Natural Coral as a Biomaterial Revisited

This paper first describes the state of the art of natural coral. The biocompatibility of different coral species has been reviewed and it has been consistently observed that apart from an initial transient inflammation, the coral shows no signs of intolerance in the short, medium, and long term. Immune rejection of coral implants was not found in any tissue examined. Other studies have shown that coral does not cause uncontrolled calcification of soft tissue and those implants placed under the periosteum are constantly resorbed and replaced by autogenous bone. The available studies show that the coral is not cytotoxic and that it allows cell growth. Thirdly, porosity and gradient of porosity in ceramics is explained based on far from equilibrium thermodynamics. It is known that the bone cross-section from cancellous to cortical bone is non-uniform in porosity and in pore size. Thus, it is hypothesized that a damaged bone containing both cancellous and cortical bone can be better replaced by a graded/gradient porous implant based on the idea of a biomimetic approach. The purpose of this article is to review and summarize all the pertinent work that has been published on natural coral as a bone graft during the last twenty years including in vitro , animal, and clinical human studies. In addition, as an illustration, we report the clinical experience of one of us using coral. It is a case study of complex femoral fracture (Table 1) where the essential role of vascularization and stabilization of the fracture site are underlined. The results are supported with more than 300 other femoral fractures treat- ed using the same modus operandi. Finally, this paper overviews the ecological and ethical concerns around the use of corals as well as discussing briefly about recent impacts of nano-pollutants. Abstract This is the story of a patient that has been hospitalized following a car accident for a complex fracture of the lower third of the femur. However, the bones non-union is confirmed. It was then necessary to perform another surgery, removing the initial material in addition to carrying out an anatomical reduction. A screwed plate- blade Poitout, et al. [95,96] and a biomaterial graft Cirotteau, [97,98] are performed to stimulate new osteosynthesis. Consolidation took 4 months; the femoral shaft is anatomically reconstructed in 1 year and there were no sequelae. The follow-up was made on a period over 2 years. This case study highlights, among other things, the essential role of vascularization. And the results are supported by more than 300 other femoral fractures treated according to the same surgical procedure. ne an emergency surgery when came the hospital. The lower third of her femur was fractured. It was a complex fracture including bone non-union. The femur was osteo-synthesized using a long plate. The patient refuses a re-operation proposed by the second

million bone grafts annually to treat some 7 million fractures at a cost of $2.5 billion. In the EU there are currently an estimated 22 million women and 5.5 million men aged between 50-84 years old with osteoporosis and the rate of hip-related fractures will increase accordingly Esther, et al. [1]. Autologous bone grafting has all the properties of the ideal graft material, being an osteoinductive and osteoconductive scaffold with no immunogenicity and containing significant numbers of osteoprogenitor cells. However, its use has several drawbacks including limited availability, variable graft quality, surgery complications, increased operative time and donor site morbidity Giannoudis, et al. [2][3][4].
Ceramics are widely used for bone repair and bone regeneration in orthopedics and dentistry. From the end of the 1980s, our biomaterials laboratory at Polytechnique Montreal got involved in the development of alumina ceramic prostheses Boutin, et al. [5]. Boutin introduced alumina ceramics for hip prostheses in the early 1970s. Before this, metal was the femoral end (head/ ball) that was used. Unfortunately, the metal system usually failed because of wear based on Cobalt-Chrome (CoCr) grinding away at the polyethylene cup. Much of this was because the metal could only be polished to a certain level of smoothness. The failure was accompanied by a large amount of wear debris in the joint space.
The result of this wear debris was characterized as particle disease.
The use of polished alumina as part of the articulating joint was motivated mainly by its exceptionally low coefficient of friction and low wear rates Lerouge, et al. [6,7]. The superb tribology properties Subsequently, our group accompanied this development during a decade of collaboration with Professor Sedel's laboratory, until the approval of alumina prostheses by the FDA in 2003 Lerouge, et al. [6][7][8][9] In parallel, our laboratory hasalso been involved later in resorbable ceramics such as coral in the early 2000s Demers, et al. [10]. Coral bone graft substitutes have been supplemented in the past with growth factors to further enhance bone regeneration in defects. Little is known, however, on the dynamics of protein release from coral. Coral particles were studied for their ability to release transforming growth factor beta 1 (TGF-β1) in vitro, under different adsorption conditions. Moreover, it was found that coral is biocompatible Petite [11]. Studies in our laboratory suggest that coral particles could be used as a delivery system for growth factors, and that the release rate may be modulated through modification of the adsorption conditions and coral particle size Demers, et al. [12].
In short, the purpose of this article is to review and summarize all the pertinent work that has been published on natural coral as a bone graft during the last twenty years including in vitro, animal, and clinical human studies. Furthermore, porosity and gradient of porosity in ceramics are explained based on far from equilibrium thermodynamics. In addition, the clinical experience of one of us is highlighted.

State of the Art Coral Materials
Over the past decade, a new generation of biomimetic or bioinspired materials has been developed to provide biophysical and biochemical cues intended to bone tissue regeneration. Coral skeleton is one of the candidates often used as bone biomaterial or a source of inspiration for designing 3D bone scaffolds Ehrlich, et al. [13][14][15][16][17]. Moreover, as recently reported by Gancz and co-workers, "the coral skeleton biomaterial may act as a strong, promotive scaffold for tissue regeneration due to its ability to reduce its rejection by inflammatory reactions such as phagocytosis" Gancz et.al. [17]. Coral-derived material is biocompatible, structurally similar to human bone, with young's modulus of 0.580 to 9.032 GN m −2 Boller et al. [18], non-toxic, biodegradable and of low immunogenicity Ehrlich, et al. [19].

Resorption and Neoformation Process
The studies by Guillemin,et al. [20] show that the process of to be dependent on the action of osteoclasts that are constantly found at the edges of implanted coral fragments. This action has been reported to the activity of the carbonic anhydrase they contain Gay, et al. [21,22] because the intervention of this enzyme in the destruction of carbonate substrates has already been demonstrated.
Indeed, animal studies have showed that the injection of specific inhibitors (such as acetazolamide) of carbonic anhydrase resulted in a significant slowing resorption of the implanted coral, compared to the case not treated with this inhibitor. This slowing down was associated with bone necrosis at the edge of the implant, leading in all cases to pseudo-osteoarthritis. Carbonic anhydrase an enzyme that assists conversion of carbon dioxide and water into carbonic acid, protons, and bicarbonate ions -would play a role in within the osteoclast, the role of proton pump increasing the extracellular pH and promoting the dissolution of minerals.

Biocompatibility
The biocompatibility of coral in different species has been evaluated by different authors. It has been consistently observed that apart from an initial transient inflammation, the coral shows no signs of intolerance in the short, medium, and long term.
None of the following reactions were reported: acute or chronic inflammation, infectious reaction with neutrophils, rejection reaction with proliferation of round cells, fibrous encapsulation.
Immune rejection of coral implants was not found in any tissue examined. Other studies have shown that coral does not cause uncontrolled calcification of soft tissue and those implants placed under the periosteum are constantly resorbed and replaced by autogenous bone. Most in vitro studies have analyzed the biocompatibility between coral and osteoprogenitors. This support material should allow the attachment, proliferation, differentiation of MSCs and osteoblasts Tran, et al. [23]. The available studies show that the coral is not cytotoxic and that it allows cell growth Shamsuria, et al. [24].
When cells are placed on the coral granules, they show a good capacity for attachment, spreading and proliferation.
Following osteogenic induction, alkaline phosphatase activity and the presence of mineral matrix have been observed in the coral material and their count were significantly higher in osteoblasts implanted in coral than in other ceramic materials. Analysis of gene expression osteoblasts implanted in Porites-like coral showed increased expression of RUNX 2, osteopontin, alkaline phosphatase and osteocalcin. The authors concluded that the coral is a favorable material for the implantation of osteogenic cells Lean, et al. [25].
Finally, a study comparing the implantation of osteogenic cells in a coral graft vs a human bone graft concluded that the osteogenic differentiation of MSCs is superior in a coral graft compared to a bone graft. An important and differentiating factor is the increased expression of osteonectin. We have tried to extend the properties of coral by adding osteoinductive molecules. Coral particles can absorb and diffuse TGF-β1 in vitro Demers, et al. [10]. Another study shows that a chitosan/coral composite material was combined with a plasmid encoding the PDGF-β protein. The proliferation of inseminated cells was increased.

Porosity of Bone Biomaterials: Lessons from Bone and Natural Coral
Most porous biomaterials developed and studied so far are homogeneous in terms of pore size distribution, porosity distribution, composition, and mechanical properties. However, bone cross-section from cancellous to cortical bone is known to be non-uniform in porosity and in pore size. Therefore, a graded/ gradient porous will mimic better the natural bone structure.
During the last decades, several methods have been developed for the fabrication of graded/gradient porous biomaterials Miao, et al. [26,27].

Gradient of Porosity
Gradients are largely present in the body leading several events and processes in the living organisms. Structural and composition gradients can be found in the body mainly at the interface between tissues. For example, a structural gradient is found in long bones in radial direction and in flat bones in axial direction, presenting a variation in bone density from the cancellous bone to the cortical bone Wang, et al. [28]. The architecture of bone is such that the resulting porosities are non-uniform in nature. Sobral, et al. [29] showed that the creation of a gradient in scaffold porosity and pore size could influence human mesenchymal stem cells differentiation by impacting cell density and nutrients availability. It was hypothesized that 3D scaffolds presenting a gradient structure could provide cues similar to the native environment and may guide stem cells to differentiate toward the lineage of the targeted tissue to be regenerated. Taken together the findings of Sobral, et al. [29] introduce pore size gradients as a structural factor that could be taken into consideration when combining scaffolds and stem cells for bone tissue engineering purposes. Interconnected porosity allows the supply of blood and nutrients for the viability of bone.
Furthermore, pore interconnectivity is defined as the following fraction: where Mechanical properties and cell/tissue ingrowth behavior depend on the pore size, total porosity and pore interconnectivity in different ways. In fact, porous biomaterials have much reduced Young's moduli compared to the dense counterparts, thus it is possible to match the Young's moduli of porous implants with those of bones. This match in young's modulus serves to minimize the problem of stress shielding. Stress-shielding is the phenomenon whereby a prosthesis having a biomaterial Young's modulus that is too high compared to that of its neighboring bone causes the degradation of this neighboring bone. Indeed, the Young's modulus of the biomaterial being greater, it provides a greater proportion of the stress support, thus leaving the bone under-stressed. The body will then respond to this under solicitation by sending osteoclasts to resorb the non-used bone.

Porous Biomaterials as Dissipative Structure
Biological structures have always been a source of inspiration for solving technical challenges in architecture, mechanical engineering, or materials science. It seems that the internal architecture of living tissue, including porosity gradients are self-organized phenomena that occur under conditions far from thermodynamic equilibrium.
According to Prigogine, irreversible processes can be thought of as thermodynamic forces and thermodynamic flows; the latter being a consequence of the thermodynamic forces. For example, a concentration gradient is the thermodynamic force that causes the flow of matter. In general, the irreversible change in entropy d i S is associated with a flow dX of a quantity, such as matter, that has occurred in a time dt. For the flow of matter, dX=dNmoles of substance that flowed in time dt.
Thus, the change in entropy can be written in the form (1): where F is the thermodynamic force. . to ordered, compartmented spatial structures Rossi, et al. [30].
Entropy undergoes a sharp reduction because it is dissipated into the wider environment in these spatial ambits. The amount of entropy dispersed is greater than that produced by the system. In this way, the process is irreversible and spontaneous. Biotic systems pass from conditions of minimum entropy production to conditions of maximum entropy production, in which high dissipation creates and maintains system order. The basics of this concept are well illustrated by low-level self-organized systems, like the formation of Benard cells Prigogine, et al. [31]. The following ( Figure 1)  Bejan, et al. [33] "For a finite-size flow system to persist in time (to live), its configuration must evolve freely in such a way that provides greater and greater access to the currents that flow through it." According to the Constructal Law, a living system is one that has 2 universal characteristics: it flows (i.e., it is a nonequilibrium system in thermodynamics), and it morphs freely toward configurations that allow all its currents to flow more easily over time. Life and evolution are a self-standing physics phenomenon, and they belong in physics Bejan, et al. [33]. Although it is currently impossible to determine the trigger, or establish where in the system the process arises, its beauty, stability and potentiality to evolve is lively testified within the biosphere. A magnificent organismic manifestation can be found in archaic invertebrates, within the Phylum cnidaria -commonly known as corals (see Figure 1). Although corallite development is rather complex, the modular morphology of Siderastrea share some essential features of Benard cells in that they tie together dissipative structures and the associated flow of energy to yield distinctive morphological phenotypes.

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Copy@ LH Yahia This similarity with Benard-Rayleigh-like cell type, along with re-organization giving rise to a honeycomb-like appearance is not accidental at all even though it is biochemically rather than thermically driven Rossi, et al. [30]. al. [38] suggested to use this dissipative structure approach to optimize the design of scaffold for tissue regeneration Ingber, et al. [38]. This will then permit the design of thermodynamically optimal scaffold geometries that could facilitate the energy dissipation and consequently the in vitro formation of multicellular patterns similar to authentic tissue structures. A scaffold design methodology, based on thermodynamically relevant indices that incorporate key design parameters, can circumvent the vast combinatorial combination of parameters that are presently tested with trial-and-error approaches, which create difficulties in deciphering their relative importance Ingber, et al. [38]. The term biomimicry was coined by biologist Janine Benyus in her 1997 book Biomimicry-Innovation Inspired by Nature, where she reintroduced the term to scientific literature and widely broadened its usage Benyus [39]. The Earth's biological materials and the processes through which they're generated represent the fruits of about 3.8 billion years of research and development.
Biomimetic materials such as coral exoskeletons possess unique architectural structures with a uniform and interconnected porous network that can be beneficial as a scaffold material for bone regeneration. We were among the first to postulate that the porosity gradient of biomaterials played a crucial role in the regeneration and growth of bone Hernandez et al. [40]. Recently, this hypothesis was confirmed in TCP scaffolds Zhim, et al. [27,41]. In another recent and better mechanical properties. One way to deal with the wide variations in pore sizes in the scaffolds is the introduction of gradient or hierarchical porosity, which is also observed in tissues such as skin, cartilage, and bone. Gradient porosity is also known to promote specific cell migration during tissue engineering, which is a requirement, e.g., for the treatment of articular cartilage defects in osteochondral tissue engineering Bretcanu et al. [42,43].

Fabrication of Porous Biomaterials
In the 1980s, in the USSR (Siberian Physical-Technical Institute), porous Nickel-Titanium (NiTi) alloys were obtained using the self-propagating high-temperature synthesis (SHS) process or combustion synthesis (CS) in an inert atmosphere, followed by successful clinical use of implant systems made of porous SHS NiTi Itin, et al. [44,45]. The SHS method to synthesize refractory ceramic compounds was initially proposed and comprehensively described by Merzhanov, et al. [46]. SHS, as a powder metallurgy method, turned out to be the most appropriate for the fabrication of the porous NiTi body having the specified characteristics Li, et al. [47,48]. Changing SHS variables such as starting powders, loose compacting degree, heating rate/schedule, ignition temperature etc., one may fabricate the different structure of porous body having a particular pore size and predetermined pore size distribution, which is known to be so crucial in cellular and tissue engineering Gunther, et al. [49]. Historically, it seems that porous NiTi was developed initially in Soviet Union during the cold war to trap hydrogen isotopes Rames, et al. [50]. Nuclear fusion constitutes a new source of energy that needs the use of significant amounts of deuterium and tritium. In fact, certain fusion reactors could generate large volumes of gas consisting of helium and hydrogen isotopes. These complex mixtures must be treated directly on site, so that tritium can be recycled. By balancing these parameters, the energy of their action is controlled to create the desired product stoichiometry, porosity, and mechanical properties. SHS provides a means to rapidly manufacture materials, saving time and production costs as well as enabling the synthesis of custom devices with individual molds.
Mold materials can range from graphite to paper or paper machete.  [62]. Coral, he thought, would make great bone grafts. The pores of the coral skeletons were uniformly sized, evenly distributed, and completely interconnected, which would allow bone and blood cells to flow through the implant and new blood vessels and bone tissue to grow into the graft. White et al. [62] outlined some of these advantages in a 1972 paper in Science and suggested that Porites coral, often called finger coral, might make a particularly good source of implant material. Chiroff and coworkers placed calcium carbonate (CC) in cancellous defects in dogs for 8 weeks and found that the material was biocompatible and that new bone could fill the pores. Some implants were left for 1 year and were observed to be almost completely resorbed Chiroff et al. [62].
The first clinical reports were published in France by the ''Institut de Recherches Orthopédiques, Université René-Descartes Paris V'' in 1980 Patel, et al. [66]. Since then, CC has been used clinically in maxillofacial surgery to correct periodontal defects [67,68] and to fill and reconstruct bony defects in cranial surgery Roux, et al. [69,70]. The craniofacial bones can be augmented by the granular form of CC Marchac, et al. [71]. In orthopaedic surgery CC has been used as a filler in tibial osteotomies Kenesi, et al. [72], in bone tumour surgery Rouvillain et al. [73] and in lower limb metaphyseal fractures to support articular surfaces de Peretti, et al. [74]. The possibly most appropriate indication at the moment is spinal fusion, where CC can be used to diminish the amount of bone grafts in conjunction with autogenous bone Pouliquen, et al. [75]. Very little exact information exists also on the resorption time of CC. It seems to depend on the animal species used. When the implant was placed in the cortex of the femur in pigs, 64% of the CC blocks were resorbed after 1 month, whereas in sheep the figure was 93% Guillemin, et al. [65]. The granular form has been observed to resorb completely at 24 weeks in a connective tissue site in pigs but, in humans, the same material placed in subcutis can still be found after several years Marchac, et al. [71]. Roux and coworkers reported almost complete resorption after 1 year in 50% of cases when coral was used to fill craniotomy burrholes in humans Roux, et al. [69]. Larger blocks used in humans have still been X-Ray positive after 4 years de Peretti, et al. [74]. Coral resorption is most active in the bone implant contact areas and proceeds centripetally Braye, et al. [76]. Carbonic anhydrase, an enzyme abundant in osteoclasts, plays a key role in the resorption process. Locally it lowers the pH at the osteoclast implant interface, dissolving the CC matrix Chétail, et al. [21,22,61,77]. Resorption can be halted by the administration of the diuretic acetazolamide, a known inhibitor of carbonic anhydrase Guillemin, et al. [61].
Moreover, according to Fricain and coworkers, data suggest that both fibroblasts and macrophages dissolve the coral, and that one of the mechanisms is the intracellular degradation in phagolysosomes [78]. A prerequisite for the process is direct contact between these cells and the coral matrix [79]. The structure of the commonly used coral Porites, is similar to that of cancellous bone, and its initial mechanical properties resemble those of bone [80].

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The exoskeleton of these high content calcium carbonate scaffolds has since been shown to be biocompatible, osteoconductive, and biodegradable at variable rates depending on the exoskeleton porosity, the implantation site, and the species Oladeji, et al. [81]. Although not osteoinductive or osteogenic, coral grafts act as an adequate carrier for growth factors and allow cell attachment, growth spreading and differentiation. When applied appropriately and when selected to match the resorption rate with the bone formation rate of the implantation site, natural coral exoskeletons have been found to be impressive bone graft substitutes Demers, et al. [10,12]. The harvested coral is purified into the intermuscular space of rabbits were observed to allow bone formation Nade, et al. [87]. The use of injectable biomaterials is currently growing as the demands for minimally invasive procedures, and more easily applicable implants become higher.
However, their clinical availability is still limited due to difficulties associated to their design. Injectable biomaterials are usually referred to as 'bone cements' if they are intended to interact mostly with bony tissue, although soft tissue applications are also becoming more important, in which case the biomaterials are generally referred to as just 'cements', or 'scaffolds' if also intended for tissue regeneration. Injectable and fully degradable radiopaque ceramics may be of interest not only for vertebroplasty Belkoff, et al. [88] with nonacrylic materials, but also for dentistry Hill, et al. [89] and other novel orthopedic applications that demand radiocontrast such as tibioplasty Pizanis, et al. [90], proximal humerus augmentation Gradl, et al. [91], and femoral head treatments Ng, et al. [92]. Strontium halides, except strontium fluoride, are watersoluble and previous investigations have indicated their potential as radiopacifiers Wiegand, et al. [93,94]. To conclude, it is important to recognize that developing materials that can be delivered through a syringe is overall a major challenge, since, assembly of the final implant is intended to proceed in vivo, where the conditions are relatively harsh, difficult to control, and sometimes unpredictable.

Case Study-Personal Series
Abstract This is the story of a patient that has been hospitalized following a car accident for a complex fracture of the lower third of the femur. However, the bones non-union is confirmed. It was then necessary to perform another surgery, removing the initial material in addition to carrying out an anatomical reduction. A screwed plate-blade Poitout, et al. [95,96] and a biomaterial graft Cirotteau, [97,98] are performed to stimulate new osteosynthesis.

Clinical history
On August 12 th , 1999, Miss Hélène G ..., aged 20, had an emergency surgery when she came at the hospital. The lower third of her femur was fractured. It was a complex fracture including bone non-union. The femur was osteo-synthesized using a long plate. The patient refuses a re-operation proposed by the second surgeon despite the practitioner's insistence and against the advice of the family. She persisted in her refusal for 7 months, before finally accepting the procedure.

Method and Technique
The operation took place on March 7 th , 2000. Using the technique described by Robert Judet, et al. [99] the surgeon dissects the nonunion area after removing the initial material. Then he wraps it in a natural coral biomaterial Guillemin,et al. [20,100]. Anatomical reduction of fracture foci is temporarily maintained by forceps and securely fixed by a plate blade Poitout et al. [95,96]. A short antibiotic therapy (5 days) was prescribed. The aftermath was simple. From the 12 th day, the patient was authorized to use partial support with 2 crutches, and she underwent a few rehabilitation sessions. After 3 months, the patient was able to let go of one of the crutches and the abandonment of the second followed after the 4 th month. At the 3 rd month clinical examination, the mobility of the knee was normal, there was no more lameness, and the femur was painless. After 8 months, the patient was able to regain normal muscle mass. After 1-year, unrestricted sports activity was allowed.
The follow-up was up to 2 years.

Equipment
Plate-Blade: The material of the plated blade is stainless steel Poitout et al. [95,96]. The 6.5cm long blade is bent at 95° and is drilled with 9 holes for 5mm diameter screws. The 3 upper screws take 6 cortices above the most proximal fracture line. Biomaterial: Natural coral Guillemin et al. [100] is Porites, with a porosity of 50% in which fluid can flow and interconnecting pores with an average pore size between 150 to 500mm. The circulation of bone marrow cells (as well as blood fluids, anions, cations ...) is favored by the volume, the thickness of the walls of the pores and the structural regularity. The biomaterial, which is 98% Calcium Carbonate, is used in the form of spheres 1.5 to 2 mm in diameter.
The architecture of natural coral is favorable to bone growth. In addition, natural coral has remarkable mechanical properties such as resistance to compressive stresses, even when the pore volume approaches 50%, which is identical to that of cancellous bone.
However, the mechanical strengths in bending and in torsion are

Evolution of vascularization of a long bone
It is well known that vascular supply changes over time Kadiyala,et al. [102]. In toddlers, the vascular supply to the cortex emanates exclusively from the bone marrow ( Figure 2). The richly vascularized periosteum is perfectly integrated with the cortex and does not penetrate. At a later age, the quality of vascular supply to bone tends to decline. In this angiographic section of a crosssection of a 42-year-old femur see Figure 3

Resorption of Natural Coral Grafts
Rejection phenomena have been observed -both clinically and histologically, in cases where the coral used was of large size (For example with osteotomy wedges greater than 5°). It has been proven that the foreign body reaction was due to insufficient purification at the core of the biomaterial. The development of a technique using supercritical fluids by Prof. Yann Le Petitcorps & coworkers, made it possible to remove the remaining proteins from the core. The biomaterial, thus purified, has been used without any rejection, nor any reaction to foreign body.

Role of Vascularization
In necrotic bone or in bone in contact with inert metallic material (such as a plate, screw, nail), there is no possibility of developing adequate vascularity. The (Figure 7) shows as an example that there is no modification of the biomaterial of the coral Porites spheres in contact with the inert metallic biomaterial.
In fact, the biomaterial remains in the mineral state, surrounded by an avascular fibrous sleeve. This supports the argument that vascularization has a fundamental role. On the contrary, adjoining the well-vascularized cancellous bone, the biomaterial is integrated and assimilated by a double mechanism: demineralization process with the osteoclast's activities, then remineralization process with the osteoblast's actions, and a newly formed bone appear as shown in Figure 8.

Methodology of the Mechanical Evolution of Coral Graft
On the frontal and lateral radiographs (taken at 1 meter distance, by the same operator and with the same device), it is visually possible to distinguish the spheres of coral infused with bone marrow which are clearly individualized. On the anterior and posterior surfaces (in lateral radiography) and on the external and internal surfaces (in frontal radiography), the spheres were counted and then recorded on a sheet of millimeter paper as shown in Figure 9. They were counted at different stages of consolidation.
In fact, there are 5 phases of reconstruction. In phases III and IV, an alignment of the external spheres along the major axis of the diaphysis was observable, suggesting the beginning of the organization of the Haversian system. This example, shown in Figure 9, is that of a young patient, 19-years-old, suffering from a non-union of the femur treated by 2 external fixators (front and side), thus allowing the counting of the balls on the 4 faces. It can be seen that the resorption is identical at the level of the 4 faces.
By plotting the number of remaining spheres as a function of time on a graph presented in Figure 10, one obtains a logarithmic plot in geometric degression, suggesting an identical and coherent physiological process of resorption.

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Radiographic Evolution of the Femur of the Young Patient
Here in this section, we go over the evolution (from 0 to 20 months) of the reconstructed bone of the 19-years-old patient. The radiograph story presented in Figure 11 depicts the new internal fixation and bone consolidation after 4 months. The reader can find in the following table1 the different stages of this remarkable reconstruction.

Evolution of the Bone Mineral Density over Time
There is a steady decrease in bone mineral density (BMD) over time. This decline begins on average in young adults around 23 years of age or as soon as the subject has reached bone maturity.
Interestingly, this natural evolution also takes place in patients whose fracture has been restored by combining osteosynthesis and biomaterial. Here in Table 2, the reader can appreciate the natural decrease of BMD in gr/cm 2 of 2 older patients of 80 and 94 years old.

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Ecological and Ethical aspects
This review paper revealed that coral has medical applications,

Nanoparticle Pollution
In recent years, TiO 2 and ZnO NPs in sun care products have received criticism for their possible adverse effects on humans and in the aquatic environment regarding the reactive oxygen species (ROS) they produce when exposed to sunlight Skocaj et al. [107].
Additionally, ZnO NPs are subjected to solubilization into harmful Zn 2+ ions in seawater due to a higher pH environment Wong, et al. [108]. Consequently, non-nano TiO 2 and non-nano ZnO (with nanoparticles measuring>100nm) are becoming increasingly popular for sunscreen formulations produced by smaller, ecoconscious sunscreen companies Maipas [109]. Interestingly, of the countries that permit the use of mineral UV filters, their percentage limit for the amount of a UV filter contained within a sunscreen

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formulation is higher compared to most chemical UV filters (20-25% or no limit for mineral UV filters versus a 10% average limit for chemical UV filters).
However, commercial sunscreen formulas often contain a unique mixture of both mineral and chemical UV filters to produce a broader spectrum of protection Sánchez-Quiles [110]. Many sunscreen manufacturers claim that their sunscreens are "reef safe", but is that true? The studies presented in this capstone clearly demonstrate that even "ecofriendly" sunscreens can have negative effects on marine organisms at very low concentrations. Some claimed "reef-safe" brands contain UV filters that have reproductive inhibition in sediment dwellers Fabrega, et al. [111] when exposed to non-nano UV filter particles. Authors from these studies indicate that these organisms may readily uptake higher concentrations of larger non-nanoparticles due to their higher bioavailability. Still, non-nano UV filters are generally lower in toxicity than other types of UV filters and seem least toxic to Scleractinian corals compared to others.
Unfortunately, there are no current regulations that enforce the integrity of "non-nano" and "reef-safe" advertisement claims, but consumer awareness has recently demanded that manufacturers should be more accurate Sobek, et al. [112]. UV filters that seem promising to the health of marine organisms are non-nano TiO 2 and non-nano ZnO, based on their larger particle size and lower solubility rates in seawater Fabrega, et al. [111][112][113][114]. Contradicting studies, however, found that non-nano UV filters were more toxic to some marine organisms compared to smaller nanoparticles Wong, et al. [108,115]. Specifically, these studies observed DNA damage in hemocytes in filter-feeders D' Agata et al. [115], oxidative stress in crustaceans and fish Wong, et al. [108] and been shown to be toxic to marine life, both mineral and chemical UV filters.

Coral protection
A coral future in medicine can be assured by proper advances  [118], one third of the world's coral species are said to be at increased risk of extinction. Thus, there is a need to look for alternatives, and cultured coral can be a suitable option.

Concluding Remarks and Futures Directions
An ideal bone graft substitute should be osteoconductive, inert, readily available and adaptable in terms of size and shape. The readers can find some of these cases and others on his site: osteoporosis-surgery.fr. Another thing to take into account with the coral material is the initial mechanical weakness. Once bone ingrowth occurs the mechanical stability improves. It is characteristic that the compressive strength of corals could be as low as 2.62 MPa when the one of bone is between 131 and 283 MPa. Even if the abovementioned issues are addressed, corals can be considered a viable solution as a bone graft material only if they are sustainable and with minimal environmental impact [120]. Porites and Goniopora corals that are used for the commercially available products derive from corals of the Pacific and Indian Oceans. These corals are not classed as endangered, however, their overexploitation together with the environmental changes, ocean warming, and acidification could put them at risk. Furthermore, some authors highlighted the negative effect or even complete cessation of the overall calcification that the rising water temperature and acidity have on these corals.
In addition, a substantial decrease in the coral reefs has been noted since 1990 and it is expected that approximately 50% of the reefs will be destroyed by 2030. These data add to the overall uncertainty when planning to explore the utilization of the corals further.

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Despite all the aforementioned concerns, we believe that some coral derived biomaterials are good void fillers with distinct role in our armamentarium. Their utilization should be performed with prior knowledge of the properties of each different product [121].
The fact that they are inner osteoconductive material, safe from a disease transmission point of view, and the need to incorporate an osteoinductive signal to safeguard the overall success, is an undisputable strength. As far as the coralline hydroxyapatite is concerned, this should be considered as a permanent implant, the effectiveness of the partially converted analogue would require further investigation in terms of their overall effectiveness and properties in clinical applications [122]. Tissue engineering approaches with graft supplementation with different osteogenic cells, bone marrow, platelet rich plasma and a number of growth factors is promising but the ideal combination enhancing the neoangiogenesis and osteogenesis needs further clarification.
Research is ongoing on strategies how to enhance and optimize bone repair strategies. Ongoing research Coralline-derived bone grafts are safe, inert osteoconductive material, which are readily available in Nature [123]. Their highly porous structure is similar to cancellous bone. Raw coralline graft products are brittle, lack mechanical strength and are resorbed by the host fast. The conversion to hydroxyapatite diminishes the resorption of the graft making it a permanent implant [124]. Our current clinical evidence is limited to well-contained voids in dental and maxillofacial surgery. Some authors report good clinical results, yet others reported devastating poor outcomes. Until further clarification and development of new coral-based implants that address the shortcomings of the current materials the utilization of such material should be limited to well contained, well vascularized defects, bearing into consideration the potential permanent nature of this graft material [125][126][127].