Nanobiomaterials in Hard Tissue Engineering: Applications of Nanobiomaterials

China

Preface of the series

Ecaterina Andronescu, Department of Science and Engineering of Oxide Materials and Nanomaterials, Faculty of Applied Chemistry and Materials Science, University Politehnica of Bucharest, Bucharest, Romania, Department of Biomaterials and Medical Devices, University Politehnica of Bucharest, Bucharest, Romania

The era of nanosized materials is now considered the center of the evolution of future tools and emerging technologies with wide applications in industry, research, health, and beyond. Despite recent scientific progress, biological applications of nanomaterials are far from being depleted and current knowledge is limited by the poor access to significant data, but also by widespread and usually unfounded speculation. Although exhaustive, the current literature is difficult to reach and understand because of the specificity and strict focuses of researchers investigating different applications of nanomaterials.

In this context, the scientific series entitled Applications of Nanobiomaterials was motivated by the desire of the Editor, Alexandru Mihai Grumezescu, and others to bring together comprehensive, up-to-date and relevant findings on the field of biological applications of nanostructured materials, to promote the knowledge and expand our vision regarding future perspectives. Even though the approached domain is quite specific and research-oriented, this multivolume set is easily intelligible for a wide audience including: under-graduate and post-graduate students, engineers, researchers, academic staff, pharmaceutical companies, biomedical sector and industrial biotechnologies. However, some basic knowledge of the field of materials science (nanobiomaterials, pharmaceutical industry, products for medicinal treatments, nanoarchitectonics for delivery of biological active molecules and release, bone implants and stomatology) and engineering is a requisite for understanding technical aspects.

The selected authors of each chapter are outstanding specialists in the field of nanobiomaterials, who have made impressive contributions in a specific area of research or applied area within the scope of this book.

Each of the 11 volumes of the series contains 15 chapters, addressing the most relevant and recent matters on the field of the volume.

The first volume, Fabrication and Self-Assembly of Nanobiomaterials, introduces the reader to the amazing field of nanostructured materials and offers interesting information regarding the fabrication and assembly of these nanosized structures. In Volume II, entitled Engineering of NanoBioMaterials, readers can easily find the most commonly investigated methods and approaches for obtaining tailored nanomaterials for a particular application, especially those with a great deal of significance in the biomedical field. In the following step, readers will discover the importance and the ways of modifying the surface of nanostructured materials to obtain bioactive materials, by reading Volume III, Surface Chemistry of Nanobiomaterials. Starting with Volume IV Nanobiomaterials in Hard Tissue Engineering and Volume V Nanobiomaterials in Soft Tissue Engineering the biomedical applications of engineered nanomaterials are revealed and discussed, focusing on one of the most impacted fields, tissue engineering. Volume VI, Nanobiomaterials in Antimicrobial Therapy, highlights the potential of different nanostructured materials to be utilized in the development of novel efficient antimicrobial approaches to fight the global crisis of antibiotic inefficiency and emerging infectious diseases caused by resistant pathogens. Volume VII moves on to another key biomedical domain—cancer therapy. This volume, Nanobiomaterials in Cancer Therapy, describes current issues of cancer therapy and discusses the most relevant findings regarding the impact of nanobiomaterials in cancer management. Medical Imaging represents the focus of Volume VIII, while Volume IX deals with applications of Nanobiomaterials in Drug Delivery. Volume X, entitled NanoBioMaterials in Galenic Formulations and Cosmetics, refers to the perspectives highlighted by the utilization of nanosized functional biomaterials in the development of improved drugs and active principles for different biomedical industries. Finally, Volume XI is dedicated to the impact of Nanobiomaterials in Dentistry, which currently represents one of the most investigated and controversial domains related to the biomedical applications of nanostructured materials.

Due to their specific organization, each volume can be treated individually or as a part of this comprehensive series, which aims to bring a significant contribution to the field of research and biomedical applications of nanosized engineered materials.

Preface

Alexandru Mihai Grumezescu, http://grumezescu.com/,

Department of Science and Engineering of Oxide Materials and Nanomaterials, Faculty of Applied Chemistry and Materials Science, University Politehnica of Bucharest, Bucharest, Romania, Department of Biomaterials and Medical Devices, Faculty of Medical Engineering, University Politehnica of Bucharest, Bucharest, Romania

About the Series (I–XI)

The increased fabrication of nanosized materials with applications on the biomedical field by using biomimetic and bio-inspired processes and formulations, has recently lead to a new concept, Nanobiotechnology. This complex research brings together significant knowledge from physical, chemical, biological, and technological sciences in a widely applied field.

Medical applications of nanobiomaterials range from the development of adequate scaffolds for tissue engineering to therapeutic nanostructures, such as targeted drug delivery systems. The purpose of this multivolume set, entitled Applications of NanoBioMaterials, is to offer a broad, updated and interdisciplinary point of view regarding the application of these materials of future medicine, starting with their fabrication, specific engineering, and characterization and ending with the most investigated applications such as tissue engineering, antimicrobial and cancer therapies, and also the development of different medical and cosmetic use products. These books bring together the work of outstanding contributors who have significantly enhanced the basic knowledge and applicative concepts of this research field in their respective disciplines.

The Multivolume SET Applications of NanoBioMaterials contains 165 chapters, organized in 11 volumes which are ready to present a novel and up-to-date approach related to this intriguing domain. Each chapter is carefully composed and illustrated to highlight the relevance of nanobiomaterials on most biomedical fields, revealing the most recent applications on a specific domain. The whole set represents a great material for the academic community, including undergraduate and postgraduate students, researchers, engineers, and medical doctors, but also pharmaceutical companies and innovative biotechnologies.

These 11 volumes cover almost all aspects related to the Applications of NanoBioMaterials as it follows:

Volume I: Fabrication and Self-assembly of NanoBioMaterials

Volume II: Engineering of NanoBioMaterials

Volume III: Surface Chemistry of NanoBioMaterials

Volume IV: NanoBioMaterials in Hard Tissue Engineering

Volume V: NanoBioMaterials in Soft Tissue Engineering

Volume VI: NanoBioMaterials in Antimicrobial Therapy

Volume VII: NanoBioMaterials in Cancer Therapy

Volume VIII: NanoBioMaterials in Medical Imaging

Volume IX: NanoBioMaterials in Drug Delivery

Volume X: NanoBioMaterials in Galenic Formulations and Cosmetics

Volume XI: NanoBioMaterials in Dentistry

About Volume IV

Volume IV, entitled NanoBioMaterials in Hard Tissue Engineering, is an comprehensive up-to-date book that highlights the most recent progress obtained in the field of hard tissue engineering. The book contains data about the fabrication and characterization of nanobiomaterials involved in hard tissue reconstruction, harmoniously describing recent progress and advantages of both conventional and computer-assisted methods. The book discusses recent applications of different classes of nanobiomaterials. In vitro and in vivo applications are also well described and exemplified. Special attention is paid to the applications of nanobiomaterials in bone regeneration and in the development of functional coatings for tailored implants to improve osseointegration. Also, future challenges and perspectives of the application of nanobiomaterials in hard tissue engineering are presented.

Volume IV contains 15 chapters, prepared by outstanding international researchers from Canada, the United States of America, Spain, Germany, Czech Republic, Serbia, Romania, India, Malaysia, and China.

In Chapter 1, Nanobiomaterials in hard tissue engineering, Gabriel Castillo et al., review the main types of resorbable biomaterials and their in vitro and in vivo applications in biomedicine. The authors also report an optimized surgical method to improve bone regeneration by using functional biomaterials.

Imran Sheikh et al., in Chapter 2, Applications of nanobiomaterials in hard tissue engineering, give an overview about the implications of nanotechnology in the fabrication of biomaterials for hard tissue engineering applications. The most utilized materials (metals, ceramics, polymers, and biocomposites) are described. The authors highlight the most recent applications of biocomposites based on biodegradable polymers and ceramic or metallic nanoparticles in bone and cartilage engineering. The chapter reviews both ordered and unordered fabrication methods of nanobiomaterials. It is revealed that while conventional, unordered methods have been optimized in terms of time and cost, computer-aided methods of ordered nanofabrication boast higher resolution, precision, and accuracy at an increased resource cost.

Chapter 3, Emerging trends of nanobiomaterials in hard tissue engineering, reviews the advantages and disadvantages of hard tissue regeneration technologies and the main nanostructures involved.

In Chapter 4, Nanostructured materials as substrates for the adhesion, growth, and osteogenic differentiation of bone cells, Lucie Bacakova et al. present the most recent advances on planar materials with nanoscale surface roughness and three-dimensional nanostructured scaffolds. Their research supports the use of nanofibrous scaffolds and scaffolds with pores decorated with nanoparticles which are considered excellent substrates for tissue engineering.

Sampada Sawant et al., in Chapter 5, Bone scaffolds: what is new in drug delivery research?, report an up-to-date overview on patented technologies approved and marketed scaffolds and some of the ongoing clinical trials. Also, this chapter performs a detailed analysis of nanoparticle effects in the area of bone scaffolding.

In Chapter 6, Nano-hydroxyapatite: novel approaches in biomedical applications, Ecaterina Andronescu et al. highlight the main synthesis methods, in vivo applications and various available forms of hydroxyapatite, with particular reference to the field of bone regeneration and functional coatings for implants to improve osseointegration.

M.A. Bakheet et al., in Chapter 7, First principles study of the physical properties of pure and doped calcium phosphate biomaterial for tissue engineering, highlight the importance of calcium phosphate bioceramics in tissue engineering as a good alternative to biocompatible ceramics to fabricate scaffolds to accommodate and improve the growth of living cells and tissue reformation in three dimensions. A theoretical overview about the mechanical properties and doping with some trace elements such as zinc, magnesium, strontium, and silicon to the considered compound is also given.

In Chapter 8, Nanostructured scaffold and its bioactive potential in bone tissue engineering, Limei Wang et al. discuss the characteristics and applications of conventional biological scaffolding materials in bone tissue engineering. Properties of nanobiomaterials and applications of nanostructured scaffolds, such as nanoscale inorganics/organics composite scaffolds, nanofibers, and nanostructured delivery systems in bone tissue engineering, are emphatically discussed. Also, future challenges of the applications of nanostructured scaffolds in bone tissue engineering are presented.

Chapter 9, Inorganic micro- and nanostructured implants for tissue engineering, aims to bring an up-to-date overview regarding the production and biological applications (orthopedic or dental implants) of ceramic scaffolds (zirconia and alumina). Other materials, such as bioactive glasses and ceramics, have been widely known as good candidates for osteoconduction. In order to achieve economical production of tailored scaffolds, it is of great technological significance to understand clinical and materials requirements.

Chapter 10, Hydroxyapatite-silver nanobiomaterial, prepared by Chao Guo et al., reviews hydroxyapatite-silver (HA-silver) nanobiomaterials and divides them into three categories: silver-substituted HA nanoparticle, HA-silver nanocomposite, and HA-silver coating. The authors summarize the fabrication method, phase composition, and corresponding properties of these biomaterials.

Mehdi Razavi et al., in Chapter 11, Nanobiomaterials in periodontal tissue engineering, review different nanobiomaterials employed in periodontal tissue engineering for the effective regeneration of lost tissues. The main benefits and drawbacks of these nanomaterials, focusing on their biological characteristics, are discussed.

Chapter 12, Application of nanobioceramics in bone tissue engineering, by Ranjana Das, discusses recent results obtained on fundamentals of bone tissue engineering, the current state of the techniques, the recent development of bioceramics, and approaches used to enhance bone regeneration.

Oana Fufă et al., in Chapter 13, Metallic nanosystems in hard tissue implants, propose a circumstantial overview with respect to the current trends in hard tissue implant therapy.

Dejan Markovic et al., in Chapter 14, Nanomaterials as scaffolds in bone tissue engineering in dental medicine, describe the main requirements for bone tissue engineering scaffolds, as well as the main types and design strategies. The authors explain the mechanism by which nanomaterials promote bone formation and reveal the current research status of the main types of nanostructured scaffolds.

Chapter 15, Magnetite nanoparticles for diagnostics and laser repair of cartilage, prepared by Yulia Soshnikova, presents an up-to-date overview of the aspects of biofunctional magnetite nanoparticles for their potential use as absorbing agents in laser diagnostics and regeneration of cartilage. The effect of impregnating nanoparticles into healthy and damaged cartilage is also discussed.

Chapter 1

Nanobiomaterials in hard tissue engineering

Gabriel Castillo Dalí and Daniel Torres Lagares,    Department of Oral Surgery, School of Dentistry, University of Seville, Seville, Spain

Abstract

In this chapter we will conduct a focused review of the main types of resorbable biomaterials like PLGA (polylactic-glycolic acid copolymer), and their surface modifications on the nanoscale (like the nanocomposites of metallic oxides, oxygen plasma, hydroxyapatite, etc.), their applications in biomedicine, and future prospects.

We will also talk about the current advances in nanotechnology, and the surgical method of guided bone regeneration where these biomaterials are used. Subsequently, we will comment on the background of the previous, more favorable, in vitro studies that used this kind of biomaterial tested in human osteoblast cell culture. On a second level, we will examine an animal histomorphometric study, in vivo, of a successful evaluation of PLGA membranes with a new surface modification consisting of a physical pretreatment using oxygen plasma to favor their degradation into bone tissue in the healing phase, together with the addition of nanocomposites of osteogenic mediators like titanium oxide. The membranes were tested at 1-month regeneration in 8 experimental rabbits with 2 surgically generated critical bone defects.

To compare the potential effect of these membranes, a number of studies and comparisons have been performed, the following analyses and methods are used: a qualitative comparison of the integral structure and bone density generated, the osteoid and the presence or absence of abnormal inflammation were performed using toluidin blue dye (BT), a quantitative histomorphometric analysis of the percentage of newly formed bone was obtained using the Von Kossa stain (VK), a quantitative study of growth in millimeters per day was made by labeling with the Calcein clinic method (C), a quantitative study of the level of bone resorption was made using enzymatic staining with sodium-tartrate-resistant acid phosphatase, the enzymatic staining with alkaline phosphatase was used to make a qualitative comparison of the normal presence of osteoblastic apposition, related to controls (with only simple PLGA).

Keywords

Biomaterials; resorbable membranes; nanocomposites; guided bone regeneration; titanium oxide; plasma

1.1 Introduction

Bone is composed of 2% of the total volume of cells (mainly osteocytes, osteoblasts, osteoclasts, coating cells, progenitor cells, and adipocytes). Also, it has a 65% dry weight of mineral matrix in its crystalline form and the most calcified fraction of calcium phosphate and other ions is in the form of rods of hydroxyapatite (HA) (Ca10(PO4)6(OH)2, which are 3 nm thick and 60 nm long) (Testut and Latarjet, 2012).

The most serious problem in human medicine is the loss of organs produced by tissue diseases, trauma or damages, and their regeneration, which costs millions of dollars globally every year. Organ donation banks do not always meet the need, and the trauma suffered by some of these patients creates social and physiological rejection (Pontoriero et al., 1987).

Moreover, guided bone regeneration (GBR), used with tissue engineering, is becoming a new and necessary field of study that is growing and producing biocompatible scaffolds in order to promote the self-reparation of damaged tissues and organs. In dentistry, bone reparation is the most necessary and requested application that can only be done using GBR, combining tissue engineering and the fabrication of a new generation of enhanced biomaterials that are able to fulfill the growing needs of morphological and functional parameters of cells in bone repair (Castillo-Dali et al., 2014a,b; Cavalcanti-Adam et al., 2007).

The development of scaffolds has been promoted to seek a better quality of life associated with increased bone regeneration in the application of oral surgery. Nowadays, new materials have been created, such as PLGA (polymers of lactic-glycolic acid), and are being used to develop scaffolds that are implemented in bone. Tissue engineering has covered the need in medicine and dentistry of new devices that allow for better bone repair (Chhabra et al., 2011; Park et al., 2012).

In this regard, tissue engineering has revolutionized orthopedic and surgical researches showing a new reorientation in this field with new scaffolds that have nanometric-scale surface modifications in order to imitate the properties of the extracellular matrix (ECM) and structural variables of the autologous tissues and organs where they are implanted (Aboudzadeh et al., 2010; Boos et al., 2010).

Currently, it is known that tissue matrix is not only a support, but also a dynamic system integrated by several molecules depending on the type of tissue where they are formed. The barrier effect and the tridimensional design of the scaffold are both vital in promoting an appropriate response of the cellular and extracellular components of bone that lead to tissue repair. The above-mentioned is one of the fundamental bases of tissue engineering and it lead to the creation of the third generation of new biomaterials that provide an ideal substrate for the graft, giving the correct development of cell differentiation. The use of resorbable and biocompatible synthetic implants combined with autologous cells, stem cells, and growth factors would be another strategy to consider as a new and interesting approach to bone repair (Anselme, 2000; Dalby, 2005; Diener et al., 2005; Iglhaut et al., 1991).

1.2 Implants and Biomaterials in Tissue Engineering

1.2.1 Implants and Grafts, Associated Problems

An ideal dental implant should promote that the peri-implant tissues remain the same as they appear in natural teeth. Implant success depends on the osseointegration between the implant and the alveolar bone. Osseointegration is a term first used by Branemark as a direct anchoring between living bone and the implant inserted. There is a general agreement that GBR is not easy to stimulate and it demands special skills and experience of the therapist. One of the main problems associated with implants and grafts is the shortage of bone in the recipient area, which is often a cause of senescence, atrophy, trauma, or genetic factors of each individual. When the treatment area does not have enough bone to hold an implant or other orthopedic treatment, autologous bone graft can be used, although it requires a difficult and risky surgical procedure. The use of non-resorbable materials does not provide a total rehabilitation of the physiological anatomy of the patient, and it may cause rejection. Nevertheless, GBR processes are much faster, painless and have less surgical and incompatibility risks. It is for this reason that surgical implantations of such absorbable polymeric membrane PLGA, using the GBR technique, can not only improve bone regeneration, it can also restore the physiological anatomy of the treated area and the loss of bone volume required to place the implant. In addition, there is no need for the patient to undertake a second surgery, as it is in the non-resorbable membranes; it also avoids rejections, discomfort, and complications associated with the use of bone grafts (Davies, 2003; Hardwick et al., 1994; Karring et al., 1980; Lin et al., 2009; Maréchal et al., 2008; Meinel et al., 2005; Winter, 1994).

1.2.2 Biomaterials

The term biomaterial means any material, not necessarily biological in nature, that is tolerated by the organism and that could be used as a scaffold or can induce a process of repair in order to achieve restitutio ad integrum of the damaged tissue (Fuchs et al., 2011; Hutmacher et al., 1996).

The biomaterials can be classified as:

• Resorbable (chitosan, synthetic collagen, PLA (polylactic acid), PGA (polyglycolic acid), PLGA (polylactic-co-polyglycolic acid), etc.)

• Non-resorbable (titanium, silicon, hydroxyapatite, ceramic, PET, etc.).

This classification depends on its durability inside the organism or its degradation. It is a non-resorbable biomaterial when degradation never takes place, but if the biomaterial degrades after a day, a week or a month, it is a resorbable biomaterial. These kinds of synthetic biocompatible biomaterials are susceptible to being used in guided tissue regeneration and particularly in the repairing phase of the three-dimensional matrix supports in GBR. The principal function of these scaffolds is to separate the fast-growing fibroblasts of connective tissue from the slow osteoblasts of new bone in the regeneration phase, in order to promote their correct adhesion, proliferation and differentiation, providing the optimum environment to achieve the maintenance of their phenotype, morphology, and physiology (Caffesse et al., 1994).

The biomaterials used to create scaffolds in guided tissue regeneration must fulfill the following characteristics (Lee et al., 2006):

• Biodegradability (degradation in body liquids allows resorbability and does not require a second surgery)

• High porosity (pores allow cell adhesion and vascularization)

• Maximum surface area (the current size is necessary to manipulate in vivo and in vitro)

• Superficial rigidity (a minimum of stability is fundamental to keep the maintenance of its functionality)

• Specific three-dimensional form (it is essential to cell fixation and convergence).

These biomaterials also favor a correct cell migration to enhance restoring the capacity of the damaged tissue architecture and its function, making a temporary supporting framework structure until the surrounding preosteoblastic cells are able to secrete their own proteins to form the bone's ECM. Furthermore, the variables of strength, porosity, and velocity of degradation should be designed according to the type of local hard tissue that is going to be restored.

It is also known that providing a suitable environment for cell and tissue development is a crucial fact to maintain the cellular function and the proper development of a newly formed tissue. Its function is to direct the cell growth, either from adjacent tissues or cells seeded on it. The porous structure of the scaffold provides two essential characteristics to the implanted material; it increases the interaction surface between the device and the body cells and it allows the entry of nutrients into the scaffold.

It is important to develop less expensive methods that can be implemented in "in vitro cellular systems and interact with materials that can be assessed inside the biological environment. The development of these methods would solve the problems of biocompatibility and reduce the number of tests on animals and humans in vivo" that are currently carried out. The extensive variety of tissues makes it necessary to create different types of scaffolds that enhance microstructures, which are being developed, evaluated and analyzed to apply in TE, such as the alphahydroxy acids and their derivatives, the PLGA (Zimerman et al., 2004a,b).

The great progress in tissue engineering has led to the development of techniques that nowadays promise advantages in surgical implantation of osteoconductive scaffolds that contain biologically active transporters, osteoinductive agents, and the possibility to isolate cells. Osteoconductive, osteoinductive and undifferentiated bone cells are often combined to treat bone defects (Kikuchi et al., 2005; Linez-Bataillon et al., 2002; Liu and Ma, 2004).

Traditionally techniques have been used for the treatment of post-traumatic segmental bone defects, such as the shortening of a limb, the use of non-vascularized autologous bone, distraction osteogenesis, and the transposition of vascularized bone allografts. Today, however, there are new systems described; the filling titanium boxes mechanisms, the synthetic bone substitutes and, for example, hydroxyapatite or tricalcium phosphate, etc. Another indication of these new technologies is their substitution in oncology bone surgery. As mentioned earlier, the high incidence of osteolysis caused by the use of biomaterials is an important source of research that is in demand because of the preservation of the tissue and for being good in regeneration when there is destruction (Kanczler & Oreffo, 2008; Rouahi et al., 2006).

The real success of tissue engineering is to overtake the limitations that standard treatments have in tissue or organ transplantation, implantation, and the use of animal origin materials by using a new generation of synthetic resorbable biomaterials, which are well tolerated, to restore the damaged organ into the living body at real time. There are a lot of modifications designed for PLA as a copolymer, and several morphologies are used in biomedical applications in this matter.

The goal of this therapy is to promote permanent tissue regeneration without excessive trauma for the patient and avoiding a second surgery or supplemental therapies that can elevate the morbidity, discomfort, and cost of the treatment (Scantlebury, 1993; Tamimi et al., 2008).

1.2.2.1 Resorbable membranes

Resorbable membranes are films of organic polymeric biomaterials used as a natural barrier to treat critical bone defects that are difficult to recover optimally. These membrane-shaped matrices have the characteristic of being reabsorbed by the body without generating an immune response or inflammation response, and in turn they act as matrices that allow cell growth therein. They are designed to inhibit or delay the apical migration of the connective tissue during the healing phase, thus reducing the formation of pockets on the outer surface of the membrane, and they have also shown good adhesion and vascularization of the tissues in which they are placed. This effect is the result of a phenomenon known as contact inhibition, which reduces the formation of pockets on the outer surface of the membrane (Pontoriero et al., 1982). The use of resorbable membranes prevents a second invasive surgery where the non-absorbable membrane is removed, as this process may damage the regenerating tissue producing unwanted damaging inflammation, trauma and patient discomfort. The use of resorbable scaffolds helps to mitigate the pain produced by a second intervention where the metallic or non-resorbable materials are removed, sometimes producing risk of contamination, infection, and the creation of fibrous tissue in the bone-restoring area. These resorbable materials can produce a precise interaction with the cells that are in the proliferation phase through molecular stimuli into the host tissue, due to their property of absorbability and biological activity. In contrast to materials of animal origin, synthetic bioresorbable materials are designed to create the ideal scaffold in order to imitate all the characteristics of the extracellular matrix of the recipient’s tissue during the reparation phase (He et al., 2008; Hild et al., 2011).

The most usual biodegradable polymers in (GBR) are represented in Figure 1.1.

Figure 1.1 Resorbable polymers in biomedical applications.

The first time that PGA or PLA were used in resorbable sutures was in 1960. In 1990 the first research about GBR was published, they were mainly based in its property of occlusivity which avoids the colonization of cells that do not belong to the healing area (Sculean et al., 2008). PLGA is a biomaterial used to create resorbable membranes. PLGA, discovered in 1954, is a biodegradable biocompatible thermoplastic and synthetic polymer made of aliphatic polyester, and is prepared using glycolic acid, lactic acid, and polycondensation or certain forms of polymerization. It is framed within the poly-α-hydroxy acids, and can also be used to release steroids, anticancer agents, peptides, proteins, antibiotics, anesthetics, and vaccines. Their physicochemical properties are determined by the architecture of the copolymer and the molecular weight. Since it is a copolymer, its composition also plays an important role. Figure 1.2 represents the synthesis of the PLGA from lactic acid and glycolic acid, which are biological natural acids in the cell (Jung et al., 2011; Lundgren et al., 1995).

Figure 1.2 Summary of the synthesis of PLGA.

Biodegradable synthetic polyesters, such as PLGA and its derivatives, are widely used in bone regeneration. Currently, the polyglycolate and their copolymers with or without lactic acid are widely used for the synthesis of absorbable sutures and are being evaluated in other areas of the biomedical field. Three-dimensional matrices are devices that provide the support that cells need to proliferate and maintain biological signals required for the conservation of specific gene expression. It also defines the architecture of the tissue providing a specific environment, acting as a reservoir for water, nutrients, cytokines, and growth factors, among others (Keller et al., 2003; Lim et al., 2007).

The three-dimensional matrices used in bone tissue engineering (ITO) must meet certain criteria (Scantlebury, 1993):

1. Biocompatibility: The materials and their degradation products should not present toxic effects (genotoxic or cytotoxic), an immune response, or allow proper integration into the host tissue. Antiseptics and sterilizing must be used.

2. Porosity: Biomaterials must have open and interconnected pores which are necessary to disseminate nutrients and gases, and are needed to remove metabolic waste that comes from cell activity. This process is essential in bone due to its metabolic characteristics; in high rates mass transfer is expected even when processes are performed in vitro.

3. Pore size: The ideal pore size according to the ITO varies between 200 and 900 microns.

4. Surface properties: Chemical changes, such as the surface topography, can control and affect cell adhesion and proliferation. Chemical properties are associated with the ability cells have to adhere to material. Topographical properties are essential for osteoconduction, which is the process that osteogenic and vascular cells use to migrate to the surface of the three-dimensional matrix through a fibrin clot, which is implemented after the material sets to promote repair.

5. Osteoinduction: This is the process in which stem cells and osteoprogenitor cells are recruited to the site of bone regeneration and stimulate the differentiation into the osteogenic line, it also segregates mineralized bone matrix.

6. Mechanical properties: These depend on the implantation site and the mechanical forces to which the implant will be exposed.

7. Biodegradability: In order to manufacture the matrices, materials with progressive resorption rates should be used to equal those of bone formation. By transferring loads gradually the lesion regenerates entirely and the three-dimensional matrix degrades completely. Other requirements that should be met are the ability to be formed into different shapes and the need for an easy process.

8. Radiolucent material: This is preferable to differentiate radiographically the new bone formed around the implanted material.

The tridimensional structure of the scaffold, at a microscopic level, has a significant effect on surrounding cells; it has influence on their morphological and functional development (mechanotransduction) (Zong et al., 2010).

The initial response of the host to the biomaterial is an inflammatory reaction triggered by surgical trauma. In these early stages, cells start the deposition of a layer of non-collagenous proteins on the surface of the biomaterial, which regulates cell adhesion and mineralization. Cells, such as osteoblasts, grow anchored to an ECM and need to adhere to the substrate before they can start their normal performance. The ability to adhere to the substrate is what allows the ECM to signal from the core using cascades with intracellular proteins that act as an intermediary. Additionally, materials capable of stimulating osteoblast adhesion may increase its functional differentiation, regulating osteospecific genes involved in the neogenesis of mineralized matrix and bone tissue formation. It is recognized that the topography of the surface plays a decisive role in the adhesion of the osteoblast, migration and metabolism, modifying and controlling the process of osteoblast differentiation. These membrane-shaped matrices have the characteristics of being reabsorbed by the body without generating an immune rejection response and, in turn, they act as matrix scaffolds that allow cell growth therein. They are designed to inhibit or delay the apical migration of the connective tissue during the healing phase by reducing the formation of pockets on the outer surface of the membrane (Riveline et al., 2001; Schwarz and Bischofs, 2005). They have also shown good adhesion to and vascularization of the tissues in which they are placed.

Improved osteoinductive properties can be achieved through surface functionalization of these polymeric membranes, these thin layers incorporate crystal deposits in a nanometric scale creating a more complex surface topography that increases the osteoinductive capacity.

1.2.2.2 Biomaterial cytotoxicity

The ability to cause cell damage by a different toxicity, cytotoxicity, will depend on the physical and chemical properties of the biomaterial. However, the final common pathway will consist of striking an enzymatic system, giving as a result a reversible or irreversible inhibition. Moreover, such action will be located selectively in specific sites of the cell, as for example lesions on the membrane, which generally alter the lipoprotein structure and hence its permeability. This leads to the output of nutrients or ions. Another level of action is the endocellular site, which in turn may result in a blockade by various mechanisms (Krebs cycle, synthesis of fatty acids, etc.). Finally, the damage to these substances can also target cell organs, such as the mitochondria (the mechanisms responsible for oxidation), the microsomal system, ribosomes, the reticuloendothelial system, endoplasmic organelles, the enzymatic biodegradation induction, or it may cause lesions of the nucleus (DNA replication and nuclear protein synthesis).

The assessment of cytotoxicity can be carried out in different phases. On the one hand, there are initial tests that include models that study the cytotoxicity in vitro, in which the materials are in direct contact with cells cultured in the laboratory. Through this methodology we can assess the morphology of the cell, changes in protein synthesis, mitochondrial respiration, and different colorimetric, metabolic, enzymatic, hormonal, and radiometric activities, which are tests that can be easily standardized. In addition, these investigations assess cell sensitivity in different biomaterials and they also reduce the probability of subsequent failures of biocompatibility in in vivo tests, which are usually more expensive and require a longer period of time. Other assessments would lead to subtests, which are made in order to assess the inflammatory and/or immune reactions to biomaterials using experimental animals. Finally, there is the evidence of clinical application; which involves assessing the effects of dental biomaterials used in specific tissues with therapeutic applications. Primates and humans are preferably used to develop these tests. The development of these biocompatibility tests allows the assessment of the properties of dental materials with maximum effectiveness and within a short time to prevent health risks to the patient (Heidemann et al., 2002).

According to Scantlebury, the ideal characteristics of PLGA resorbable membranes are (Scantlebury, 1993):

• Biocompatibility: The capacity of being a non-allergenic and non-inflammatory material that is well accepted into the living body.

• Biodegradability: The property of assimilation, it is assimilated in the tissue where it is located through temporal degradation.

• Tissue integration: The capacity of local assimilation in the specific organ where it is implanted.

• Cell occlusion: The property to promote the recruitment and junction of the cells that are forming the new tissue.

• Clinical manageability: The capacity to work effectively in a clinic intervention.

• Maintenance of space: Being able to keep the different tissues separated.

The scaffold is usually made with degradable materials so it can be used inside the body. The degradation of these materials cannot affect the functional capacity of the organ. To maintain it, the degradation must be made gradually and in order. It is important that the degradation leads to the formation of bioactive materials without forming toxic materials.

These materials degrade as non-toxic waste in the organism, and they are subsequently removed. An example of PLGA membrane is shown below in Figure 1.3a and b.

Figure 1.3 (a) Surface micrography of a PLGA membrane (SEM). (b) High-resolution internal microstructure of a PLGA membrane (FSEM).

Whereas PLA–PGA polymers have been used primarily with bone and cartilaginous tissue, other polymers like polyanhydrides, polyorthoesters, polycaprolactones, polycarbonates, and polyfumarates have been applied in other kinds of tissues (Nyman et al., 1980a,b; Papalexiou et al., 2004).

1.3 Advances in Nanotechnology

One of the main goals of biomaterials used for bone regeneration is the capacity to imitate and perform an excellent interaction with the natural components of the body.

PLGA and other derivatives generally offer good biocompatibility and processing, however, there have been numerous problems with the use of these biopolymers when they are applied in tissue engineering. One of these problems is the hydrophobicity associated with PLGA. In order to overcome these disadvantages, inorganic materials can be incorporated in PLGA in the form of composites, such as titanium oxide (TiO2) or silicon (SiO2), hydroxyapatite (HA), tricalcium phosphate (TCP), or bioactive glasses which are currently under study (Barranco et al., 2001; Caffesse et al., 1990; Borrás et al., 2007).

There are three reasons which justify the inorganic component added to the polymeric portion of the scaffolds in almost all its uses. By adding this inorganic component, the polymeric matrix can be established as a substrate used in tissue engineering.

• The first reason is to reinforce the scaffold's structural integrity by changing the mechanical properties of the material when the inorganic component is added.

• Secondly, the polymer intensifies its bioactivity with the inorganic component.

• Finally, this component can help reduce the degradation of the polymer positively.

Apart from the qualities of biopolymers mentioned above, they have special properties in their physical structure, as for example: mechanical properties that can be applied in different tissues, macromolecular permeability, the ability to attract or repel proteins, the capacity to adhere to the tissue and lubricate it, and they also help facilitate cell processes. Thanks to the characteristics biopolymers have to interact with cells, they have different applications when used to make scaffolds in biomedicine, such as (Ngiam et al., 2009; Escobar and Vasseur, 1996):

1. Bioabsorbable polymers

a. Poly-amino acids (controlled release, cell adhesion peptides)

b. Polyanhydrides (controlled release)

c. Polycaprolactones (sutures and controlled release)

d. Lactic acid and glycolic acid copolymers (sutures, controlled release, bone discs)

e. Polyhydroxybutarates (controlled release, bone discs)

f. Polyorthoesters (controlled release)

g. Collagen (tissue reconstruction and coatings)

2. Biologically derived macromolecules

a. Crosslinked albumin (coatings for vascular grafts and a contrast dye for ultrasound)

b. Cellulose acetates (hemodialysis membranes)

c. Copper ammonia cellulose (hemodialysis membranes)

d. Cytosine (coatings and controlled release)

e. Collagen (coatings and hybrid organs)

f. Elastin (coatings)

g. Crosslinked gelatin (coatings for artificial hearts)

h. Hyaluronic acid (coatings, antiadhesives, ocular and auricular anti-inflammatory agents)

i. Phospholipids (liposomes)

j. Silk (sutures, experimental silk protein coatings)

3. Passive coatings

a. Albumin (thrombo-resistance)

b. Alkyl chains (absorbs albumin for thrombo-resistance)

c. Fluorocarbon (reduces catheter chaffing)

d. Hydrogels (reduce catheter chaffing)

e. Silica-free silicones (thrombo-resistance)

f. Silicone oils (lubrication for needles and catheters)

4. Bioactive coatings

a. Anticoagulants; e.g., heparin (thrombo-resistance)

b. Antimicrobials (resistance against infection)

c. Cell adhesion peptides (improve cell adhesion)

d. Cell adhesion proteins (improve cell adhesion)

5. Tissue adhesives

a. Cyanoacrylates (microsurgery)

b. Fibrin glue (coatings for vascular grafts and microsurgery).

The design and manufacture of three-dimensional porous matrix scaffolds have been improved using different techniques making them more functional in the final outcome of the scaffold (Shin et al., 2008; Galgut et al., 1991).

Polymeric structures, specially the ones with large porosities, can be made in different forms: gel casting, particle dissolution and release, membrane lamination, phase separation, gas saturation, high-pressure foamed particle release, lyophilization, fiber bonding or three-dimensional printing (Aboudzadeh et al., 2010; Ge et al., 2009).

Scaffolds for osteogenesis must mimic bone morphology, structure, and function in order to optimize integration into the implanted bone tissue. An appropriate porosity is one of the main characteristics of the material since a structure with reduced porosity applied in bone regeneration may cause demineralization. An appropriate size of porosity generates the correct entrance for blood vessels and any other substance into the cell, whereas a wide porosity allows the entrance of fibrous tissue that may block it.

Porosity and densities in the biomaterial can be controlled by modifying temperature and time intervals in the preparation of solvents and varying the density of solutions, as well as modifying the viscosity of polymers.

The time of degradation of the polymeric scaffold is also very important in the regeneration phase, and it should be designed and defined depending on the tissue where it will be implanted. The biomaterial has to be stable in order to act as a barrier to separate the different types of tissues with different growing speeds, but the barrier occurs only until the damaged tissue is nearly repaired, and it is then resorbed physiologically.

To improve osseointegration, many researchers have tried to make changes to the scaffolds in many ways. The membrane surface has been the main focus of these modifications, but it does so without modifying its internal structure since it can modify its surface. Some modifications try to create the capacity of the scaffold to generate chemical bonds between the recipient area and the device material, this can be achieved by using layers of specific metal oxides.

Nanometric improvements are within these modifications and they are usually made at room temperature, adding an advantage since it does not modify the microscopic characteristics of the material.

Nowadays, scientists study many functionalities to promote the osteoinduction of osteoblasts in the anatomic area where they are surgically inserted, thus favoring the synthesis of mineral matter and regenerating the defect of the bone with newly formed autogenous bone. Some of these modifications or improvements made to the scaffold surface are based on extracellular signaling, regulation, metabolism, proliferation, differentiation, and function in bone regeneration and are currently under study. These modifications are shown in Figure 1.4 (Emsley, 2003; Ivanoff et al., 2001).

Figure 1.4 Kinds of enhancers nanocomposites in PLGA scaffolds.

The principal techniques used to establish porosity in biomaterials in order to add nanocomposite enhancers are (Shokrgozar et al., 2010; Lobo et al., 2009):

• Salt leaching

• Gas foaming

• Crosslinking

• Phase transformation

• Sintering

• Magnetron sputtering

• Solvent casting/particulate leaching

• Rapid prototyping

• Freeze-drying

• Laminated manufacturing

• Foaming object H2O2.

Some of these techniques allow processing and add metallic materials, ceramics, and organic polymers found in nature (collagen, hyaluronic acid, fibrinogen, chitin, chitosan) or are synthetic (polycarbonates, polymers PLA, PGA derivatives, and copolymers thereof) (Vigier et al., 2011; Parodi et al., 1996).

1.3.1 Titanium Oxide (TiO2)

Titanium is a transition metal element with a much lighter density than steel. It has high corrosion resistance and high mechanical strength. It is a refractory metal that is abundant in nature and has a low thermal and electrical conductivity. Its use is widespread, depending on its characteristics, since it is able to withstand extreme temperatures, cold and heat, and it resist acid attack. This metal has biocompatible properties, since body tissues tolerate its presence, this is why it is feasible to manufacture many prostheses and implants with this metal. In addition, both titanium and titanium dioxide have a low level of toxicity, the International Agency for Research on Cancer (IARC) states that titanium is not classifiable as carcinogenic for humans.

Previous studies indicate that by adding crystals to this material its osteoconductive characteristics improve (Park et al., 2005; Popat et al., 2007; Sader et al., 2005).

1.3.2 Oxygen Plasma (P-O2)

Oxygen plasma is a physical surface treatment that increases the roughness of the membranes to promote cell adhesion, cell contact, and the degradation of polylactic in the tissue of the organism where it is implanted. The feasibility of the GBR treatment and the capacity of osteoinduction are also activated in this way. The working conditions of oxygen plasma and its functionalization process can be found in various known references (Biggs et al., 2007; López-Santos et al., 2008).

Different types of macroscopic scaffold designs are used in biomedicine and guided tissue regeneration and are being applied today. At first, two-dimensional membranes were created to use as a barrier, later on a design with fiber networks in carpets or porous meshes came out, and recently, there are complex scaffolds with three-dimensional structure matrices such as cylinders, discs, and 3D high-porosity spheres (Kreder et al., 1994).

1.3.3 Three-Dimensional Discs and Cylinders

This type of scaffold is a porous structure with cylindrical masses used to fill pre-implant mandibular cavities. The curvature and size of the matrix depends on the area of application.

1.3.4 Three-Dimensional Spheres

Microspheres are highly porous 3D homogeneous masses used for drug release or to fill bone cavities. The application of this type of scaffold when using polymers has increased in the last few years even in the injectable form. The fabrication of microspheres shows an excellent reproducibility in terms of the microencapsulation process and solutes release, such as proteins, peptides, antibiotics, antigens, and hormones, which may present a reduced activity a few days later due to interactions between the hormone and the polymer (Wang et al., 2010).

Other new interesting types of biomaterials structures recently designed are follows.

1.3.4.1 Controlled release gels

Time-release gels are the third generation of biomaterials, they have many variants depending on the nature of the substances that are released, the time they take, and the target tissue that is going to be implanted and treated. Having a controlled release not only prolongs action but also attempts to maintain drug levels to avoid potentially hazardous peaks in drug concentration, ingestion, or injection and it maximizes therapeutic efficiency. The release is usually affected by degradation, disintegration, or dissolution of the excipient in which the active compound is formulated. The stimuli used to allow drug release include: light, pH, enzymes, temperature, ultrasonics, osmosis, magnetic fields, and an electronic control such as MEMS and NEMS. Examples of personal care, cosmetics, food and science applications are often focused on flavor or odor