Composite biomaterials

Keywords

Biomaterials, Tissue Engineering, Composites, Scaffolds,

1.    Introduction

-    General Background

The term “composite” refers to a material system composed of a mixture or a combination of two or more materials on a macroscopic scale, in which the materials are of different composition, morphology and general physical properties. The constituents that are combined to make up composite materials and also to form a material with a balance of properties include polymers, ceramics, and metals. One of the most commonly used composites is fiberglass, a mixture of glass fibers coated with a polymeric matrix. Fiberglass is commonly used because it provides a material that is strong and lightweight suitable for use in many industries. The design of composites in most cases, and depending on the constituent properties of the composites, is done to develop materials with properties tailored to meet certain chemical, physical or mechanical requirements. The use of composites has progressively increased over the past few decades (approximately 40 years now) due to the capabilities linked to its use in many different applications that include in aeronautic industry, automotive, medicine, electronics, military and naval and so on (Gay, Daniel., 9). Most of the composite materials get fabricated to provide the desired mechanical properties of materials such as stiffness, strength, toughness and fatigue resistance. Consequently, numerous tests and studies have been conducted on composite biomaterials, including their application in medical application and tissue engineering.

Biomaterials get defined as materials, either natural or artificial material that comprises of whole or part of a living structure or function in intimate contact with living tissue. Biomaterial composites get developed in a manner that allows for the fabrication of real composites comprising of both proteins and nonliving constituents such as glasses and polymers. Composite biomaterials are preferred when compared to single-phase or monolithic biomaterials since they do not always give all the properties required for a given application. In the medical application, biomaterials interface with biological systems so as to evaluate, treat, augment or replace a tissue, organ or a body function. Composite biomaterials are classified into three groups namely bioinert, bioactive and bioresorbable (Sreeram Ramakrishna, Murugan Ramalingam, TS Sampath Kumar, and Winston O. Soboyejo, 20). It is because composites implanted into the human body tend to elicit a biochemical and biological response by the host tissue depending on their surface characteristics. All the kinds of composite biomaterials with different matrices (such as ceramic, metallic and polymeric) got created and tested for biomedical applications.  Current applications of composite biomaterials in the biomedical sector include dental composites used in dental filler materials, coated metallic implants and reinforced polymethyl methacrylate bone cement utilized in joint replacement surgeries (Seal, Sudipta, 202). The benefits of composite materials include their ability to tailor their properties as per the need; hence, are beneficial when compared to homogenous biomaterials.

Biomaterials get commonly used in tissue engineering as scaffolds and carriers of cells, growth factors and genes. All kinds of biomaterials including composites, ceramics, polymers and metals get used in tissue engineering. A scaffold is used to offer the required mechanical support and a physical structure for the transplanted cells so as to attach, grow and maintain differentiated functions. Biomaterials are used as carriers for delivery cells and bioactive substances, in which they should possess the required surface chemistry for cell attachment and proliferation and biological substance immobilization (Mauck, Robert L, 158). The use of scaffolds that are produced from composite biomaterials and manufactured utilizing a plethora of fabrication strategies has assisted to regenerate different tissues and organs in the body. Various considerations are essential in designing and determining the suitability of a scaffold for use in tissue engineering.

One of the critical aspects to consider in biomaterials is biocompatibility, which is its ability to function with a suitable host response in a particular biomedical application. Examples of ‘suitable host response’ are the resistance to blood clotting, normal, uncomplicated healing and resistance to bacterial colonization. Examples of specific applications can include a urinary catheter, a hemodialysis, and hip-joint replacement prosthesis.  Any scaffold intended to be used in tissue engineering must be biocompatible that is cells should adhere, function normally and migrate onto the surface as well as through the scaffold and start to proliferate before the laying down new matrix (O'Brien, Fergal J., 90). The development of composite biomaterials is not an easy task since all constituent phases in the composite must be proved biocompatible. Composites provide the probability of causing severe tissue reaction since they are made up of two or more constituents. Another aspect to consider is biodegradability as the tissue engineering aims at allowing body cells to replace the implanted scaffold over time. Therefore, since they are not intended to be permanent implants must be biodegradable to allow cells to generate own extracellular matrix (Gil, Sara, and João F. Mano, 815). Besides, the byproducts of the degradation process should be non-toxic and be able to exit the body. A third aspect that must be considered is the mechanical properties of the biomaterial. For the scaffold to be used in tissue engineering, it should have mechanical properties consistent with the anatomical site where it will be implanted and also must have a high strength to allow surgical handling during implantation (Hussein, Mohamed A., Abdul Samad Mohammed, and Naser Al-Aqeeli, 250). The fourth consideration when planning to use scaffolds in tissue engineering is scaffold architecture, in which the scaffolds should have an interconnected pore structure and high porosity so as to ensure cellular penetration and diffusion of nutrients to cells within the construct and the extra-cellular matrix formed by the cells. High corrosion resistance is also an important consideration when selecting a biomaterial for use in tissue engineering. When a biomaterial with a low corrosion resistance is used for an implant, it can release metal ions into the human body that in turn lead to toxic reactions.

-    Motivation

There has been numerous advances and research in the field of tissue engineering and regenerative medicine in which they have enhanced the potential of regenerating all tissues and organs of the body. Tissue engineering aims at restoring maintaining and improving the functionality of tissues that are defective or affected by pathological conditions. Tissue engineering usually prefers the use of biodegradable biomaterial since as the new tissue develop, it gradually replaces the scaffold (Shi, Donglu, 199). Due to the preference of using biodegradable materials as supports since they disappear from the transplantation site over a period, leaving behind a perfect patch of the natural tissue, considerable interest and research have been done on them. This paper is based on the preparation and characterization of bioactive, biodegradable and bioactive glass composites.

-    Objectives

The primary objective of the paper is to synthesize composite biomaterials so that they can be used for further tissue engineering purposes.

2.    Literature Review

In engineering design, composite materials are material systems that consist of constituents in the micro- to macro- size range, favoring the macro size range (Ratner, Buddy D., et al., 182). This definition encompasses the fiber and composite materials of primary interest as biomaterials. This kind of composites comprises of one or more discontinuous phases embedded within a continuous phase. The discontinuous phase is harder and stronger when compared to the continuous phase and is known as the reinforcement or reinforcing the material, while, on the other hand, the continuous material is known as matrix. The application of composite materials provide a wide variety of advantages over ceramics, metals, and polymers that incorporate the desirable properties of each of the constituent materials, and at the same time mitigating the more limited features of all components (King, Michael R., 178). The constituents that combine to develop composite materials differ in both form and chemical composition and are also insoluble in each other. The composite materials properties are dependent upon the shape of the heterogeneities, upon the interfaces among the constituents and also upon the volume fraction occupied by them. Most of the biological materials found in nature are natural composites and include skin, wood, dentin, bone and cartilages (Park, Joon B., and Roderic S. Lakes, 208). Natural foams include cancellous bone, wood, and lung. Natural composites often portray hierarchical structures in which particulate, porous and fibrous structural characteristics get viewed on different length scales.

Reinforcing systems

The properties of composite materials are dependent upon the structure as they do in homogenous materials. Composites are different in that significant control can be exerted over the larger scale structure and therefore above the required properties. The main reinforcing materials used in biomedical composites include carbon fibers, polymer fibers, glasses, and ceramics. The reinforcements are either inert or absorbable depending upon the application.

Carbon fiber

Carbon fiber is a lightweight, high-strength, flexible and a high tensile modulus material that is produced by the pyrolysis of organic precursor fibers that include rayon, polyacrylonitrile (PAN) and pitch in an inert environment. Carbon fibers have a low density (ranging from 1.7 to 2.1 g/cm³ depending on the precursor) and high mechanical properties (elastic modulus up to 900 Gpa and strength up to 4.5 Gpa, depending on the precursor and the fabrication process, therefore are much stiffer and stronger when compared to steel). Due to these properties, composites are used in a wide range of applications that require lightness and high mechanical properties. However, carbon fibers are disadvantageous since they have poor shear strength. In medicine, different commercial products are made using carbon fibers.

Polymer fibers

Polymer fibers do not have comparable strength or stiffness as reinforcements for other polymers when compared to carbon fibers. However, there is a possible exception of aramid fibers or ultrahigh molecular weight polyethylene (UHMWPE) fibers. Biocompatibility, high strength, and fatigue resistance are necessary for biomedical applications, whereas stiffness is a design parameter to get adapted to the specific conditions. Aramid is the generic name for aromatic polyamide fibers. The most common aramids are Nomex, Twaron, and Kevlar (Robertson, James R., Claude Roux, and Ken Wiggins, 19). Aramid fibers are light (density of 1.44 g/cm³), stiff (the modulus can be as high as 190 Gpa), strong (tensile strength of about 3.6 Gpa) and also can resist impact and abrasion damage. The commercial applications of Aramid fiber composites are suitable where high tensile strength and stiffness, damage resistance and resistance to fatigue and stress rupture are necessary. The major applications in medicine are in dentistry and ligament prostheses.

Ceramics

Ceramic materials are used to reinforce biomedical composites. Biocompatible ceramics are relatively weak and brittle materials when compared to metals especially when loaded in tension or shear; therefore, the preferred form of this reinforcement is usually particulate (Ratner, Buddy D., et. al. 281). Tricalcium phosphates and hydroxyapatite are commonly known as bioceramics that are bioactive ceramics that can elicit a certain biological response that leads to the formation of a bond between the tissues and material. In biomedical applications, tricalcium phosphates and hydroxyapatite are applied in orthopedics and dentistry on their own, or in combination with other materials, or as a coating of metal implants (Jaboro, Claudine, 14).

Glasses

Glass fibers are applied in the reinforcement of plastic matrices so as to form structural composites and molding compounds. The desirable characteristics of commercial glass fiber plastic composite materials are high-strength to weight ratio, adequate resistance to heat, moisture, cold and corrosion, excellent dimensional stability, good electrical insulation properties, low cost and ease of fabrication. In biomedical applications, glass fibers are used to increase the mechanical properties of acrylic resins for use in dentistry.

Applications of Biomaterials

The primary application of biomaterials is medical application, although they also get used for growing cells in culture, to assay for blood proteins in the clinical laboratory, for implants in fertility regulation in cattle, in equipment for processing biomolecules for biotechnology applications, in diagnostic gene arrays, in the aquaculture of oysters and for investigational cell-silicon ‘biochips’. Biomaterials, in medical applications, are rarely utilized as isolated materials but get integrated into devices or implants. The various medical applications of composite biomaterials get described below.

Heart Valve Prostheses

Diseases affecting heath valves and damage of heart valves caused by the opening and closing of heart valves for many times (say over 40 million times a year) usually require surgical repair or replacement. The cases of surgical repair or replacement of heart valves are over 80, 000 each year in the United States due to acquired damage of the natural valve and congenital heart anomalies (Ratner, Buddy D, 2). Different types of heart valve prostheses are fabricated from metals, carbons, plastics, fabrics, elastomers and animal or human tissues chemically pretreated so as to minimize their immunologic reactivity and to improve durability.

Artificial Hip Joints

The human hip joint gets subjected to high levels of mechanical stress and also significantly gets abused during its normal operation (Hench, L., and J. Jones, 102). It is due to this abuse or stress that after 50 or more years of cyclic mechanical stress or due to degenerative or rheumatological illness that the natural joint wears out; hence, causing a considerable loss of mobility and wheelchair confinement. Common hip joint prostheses get fabricated from materials such as stainless steel, titanium, special high-strength alloys, ceramics and also ultrahigh molecular weight polyethylene.  Some kinds of replacement hip joints, as well as surgical procedures, use polymeric cement, in which the ambulatory function gets restored with a few days after surgery. Other types of replacement hip joints and surgical procedures have a varying healing period necessary for the integration of the bone and the implant before the joint can bear the full body weight.

Dental implants

The introduction of titanium implants has revolutionized the dental implantology, in which they are used to create an implanted artificial tooth anchor upon which a crown gets affixed. The approximate number of dental implants in the US is about 300, 000 people, with some people having over 12 implants (Park, Joon, and Roderic S. Lakes, 443). For a material to be used for a dental implant, it requires having the ability to form a tight seal against bacterial invasion where the implant traverses the gingival (gum).

Intraocular lenses

There have been many intraocular lenses (IOLs) that have been fabricated of poly (methyl methacrylate), silicone elastomer, hydrogels or soft acrylic polymers and get used in the replacement of natural lens when it is cloudy because of a cataract formation. Approximately 50 percent of the population suffers from cataracts by the age of 75 and can be repaired through IOL implantation. The number of IOL implantations done in the US each year is about 4 million implantations, and globally it is double the number. The success rate of the IOLs is high as good vision is restored immediately.

Left Ventricular Assist Device

An efficient and also a safe mechanical cardiac assist or replacement has become an attractive goal since a large population today is affected by failing hearts (approximately 50, 000 annually) of people who need cardiac assist or replacement, and also because of the many heart donors (approximately 3, 000 annually). Left ventricular assist devices (LVADs) that can get considered as one-half of the total artificial heart is used to maintain a patient with a failing heart as the patient awaits the availability of a transplant heart and also is used as a permanent therapy for some patients.

Applications in Tissue Engineering

Tissue engineering aims at promoting cell regeneration and healing of defective or lost natural tissues or assisting in the development of biological substitutes that restore, maintain as well as enhance tissue and organ functions (Dhandayuthapani, Brahatheeswaran, Yasuhiko Yoshida, Toru Maekawa, and D. Sakthi Kumar, 1). The fundamental principle of tissue engineering gets guided by the application and control of cells, materials and the microenvironment where they will be delivered. Tissue or organs in tissue engineering method are created in vivo, in vitro and ex vivo and then implanted at the diseased or damaged site in the body. For a successful replacement or treatment, the components employed in a tissue engineering approach to fabricating biological tissues are viable, responsive cells, a scaffold to support tissue formation as well as a growth inducing stimulus (Laurencin, Cato T., and Lakshmi S. Nair, 195). The combination of synthetic or natural scaffolds with cells offers an alternative to organ transplantation and implantation of mechanical devices that cannot perform all the functions of tissue (Salernitano, E., and C. Migliaresi, 7). Tissue engineering relies extensively on the use of porous 3D scaffolds so as to provide the suitable environment for the regeneration of tissues and organs in the human body. Bone tissue engineering combines both the cells and a biodegradable 3D scaffold so as to repair damaged or an ill bone tissue (Chen, Q., J. A. Roether, and A. R. Boccaccini, 2). The scaffolds act as a template for tissue formation and get typically seeded with the cells and growth factors, or subjected to biophysical stimuli in the bioreactor form. The cell-seeded scaffolds are either cultured in vitro so as to synthesize tissues that can then get implanted into a diseased or damaged organ or tissue, or get implanted directly into a diseased or damaged organ or tissue, using own body systems, where regeneration of tissues or organs is induced in vivo. Culturing isolated cells on a biomaterial scaffold that is to be transplanted for regeneration of a target tissue or organ is a significant concept in tissue engineering (Franulović, Marina, et al., 1). Much of the research in tissue engineering focuses on the selection of suitable biomaterials as scaffolds for cellular attachment, proliferation as well as differentiation, so as to improve the potential of the technology.

3.    Experimental

-    Materials Preparation

The bioactive property of the selected composite is provided by a bioactive glass in the formula given as 56SiO₂∙ (40-x) CaO•4P₂O₅•xAg₂O with x = 0, 2, 4, 6, 8 and 10 mol%. The preparation method that was employed for the bioactive glasses is sol-gel method since it allows for the test to take place at low temperature and has a better controllability of the bioactive glass structure and morphology. The use of the sol-gel method was also appropriate since it allowed for the introduction of the antibacterial agent in the glass composition providing the samples a composite nature with metallic silver as dispersed phase in an almost amorphous glass structure. The sol-gel process entails of the transition of a system from a liquid “sol” into a solid “gel” form. The experiment then evaluates the nature of the silver species embedded into the bioactive glass network before as well as after immersion in simulated body fluid (SBF) and also to characterize the structural, morphological and textural changes that occur after addition of silver to the bioactive glass matrix.  The gels in the experiment were obtained by hydrolysis and condensation of calcium nitrate tetrahydrate (Ca(NO₃)₂.4H₂O), tetraethyl orthosilicate (TEOS), ammonium phosphate dibasic ((NH₄)2HPO₄) and silver nitrate (AgNO₃). The biodegradable component of the tested samples was given by a Poly-96L/4 D-lactide copolymer highly porous structures.

The bioactive glass matrix sample belonging to the composition 56SiO₂∙40CaO•4P₂O₅ was prepared and labeled x=0. The samples containing the silver were also developed and marked X=2, x=4, x=6, x=8 and x=10. Ag₂O was then added by partial substitution of CaO. The samples were then dried, grounded and were heat treated at 580 ^oC for half an hour. The samples were then introduced into the preheated oven and taken out after another half an hour. 

-    Methods

Characterization of the prepared sol-gel glasses

Differential thermal analysis (DTA) and thermogravimetric analysis (TGA) methods were used to understand the way the silver presence reacts and affects the thermal events and also the XRD and FTIR spectroscopy were used so as to determine the developed crystalline phases and the structural changes generated inside the bioactive glass matrix by silver addition.

Differential Thermal Analysis (DTA)

DTA comprises of heating and cooling a sample that is being tested and an inert reference under identical conditions. The temperature changes that occur between the sample and reference are recorded. The differential temperature is then plotted against time or against temperature. The variations in the test sample that lead to the absorption or evolution of heat can get detected about the inert reference.

The test is conducted using a sample holder that comprises of thermocouples, sample containers, and a ceramic block. Each of the sample and references is surrounded by a block so as to ensure an even heat distribution. The sample gets contained in a small crucible designed with an indentation on the base so as to ensure a snug fit above the thermocouple bead. The thermocouples are not placed in direct contact with the sample so as to avoid contamination and degradation; however, it may lead to a compromise in sensitivity.

Thermal Gravimetric Analysis (TGA)

The TGA is the method used to determine the characteristics of polymer and plastic materials and to determine degradation temperatures. The TGA measurements are used to determine the thermal as well as oxidative stabilities of materials and their compositional properties.

The weight percentage of the glasses were estimated by determining the difference in percentage mass of the composites at the start and at the end of the DTA test, where the polymer had to be assumed to be totally thermally degraded.

X-ray diffraction (XRD)

The carrying out of XRD analysis on composite samples assists to get their solid-state structural information that includes a degree of crystallinity.

Bioactivity Studies

The bioactivity of the samples was investigated through soaking of the materials in SBF at 37 ^oC for the purpose of studying the de hydroxyapatite/carbonated hydroxyapatite (HA/HCA) formation by XRD, FTIR spectroscopy, SEM observation, EDS analysis and XPS. In vitro experiments were undertaken by soaking the samples in SBF according to the Kokubo composition up to 14 days.

Pieces of size 5 mm by 5 mm of coated polymer fabrics were soaked in 25 ml of SBF in clean conical flasks. The conical flasks were placed on an orbital shaker that rotates at 100 rpm at a controlled temperature of 37 ^oC. The samples were the extracted from the SBF solution after a given period that is 1, 7, 14 and 21 days. So as to maintain the cation concentration in the SBF, it was replaced twice each week as it concentration decreased with time leading to change in the chemistry of the investigated samples. After extraction of the samples from the incubation flasks, they were rinsed in pure ethanol and then using deionized water and dried at ambient temperature. The test then investigated the formation of HA on the surface of samples after immersion in SBF using analytical methods namely XRD, FTIR spectroscopy, SEM observation, EDS analysis and XPS. The XPS survey spectra were used to determine the elemental composition recorded on the surface of the polymers and the samples before and after immersion in SBF.

4.    Results and Discussion

Characterization of the prepared sol-gel glasses

In the characterization of the prepared sol-gel glasses, the DTA and TGA methods were utilized for verification of the thermal degradation temperatures of the composite materials and also in the quantification of the bioactive glass content on the polymer meshes.

DTA

The DTA curves of the test are shown in the figures below.

The first endothermic peak situated about 60-80 ^oC and linked with the weight loss gets observed in the tested samples and corresponds to the release of physisorbed water and the pore liquor. The exothermic peak with an onset of about 277 ^oC is assigned to the removal of organic residues. A relative intense endothermic signal is seen in the DTA curves of about 485 ^OC for the tested samples and could be linked to the silver oxide decomposition and possibly the formation of metallic silver nanocrystals. The presence of higher silver content cannot get excluded as long as the peak located approximately at 500 ^oC becomes over pronounce and the temperature range is close to 485 ^oC.

The endothermic peaks from the 540-550 ^oC temperature range that occur for the samples are due to dehydroxylation and get associated with weight loss from the corresponding TGA curves. An analysis of the endothermic peaks position between 540 and 550 ^oC show that the decrease of the temperature where the thermal event occur, as the silver content increases.

XRD

The XRD patterns that were obtained for the investigated samples are shown in the diagram 2 below;

Figure 2: XRD patterns of the tested samples

The XRD patterns shown in the diagram 2 above show mainly amorphous characteristics corresponding to glass, although they demonstrate incipient crystallization of a tricalcium phosphate (TCP) phase identified as Ca₃(PO₄)₂.centered at 2θ=32. The result of the experiment suggests that the addition of high silver amounts into the investigated SiO₂-CaO-P₂O₅ system has no effect on its bioactivity. XRD patterns of the samples with x ≥ 4 mol percentage demonstrate the composites nature with metallic silver as dispersed phase. The XRD patterns however also show an Ag₂O phase comprising of tiny crystallites that become less visible when the silver content increases. This result concurs with the DTA analysis assumption concerning the endothermic event situated around 485 ^oC that got associated with the metallic silver nanocrystals formation. The experiment also shows that the increase of silver content incorporated in the sample causes an expected increase of crystallized metallic silver amount.

Bioactivity Studies

XRD Analysis

XRD analyses carried out after SBF immersion permit the verification of how silver content affects the self-assembling process on samples surface induced by the ionic exchange between the SBF solution and the bioactive glasses. The figure 3 below shows the XRD patterns of the investigated samples in which (a) curve represent x=0, (c) curve represent x=2, (e) curve represent x=8 all before SBF soaking while after soaking (b) curve represent x=0, (d) curve represent x=2, and (f) curve represent x=8.

Figure 3: XRD pattern of the investigated samples

The XRD patterns of non-immersed samples demonstrate a wide halo of the non-crystalline calcium phosphor silicate matrix (x=0) that has maximum centered at 2θ = 32 ^oC and weak characteristics of an apatite such as phase with nanosized crystallites. The standard hydroxyapatite (HA) pattern in the figure above is inserted so as to assist in distinguishing the apatite phase. For the sample (x=0) without silver content, only the strongest lines about HA pattern is evident, while after SBF soaking for 14 days there appears new peaks corresponding to crystallized HA phase. For the sample (x=2) that has the smallest concentration of silver, small peaks were observed that can get attributed to metallic silver content, but also to silver oxide crystals. After the immersion in SBF, the signals grow in intensity, and new peaks appear that are attributed to Ag₃PO₄ phase. The sample (x=8) with high silver concentration has the same behavior as the sample with the lowest (x=2), although the new crystalline phases formation is more apparent when compared.

FTIR Spectroscopy

The FTIR spectra of the silver free sample and silver comprising samples before as well as after immersing in SBF for in vitro bioactivity tests together with the spectrum of pure HA are displayed in the figure 4 below.

Figure 4: FTIR spectra of the tested samples

The curves in the figure are divided into two that is after and before SBF immersion in which (a) curve represent x=0, (c) curve represent x=2, (e) curve represent x=8 all before SBF soaking while after soaking (b) curve represent x=0, (d) curve represent x=2, and (f) curve represent x=8. The presence of the bands at 569, 605, 1040 cm^-1 are as a result of the [PO₄) unit vibrations corresponding to the crystalline HA and also are visible for non-immersed samples. In the spectra of non-immersed samples, peaks are observed as well as the presence of the band at 1080 cm^-1 that are attributed to the stretching vibration of Si-O bonds and of the shoulder about 950CM^-1 caused by the vibration of Si-O-Si groups. The Si-O-Si bond bending motion led to the strong band of around 470 cm^-1. In addition to the phosphate bands, the FT-IR spectra of the samples that had been incubated in SBF solution show a new band at 870 cm^-1, which had been assigned to carbonate ions that signify the formation of a carbonate apatite phase. The addition of carbonate ions in the HA layer takes place through ion exchange mechanism, where CO₃-2 ions from the SBF partially replace the PO₄-3 ions, and these phosphate ions react with silver to form Ag₃PO₄ crystals that are clearly visible in SEM images.

SEM

The figure 5 below shows the SEM images of the three samples before and after immersion in SBF.

Figure 5: SEM images of the bioactive glass samples that have different silver amount

The image (a) represent x=0, (d) represent x=2, (g) represent x=8 all before SBF immersion while after soaking the samples are represented as follows; image (b) represent x=0, (e) represent x=2, and (h) represent x=8. Also, the images (c) represent x=0, (f) represent x=2, and (i) represent x=8 are backscattered electron images after SBF immersion. The HA/HCA layer is visible on the surface of the SBF immersed samples as indicated in images (b), (e) and (h) and the HA crystals size increases with the increase of silver concentration in the sample as indicated in the image (h). The analysis of the SEM images performed with backscattered electrons, well-defined Ag3PO4 sub-microcrystals got observed in the silver containing the sample.

XPS

The figure 6 below shows the XPS survey spectra for bioactive glass with different silver content before and after soaking in SBF.

In the figure, the recorded data is presented in such a way that a) curve represent x=0, (c) curve represent x=2, (e) curve represent x=8 all before SBF soaking while after soaking (b) curve represent x=0, (d) curve represent x=2, and (f) curve represent x=8. For the samples containing silver, the results show that there is a significant increase of the relative atomic percent of carbon thus suggesting that the silver content favors the formation of carbonated apatite.

The XPS was also used to investigate the protein-binding capability of the silver-containing bioactive glasses. The figure 7 below shows the representation of the data obtained from the XPS survey spectra.

The influence of the silver content in the samples on the adsorption of methemoglobin from solution get reflected by the evolution of the new N 1s and S 2p photoelectron peaks and by the considerable amount of C 1s that were recorded after immersion in the protein solution. The study found out that the amounts of each of the elements increased significantly with the silver content, whereas, the amounts of the main elements reduced due to the coverage of the surface by the protein. The results indicate that the higher the silver content, the larger the coverage of the sample with protein.

5.    Conclusions

Tissue engineering has a great potential of transforming the clinical and biomedical field by enabling the regeneration and replacement of all tissues and organs in the human body. Numerous naturally occurring as well as synthetic materials have been introduced in different clinical and biomedical applications. The selection of appropriate biomaterials as scaffolds for cellular attachment, proliferation, and differentiation is an important part of tissue engineering research. Due to increased attention and research in tissue engineering, there has been a fabrication, synthesis, and processing of many biomaterials into a wide range of shapes and forms for specific applications necessary so as to interface with biological systems.

The sol-gel process was used successfully to obtain new silicate based composite samples containing silver amounts that varied. The XRD, FTIR spectroscopy, SEM images and XPS analysis revealed that the addition of silver content to SiO₂-CaO-P₂O₅ bioactive glass matrix favors the formation of HA/HCA layer and also influenced the formation of Ag₃PO₄ crystals on the surface of the samples after SBF immersion. The SEM analysis demonstrated that the porous structure of the obtained composites, while the thermal analyses estimated the amount of bioactive glass from composites. The experiment successfully obtained a biodegradable polymeric phase and a bioactive inorganic phase. The composites in this study can be used for scaffold production especially concerning bone tissue engineering. The composite biomaterial is a silicate based bioactive glass that can stimulate osteogenesis and also facilitate bone growth during regeneration. The composite is a potent antibacterial agent that forms Ag₃PO₄ crystals on the bioactive glass thus allowing an extended period of the antibacterial agent. The composite is biodegradable thus can support bone formation as well as permit tissue ingrowths and vascularisation. The outcome of the study significantly contributes to improving the potential of tissue engineering in regeneration or replacement of diseased or damaged tissues or organs through the preparation and also the characterization of bioactive, biodegradable and bioactive glass composite.

6.    Future Work

Tissue engineering has a significant potential to be used as an alternative or complementary technique in different clinical and biomedical applications especially in enabling regeneration or replacement of tissues in the body. Numerous materials have been researched and currently used in tissue engineering procedures; however, further research is necessary so as to improve various aspects of the materials such as biocompatibility, mechanical properties, biodegradability and so on. In the study, the preparation and the characterization of bioactive, biodegradable and bioactive glass composite were accomplished successfully; however, further study should be carried out to determine its suitability for use in regeneration of different tissues and organs in the human body.

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