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.

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.

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.