Orthopaedic or bone tissue engineering is a fast growing field in medical science to treat the people suffering from severe pain due to bone loss and damage. In this area the artificial extracellular matrix called scaffold play a critical role in cell seeding, proliferation and new 3D tissue formation. So the key challenge in bone tissue engineering is the development of scaffold with desirable characteristics like biocompatibility, biodegradability, bioresorbability and surface characteristics. In most cases the response of host organism in macroscopic, cellular and protein levels to biomaterials is closely associated with the materials' surface properties. Traditional scaffolds based on biodegradable polymers such as PLA and PLGA are weak and non-osteo-conductive. Whereas the natural polymers are more biocompatible, bioresorbable & induces mineralization of bone tissue. Naturally derived collagen, Chitosan, Alginates, Silk from silkworm cocoon & spider dragline are promising substitutes for bone tissue engineering. The nature of the surface can directly affect cellular response that ultimately influence the rate and quality of new tissue formation by determining whether protein molecules can adsorb and how cell attach and align themselves. Surface properties of scaffolds are generally characterized by Equilibrium Water Content(EWC), Air-surface contact angle, Surface free energies, SEM photography & FTIR-ATR spectral characterization This paper describes the various types of Natural biopolymers, Techniques for better morphology of scaffold, Surface modification & techniques to analyze the surface characteristics.
Key Words: Surface modification, Natural polymers, Scaffold, Tissue engineering, Plasma treatment, SEM,
FTIR
Introduction
Most frequent, traumatic and expensive ailments in human health care is caused due to the loss or failure of an organ or tissue. Severe damage to tissue or organ caused by developmental abnormalities, trauma, infection or aging-related degeneration result in disability and extensive pain (e.g. osteoarthritis). Millions of people every year suffer from these tissue-related diseases like bone, cartilage, skin, burn etc worldwide [1,2].Current clinical treatments such as auto grafting or allografting and the use of synthetic materials such as metals and bone cements for bone and cartilage defects have several limitations such as improper and incomplete defect healing [3]. Though autograft transplantation is the preferred treatment but suffers limited supply donor site morbidity [4,5]. Whereas allografting introduces the risk of immunorejection that may cause lessening or complete loss of the bone inductive factors [6,7]. Metals or bone cements have often resulted in complications such as stress, shielding-induced resorption of the surrounding bone and fatigue of the implant [8]. Therefore, in recent years research work has been directed towards the development of celluralized scaffolds for the regeneration of various tissues including bone and cartilage to treat the patient suffering from these diseases by the tissue engineering approach [9, 10]. However, there is need to develop bone tissue engineering including need for better filler materials that can be used in the reconstruction of large orthopaedic defects and the need for orthopaedic implants that are mechanically more suitable to their microenvironment [11]. This paper describes the various types of Natural biopolymers , Techniques for better morphology of scaffold, Surface modification & techniques to analyze the surface characteristics to prepare the scaffold for orthopaedic tissue engineering.
It is notable that polymer surface science has got broad spectrum of techniques & approaches, full of controversies , thus unmanageable to give a complete review at a place. This review is by no means includes all previous works, rather the aim of the paper is to make better understanding of the polymer application in tissue engineering area.
Scaffold Materials for Bone Tissue Engineering
The potential tissue engineering materials for bone defects are osteoinductive and osteoconductive. Osteoinduction causing pluripotent cells to differentiate into new bone formation [12,13]. These materials are necessary for bone repair in a location that would normally not heal itself if left untreated [7]. Whereas osteoconductive materials give support to host body for ingrowth of three dimensional structure for bone formation in which healing can occur if left untreated. Researchers have found that osteoconductive properties of biopolymers depend on their location and the structure of the polymers[14] .The absorbable polymers for long bone defects are believed to promote bone growth by excluding surrounding soft tissue and undesired cell components from the defect location by maintaining an osteogenic-rich environment and allowing bone growth onto the polymer skeleton[14] . Cellular phenotype is affected by their relative hydrophobicity and percent crystallinity whereas Variations in surface charge affects cellular spreading or affinity for the surface, causing changes in phenotypic expression [15]. There are several methods of processing the polymeric matrices including the most common method ,solution cast, particulate-leached,developed by Mikos [16]. In order to refine this method, it is quite possible to modulate the pore topography and size to suit a particular cell type, e.g., osteoblasts. Since bone has very different structures depending on its function and location, pore size and tortuosity must be carefully modulated to control the release of a material complexed to the polymer [17,18]. Osteoblast proliferation is sensitive to surface topography [19], strain or other mechanical stimuli. The adhesive, proliferative, and phenotypic properties of cells is affected by particle size, shape, and surface roughness. Cells can change topography, and they are most obviously sensitive to chemistry, topography, and surface energy. Particularly for an absorbable material such surface features becomes interesting since this is a dynamic material, always presenting a new surface. Desirable qualities of a bone tissue-engineering scaffold [20, 21] are as follows-
Correct mechanical and physical properties for application
Absorbs in predictable manner in concert with bone growth
Does not induce soft tissue growth at bone/implant interface
Average pore sizes approximately 200-400 µm
Maximal bone growth through osteoinduction and/or osteoconduction
Support to mineralization
Adaptability for irregular wound sites
No detrimental effects to surrounding tissue due to processing
Sterilizable without loss of properties
Types of natural polymers
The biomaterial used for the development of scaffolds should be biocompatible, biodegradability & bioresorbability, non-toxic supporting cellular interactions & in vivo tissue formation possessing the required physical and mechanical properties. Animal or plant derived proteins have been shown to be used as scaffold material for tissue engineering applications. An important aspect of natural materials is the induction of undesirable immune response due to the presence of endotoxins & impurities depending upon the source of material Also, their quality may differ batch wise during large scale isolation & processing. Polymers from natural sources include (i) proteins (ii) polysaccharides and (iii) polyesters are considered to be potential for scaffold preparation.
Protein-derived polymers:
The most important protein derived polymers are Collagen and Silk proteins. Type I Collagen being the most abundant & investigated for tissue engineering application [22]. Heat treatments and/or Chemical glycation procedures are used to fabricate matrices with adequate mechanical properties. Collagen microsponges into synthetic polymeric scaffolds increase their mechanical performance [23]. Collagen-based scaffolds, combined with active agents like growth factors have more therapeutic influence on tissue engineering approaches [24]. Silk proteins contain a highly repetitive primary sequence of simple amino acids that leads to a high content of β-sheets and responsible for good mechanical properties. Research have shown that the utilization silk fibroin in tissue engineering applications, particularly where high mechanical strength and slow biodegradation is required [25]. Experimental studies of MSCs seeded highly porous silk scaffolds for in vitro cartilage [26] and bone [27] regeneration have given positive results.
Polysaccharides:
Polysaccarides have got wide application in tissue engineering particularly for the inhancement of mechanical properties. Thee biopolymers can broadly be divided into four major groups according to the source from which these are obtained. These are subdivided ,based on their chemical structure. Starch & cellulose are the most important plant saccarides finding special application in tissue engineering . The cohesive & hydrogen-bonded structure of cellulose fibres makes it exceptionally water insoluble & great strength exhibiting poor degradation in vivo. Alginate, an algal polysaccharide has potential to combine with calcium [28] and this property can be utilized for mineralization for orthopaedic tissue engineering. Galactose are also obtained from algae. Chitin and its derivative chitosan from animal exoskeleton have been studied for bone regeneration and have been found excellent biodegradability & cell adhesion. Hyaluronic acid, an important GAG component has been used for finishing of scaffold surface for improving its biocompatibility. Non-toxicity of Pullulan and unique rheological properties of xantan gum from microbial culture also finds application in vivo. Bacterial cellulose (BC) has high water- holding capacity, crystallinity & biocompatibility giving it high tensile strength in wet state.
Naturally derived polyesters:
Polyhydroxyalkanoates (PHAs) are thermoplastic polyesters showing good biodegradability &, biocompatible are obtained from micro-organisms. Among these, poly-hydroxybutyrate (PHB) and poly-hydroxybutyrate- co-valerate (PHBV) are commonly used for tissue engineering application. Materials more flexible, less crystalline and easy to process can be produced by copolymerization from pure PHB. These materials can be used as support material for tissue engineering application.
Bone regeneration
The major challenge concerned with polymeric scaffolds for application in bone tissue engineering is due to low mechanical strength and shape retention failure. It has been shown that silk-based scaffolds support human MSC (hMSC) adherence and proliferation in vitro. However, different responses were observed for bone-like tissue formation depending on materials and processing conditions. Due to fast degradation of collagen scaffolds, isomorphous replacement with a newly formed bone doesn't take place. In comparison with collagen scaffolds, silk and RGD-silk scaffolds were more supportive to osteogenesis due to their stable macroporous structure and slow degradation [29]. Other studies have proved that the crosslinking [29] or the composite of collagen with materials having improved mechanical properties [30] forms scaffold robust enough to support bone regeneration. Due to high degree of swelling some chitosan scaffolds are mechanically weak and instable. The increase in mechanical strength can be achieved not only by changing the processing methodology [31], but also by chemical bonding with other polymers such as alginate [32]. A nano- and microfiber combined starch-based scaffold [33] showed unique architecture, being able to support cells as well as to provide supply of nutrient and gas to cells for bone regeneration.
Mode Of Application Of Naturally Derived Polymers For Bone Tissue Engineering
BMP systems:
Yasko and coworkers created 5-mm defects in the femora of 45 adult rats. By showing a 100% union rate using a combination of rhBMP 2 and DBM as a carrier, they concluded that BMP might prove to be a bone graft substitute of unlimited quantity [34]. Reddi and Levine both cite insoluble collagen as a potential carrier for BMP, but point out that data are limited for this matrix [35]. The implantation of purfied, hydrophilic BMP promotes their dispersion shortly after implantation and before osteoinduction can occur [36]. They are normally complexed with carriers to prevent the dispersion and to deliver the BMPs slowly to the desired sites.
Cellular systems:
The collagen materials have been applied as cellular scaffolding systems. Since collagen possesses no inherent structural mechanical properties, engineering modifications can form a stiffer polymer to enhance load bearing capacity of bone during the regenerative phase of healing. When treated with calcium solution, deposition of calcium phosphate improves mechanical integrity [37]. This technique have shown great promise in chondrocyte culture and bone tissue engineering as well. Collagen sheets used to fabricate composite bone tissue-engineering scaffolds have already been reported [38].
Surface Nano-Patterning Of Scaffolds
The types of nanotopographical features that can be created on materials of two categories: unordered topographies and ordered topographies. Unordered topographies are typically those that spontaneously occur during processing. The techniques include -polymer demixing, colloidal lithography and chemical etching. These techniques are usually simpler, quicker, and less costly than the more complex equipment and processes needed to create ordered topographies. Ordered topographies are those that can be created with techniques like photolithography and electron beam lithography. Table 1 compares different Nanoscale topography fabrication methods
Electrospinning:
Electrospinning can be use to create a simple ordered topography of aligned fiber bundles controlled fabrication of model substrates that will allow for a systematic study of surface topographies and their effects on a variety of growth parameters. Study by Sepideh et. Al reveals that no additional surface modifications is required for electrospun fibers since they support attachment and proliferation of mesenchymasl stem cells [39].
Electron Beam Lithography:
Electron beam lithography has been used to create surface topographies at the nanometer scale for studying cellular growth and behavior on these surfaces [40]. This method involves the use of high-energy electrons to expose an electron-sensitive resist [41] and has the ability to create single surface features down to about 3-5 nm [40].
Photolithography:
This method can create precise geometries and patterns. This method allows for investigators to observe a large population of cells in order to gain a significant understanding of the mechanisms of single-cell surface interactions. Dalby et al. investigated fibroblast response to arrays of nanopits created using EBL [42]. Fabrication using EBL can be time consuming and costly. To overcome this, nanometer patterns can be replicated in polymeric materials making the ability to mass reproduce the patterns created a much faster and inexpensive process[43]. This can dramatically cut the time and Methods for Fabrication of Nanoscale Topography for Tissue Engineering Scaffolds .
Polymer Demixing:
A unique method for creating nanoscale topographical features for use as cell substrates is through the use of polymer demixing. It involves the spontaneous phase separation of polymer blends which occurs under conditions such as spin casting onto silicon wafers [44]. Polymer demixing can be used to create topographies similar to those commonly used to study cell growth on nanostructured surfaces. Polymer demixing may not be ideal for creating model surfaces to study cellular interactions with nanoscale features. Despite this limitation, nanotextured surfaces created by polymer demixing have been used to study the interactions of various cell types to nanotopographies of various heights [45,46].
Colloidal Lithography:
Colloidal lithography is an inexpensive method for creating nanoscale topographies. This technique allows for the production of surfaces with controlled heights and diameters. Colloidal lithography involves the use of nanocolloids as an etch mask. These nanocolloids are dispersed as a monolayer and are electrostatically self assembled over a surface. To vary the surface structure, the coverage of the monolayer of colloid and the size of the colloid can be varied. The spacing between particles can be controlled by changing the ionic strength of the colloid solution. Patterning with particle sizes of 20 nm has been demonstrated.[ 47] With this technique, large surface areas can be patterned (∼cm2), making it a suitable technique for creating functional biomaterials for cell studies.[ 48]. Repeated adsorption of particles and charging of the substrate results in multilayer formation, finally fabrication of controlled 3D nanoporous particle films. Research is under way for in vitro cell studies of how nanotopography influences osteoblast (bone forming cell) adhesion and morphology.
Chemical Etching:
Chemical etching is a means of producing nanoscale features on the surface of a material by soaking it in an etchant. Typical etchants are hydrofluoric acid (HF) and sodium hydroxide (NaOH). As the material is etched away, the surface is roughened creating pits and protrusions at the nanometer scale. This process is essentially a surface treatment and cannot create structures with any prescribed geometry or organization. It can, however, provide a very quick, easy, and inexpensive means of creating a nanostructured surface by changing the scale of the roughness on the material surface. Three dimensional scaffolds with nanoscale features can be made by this method. The nanoroughened surface not only increase adhesion but provides more room for cell adhesion and population to grow into. Increased porosity also allows for greater infiltration of the scaffold by cells as well as increased nutrient and waste diffusion.
Surface Modification Methods
The success of tissue growth depends inevitably on the cellular interaction with scaffold material since the nature of surface directly affects the cellular behavior. Cell adhesion on the surface is determined by surface chemistry as well as its topography. The natural polymers provide the ideal environment for cell-material interactions. Therefore, various surface modification methods for natural biopolymers are being studied and is an active area of research. Two main strategies in surface engineering of biomaterials are often employed based on the understanding of the dominance of the biorecognition process on cell behaviors. The normal bioactivities of the adsorbed proteins is maintained by surface properties such as chemical composition, hydrophilicity/hydrophobicity, surface charge and roughness, etc.. Such nonspecific protein absorption doesn't induce specific cell behaviors. So the strategy to directly immobilize certain biomolecules on the biomaterial surfaces is more effective to induce specific cellular response [49]. For bone tissue engineering common methods include abrasion and sand blasting, chemical treatment and surface activation. Several surface activation techniques are atom bombardment, plasma treatment, ion implantation, laser treatment, electron beam and welding. The various methods of surface activation have been categorized in Table 2 whereas Table 3 compares the surface modification methods in terms of various topographical, cell/proteins and interfacial properties.
Chemical modification:
Biomaterial surface chemistry can influence surface adhesion as well physiological pathways regulating cellular proliferation, differentiation, and survival. Improved surface chemistry of a biomaterial also aid in construction of an artificial tissue mimicking with native tissue in physical properties [50]. For bone tissue engineering, polymeric scaffolds modified with charged groups that are conducive to mineralization can facilitate premineralization of the scaffold with hydroxyapatite [51] and induce differentiation of stem cells into an osteogenic lineage [52]. Likewise, cartilage tissue formation can be induced by use of sulfated polymers [53]. For silk, the derivatization of the carboxylic acid residues through carbodiimide coupling with primary amines is the most commonly used chemical modification method [54]. A simpler and effective method to tailor the structure and hydrophilicity of silk fibroin protein was developed using diazonium coupling chemistry [55]. Introducing hydrophobic functional groups cause rapid conversion of the protein from a random coil to a β-sheet structure, while addition of hydrophilic groups inhibits the process. Novel surface chemical modification appeoaches are under way, including [56]-
Plasma polymerization technology
Tailoring di-peptide monolayers to induce surface wettability
Self-assembly of nanoscale templates to induce biofunctionality of surface
Gamma irradiation:
Gamma irradiation of surface inhance its surface properties by crosslinking long chains . Fei Yang et al did experiment on the chitosan membranes irradiated by gamma rays when processed using infrared spectroscopy ,mechanical properties was found improved. The infrared spectra of the chitosan membranes with and without irradiation were compared to investigate the mechanism for modification of the chitosan membranes induced by the gamma irradiation.
The experimental results show that the gamma irradiation in the specified dose range can improve the mechanical properties of chitosan membranes while maintaining their excellent biocompatibility. Generally, two factors induce changes of mechanical properties in polymeric membranes: crosslinking between molecules and crystallinity [57,58]. According to previous reports [58,59], gamma irradiation affects the geometric regularity of the structure and the material crystallinity is closely related to its mechanical properties. Therefore, the gamma radiation effect on the crystallinity is one factor which improves the chitosan mechanical properties. Free groups, such as amido, alkyl, hydroxyl etc. in this polymeric saccharide long chain produce radicals. So crosslinking between radicals is another possible factor inducing the mechanical property improvements. The crystallinity reaches a maximum value for the gamma irradiation dose in an appropriate range. For macromolecular materials, enhanced crystallinity improves their rupture intensity, breaking elongation, Young's modulus and other mechanical properties [60]. Water contact angles on the materials surface shows the degree of hydrophilicity.
Plasma surface modification:
Plasma processes have been developed to attain specific surface properties of biomaterials. Plasma treatment offers flexibility, effectiveness, safety and environmental friendliness. The plasma is effective at near-ambient temperature without damage for most heat-sensitive biomaterials. Plasma treatment modifies only the near surface of treated substrates and does not change the bulk material properties. Plasma surface treatment improves interfacial adhesion by creating chemically active functional groups, such as amine, carbonyl, hydroxyl, and carboxyl groups. The process can also be used to alter surface energies according to the application. Polymers interaction with gas plasma can create hydrophilic and hydrophobic surfaces whereas the wettability of the surface will be increased by creating hydroxyl functionality using oxygen. In a similar way, surfaces can be specifically engineered to modify protein binding and improve blood compatibility. Plasma processes have been employed to modify the surface of metal implants for adhesion promotion to bone cements, or enhancing cell attachment and growth. The degradation rate of bioabsorbable polymer has been shown to be regulated by Plasma crosslinking at surface [61].
Medical implants and devices, sensitive to temperature, radiation and chemicals can be effectively sterilized by Plasma process.
There are also a number of obstacles opposing the commercial use of plasma depositions and reactions in the biomedical field. One major barrier is the lack of clearly defined guidelines on how surface modifications will be regulated. In addition, although plasma processes and applications have been proposed and studied extensively in academic settings, process reproducibility is matter of concern.
Atomic oxygen surface modification:
Atomic oxygen surface modification and texturing technology is being evaluated for use in a number of biomedical applications. It is useful for changing the wetting characteristics of surfaces, improving cell growth and adhesion and removing biologically active contaminants such as endotoxins, from the surfaces of orthopaedic implants. Benefits include Surface texturing of polymers for cell growth & endotoxin removal. Researchers at the NASA Glenn Research Center also have used this process as an innovative approach to solve the problem of endotoxin contamination on the surfaces of orthopaedic implants. NASA's approach to remove endotoxin is an atomic oxygen treatment process that occurs in a low-pressure (4-100microns) air plasma and at a relatively low temperature (<60ËšC) converting endotoxins into harmless gases [62].
Surface Characterization and Analysis
The main surface characterization techniques for polymers are contact angle measurements, XPS, FTIR, and differential scanning calorimetry. The contact angle values can successfully be used as an indirect measure of protein adsorption, XPS for surface chemistry and FTIR for subsurface composition. Two general principles for sample analysis are: Firstly, all surface analytical methods have potential to alter surface properties. So we should aware of any kind of damage to surface characteristics. Secondly, no one method is sufficient to derive all data. So, two or more analytical methods may be required. These analytical methods for surface characteristics can be broadly divided into spectroscopy, imaging and optical techniques. Here we are taking spectroscopy and imaging techniques for consideration due to its utility for bone tissue engineering.
Spectroscopic techniques for surface morphology and chemistry identification:
Spectroscopic techniques as used in the evaluation of polymers as applied to biomedical devices and the focus area of cardiovascular bypass grafts are predominantly based on the principle that when energy is directed at an object, the resultant energy is either reflected, transmitted, scattered, or absorbed as illustrated by equation :
r + a + s + t = 1
Where, 'r' is the reflection coefficient, 'a' is the absorption coefficient,'s' is the scattering coefficient and 't' is the transmission coefficient.
Infrared (IR) and Raman spectroscopy are two very closely related spectroscopic techniques which belong to the group of molecular vibrational spectroscopies. Attenuated total reflectance spectroscopy-Fourier transform infrared spectroscopy (ATR-FTIR) has been developed specifically for the analysis of surfaces [63,64]. Raman spectroscopy highly complements IR spectroscopy as the former is based on polarizability and the latter, on dipole moments [65].
The most widely used surface analysis techniques in polymer chemistry are electron spectroscopy for chemical analysis (ESCA) and X-ray photoelectron spectroscopy (XPS); a methodology for determining surface elemental composition to a depth of several Angstroms (AËš) (or several nanometers [66,67]. Previous studies showed that XPS can access the extent and thickness of adsorbed proteins on a surface[68,69].
Circular dichroism spectroscopy (CD) is a routinely employed technique to study protein conformation. Different secondary structure of proteins visually, α-helix, β-sheet and random coil structures generate different absorbance and refractive indices to both right and left circular polarized lights, resulting in formation of different CD spectra. [70, 71]
Time-of-flight secondary-ion mass spectrometry (TOF-SIMS) based on static, surface characterization of the polymer surfaces using ultrahigh vacuum (UHV), which generates both positive and negative spectra from the outer 10-20AËš, this being read by mass spectroscopy or recorded as an image [72]. The limitations of TOF-SIMS include low-depth penetration (1 nm) [73], extreme sensitivity to contaminants, need for the sample to withstand high negative pressures (108 Torr) and the variability of secondary ion yields in different polymer environments ('matrix effect') [72]. In the case of polymers and the biological tissue present on the surface of these, the 'matrix effect' is minimal.
Variations of SIMS exist whereby the primary ion beam source is altered: electron ionization, chemical ionization, fast atom bombardment, electro-spray ionization, and matrix-assisted laser de-sorption/ ionization (MALDI). The MALDI is augmented by the delayed extraction technique and reflection instruments are able to ionize a wider class of polymers with a greater mass range detection but are unsuitable for small molecular weight polymers due to the 'matrix effect' [74]. This can be overcome by using surface-assisted laser de-sorption/ionization [75] or specific patterned platforms which allow protein detection [76].
Auger electron spectroscopy (AES) is used to assess surface morphology as well as to determine elemental composition. Advantages of AES include surface sensitivity combined with high lateral resolution (20 AËš) . A limitation, however, is its inability to characterize organic surfaces as the electron beam thermally damages biomolecules.
When a neutron beam is incident on a surface at an angle, a proportion of it is reflected and this is the principle of specular neutron reflectivity (SNR) and diffuse reflectance spectroscopy. SNR is directly dependent on the material's refractive index, angle of incidence, surface topography, and the proportion absorbed, which vary between different polymeric materials. The principal limitation associated with SNR is the prerequisite of excessively large surface areas. In the related technique, namely the diffuse reflectance spectroscopy, as incident energy on a polymeric surface penetrates its particles, the energy is reflected in all directions (diffuse reflectance). If the polymeric sample is coated with a powder such as potassium bromide (KBr) then this causes the reflection of outgoing electromagnetic energy and, in so doing, collects the energy over a wide angle. Diffuse reflectance spectroscopy allows complementary information to be obtained and is, therefore, particularly useful in the study of polymeric based powders and fibers.
Polarization-modulation infrared reflection-absorption spectroscopy (PM-IRRAS) involves a primary, polarized infrared light source being incident on a polymeric surface, which intensifies when its dipole moment is in the surface normal direction.
In photo-acoustic spectroscopy, the incident energy is modulated infrared radiation which, after being absorbed by the polymeric material, is converted to heat or mechanical energy which is in turn transferred to a microphonic device as a pressure wave [77].
Colorimetric method for surface density of functional groups:
ATR-FTIR, XPS and SIMS are powerful techniques to understand surface chemistry quantitatively or qualitatively. A complement approach for quantification of surface functional groups on a biomaterial surface is colorimetric method, particularly when a large amount of functional groups exist on the surface. Several colorimetric methods have been developed to effectively measure the surface density of carboxyl groups and amino groups on polymer surfaces, based on either ion exchange mechanism or particular chemical reactions. The main disadvantage of the colorimetric methods, however, is that they are far less sensitive than XPS and SIMS, and thus is only useful when a large amount of surface functional groups are present. Rhodamine 6G (Rd6G) [78] & toludine blue (TBO) colorimetry [79] can be used to determine trace amount of carboxyl groups while methyl orange (MO) [80] & ninhydrin[81] are employed for quantification of amino groups on material surface under acidic condition based on ion exchange mechanism.
Contact angle measurement:
Surface free energy is an important parameter in polymer based systems, in particular, whenever they are applied in biomedical devices .The surface free energy of a polymer is defined as the unit surface area of the polymer at constant pressure, temperature, and chemical composition. Using contact angle measurements (sessile drop method), the surface free energy is calculated by using Young's equation:
Cos(θ)= (γSVγSL)/γsv
where γ is the contact angle, γSV is the solid-vapour interfacial tension and γSl is the solid-liquid interfacial tension. A variety of methods such as the Wilhelmy plate, sessile drop, and captive bubble techniques [82] have been used to determine surface energetics. Modifications like acid-base contact angles (for surface chemistry) can also be used [83]. Very few works have been contributed to detailed understanding of the optimal hydrophilicity/ hydrophilicity equilibrium for a specific cell's behavior on a specific material. Studies have revealed that optimal surface hydrophilicity is a variable value depending on cell types and specific material surfaces. For example, it was found the best water contact angle for endothelial is for chondrocytes cell attachment and proliferation is 76â-¦ (COOH density, 3.8Ã-10−7 mol/cm2) on the PLLA-g-PMAA prepared by the same grafting method [84].
Measurement of wettability:
Microcalorimetry is another technique used to determine the wettability of a polymer surface by using heat. The energy required to displace solvent molecules from a pre-wetted surface is assessed using probe molecules. These devices are sub-classified into titration, adiabatic, and heat conduction modes [85]. The underlying principle involves differential measurement whereby differences in enthalpy between the polymeric sample and medium over time are measured. In these terms this is an indirect methodology but still extremely sensitive and a direct measure of any degradation occurring within the sample. [86].
Imaging Techniques:
Similar to spectroscopy in principle, an incident electron beam is focused onto the polymeric sample surface that has normally been coated with sputtered gold or platinum this resulting in the formation of secondary electrons and an electromagnetic wave is subsequently emitted [87]. When used in conjunction with energy dispersive X-ray analysis (EDXA), it can detect the surface elemental composition [88] and with IR or Raman spectroscopy, it detects surface modifications. Although not strictly a surface technique, transmission electron microscopy (TEM) [87] can also give us further insight into the composition of the vascular device. In the case of nanomaterials, it allows us to visualize the nanoscale interactions between the polymeric components. In scanning tunneling microscopy (STM) the three-dimensional surface topography is obtained by interpreting data from a current tunneled between a conducting probe and the surface. The underlying principle is that the current is inversely proportional to the distance between the probe and its surface where the probe tip end in a single atom is placed within 5-10A Ëš of the surface (the quantum tunneling distance) [88] . A limitation is that the sample needs to be conductive or otherwise, needs to be coated with a thin metallic coating [87]. In atomic force microscopy (AFM), topographic images of surfaces are obtained with the added advantage of not needing preliminary preparation, thereby preventing any direct damage to the polymer surface.The AFM has a resolution high enough to image surface proteins and nanometer-scale structures.This technique enables the determination of fundamental interactions between biomolecules [89].
Combinations of these techniques such as SEM and SERS have been used to characterize surface topography and molecular integrity [90]. Although the common techniques used both in vitro and in vivo are XPS [91], FTIR microscopy [92], gas permeation chromatography (GPC), and static ion mass spectroscopy (SIMS) [93], imaging techniques such as SEM [94] has also been used to assess for degradation. Measurement of Surface Kinetics When materials are implanted in vivo, they interact with proteins depending on their surface characteristics and composition [95]. More newer biomimetic materials are being developed either as grafts or as coatings to improve biocompatibility [96]. No set methodology exists as of yet to elicit the quantitative in vitro biocompatibility of a polymeric surface; however, combinations such as XPS, Raman spectroscopy and surface free energy have been used. Protein adsorption analyses may also be performed by SNR, photoelectron spectroscopy, SIMS, XPS, electro-osmosis, MALDI-TOFS, inverted confocal Raman microscopy and SERS.
However, spectroscopic analysis is very sensitive to surface chemistry, it is damaging to reactive moieties on organic surfaces which hampers real-time analyses. An alternative method is the usage of colorimetric assays. The reactant, usually a dye such as tri-nitrobenzene sulphonate (TNBS) is placed in contact with the polymeric/biological surface and the color change is then measured spectroscopically over a time period. Like colorimetry, ellipsometry is another non-destructive analytical method, that is ideal in monitoring the in vitro surface kinetics of protein adsorption such as fibrinogen [97] and antibody interactions [98]. From the sample, change in polarization upon reflection from the surface of the sample-media interface [99]are then detected and interpreted based on a common database of all polymers.
Conclusion:
For enhancing the properties of polymers , biocomposites with other natural or synthetic polymers or ceramic materials, particularly hydroxyapatite (HA) are prepared for bone tissue engineering. Efforts are being made to optimize the composition ratio and components of scaffold biomaterial for the above application. Surface modification, analysis and characterization of biopolymers for tissue engineering applications were reviewed from our particular perspectives. To improve biomaterial's cytocompatibility and adherence of specific protein biomolecules , increase in surface hydrophilicity is the most effective way to prepare surfaces for biomimetic response. Different surface chemical composition analysis techniques have very different sampling depth, results in better interpretation. Calorimetric approach for protein investigation over surface may be less sensitive than ATR-FTIR, XPS, SIMS but seems to be useful when a large number of functional groups over surface are under consideration. Reactive groups such as -COOH and -NH2 are usually introduced onto material surfaces as coupling sites to covalently attach proteins. Conformational study of protein secondary structure interacting with polymer surface can be studied using circular dichroism while contact angle measurement proves significant for measurement of hydrophilicity and orientation of protein over biopolymer surfaces. Despite of controversies over the application of particular techniques for characterization and analysis of polymers for application in bone tissue engineering, a set of techniques are more useful for interpreting the best result for the purpose.
