Nacre-mimetic cerium-doped nano-hydroxyapatite/chitosan layered composite scaffolds regulate bone regeneration via OPG/RANKL signaling pathway.

Autogenous bone grafting has long been considered the gold standard for treating critical bone defects. However, its use is plagued by numerous drawbacks, such as limited supply, donor site morbidity, and restricted use for giant-sized defects. For this reason, there is an increasing need for effective bone substitutes to treat these defects. Mollusk nacre is a natural structure with outstanding mechanical property due to its notable "brick-and-mortar" architecture. Inspired by the nacre architecture, our team designed and fabricated a nacre-mimetic cerium-doped layered nano-hydroxyapatite/chitosan layered composite scaffold (CeHA/CS). Hydroxyapatite can provide a certain strength to the material like a brick. And as a polymer material, chitosan can slow down the force when the material is impacted, like an adhesive. As seen in natural nacre, the combination of these inorganic and organic components results in remarkable tensile strength and fracture toughness. Cerium ions have been demonstrated exceptional anti-osteoclastogenesis capabilities. Our scaffold featured a distinct layered HA/CS composite structure with intervals ranging from 50 to 200 µm, which provided a conducive environment for human bone marrow mesenchymal stem cell (hBMSC) adhesion and proliferation, allowing for in situ growth of newly formed bone tissue. In vitro, Western-blot and qPCR analyses showed that the CeHA/CS layered composite scaffolds significantly promoted the osteogenic process by upregulating the expressions of osteogenic-related genes such as RUNX2, OCN, and COL1, while inhibiting osteoclast differentiation, as indicated by reduced TRAP-positive osteoclasts and decreased bone resorption. In vivo, calvarial defects in rats demonstrated that the layered CeHA/CS scaffolds significantly accelerated bone regeneration at the defect site, and immunofluorescence indicated a lowered RANKL/OPG ratio. Overall, our results demonstrate that CeHA/CS scaffolds offer a promising platform for bone regeneration in critical defect management, as they promote osteogenesis and inhibit osteoclast activation.

The Role of Marine Organic Extract in Bone Regeneration: A Pilot Study.

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.

Bioactive Compounds from Marine Organisms: Potential for Bone Growth and Healing.

Marine organisms represent a highly diverse reserve of bioactives which could aid in the treatment of a wide range of diseases, including various musculoskeletal conditions. Osteoporosis in particular would benefit from a novel and effective marine-based treatment, due to its large disease burden and the inefficiencies of current treatment options. Osteogenic bioactives have been isolated from many marine organisms, including nacre powder derived from molluscan shells and fucoidan-the sulphated polysaccharide commonly sourced from brown macroalgae. Such extracts and compounds are known to have a range of osteogenic effects, including stimulation of osteoblast activity and mineralisation, as well as suppression of osteoclast resorption. This review describes currently known soluble osteogenic extracts and compounds from marine invertebrates and algae, and assesses their preclinical potential.

Water­soluble nano­pearl powder promotes MC3T3­E1 cell differentiation by enhancing autophagy via the MEK/ERK signaling pathway.

Nacre (mother of pearl) is a bioactive material capable of facilitating osteoblast proliferation and differentiation; however, further investigation into the mechanism underlying the effects of nacre on the stimulation of bone differentiation is required. The present study aimed to elucidate the effects of water­soluble nano­pearl powder (WSNNP) on osteoblast differentiation and to examine the underlying mechanisms. A MTT assay revealed that WSNNP (10, 25 and 50 µg/ml) may stimulate the viability of preosteoblastic MC3T3­E1 cells and 50 µg/ml WSNNP exhibited the maximum stimulatory effect. Furthermore, WSNNP significantly enhanced the protein expression levels of differentiation markers, including collagen I, runt­related transcription factor 2 (RUNX2), secreted phosphoprotein1 (SPP1) and alkaline phosphatase (ALP) in a dose­dependent manner, which indicated that WSNNP may promote osteoblast differentiation. Subsequently, whether autophagy serves a role in WSNNP­mediated differentiation of osteoblasts was investigated via western blotting and immunofluorescence. The results of the present study demonstrated that WSNNP treatment significantly evoked the expression of autophagy markers, including microtubule­associated light chain 3 (LC3)II/I, Beclin1 and autophagy­related 7 (ATG7), whereas the autophagy inhibitor 3­methyladenine significantly inhibited WSNNP­induced osteoblast differentiation. Furthermore, the role of WSNNP on the potential signaling pathways that activate autophagy was investigated. The present study reported that WSNNP may significantly upregulate the mitogen­activated protein kinase kinase (MEK)/extracellular signal­regulated kinase (ERK) signaling pathway. Treatment with the MEK inhibitor U0126 significantly inhibited the protein expression levels of WSNNP­induced differentiation markers, including collagen I, RUNX2, SPP1 and ALP, and autophagy markers, including LC3II/I, Beclin1 and ATG7. Therefore, the findings of the present study suggested that WSNNP may contribute to osteoblast differentiation by enhancing autophagy via the MEK/ERK signaling pathway, thus suggesting a novel direction for optimizing the biological materials in bone implants.

Design strategies and applications of nacre-based biomaterials.

The field of tissue engineering and regenerative medicine relies heavily on materials capable of implantation without significant foreign body reactions and with the ability to promote tissue differentiation and regeneration. The field of bone tissue engineering in particular requires materials capable of providing enhanced mechanical properties and promoting osteogenic cell lineage commitment. While bone repair has long relied almost exclusively on inorganic, calcium phosphate ceramics such as hydroxyapatite and their composites or on non-degradable metals, the organically derived shell and pearl nacre generated by mollusks has emerged as a promising alternative. Nacre is a naturally occurring composite material composed of inorganic, calcium carbonate plates connected by a framework of organic molecules. Similar to mammalian bone, the highly organized microstructure of nacre endows the composite with superior mechanical properties while the organic phase contributes to significant bioactivity. Studies, both in vitro and in vivo, have demonstrated nacre's biocompatibility, biodegradability, and osteogenic potential, which are superior to pure inorganic minerals such as hydroxyapatite or non-degradable metals. Nacre can be used directly as a bulk implant or as part of a composite material when combined with polymers or other ceramics. While nacre has demonstrated its effectiveness in multiple cell culture and animal models, it remains a relatively underexplored biomaterial. This review introduces the formation, structure, and characteristics of nacre, and discusses the present and future uses of this biologically-derived material as a novel biomaterial for orthopedic and other tissue engineering applications. STATEMENT OF SIGNIFICANCE: Mussel derived nacre, a biological composite composed of mineralized calcium carbonate platelets and interplatelet protein components, has recently gained interest as a potential alternative ceramic material in orthopedic biomaterials, combining the integration and mechanical capabilities of calcium phosphates with increased bioactivity derived from proteins and biomolecules; however, there is limited awareness of this material's potential. Herein, we present, to our knowledge, the first comprehensive review of nacre as a biomaterial. Nacre is a highly promising yet overlooked biomaterial for orthopedic tissue engineering with great potential in a wide variety of material systems. It is our hope that publication of this article will lead to increased community awareness of the potential of nacre as a versatile, bioactive ceramic capable of improving bone tissue regeneration and will elicit increased research effort and innovation utilizing nacre.

A new method for the separation and purification of the osteogenic compounds of nacre Ethanol Soluble Matrix.

Nacre is able to induce bone-forming cells mineralization, and gains widely interest in bone regeneration. While, the osteoinductive compounds are not yet identified. ESM (Ethanol Soluble Matrix), a nacre extract from powder of Pinctada margaritifera pearl oyster shell, has been firstly proven having the capacity to induce mineralization and to restore mineralization defect in vitro. It is suitable to treat ESM as a source of osteoinductive compounds. Herein, we develop a new method for separating and purifying nacre extracts by an ionic approach. At first, cationic ESM (ESMc) and anionic ESM (ESMa) were achieved with ion-exchange resin. Then, ESM was separated and collected on cation exchange HPLC. Scanning Electron Microscopy coupled with Energy Dispersive X-ray Spectrometry (EDS) was used to reveal the concentrated elements in ESM fractions. A coupled cell models were used to test the ESM fractions. Alizarin Red staining was performed and quantified to evaluate the mineralization level. ESMc and 2 HPLC fractions stimulated the mineralization in both cells. EDS demonstrated the abundant presence of calcium and chloride in the osteogenic fractions. To validate, pure CaCl2 was tested and proven having an osteogenic effect in both cells, but less stable than ESM. The mineralization nodules induced by ESM fractions and CaCl2 differed in both cells. In conclusion, a new method was developed for separating and purifying nacre extracts by an ionic approach. By which, the osteoinductive compounds in ESM were proven cationic, and calcium in ESM was demonstrated to play a role in inducing the cell mineralization.

Anticonvulsant and sedative-hypnotic activity screening of pearl and nacre (mother of pearl).

ETHNOPHARMACOLOGICAL RELEVANCE: Pearl and nacre are valuable traditional medicines to treat palpitations, convulsions or epilepsy in China for thousands of years. However, the active ingredients are not clear till now. AIM OF THE STUDY: The main purpose of the current investigation was to assess the anticonvulsant and sedative-hypnotic activity of pearl powder and nacre powder, including their corresponding 6 protein extracts. MATERIAL AND METHODS: Determination of the amino acid composition of the obtained protein was carried out by ultra-performance liquid chromatography (UPLC) combined with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) pre-column derivatisation. The influence of the tested drugs on locomotor activity and convulsions latency was recorded. The contents of 5-Hydroxytryptamine (5-HT) and γ-aminobutyric acid (GABA) in brain were detected by enzyme-linked immunesorbent assay (ELISA) kits. In addition, immunohistochemistry was carried out to evaluate the changes of 5-HT3 and GABAB. In parallel, the expressions of them were demonstrated by western blot. RESULTS: The obtained data suggested that pearl original powder (1.1g/kg), pearl water-soluble protein (0.2g/kg), pearl acid-soluble protein (0.275g/kg), pearl conchiolin protein (1.1g/kg), nacre original powder (1.1g/kg), nacre water-soluble protein (0.2g/kg), nacre acid-soluble protein (0.7g/kg) and nacre conchiolin protein (1.1g/kg) could down-regulate the expression of 5-HT3 and up-regulate the level of GABAB to varying degrees compared with the control group. Besides, drug administration also reduced the locomotor activity and increased convulsions latency with a certain mortality. CONCLUSIONS: These findings correlated with the traditional use of pearl and nacre as sedation and tranquilization agents, thus making them interesting sources for further drug development and also providing critical important evidence for the selection of quality control markers.

Nacre extract restores the mineralization capacity of subchondral osteoarthritis osteoblasts.

Osteoarthritis (OA) is the most common cause of joint chronic pain and involves the entire joints. Subchondral osteoarthritic osteoblasts present a mineralization defect and, to date, only a few molecules (Vitamin D3 and Bone Morphogenetic Protein2) could improve the mineralization potential of this cell type. In this context, we have tested for the first time the effect of nacre extract on the mineralization capacity of osteoblasts from OA patients. Nacre extract is known to contain osteogenic molecules which have demonstrated their activities notably on the MC3T3 pre-osteoblastic cell line. For this goal, molecules were extracted from nacre (ESM, Ethanol Soluble Matrix) and tested on osteoblasts of the subchondral bone from OA patients undergoing total knee replacement and on MC3T3 cells for comparison. We chose to investigate the mineralization with Alizarin Red staining and with the study of extracellular matrix (ECM) structure and composition. In a complementary way the structure of the ECM secreted during the mineralization phase was investigated using second harmonic generation (SHG). Nacre extract was able to induce the early presence (after 7 days) of precipitated calcium in cells. Raman spectroscopy and electron microscopy showed the presence of nanograins of an early crystalline form of calcium phosphate in OA osteoblasts ECM and hydroxyapatite in MC3T3 ECM. SHG collagen fibers signal was present in both cell types but lower for OA osteoblasts. In conclusion, nacre extract was able to rapidly restore the mineralization capacity of osteoarthritis osteoblasts, therefore confirming the potential of nacre as a source of osteogenic compounds.

Osteogenic potency of nacre on human mesenchymal stem cells.

Nacre seashell is a natural osteoinductive biomaterial with strong effects on osteoprogenitors, osteoblasts, and osteoclasts during bone tissue formation and morphogenesis. Although nacre has shown, in one study, to induce bridging of new bone across large non-union bone defects in 8 individual human patients, there have been no succeeding human surgical studies to confirm this outstanding potency. But the molecular mechanisms associated with nacre osteoinduction and the influence on bone marrow-derived mesenchymal stem cells (BMSC's), skeletal stem cells or bone marrow stromal cells remain elusive. In this study we highlight the phenotypic and biochemical effects of Pinctada maxima nacre chips and the global nacre soluble protein matrix (SPM) on primary human bone marrow-derived stromal cells (hBMSCs) in vitro. In static co-culture with nacre chips, the hBMSCs secreted Alkaline phosphatase (ALP) at levels that exceeded bone morphogenetic protein (rhBMP-2) treatment. Concentrated preparation of SPM applied to Stro-1 selected hBMSC's led to rapid ALP secretions, at concentrations exceeding the untreated controls even in osteogenic conditions. Within 21 days the same population of Stro-1 selected hBMSCs proliferated and secreted collagens I-IV, indicating the premature onset of an osteoblast phenotype. The same SPM was found to promote unselected hBMSC differentiation with osteocalcin detected at 7 days, and proliferation increased at 7 days in a dose-dependent manner. In conclusion, nacre particles and nacre SPM induced the early stages of human bone cell differentiation, indicating that they may be promising soluble factors with osteoinductive capacity in primary human bone cell progenitors such as, hBMSC's.

Osteogenic differentiation of human bone marrow mesenchymal stem cells in hydrogel containing nacre powder.

Nacre (or mother of pearl) can facilitate bone cell differentiation and can speed up their mineralization. Here we report on the capability of nacre to induce differentiation of human bone marrow mesenchymal stem cells (hBM-MSCs) and the production of extracellular matrix. hBM-MSCs were encapsulated in an alginate hydrogel containing different concentrations of powdered nacre and cultured in the same environment until Day 28. Analysis of osteogenic gene expression, histochemistry, second harmonic generation (SHG) microscopy, and Raman scattering spectroscopy were used to characterize the synthesis of the extracellular matrix. In the presence of nacre powder, a significant increase in matrix synthesis from D21 in comparison with pure alginate was observed. Histochemistry revealed the formation of a new tissue composed of collagen fibers in the presence of nacre (immunostaining and SHG), and hydroxyapatite crystals (Raman) in the alginate beads. These results suggest that nacre is efficient in hBM-MSCs differentiation, extracellular matrix production and mineralization in alginate 3D biomaterials.

The role of nacreous factors in preventing osteoporotic bone loss through both osteoblast activation and osteoclast inactivation.

Excessive bone resorption by osteoclasts relative to bone formation by osteoblasts results in the development of osteoporosis. Anti-osteoporotic agents that are able both to inhibit bone resorption and to stimulate bone formation are not available. We now show that water-soluble nacreous factors prepared from the pearl oyster Pteria martensii prevent osteoporotic bone loss associated with estrogen deficiency in mice mainly through osteoclast inactivation. Nacreous factors stimulated osteoblast biomineralization in vitro in association with activation of signaling by c-Jun NH(2)-terminal kinase (JNK) and Fos-related antigen-1 (Fra-1). They also suppressed both osteoclast formation by blocking up-regulation of nuclear factor of activated T cells cytoplasmic 1 (NFATc1) as well as bone pit formation mediated by mature osteoclasts, likely by disrupting the actin ring of these cells. Our findings thus show that the components of a natural material have beneficial effects on bone remodeling that are mediated through regulation of both osteoblast and osteoclast function. They may thus provide a basis for the development of biomimetic bone material as well as anti-osteoporotic agents.

Repair of critical size bone defects with porous poly(D,L-lactide)/nacre nanocomposite hollow scaffold.

OBJECTIVE: To generate a novel porous poly(D,L-lactide)/nacre nanocomposite hollow scaffold. METHODS: This study was performed in the Department of Spine Surgery, Southern Medical University, Guangzhou, China from September 2010 to September 2011. Nacre nanoparticles were prepared using a physical process and identified by x-ray diffraction and transmission electron microscopy, to generate a novel scaffold though the salt leaching processing technique. The morphology and structure properties of this scaffold were further investigated under scanning electron microscope and mechanical property testing. Additionally, the biological characteristics were evaluated by cell culture experiments in vitro. Thirty-six rabbits were randomly divided into 3 groups. The defects were implanted with/without poly(D,L-lactide)/nacre scaffold or poly(D,L-lactide) scaffold. The results were assessed by radiographs and bone mineral density to monitor bone repairing. RESULTS: The nacre nanoparticles were spherical in shape, with a diameter range from 45-95 nm. The scaffolds possessed an interconnected porous structure with an average pore size of 322.5+/-50.8 µm, and exhibited a high porosity (82.5 +/-0.8%), as well as good compressive strength of 4.5+/-0.25 Mpa. Primary biocompatibility experiments in vitro showed that cells adhered and proliferated well on the scaffolds. The animal study further demonstrated that the scaffolds could repair the critical size segmental bone defects in 12 weeks. CONCLUSION: Newly established scaffolds may serve as a promising biomaterial for bone tissue engineering.

Nacre-driven water-soluble factors promote wound healing of the deep burn porcine skin by recovering angiogenesis and fibroblast function.

To assess the recovery effect of water-soluble components of nacre on wound healing of burns, water-soluble nacre (WSN) was obtained from powdered nacre. Alterations to WSN-mediated wound healing characteristics were examined in porcine skin with deep second-degree burns; porcine skin was used as a proxy for human. When WSN was applied to a burned area, the burn-induced granulation sites were rapidly filled with collagen, and the damaged dermis and epidermis were restored to the appearance of normal skin. WSN enhanced wound healing recovery properties for burn-induced apoptotic and necrotic cellular damage and spurred angiogenesis. Additionally, WSN-treated murine fibroblast NIH3T3 cells showed increased proliferation and collagen synthesis. Collectively, the findings indicate that WSN improves the process of wound healing in burns by expeditiously restoring angiogenesis and fibroblast activity. WSN may be useful as a therapeutic agent, with superior biocompatibility to powdered nacre, and evoking less discomfort when applied to a wounded area.