Biomaterials in search of a meniscus substitute

The menisci fulfill key biomechanical functions in the tibiofemoral (knee) joint. Unfortunately meniscal injuries are quite common and most often treated by (partial) meniscectomy. However, some patients experience enduring symptoms, and, more importantly, it leads to an increased risk for symptomatic osteoarthritis. Over the past decades, researchers have put effort in developing a meniscal substitute able to prevent osteoarthritis and treat enduring clinical symptoms. Grossly, two categories of substitutes are observed: First, a resorbable scaffold mimicking biomechanical function which slowly degrades while tissue regeneration and organization is promoted. Second, a non resorbable, permanent implant which mimics the biomechanical function of the native meniscus. Numerous biomaterials with different (material) properties have been used in order to provide such a substitute. Nevertheless, a clinically applicable cartilage protecting material is not yet emerged. In the current review we provide an overview, and discuss, these different materials and extract recommendations regarding material properties for future developmental research.     

Simulation of the in vivo resorption rate of β-tricalcium phosphate bone graft substitutes implanted in a sheep model

A few years ago, a model was proposed to predict the effect of the pore architecture of a bone graft substitute on its cell-mediated resorption rate. The aim of the present study was to compare the predictions of the model with the in vivo resorption rate of four β-tricalcium phosphate bone graft substitutes implanted in a sheep model. The simulation algorithm contained two main steps: (1) detection of the pores that could be accessed by blood vessels of 50 μm in diameter, and (2) removal of one solid layer at the surface of these pores. This process was repeated until full resorption occurred. Since the pore architecture was complex, μCT data and fuzzy imaging techniques were combined to reconstruct the precise bone graft substitute geometry and then image processing algorithms were developed to perform the resorption simulation steps. The proposed algorithm was verified by comparing its results with the analytical results of a simple geometry and experimental in-vivo data of β-TCP bone substitutes with more complex geometry. An excellent correlation (r(2)>0.9 for all 4 bone graft substitutes) was found between simulation results and in-vivo data, suggesting that this resorption model could be used to (i) better understand the in vivo behavior of bone graft substitutes resorbed by cell-mediation, and (ii) optimize the pore architecture of a bone graft substitute, for example to maximize its resorption rate.     

Osteoclast resorption of beta-tricalcium phosphate controlled by surface architecture

A resorbable bone graft substitute should mimic native bone in its capacity to support bone formation and be remodeled by osteoclasts (OCl) or other multinucleated cells such as foreign body giant cells (FBGC). We hypothesize that by changing the scale of surface architecture of beta-tricalcium phosphate (TCP), cellular resorption can be influenced. CD14⁺ monocyte precursors were isolated from human peripheral blood (n = 4 independent donors) and differentiated into OCl or FBGC on the surface of TCP discs comprising either submicron- or micron-scale surface topographical features (TCPs and TCPb, respectively). On submicrostructured TCPs, OCl survived, fused, differentiated, and extensively resorbed the substrate; however, on microstructured TCPb, OCl survival, TRAP activation, and fusion were attenuated. Importantly, no resorption was observed on microstructured TCPb. By confocal microscopy, OCl formed on TCPs contained numerous actin rings allowing for resorption, but not on TCPb. In comparison, FBGC could not resorb either TCP material, suggesting that osteoclast-specific machinery is necessary to resorb TCP. By tuning surface architecture, it appears possible to control osteoclast resorption of calcium phosphate. This approach presents a useful strategy in the design of resorbable bone graft substitutes.     

Art of replacing craniofacial bone defects

In the history of medicine, many surgeons have been tried to reconstruct lost tissue and correct deformity, attempts to use implant materials have probably paralleled those involving autogenous tissue. Recently there has been an acceleration in the understanding of the requirements and potentials of implant materials caused by collaboration between material scientists, biomaterials engineers, clinicians, and clinical investigators. Alloplastic materials have become an essential part of reconstructing the function and contour of the craniofacial skeleton. Bone is a specialized form of connective tissue, which provides support, and protects vital and delicate organs. Bone is embryologically derived from mesenchymal tissue through membranous and endochondral ossification. In the clinical field, the need for bone graft has been increased due to trauma, tumor, craniosynostosis, and pure esthetic bone surgery. Various types of bone grafts have been used to repair craniofacial bone defects over many years, but the autogenous graft has many disadvantages, such as, limited donor sites, donor morbidity, pain, growth deformity and resorption. Many surgeons working in a number of centers around the world have created substitutes and simpler methods for bone replacement. As the alloplatic bone substitute has been advanced, many synthetic substitutes are replaced by bone in vivo over time. The ideal material should be cost effective, non-toxic, non-antigenic, non-carcinogenic, and inert in the body fluids, be easily shaped at the operating table, and maintain its desired form and consistency in situ. This article reviews several of the more commonly used materials for craniofacial reconstruction and summarizes their mechanical properties and clinical aspects.     

Resorbability of bone substitute biomaterials by human osteoclasts

Third generation biomaterials are being designed with the aim that once implanted they will help the body to heal itself. One desirable characteristic of these materials in bone is their ability to be remodeled, i.e. that osteoclasts resorb the material and it is subsequently replaced by newly formed bone through osteoblastic activity. So far the only way to test this biological property of bone substitutes are animal experiments with all their limitations like ethics, costs and limited transferability to man. The present study was designed, to develop a human in vitro assay, allowing to generate human osteoclasts directly on the biomaterial. The assay was validated using calcium phosphate cement and PMMA as biomaterials. Quantification was performed by raster electron microscopy and computer assisted image analysis. Dentin was used as internal standard. Our assay shows iso-bone resorbability of calcium phosphate cement in comparison to unresorbable PMMA cement. Both current clinical orthopedic practice and future skeletal engineering may profit from the availability and use of a test system for the assessment of resorption quality. The assay presented here allows to address this question of resorbability and to select the best materials for the use as bone substitutes in specific patients.     

A novel hydroxyapatite ceramic bone substitute transformed by ostrich cancellous bone: characterization and evaluations of bone regeneration activity

Various biomaterials have been used for bone repair and reconstruction of bone defects. Inorganic xenogenic bone substitutes have been intensively studied because they possesses favorable regenerative properties. The purpose of this study was to evaluate the properties of a novel inorganic xenogenic bone substitute, sintered ostrich cancellous bone (SOCB). Bone regeneration capability was also comparing to that of other bone substitutes in rabbit calvarial defects. Biochemical and biomechanical properties of the SOCB ceramic closely resembled those of human bone. Bone regeneration was evaluated by radiograph, histology, and histomorphometry. Bone regeneration was significantly enhanced in defects treated with SOCB when compared with other bone substitutes. The biochemical and biomechanical properties of SOCB are favorable for bone regeneration. SOCB might be a promising biomaterial for the repair of bone defects.     

Osteoclastogenesis and osteoclastic resorption of tricalcium phosphate: effect of strontium and magnesium doping

Bone substitute materials are required to support the remodeling process, which consists of osteoclastic resorption and osteoblastic synthesis. Osteoclasts, the bone-resorbing cells, generate from differentiation of hemopoietic mononuclear cells. In the present study, we have evaluated the effects of 1.0 wt % strontium (Sr) and 1.0 wt % magnesium (Mg) doping in beta-tricalcium phosphate (β-TCP) on the differentiation of mononuclear cells into osteoclast-like cells and its resorptive activity. In vitro osteoclast-like cell formation, adhesion, and resorption were studied using osteoclast precursor RAW 264.7 cell, supplemented with receptor activator of nuclear factor κβ ligand (RANKL). Osteoclast-like cell formation was noticed on pure and Sr-doped β-TCP samples at day 8, which was absent on Mg-doped β-TCP samples indicating decrease in initial osteoclast differentiation due to Mg doping. After 21 days of culture, osteoclast-like cell formation was evident on all samples with osteoclastic markers such as actin ring, multiple nuclei, and presence of vitronectin receptor α(v)β(3) integrin. After osteoclast differentiation, all substrates showed osteoclast-like cell-mediated degradation, however, significantly restricted for Mg-doped β-TCP samples. Our present results indicated that substrate chemistry controlled osteoclast differentiation and resorptive activity, which can be used in designing TCP-based resorbable bone substitutes with controlled degradation properties.     

Controlling the biological function of calcium phosphate bone substitutes with drugs

There is a growing interest in bone tissue engineering for bone repair after traumatic, surgical or pathological injury, such as osteolytic tumor or osteoporosis. In this regard, calcium phosphate (CaP) bone substitutes have been used extensively as bone-targeting drug-delivery systems. This localized approach improves the osteogenic potential of bone substitutes by delivering bone growth factors, thus extending their biofunctionality to any pathological context, including infection, irradiation, tumor and osteoporosis. This review briefly describes the physical and chemical processes implicated in the preparation of drug-delivering CaPs. It also describes the impact of these processes on the intrinsic properties of CaPs, especially in terms of the drug-release profile. In addition, this review focuses on the potential influence of drugs on the resorption rate of CaPs. Interestingly, by modulating the resorption parameters of CaP biomaterials, it should be possible to control the release of bone-stimulating ions, such as inorganic phosphate, in the vicinity of bone cells. Finally, recent in vitro and in vivo evaluations are extensively reported.     

Human osteoclast formation and activity on a xenogenous bone mineral

To date, the majority of studies on bone substitute materials have investigated their regenerative properties; however, little is known about their resorption processes, forasmuch as it is believed that the ideal biomaterial for bone regeneration must be completely resorbable. This study is aimed at defining the in vitro resorption potential of human osteoclasts (OCLs) on a xenogenous bone mineral (XBM). Peripheral blood mononuclear cells from healthy volunteers were used to generate OCLs in vitro in the presence of macrophage colony stimulating factor and receptor activator of NF-kappaB ligand on bovine bone slices and XBM. By using morphologic and biochemical methods, we observed that OCL formation occurred on XBM and these cells were positive for the major OCL markers. Regarding OCL activity, resorption pits were detected on XBM by reflection and confocal microscopy. However, biochemical analysis revealed that collagen degradation at day 14 and 21 was significantly lower in XBM supernatants when compared to bovine bone, suggesting that XBM underwent a much slower resorption over time. These findings demonstrate that OCLs are generated on, attach to, and resorb XBM though more slowly than native bone, and suggest that cultured human OCLs could be used as a model for comparing resorption rates of bone substitute materials.     

Effect of Osteoclast Co-culture on the Differentiation of Human Mesenchymal Stem Cells Grown on Bone Graft Granules

Traditional approaches to bone repair are currently being integrated with innovative tissue-engineering techniques, as researchers and clinicians shift their treatment focus toward regenerating functional tissue rather than just filling a defect to provide structural support. Cells are expanded and incorporated into implantable systems in hopes of enhancing the bone-forming capabilities of traditional bone graft substitutes. The present study examined how osteoclasts might be used to stimulate the differentiation of human mesenchymal stem cells (hMSCs) into bone forming cells. The two cell types were co-cultured on a resorbable, three-dimensional bone graft substitute. Osteoclasts were seeded prior to the addition of hMSCs, as well as simultaneously, to determine if resorption of the scaffold would have any bearing on observed response by hMSCs. When seeded directly with hMSCs on the 3-D substrates, the osteoclasts had an increase in TRAP expression over time if seeded simultaneously. The co-culture setup had a positive influence on the proliferation of hMSCs. Late stage osteoblast differentiation markers (bone sialoprotein) were positively affected by direct co-culture with osteoclasts. The addition of RANKL to the culture medium for osteoclastogenesis appears to be a factor in the observed responses by hMSCS, but is not the only factor influencing the MSCs. Osteoclasts were shown to have an influence on the development of mesenchymal stem cells into osteoblasts when cultured in vitro. Findings from this study, coupled with the knowledge obtained from our previous work, will aid in the development of a clinically viable mesenchymal stem cell based bone graft system.     

Effect of rapidly resorbable calcium phosphates and a calcium phosphate bone cement on the expression of bone-related genes and proteins in vitro

The use of biodegradable bone substitutes is advantageous for alveolar ridge augmentation because it avoids second-site surgery for autograft harvesting. This study examines the effect of novel, rapidly resorbable calcium phosphates and a calcium phosphate bone cement on the expression of bone-related genes and proteins by human bone-derived cells (HBDCs) and compares this behavior to that of tricalciumphosphate (TCP). Test materials were alpha-TCP, two materials with a crystalline phase Ca(2)KNa(PO(4))(2) and with a small amorphous portion containing either magnesium potassium phosphate (material denominated GB14) or silica phosphate (material denominated GB9), and a calcium phosphate bone cement (material denominated Biocement D). HBDCs were grown on the substrata for 3, 7, 14, and 21 days, counted, and probed for various mRNAs and proteins (type I collagen, osteocalcin, osteopontin, osteonectin, alkaline phosphatase, and bone sialoprotein). All substrates supported continuous cellular growth for 21 days. In the presence of GB14 and Biocement D specimens cell proliferation was reduced and cell differentiation increased. At day 21, the greatest number of cells was found on GB9 expressing significantly higher levels of bone-related proteins than cells grown on all other surfaces. Because all novel materials facilitated the expression of the osteoblastic phenotype at least as much as TCP and the polystyrene control, these biomaterials can be regarded as excellent candidate bone substitute materials. GB9 induced the highest proliferation and cellular differentiation after 21 days of incubation, suggesting that this material may possess a higher potency for enhancing osteogenesis than TCP.     

Epithelial and stromal developmental patterns in a novel substitute of the human skin generated with fibrin-agarose biomaterials

Development of human skin substitutes by tissue engineering may offer new therapeutic alternatives to the use of autologous tissue grafts. For that reason, it is necessary to investigate and develop new biocompatible biomaterials that support the generation of a proper human skin construct. In this study, we generated a novel model of bioengineered human skin substitute using human cells obtained from skin biopsies and fibrin-agarose biomaterials and we evaluated this model both at the ex vivo and the in vivo levels. Once the dermal fibroblasts and the epithelial keratinocytes were isolated and expanded in culture, we used fibrin-agarose scaffolds for the development of a full-thickness human skin construct, which was evaluated after 1, 2, 3 and 4 weeks of development ex vivo. The skin substitutes were then grafted onto immune-deficient nude mice and analyzed at days 10, 20, 30 and 40 postimplantation using transmission electron microscopy, histochemistry and immunofluorescence. The results demonstrated that the fibrin-agarose artificial skin had adequate biocompatibility and proper biomechanical properties. A proper development of both the bioengineered dermis and epidermis was found after 30 days in vivo, although the tissues kept ex vivo and those implanted in the animal model for 10 or 20 days showed lower levels of differentiation. In summary, our model of fibrin-agarose skin equivalent was able to reproduce the structure and histological architecture of the native human skin, especially after long-term in vivo implantation, suggesting that these tissues could reproduce the native skin.     

Synchrotron X-ray microtomography (on a micron scale) provides three-dimensional imaging representation of bone ingrowth in calcium phosphate biomaterials

This study used synchrotron X-ray microtomography on a micron scale to compare three-dimensional (3D) bone ingrowth after implantation of various calcium phosphate bone substitutes in a rabbit model. The advantage of using this new method for the study of biomaterials was then compared with histomorphometry for analysis of interconnection and bone ingrowth. The study focused on the newly formed bone-biomaterial interface. Macroporous Biphasic Calcium Phosphate (MBCP) ceramic blocks and two different injectable calcium phosphate biomaterials [an injectable bone substitute (IBS) consisting of a biphasic calcium phosphate granule suspension in hydrosoluble polymer and a calcium phosphate cement material (CPC)] were studied after in vivo implantation.Absorption or phase-contrast microtomography was performed with the dedicated set-up at beamline ID22. Experimental spatial resolution was between 1 and 1.4 microm, depending on experimental radiation. All calcium phosphates tested showed osteoconduction. IBS observations after 3D reconstruction showed interconnected bioactive biomaterial with total open macroporosity and complete bone ingrowth as early as 3 weeks after implantation. This experimentation was consistent with two-dimensional histomorphometric analysis, which confirmed its suitability for biomaterials. This 3D study relates the different types of bone substitution to biomaterial architecture. As porosity and interconnection increase, bone ingrowth becomes greater at the expense of the bone substitute: IBS>MBCP>CPC.     

Hydroxy apatite microspheres enhance gap junctional intercellular communication of human osteoblasts composed of connexin 43 and 45

The aseptic loosening of artificial joints with associated periprosthetic bone resorption may be partly due to the suppression of osteoblast function to form new bone by wear debris from the joint. To assess the effect of wear debris on osteoblasts, effects of model wear debris on gap junctional intercellular communication (GJIC) of normal human osteoblasts were estimated. The GJIC activity of the osteoblasts after a 1-day incubation with the microspheres was similar to that of normal osteoblasts. However, hydroxy apatite particles, which have been reported to enhance the differentiation of osteoblasts in contact with them, enhanced the GJIC function of the osteoblasts. From RT-PCR studies, not only connexin 43 but also connexin 45 is suggested to play a role in the GJIC of the osteoblasts in an early stage of coculture with the microspheres, although it is still unclear how these connexins work and are regulated in the GJIC and differentiation. However, this study suggests that there is a relationship between the early levels of GJIC and the differentiation of the cells. Therefore, estimating the effect of biomaterials, even in the microsphere form, on the GJIC of model cells, with which the biomaterials may be in contact in vivo, can provide important information about their biocompatibility.     

Supercritical carbon dioxide processed resorbable polymer nanocomposite bone graft substitutes

The development of synthetic bone graft substitutes is an intense area of research due to the complications associated with the harvest of autogenous bone and concerns about the supply of allogenic bone. Porous resorbable polymers have been used extensively in hard tissue engineering applications, but currently lack load-bearing capacity. Supercritical carbon dioxide (scCO(2)) processing is used as a novel method to simultaneously impart a porous structure and disperse a nano-clay in a resorbable polymer matrix suitable for load-bearing applications. Porous resorbable polylactic acid (PLA)/cloisite clay nanocomposite constructs prepared using scCO(2) processing exhibit a 2.5-fold increase in compressive strength compared with pure polymer constructs. The resulting mechanical properties are comparable with human cancellous and cortico-cancellous bone. In addition to the significant improvements in mechanical properties, the nanocomposite constructs display a biocompatibility greater than that of polystyrene culture plate controls. Furthermore, calcium phosphate-rich deposits could clearly be seen on the surface of the constructs, as well as at the center of the cultured constructs, indicating that osteoblasts are able to penetrate the porous network of the nanocomposite constructs. Cellular infiltration of these constructs is important for their in vivo use as bone graft substitutes. The diameter of the pores suggests that these constructs would also support neovascularization, which is integral for nutrient transport.     

Extensive H(+) release by bone substitutes affects biocompatibility in vitro testing

Bone substitutes are widespread in orthopedic and trauma surgery to restore critical bony defects and/or promote local bone healing. Cell culture systems have been used for many years to screen biomaterials for their toxicity and biocompatibility. This study applies a human bone marrow cell culture system to evaluate the toxic in vitro effects of soluble components of different bone substitutes, which are already in clinical use. Different specimens of tricalcium phosphates (TCP) (Vitoss, Cerasorb), nondecalcified bovine bone (Lubboc), demineralized human bone matrices (DBM) (Grafton Flex/Putty), and collagen I/III matrix (ACI-Maix) were tested in Dulbecco's modified Eagle's medium (DMEM) and MesenCult culture solution and compared with a biomaterial-free cell culture. Biocompatibility parameters were cell viability evaluated by phase-contrast microscopy and laser flow cytometry, morphology, and the local H(+) release by bone substitutes. There were significant differences (p < 0.05) between the tested biomaterials and culture solutions. Collagen I/III, non-demineralized bovine bone, and TCP materials showed advantages for cell survival over other tested biomaterials (average values of vital cells/mL MesenCult/DMEM: Collagen I/III: 1090/1083; Vitoss: 893/483; Cerasorb: 471/523; Lubboc: 815/410; Grafton Putty: 61/44; Grafton Flex: 149/57). Especially the DBM materials lead to a significant decrease of pH, which is considered to be a major factor for cell death. DMEM culture solution supports cell survival for those bone substitutes that induce an alkaline reaction, whereas MesenCult media promotes cell vitality in biomaterials, which leads to an acidification of culture solution.     

Soluble silica inhibits osteoclast formation and bone resorption in vitro

Several studies have suggested that silicon (Si) may be essential for the normal development of connective tissue and the skeleton. Positive effects of Si from the diet as well as from Si-containing biomaterials, such as bioactive glass 45S5 (BG), have been demonstrated. Studies have reported that Si stimulates osteoblast proliferation and differentiation. However, the effects of Si on osteoclasts have not been directly addressed. The purpose of the present in vitro study was to clarify if Si has regulatory effects on osteoclast formation and bone resorption. The effects of BG, BG dissolution extracts and Si containing cell culture medium were investigated in a mouse calvarial bone resorption assay and osteoclast formation assays (mouse bone marrow cultures and RAW264.7 cell cultures). We conclude from our results that Si causes significant inhibition of osteoclast phenotypic gene expressions, osteoclast formation and bone resorption in vitro. In conclusion, the present study suggests that Si has a dual nature in bone metabolism with stimulatory effects on osteoblasts and inhibitory effects on osteoclasts. This suggested property of Si might be interesting to further explore in future biomaterials for treatments of bone defects in patients.     

Ectopic bone formation by 3D porous calcium phosphate-Ti6Al4V hybrids produced by perfusion electrodeposition

Successful clinical repair of non-healing skeletal defects requires the use of bone substitutes with robust bone inductivity and excellent biomechanical stability. Thus, three-dimensionally functionalised porous calcium phosphate-Ti6Al4V (CaP-Ti) hybrids were produced by perfusion electrodeposition, and the in vitro and in vivo biological performances were evaluated using human periosteum derived cells (hPDCs). By applying various current densities at the optimised deposition conditions, CaP coatings with sub-micrometer to nano-scale porous crystalline structures and different ion dissolution kinetics were deposited on the porous Ti6Al4V scaffolds. These distinctive physicochemical properties caused a significant impact on in vitro proliferation, osteogenic differentiation, and matrix mineralisation of hPDCs. This includes a potential role of hPDCs in mediating osteoclastogenesis for the resorption of CaP coatings, as indicated by a significant down-regulation of osteoprotegerin (OPG) gene expression and by the histological observation of abundant multi-nucleated giant cells near to the coatings. By subcutaneous implantation, the produced hybrids induced ectopic bone formation, which was highly dependent on the physicochemical properties of the CaP coating (including the Ca(2+) dissolution kinetics and coating surface topography), in a cell density-dependent manner. This study provided further insight on stem cell-CaP biomaterial interactions, and the feasibility to produced bone reparative units that are predictively osteoinductive in vivo by perfusion electrodeposition technology.     

Diversity of fibroblasts--a review on implications for skin tissue engineering

Enormous advances in the development of skin substitutes have occurred in the past 3 decades. Major obstacles yet to be overcome in the quest for an optimal skin substitute include controlling scar formation, contraction and the loss of adnexal structures. Mesenchyme-derived signals are essential for epithelial proliferation, skin morphogenesis, homeostasis and differentiation. Having previously shown that fibroblasts differentiate along a lineage from highly proliferative progenitor fibroblasts with characteristic spindle-shaped appearance to differentiated postmitotic polygonal fibrocytes, we have now established that the different subsets of fibroblasts exert significantly different patterns of cytokine release and that the highest levels of keratinocyte growth factor and transforming growth factor-beta1 expression result from differentiated fibroblasts. Coculture studies with keratinocytes reveal that postmitotic fibroblasts stimulate keratinocyte proliferation to a greater extent than progenitor fibroblasts. Acellular and fibroblast-seeded dermal substitutes have been shown to improve scarring and contraction in animal studies, the latter substitutes yielding the most favorable results. Fibroblasts from different body sites display different functional properties which may affect their suitability for dermal substitutes. Future in vivo human studies in tissue-engineered dermal substitutes will likely focus on fibroblast-seeded lattices and the impact of fibroblast subpopulations and bone marrow-derived mesenchymal stem cells on dermal regeneration.     

Natural coral exoskeleton as a bone graft substitute: a review

Natural coral graft substitutes are derived from the exoskeleton of marine madreporic corals. Researchers first started evaluating corals as potential bone graft substitutes in the early 1970s in animals and in 1979 in humans. The structure of the commonly used coral, Porites, is similar to that of cancellous bone and its initial mechanical properties resemble those of bone. 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. 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. 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 including in vitro, animal and clinical human studies. Preliminary report of our own experiments as well as our recommendations on the use of coral are also included.