrane resorption in guided bone regeneration using deproteinized bovine bone mineral versus calcium sulfate

Guided Bone Regeneration (GBR) is a technique based on the use of a physical barrier that isolates the region of bone regeneration from adjacent tissues. The objective of this study was to compare GBR, adopting a critical-size defect model in rat calvaria and using collagen membrane separately combined with two filling materials, each having different resorption rates. A circular defect 8?mm in diameter was made in the calvaria of Wistar rats. The defects were then filled with calcium sulfate (CaS group) or deproteinized bovine bone mineral (DBBM group) and covered by resorbable collagen membrane. The animals were killed 15, 30, 45 and 60 days after the surgical procedure. Samples were collected, fixed in 4% paraformaldehyde and processed for paraffin embedding. The resultant sections were stained with H&E for histological and histomorphometric study. For the histomorphometric study, the area of membrane was quantified along with the amount of bone formed in the region of the membrane. Calcium sulfate was reabsorbed more rapidly compared to DBBM. The CaS group had the highest percentages of remaining membrane at 15, 30, 45 and 60 days, compared to the DBBM group. The DBBM group had the highest amount of new bone at 45 and 60 days compared to the CaS group. Based on these results, it was concluded that the type of filling material may influence both the resorption of collagen membrane and amount of bone formed.

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Enhanced bioactivity of polyvinylidene chloride films using argon ion bombardment for guided bone regeneration

Polyvinylidene chloride (PVDC) is a long chain carbon synthetic polymer. The objective of this study was to improve the bioactivity of PVDC films through surface modification using argon (Ar) ion bombardment to create Ar-modified PVDC films (Ar-PVDC) to address the clinical problems of guided bone regeneration (GBR), which is technique-sensitive, and low bone regenerative ability. First, the effects of Ar ion bombardment, a low temperature plasma etching technique widely used in industry, on PVDC film wettability, surface chemistry, and morphology were confirmed. Next, fibroblast-like and osteoblast-like cell attachment and proliferation on Ar-PVDC were assessed. As a preclinical in vivo study, Ar-PVDC was used to cover a critical-sized bone defect on rat calvaria and osteoconductivity was evaluated by micro-computed tomography analysis and histological examinations. We found that the contact angle of PVDC film decreased by 50° because of the production of –OH groups on the PVDC film surface, though surface morphological was unchanged at 30 min after Ar ion bombardment. We demonstrated that cell attachment increased by about 40 % and proliferation by more than 140 % because of increased wettability, and 2.4 times greater bone regeneration was observed at week 3 with Ar-PVDC compared with untreated PVDC films. These results suggest that Ar ion bombardment modification of PVDC surfaces improves osteoconductivity, indicating its potential to increase bone deposition during GBR.     

Development and cell response of a new biodegradable composite scaffold for guided bone regeneration

Composites of biodegradable polymers with different calcium phosphate ceramics and glasses, have been developed as scaffolds for applications in bone-tissue engineering. In this work, phosphate glass particles have been incorporated into the polymer, poly(95L/5DL) lactic acid (PLA) and porous structures were elaborated. Their porosity, compressive mechanical properties and biological response were evaluated. Interconnected structures with evenly distributed pores and a porosity as high as 97% were obtained. The incorporation of glass particles into the polymer showed to have a positive effect in the mechanical properties of the foams. Indeed, the compressive modulus increased from 74.5 to 120 KPa and the compressive strength from 17.5 to 20.1 KPa for the PLA and the PLA/glass foams, respectively. The biological response was evaluated by means of the MTT test, the materials resulted to be noncytotoxic.     

Injectable silk/hydroxyapatite nanocomposite hydrogels with vascularization capacity for bone regeneration

Localized and sustained osteogenic-angiogenic stimulation to bone defects is critical for effective bone repair.Here,desferrioxamine(DFO)was loaded on silk fibroin nanofibers and blended with hydroxyapatite nanorods(HA),forming injectable DFO-loaded silk fibroin-HA nanocomposite hydrogels.The composite hydrogels remained homogeneous distribution of HA with high ratio(60%)and also higher stiffness than that of pure silk fibroin nanofiber hydrogels,which provided stable osteogenic stimulation niches for tissue regeneration.Without the scarify of injectability,the hydrogels achieved slow delivery of DFO for above 60 days,resulting in suitable angiogenesis in vitro and in vivo and better osteogenesis than DFO-free systems.Compared to previous injectable silk fibroin-HA hydrogels,the introduction of vascularization capacity further stimulated the osteogenic differentiation of stem cells and accelerated new bone formation.Quicker and better bone healing were detected at defect sites after the injection of DFO-loaded nanocomposite hydrogels,indicating the effective synergistic effect of osteogenic and angiogenic cues.This work provides a simple and effective strategy of introducing angiogenic cues to bone matrices.We believe that the injectable nanocomposite hydrogels are suitable for the regeneration of bone tissues.     

基于超声导波反演参数定征皮质骨骨质状况研究

骨质疏松发生时,人体皮质骨层的孔隙度将增大。为研究皮质骨参数（厚度、横波速度、纵波速度）与骨质疏松症的关系,文章利用有限元方法对不同孔隙度（0∶3%∶27%）的单层皮质骨进行仿真,以3个周期的高斯包络正弦波作为激励,将采集到的超声导波信号先后经过二维傅里叶变换和Burg算法处理后得到频散数据,与基于FloquetBloch理论建立的理论频散曲线数据库进行匹配反演,得到皮质骨厚度、横波速度、纵波速度参数。结果显示皮质骨厚度反演准确,皮质骨孔隙度与横波速度和纵波速度呈负相关,横波速度敏感度为19.0%,纵波速度敏感度为5.5%。横波速度敏感度更高,临床诊断潜力更大。并对6组牛胫骨进行了离体实验,结果显示,反演得到的皮质骨厚度与其实际测量值的平均相对误差为4.0%,且实验频散曲线与理论频散曲线相吻合,验证了文中算法在真实皮质骨参数反演上的可行性和准确性。文中的研究在骨质疏松超声检测中具有应用潜力。     

壳聚糖/羟基磷灰石引导组织再生膜材料的制备和性能

用生物矿化交替沉积法成功在CS基质膜表面构建HA涂层,并进行了表征.结果表明,HA含量随着沉积循环数增加而增加,且在4个循环后基本达到饱和,饱和含量为8.8%;随着沉积循环数增加HA由均一颗粒状转变为无规则平板状;拉伸强度由纯CS膜的68.6 MPa降低到沉积5个循环后的46.9 MPa.控制沉积数可调控HA的含量、形貌和力学强度,以满足引导组织再生术需要.     

溶胶-凝胶法甲基化改性SiO2膜的气体渗透性和再生性研究

以甲基三乙氧基硅烷替代部分正硅酸乙酯作为前驱物,用溶胶-凝胶法对亲水性SiO2膜进行改性,制备了甲基化改性SiO2膜.通过FTIR、SEM和气体渗透实验考察了甲基化改性对SiO2膜形貌、化学结构、气体渗透性及再生性的影响.结果表明,压差、温度对两种SiO2膜渗透分离性能的影响相似;甲基化改性使SiO2膜气体渗透速率显著增加,而H2/CO2选择性却没有明显下降;在水气环境中陈化后,虽然两种SiO2膜的H2/CO2选择性均未显著降低,但亲水性SiO2膜气体渗透速率下降明显,而甲基化改性SiO2膜则下降较小.再生可以部分恢复SiO2膜的渗透分离性能,与陈化前相比,再生使两种SiO2膜的渗透性能降低,H2/CO2选择性增大,甲基化改性大大减小了再生对膜渗透性能的劣化.     

Formation and bioactivity of porous and nanostructured TiO2/beta-TCP coating on titanium

Titanium and its alloys have been widely used as hard tissue implants due to their excellent mechanical properties and biocompatibility. However, their near bio-inertness and metallic ion release are still the problems with clinical uses. In this paper, porous and nanostructured TiO2/beta-tricalcium phosphate (beta-TCP) composite coatings were prepared on titanium substrates by plasma electrolytic oxidation (PEO) in a Ca and P-containing electrolyte. The influence of PEO electric current density on phase composition and bioactivity of the coatings were studied. X-ray diffraction, scanning electron microscopy and Fourier transfer infrared spectroscopy were utilized to characterize the phase composition and microstructure of the coatings. Simulated body fluid immersion tests were employed on the coatings to evaluate their bioactivity. The results reveal that TiO2/beta-TCP composite coating with pores size less than 10 microm and grains of 50-100 nm in size was prepared. The electric current density of PEO is an important factor in the formation of the composite coating. The TiO2/beta-TCP composite coating shows good bioactivity, which are attributed to the incorporation of beta-TCP.     

In vivo bone regeneration with injectable chitosan/hydroxyapatite/collagen composites and mesenchymal stem cells

For reconstruction of irregular bone defects, injectable biomaterials are more appropriate than the preformed biomaterials. We herein develop a biomimetic in situ-forming composite consisting of chitosan (CS) and mineralized collagen fibrils (nHAC), which has a complex hierarchical structure similar to natural bone. The CS/nHAC composites with or without mesenchymal stem cells (MSCs) are injected into cancellous bone defects at the distal end of rabbit femurs. Defects are assessed by radiographic, histological diagnosis and Raman microscopy until 12 weeks. The results show that MSCs improve the biocompatibility of CS/nHAC composites and enhance new bone formation in vivo at 12 weeks. It can be concluded that the injectable CS/nHAC composites combined with MSCs may be a novel method for reconstruction of irregular bone defects.     

振动声激发超声导波评估皮质骨厚度的研究

利用振动声激发超声导波来评价长骨是一种较新的思路。基于超声轴向传播方法,首先通过三维有限元法对导波在长骨中的传播进行建模仿真,再根据接收波形求解0A导波模式的相速度,最后依据理论相速度频散曲线得出皮质骨厚度。对不同厚度(2~6 mm)的骨板仿真的结果表明,皮质骨厚度估计的平均误差仅为2.61%。所提出的方法能够有效评估皮质骨厚度,对发展超声导波反演长骨的新技术具有一定意义,同时为临床超声导波长骨诊断的研究提供了新的思路。     

Material design of bioabsorbable inorganic/organic composites for bone regeneration

Two new bioabsorbable inorganic/organic composite materials were developed for bone regeneration. One material used was beta-TCP/PLGC in which poly(L-lactide-co-glycolide-co-epsilon-caprolactone) and beta-tricalcium phosphate were used as the matrix and filler, respectively. The other material used was HAp/Col-a soft nanocomposite of hydroxyapatite and type I collagen. Using these composites, two bone implants were designed. The efficacy of these implants was investigated by applying them to the critical-sized bone defects that were created in the canine tibia. Although no tissue engineering techniques such as application of growth factors or stem cells was utilized, successful healing was observed. These results suggested that bone regeneration in the critical-sized defects is possible without the use of growth factors or stem cells if the materials and the bone implants are suitably designed.     

超声造影评估早期骨再生血管化的实验研究

目的探讨超声造影(Contrast Enhanced Ultrasound,CEUS)在骨再生过程中骨移植材料早期血管化的应用价值,为临床骨再生不良提供早期诊断信息。方法将25只斯泼累格·多雷(Sprague Dawley,SD)级雄性大鼠,于左侧后肢近股骨头处肌间隙内植入磷酸钙骨水泥/重组人骨形态蛋白2(Calcium Phosphate Cement/recombination huaman Bone Morphogenetic Protein-2,CPC/rh BMP-2),建立大鼠肌内异位成骨模型。在术后3、7、14、21、28 d分别进行高频超声、CEUS及病理学检测。观察不同时间CPC/rh BMP-2内的血流灌注情况及骨生成情况,并与病理结果进行对照。结果 CEUS显示,术后3 d,支架外周见少许星点状造影剂灌注,随着时间的增加,造影剂逐步向中央填充,且时间-强度曲线定量分析显示,峰值强度(Peak Intensity,PI)及曲线下面积(Area Under the Curve,AUC)逐渐上升;术后14确d,PI和AUC均达到最大值,术后21、28 d造影剂的PI、AUC降低。各组间差异具有统计学意义(p0.05),各组间达峰时间(Tine To Oeak,TTP)差异无明显统计学意义(p0.05)。苏木精伊红(Hematoxylin-Eosin,HE)染色显示,术后3、7、14、21、28 d支架内可见不同程度的新生血管形成,术后14 d为显著,与CEUS检查相符。结论 CEUS可早期显示CPC/rh BMP-2的新生血管形成情况,为骨移植的生长评估及骨再生不良提供诊断依据。