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Cadherins are cell-surface proteins that mediate cell-cell adhesion. By regulating their grip formation and strength, cadherins play a pivotal role during normal tissue morphogenesis and homeostasis of multicellular organisms. However, their dysfunction is associated with cell migration and proliferation, cancer progression and metastasis. The conserved amino acid sequence His-Ala-Val (HAV) in the extracellular domain of cadherins is implicated in cadherin-mediated adhesion and migration. Antagonists of cadherin adhesion such as monoclonal antibodies and small molecule inhibitors based on HAV peptides, are of high therapeutic value in cancer treatment. However, antibodies are not stable outside their natural environment and are expensive to produce, while peptides have certain limitations as a drug as they are prone to proteolysis. Herein, we propose as alternative, a synthetic antibody based on molecularly imprinted polymer nanogels (MIP-NGs) to target the HAV domain. The MIP-NGs are biocompatible, have high affinity for N-cadherin and inhibit cell adhesion and migration of human cervical adenocarcinoma (HeLa) cells, as demonstrated by cell aggregation and Matrigel invasion assays, respectively. The emergence of MIPs as therapeutics for fighting cancer is still in its infancy and this novel demonstration reinforces the fact that they have a rightful place in cancer treatment.

The hydrozoan Millepora dichotoma (MD) is a typical Red Sea species containing a porous skeleton in the form of aragonite crystalline calcium carbonate. Due to environmental considerations, the artificial production of coralline species under controlled conditions is pertinent and underway. Artificially grown MD was used as a raw material for the production of calcium phosphate, mainly hydroxyapatite bioceramics, to be used in the drug delivery systems as a drug carrier or in the tissue engineering such as bone graft. DTA-TGA, XRD, FT-IR, Raman, and SEM analysis were carried out to analyze both unconverted and converted artificial corals. Hydrothermally converted coral fine powders were loaded with gentamicin (Gm) antibiotic, and the drug-loaded particles were analyzed by SEM. Unconverted coral was mainly aragonite, while hydrothermally treated coral was completely converted to hydroxyapatite. Hydrothermally treated coral was showing agglomerated nodules up to 1-μm size consisting of nanocrystalline hydroxyapatite platelets in the size range of less than 100 nm. The general macropore size of the coral was found to be appropriate for osteoid growth, which is 100 to 600 μm range. These artificially grown corals can be easily produced and used for bone growth and repair and other biomedical applications.

Metastasis is a major contributor to cancer-associated deaths. It involves complex interactions between primary tumorigenic sites and future metastatic sites. Accumulation studies have revealed that tumour metastasis is not a disorderly spontaneous incident but the climax of a series of sequential and dynamic events including the development of a pre-metastatic niche (PMN) suitable for a subpopulation of tumour cells to colonize and develop into metastases. A deep understanding of the formation, characteristics and function of the PMN is required for developing new therapeutic strategies to treat tumour patients. It is rapidly becoming evident that therapies targeting PMN may be successful in averting tumour metastasis at an early stage. This review highlights the key components and main characteristics of the PMN and describes potential therapeutic strategies, providing a promising foundation for future studies.

Despite the significant progress made over the past decade with combination of molecular profiling data and the development of new clinical strategies, our understanding of metastasis remains elusive. Bone metastasis is a complex process and a major cause of mortality in breast and prostate cancer patients, for which there is no effective treatment to-date. The current review summarizes the routes taken by the metastatic cells and the interactions between them and the bone microenvironment. We emphasize the role of the specified niches and cues that promote cellular adhesion, colonization, prolonged dormancy, and reactivation. Understanding these mechanisms will provide better insights for future studies and treatment strategies for bone metastatic conditions.

Background: Wnt signaling pathways are taking a part in regulation of cell fate decisions in normal and cancerous cells. In some cancer types, a transition from canonical to non-canonical Wnt signaling pathways was identified, a phenomenon, that in return led to increase proliferation, invasiveness and metastasis. Methods: In the current in vitro study we investigated the influence of MSCs, co-cultured in direct and indirect contact with OS cells, on the role of Wnt signaling pathways and tumor aggressiveness. Sub-populations were separated using Boyden chambers. Gene expression profiles were determined by qPCR. Results: The results revealed that interactions with MSCs increased migration and invasion capacities along with OS proliferation. Moreover, canonical Wnt signaling activity was low in OS, and co-culture with MSC. However, MSCs did not trigger a switch between the canonical to the no-canonical Wnt pathways. In addition, a more aggressive OS sub-population tend to undergo a transition towards the non-canonical pathway. Moreover, this aggressive subtype presented cancer stem-cells like characteristic. Conclusions: We submit that the progression in OS aggressiveness is attributed to a transition in Wnt signaling from canonical to non-canonical pathways, although MSCs are likely to take a part during the tumor progression, in the case of OS, they did not affect the Wnt switch. These complex tumor promoting interactions may be found in the natural and tumorigenic bone microenvironment. A better understanding of the molecular signaling mechanisms involved in the tumor development and metastasis may contribute to development of new cancer therapies.

Distinct regions harboring cancer stem cells (CSCs) have been identified within the microenvironment of various tumors, and as in the case of their healthy counterparts, these anatomical regions are termed “niche.” Thus far, a large volume of studies have shown that CSC niches take part in the maintenance, regulation of renewal, differentiation and plasticity of CSCs. In this review, we summarize and discuss the latest findings regarding CSC niche morphology, physical terrain, main signaling pathways and interactions within them. The cellular and molecular components of CSCs also involve genetic and epigenetic modulations that mediate and support their maintenance, ultimately leading to cancer progression. It suggests that the crosstalk between CSCs and their niche plays an important role regarding therapy resistance and recurrence. In addition, we updated diverse therapeutic strategies in different cancers in basic research and clinical trials in this review. Understanding the complex heterogeneity of CSC niches is a necessary pre-requisite for designing superior therapeutic strategies to target CSC-specific factors and/or components of the CSC niche.

Zinc-based alloys hold promise as next generation biodegradable implants. Although zinc implants exhibit excellent biocompatibility in vivo, they have been found to inhibit cell attachment and viability under in vitro conditions. In order to clarify the cell response disparity and improve the biocompatibility of zinc implant materials, Zn-1Mg and Zn-1Mg-0.5Ca alloys underwent surface stabilization treatment in cell culture medium. Surface processing resulted in a stable surface oxide film of ∼300 nm thickness. The stability of the oxide layer substantially increased the viability of cells in both direct and indirect contact assays. It is consequently believed that the surface film characteristics of zinc implants may be an important determinant of biocompatibility.

Polymeric nanoparticles may enable delivery of drugs with lower systemic toxicity to solid tumors. Wnt signaling are evolutionary conserved pathways, involved in proliferation and fate decisions. Alterations in Wnt signaling play a pivotal role in various cancer types that promote cancer initiation, growth, metastasis and drug resistance. We designed a new strategy to allow an efficient targeting of both the canonical and the non-canonical Wnt pathways using nanoparticles loaded with inhibitor of Wnt productions-2 (IWP-2). This hydrophobic drug was successfully co-assembled into NPs composed of poly gamma-glutamic acid and a cationic and amphiphilic b-sheet peptide. Aggressive 4T1 breast cancer cells that were treated with IWP-2 loaded NPs gained a significant decrease in tumorigenic capacities attributed to improved IWP solubility, cellular uptake and efficacy.

One of the most promising strategies to treat cancer is the use of therapeutic antibodies that disrupt cell-cell adhesion mediated by dysregulated cadherins. The principal site where cell-cell adhesion occurs encompasses Trp2 found at the N-terminal region of the protein. Herein, we employed the naturally exposed highly conserved peptide Asp1-Trp2-Val3-Ile4-Pro5-Pro6-Ile7, as epitope to prepare molecularly imprinted polymer nanoparticles (MIP-NPs) to recognize cadherins. Since MIP-NPs target the site responsible for adhesion, they were more potent than commercially available therapeutic antibodies for inhibiting cell-cell adhesion in cell aggregation assays, and for completely disrupting three-dimensional tumor spheroids as well as inhibiting invasion of HeLa cells. These biocompatible supramolecular anti-adhesives may potentially be used as immunotherapeutic or sensitizing agents to enhance antitumor effects of chemotherapy.

We have developed a novel bioactive hybrid metallic implant that integrates the beneficial characteristics of a permanent matrix and a biodegradable substance. Such a combination may generate a material system that evolves into a porous structure within weeks to months following implantation and can be used to form strong interfacial bonding and osseointegration for orthopedic and dental applications. Presently, traditional technologies such as casting, powder metallurgy and plastic forming have limited ability to produce the complex bioactive implant structures that are required in practical applications. The present study aimed to develop an innovative bioactive Ti Mg (BTiMg) hybrid system using a Ti-lattice (Ti-6Al-4 V) produced by an additive manufacturing (AM) process, in combination with a new Mg-based alloy (Mg-2.4%Nd −0.6%Y -0.3%Zr) as a biodegradable filling material. We evaluated the in-vitro behavior of the BTiMg system in a simulated physiological environment, along with cytotoxicity assessment. The microstructure was evaluated by scanning electron microscopy and X-ray diffraction, mechanical properties were examined in terms of compressive strength, environmental performance analysis was conducted by electrochemical testing using potentiodynamic polarization and impedance spectroscopy (EIS), and cytotoxicity characteristics were assessed by indirect cell viability analysis. The results demonstrated the feasibility to produce geometrically complex implants by AM technology, as well as the strength and non-cytotoxic effects of the BTiMg system. Benefits included a relatively high ultimate compressive strength (UCS) and a high yield point (YP), along with an adequate cell viability response in the range between 70 and 120%.

The term “osseointegrated implants” mainly relates to structural systems that contain open spaces, which enable osteoblasts and connecting tissue to migrate during natural bone growth. Consequently, the coherency and bonding strength between the implant and natural bone can be significantly increased, for example in operations related to dental and orthopedic applications. The present study aims to evaluate the prospects of a Ti–6Al–4V lattice, produced by selective laser melting (SLM) and infiltrated with biodegradable Zn2%Fe alloy, as an OI–TiZn system implant in in vitro conditions. This combined material structure is designated by this study as an osseointegrated implant (OI–TiZn) system. The microstructure of the tested alloys was examined both optically and using scanning electron microscopy (SEM) and X-ray diffraction (XRD) analysis. The mechanical properties were assessed in terms of compression strength, as is commonly acceptable in cases of lattice-based structures. The corrosion performance was evaluated by immersion tests and electrochemical analysis in terms of potentiodynamic polarization and electrochemical impedance spectroscopy (EIS), all in simulated physiological environments in the form of phosphate buffered saline (PBS) solution. The cytotoxicity was evaluated in terms of indirect cell viability. The results obtained demonstrate the adequate performance of the OI–TiZn system as a non-cytotoxic structural material that can maintain its mechanical integrity under compression, while presenting acceptable corrosion rate degradation.

The growing interest in Zn based alloys as structural materials for biodegradable implants is mainly attributed to the excellent biocompatibility of Zn and its important role in many physiological reactions. In addition, Zn based implants do not tend to produce hydrogen gas in in vivo conditions and hence do not promote the danger of gas embolism. However, Zn based implants can provoke encapsulation processes that, practically, may isolate the implant from its surrounding media, which limits its capability of performing as an acceptable biodegradable material. To overcome this problem, previous research carried out by the authors has paved the way for the development of Zn-Fe based alloys that have a relatively increased corrosion rate compared to pure Zn. The present study aims to evaluate the effect of 0.3&ndash, 1.6% Ca on the in vitro behavior of Zn-Fe alloys and thus to further address the encapsulation problem. The in vitro assessment included immersion tests and electrochemical analysis in terms of open circuit potential, potentiodynamic polarization, and impedance spectroscopy in phosphate buffered saline (PBS) solution at 37 &deg, C. The mechanical properties of the examined alloys were evaluated by tension and hardness tests while cytotoxicity properties were examined using indirect cell metabolic activity analysis. The obtained results indicated that Ca additions increased the corrosion rate of Zn-Fe alloys and in parallel increased their strength and hardness. This was mainly attributed to the formation of a Ca-rich phase in the form CaZn13. Cytotoxicity assessment showed that the cells&rsquo, metabolic activity on the tested alloys was adequate at over 90%, which was comparable to the cells&rsquo, metabolic activity on an inert reference alloy Ti-6Al-4V.

Porous Mg scaffolds are considered as potential bone growth promoting materials. Unfortunately, the high rate of biocorrosion inherent to Mg alloys may cause a premature loss of mechanical strength, excessive evolution of hydrogen gas, and a rapidly shifting surface topography, all of which may hinder the ability of native cells to attach and grow on the implant surface. Here we investigated the cell cytotoxicity effects during corrosion of a novel magnesium alloy, EW10X04 (Mg-1.2%Nd-0.5%Y-0.5%Zr-0.4%Ca), following diffusion coating (DC) and heat treatment to reduce the corrosion rate. Cells were exposed either to corrosion products or to the corroding scaffold surface, in vitro. The microstructure characterization of the scaffold surface was carried out by scanning electron microscopy (SEM) equipped with a Noran energy dispersive spectrometer (EDS). Phase analyses were obtained by X-ray diffraction (XRD). We found that cell viability, growth, and adhesion were all improved when cultured on the EW10X04+DC surface or under corrosion product extracts due to lower corrosion rates relative to the EW10X04 control samples. It is therefore believed that the tested alloy after Nd coating and heat treatment may introduce a good balance between its biodegradation characteristics and cytotoxic effects towards cells.

Increasing interest in biodegradable metals (Mg, Fe, and Zn) as structural materials for orthopedic and cardiovascular applications mainly relates to their promising biocompatibility, mechanical properties and ability to self-remove. However, Mg alloys suffer from excessive corrosion rates associated with premature loss of mechanical integrity and gas embolism risks. Fe based alloys produce voluminous corrosion products that have a detrimental effect on neighboring cells and extracellular matrix. In contrast, Zn does not appear to exhibit a harmful mode of corrosion. Unfortunately, pure zinc possesses insufficient mechanical strength for biomedical structural applications. The present study aimed at examining the potential of two new zinc based alloys, Zn-1%Mg and Zn-1%Mg-0.5%Ca to serve as structural materials for biodegradable implants. This examination was carried out under in vitro conditions, including immersion testing, potentiodynamic polarization analysis, electrochemical impedance spectroscopy (EIS), and stress corrosion cracking (SCC) assessments in terms of slow strain rate testing (SSRT). In order to assess the cytotoxicity of the tested alloys, cell viability was evaluated indirectly using Saos-2 cells. The results demonstrate that both zinc alloys can be considered as potential candidates for biodegradable implants, with a relative advantage to the Zn-1%Mg alloy in terms of its corrosion resistance and SCC performance.

The microstructure and chemical composition of the calcite shell of the sea barnacle Tetraclita rufotincta (Pilsbry, 1916) were investigated using microscopic and analytical methods. The barnacle shell was separated mechanically into its three substructural units: outer, interior, and inner layers. The organic matrices of these structural parts were further separated into soluble and insoluble constituents and their characteristic functional groups were studied by FTIR. Investigation of the mechanical properties of the interior mass of the shell reveals remarkable viscoelastic behavior. In general, the mechanical behavior of the shell is a function of its geometry as well as of the material, of which it is constructed. In the case of T. rufotincta, as calcite is a brittle material, the elastic behavior of the shell is apparently related to its micro- and macroarchitecture. The latter enables the shell to fulfill its primary function which is to protect the organism from a hostile environment and enables its survival. Our detailed identification of the similarities and differences between the various structural components of the shell in regard to the composition and properties of the organic component will hopefully throw light on the role of organic matrices in biomineralization processes.

In the present research we study the effects of surface chemistry of an aragonite crystalline biomatrix on the chondrogenesis of mesenchymal stem cells (MSCs). An aragonite matrix obtained from the coral Porites lutea and a gold-coated P. lutea matrix were seeded with MSCs, with and without the addition of growth factors (GFs). Scanning electron microscopy, histochemical staining, immunofluorescence, biochemical analyses and quantitative polymerase chain reaction showed that the chemistry of the matrix influenced the differentiation process of the MSCs. The calcium carbonate composition of the coral promoted osteogenesis, while impeding cell-material contact (by gold coating) altered the differentiation lineage of MSCs towards chondrogenic fate. Supplementation of the culture medium with GFs intensified the influence of the surface composition on the differentiation of MSCs, and the synergistic effect of the biomatrix surface composition and the GFs induced chondrogenesis and facilitated maintenance of the chondrocyte phenotype. Therefore, we suggest that scaffolding material candidates for tissue engineering should be examined for their effects on the MSCs differentiation process and their effect on signal transduction events in the cells.

Bovine articular chondrocytes were seeded on either polyglycolic acid (PGA) non-woven mesh scaffolds or a biomatrix from the species Porites lutea (POR). These constructs were cultured for 6 weeks in the presence of insulin-like growth factor (IGF)-I (10 ng/ml or 100 ng/ml) or transforming growth factor (TGF)-β1 (5 ng/ml or 30 ng/ml) to determine the in-vitro articular cartilage regeneration capacity of each. Histology, deoxyribonucleic acid content, collagen I and II (immunohistochemistry and enzyme-linked immunosorbent assay), and glycosaminoglycan (GAG) contents were measured at 0 weeks, 2 weeks, and 6 weeks to assess the characteristics of chondrogenesis. Both scaffolds supported the maintenance of the chondrocytic phenotype, as evidenced by the predominance of collagen II and the presence of rounded chondrocytes embedded in lacunae. Regardless of growth factor treatment, cells cultured on PGA scaffolds produced more collagen type II than those cultured on POR. Conversely, by 6 weeks, cells cultured on POR scaffolds produced more GAG than those cultured on PGA scaffolds, again regardless of the growth factor used. Across the two groups, 100 ng/ml of IGF-I had the greatest overall effect in GAG content. This work indicates that PGA and the POR scaffolds are both effective growth matrices for articular cartilage, with each scaffold exhibiting different yet desirable profiles of articular cartilage growth.

This study examines the suitability of marine origin coral species, Porites lutea (POR) and the hydrozoan Millepora dichotoma (MIL), for use as novel three dimensional growth matrices in the field of articular cartilage tissue engineering. Therefore, mesenchymal stem cells (MSCs) and chondrocytes were grown on the skeletal material obtained from each of these two organisms to investigate their potential use as three dimensional scaffolding for cartilage tissue growth. Chondrogenic induction of MSCs was achieved by addition of transforming growth factor-β1 (TGF-β1) and insulin growth factor-I (IGF-I). Cell adherence, proliferation, differentiation and tissue development were investigated through six weeks of culture. Cartilage tissue growth and chondrocytic phenotype maintenance of each cell type were examined by cell morphology, histochemical analyses, expression of collagen type II and quantitative measures of glycosaminoglycan (GAG) content. The MSCs and the chondrocytes were shown good adherence to the scaffolds and maintenance of the chondrocytic phenotype in the initial stages of culture. However after two weeks of culture on MIL and three weeks on POR these cultures began to exhibit signs of further differentiation and phenotypic loss. The shown results indicated that POR was a better substrate for chondrocytes phenotype maintenance than MIL. We believe that surface modification of POR combined with mechanical stimuli will provide a suitable environment for chondrogenic phenotype maintenance. Further investigation of POR and other novel coralline biomatrices is indicated and warranted in the field of cartilage tissue engineering applications.

Biomineralization is the process by which organisms precipitate minerals. Crystals formed in this way are exploited by the organisms for a variety of purposes, including mechanical support and protection of soft tissue. Skeletal precipitation, via millions of years of evolution, has produced a wide variety of architectural configurations and material properties. It is exactly these properties that now attract the attention of researchers searching for new materials for a variety of biomedical applications.

Biomaterials play a pivotal role as scaffolding materials in the study of three-dimensional (3D) cell and tissue development and in a variety of bioengineering strategies for the restoration of damaged and malfunctioning tissues. Both in vitro and in vivo (e.g. cell-based therapies) bioengineering strategies deliver cells or a mixture of cells and signaling factors to a target tissue together with an acellular scaffolding material that triggers tissue ingrowth and regeneration. Bioengineering strategies in which tissue-like constructs are produced under controlled conditions are based on 3D lattices as vehicles for the cells that regenerate into the required tissues. Fabrication of engineered tissue is a complicated task, since tissue function is controlled by complex spatially and temporally ordered cues, each of which is the product of a myriad of cell-to-cell and cell-to-extracellular matrix (ECM) interactions.

The hermatypic coral Stylophora pistillata has a wide bathymetric distribution (0 to 70 m). Within this range, light intensity decreases exponentially. Deep-water colonies are generally planar in morphology, with the upper part being dark and the bottom-facing part pale. Shallow-water colonies are generally subspherical and ivory in coloration. We studied the effects of photoacclima- tion on photosynthesis, respiration, and calcification in S. pistillata colonies along its bathymetric range over a reef profile (5 to 65 m) in Eilat, Gulf of Aqaba, Red Sea, during winter and summer, using a submersible respirometer. Respiration rate, light-saturated rate of photosynthesis (Pmax), compensa- tion light intensity (Ec), and light intensity of incipient saturation (Ek), all decreased with depth. In contrast, the efficiency of photosynthesis (α) increased with depth. All colonies displayed 'light- enhanced calcification' during daytime and decreasing calcification rates with depth. These results indicate an adjustment in harvesting and utilization of light by the algal symbionts to the light envi- ronment. At all light intensities except the lowest ones, there was a consistent ratio of calcification to photosynthesis, in agreement with the concept of light-enhanced calcification. In the deepest, low- light corals, there was no evidence for support of calcification by photosynthesis, and we assume that these colonies subsist mainly by preying on zooplankton.

Mesenchymal stem cells (MSCs) can differentiate into various cellular lineages, including osteoblasts (that deposit hydroxyapatite, the main mineral constituent of bone), and also exhibit a high morphological plasticity. Here we grew for the first time MSCs on micropatterned silicon chips, in order to induce topography-guided alignment. Light microscopy, scanning electron microscopy and atomic force microscopy were used to characterize the cell response on various length scales. A notable alignment and movement of MSCs along the microgrooves on the chips was revealed. The cells were shown to inhabit the grooves rather than ridges and exhibited an elongated shape, with unusually long processes. On these cells, we revealed rhizome structures arranged along the extensions, which may serve as adhesion centers and participate in elongation and locomotion.

Magnesium alloys have been widely investigated for biodegradable medical applications. However, the shielding of harmful cells (eg. bacteria or tumorous cells) from immune surveillance may be compounded by the increased porosity of biodegradable materials. We previously demonstrated the improved corrosion resistance and mechanical properties of a novel EW62 (Mg-6%Nd-2%Y-0.5%Zr)) magnesium alloy by rapid solidification followed by extrusion (RS) compared to its conventional counterpart (CC). The present in vitro study evaluated the influence of rapid solidification on cytotoxicity to murine osteosarcoma cells. We found that CC and RS corrosion extracts significantly reduced cell viability over a 24-h exposure period. Cell density was reduced over 48 h following direct contact on both CC and RS surfaces, but was further reduced on the CC surface. The direct presence of cells accelerated corrosion for both materials. The corroded RS material exhibited superior mechanical properties relative to the CC material. The data show that the improved corrosion resistance of the rapidly solidified EW62 alloy (RS) resulted in a relatively reduced cytotoxic effect on tumorous cells. Hence, the tested alloy in the form of a rapidly solidified substance may introduce a good balance between its biodegradation characteristics and cytotoxic effect towards cancerous and normal cells.

Astrocytes play a pivotal role in the development and function of the central nervous system by regulating synaptic activity and supporting and guiding growing axons. It is therefore a central therapeutic and scientific challenge to develop means to control astrocytic survival and growth. We cultured primary hippocampal astrocytes on a crystalline three-dimensional (3D) aragonite biomatrix prepared from the exoskeleton of the coral Porites lutea. Such culturing led to the formation of astrocytic tissue-like 3D structures in which the cells had a higher survival rate than astrocytes grown in conventional cell culture. Within the pore void areas, multiple layers of astrocytic processes formed concave sheet structures that had no physical contact with the surface. The astrocytes attached to the crystalline perpendicular edges of the crystalline template surface extended processes in 3D and expressed glial fibrillary acidic protein. The astrocytes also expressed gap junctions and developed partly synchronized cytosolic Ca2+ oscillations. Preliminary in vivo models showed that astrocytic networks were also developed when the matrices were implanted into cortical areas of postnatal rat brains. Hence, we suggest that the biomatrix is a biocompatible supportive scaffold for astrocytes and may be exploited in applications for neuronal tissue restoration in injured or diseased central nervous system.

Macroporous composites made of coralline:chitosan with new microstructural features were studied for their scaffolding potential in in vitro bone regeneration. By using different ratios of natural coralline powder, as in situ gas forming agent and reinforcing phase, followed by freeze-drying, scaffolds with controlled porosity and pore structure were prepared and cultured with mesenchymal stem cells (MSCs). Their supportive activity of cellular attachment, proliferation and differentiation were assessed through cell morphology studies, DNA content, alkaline phosphatase (ALP) activity and osteocalcin (OC) release. The coralline scaffolds showed by far the highest evaluation of cell number and ALP activity over all the other chitosan-based scaffolds. They were the only material on which the OC protein was released throughout the study. When used as a component of the chitosan composite scaffolds, these coralline's favourable properties seemed to improve the overall performance of the chitosan. Distinct cell morphology and osteoblastic phenotype expression were observed depending on the coralline-to-chitosan ratios composing the scaffolds. The coralline-chitosan composite scaffolds containing high coralline ratios generally showed higher total cell number, ALP activity and OC protein expression comparing to chitosan scaffolds. The results of this study strongly suggest that coralline:chitosan composite, especially those having a high coralline content, may enhance adhesion, proliferation and osteogenic differentiation of MSCs in comparison with pure chitosan. Coralline:chitosan composites could therefore be used as attractive scaffolds for developing new strategies for in vitro tissue engineering.

Adipogenic and osteogenic cells share part of the early differentiation cascade of mesenchymal stem cells (MSCs). The choice of a mesenchymal precursor cell to differentiate into a particular cell type is dictated by many spatial and temporal cues, including growth factors, neighboring mature cells, and the extracellular matrix (ECM), which plays an important role in bone formation. Whether adipocytes that have initiated differentiation along one lineage can convert into osteogenic lineage by merely interacting with materials having specific surface parameters is unknown. Using crystalline three-dimensional (3D) biomatrices of marine origin (CaCO(3)), we explored whether preadipocytes can convert into osteoblasts. Cells (3T3F442A) were seeded on 3D biomatrices of marine origin (Porites lutea). Analyses were made at different time intervals-1, 2, 5, 7, 14, 21, and 28 days post-seeding. Cell characterizations were done using morphological (light microscopy and scanning electron microscopy), histological (Alizarin red, von Kossa and Oil red O staining), enzymatic (alkaline phosphatase activity, and quantitative PCR testing transcript levels of osteocalcin, alkaline phosphatase, core binding factor- 1 (Cbfa1), and fatty acid binding protein (aP2). We demonstrated 3T3F442A preadipocyte modulation and differentiation into bone-forming cells when grown on biomatrix of marine origin without addition of other bone morphogenesis inducers. We found an active ossification process typical of osteogenic phenotype as early as 2 days after seeding. It is suggested that this crystalline biomatrix having a particular 3D topology or surface parameters supports fast cellular adhesion, proliferation, and differentiation of preadipocytes to osteogenic phenotype.

The first in vitro study testing the effect of a natural mixture extract of quality elk velvet antler (QEVA) on the development of bone marrow-derived mesenchymal stem cells (MSCs) is reported. To examine cellular responses, MSCs were seeded on gold-coated glass surfaces and grown in a medium supplemented with QEVA extract or with the osteogenic agent dexamethasone (Dex), or in unsupplemented medium as control. The MSCs were analyzed by light microscopy and confocal Raman microspectroscopy at different time intervals in the culture. The microscopy revealed that the proliferation, up to day 4, of cells treated with QEVA is higher than that of cells treated with Dex or of the control group. Raman spectroscopy revealed deposition of hydroxyapatite (HA) mineral by cells exposed to QEVA and HA precursors in cells treated with Dex, but no mineralization in the control group. The extent of mineralization for MSCs treated with QEVA increased systematically with time, up to day 14. These results indicate that QEVA enhances proliferation and promotes differentiation toward osteogenic fate more effectively than Dex, suggesting that addition of QEVA to culture media might be advantageous to bone-tissue-engineering implications. Copyright © 2005 John Wiley & Sons, Ltd.

Mesenchymal stem cells (MSCs) seeded on three-dimensional (3D) coralline (Porites lutea) biomatrices were irradiated with low-level laser irradiation (LLLI). The consequent phenotype modulation and development of MSCs towards ossified tissue was studied in this combined 3D biomatrix/LLLI system and in a control group, which was similarly grown, but was not treated by LLLI. The irradiated and non irradiated MSC were tested at 1-7, 10, 14, 21, 28 days of culturing via analysis of cellular distribution on matrices (trypan blue), calcium incorporation to newly formed tissue (alizarin red), bone nodule formation (von Kossa), fat aggregates formation (oil red O), alkaline phosphatase (ALP) activity, scanning electron microscopy (SEM) and electron dispersive spectrometry (EDS). The results obtained from the irradiated samples showed enhanced tissue formation, appearance of phosphorous peaks and calcium and phosphate incorporation to newly formed tissue. Moreover, in irradiated samples ALP activity was significantly enhanced in early stages and notably reduced in late stages of culturing. These findings of cell and tissue parameters up to 28 days of culture revealed higher ossification levels in irradiated samples compared with the control group. We suggest that both the surface properties of the 3D crystalline biomatrices and the LLLI have biostimulatory effects on the conversion of MSCs into bone-forming cells and on the induction of ex-vivo ossification.

Tissue-like structures of hippocampal neurons were established in a crystalline three-dimensional (3D) aragonite biomatrix obtained from the exoskeleton of the coral Porites lutea. Cultures were maintained in vitro for up to 5 weeks. Cell viability and regeneration of neuronal properties were studied by immunocytochemical methods, light microscopy image analysis techniques, and scanning electron microscopy. Some portions of the cell population acquired the morphological characteristics of hippocampal pyramidal or granule neurons with axons and dendrites extending in a 3D manner along the surfaces of the crystalline biomatrix. The neurons usually grew on a sheet of glial cells. Within the pore void areas, multiple layers of neurons were formed, many of the neurons growing with no attachment to the crystalline surfaces. The neurons developed mature synaptic connections, with presynaptic sites expressing the synaptic vesicle protein 2 and postsynaptic sites having the shape of dendritic spines and expressing type 1 glutamate receptors, as these cells do under conventional culture conditions. The findings of the present study suggest that neuronal networks growing in a strong 3D aragonite support may find application as tissue replacement material for the central nervous system.

We studied the effect of light availability on the skeletal aragonite microstructure of the reef-building coral Fungia simplex. Scanning electron microscopy (SEM) of samples transplanted from light to shade conditions showed that the latter promoted a well-defined crystal alignment and dense packing. An X-ray diffraction (XRD) study of newly formed coral skeleton tissue showed a twofold increase in coherence length along the c-axis under shade conditions, while coherence length along the perpendicular direction remained essentially unchanged. These findings indicate that under shade conditions controlled calcification occurred by extension of the crystalline domains along the c-axis of the prismatic aragonite crystals. The reduced calcification rate under shade conditions was in keeping with the lower algal densities and the higher chlorophyll a content of the zooxanthellae that mediate the calcification process.

An attempt at using laser ablation of biofabricated coralline skeletal material Porites lutea for particle deposition is described. The deposition was performed in an evacuated chamber by pulses of the second harmonic (532 nm) of a Nd:YAG laser. The morphology and the local structure of the deposited films were characterized by scanning electron microscopy, X-ray diffraction, confocal Raman microscopy, and transmission electron microscopy. The as-deposited coatings partially retained the inorganic constituent of the CaCO3 target and mostly transformed to CaO. The crystalline phase was changed from solely aragonite in the target to a mixture of aragonite and calcite microparticles and CaO nanofeatures.

This study presents the characterization of the skeletal structure of the coral Fungia simplex, a hermatypic resident of the shallow zones of tropical reefs. In contrast to most cnidarians, this species has a unique multi-septal skeletal architecture. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) showed the skeletal microstructure to be made up of bundles of thin needles of prismatic crystals. X-ray diffraction (XRD) indicated that the crystals are composed of aragonite with a preferred orientation exposing the {221} planes parallel to the septal surface and tilted by 20° with respect to the growth (c-) direction.

Biotechnical and biomedical approaches were combined in an attempt to identify potential uses of biofabricated marine carbonate materials in biomedical applications, particularly as biomatrices for remodeling bone and cartilage tissue. After grafting, it is desirable for bone ingrowth to proceed as quickly as possible because the strength of the implanted region depends on a good mechanical bond forming between the implant and surrounding regions in the body. Ingrowth can take place as a result of growth of tissue and cells into the implanted porous material, or it may be promoted by transplanting cells seeded onto such a material. The rate at which ingrowth occurs is dependent on many factors, including pore size and the interconnectivity of the implanted structure. In vivo graftings into osteochondral defects demonstrated that our biofabricated porous material is highly biocompatible with cartilage and bone tissue. The biofabricated matrix was well incorporated into the biphasic osteochondral area. Resorption was followed by bone and cartilage formation, and after 4 months, the biomaterial had been replaced by new tissue. Ossification was induced and enhanced without introduction of additional factors. We believe that this is the first time that such biofabricated materials have been used for biomedical purposes. In face of the obvious environmental disadvantages of harvesting from limited natural resources, we propose the use of bioengineered coralline and other materials such as those cultured by our group under field and laboratory conditions as a possible biomatrix for hard tissue remodeling.

Mediated by algal symbionts, calcification in reef building corals is one of the important processes, which enable coral's growth. In the present study, we used a buoyant weighing technique to study calcification of two coralline species, Stylophora pistillata and the hydrocoral Millepora dichotoma. The colonies were grown in a tank system, in which light, nutrition and water motion were kept constant and temperature was elevated by means of a computerized controlled apparatus. An almost constant rate of calcification was observed in the two species at 22-28 degrees C. Elevation of the temperature above this range to 29-31 degrees C caused a slow down in calcification in both species. A grater number of S. pistillata colonies became bleached at temperatures ofor=29 degrees C, whereas M. dichotoma colonies suffered from bleaching only after three days at 31 degrees C. For both species, control groups, remained viable during the experimental period. The differences in responses to changes in temperature of the two species may be as a consequence of different adaptive mechanisms or to different susceptibilities of the corals to elevated temperatures. We have shown that elevating temperatures above annual maximal ranges have a significant effect on coral calcification. We also demonstrated that sessile calcified marine organisms having ecological and biomedical significance could be cultured and manipulated under laboratory conditions.

For many decades, fundamental cancer research has relied on two-dimensional in vitro cell culture models. However, these provide a poor representation of the complex three-dimensional (3D) architecture of living tissues. The more recent 3D culture systems, which range from ridged scaffolds to semiliquid gels, resemble their natural counterparts more closely. The arrangement of the cells in 3D systems allows better cell-cell interaction and facilitates extracellular matrix secretion, with concomitant effects on gene and protein expression and cellular behavior. Many studies have reported differences between 3D and 2D systems as regards responses to therapeutic agents and pivotal cellular processes such as cell differentiation, morphology, and signaling pathways, demonstrating the importance of 3D culturing for various cancer cell lines.

Field observations show that some coral species may assume different growth forms, or morphotypes, within apparently uniform habitats. The present paper examines the causes of this morphological diversity in hermatypic cnidarians and more particularly the following questions: Are there stable morphotypes among all the possible growth forms of a coral; if so, can a colony belonging to a certain stable morphotype change its growth form, and what can induce transition from type to type when the environment is relatively steady and only limited random (and short-term) variations of the external conditions occur? The approach adopted was to construct a mathematical model of the form dynamics of a coral colony and validate it by field data on the Red Sea hydrocoral Millepora dichotoma. The ratio between the volume and the surface area of a coral served as an index of its form and enabled treatment of the coral morphology in terms of dynamical systems. Such a dynamical system must have certain stable states corresponding to specific growth forms of a coral colony. Computer simulations showed that limited stochastic disturbances in the processes of biomass and skeletal growth caused by random fluctuations in environmental conditions may induce transitions of a coral from one stable growth form to another.

HAp derived from converted coral has been used as a bone graft substitute in orthopaedic surgery for nearly twenty years. After grafting it is desirable for bone ingrowth to occur as quickly as possible as the strength of the region which has been implanted is dependent on a good mechanical bond forming between the implant and the surrounding regions in the body. The rate at which ingrowth occurs is dependent on many factors, including the pore size and interconnectivity of the implanted structure. It is therefore necessary to develop a precise knowledge of the factors, which influence the microstructure and the pore size in the converted HAp structure. A specific Australian coral were used for analysis. A modified and improved hydrothermal conversion process was used to convert the corals from calcium carbonate to HAp. The effects of heat treatment and hydrothermal conversion on the pore size and the surface morphology of HAp structure have been studied using scanning electron microscopy. Samples after hydrothermal conversions were further dip-coated via an alkoxide sol-gel method were also examined to determine the morphology. The sol-gel/coralline hydroxyapatite interface and the mechanical properties have also been studied.

In the Gulf of Elat, the hydrozoan coral, Millepora dichotoma Forskal exhibits four main morphotypes: encrusting, delicate lace-like, leaf-like bladed and robust “box-work”. The distribution of these morphotypes was found to vary with depth and between locations. The encrusting form was found in all sites and appeared to be the initial growth form adopted by M. dichotoma. The distribution of the other three growth forms suggested that further development from the encrusting form depends upon environmental conditions. The encrusting form was found at all sites but was dominant in highly energetic environments. The lace-like form develops from the encrusting form in low energy environments. In somewhat higher energy environments, repeated damage to the lace-like form moves development towards the bladed growth form and the box-work growth form. A model of this development is presented in which critical factors controlling the development of the different growth forms are sediment levels and damage to colonies. Both of these factors are linked with levels of turbulence in particular environments.