OBJECTIVE: Nano-hydroxyapatite (nHA)/ gel porous scaffolds loaded with WSM carriers are promising bone replacement materials that can improve osseointegration ability. This investigation aimed to evaluate the osteoinductive activity by implanting the composition of nano-hydroxyapatite (nHA)/ Gel porous scaffolds as a carrier of WSM via an animal model.
METHODS: WSM was extracted and nHA was added to the matrix to construct porous composite scaffolds. The dose-effect curve of WSM concentration and alkaline phosphatase (ALP) activity was made by culturing rat osteoblasts and examining the absorbance. Three different materials were implanted into critical size defects (CSD) in the skulls of rats, which were further divided into four groups: WSM nHA /Gel group, n-WSM nHA /Gel group, HA powder group, and control group.
RESULTS: WSM (150 μg/mL-250μg/mL) effectively improved the activity of ALP in rat osteoblasts. All rats in each group had normal healing. WSM-loaded nHA /Gel group showed better performance on newly-formed bone tissue of rat skull and back at 4th week and 8th week, respectively. At the 4th week, the network of woven bone formed in the WSM-loaded nHA/Gel scaffold material. At 8th week, the reticular trabecular bone in the WSM-loaded scaffold material became dense lamellar bone, and the defect was mature lamellar bone. In the subcutaneous implantation experiment, WSM-loaded nHA/Gel scaffold material showed a better performance of heterotopic ossification than the pure nHA/Gel scaffold material.
CONCLUSIONS: WSM promotes osteoblast differentiation and bone mineralization. The results confirm that the nHA/ Gel Porous Scaffold with Nacre Water-Soluble Matrix has a significant bone promoting effect and can be used as a choice for tissue engineering to repair bone defects.

Organisms hold an extraordinarily evolutionary advantage in forming complex, hierarchical structures across different length scales that exhibit superior mechanical properties. Mimicking these structures for synthesizing high-performance materials has long held a fascination and has seen rapid growth in the recent past thanks to high-resolution microscopy, design, synthesis, and testing methodologies. Among the class of natural materials, nacre, found in mollusk shells, exhibits remarkably high mechanical strength and toughness. The highly organized \"brick and mortar\" structure at different length scales is a basis for excellent mechanical properties and the capability to dissipate energy and propagation in nacre. Here, we employ large-scale atomistic coarse-grained molecular dynamics simulations to study the mechanical and viscoelastic behavior of nacre-like microstructures. Uniaxial tension and oscillatory shear simulations were performed to gain insight into the role of complex structure-property relationships. Specifically, the role played by the effect of microstructure (arrangement of the crystalline domain) and polymer-crystal interactions on the mechanical and viscoelastic behavior is elucidated. The tensile property of the nanocomposite was seen to be sensitive to the microstructure, with a staggered arrangement of the crystalline tablets giving rise to a 20-30% higher modulus and lower tensile strength compared to a columnar arrangement. Importantly, the staggered microstructure is shown to have a highly tunable mechanical behavior with respect to the polymer-crystal interactions. The underlying reasons for the mechanical behavior are explained by showing the effect of polymer chain mobility and orientation and the load-carrying capacity for the constituents. Viscoelastic responses in terms of the storage and loss moduli and loss tangent are studied over three decades in frequency and again highlight the differences brought about by the microstructure. We show that our coarse-grained models offer promising insights into the design of novel biomimetic structures for structural applications.

The lesson learnt from nature is presented through the examination of shells from four mollusc species (two gastropods and two bivalves). Individual aragonite tablets together with the organic network have been studied in terms of tablet dimensions, tablet stacking sequence, texture and the weight fraction of the organic present in these bio-composite shells. Mechanical properties viz. elastic modulus and hardness at the macro (bulk), micro (tablet) and nano (aragonite nanograins) levels have been determined using the experimental technique of nanoindentation together with the theoretical formulations offered by the Mori-Tanaka method and other such mechanical models for composites. XRD studies confirm the strong c-axis crystallographic orientation - aligned parallel to the direction of growth and crystallization of nacreous tablets, and fractography performed on the shell specimens suggests a difference in the stacking of individual aragonite layers for the gastropod and bivalve molluscan class, respectively. DSC and TG-DTA data helped in characterizing the organic material present in the shell specimens and in calculating the organic weight and volume fractions across species. Further to this, the DSC data revealed that the nature of the organic in the brick and mortar assembly of nacre, was similar in nature across the two molluscan classes reported in this study. EBSD studies performed on our system revealed a difference in crystallographic texture for the two different tablet stacking (columnar and sheet nacre). It has been shown experimentally that the shells of gastropods (columnar nacre) are orthotropic in nature, while the bivalve mollusc shells (sheet nacre) are anisotropic in nature.

Novel biomaterials capable of accelerating the healing process of skeletal tissues are urgently needed in dentistry. The present in vivo study assessed the osteoconductive and osteoinductive properties of experimental biphasic bioceramics (HA-TCP) modified or not by a nacre extract (marine organic extract, MOE) in a sheep model. Fabrication of MOE involved mixing ground nacre (0.05 g, particle sizes < 0.1 mm) with glacial ethanoic acid (5 mL, pH 7) for 72 hours using external magnetic stirring (25°C). Nonreactive carriers (sterile polythene tubes; 3/animal, radius: 2.5 mm, length: 10.0 mm) pertaining to the control (empty) or experimental groups (HA-TCP or MOE-modified HA-TCP) were implanted intramuscularly into the abdominal segment of the torso in sheep (n = 8, age: 2 years, weight: 45 kg). Euthanization of animals was performed at 3 and 6 months after surgery. Tissues harvested were subjected to macroscopic and radiographic assessments. Specimens were then stained for histological analysis. Both control and experimental animals were capable of inducing the neoformation of fibrous connective tissue at both time points where superior amounts of tissue formation and mineralization were detected for experimental groups (unaltered (at 3 and 6 mos) and MOE-modified HA-TCP (at 3 mos)). Histological results, however, revealed that mature bone formation was only observed for specimens fabricated with MOE-modified HA-TCP in a time-dependent manner. The present study has successfully demonstrated the in vivo utility of experimental biphasic bioceramics modified by MOE in an ectopic grafting sheep model. Promising osteoconductive and osteoinductive properties must be further developed and confirmed by subsequent research.

As a natural composite, nacre has an elegant staggered \'brick-and-mortar\' microstructure consisting of mineral platelets glued by organic macromolecules, which endows the material with superior mechanical properties to achieve its biological functions. In this paper, a microstructure-based crack-bridging model is employed to investigate how the strength of nacre is affected by pre-existing structural defects. Our analysis demonstrates that owing to its special microstructure and the toughening effect of platelets, nacre has a superior flaw-tolerance feature. The maximal crack size that does not evidently reduce the tensile strength of nacre is up to tens of micrometres, about three orders higher than that of pure aragonite. Through dimensional analysis, a non-dimensional parameter is proposed to quantify the flaw-tolerance ability of nacreous materials in a wide range of structural parameters. This study provides us some inspirations for optimal design of advanced biomimetic composites.

Nacre tablets from the shell of Pinctada maxima were studied with SEM, TEM and STEM. The systematic nanolath morphology on the (001) surface of nacre tablets was observed after acidic etching and mechanical polishing. The nanolaths were along the [100] crystallographic orientation of aragonite crystal. The (010) and (100) cross section surfaces of the nacre tablets showed nanolath and nanograin morphologies, respectively, which was consistent with [100] crystallographic orientation of nanolath on the (001) surface. Sheet-like defects with low mass density were observed on the (001) plane inside nacre tablets and were considered to be the cause of nanolath morphology revealed on the surfaces by acidic etching and mechanical polishing. On the other hand, large block [110] twins that divide the nacre tablets into two sectors were identified. The implication of these twins on the understanding to the crystallization mechanism of nacre tablets was discussed.

Aragonite pearl, vaterite pearl and shell nacre of the freshwater mollusc Hyriopsis cumingii (Zhejiang province, China) were chosen to analyze microstructure and organic composition in the different habits of calcium carbonate. SEM and TEM were used to reveal the microstructure and mineralogical phase. We found that tablets in vaterite exhibited more irregular texture and were packaged with more organic matrices than in aragonite forms. Then a peculiar method was introduced to extract water soluble matrix (WSM), acid soluble matrix (ASM) and acid insoluble matrix (AIM) from the three samples, and biochemical analysis of these organic matrixes involved in crystal formation and polymorph selection was carried out. High performance liquid chromatography (HPLC) confirms the hydrophobic pattern of the organic matrix intermingled with mineral, the opposite of the early mobilizable water soluble fraction. Amino acid composition confirms hydrophobic residues as major components of all the extracts, but it reveals an imbalance in acidic residues rates in WSM vs. ASM and in aragonite vs. vaterite. Electrophoresis gives evidence for signatures in proteins with a 140 kDa material specific for aragonite in WSM. Conversely all ASM extracts reveal the presence of about 55 kDa components, including a discrete band in vaterite extract.

The present study was designed to analyze the intra-articular behaviour of nacre, when implanted in the subchondral bone area in the sheep knee. We implanted nacre blocks in sheep\'s trochlea by replacing the half of the femoral trochlea (nacre group). For comparison we used complete cartilage resection (resection group) down to the subchondral bone. In the \"nacre group\", implants were well tolerated without any synovial inflammation. In addition, we observed centripetal regrowth of new cartilage after 3 months. In the \"resection group\", no chondral regrowth was observed, but, in contrast, a thin layer of fibrous tissue was formed. After 6 months, a new tissue covered the nacre implant formed by an osteochondral regrowth. Nacre, as a subchondral implant, exerts benefic potential for osteochondral repair.

Mimicry of the tough natural composite nacre in future bioengineering requires knowledge of the biomineralisation process. The insoluble organic matrix isolated from the shell of the gastropod Haliotis laevigata was characterised by protein chemistry, topographical and mechanical measurements. Demineralisation of nacre in dilute acetic acid or ethylenediaminetetraacetic acid revealed a set of soluble proteins and the insoluble matrix. The insoluble matrix contains a chitin core and firmly attached proteins, which could be removed by sodium dodecyl sulfate and glycerol indicating a hydrophobic interaction. Atomic force microscopy images of the native insoluble matrix showed a filamentous network with pores or holes, where the filaments showed globular attachments of different sizes, possibly the attached protein molecules. During direct observation of protein degradation imaged by atomic force microscopy the insoluble matrix gets smooth and flat indicating the removal of the attached proteins by proteases. We propose a model of protein coated chitin filaments for the insoluble matrix of nacre. Mechanical measurements by force mapping revealed a Young\'s modulus depending on the hydration state of the organic layers. The fully hydrated organic matrix has an elastic modulus below 1 MPa comparable to some hydrogels.