\r\n\t
\r\n\tThe aim of this book project is to compile the updated research work on medicinal applications of noble metal complexes mainly focusing the structure activity relationship of metal complexes with targeting biological components.
Ti and Ti alloys are corrosion resistant, light, yet sufficiently strong for utilization as load-bearing and machinable orthopaedic implant materials. They are one of the few biocompatible metals which osseo-integrate, provides direct chemical or physical bonding with the adjacent bone surface without forming a fibrous tissue interface layer. For these reasons, they have been used successfully as orthopaedic and dental implants (Ratner 2004). To impart even greater bioactivity to the Ti surface and enhance integration properties, surface treatments such as surface roughening by sand blasting, formation of anatase phase TiO2 (Uchida et al. 2003), hydroxyapatite (HAp) coating, or chemical treatments (Ducheyne et al. 1986; Cooley et al. 1992) have been employed. However, these treatments are generally on the micron scale. Webster et al. (Webster et al. 2001; Webster, Siegel, and Bizios 1999) reported that it is even more advantageous to create nanostructured, in particular in the less than 100nm regime, surface designs for significantly improved bioactivity at the Ti implant interface and for enhanced cell adhesion. Since then, advances in biomaterial surface structure and design, specifically on the nanoscale, have improved tissue engineering in general. This chapter is a report on titanium dioxide (TiO2, or Titania) nanotube surface structuring for optimization of titanium (Ti) implants utilizing nanotechnology.
The main focus will be on the unique 3-D tube-shaped nanostructure of TiO2 and its effects on creating profound impacts on cell behavior. We will also shed light on the effects of changing the nanotube diameter size and optimizing the geometry for enhanced cell behavior. This work focuses on the tissue specific areas of cartilage and bone. Specifically, we will discuss how the desired cell behavior and functionality are enhanced on surfaces with TiO2 nanotube surface structuring. Here we reveal how the TiO2 surface nano-configurations are advantageous in various tissue engineering and regenerative medicine applications, for osteo-chondral, orthopedic, and osteo-progenitor implant applications discussed here and beyond. This chapter will also shed light on future applications and the direction of nanotube surface structuring.
In general, the mechanism of TiO2 nanotube formation in fluorine-ion based electrolytes is said to occur as a result of three simultaneous processes: the field assisted oxidation of Ti metal to form titanium dioxide, the field assisted dissolution of Ti metal ions in the electrolyte, and the chemical dissolution of Ti and TiO2 due to etching by fluoride ions, which is enhanced by the presence of H+ ions (Shankar et al. 2007). TiO2 nanotubes are not formed on the pure Ti surface but on the thin TiO2 oxide layer naturally present on the Ti surface. Therefore, the mechanism of TiO2 nanotubes formation is related to oxidation and dissolution kinetics. Schematic diagram of the formation of TiO2 nanotubes by anodization process is shown in Figure 1. For a description of the process displayed in Figure 1, the anodization mechanism for creating the nanotube structure is as follows:
Before anodization, a nano scale TiO2 passivation layer is on the Ti surface.
When constant voltage is applied, a pit is formed on the TiO2 layer.
As anodization time increases, the pit grows longer and larger, and then it becomes a nanopore.
Nanopores and small pits undergo continuous barrier layer formation. (e) After specific anodization time, completely developed nanotubes are formed on the Ti surface.
Schematic illustration of TiO2 nanotube formation.
Furthermore, based on the mechanism of nanotube formation, it is inherent that the nano-tubular structure formation depends on both the intensity of applied voltage and the concentration of fluorine ions in the electrolyte solution. It is well-known that by increasing the applied voltage, larger diameter nanotubes can be formed. This aspect of diameter manipulation using applied voltage will be further emphasized and the effects on cell function and fate is also discussed.
The Jin lab was the first to demonstrate that TiO2 nanotubes can significantly accelerate osteoblast (bone cell) adhesion and proliferation at the biomaterial/tissue interface and enhance bone mineral formation. The TiO2 nanotubes are formed as vertically aligned configuration, with an average diameter of ~100 nm, a height of ~300 nm, and a wall thickness of ~10 nm. According to published research (Oh S 2006), nanotube arrays on titanium surfaces induced proliferation of osteoblasts by as much as 300 – 400% compared to non-modified titanium surfaces. In other research groups studying nanoporous materials, major accomplishments have been made in the generation of geometrically defined surfaces with the fabrication of Al and Si nanostructured surfaces. There is rapidly increasing evidence that the lateral spacing of features on the nanoscale can impact and change cell behaviour (Boyen et al. 2002; Cavalcanti-Adam et al. 2006; Popat et al. 2006). Therefore, in order to optimize the lateral spacing of the TiO2 nanotube system, by changing the geometry of the nanotubes, four different pore sizes (30, 50 70, and 100nm in diameter) were created (Figure 2) for examination of cartilage chondrocyte cells, bone osteoblast cells, and osteo-progenitor mesenchymal stem cells.
Physical characterization of different size nanotube surfaces. (a) SEM micrographs of self-aligned TiO2 nanotubes with different diameters. The images show highly ordered nanotubes with four different pore sizes between 30-100nm created by controlling the voltage from 5-20V. (b) Table with the applied voltage parameter, estimated inner pore size from SEM images, average roughness (Ra) and surface contact angle measurements for Ti and 30-100nm TiO2 nanotube surfaces.
In terms of current biologically active implants, enhanced surface roughness is one of the important factors in providing the proper cues for a positive cell response to implanted materials. However, much of the research related to the effect of macro and micro-roughness on cellular responses and tissue formation are inconclusive due to the non-uniformity of macro and micro-roughness stemming from crude fabrication methods like polishing, sand blasting, chemical etching and so on. An important aspect of our nanotube system shown in the SEM images (Figure 2) is that the nano-topography can feature a more defined, reproducible and reliable roughness than micro and macro-topography for enhanced bone cell function in vivo. Although, the heights of the nanotube walls increase proportionally to the increasing diameter, there is no evidence of changes in surface roughness between the different sized nanotubes based on atomic force microscopy (AFM) data (Figure 2 (b)). As expected, the nanotube surfaces have a slightly higher roughness over flat Ti, but between the nanotubes, there appears to be no difference. The AFM data was performed because it is a somewhat standard surface analysis technique as it is useful for coarser or microscale roughness measurements, say for other convential coatings, but for the nanotube dimensions it may not always represent the true roughness when the probe tip radius is not substantially finer than the nanotube dimensions such as in the TiO2 nanotube case. The wall thickness, pore diameter, nanotube spacing, etc can be as small as ~10 nm, while the AFM probe tip diameter can be as large as 30 - 50 nm.
Furthermore, it can be assumed that the surface area on the nano-scale may be affected based on the various sizes and the surface area probably increases proportionally with increasing nanotube size. It is expected that the surface area to be 3 times higher on the 100nm diameter nanotubes compared to the 30nm diameter nanotubes, respectively. Additionally, the contact angle describing the wettability of the surface is enhanced, more hydrophilic, on the nanotube surfaces (showing contact angles between 4-11°), which can been advantageous for enhancing protein adsorption and cell adhesion.
Cells respond to the amount and area of proteins that are available for binding. In fact, cells do not see a naked material, in vivo or in in vitro culture. At all times, the material is conditioned by the components of the fluid in which the material is immersed, whether it is serum, saliva, cervicular fluid or cell culture media. As the cell begins to adhere and spread on the nanotubes, there will be a dissimilar protein density and extra cellular configuration based on the nanotube diameter. The behaviour of protein adsorption on the nanotube surfaces are shown in Figure 3. On the 30nm diameter nanotubes there is a large number and thorough distribution of protein nanoparticles covering the whole surface of the nanotubes after just 2 hours of incubation in culture media. However, proteins on 100 nm TiO2 nanotubes can only adhered sparsely at the top wall surface owing to the presence of large empty nanotube pore spaces. This inherent protein adsorption property of the nanotubes based on poresize is hypothesized to influence cell shape and fate. It is shown in the next sections that the changes in poresize even in such a small range of dimensions (30-100nm) will have huge impacts on downstream cell morphology and behavior.
SEM micrographs of flat Ti and 30, 50, 70, 100nm diameter TiO2 nanotube surfaces after 2 hours of culture showing protein adsorption from media.
Artificial cartilage prepared from cultured chondrocytes offers promise as a treatment for cartilage defects (Fedewa et al. 1998), but connecting this artificial soft tissue to bone in the attempts to restore the defected cartilage is difficult. One strategy employed in this section is to develop a dually functional substrate that supports the growth and attachment of cartilage tissue on one extremity and encourages osseointegration, a direct structural and functional connection to living bone, on the other. This substrate should be an engineered interface between artificial cartilage and native bone (Zhang, Ma, and Francis 2002).
In recent studies, Ti has emerged as a candidate material in cartilage tissue formation as well. It has been demonstrated that a micrometer porous substrate of Ti-6Al-4V provided conditions that favored cartilage tissue formation by influencing cell attachment, spreading and the amount and composition of cartilaginous tissue that forms (Spiteri, Pilliar, and Kandel 2006; Bhardwaj et al. 2001; Ciolfi et al. 2003). Not only porosity, but also surface geometry and topography have been found to have positive effects on the behaviour of chondrocytes (Bhardwaj et al. 2001).
Nanoscale topographic effects have been illustrated in nanostructured poly-lactic-co-glycolic acid (PLGA)/nanophase Titania (TiO2) composites, which have elicited an enhanced chondrocyte response compared to surfaces with a conventional or micrometer topography (Savaiano and Webster 2004). We have recently reported on our hypothesis that the nanotopographical cues, from porous nanotubular structured substrates made of TiO2, already being an osseointegrating biomaterial (Bjursten 2009; Oh et al. 2006), may also be a candidate for providing an alternative way to positively influence cartilage formation and the cellular behaviour of cartilage chondrocytes.
The dimensions of the nanotubes were varied in order to determine if the size of the nanotube diameters would play a role in the chondrocyte behaviour. For this comparative chondrocyte cell culture study, a commercially pure Ti surface, without surface modification was used as a control, as it commonly used as implant material.
It is well known that chondrocytes, the primary cells of cartilage, are extremely active cells. They produce a large amount of extracellular matrix (ECM) that is critical for the mechanical properties and joint lubrication characteristics of cartilage. In the SEM micrographs in Figure 4, the nanotubes substrates appear that they are inducing a positive response from the chondrocytes because the cells begin synthesizing abundant ECM deposition and fibril organization. In the SEM observations of chondrocytes a striking difference in the production of ECM fibrils between the flat Ti without a nanostructure vs. TiO2 nanotube surfaces is revealed. Fibrils are abundant and extending from all areas of the chondrocyte cell creating a dense network of ECM on the nanotube substrates.The flat Ti most likely lacks surface structuring cues for signaling ECM fibril production and organization. One possibility is that ECM protein formation into dense fibrils on the surface may be “nano-inspired” to form on the nanotube structure because of the precise dimensions or fine scale cues of the top surface (tip of the vertical wall) of TiO2 nanotubes having a physically confined geometry which could aid in fibril formation. It was demonstrated previously that the nanotubes produced bio-active nanostructured formations of sodium titanate nanofibers directly on the top of TiO2 nanotube walls when the nanotubes were exposed to NaOH solution (Oh S 2005). ECM proteins once secreted, in this study, may also self-assemble according to the top-wall surface geometric nanocues.
In the lower panel of Figure 4, immunofluorescent images for collagen Type II (red color) are illustrated for flat Ti vs. TiO2 nanotubes. Both surfaces stained positive for collagen type II, but there was large networks of connected bundles expressed across the surface of the nanostructure.
When a morphological analysis was conducted, it was determined that nanotubes induce a more spherical chondrocyte cell shape (data not shown). Fibroblastic shaped cells were observed on the flat controls. The percentage of round shaped, spherical cells was significantly lower for chondrocytes on the polystyrene, Ti, and the smallest diameter (30nm) nanotube substrates compared to the larger diameter 50, 70, and 100nm TiO2 nanotube surfaces respectively.
Extracellular matrix (ECM) production on experimental surfaces Ti vs. TiO2 nanotubes. High magnification SEM observations of chondrocytes reveal a striking difference in the production of ECM fibrils between the flat Ti without a nanostructure vs. TiO2 nanotube surfaces. Fibrils are abundant and extending from all areas of the chondrocyte cell creating a dense network of ECM on the nanotube substrates. (b) Immunofluorescent images of collagen type II (red) ECM fibrils produced by chondrocytes on flat Ti and nanotube surfaces (100nm diameter shown in this image).
It was also determined that all diameter nanotube surfaces were significantly higher than flat Ti which probably indicates that the cell shape was influenced by the presence of the nanostructure itself. The nanotube geometry seen in Figure 2 most likely aids in preserving the chondrocyte spherical morphology because of the distinct structure of the surface. Cells may be localized atop the pores, anchored possibly at the tip of the nanotube walls and confined by the tube contour. The chondrocytes on the flat Ti seem to be spread along the surface probably because the necessary structuring cues and nanopores needed for shape confinement are absent. To further describe the chondrocyte shape phenomenon found the experimental surfaces, a schematic is shown in Figure 5.
It was formerly assumed that focal contacts should be a specific length in order to promote adhesion and that the maximum overall contact of the cell with its substrate was most favourable (Ohara and Buck 1979). Yet, more recent studies suggest that cell-flattening or spreading is not always compatible with differing cell types, particularly in the case of chondrogenesis (Solursh 1989; Benya and Shaffer 1982; Solursh 1982; Zanetti and Solursh 1984).
Chondrocyte cell adhesion and spreading schematic determined by the size of the nanotube diameter and focal attachment sites.
Thus, the type of focal adhesion and its geometry can influence the shape the cell assumes, ultimately influencing the phenotypic expression. It has been reported that the dedifferentiation of chondrocytes in culture is usually associated with changes in cell morphology, from a rounded to a spread one (Costa Martinez et al. 2008). The results reported here suggest that creating pores by fabricating nanotubes on Ti surfaces provides a more favorable environment for the retention of the rounded morphology and the prevention of chondrocyte spreading, reducing the risk of a loss of phenotype. It should be noted that although chondrocyte cells retain this type of spherical morphology in response to the nanotube pores, different cell types will differ in size, shape, function, and how they operate on the nanotube surfaces. It is well known that different cell types elicit their own unique responses to environmental cues. The chondrocyte cells with their spherical morphology are much different than mesenchymal stem cells and osteoblast cells, described in later sections, and therefore will adhere differently to the topography and form different morphologies.
Because chondrocytes are very dynamic cells that produce and maintain the cartilaginous matrix, which consists mainly of collagen and proteoglycans, it is important to test the biochemical ECM production on the different experimental surfaces. Therefore, to further evaluate the response of BCCs for this comparative report, the glycosaminoglycan (GAG) secretion in the media was also studied and shown in Figure 6.
Naturally, aggrecan draws water into the tissue and swells against the collagen network, thereby resisting compression and allowing for proper joint movement (Muir 1995). An interesting concept worthy to note is that the up-regulation of GAG chains indicative of increased aggrecan production observed on the larger sized nanotube pores could imply that because there are increased storage volume capabilities as pore size increases it triggers a higher rate of production because the molecule retention ability of the cellular environment has been inflated.
It was determined that there was a correlation in the increased GAG secretion in the media and the reduction of fibroblastic shaped cells. Specifically, TiO2 nanotubes with diameters in the range of 50-100nm had significantly higher levels of both round, spherical shaped cells (more phenotypic) and GAG secretion over flat Ti and small 30nm TiO2 nanotube surfaces.
Glycosaminoglycan (GAG) secretion in the media.
In this study, the larger diameters (50nm-100nm) nanotubes were revealed to be most suitable for chondrocyte culture in vitro. It would be interesting to investigate nanotube diameter sizes larger than 100nm by other fabrication means so as to elucidate the possible effect of large pore size and find an ultimate productivity saturation limit; this would certainly allow more light to be shed on the beneficial nature of nanotopography.
As Ti is the well accepted orthopaedic implant material, the results obtained are very encouraging and suggest that the use of nanotube structures could up-regulate production of extracellular matrix by chondrocytes. In clinical applications the TiO2 nanotube surface can be utilized as an in-vitro culture surface to enhance chondrocyte cell behavior and extracellular matrix production during patient-specific in-vitro chondrocyte expansion, which can then be transplanted to the defective cartilage areas. In addition, the TiO2 nanotube surface exhibits significantly augmented, mechanically and chemically strong osseo-integration with existing bones with a minimal chance of bone loosening evidenced by in vitro data [22], and our preliminary in vivo animal data indicating a strong new bone integration on the nanotube surface with reduced soft tissue trapping (data not shown). Therefore, a Ti implant with all surfaces covered with the nanotubes can be potentially utilized, for some specific types of articular cartilage injuries, to serve with dual function of accelerated osseointegration to the existing articular bone surface at the bone-facing contact interface while the exposed nanotube surface can accelerate the cartilage tissue regeneration by providing positive surface nanostructuring effects on chondrocytes, as illustrated in Figure 7.
Schematic illustration of TiO2 nanotubes for dual function of osseointegration and enhanced chondrocyte function and ECM production.
As mentioned earlier, while a thin TiO2 passivation layer on the Ti surface can impart improved bioactivity and better chemical bonding to the bone (Feng et al. 2003), other techniques have been developed to further enhance the bioactivity of a pure Ti surface, such as direct coating of bioactive materials like hydroxyapatite and calcium phosphate (Puleo et al. 1991; Salata 2004; Satsangi et al. 2003). However, even though these surface modified layers have good bioactivity and high surface area, they tend to delaminate at the interface between the implant and the bone due to the relatively large, micrometer-regime thickness of the coated layer on Ti (Ong et al. 1992), presumably due to the stress accumulation commonly seen in a thick coating of foreign material. This ultimately leads to implant failure. In order to overcome this problem, some plasma spray Ca-Si based ceramic coatings have been developed but still have roughness and layer thickness in the micrometer range. For the purposes of this study, the focus is on nanoscale thickness surface coatings. Therefore, developing an implant bioactive surface layer having high surface area for enhanced bonding yet thin enough, in the nanometer range per se, to minimize delamination would be desirable.
Recent reports indicate that modifying Ti surfaces with TiO2 nanotubes for orthopedic applications significantly enhances the mineral formation (Oh et al. 2005), adhesion of osteoblasts in vitro (Oh et al. 2006), and strongly adherent bone growth in vivo (Bjursten 2009), showing better bone bonding characteristics than conventional micro-roughened Ti surfaces by sandblasting. One physical advantage of the TiO2 nanotube surface system is that it is composed of and created directly from the native underlying Ti constituent, unlike the foreign ceramic and spray coatings on Ti or Ti alloyed surfaces mentioned previously. As well, the nanotube layer is at most ~300nm tall (for the purposes of this work) which in the scheme of things is a much thinner layer and this nanometer length scale eliminates the tendency of delamination prevalent in thick micrometer layers.
Because orthopedic implants encounter two types of cells, osteoblast cells in bone tissue and osteo-progenitor cells also know as mesenchymal stem cells (MSCs) present in bone marrow, it is advantageous to look at the differentiation potential of orthepeadic implant surfaces in order to initiate a mature population of bone building cells for enhanced osseointegration. One key principle in terms of orthepaedic implant technologies, to initiate the differentiation of bone in the absence of chemical factors, hormones, or any other synthetic, possibly toxic chemicals traditionally used by biochemists in in vitro differentiation. The two cell types are illustrated and described in Figure 8.
Schematic illustration showing osteo-progenitor cells developing into osteoblast cells. A description of the two cell types is also portrayed in list format.
Stem cells, which have the potential to differentiate into multiple cell types, provide great promises in advances in regenerate medicine. The differentiation of stem cells into the appropriated lineages is temporal- and spatial-specific, with the surrounding microenvironment playing critical roles in governing the stem cell fate. For generation of osteoblasts, the MSCs need to be guided to selectively differentiate to osteoblasts, rather than differentiating into other types of cells. The use of nanostructures and surface topographical features have recently been shown to have positive effects on specific differentiation of mesenchymal stem cells (Dalby, Andar et al. 2008), illustrating that the surface topography alone can stimulated osteogenic differentiation.
In terms of the dimensions of the nanotubes in our osteoblast (bone cell) and mesenchymal stem cell (osteo-progenitor cell) studies, we have reported a unique variation in cell behavior even within a narrow range of nanotube diameters from 30-100nm (Brammer et al. 2009; Oh et al. 2009) and it seems that a similar trend is established for both cell types.
When cells were grown on four different diameter nanotubes, shown in Figure 2, osteoblast functionality in terms of bone forming ability, or alkaline phosphatase activity (ALP), and mesenchymal stem cell (MSC) osteogenesis (bone cell differentiation) in terms of osteogenic gene expression (osteopontin (OPN), osteocalcin (OCN), and alkaline phosphatase (ALP)) were most prominent on the large 100 nm diameter nanotubes, Figure 9 (a and b). During periods of active bone growth, ALP activity levels are elevated in osteoblast cells so it is beneficial to design an implant surface that would enhance the ALP activity to initiate the formation of new bone. As well, it is critical to design a surface that is capable of allowing the attachment of MSCs and promote osteogenic differentiation of cells for delivering a mature osteoblastic cell population capable of rapidly forming bone. On the nanotube surfaces, a reoccurring trend was revealed that as we increased the diameter of the nanotubes, there was an increase in osteogenic biochemical activity and relative gene expression, Figure 9 (c).
Comparative graphs showing the influence of TiO2 nanotube diameter on osteogenic cell behavior. (a) Osteoblast functionality in terms of alkaline phosphatase activity or bone forming ability. (b) Osteogenic gene expression. RNA levels of alkaline phosphatase (ALP), osteocalcin (OCN), and osteopontin (OPN) are given for mesenchymal stem cells grown on different size diameter nanotubes. (c) Overall all trend for diameter effect on osteogenic cell behavior. As the nanotube diameter increases the osteogenic cell behavior is enhanced.
In a recent review article, Bettinger et al. claimed that the most palpable effect of nanotopography on cells is the distinct changes in cell geometry or shape (Bettinger, Langer, and Borenstein 2009). In fact, on the nanotube substrates with varying diameters, it was revealed that the osteoblast and mesenchymal stem cells have reacted to the nanostructures by changing shape. As the nanotube diameter increases we found a clear trend of increasing cell elongation, Figure 10. The stretching aspect ratio was as great as 12:1 (length:width) on the 100 nm diameter nanotubes. It can be assumed that the initial cell stretching and elongated shape of the adhering cells on the large nanotubes impacted the cytoskeletal (actin) stress. This is supported by the general notion that nanostructures (and the adhered protein configuration for example, Figure 3) act as an extracellular matrix which imposes physical forces and morphological changes to the cell (Chen 2008). It is probable that cells must elongate their bodies to find a protein deposited surface, extending across larger areas and thus eventually forming an exceedingly elongated shape on the 100nm diameter nanotubes (because of the sparse distribution, Figure 3). Thus altering the density of extracellular matrix (ECM) attachment sites or initial protein adhesion density affects the shape of adhered cells. It has been reported that the focal attachments made by the cells with their substrate determine cell shape which, when transduced via the cytoskeleton to the nucleus, result in expression of specific phenotypes (Boyan et al. 1996). In our results, we hypothesize that increasing nanotube diameters, changes the focal adhesion sites of the osteoblast and mesenchymal stem cells, increases the cell elongation, and increases osteogenic potential.
Interestingly, the cell nuclei also exhibit a somewhat similar trend of increased elongation with increasing nanotube diameter, with the 100nm TiO2 nanotubes showing the most significantly elongated nuclear shape (by ~20-25%) (data not shown). It can be hypothesized that the nucleus organelle elongation on the TiO2 nanotube surfaces is in part due to the gross elongated cytoskeletal morphology of the cell.
Quantification of cell elongation for osteoblast and mesenchymal stem cells on nanotubes with different diameters ranging from 30-100nm. There is a trend of increased elongation with increasing nanotube diameter.
It has been reported that cell shape maintained by the cytoskeletal assembly may also facilitate nuclear shape distortion which may promote DNA synthesis by releasing mechanical restraints to DNA unfolding, changing nucleocytoplasmic transport rates, or alternating the distribution and function of DNA regulatory proteins that are associated with the nuclear protein matrix (Maniotis, Chen, and Ingber 1997). A change in the nuclear structure has an effect on the 3-dimensional internal organization (Getzenberg et al. 1991). It appears that the osteoblasts and mesenchymal stem cells are adapting to the nanotube substrate nanotopography by organizing both external and internal shapes.
A concept developed by Dalby et al. (Dalby et al. 2008) suggests that MSC osteo-differentiation is determined by mechanotransductive pathways that were stimulated because the cell was under tension caused by way the cell was adhering and the shape it assumed due to the underlying nanostructure surface. Cell morphology/spreading dominates cell fate. McBeath et al. showed that commitment of stem cell differentiation to specific lineages is dependent upon cell shape (Mc Beath et al. 2004). In a single cell experiment with micropatterned surfaces the critical role of cell spread/shape in regulating cell fate was determined.
Nonetheless there is a need to better understand the mechanism by which such nanosurfaces direct MSC osteogenesis, and to optimize the culture conditions in order to maximize MSC expansion and differentiation. For bone growth, it requires cell proliferation and selective differentiation, and these processes are found to occur at different but discrete nanosurface topography conditions such as variations in nanotube diameter.
Establishing possible connections between mechanical properties of MSCs in differentiation is valuable to the field of mechanobiology. It can be speculated that natural forces that MSCs encounter in a physical environment does not need a strong cytoskeleton, however upon osteogenic differentiation, in which differentiation of MSCs become part of a larger bone structure that functions to provide both form and strength, the supporting structure of the cells are enhanced to enable function and withstand load bearing wear that bones endure. Understanding the physical characteristics of MSCs during differentiation may aid in the development of new biomaterials, which can potentiate the necessary mechanics of the cells for the advancement of tissue engineering.
In terms of the dimensions of the nanotubes in the osteoblast (bone cell) and mesenchymal stem cell (osteo-progenitor cell) studies, it was reported that a unique variation in cell behaviour even within a narrow range of nanotube diameters (Brammer et al. 2009; Oh 2009).The results of the previous research can be simply summarized: osteogenic functionality, both biochemical activity in osteoblasts and internal gene regulation of osteo-progenitor cells, were altered by the size/diameter of TiO2 nanotubes, as the nanotube diameter increased, the osteogenic function also increased. Such a trend can be utilized for improvement and control of the bone forming functionality for advanced orthopaedic implant technologies.
In these studies however, TiO2 nanotubes having a 1: 3 diameter: height aspect ratio was used, which was determined by the electrochemical anodization conditions including electrolyte solution, voltage, time, etc. While this current-state-of-the-art self assembly process of TiO2 anodization does not easily allow fabrication of TiO2 nanotubes with the diameter larger than ~100nm with the electrolyte used in this study, it would be interesting to study the effect of even larger diameter TiO2 nanotubes, possibly using a modified chemical process, on osteogenic cells.
Large diameter nanotubes prepared in 0.25 w/v% NH4F with various applied voltages. (A) SEM micrographs showing nanotube morphology by top view (a, c, e) and cross-sectional view (b, d, f). (B) Chart describing the effect of applied voltage on the physical nature of the nanotube dimensions.
Other methods for making large diameter (>100nm) TiO2 nanotubes using aqueous organic electrolytes with a future potential use as orthopaedic implant surfaces have been explored. Previously the anodization electrolyte method included aqueous dilute hydrofluoric acid. In an alternative fabrication method ammonium fluoride (NH4F) in an ethylene glycol solution as an electrolyte can be investigated. Figure 11 illustrates the SEM figures of TiO2 nanotubes prepared by 0.25 w/v% NH4F electrolyte at various anodization voltage for 17 hrs. The table in Figure 11 indicates the variations in applied voltage and the resultant physical dimensions of the fabricated nanotubes. By controlling the applied voltage, the diameter of the nanotubes could also be controlled. Nanotubes with diameters from ~130-225nm have been successfully prepared. Future work should include the use of large size nanotubes to find the most optimal stem cell osteogenesis and bone cell/tissue function.
In the future, nanostructured ceramics will be created that demonstrate even higher degrees of integration between materials and biology (Narayan et al. 2004). Future ceramic nanostructures may possess even more precisely tailored grain and/or pore sizes in order to obtain specific tissue interactions. For instance, loading nanoporous structures can provide an implant with biological functionalities (Cowan et al. 2003) are key prospects in deriving therapeutic based nanostructures that integrate and for wound healing, bone repair, and cardio vascular restoration, to name just a few. For example, silver and zinc-containing zeolites, marketed under the trade name AgION®, are currently being assessed for use in wound dressings. Figure 12 shows the idea of a “nanodepot” using the nanopores of the TiO2 nanotubes for loading biological agents.
a) Schematic illustration of “nanodepot” concept in TiO2 nanotubes. (b) Example of albumin protein elution from inner pores of TiO2 nanotube structures.
In the previous sections it has been shown that the physical environment of nanotopography has positive effects on cell behaviour, yet direct comparisons of nanotopographic surface chemistry has not been fully explored. For instance the possibility of nanotubes made of different materials, i.e. carbon, gold-Pd, zirconia, or tantalum for instance.
To date, a large part of the interest has remained on titanium oxide (TiO2) nanotubes because it is well known that titanium (Ti) is a biocompatible orthopedic material which provides an excellent osseointegrative surface. However, little notice has been given to zirconium oxide (ZrO2) nanotubes, which are formed via the similar self-assembled mechanism as TiO2 nanotubes, through an electrochemical anodization process (Berger et al. 2008). Zirconium (Zr) is similar to titanium in that it possesses a thin passivation oxide layer which makes it highly resistant to corrosion in bodily fluids (Oliveira et al. 2005). In fact, while the corrosion resistance and biocompatibility of certain Zr alloys are as good as those of Ti alloys, the mechanical properties have been found to be superior to those of the commonly used Ti-6Al-4V alloy (Kobayashi et al. 1995). Furthermore, a recent study by Bauer and co-workers demonstrated that mesenchymal stem cells react in the same manner to ZrO2 nanotubes, AuPd-coated TiO2 nanotubes, and as-formed TiO2 nanotubes (Bauer et al. 2009). Their results indicate that the cell response is chiefly due to nanotopographical cues instead of a specific surface chemistry pertaining only to TiO2.
There is a vast parametric space in which to explore novel nanostructures, nanopore dimensions, and material compositions for optimizing and advancing implant designs for desired tissue interactions.
The authors acknowledge financial support of this research by Iwama Fund at UC San Diego and UC Discovery Grant No. ele08-128656/Jin. This work was also partially supported by Postdoctoral Fellowship (for S. Oh) from the California Institute for Regenerative Medicine (CIRM).
There is a noticeable increase in major incidents (MI) of different types affecting the world in the last three decades with increased human and financial losses. There are different types of incidents: natural, man-made, and infectious epidemics. For optimum response to those incidents, there should be multidisciplinary coordinated teams’ response [1].
Shortly after I started writing this chapter, the COVID-19 epidemic in China expanded to be a pandemic, affecting too many countries and hundreds of thousands of people and killing more than 50,000 all over the world. This has a clear effect on my chapter to concentrate on this type of major incidents.
Primary health care (PHC) system scope of service was concentrated on disease consultations and prescription of medication. This scope had been changed in 1978 by the Declaration of Alma-Ata, which considered health care as fundamental human right. Section V in the declaration stated clearly that the primary health care system is the key to achieve the targets of the declaration. This declaration leads to the change of the scope of work of the PHC from giving advice about patients’ symptoms to the comprehensive health care of the community [2]. This aim of comprehensive health care of the PHC was reviewed in WHO report in 2008 [3], which centered on taking the PHC service from hospitals and specialized centers to be nearby people in general walk-in clinics that are easily reached by community. This proved to have many benefits in:
Relief suffering
Prevention of illness and death
Improved health equity
The PHC system, with its distribution in all areas to be near to large population, can play an important role in response to any MI affecting its catchment area. The PHC staff have better knowledge in their area population and the special needs categories. Those abilities enable the PHC system to play a crucial role in response to major incidents in all their stages.
PHC is a whole-of-society approach to health that aims to ensure the highest possible level of health and well-being and their equitable distribution by focusing on people’s needs and preferences (as individuals, families, and communities) as early as possible along the continuum from health promotion and disease prevention to treatment, rehabilitation, and palliative care and as close as feasible to people’s everyday environment [4].
“The practice of continuing comprehensive care is the concurrent prevention and management of multiple physical and emotional health problems of a patient over a period of time in relationship to family, life events and environment” [5].
In health care system, “A major incident can be defined as any incident where the location, number, severity or type of live casualties requires extraordinary resources” [6].
Vulnerable populations include patients who are racial or ethnic minorities, children, elderly, socioeconomically disadvantaged, underinsured, or those with certain medical conditions. Members of vulnerable populations often have health conditions that are exacerbated by unnecessarily inadequate health care [7].
Work concept: Changing to comprehensive health care of the community makes the PHC responsible to prepare people to a disaster if there is time to and help them during its occurrence and in the post-incident stage.
Community based: The responsibility is for the welfare of the whole community and not for medical advice when asked only.
Easy accessibility: The presence of PHC centers in close proximity to people makes them reachable with little efforts. During a major incident, there are many things people are busy with, or some have lost helpers; the presence of a nearby health care facility will decrease patients and people suffering in attending and getting the proper health advice they need.
Dealing with all types of diseases: The PHC centers are run by family medicine physicians (FP) or general practitioners (GP), and they are trained to deal with all types of diseases and possess the ability to deal with all patients or persons asking different types of consultations in many specialties.
Filtering ability: The doctors in primary care are trained to deal with patients in all specialties. This practice gives them the ability to work in the early step of sorting cases. PHC can deal with simple cases that constitute 50 to 60% of cases and keep hospitals and specialized centers for more severe cases.
Decrease cost: FP/GP use less tools and request less investigations for their work, and this cuts the cost the patient should pay for care. This is exaggerated during major incident circumstances, which enable the health system to deal with larger number of patients in the same cost if the patients have been managed by hospitals only.
Stages of major incidents: Most of the researchers divide the major incidents into four stages:
Mitigation and prevention
Preparedness
Response
Recovery
Although this has many positive implications, Quarantelli in 1980 [8] put major incident phases depending on the time factor. He put three phases as follows:
Pre-impact phase
Trans-impact phase
Post-impact phase
In the coming sections, I will discuss the role of the PHC system according to the phase and regarding the staff, space, stuff, and communication roles. The discussion will be divided into two parts as follows:
Effect of major incident on primary health care. This will be under the title “Challenges.”
Actions by the primary health care in response. This will be under the title “Actions.”
PHC system is the nearest health facility to most people and is easily accessible; therefore, it is, mostly, the first place that the patients will attend to get information and advice for their conditions and worries about any new threat or risk.
The challenges will affect PHC staff and PHC facility. The challenges will depend on the risks more suspected in the geographical area in which the health facility is present.
The PHC staff are holding double personality during major incidents; from one side they are part of families and they have responsibilities for them, and on the other side, they have commitment to work to help people and other community services.
The staff will be under pressure to have answers to the new threat and its relations to the different diseases and medications.
Dealing with major incidents is not part of day-to-day activity of the health sector; therefore, there is lack of knowledge of the major incident response plan (MIRP) to the facility and their specific roles. All staff in the PHC in all levels whether clinical or nonclinical should have specific training on the MIRP and their specific role in it, so everybody will speak the same language during the response and can harmonize work better.
Psychiatric aspect of the major incident will affect both PHC staff and people visiting the health facility. Staff should have basic training to deal with those difficult situations.
There will be an increase in visits to PHC from people for different consultations, which will lead to overcrowding in the PHC.
More medications and stuff are requested by patients to have stocks during the expected incidents. This will impose pressure on the stocks of the medications and consumable stuffs.
How to change the layout of the facility to accommodate more visitors if needed? This is depending on the blue print of the facility and whether it is manageable or not.
Is the facility in danger of the incident or not? This is more applicable in the natural disasters like earthquake, tornados, and hurricanes. What to do if it is affected and cannot provide service?
The infrastructures like electricity, water, or network may be affected by the incident. What are the substitutes or mitigation solutions for this breakdown?
The period before expected incidents is a suitable situation to do improvements in planning, training for staff, and stockpiling stuff. PHC administrators need to achieve the following aims:
Orient the local and national health system on the PHC active involvement in protecting community, and strengthen the skills of risk management in the PHC directors.
Concentrate on efforts to support communities, individuals, and health workers to better respond to major incidents. This is done by providing enough information about the disease and protective measures, vaccinations, and actions to do if any family member or oneself is affected.
Financial investment in the infrastructures of the PHC regarding water, electricity, information, communications, and supply network.
The abovementioned aims will be accomplished through the following:
PHC should work in close collaboration with local health authority and other emergency services in developing response plans.
They should look to the following issues:
Define hazards and risk factors to the area, in collaboration with other emergency services. This will help in predicting expected incidents and their effects on the health of the community they are serving. Also, it will help in preparing the type of medications and consumables.
Make lists with addresses of the vulnerable population and those using regular medications in their area. This will allow the health and other authorities to prepare shelter, medications, and other care plans during MI.
The effect of the expected MI on different diseases should be prepared with the help of the specialized sections in local, state, or federal (or ministerial) health authorities. Information should be relayed to the concerned patients along with best mitigation actions.
Setting agreements with ambulance services to transfer patients (who need hospital transfer) received in PHC during MI.
Information regarding any MI expected, its nature, the expected size, and the need to activate the MI response plan. That critical information should be verified, i.e., the source of information should be trusted and accurate.
Allocate area to be an alternative place, and make pre-arrangement for rapid conversion into PHC facility if the original building became unsuitable for work.
Staff should have training on several aspects of MI response including the needed information to help the community and individuals.
This includes the following:
Training the staff on the MI response plan set for the area. The response plan should be flexible to meet the different types and effects of different incidents. The plan should rhyme with the local and state health response plan.
Training the staff on communication with people including agitated and aggressive people. Communication training should include breaking bad news. They may have to visit families and inform them about their relatives, good or bad news.
Training on working in austere conditions in case there is damage to the infrastructure of the PHC facility and a need to work in small or large teams depending on the incident circumstances and decision of the local authority or state’s incident response leaders.
Training on more than one method of communication including radio communication protocols. Radio communication will be used if other methods are lost by the effect of the incident.
Electricity failure is one of the major failures during MI. It has serious negative effects on patients’ care. Electricity generator working on petrol should be prepared and maintained regularly to be ready anytime electricity cut occurs.
Water loss. Large water reservoir should be ready, and the water should be replaced on a daily basis. In addition, drinking water bottles should be part of stockpiling in preparation to a major incident. Potable drinking water in addition to clean water suitable for different uses in the facility and patients’ care is crucial for work in any place and especially in PHC.
Communications: It is important to have at least two ways to contact the staff. If one method failed, the other will be the backup. A third wireless radio communication should be prepared for extreme loss of any sort of communication.
Network: Failure of network can occur alone or accompanying electricity failure. Server backup should be applied to keep the important information regarding patient, administrative, pharmacy, and store documents.
During daily work, the PHC centers are stocking their expected needs of drugs and consumables for a certain period depending on the chain of supply. If there is a risk or threat of any type that needs special medications or protective equipment, the PHC centers should bring these stocks according to the population supported with the coordination of the local or national health authority.
As mentioned above, the PHC will be the first point people will reach to seek help and advice regarding injuries or information regarding an epidemic infection. This includes many challenges.
The WHO in their briefing about PHC and emergencies [9] mentioned some of the challenges as follows:
Without earlier warning system, case identification and escalation are a challenge that PHC staff face. Most epidemics usually start as patients come to seek medical advice, which is a day-to-day work in PHC. Case definition and raising the suspicion of a disease that may be an early epidemic or even pandemic (like the COVID-19) need to be reported to more central and higher authority with experts in epidemiology and infectious diseases.
Geographical accessibility:
Several types of natural disasters like earthquake, floods, etc. may affect the PHC and its surrounding area, preventing health workers, people, or supply chain from reaching it. This will render the PHC useless, and alternative methods should be placed to mitigate this challenge.
In the same context, geographical accessibility to patients and survivals will be sometimes difficult if not impossible and need special help.
People with special need will face more difficulty in major incidents in accessing the PHC. The rescuers will face more difficulty in moving people with special needs in case there is need for it.
Transferring patients and staff to an alternative place or transferring patients to hospital may be a challenge in certain circumstances.
Skillful health workers are part of the community and may be affected by the incidents either themselves or their families, and in both situations, they cannot be available to treat and help people. PHC may replace them by less skilled staff which will affect the care in this difficult situation.
Health facility infrastructure may be affected by the incident, and loss of water, electricity, and network supply, for example, will also affect the ability of PHC to offer help and services.
The incidents may affect the chain of supply, leading to a decrease in resources and limitations in the numbers and types of medications and services available.
If there is an incident that is big enough to affect large cities and states, the government will distribute the funds and resources to all areas. This mostly will make the amount given to a certain PHC center below its actual needs. This has effects on the availability of staff and supplements and decreases in the PHC center’s effective services.
Ensuring quality of care to be in acceptable level. In day-to-day work, the quality should be in the optimum; this is not applicable in most MI, but there should be an agreed acceptable level of quality during major incident, so the PHC centers do not go below it. This is extremely important to decrease the spread of infectious diseases that follow major incidents and decrease mortality and morbidity for all patients.
There are many actions needed during the response phase. Those actions are classified according to the condition of the facility.
Health facility is intact, accessible, and functioning:
Activate the major incident response plan. This is the decision taken by PHC facility leader in liaison with the local and state health authority.
Change the layout of the facility and the patients’ flow to permit faster management of patients with acceptable standards.
Call the staff who are in their off or vacation to join work in the facility to deal with the increased numbers of patients and visitors.
Stop elective visits of patients, and replace it by telephone or online consultations. If there is a need to see the patient and examine him physically, then go to him/her or call the patient to the center; examine the patient in area away from the incident management venue. This will decrease the number of patients attending to the health center and create surge capacity to examine patients related to the major incidents.
Contact local health authority and nearby hospitals to liaise about the situation and work distribution. The PHC can have an important role in dealing with the well and worried people and patients with mild symptoms to decrease the load on hospitals and minimize people’s need for transportation. The movement restriction is an important factor in controlling epidemics and makes movement of emergency services easy and fast.
Make special documentation for the incident patients and other related issues like questionnaires, asking about relatives, etc. This will help in better preparedness for future incidents.
PHC director and supervisors should observe staff for signs of PTSD. Daily debriefing session should be done by the end of the shift. The management of PTSD staff will depend on the severity of symptoms.
Mild PTSD usually is solved by the support of colleagues and the daily debriefing session.
They may change the type or place of work of the staff if they noticed moderate symptoms of PTSD.
Staff with severe symptoms of PTSD should be stopped from dealing with patients, and psychologist or psychiatrist consultation is requested.
If the primary health facility is not accessible or not functioning:
The alternative site should be activated, and directional signs to the new site, people, and authorities should be informed about the new site.
The concept of emergency medical teams applied in disaster management is well known worldwide and applied by the WHO and other bodies interested in disaster response. We can apply this concept to distribute the primary health staff into small teams and direct them (as forward teams) to different residential areas in conjunction with other emergency services. Their duty is to:
Define the special needs and vulnerable population.
Supply chronic medications to the needed.
Defining affected people and do baseline life support interventions.
Contact health authority or hospitals for patients who need to transfer to higher level of health care.
This concept, going to patients in their residency, can be used even if the facility is accessible and functioning. It is an extra service for people in catchment area aiming to bring health care very near to people and help in stopping the spread of epidemic infections.
Start thinking of recovery for the health centers during a long-lasting major incident, for example, the COVID-19 pandemic, which is affecting the whole world now. There should be regular thinking of how to resume work in the PHC to serve the patients who need regular follow-up. This can be done by telephone or network meeting (as mentioned above), and medications can be delivered to the patient at their homes through the post. Fees can be paid by electronic payment methods.
Live experience on the role of PHC in epidemic.
During the writing of this chapter, there is a COVID-19 epidemic. In the beginning of the epidemic, many people came to the emergency department asking for checkup and PCR test for COVID-19. This issue created overcrowding in emergency departments all over the country, and many people present are requesting the test. This was dangerous for spreading the infection if someone is really infected. Two days later, the PHC sets up centers for dealing with well and worried persons and provided COVID-19 PCR test. This action by the PHC did a huge decompression to emergency departments, decreased mix between well and feverish persons, and gave us opportunity to concentrate on symptomatic patients.
Recovery from the effects of the MI occurs in this phase. Sometimes, when the MI takes long time, the PHC should resume receiving patients other than MI. There is merging between trans-impact and post-impact phases. Recovery of the PHC will be better if it was part of the pre-incident plan [12].
Many times, there are epidemics after major incidents especially if the incident affected the infrastructures of basic services like potable water and electricity. This effect is aggravated if people need to migrate from their area for any reason and assemble in a new area, which is usually less suitable and overcrowded. This permits for disease transmission creating an epidemic between the migrants. Acute respiratory infections and cholera are among the most epidemics that occur in immigrants [13]. Some of these diseases are not present in the PHC area previously.
New patients may be added to the PHC from two sources:
In the first source, there is an epidemic in the area covered by the PHC after the major incident. More patients are presented to PHC for consultations.
The second source is the new people who moved to their area from other places, which rendered unsuitable for living (temporarily or permanently) by the major incident.
New diseases may occur due to loss of infrastructure and sanitation or due to earth changes, leading to bacterial, fungal, and other infectious organisms that are not common in the area. It is found in one study that there was an increase in visits for patients complaining of respiratory symptoms (mostly asthma) and diabetes [14].
In the last 40 years, all the studies showed that there is a significant increase in stress levels of both health care staffs and patients in post-major incident phase [15]. This will decrease the working staff on the one hand and increase the patients visiting the PHC on the other hand.
Delayed appointments for patients already registered in the PHC due to MI response actions. After the end of the incident, work need to be back to normal, and the patients whom appointments were postponed in the response period need to be rescheduled in addition to the regular appointments and providing appointments to the new patients.
Gathering information and statistics about the MI and its effects on the PHC in all aspects. Information regarding response to the major incidents, patients’ flow, number of patients, types of complaints, areas of crowding and delay, etc. all need to be collected, summarized, studied, and used in this phase to prepare the PHC for improved response in the next major incident.
Funding. During the trans-impact phase, the media concentrate on every activity done by all emergency services. This media coverage is a good motivation for providing funds by governments, nongovernmental organizations, and personnel. In the post-impact phase, this media coverage will decrease a lot especially if there is another major incident in other parts of the world.
This stage is an opportunity to improve the PHC and rebuild it better than before, by applying risk reduction and optimizing work protocols [16]. The actions in the post-impact phase can be divided into three stages [17]:
Early recovery stage
In this stage there is emphasis on clinical services; the PHC is trying to go back actively again. It is trying to open its services to people if the facility was closed during the incident’s response phase or increase work if it was working in limited scope during the previous phase. The following are the steps to do this objective:
Maintain the PHC building, especially if it is affected by the incident. If there was an epidemic, then there is a need to deep cleaning and sterilization before allowing entrance and providing service to usual patients.
Debriefing to staff after the trans-impact phase. This may need psychologist or psychiatrist sometimes. The PHC director should evaluate the staff during all the phases. In this phase there is time to deal with the staff who shows mild-to-moderate PTSD.
Rearrange duty roasters for staff in a way that they can work and have time to look after their families.
Education to staff regarding new diseases that occurs in the post-impact phase. This should be arranged with local health authority and hospitals. There will be many questions regarding the new disease and relations to the chronic diseases they have.
Rehabilitation stage
The aim of this stage is to reach the pre-impact stage level of work and go back to normal activity or put a new norm to help in better response in the future. Activities in this stage are the following:
Maintain used equipment and fix the damaged ones. Electricity generators, water tanks, and ultrasound machines all should be checked by specialized teams.
Refill stores with medicines and consumables. When there is MI, there is large number of patients which is more than the present resources. After the end of the trans-impact phase, stores should be rearranged to have enough supply of the previous drugs and stuff for the new emerging diseases.
Rearrange patients’ visits to decrease delay, and catch the condition before the incident.
Make appointments for the new patients added to the PHC facility after the impact. This may need recruitment of new staff to accommodate those patients in addition to the delayed patients mentioned in the previous point.
If an alternative place has been used and proven to be better than the original one, try using it permanently. All actions, official documentations, and addresses should be changed to the new place.
Development stage
This is a long-term stage, and its aim is to prepare the PHC for the next major incident. It will be mixed with the mitigation and preparedness stages in the pre-impact phase. To achieve this stage properly, there is a need to:
Collect data through statistics regarding different aspects of the incident, for example, numbers, diagnosis, age, etc.
Make plans for better facility; this may need change or an increase in facility building. Make plans to divide the facility into sectors if needed to maintain isolation for containing infections and easy sterilization of the parts of the facility. This should include the ventilation system of different areas.
Improve communication plan to the staff. All staff should have their contact addresses including the social media addresses and landlines if present.
Arrange with the local health authority to have any specific PHC plan to be part of a master plan and any PHC capabilities to add to the total state or national health abilities, and there is no need for doubling the work.
Fund raising should be started and requested from the local and state health authority to do all these changes in the facility and train the staff on different aspects of the MI response.
The primary health care system has big and important roles that can be played in response to major incidents. They are the most nearby health care facility and well known to people in each area, making it the nearest health facility that is ready to provide help when people are exposed to a sudden incident.
Their role is not limited to any stage in the incident, but it is in all phases. In some aspects of response, the primary health care staff will guide the state or national efforts to find and help vulnerable population in their catchment area.
More concentration on the primary health care system in terms of staff training, facility floor plans, and stuff stockpiling will yield a remarkable improvement in response to any major incidents.
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The Corresponding Author (acting on behalf of all Authors) and INTECHOPEN LIMITED, incorporated and registered in England and Wales with company number 11086078 and a registered office at 5 Princes Gate Court, London, United Kingdom, SW7 2QJ conclude the following Agreement regarding the publication of a Book Chapter:
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\n\n6.1 Unless prevented from doing so by events outside its reasonable control, IntechOpen, in its discretion, agrees to publish the Chapter attributing it to the Corresponding Author and any Co-Author.
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\n\n6.3 IntechOpen is granted the authority to enforce the rights from this Publication Agreement, on behalf of the Corresponding Author and any Co-Author, against third parties (for example in cases of plagiarism or copyright infringements). In respect of any such infringement or suspected infringement of the copyright in the Chapter, IntechOpen shall have absolute discretion in addressing any such infringement which is likely to affect IntechOpen's rights under this Publication Agreement, including issuing and conducting proceedings against the suspected infringer.
\n\n7. MISCELLANEOUS
\n\n7.1 Further Assurance: The Corresponding Author shall and will ensure that any relevant third party (including any Co-Author) shall, execute and deliver whatever further documents or deeds and perform such acts as IntechOpen reasonably requires from time to time for the purpose of giving IntechOpen the full benefit of the provisions of this Publication Agreement.
\n\n7.2 Third Party Rights: A person who is not a party to this Publication Agreement may not enforce any of its provisions under the Contracts (Rights of Third Parties) Act 1999.
\n\n7.3 Entire Agreement: This Publication Agreement constitutes the entire agreement between the parties in relation to its subject matter. It replaces and extinguishes all prior agreements, draft agreements, arrangements, collateral warranties, collateral contracts, statements, assurances, representations and undertakings of any nature made by or on behalf of the parties, whether oral or written, in relation to that subject matter. Each party acknowledges that in entering into this Publication Agreement it has not relied upon any oral or written statements, collateral or other warranties, assurances, representations or undertakings which were made by or on behalf of the other party in relation to the subject matter of this Publication Agreement at any time before its signature (together "Pre-Contractual Statements"), other than those which are set out in this Publication Agreement. Each party hereby waives all rights and remedies which might otherwise be available to it in relation to such Pre-Contractual Statements. Nothing in this clause shall exclude or restrict the liability of either party arising out of its pre-contract fraudulent misrepresentation or fraudulent concealment.
\n\n7.4 Waiver: No failure or delay by a party to exercise any right or remedy provided under this Publication Agreement or by law shall constitute a waiver of that or any other right or remedy, nor shall it preclude or restrict the further exercise of that or any other right or remedy. No single or partial exercise of such right or remedy shall preclude or restrict the further exercise of that or any other right or remedy.
\n\n7.5 Variation: No variation of this Publication Agreement shall be effective unless it is in writing and signed by the parties (or their duly authorized representatives).
\n\n7.6 Severance: If any provision or part-provision of this Publication Agreement is or becomes invalid, illegal or unenforceable, it shall be deemed modified to the minimum extent necessary to make it valid, legal and enforceable. If such modification is not possible, the relevant provision or part-provision shall be deemed deleted.
\n\nAny modification to or deletion of a provision or part-provision under this clause shall not affect the validity and enforceability of the rest of this Publication Agreement.
\n\n7.7 No partnership: Nothing in this Publication Agreement is intended to, or shall be deemed to, establish or create any partnership or joint venture or the relationship of principal and agent or employer and employee between IntechOpen and the Corresponding Author or any Co-Author, nor authorize any party to make or enter into any commitments for or on behalf of any other party.
\n\n7.8 Governing law: This Publication Agreement and any dispute or claim (including non-contractual disputes or claims) arising out of or in connection with it or its subject matter or formation shall be governed by and construed in accordance with the law of England and Wales. The parties submit to the exclusive jurisdiction of the English courts to settle any dispute or claim arising out of or in connection with this Publication Agreement (including any non-contractual disputes or claims).
\n\nLast updated: 2020-11-27
\n\n\n\n
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