Ultrastructure of the bone-titanium interface in rabbits

The interface zone between cortical bone and threaded non-alloyed titanium implants inserted in the rabbit tibia for 12 months was examined by light and electron microscopy. The implants were removeden bloc with the perfusion-fixed surrounding bone and the undecalcified specimens were, after osmification, dehydrated and embedded in plastic resin (LR White). In ground sections (about 10 µm thick) cortical bone appeared to be in direct contact with the implant surface and the implants were thus osseointegrated. Sections for light microscopy (1 µm thick) and electron microscopy (40 nm to 0.5 µm) were prepared by using an electropolishing technique by which the bulk part of the metal was electrochemically removed and a fracture technique by which the implant was separated from the embedded tissue before sectioning. In the electropolished specimens an unmineralized zone, 2–10 µm wide, was observed at the interface. The interface zone contained osteoid-like tissue (densely packed collagen fibrils, osteocyte canaliculi) but in general no deposits of calcium mineral. This feature of the interface could not be observed in specimens prepared by the fracture technique, indicating that the electropolishing technique had induced serious artefacts, including decalcification of the interface bone. In sections prepared by the fracture technique, mineralized bone was present very close to the implant surface. No gradient of mineral was observed. A thin layer of amorphous material (100–200 nm wide) was present peripheral to the mineralized bone. An electron dense line about 100 nm wide was formed at the border between the mineralized bone and the amorphous layer. The dense layer had the same characteristics as the lamina limitans observed around osteocyte lacunae and canaliculi or the zone between areas of bone with different degree of mineralization.Our observations suggest that mineralized bone reached close to the surface of titanium implants inserted in the rabbit tibia for 12 months but that a direct contact is not established.     

Effect of microstructure on micromechanical performance of dry cortical bone tissues

The mechanical properties of bone depend on composition and structure. Previous studies have focused on macroscopic fracture behavior of bone. In the present study, we performed microindentation studies to understand the deformation properties and microcrack–microstructure interactions of dry cortical bone. Dry cortical bone tissues from lamb femurs were tested using Vickers indentation with loads of 0.245–9.8 N. We examined the effect of bone microstructure on deformation and crack propagation using scanning electron microscopy (SEM). The results showed the significant effect of cortical bone microstructure on indentation deformation and microcrack propagation. The indentation deformation of the dry cortical bone was basically plastic at any applied load with a pronounced viscoelastic recovery, in particular at lower loads. More microcracks up to a length of approximately 20 μm occurred when the applied load was increased. At loads of 4.9 N and higher, most microcracks were found to develop from the boundaries of haversian canals, osteocyte lacunae and canaliculi. Some microcracks propagated from the parallel direction of the longitudinal interstitial lamellae. At loads 0.45 N and lower, no visible microcracks were observed.     

Multifunctional Nanoengineered Hydrogels Consisting of Black Phosphorus Nanosheets Upregulate Bone Formation

Tissue‐engineered hydrogels have received extensive attention as their mechanical properties, chemical compositions, and biological signals can be dynamically modified for mimicking extracellular matrices (ECM). Herein, the synthesis of novel double network (DN) hydrogels with tunable mechanical properties using combinatorial screening methods is reported. Furthermore, nanoengineered (NE) hydrogels are constructed by addition of ultrathin 2D black phosphorus (BP) nanosheets to the DN hydrogels with multiple functions for mimicking the ECM microenvironment to induce tissue regeneration. Notably, it is found that the BP nanosheets exhibit intrinsic properties for induced CaP crystal particle formation and therefore improve the mineralization ability of NE hydrogels. Finally, in vitro and in vivo data demonstrate that the BP nanosheets, mineralized CaP crystal nanoparticles, and excellent mechanical properties provide a favorable ECM microenvironment to mediate greater osteogenic cell differentiation and bone regeneration. Consequently, the combination of bioactive chemical materials and excellent mechanical stimuli of NE hydrogels inspire novel engineering strategies for bone‐tissue regeneration.     

Parathyroid Hormone Derivative with Reduced Osteoclastic Activity Promoted Bone Regeneration via Synergistic Bone Remodeling and Angiogenesis

Osteogenesis, osteoclastogenesis, and angiogenesis are the most important processes in bone repair. Parathyroid hormone (PTH) has pro‐osteogenic, pro‐osteoclastogenic, and proangiogenic effects and may be a candidate for use in bone defect repair. However, the local application of PTH to bone defects is counterproductive due to its excessive osteoclastic and bone resorptive effects. In this study, a PTH derivative, PTHrP‐2, is developed that can be applied to local bone defects. First, a modified peptide with a calcium‐binding repeat glutamine tail undergoes controlled local release from a ceramic material and is shown to be a better fit for the repair process than the unmodified peptide. Second, the modified peptide is shown to have strong pro‐osteogenic activity due to mineralization and its facilitation of serine (Ser) phosphorylation. Third, the modified peptide is shown to maintain the pro‐osteoclastogenic and proangiogenic properties of the unmodified peptide, but its pro‐osteoclastogenic activity is reduced compared to that of the unmodified peptide. The reduced pro‐osteoclastogenic and increased pro‐osteogenic properties of the modified peptide reverse the imbalance between osteoblasts and osteoclasts with local PTH application and shift bone resorption to bone regeneration.     

Hierarchical and Heterogeneous Bioinspired Composites—Merging Molecular Self‐Assembly with Additive Manufacturing

Biological composites display exceptional mechanical properties owing to a highly organized, heterogeneous architecture spanning several length scales. It is challenging to translate this ordered and multiscale structural organization in synthetic, bulk composites. Herein, a combination of top‐down and bottom‐up approach is demonstrated, to form a polymer‐ceramic composite by macroscopically aligning the self‐assembled nanostructure of polymerizable lyotropic liquid crystals via 3D printing. The polymer matrix is then uniformly reinforced with bone‐like apatite via in situ biomimetic mineralization. The combinatorial method enables the formation of macrosized, heterogeneous composites where the nanostructure and chemical composition is locally tuned over microscopic distances. This enables precise control over the mechanics in specific directions and regions, with a unique intrinsic–extrinsic toughening mechanism. As a proof‐of‐concept, the method is used to form large‐scale composites mimicking the local nanostructure, compositional gradients and directional mechanical properties of heterogeneous tissues like the bone‐cartilage interface, for mechanically stable osteochondral plugs. This work demonstrates the possibility to create hierarchical and complex structured composites using weak starting components, thus opening new routes for efficient synthesis of high‐performance materials ranging from biomaterials to structural nanocomposites.     

Flexoelectricity in Bones

Bones generate electricity under pressure, and this electromechanical behavior is thought to be essential for bone's self‐repair and remodeling properties. The origin of this response is attributed to the piezoelectricity of collagen, which is the main structural protein of bones. In theory, however, any material can also generate voltages in response to strain gradients, thanks to the property known as flexoelectricity. In this work, the flexoelectricity of bone and pure bone mineral (hydroxyapatite) are measured and found to be of the same order of magnitude; the quantitative similarity suggests that hydroxyapatite flexoelectricity is the main source of bending‐induced polarization in cortical bone. In addition, the measured flexoelectric coefficients are used to calculate the (flexo)electric fields generated by cracks in bone mineral. The results indicate that crack‐generated flexoelectricity is theoretically large enough to induce osteocyte apoptosis and thus initiate the crack‐healing process, suggesting a central role of flexoelectricity in bone repair and remodeling.     

Viscoelastic properties of mineralized alginate hydrogel beads

Alginate hydrogels have applications in biomedicine, ranging from delivery of cells and growth factors to wound management aids. However, they are mechanically soft and have shown little potential for the use in bone tissue engineering. Here, the viscoelastic properties of alginate hydrogel beads mineralized with calcium phosphate, both by a counter-diffusion (CD) and an enzymatic approach, are characterized by a micro-manipulation technique and mathematical modeling. Fabricated hydrogel materials have low mineral content (below 3?% of the total hydrogel mass, which corresponds to mineral content of up to 60?% of the dry mass) and low dry mass content (<5?%). For all samples compression and hold (relaxation after compression) data was collected and analyzed. The apparent Young's modulus of the mineralized beads was estimated by the Hertz model (compression data) and was shown to increase up to threefold upon mineralization. The enzymatically mineralized beads showed higher apparent Young's modulus compared to the ones mineralized by CD, even though the mineral content of the former was lower. Full compression-relaxation force-time profiles were analyzed using viscoelastic model. From this analysis, infinite and instantaneous Young's moduli were determined. Similarly, enzymatic mineralized beads, showed higher instantaneous and infinite Young's modulus, even if the degree of mineralization is lower then that achieved for CD method. This leads to the conclusion that both the degree of mineralization and the spatial distribution of mineral are important for the mechanical performance of the composite beads, which is in analogy to highly structured mineralized tissues found in many organisms.     

Effects of direction and shape of osteocyte lacunae on resisting impact and micro-damage of osteon

Animal bones bear varied impact loadings during in the movements of animals. The impact resistance and micro-damage of bones are influenced by their various microstructures at different length scales. In this paper, according to the microstructure of osteon, three 2-D microstructure models (circumferential ellipse lacunae (Model A), radial elliptical lacunae (Model B) and circular lacunae (Model C) were constructed for investigating the influences of the arranged direction and shape of osteocyte lacunae on resisting impact and micro-damage. Impact analytical results show that the maximal stress of the Model A is the minimum and that of the Model B is the maximal under same boundary conditions, which indicates that the circumferentially elliptical lacunae, whose minor axis is along the radial direction of the osteon (Model A), can enhance impact resistance of osteons effectively. The investigated results of the progressive damage show that the circumferentially ellipse lacunae (Model A) are more benefit to resist micro-damage and that the micro-cracks in the model are mainly along the circumferential direction of the osteon. These investigated results for the novel microstructures found in osteon can serve engineers as guidance in the designs of biomimetic and bioinspired tubular structures or materials for engineering applications.     

不同模拟体液对硼硅酸盐生物活性玻璃基骨水泥矿化性能的影响

硼硅酸盐生物活性玻璃基(Borosilicate bioactive glass-based, BBG)骨水泥由于其优异的生物活性和生物降解性, 在治疗骨质疏松性骨折以及骨肿瘤、骨创伤、骨髓炎等疾病方面具有重要的应用前景, 受到人们的广泛关注。为进一步了解氨基酸对其植入生物体内后的矿化影响, 本研究在常规的SBF溶液中添加了不同种类及浓度的氨基酸物质, 重点研究对植入体表面形貌的影响。同时为在矿化过程中同步形成白磷钙矿(Whitlockite, WH)和羟基磷灰石(hydroxyapatite, HA), 调整了SBF溶液的温度以及酸碱度和Mg²⁺浓度, 研究了不同SBF溶液中BBG骨水泥表面形成的矿化产物。研究结果表明, 不同的氨基酸及浓度的变化对矿化产物的影响有较大差异, 天冬氨酸和赖氨酸的浓度变化影响矿物的长径比, 而甘氨酸对矿物形貌的影响较小。将硼硅酸盐生物活性玻璃压片放置在70 ℃下的高Mg²⁺浓度的酸性(pH=3.5)SBF溶液中浸泡一定时间后, 能够获得HA/WH的复相矿物。     

New evidence of surface mineralization of collagen fibrils in wild type zebrafish skeleton by AFM and TEM

Zebrafish (Danio rerio) has been generally accepted as a simple model to investigate the vertebrate tissues. In this study, comparative observations to the structural characteristics of the wild type zebrafish skeletal bone before and after decalcification were performed to gain information about surface mineralization in the collagen-mediated mineralization system using Transmission Electron Microscope (TEM) and Atomic Force Microscope (AFM). TEM observations to the decalcified samples showed empty space between the surfaces of the collagen fibrils. However in the TEM micrographs of the sections without staining and decalcification, hydroxyapatite (HA) crystals are observed to deposit on the surfaces, filling in the space and making the mineralized fibrils compactly aligned, with the diameter increasing to more than 150 nm. Consistent evidence was supplied by the AFM observations, which also indicated that the mineralized fibrils become thicker and more compactly aligned. Moreover, it was revealed that the diameters of the mineralized fibrils increase, as mineralization becomes heavier. In highly mineralized areas, they aggregate into thicker fibers. All of these data have provided the first visual evidence supporting the concept of surface mineralization on the collagen fibrils in the zebrafish skeleton system, by which the structural complexity is achieved.     

A multiscale analytical approach to evaluate osseointegration

Osseointegrated implants are frequently used in reconstructive surgery, both in the dental and orthopedic field, restoring physical function and improving the quality of life for the patients. The bone anchorage is typically evaluated at micrometer resolution, while bone tissue is a dynamic composite material composed of nanoscale collagen fibrils and apatite crystals, with defined hierarchical levels at different length scales. In order to understand the bone formation and the ultrastructure of the interfacial tissue, analytical strategies needs to be implemented enabling multiscale and multimodal analyses of the intact interface. This paper describes a sample preparation route for successive analyses allowing assessment of the different hierarchical levels of interest, going from macro to nano scale and could be implemented on single samples. Examples of resulting analyses of different techniques on one type of implant surface is given, with emphasis on correlating the length scale between the different techniques. The bone-implant interface shows an intimate contact between mineralized collagen bundles and the outermost surface of the oxide layer, while bone mineral is found in the nanoscale surface features creating a functionally graded interface. Osteocytes exhibit a direct contact with the implant surface via canaliculi that house their dendritic processes. Blood vessels are frequently found in close proximity to the implant surface either within the mineralized bone matrix or at regions of remodeling.     

In vivo skeletal response and biomechanical assessment of two novel polyalkenoate cements following femoral implantation in the female New Zealand White rabbit

Glass-ionomer cements (GIC) offer several advantages over the conventional acrylic-based bone cements. The formation of an adhesive bond with bone and metals, a low setting exotherm and no systemic or local toxicity are some of the advantages cited. This study examines the in vivo biological and biomechanical behavior of two polyalkenoate cements (LG26 and LG30) implanted for 6 wk into the submetaphyseal spongiosa of the rabbit femur. Cements were implanted as both set cement rods and unset cement dough. Implantation of set rods resulted in the formation of variably mineralized osteoid/woven bone at the bone–cement interface. Mechanical (push-out) testing revealed the strength of this bone–cement interface was of similar magnitude to control (PMMA-rod implanted) animals. The bone of LG cement-dough implanted animals exhibited demineralization of pre-existing bone local to the site of implantation, accumulation of aluminum both locally and at a distance from the site of implantation, and defective mineralization of newly formed osteoid. The histological picture following LG implantation was strikingly similar to human renal osteodystrophy, in which skeletal accumulation of aluminum is a noted feature. The development of a GIC with low/no aluminum release from the unset cement dough is a priority in the further development of these cements for possible orthopaedic applications. © 1998 Kluwer Academic Publishers     

仿生矿化法制备可降解羟基磷灰石/氧化细菌纤维素复合材料

细菌纤维素是具有天然纳米网状结构的支架材料,对其进行氧化改性后可获得可调控的降解性能。通过仿生矿化氧化改性的细菌纤维素支架,制备了可降解羟基磷灰石/氧化细菌纤维素复合骨组织工程支架材料。观察并分析了仿生矿化过程氧化细菌纤维素的降解和羟基磷灰石的形成,并通过SEM、EDS、XRD对羟基磷灰石在可降解氧化细菌纤维素支架上沉积进行了表征,矿化7天的羟基磷灰石/氧化细菌纤维素复合材料表面和内部均有磷灰石形成,测得磷灰石的钙磷比为1.75,主要为羟基磷灰石,伴有少量碳羟磷灰石。结果表明,使用仿生矿化法成功获得了一种新型可降解羟基磷灰石/氧化纤维素复合材料支架。     

The role of nanoscale architecture in supramolecular templating of biomimetic hydroxyapatite mineralization

Understanding and mimicking the hierarchical structure of mineralized tissue is a challenge in the field of biomineralization and is important for the development of scaffolds to guide bone regeneration. Bone is a remarkable tissue with an organic matrix comprised of aligned collagen bundles embedded with nanometer-sized inorganic hydroxyapatite (HAP) crystals that exhibit orientation on the macroscale. Hybrid organic-inorganic structures mimic the composition of mineralized tissue for functional bone scaffolds, but the relationship between morphology of the organic matrix and orientation of mineral is poorly understood. Herein the mineralization of supramolecular peptide amphiphile templates, that are designed to vary in nanoscale morphology by altering the amino acid sequence, is reported. It is found that 1D cylindrical nanostructures direct the growth of oriented HAP crystals, while flatter nanostructures fail to guide the orientation found in biological systems. The geometric constraints associated with the morphology of the nanostructures may effectively control HAP nucleation and growth. Additionally, the mineralization of macroscopically aligned bundles of the nanoscale assemblies to create hierarchically ordered scaffolds is explored. Again, it is found that only aligned gel templates of cylindrical nanostructures lead to hierarchical control over hydroxyapatite orientation across multiple length scales as found in bone.     

基质囊泡及其在骨矿化过程中的作用

基质囊泡(Matrix vesicles,MVs)是骨矿物初始形核生长的场所。骨矿化过程中,钙、磷酸根等离子经通道蛋白跨膜运输进入MVs内,当局部浓度达到一定值时,磷酸钙晶体开始沉积。磷酸钙的存在形态包括无定形磷酸钙、磷酸八钙及羟基磷灰石等。MVs可以调节细胞内外基质中的钙和磷酸根离子的稳态及无机磷酸盐/无机焦磷酸盐的比值,提供磷酸钙晶体成核位点,在骨矿化的初始启动过程中发挥重要作用。本文概述了MVs的生物来源、分子组分、提取方法,MVs介导的骨矿化过程,以及近年来利用囊泡作为体外矿化模型模拟MVs矿化过程的研究进展。     

Microstructural evolution in external callus of human long bone

Accurate knowledge of bone fracture healing process is of clinical and theoretical importance in bone repair and regeneration, and biomineralization. It is well known that the histological healing occurs via the formation of hematoma, fibrocartilage, bony callus and bone modeling/remodeling. However, the detailed process from fracture to healing at the microstructural level remains unclear. In the present study, an evolutionary model of external callus is proposed, in which five representative stages are presented in terms of the organization of collagen and minerals during the formation of bony callus. The first stage is the formation of loose, disordered collagen fibrils, which is followed by mineralization on some of these individual microfibrils. Then the matrix is characterized by the fusion of mineralized individual fibrils into bundles. In the third stage, the absorption of disordered matrix occurs. This is gradually replaced by ordered collagen in stage four. Finally, completely ordered mineralized tissue is formed. The proper sequence of the process plays an important role in deciding the success of healing.In addition to the common mineral phase of hydroxyapatite (HA), dicalcium phosphate dihydrate (DCPD) phase was also found in early stage of healing, especially in rapid healing (children's callus). It vanished in the following process of healing. The deposition of DCPD is supposed to be brought about by some non-collagenous protein.     

Synthesis and characterization of mechanically strong carboxymethyl cellulose–gelatin–hydroxyapatite nanocomposite for load-bearing orthopedic application

Novel three-dimensional hybrid polymer–hydroxyapatite nanocomposites have been developed as load-bearing synthetic bone graft through in situ mineralization process, using natural polymers carboxymethyl cellulose (CMC) and gelatin (Gel) as matrix. This process is simple and does not involve any chemical cross-linker. Detailed structural and physicochemical characterization of the samples disclosed that incorporation of gelatin with CMC assists the formation of CMC-Gel polymeric network of new conformational structure through non-covalent interactions (H-bond). The formation of hydroxyapatite (HA) in this polymeric network was occurred in such a fashion that the HA serves as bridging molecule which strengthen the polymeric network more and formed a mechanically strong three-dimensional CMC-Gel-HA nanocomposite. The synthesized CMC-Gel-HA nanocomposites have compressive strength and modulus in the range of 40–86 MPa and 0.4–1.2 GPa, respectively, analogous to human cancellous as well as cortical bone. In vitro cell interaction of the synthesized nanocomposites with osteoblast-like MG-63 cells has been evaluated. Results showed that synthesized CMC-Gel-HA nanocomposite promote cells for high alkaline phosphatase activity and extracellular mineralization. Extracellular mineralization ability of nanocomposite was investigated by alizarin red staining and von Kossa staining. Biodegradable nature and bone apatite formation ability of CMC-Gel-HA nanocomposite under simulated physiological environment were investigated by different characterization processes. Results indicated that the synthesized CMC-Gel-HA nanocomposite has great potential to be used as regenerative bone graft in major load-bearing region.     

Microstructural characteristics and nanomechanical properties across the thickness of the wild-type zebrafish skeletal bone

In this study, both atomic force microscopy (AFM) and transmission electronic microscopy (TEM) were used to explore the microstructural characteristics across the thickness of the wild-type zebrafish skeletal bone wall. Evident variations of the microstructure were observed from the inner to outer side of the bone by AFM, indicating that the mineralized fibrils become thinner and more confusing across the thickness. Correspondingly, similar results could be concluded from the TEM investigations. Both of the AFM and TEM results imply that the degree of mineralization from near the center of the skeletal bone were greater than those from the outer layers, which was supported by the TEM electron diffraction results. Nanoindentation tests displayed that there is a decline in both nanohardness and elastic modulus from the center of the skeletal bone outward. These alterations of mechanical properties across the thickness can be visually reflected by the AFM topographies of the residual indent impressions. Also, the characteristics of biomineralization and microstructures of zebrafish skeletal bone are similar with those of human Haversian bone. Zebrafish skeletal bone could be viewed as a simple model to study mineralization characteristics of the human Haversian system and human bone diseases.     

Direct communication between osteocytes and acid-etched titanium implants with a sub-micron topography

The osteocyte network, through the numerous dendritic processes of osteocytes, is responsible for sensing mechanical loading and orchestrates adaptive bone remodelling by communicating with both the osteoclasts and the osteoblasts. The osteocyte network in the vicinity of implant surfaces provides insight into the bone healing process around metallic implants. Here, we investigate whether osteocytes are able to make an intimate contact with topologically modified, but micrometre smooth (S _a?<?0.5?µm) implant surfaces, and if sub-micron topography alters the composition of the interfacial tissue. Screw shaped, commercially pure (cp-Ti) titanium implants with (i) machined (S _a?=?~0.2?µm), and (ii) two-step acid-etched (HF/HNO₃ and H₂SO₄/HCl; S _a?=?~0.5?µm) surfaces were inserted in Sprague Dawley rat tibia and followed for 28?days. Both surfaces showed similar bone area, while the bone-implant contact was 73?% higher for the acid-etched surface. By resin cast etching, osteocytes were observed to maintain a direct intimate contact with the acid-etched surface. Although well mineralised, the interfacial tissue showed lower Ca/P and apatite-to-collagen ratios at the acid-etched surface, while mineral crystallinity and the carbonate-to-phosphate ratios were comparable for both implant surfaces. The interfacial tissue composition may therefore vary with changes in implant surface topography, independently of the amount of bone formed. Implant surfaces that influence bone to have higher amounts of organic matrix without affecting the crystallinity or the carbonate content of the mineral phase presumably result in a more resilient interfacial tissue, better able to resist crack development during functional loading than densely mineralised bone.     

Strain amplification in bone mechanobiology: a computational investigation of the in vivo mechanics of osteocytes

The osteocyte is believed to act as the main sensor of mechanical stimulus in bone, controlling signalling for bone growth and resorption in response to changes in the mechanical demands placed on our bones throughout life. However, the precise mechanical stimuli that bone cells experience in vivo are not yet fully understood. The objective of this study is to use computational methods to predict the loading conditions experienced by osteocytes during normal physiological activities. Confocal imaging of the lacunar–canalicular network was used to develop three-dimensional finite element models of osteocytes, including their cell body, and the surrounding pericellular matrix (PCM) and extracellular matrix (ECM). We investigated the role of the PCM and ECM projections for amplifying mechanical stimulation to the cells. At loading levels, representing vigorous physiological activity (3000 µɛ), our results provide direct evidence that (i) confocal image-derived models predict 350–400% greater strain amplification experienced by osteocytes compared with an idealized cell, (ii) the PCM increases the cell volume stimulated more than 3500 µɛ by 4–10% and (iii) ECM projections amplify strain to the cell by approximately 50–420%. These are the first confocal image-derived computational models to predict osteocyte strain in vivo and provide an insight into the mechanobiology of the osteocyte.