Characterization of biodegradable and cytocompatible nano-hydroxyapatite/polycaprolactone porous scaffolds in degradation in vitro

Porous nano-hydroxyapatite/polycaprolactone (nHA/PCL) scaffolds with different composition ratios of nHA/PCL were fabricated via a melt-molding/porogen leaching technique. All scaffolds were characterized before and after degradation in vitro for six months. The original scaffolds had high porosity at around 70% and showed decreasing compressive modulus (from 24.48 to 2.69 MPa for hydrated scaffolds) with the introduction of nHA. It was noted that the scaffolds could retain relatively stable architecture and mechanical properties for at least six months, although some slight changes happened with the nHA/PCL scaffolds in the mass, the nHA content, the PCL molecular weight and the crystallinity. Moreover, during the 7 days culture of bone marrow stromal cells (BMSCs) on scaffolds, the cell adhesion and proliferation of BMSCs were presented well on both the surface and the cross-section of the scaffolds. All of these results suggested the nHA/PCL scaffolds to be promising in bone tissue engineering.     

Time-of-flight secondary ion mass spectrometric analysis of the interface between bone and titanium implants

Implant healing into bone tissue is a process where the mature bone grows towards and eventually fuses with the implant. In this study we investigated implant healing during 4 weeks with focus on the implant-tissue interface. Our main interest was to study the mineralization process around the implant. Titanium discs were implanted in rat tibia for 2 and 4 weeks. After implantation cross sections of bone and implant were made using a low-speed saw equipped with a diamond wafering blade. One section from each sample was stained with basic fuchsin and micrographed by light microscopy (LM). The other section was analyzed with imaging time-of-flight secondary ion mass spectrometry (TOF-SIMS) using a Bi(3)(+) cluster ion source. This ion source has recently been shown to enable identification of high-mass hydroxyapatite (HA) fragment ions (m/z 291-653) in bone samples. The LM images were used to identify areas suitable for TOF-SIMS analysis. Three areas were selected for mass spectral analysis, corresponding to interface region, bone and soft tissue, from which positive ion spectra were recorded. In the areas identified as bone, high-mass HA fragments ions were found after both 2 and 4 weeks. In the soft tissue area, no high-mass ions were found after 4 weeks. However, after 2 weeks HA-related ions were identified in mineralized spots in areas defined as soft tissue. After 4 but not after 2 weeks, high-mass HA fragment ions were found in the interface region. In conclusion, differences were observed regarding mineralization between 2 and 4 weeks of implantation and between different regions surrounding the implants. Imaging TOF-SIMS analysis using a Bi(3)(+) cluster as ion source enables identification of high-mass HA fragment ions at implant-tissue interfaces in bone. This technique might therefore be useful for biocompatibility assessment and for studying the mineralization process at implant surfaces.     

Nanoscale topography reduces fibroblast growth, focal adhesion size and migration-related gene expression on platinum surfaces

Controlling cellular responses on biomaterial surfaces is crucial in biomedical applications such as tissue engineering and implantable prosthetics. Since cells encounter various nanoscale topographic features in their natural environment, it has been postulated that surface nanotopography may be an alternative route to fabricate biomaterials with a desirable cellular response. In this framework, we investigated the responses of primary human fibroblasts to platinum substrates with different levels of surface roughness at the nanoscale. The nanorough surfaces were fabricated by using the glancing angle deposition technique (GLAD). We found that levels of cellular responses depended on the surface roughness and the size of the nanoscale features. We showed that in response to nanotopography cells spread less and have an elongated morphology, displaying signs of actin cytoskeleton impairment and reduced formation of focal adhesion complexes. Although cell growth and adhesion were impaired on the nanorough substrates, cell viability was not affected by topography. To a minor extent our results also indicate that cell migration might be reduced on the nanorough surfaces, since a significantly lower gene expression of migration related genes were found on the roughest surfaces as compared to the flat reference. The results presented here demonstrate that surface nanotopography influences fibroblasts responses on platinum, which may be used to reduce cellular adhesion on platinum implant surfaces such as implantable neural electrodes.     

Adsorption and conformational change of myoglobin on biomimetic hydroxyapatite nanocrystals functionalized with alendronate

The chemical conjugation of bisphosphonates (BPs), specifically alendronate, to hydroxyapatite could be an effective means to impart to it fine-tuned bioactivity. Horse heart myoglobin (Mb), a well-characterized protein, has been adsorbed onto biomimetic hydroxyapatite nanocrystals (nHA) and onto the nHA/alendronate conjugate powdered samples. The obtained materials have potential use in bone implantation and as prospective drug-delivery devices. The kinetic absorption of Mb onto nHA is dramatically affected by its functionalization with alendronate. The covering of the nHA surface by alendronate inhibits the adsorption of myoglobin. The adsorption mechanisms of the protein were studied by spectroscopic techniques (UV-vis and surface-enhanced Raman spectroscopy). The results indicate that the protein changes conformation upon adsorption on the inorganic substrate. In particular, the interaction with nHA alters the coordination state of the iron in the heme through the formation of a hexacoordinated low-spin Mb heme, possibly involving the distal histidine. Instead, the covering of the nHA surface by alendronate does not adsorb the protein but preserves the coordination state of the heme moiety. This study could be of significance either in the field of biomaterials science, in particular, to fine tune a bone-specific drug delivery device and to test nHA as a new support for heterogeneous catalysis, improving the understating of enzyme immobilization.     

Applications of Polydopamine in Implant Surface Modification

There is great clinical demand for orthopedic and dental implant surface modification methods to prevent osseointegration failure and improve implant biological functions. Notably, dopamine (DA) can be polymerized to form polydopamine (PDA), which is similar to the adhesive proteins secreted by mussels, to form a stable bond between the bone surface and implants. Therefore, PDA has the potential to be used as an implant surface modification material with good hydrophilicity, roughness, morphology, mechanical strength, biocompatibility, antibacterial activity, cellular adhesion, and osteogenesis. In addition, PDA degradation releases DA into the surrounding microenvironment, which is found to play an important role in regulating DA receptors on both osteoblasts and osteoclasts during the bone remodeling process. Furthermore, the adhesion properties of PDA suggest its use as an intermediate layer in assisting other functional bone remodeling materials, such as nanoparticles, growth factors, peptides, and hydrogels, to form “dual modifications.” The purpose of this review is to summarize the recent progress in research on PDA and its derivatives as orthopedic and dental implant surface modification materials and to analyze the multiple functions of PDA.     

Functionalized surface arrays for spatial targeting of immune cell signaling

The effect of surface topography and chemistry on cellular response is of fundamental importance, especially where living systems encounter device surfaces as in medical implants, tissue engineering, and cell-based sensors. To understand these biological processes on surfaces, there is a widespread interest in tailored surface-active materials produced by a combination of surface chemistry coupled to advanced patterning processes. We utilize self-assembled monolayers (SAMs) as molecular templates with submicrometer-scale spatial resolution to engage and cluster IgE receptors on rat basophilic leukemia (RBL) mast cells. Bioactive templates consisted of gold arrays on silicon with patterns from 1 mum down to 45 nm. These gold arrays served as molecular tethering sites, enabling covalent binding of functionalized self-assembled monolayers of alkanethiols. The free ends of the monolayers were functionalized with 2,4-dinitrophenyl(DNP)-caproate-based ligands which interact specifically with anti-DNP IgE bound to its high affinity cell surface receptor, FcepsilonRI on RBL mast cells. Present results on structures 1 mum down to 600 nm in size indicate that these ligand-immobilized patterned arrays can function as a powerful tool for visualization and systematic characterization of cell membrane involvement in IgE receptor-mediated immune cell signaling.     

Uniform silver nanoparticles on tunable porous N‐doped carbon nanospheres for aerobic oxidative synthesis of aryl nitriles from benzylic alcohols

Tunable N‐doped carbon nanospheres from sucrose as carbon source and Tris(2‐aminoethyl)amine (TAEA) as nitrogen source by a simple and easily reproducible method were prepared. It was demonstrated that the tunable N‐doping of carbon spheres could be realized by altering the ratio of TAEA in the raw materials. The content of doped nitrogen, surface area, pore volume and pore size of carbon nanospheres were increased with the increasing of TAEA amount in the hydrothermal process. Prepared N‐doped carbon nanospheres act as solid ligand for anchoring of Ag NPs which generated via chemical reduction of Ag ions. Benzylic alcohols and aldehydes were converted into the aryl nitriles by using Ag/N‐CS‐1 nanospheres as the catalyst and O₂ as the oxidant, efficiently. This catalyst was stable and could use for 6 successful runs.     

Temperature Controlled Lateral Pattern Formation in Confined Polymer Thin Films

The thermal induced topography change in a model system consisting of a polymer film on a Si substrate capped by a thin metal layer has been studied by using AFM. Regular lateral patterns over large areas were observed on the surface when the system was heated to a sufficiently high temperature. 2D-FFT analysis to the AFM images indicates that the patterns are isotropic and have well defined periodicities. The periodicities of the characteristic patterns are found to depend strongly on the annealing temperature. The study of the ki-netics of the formation reveals that such a topography forms almost instantaneously once the critical tempera-ture is reached. It is suggested that this wave-like surface morphology is driven by the thermal expansion co-efficient mismatch of the different layers. This method for generating regular wave-like patterns could be used as a general method for patterning various organic materials into micro/nanostructures.     

聚醚醚酮复合材料表面生物活性涂层的制备与性能

以聚醚醚酮/钡玻璃粉(PEEK-BGF)复合材料为基体, 通过硅烷偶联剂, 在复合材料表面构建具有生物活性的纳米羟基磷灰石(nHA)和甲基丙烯酸酯基的光固化树脂复合涂层. 采用扫描电子显微镜(SEM)和X射线光电子能谱(XPS)分析了材料表面形貌和元素分布, 测试了涂层与复合材料之间的粘接强度. 通过检测大鼠成骨细胞总蛋白含量和碱性磷酸酶表达水平, 评价新型光固化纳米羟基磷灰石/聚甲基丙烯酸酯(nHA/PMMA)复合涂层的生物活性. 研究结果表明, nHA填充的光固化复合材料形成粗糙的表面, 随着nHA的填充量提高, 涂层表面生物学活性得到提高.     

Incorporation of nanohydroxyapatite and vitamin D3 into electrospun PCL/Gelatin scaffolds: The influence on the physical and chemical properties and cell behavior for bone tissue engineering

Scaffold, an essential element of tissue engineering, should provide proper physical and chemical properties and evolve suitable cell behavior for tissue regeneration. Polycaprolactone/Gelatin (PCL/Gel)‐based nanocomposite scaffolds containing hydroxyapatite nanoparticles (nHA) and vitamin D3 (Vit D3) were fabricated using the electrospinning method. Structural and mechanical properties of the scaffold were determined by scanning electron microscopy (SEM) and tensile measurement. In this study, smooth and bead‐free morphology with a uniform fiber diameter and optimal porosity level with appropriate pore size was observed for PCL/Gel/nHA nanocomposite scaffold. The results indicated that adding nHA to PCL/Gel caused an increase of the mechanical properties of scaffold. In addition, chemical interactions between PCL, gelatin, and nHA molecules were shown with XRD and FT‐IR in the composite scaffolds. MG‐63 cell line has been cultured on the fabricated composite scaffolds; the results of viability and adhesion of cells on the scaffolds have been confirmed using MTT and SEM analysis methods. Here in this study, the culture of the osteoblast cells on the scaffolds showed that the addition of Vit D3 to PCL/Gel/nHA scaffold caused further attachment and proliferation of the cells. Moreover, DAPI staining results showed that the presence and viability of the cells were greater in PCL/Gel/nHA/Vit D3 scaffold than in PCL/Gel/nHA and PCL/Gel scaffolds. The results also approved increasing cell proliferation and alkaline phosphatase (ALP) activity for MG‐63 cells cultured on PCL/Gel/nHA/Vit D3 scaffold. The results indicated superior properties of hydroxyapatite nanoparticles and vitamin D3 incorporated in PCL/Gel scaffold for use in bone tissue engineering.     

纳米羟基磷灰石/丝素蛋白复合支架材料的降解特性及生物相容性研究

应用共混法制备了纳米羟基磷灰石/丝素蛋白复合支架材料, 通过体外降解和细胞培养实验研究了复合支架材料的降解特性和生物相容性. 体外降解实验结果显示, 复合支架材料具有稳定的降解能力; 在降解过程中, 羟基磷灰石由于与降解液发生钙、磷等离子的交换, 使其结晶得到了进一步生长和完善. 利用细胞计数法、四甲基偶氮唑盐(MTT)比色法和碱性磷酸酶(ALP)活性测定等分析了复合支架材料的生物相容性, 结果表明, MG63细胞在复合支架材料上具有良好的粘附、增殖能力, 并可引起早期的骨分化. 因此, 纳米羟基磷灰石/丝素蛋白复合支架作为骨组织工程的支架材料具有良好的应用前景.     

Influence of surface topography on osteoblast response to fibronectin coated calcium phosphate thin films

The ability to engineer biomaterial surfaces that are capable of a dynamic interaction with cells and tissues is central to the development of medical implants with improved functionality. An important consideration in this regard is the role played by the extracellular proteins that bind to an implant surface in vivo. Deliberate use of an ad-layer of such proteins on an implant surface has been observed to guide and direct cell response. However, the role that changes in surface topography might play in determining the nature of this cell–protein–surface interaction has not been investigated in detail. In this study, calcium phosphate (CaP) thin films have been deposited onto substrates with varying topography such that this is reflected in the (conformal) CaP surface features. A fibronectin (FN) ad-layer was then deposited from solution onto each surface and the response of MG63 osteoblast-like cells investigated. The results revealed that in all cases, the presence of the adsorbed FN layer on the CaP thin films improved MG63 cell adhesion, proliferation and promoted early onset differentiation. Moreover, the nature and scale of the response were shown to be influenced by the underlying CaP surface topography. Specifically, MG63 cell on FN-coated CaP thin films with regular topographical features in the nanometer range showed statistically significant differences in focal adhesion assembly, osteocalcin expression and alkaline phosphase activity compared to CaP thin films that lacked these topographical features. As such, these data indicate that surface topography can be used to further influence cell adhesion and downstream differentiation by enhancing the effects of a surface adsorbed FN layer.     

Microdeformation in Poly-L-lactide and Poly-L-lactide + Hydroxyapatite Nanocomposite Thin Films

In order to gain new insight into failure mechanisms in poly-L-lactide (PLLA) and PLLA + hydroxyapatite nanocomposites, transmission electron microscopy has been used to investigate room temperature microdeformation in electron transparent thin films of these materials subjected to various heat treatments and deformed in tension using the “copper grid” technique. In amorphous PLLA the dominant microdeformation mechanism was crazing. Localized fibrillar deformation zones (DZs) were also observed in semicrystalline films, tending to propagate in regions where the lamellar trajectories were at high angles to the tensile axis. Thus, in spherulitic films, in which the lamellae formed well-defined stacks with approximately straight trajectories at the scale of the spherulite radii, individual DZs were observed to propagate over relatively large distances, as in the amorphous films. On the other hand, films cold crystallized by heating from the glassy state showed more homogeneous lamellar textures. These were associated with a relatively high density of low aspect ratio DZs. Addition of well dispersed nanosized hydroxyapatite (nHA) to the amorphous PLLA films was also found to result in an increase in the craze density, attributed to stress concentrations associated with void formation at the PLLA-particle interfaces during deformation. However, interpretation was less straightforward in cold crystallized PLLA + nHA thin films, owing to a correlation between the lamellar texture and the nHA particles. In this case, both void formation and favorable lamellar orientations may have contributed to initiation of the DZs in the vicinity of the particles. The results are argued to be broadly consistent with previous observations of the behavior of bulk PLLA and PLLA + nHA films with a range of microstructures, in which there was evidence for an improvement in ductility in the presence of the nHA, again attributed to voiding at the PLLA-particle interfaces.     

Nanostructured Materials for Skeletal Repair

The treatment of bone and cartilage defects with bioengineered constructs of artificial scaffolds and autogenous cells became the main challenge of contemporary regenerative medicine. Early defect repair may prevent secondary injury. Recent studies could prove that bone and cartilage cells are sensitive to microscale and nanoscale patterns of surface topography and chemical structure. Nanostructured materials provide an environment for tissue regeneration mimicking the physiological range of extracellular matrix. The article reviews several studies substantiating the superiority of nanostructured materials for bone and cartilage repair along with own results on cell attachment.     

Carbon nanotubes reinforced with natural/synthetic polymers to mimic the extracellular matrices of bone – a review

The persistent failure of conventional materials used in manufacturing orthopedic implants was due to the deficiency or poor integrations of implant materials to the juxtaposed bone and stress-strain imbalances between the interfaces of tissues and implant materials. Therefore, the fabrication of a suitable bioactive scaffold for bone tissue engineering is considered a vital requisite to mimic the extracellular bone matrix. Numerous researches were reported concerning the fabrication of a suitable bioactive scaffold to improve cell adhesion, proliferation, and differentiation so far. However, for the past two decades, the research on carbon nanotubes (CNTs)-reinforced composites employed in the biomedical field is increasing day-by-day because of its outstanding properties. Moreover, it is essential to choose a biocompatible polymer with greater affinity to act as an extracellular matrix as well as to attract CNTs and in facilitating the homogeneous distribution of CNTs in aqueous and organic solvents. The development of CNTs-based composites in bone tissue engineering is presented in this review based on the last 10 years of research. The detailed information about the structural-functions and defects of bone, and the importance of CNTs-functionalized natural and synthetic polymers, and their potential activity in bone regenerations and bone replacements have been reviewed.     

Bone Dimensional Change Following Immediate Implant Placement in Posterior Teeth with CBCT: A 6-Month Prospective Clinical Study

This prospective clinical study aimed to evaluate the peri-implant hard tissue dimensional change at 6 months of immediate implant placement with bone graft materials in the posterior area using cone-beam computed tomography (CBCT). Twelve dental implants were placed concurrently following tooth extraction in the posterior area and filled with xenograft particles. The CBCT images were taken immediately after surgical procedures and then at 6 months follow-up. To evaluate the hard tissue changes, the vertical and horizontal bone thickness were analyzed and measured using ImageJ software. Paired t-test or Wilcoxon match-pair signed-rank test was done to analyze the changes of hard tissue values at the same level between immediately and 6 months following immediate implant placement. Independent t-test or Mann–Whitney U test was used to analyze the dimensional change in the vertical and horizontal direction in buccal and lingual aspects. The level of significance was set at p value = 0.05. All implants were successfully osseointegrated. At 6 months follow-up, the vertical bone change at the buccal aspect was −0.69 mm and at the lingual aspect −0.39 mm. For horizontal bone thickness, the bone dimensional changes at 0, 1, 5, and 9 mm levels from the implant platform were −0.62 mm, −0.70 mm, −0.24 mm, and −0.22 mm, respectively. A significant bone reduction was observed in all measurement levels during the 6 months after implant placement (p value < 0.05). It was noted that even with bone grafting, a decrease in bone thickness was seen following the immediate implant placement. Therefore, this technique can be an alternative method to place the implant in the posterior area.     

原位沉淀法制备CS/nHA多孔复合支架及其性能

通过原位沉淀法和冷冻相分离技术得到含有钙磷前驱体(CaP)的初始多孔支架, 利用多孔支架表面原位生成的壳聚糖(CS)膜减缓NaOH溶液中OH^-离子的渗透速率, 以达到纳米羟基磷灰石(nHA)缓慢形成的目的, 从而制得nHA 分布均匀的CS/nHA多孔复合支架. 利用扫描电镜(SEM)和万能试验机研究复合支架的结构和性能, 发现nHA为针状结构, 长度为80200 nm, 宽度为2050 nm. 随着nHA含量的增加, 复合支架的孔隙率下降, 由(93.8±3.3)%降至(87.7±3.8)%, 压缩强度则逐渐提高, 由(0.5±0.09) MPa增加至(1.5±0.06) MPa. 当复合支架中nHA质量分数为25%时, 未发现nHA团聚现象, nHA均匀地分布于CS基体中. 通过红外光谱(FTIR)、 X射线衍射(XRD)及X射线光电子能谱(XPS)等分析推断, nHA与CS之间可能存在配位和氢键作用. 细胞实验结果表明, CS/nHA多孔复合支架具有良好的生物相容性, 细胞在支架内部贴壁黏附生长. CS/nHA多孔复合支架有望在骨组织工程领域具有良好的应用前景.     

基于纳米羟基磷灰石溶胶的nHA/PA66复合粉体制备与表征

本文开发了一种新的制备纳米羟基磷灰石(nHA)/聚酰胺66(PA66)复合材料的方法。先用明胶包覆nHA棒状颗粒,再将其制备成以N,N-二甲基乙酰胺和甘油的混合液为溶剂的稳定溶胶。当nHA溶胶与PA66溶液混合时,相同的酰胺基团保证了二者之间良好的共混相容性,成功制备了nHA/PA66复合材料。X射线衍射(XRD)、傅立叶红外光谱分析(FTIR)、透射电镜(TEM)、扫描电镜-能谱分析(SEM-EDS)以及差热分析(DTA)和热重分析(TG)等表征了产物的形貌、结构及成分分布。结果表明：明胶对nHA颗粒具有显著的化学包覆作用,使nHA溶胶与PA66溶液得以均匀混合,均匀分布在PA66基体中,二者以氢键结合为一体。     

Cycloolefin-Copolymer/Polyethylene (COC/PE) Blend Assists with the Creation of New Articular Cartilage

It is shown that the initial biomechanical stability of the applied COC/PE blend in the treated tissue locality, the initial integrity of biomaterials substituting the subchondral bone by polymer implant, the initial bearing capacity and the vertical position of these biomaterials have a mayor influence on the regeneration of new articular cartilage and subchondral bone. These aspects are essential for a prosperous treatment of osteochondral defects. Results reveal that the initial biomechanical stiffness of materials (substituting the subchondral bone) has a fundamental influence on both the quality and the quantity of new articular cartilage and subchondral bone. Research is also aimed at the application of suitable biologically tolerated polymer material, its surface modifications, verifications of the vertical position of implants in relation to the articular cartilage surface and ensuring the initial biomechanical stability of the polymer implant.     

Bone‐guided regeneration: from inert biomaterials to bioactive polymer (nano)composites

Natural bone is a unique nanostructured material made of collagen fibre matrix and hydroxyapatite (HA) nanocrystals, providing mechanical support and protection from the vertebrate skeleton. However, in severe cases like bone‐deficiencies, bone needs to be “externally” repaired. Initially, different biological solutions were developed in bone‐guided regeneration. However, due to the limitations with the existing biological grafts, a lot of researches have been devoted toward biomaterials including metals, ceramics, and polymers. On the basis of the interface reactions between the implant and the surrounding tissues, these biomaterials may be classified as “nearly inert” or bioactive. Interestingly, the bioactive materials exhibit a specific biological response, leading to the formation of a natural bonding junction between the bone and the implant during bone regeneration. Recently, a special attention has been paid to a new generation of bioactive materials, i.e. (nano)structured biomaterials composed of a bioresorbable polymer matrix reinforced with bioactive inorganic compounds. While (bio)ceramic component provides the bioactivity, these materials can be readily engineered in such a way that their resorption rate in the body match the formation rate of the new tissue. This review hence reports the different biological and non‐biological solutions developed in bone‐guided regeneration, with a special emphasis on polymer‐based materials, and our recent results obtained in osseointegration The bone physiology, and its natural regeneration are also described. Copyright © 2011 John Wiley & Sons, Ltd.