Biomaterial-based scaffolds--current status and future directions.

Review

Biomaterial-based scaffolds -- current status and future directions 1.

Introduction

2.

Types of biomaterial-based scaffolds

3.

Ideal characteristics of

Expert Opin. Drug Deliv. Downloaded from informahealthcare.com by University of Groningen on 04/29/14 For personal use only.

biomaterial-based scaffolds 4.

Fabrication of biomaterial-based scaffolds

5.

Characterisation of biomaterial-based scaffolds

6.

Applications of biomaterial-based scaffolds

7.

Commercial status of biomaterial-based scaffold formulations

8.

Conclusion and future directions

9.

Expert opinion

Tarun Garg† & Amit K Goyal ISF College of Pharmacy, Department of Pharmaceutics, Moga, Punjab, India

Introduction: Biomaterial-based scaffold formulations (three-dimensional Porous matrix, nano-fibre mesh, hydrogels and microspheres) are the major components that are used to deliver the bioactive molecules into the body organs through different routes for an effective treatment of various diseases. Areas covered: Various fabrication techniques such as freeze-drying, polymerisation, spray drying, gas foaming, supercritical fluid technology, etc., are successfully used for fabrication of scaffold formulations. Due to their unique characteristics, these formulations are widely used against various diseases such as tuberculosis, bone defects, cartilage repair, skin diseases, cardiovascular diseases, periodontal diseases, wound dressing, etc. Expert opinion: The study of biomaterial-based scaffold formulations is exhilarating with novel approaches to drug/cell/gene delivery being developed all the time. At present, there is a huge extent of research being performed worldwide on all aspects of tissue engineering/drug or gene delivery. In the future, the main focus will be on the development of more patient compliant, sustained and controlled delivery systems against various diseases by modification of polymers, manufacturing technologies as well as carrier systems. Keywords: biomaterial-based scaffold, hydrogels, microspheres, nano-fibre, porous matrix Expert Opin. Drug Deliv. (2014) 11(5):767-789

1.

Introduction

A biomaterial-based scaffold is the principal component that is used to deliver bioactive molecules to the target place into the body. Biomaterial-based scaffolds are mainly available in four different forms such as three-dimensional (3D) porous matrix, nano-fibre mesh, hydrogels and microspheres [1]. Among these forms, the first two forms such as 3D porous matrix and nano-fibre mesh are mainly administered by implantable route and the rest two forms such as hydrogels and microspheres are given by parenteral route [2]. These systems mainly provide a platform to grow the cells with the help of growth factors. So, it is widely used in tissue engineering or regenerative medicine. The major advantages of biomaterial-based scaffolds are high loading efficiency, high surface area, greater stability, control on content release, provide suitable environment for cell growth, provide protection of encapsulated molecules, longer duration of action, target ability and increase the therapeutic effectiveness of the entrapped substances [3]. Due to their above unique characteristic properties, it becomes an attractive candidate for cell and drug (includes low-molecular-weight compounds, peptides, proteins and nucleic acids) delivery [4,5]. Nowadays, scaffold formulations are successfully used in the treatment of various diseases such as diabetes, hypertension, glaucoma, cancer, brain tumour, gout, rheumatoid arthritis, tuberculosis, bone defects, cartilage repair, skin diseases, cardiovascular diseases, periodontal diseases, wound dressing, liver diseases, act as artificial pancreas, spinal cord injury, etc. [6]. Today, biomaterial-based 10.1517/17425247.2014.891014 © 2014 Informa UK, Ltd. ISSN 1742-5247, e-ISSN 1744-7593 All rights reserved: reproduction in whole or in part not permitted

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The development of conventional drug delivery systems is limited by its poor bioavailability, high dosing frequency, poor loading efficiency, uncontrolled release behaviour, high fluctuations in circulating drug levels, toxicity and high cost, which makes productivity more difficult and the reduced ability to adjust the dosages. Biomaterial-based scaffold formulations such as porous matrix, nano-fibre, hydrogels and microspheres are the best known novel delivery systems, now used for the treatment of various diseases in clinical medicine. A biomaterial-based scaffold is the central component that is used to deliver drug/cell/gene to the target place into the body. Its biocompatible and biodegradable nature, non-toxic behaviour and similarity to extra vascular tissues provide a higher loading efficiency, sustained and controlled release behaviour, and suitable platform for cell growth as well as drug/cell/gene delivery on the target site. Various fabrication techniques such as freeze-drying, polymerisation, spray drying, gas foaming, supercritical fluid technology, etc. are successfully used for fabrication of biomaterial-based scaffold formulations against various diseases such as tuberculosis, bone defects, cartilage repair, skin diseases, cardiovascular diseases, periodontal diseases, wound dressing, etc. Commercialised biomaterial-based scaffold products such as Aquamere, Arestin, Apligraf, Biomend, Healos, Vivitrol, Trelstar, etc., successfully take place in the global market to control or treat numerous diseases.

This box summarises key points contained in the article.

scaffold products become the first choice in global market for prevention and control of the diseases. 2.

Types of biomaterial-based scaffolds

Biomaterial-based scaffolds are mainly categorised into four types according to their nature of polymers as well as their applications (Figure 1). 3D porous matrix A typical 3D porous matrix scaffold is an interconnected open porous structure, which allows high drug-loading and high cell seeding density. It also provide suitable platform for growing the cells. The pore size of porous matrix is < 1 µm. Various polymers such as chitosan, alginate, starch, dextran, polyvinyl pyrrolidone, polyvinyl alcohol, gelatin, hyaluronic acid, agar, poly lactic acid, poly glycolic acid, Pluronic F-127, polypyrrole, etc., are successfully used for fabrication of porous matrix scaffold [6]. These implantable devices are mainly used for cell growth, controlled and sustained drug delivery for longer duration of period in the body. The main mechanisms of drug release from these systems are degradation, erosion and diffusion. These systems are widely used on those conditions when long-term therapy is needed [7]. 2.1

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Nano-fibre mesh In the recent decade, the use of nano-fibre in the health care systems has increased as a tool for drugs, proteins and DNA delivery against various diseases. Nano-fibres are defined as fibres with diameter < 1000 nm [8]. They show various characteristic properties such as very large surface area to volume ratio, flexibility in surface functionalities and superior mechanical performance such as stiffness and tensile strength [9]. Various polymers are successfully used for the fabrication of nano-fibres such as polycaprolactone, poly (methacrylate), gelatin, polyacrylonitrile, poly (ethylene terephthalate), polyL-lactide, chitosan, poly (glycolic acid), polybenzimidazole, poly (vinyl alcohol), polyurethane, polystyrene, nylon 6, polycarbonate, cellulose acetate, polyvinyl chloride, polyethylene oxide, poly (vinyl acetate), sodium alginate, polyethylene and dextran. The active and passive drug-loading process in nano-fibres is done by two methods namely adsorption and dipping techniques [10]. 2.2

Article highlights.

Hydrogel Hydrogels are 3D hydrophilic structures (formed by physical or chemical cross-linking) and polymeric networks, capable of absorbing huge amounts of water or biological fluids along with satisfactory mechanical strength as well as physical integrity [11]. Hydrogels are existing in numerous physical forms for different purposes such as solid moulded (soft contact lenses), pressed powder matrices (pills for oral ingestion), microparticles (bio-adhesive carriers), coating (on implants) and membrane sheet (as reservoirs) [12]. Hydrogels network structures can be macroporous (size 0.1 -- 1 µm), microporous (size 100 -- 1000 A˚) or non-porous (size 10 -- 100 A˚) [13]. The drug releases from hydrogel follow different mechanisms such as molecular diffusion, chemical and swelling-controlled [14]. A number of different categories of biomaterials such as natural (alginic acid, collagen, pectin, chitosan, etc.), synthetic (poly (ethylene glycol), poly (lactic acid), poly (lactic-coglycolic acid), polycaprolactone, etc.) and combinational (gelatin--alginate, chitosan--alginate, etc.) are commonly used in hydrogels preparation for cell, gene and drug delivery [15]. Based on the type of stimuli, hydrogels are classified into various categories such as temperature-sensitive, pH-sensitive, glucose-sensitive, electric signal-sensitive, light-sensitive, pressure-sensitive, ion-sensitive, antigen-responsive, thrombininduced infection-sensitive, nano-hydrogels, enzyme-sensitive and photopolymerisable-sensitive hydrogels [16]. 2.3

Microspheres Microspheres (spherical empty particles) are characteristically free-flowing powders consisting of natural or synthetic polymers (biodegradable in nature), and ideally having a particle size < 200 µm [17]. The drugs are dispersed or dissolved throughout solid biodegradable microspheres matrix, which have the potential for controlled as well as sustained release of drug [18]. Various biodegradable as well as non2.4

Expert Opin. Drug Deliv. (2014) 11(5)

Biomaterial-based scaffolds -- current status and future directions

Porosity A scaffold with open and interconnected pore structure allow for optimal interaction with bioactive molecules. Small pores inhibit the cells from penetrating the scaffold, whilst large pores prevent cell attachment due to a reduced area. So, scaffold should have an adequate porosity that includes the magnitude of the porosity, the pore size distribution and its interconnectivity. This also will allow cell ingrowth as well as vascularisation and promote metabolite transport. 3.3

A.

B.

C.

D.

Figure 1. Types of biomaterial-based scaffolds (A) 3D porous matrix, (B) nano-fibre mesh, (C) hydrogels and (D) microspheres.

Stability The stability of the incorporated drug/cell/gene into scaffold formulations at physiological temperature with respect to physical, chemical and biological activity is to be evaluated. They should possess dimensional stability, chemical stability and biological activity over a prolonged period of time at different atmospheric conditions.


3.4

biodegradable natural and synthetic polymers have been widely used for the fabrication of microspheres [19]. Natural polymers included as carrier materials are albumins, gelatin, collagens (proteins), starch, agarose, carrageenan, chitosan (carbohydrates), poly (acryl) dextran, poly (acryl) starch (chemically modified carbohydrates) and synthetic polymers employed as carrier materials are PMMA, acrolein, glycidyl methacrylate, epoxy polymers (non-biodegradable), polyanhydrides, polyalkyl cyano acrylates, lactide and glycolide and their co-polymers, etc. [20,21]. The major advantages of biomaterial-based scaffolds are described in Table 1.

Ideal characteristics of biomaterial-based scaffolds [22-26]

3.

Biomaterial-based scaffolds play an important role in drug, cell as well as gene delivery to the target place. So, it must be necessary to control their characteristic properties. An ideal biomaterial-based scaffold should fulfil the following requirements such as: Biocompatibility and biodegradability Biocompatibility is the capability of the scaffold to accomplish in a definite application without provoking harmful immune or inflammatory reactions. Biodegradability means degradation of scaffold formulation to non-toxic materials into the body. So, scaffold formulation should possess acceptable biocompatibility as well as biodegradability profile. The degradation products should not be toxic, eliminated easily from implantation site of the body and integrated with surrounding tissues. 3.1

Mechanical properties Mechanical properties means the characteristics of a material that determine how it reacts when it is subjected to some type of force that attempts to stretch, dent, scratch or break it. Mechanical properties of the scaffolds should match with the implantation site tissues, or should be sufficient to shield cells from destructive compressive or tensile forces without inhibiting suitable biomechanical signals and to persist under physiological conditions. 3.2

Binding affinity and loading capacity Binding affinity is defined as how tightly the drug binds to the scaffold formulations and the amount of drug that can be mixed into the scaffold is known as loading capacity. The binding affinity must be sufficiently low to allow release of the drug but should have a maximum loading capacity so the drug is released continuously for longer duration after insertion into the body. 3.5

Structure and interface adherence Biomaterial-based scaffold formulation should have a reproducible microscopic and macroscopic structure with a high surface: volume ratio, which is suitable for cell/drug attachment. Interface adherence is how cells or proteins attach to a scaffold’s surface. The scaffold should support cell adhesion and proliferation, facilitating cell--cell contact and cell migration. 3.6

Nature Impersonating the inherent extracellular matrix (ECM), an endogenous constituent that ambiances cells/drug/gene, permit them to impasse into tissues and deliver signals that support cellular development and morphogenesis. 3.7

Processability The biomaterial-based scaffold should possess comparatively easy processability and flexibility into the preferred shape, rendering to the necessity. They should be proficient of being formed into a sterile product. 3.8

4.

Fabrication of biomaterial-based scaffolds

A variety of techniques have been used for processing biodegradable polymers into different types of porous biomaterial-based scaffolds. The conventional as well as new advanced technologies are widely used for fabrication of


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Table 1. Major advantages of biomaterial-based scaffold delivery systems.


3D porous matrix Good mechanical properties Biocompatible and biodegradable Highly porous Well-inter connected open pore structure Homogenous drug dispersion Protection of drugs and cells Predetermined bio adsorption Minimal immune or inflammatory responses Biocompatible compositions Support cell adhesion and proliferation Sustained and controlled release Low drug-binding affinity Stability Easy to modifications Easy to manufacture

Nano-fibre mesh High surface area High porosity Small pore size Woven fabric ability Economic Easy to manufacture Improved drug-loading capacity Stable Suitable for thermolabile drugs Sustained and controlled release Good mechanical properties Enhance dissolution Better targeting Better adsorption Flexible in nature Minimal toxicity Well interconnected pore structure Sterile Encapsulated various ranges of drugs

Hydrogels Biocompatible and biodegradable Delivered by various routes Easy to processability Easy to modifications Controlled release rate High loading efficiency Minimal toxicity Delivered by various routes Increase therapeutic efficiency Easy to modifications Sustained and controlled release Reduction of toxicity Protection of drugs Suitable for wide range of drugs Minimal immune or inflammatory responses

Microspheres Biocompatibility Target ability Bioresorbability Sterilisability Stability Water solubility Polyvalent Reduction of toxicity Protection of drugs Increase therapeutic efficiency Control of content release Longer duration of action Delivered by various routes Suitable for wide range of drugs Easy to manufacture Easy to modifications Sustained and controlled release Biocompatible compositions Minimal inflammatory responses

Data taken from [51-54].

scaffolds such as freeze-drying, super critical fluid technology, fibre bonding, melt moulding, solvent casting/particulate leaching, gas foaming/particulate leaching, rapid prototyping, etc. (Figure 2). Some of the important techniques for fabrication of 3D porous matrix (Table 2), nano-fibre mesh (Table 3), hydrogels (Table 4) and microspheres (Table 5) are described. 5. Characterisation of biomaterial-based scaffolds

Generally, biomaterial-based scaffolds are characterised for their morphology (porous structure), swelling property (determines the release mechanism of the drug), entrapment efficiency, loading capacity, muco-adhesive strength, release study, elasticity (affects the mechanical strength of the network and determines the stability of carriers) and biocompatibility studies. Some of the important features for characterisation of scaffolds are as follows: Surface morphology Scanning electron microscopy (SEM) is mainly used to examine the structure, shape and surface morphology of scaffold formulations. In this technique, a small section of the sample was placed on the SEM sample holder and sputter coated with platinum. Accelerating voltage of 15 -- 20 kV was employed to take SEM images. Atomic force microscopy is used for 5.1

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determining the surface properties and vertical scanning interferometer mode is used for scanning the surface characteristics with nanometric resolution of the sample [27]. Chemical/physical analysis Various chemical and physical analysis techniques such as infrared spectroscopy, UV--visible spectroscopy, NMR and mass spectrometry can provide some information on the composition of the polymeric network and water holding capacity of the scaffold formulation. X-ray diffraction process technique provides information on the crystallite size and the micro-strain developed within a specimen [28,29]. 5.2

Thermal analysis Differential scanning calorimetry (DSC) is a thermodynamic technique widely used to examine the miscibility of polymers blends through measuring the thermal properties. DSC of samples may confirm the stability of polymers in the formulation. In this technique, samples (3 -- 5 mg) were heated from 0 to 250 C at the rate of 10 C/min under a nitrogen atmosphere [30]. 5.3

Drug-loading capacity Drug-loading capacity describes that how many drug incorporated into the carrier systems. It can be described as the ratio of the weight of the drug to the weight of the excipients 5.4



Fabrication of scaffold


(A) 3D porous matrixes

(B) Nano-fiber mesh

(C) Hydrogels

(D) Microsphers

Freeze drying

Electrospinning

Isostatic ultra high pressure

Single emulsion

Particulate leaching

Drawing

Use of cross linkers

Double emulsion

Gas foaming

Template synthesis

Use of water and drying

Normal polymerization

Supercritical fluid

Phase separation

Nucleophilic substitution

Interfacial polymerization

Melt molding

Self-assembly

Use of gelling agents

Phase separation

Emulsion template

Fiber mesh

Irradiation and freeze thawing

Spray drying and spray congealing

Thermally phase separation

Fiber bonding

Bottom up and top down

Solvent extraction

Emulsification

Molding

Photolithogra PHY

Micro fluidic

Figure 2. Biomaterial-based scaffolds fabrication techniques.

used in the carrier systems. The loading capacity of scaffold formulations was calculated according to the following equations [31]: (1) Drug loading (%) = Mass of drug /

Mass of total polymers × 100 5.5

Entrapment efficiency

Entrapment efficiency describes the entrapment efficiency of the molecules into the carrier systems. The entrapment efficiency of scaffold was calculated by placing the sample into a suitable solvent in shaking incubator. The amount of drug in the respective solutions was calculated by UV--visible spectrophotometer. The percentage entrapment efficiency of the scaffold formulations were calculated from the following equation.

% Entrapment efficiency = DL - DF/DL × 100

(2)

where: DL = Initial drug loaded (mg), DF = Free drug (mg) [32]. Degree of swelling Degree of swelling represents the relative measure of crosslinking between the polymers of the formulations. The sample was placed into suitable solvent, pH as well as temperature conditions and checked at different time intervals. Degree of swelling was calculated according to the following equations: (3) Degree of swelling (%) = M - Md /Md × 100 where, M is the weight of swollen sample and Md is the weight of dried sample [33]. 5.6

Mucoadhesive strength Mucoadhesive strength represents the adhesiveness of the formulation with the mucous membrane. Mucoadhesive 5.7


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Table 2. Details of fabrication techniques of 3D porous matrix scaffold along with their advantages and limitations.


Advantages of fabrication techniques

Limitations of fabrication techniques

Freeze-drying technique [55]: Suspension of polymers poured into suitable mould and solvents were removed by using lyophiliser, resulting porous structure was obtained. This technique is mainly based on the sublimation process Highly porous structures Limited to small pore size High pore interconnectivity’s Porosity is often irregular Not require high temperature Longer processing time Not require separate leaching step Expensive technique Particulate leaching method [56]: Polymer solution poured into mould along with suitable porogen. At high pressure, porous scaffold was obtained after the evaporation of organic solvents Simple and reproducible technique Longer processing time Not require sophisticated apparatus High amount of drug lost Ease of control of porosity and geometry Contain residual salt particles Ability to tailor crystallinity Limited mechanical properties Gas foaming method [57]: Polymer gel paste along with sieved effervescent salt particles poured into mould and immersed into hot water. Formation of porous matrix after evolution of ammonia and carbon dioxide gas from salt particles of the solidifying polymer matrix Minimum loss of bioactive molecules Remove skin layer No need of leaching process Poor pore inter-connectivity No residual organic solvent Longer processing time Porosity high > 90% Limited mechanical properties Supercritical fluid technology [58]: Dry polymer is dissolved in supercritical carbon dioxide to form a single-phase polymer/gas solution and convert into porous matrix after applying reduced pressure Preserve protein drug activity Nonporous external surface Avoid high-temperature condition Closed pore structure Avoid presence of aqueous/organic interfaces Insufficient pore interconnectivity Higher mechanical strength Closed pore structure Longer processing time Melt moulding technique [59]: In this technique, polymers were melt along with suitable porogen followed by cooling of polymer mixture. Porosity is achieved by dissolving the porogen in water Controlled pore size Require high temperature Controlled macro shape Require residual porogen Controlled pore interconnectivity and geometry Longer processing time Preserve protein drug activity Limited mechanical properties Expensive technique Emulsion template techniques [60]: The internal phase of the emulsion acts as a template for the pores and polymerisation occurs in the continuous phase. The porous matrix scaffold obtained after the removal of internal phase Controlled pore interconnectivity and geometry Longer processing time High mechanical integrity Stability problems Ability to absorb larger particles Expensive technique Controlled pore size Difficult to processing Needed polymerisation steps Thermally induced phase separation technique [61]: First concentrated polymer phase produced by lowering the temperature of polymer molten solvent. Cooling below the melting point followed by vacuum drying create porous matrix scaffold Highly porous structure Use toxic solvents Easy fabrication technique Poor control over internal architecture Controlled pore morphology Limited range of pore sizes Thermodynamically stable Expensive technique

strength was determined by measuring force required to detach mucosal membrane from the formulation by using texture analyser. In this technique, sample was applied to the upper probe and mucosal membrane was fixed on the lower disc. The upper probe was then lowered to touch the sample surface. A force (0.1 N) was applied for 5 min then upper probe was moved up. The force required to detach the sample from mucosal surface was determined [34]. 772

Rheological analysis Viscosity and molecular weight of unknown sample can be determined by its rheological analysis. These studies give the basic ideas of the formation of intermolecular hydrogen bonding among the molecules and their Newtonian and nonNewtonian flow behaviour. Viscosity of samples is evaluated under constant temperature of usually 4 C by using viscometer or rheometer [35]. In this technique, the gel sample (about 5.8




Table 3. Details of fabrication techniques of nano-fibre mesh along with their advantages and limitations. Electro spinning technique: Polymer and drug solution passed through a high voltage (10 -- 20 kV) of electro spinning machine, resulting the formation of fine fibres [62]. The characteristics of nano-fibre mainly depends upon polymer nature, concentration of polymer, voltage applied, etc. High surface area Require electro spinning machine Control over pore geometry Not applicable for all polymers High entrapment efficiency Not sufficient for cell seeding Cheap and simplicity Not sufficient for cell infiltration Drawing technique [63]: Micropipette moves toward contact line to millimetric droplets of the mixture of polymer as well as drug and nano-fibre produced after withdrawal of it through micropipette Simple technique Time-consuming process Inexpensive Not applicable for all polymers No expert required on this field Not uniform shape Easy to modification Obtain very long single fibre Template synthesis technique [64]: The polymer solution was passed into a solidification solution through a fibril solid or hollow shape tubule. Through this technique, various raw materials such as metals, semiconductors and carbon can be fabricated Various nature of nano-fibre can be fabricated Can’t make one by one continuous nano-fibres Easy to modification Time-consuming process No expert required on this field Use various steps Phase separation technique [65]: It consists of dissolution, gelation and extraction using a different suitable solvents, followed by freezing and drying conditions, resulting in a formation of nanoscale porous fibres Simple technique Time-consuming process Inexpensive Lack of structural stability Mass production possible Difficult to maintain porosity Make one by one continuous nano-fibres Not applicable for all polymers Self-assembly techniques [66]: Individual, pre-existing components organises or arranged themselves into a desired patterns and functions, resulting the formation of nano-fibre mesh Mass production possible Time-consuming process Make one by one continuous nano-fibres Not applicable for all polymers Simple technique Not uniform shape Inexpensive Lower loading efficiency Fibre mesh technique [67]: In this technique, deposition of a polymer solution over a nonwoven mesh of another polymer followed by subsequent evaporation, resulting in formation of nano-fibre mesh Rapid diffusion of nutrient Lack of structural stability High cell attachment Difficult to maintain porosity Large surface area Not applicable for all polymers Favourable for cell survival and growth Lower loading efficiency Fibre bonding technique [68]: Different polymeric nano-fibres are joined at their cross-linking points by ‘sintering’ above their melting point temperature, resulting formation of nano-fibre mesh High surface area Poor mechanical integrity Easy fabrication technique Residual organic solvents Inexpensive method Lack of structural stability Control pore size Difficult to maintain porosity

10 ml) was placed in small sample adapter at low temperature and the temperature was raised above 40 C followed by cooling. The viscosity at various temperatures was recorded using suitable spindle [36]. Dynamic mechanical spectroscopy technique is also used for studying the viscoelastic behaviour of polymers. Mainly two types of dynamic mechanical analysis analysers are used currently: forced resonance analysers (forced RA) and free resonance analysers (free RA). Forced RA force the sample to oscillate at a certain frequency and are reliable for performing a temperature sweep, whereas free RA measure the free oscillations of damping of the sample being tested by suspending and swinging the sample. Analysers are made for both stress (force) and strain (displacement) control. In strain control, the probe is displaced and the resulting stress of the sample is measured by implementing a

force stress other time)

balance transducer, which utilises different shafts. In control, a set force is applied to the same and several experimental conditions (temperature, frequency or can be varied.

Gelation and gel melting Gelation and gel melting was assessed using an aliquot of gel that was transferred to test tubes, immersed in a water bath at 4 C and sealed with aluminium foil. The temperature of water circulation bath was increased in increments of 1 C and left to equilibrate for 5 min at each new setting. The samples were then examined for gelation and gel melting temperature when the gel starts flowing upon sloping through 90 was recorded [37]. 5.9


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Table 4. Details of fabrication techniques of hydrogels along with their advantages and limitations. Isostatic ultra-high pressure: Ultra-high pressure (300 -- 700 MPa) applied for 5 or 20 min. on a suspension or solution of suitable polymers (natural or synthetic) in a chamber. The above reaction causes gelatinisation and brings changes in ordered state of polymer [69] Uniformity maintain for longer time Require ultra-high pressure Short time process Not applicable for all polymers Easy processability Lower loading efficiency Sustained and controlled release Poor control on release pattern Use of cross-linkers: Various chemical (calcium chloride) as well as physical cross-linking agents (reversible ionic cross linking agents) are used to prepare hydrogels by forming hydrogen or covalent bonds between polymers [28] Control the toxicity Require physical and chemical cross-linking agents Prevent burst release Lower loading efficiency Biodegradable in nature Not applicable for all polymers Sustained and controlled release Use of water and drying condition: Suspension of polymers convert into aerogel by applying solvent (water), pH regulator (sodium carbonate) and critical drying conditions [70] Sufficient mechanical strength Require specific drying condition Biodegradable in nature Require pH-regulating agents No shrinkage Lower loading efficiency Release maintain up to several days Poor control on release pattern Use of nucleophilic substitution reaction: Hydrogels prepared by free radical polymerisation of the polymers suspension followed by nucleophilic substitution reactions [71] Sufficient mechanical strength Require nucleophilic substitution reaction Gel formation occur at pH 7.4 Not applicable on all polymers Reduced viscoelastic flow at low frequency stress Lower loading efficiency Time-consuming process Use of gelling agents: Suspension of polymers convert into mini hydrogels in the presence of various ion-sensitive, temperature-sensitive gelling agents such as sodium alginate, poloxamer, etc. [72]. Therapeutically efficacious Require gelling agents Stable and non-toxic Difficult of interaction between drug and gelling agents Sustained and controlled release Time-consuming process Release maintain up to several days Irradiation and freeze thawing techniques: Recombination of macro-radicals (generated by irradiation and freeze thawing of polymers) through covalent bonds, resulting cross-linked structure is formed [73] Sufficient mechanical strength and stability Require high-energy radiation Provide porous structure Additional freeze-thawing steps involve Useful for removal of residue Time-consuming process Expensive technique Bottom-up and top-down approaches: Cells inserted into polymer scaffold along with bioactive materials and placed into suitable place, where cells were grown. These approaches mainly used for cell delivery to the target place [74] Delivery of various types of biomaterials and cells Requires cell sheets Retain the different types of drug or cells in a single moiety Require cell printing machine Time-consuming process Expensive technique Emulsification techniques: The hydrogel precursors poured into hydrophobic medium (such as oil) with continuous agitation. The phase of hydrogels breakup into small droplets, resulting micro gels prepared [75] Easy processability Difficult to control its spherical shape No need to expert Difficult to control its size Inexpensive technique Difficult to control its degree of homogeneity Short time process Moulding approach: Hydrogels prepared by pressing the polymer solution (alginate, chitosan, fluorinated polymers) on mould of polymer plate by using such techniques like lithography, etching, etc. [76]. Controlled size and shape Require polymer plate and mould Regulate the targeting of drug carriers Not applicable for all polymers Improved mechanical properties Require constant pressure Targeted and controlled drug delivery Time-consuming process Photolithography technique: Photo cross-linkable hydrogels are situated underneath facade arrangements on equally sides of sub-micrometres to millimetre and cross-link in the existence of light (blue light, laser light) [77]

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Table 4. Details of fabrication techniques of hydrogels along with their advantages and limitations (continued). Require photoinitiator and UV light Sometime causes drug toxicity Sometime change the drug behaviour Expensive technique Microfluidic technique: Microscale structures were produced from hydrogels by generating single- or multi-phase flows within microfluidic channels, resulting complexity is generated by layering microgels [78] Controls the resulting shape Sometime complexity generate High encapsulation efficiency Sometime causes drug toxicity Create microscale structure Time-consuming process Show target ability Require specialised equipment’s


Compatible with variety of polymers as well as bioactive molecules Convenient and fast approach Target ability

5.10

Density determination

The density of the sample can be measured by using a multivolume pycnometer. In this technique, sample was placed into the multivolume pycnometer and helium is introduced at a constant pressure in the chamber and allowed to expand. This expansion decreases the pressure within the chamber and two consecutive readings of reduction in pressure at different initial pressure are noted. From the two pressure readings, the volume and density of sample are determined [38].

incubator. At designated time points, the release medium was withdrawn and replaced with pre-warmed fresh solvent. The released amounts of drug were analysed at optimum wavelength by using UV spectrophotometer [41]. Fluorescence spectrophotometer, atomic absorption photometer and enzyme-linked immunosorbent assay were also used for detection of drugs. Some important characteristics of above techniques are shown in Table 6. In-vivo studies These studies were carried out in accordance to the guidelines of the council for the purpose of control and supervision of experiments on animals, Ministry of Social Justice and Empowerment, Government of India. In these studies, the effects of formulation or samples on disease-induced animal models were evaluated. The different organ distribution studies, stability study, toxicity studies and therapeutic studies are performed under in-vivo studies. At last, all these evaluation parameter results are compared with available standard as well as marketed products [42]. 5.14

Diffusion studies Drug diffusion is defined as the amount of drug diffused or permeated through the membrane into the blood. In this study, sample was applied on the skin or synthetic cellulose membrane and mounted on the Franz diffusion cell, and the top cell was clamped and covered with a parafilm to prevent the evaporation of the receptor medium. The receptor medium was filled with phosphate buffer (pH 7.4), which was maintained at constant temperature by a circulating water bath. The temperature was maintained at 37 C in all diffusion studies. At predetermined time intervals, the samples were withdrawn from the receptor compartment and replaced by an equal volume of fresh buffer solution. The samples were analysed by a UV spectrophotometer [39]. 5.11

Stability studies Scaffold formulations were exposed at different temperature as well as pH conditions and the changes after storage for a definite period of time were evaluated. In these techniques, samples were stored for 3 -- 6 months in stability chamber at different pH and temperature condition and after 3 -- 6 months the evaluation parameters were checked. If no sign of change is observed, it means the formulation is stable [40]. 5.12

In-vitro release studies The release profiles of scaffold formulations were investigated in suitable solvents. In this technique, sample was placed within a dialysis membrane, which was kept in a conical flask containing solvent. The conical flask was covered with aluminium foil and shaken at optimum speed at 37 C in shaking 5.13

6.

Applications of biomaterial-based scaffolds

Biomaterial-based scaffolds are broadly applied in biomedical applications, as tissue engineering, in wound healing, drug delivery, gene/DNA/plasmid delivery, in filtration, as affinity membrane, in enzyme immobilisation, as vascular graft implants, in health care, biotechnology, in environmental engineering, in defence security, in energy storage and in various researches. Nowadays, biomaterial-based scaffolds are widely used in the treatment of various diseases [43,44]. Scaffolds in drug/cell/gene delivery Biomaterial-based scaffolds are mainly used for delivery of drug, cell and gene to the target site. These formulations are administrated into the body by various routes such as parenteral, ocular, peroral, rectal, vaginal, transdermal, nasal, colon, breast and topical. These formulations act as attractive scaffolding materials that provide bulk and mechanical constitution to a tissue construct where cells are adhered to or suspended within the 3D gel framework. Successful implantation of various cell types such as fibroblasts, osteoblasts, 6.1


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Table 5. Details of fabrication techniques of microspheres along with their advantages and limitations. Single emulsion technique [79]: An aqueous solution of polymer dispersed in organic phase and cross-linked with heat as well as chemical cross-linking agents followed by centrifugation, washing and separation creates microspheres Biocompatible and biodegradable Not suitable for thermolabile drugs Control of content release Excessive exposure of drugs with chemical cross-linking agents Increase the therapeutic efficiency Time-consuming process Protection of drug Double emulsion technique [80]: An aqueous solution of polymer dispersed in organic phase, followed by the addition of aqueous solution of PVA, resulting in the formation of multiple emulsions. Addition of hardening agents followed by separation, washing and drying creates microspheres Various drugs incorporated in these systems Time-consuming process Biocompatible and biodegradable Expensive technique Increase the therapeutic efficiency Stability problem Sometime complexity generates Normal polymerisation technique [81]: Monomers, bioactive agents, initiator dispersed into water along with stabilisers. Polymerisation occurs in the presence of vigorous agitation and heat/irradiation, resulting microspheres prepared Formation of the pure polymer Not suitable for thermolabile drugs Reduction of toxicity Association of polymers Various drugs incorporated Time-consuming process Biocompatible and biodegradable Expensive technique Interfacial polymerisation technique [82]: Two reacting monomers (one of which is dissolved in continuous phase and another one dispersed in continuous phase) phases diffuse rapidly at the interface and microsphere formation after separation, washing and drying steps Control of content release Toxicity associated with the unreacted monomers Various drugs incorporated High degradation of drug Relative stability Non-biodegradability Sustained release behaviour Phase separation coacervation technique [83]: Drug dispersed in aqueous/organic solution of polymer followed by phase separation by different means, resulting polymer-rich globules formed. Finally microsphere formation after separation, washing and drying steps Biocompatible and biodegradable Agglomeration of polymers Control of content release Time-consuming process Increase the therapeutic efficiency Expensive technique Protection of drug Stability problems Spray drying and spray congealing technique [84]: The polymer dissolved in volatile organic solvent and dispersion is then atomised in a stream of hot air by using spray drying machine. It leads to the formation of microsphere after the solvent evaporation Feasibility of operation under aseptic conditions Low yield Free-flowing preparation Machine cost very high Time-saving process Washing after each batch preparation Limited use of solvents Solvent extraction technique [85]: Water-miscible organic solvents such as isopropanol are removed by extraction with water. The process involves direct addition of drug to polymer organic solution Decrease the hardening time Stability problems Control of content release High degradation of drug Increase the therapeutic efficiency Non-biodegradability Protection of drug Limited use of solvents

hepatocytes, chondrocytes, myoblasts, smooth muscles, etc., on the target site is possible due to unique characteristics of scaffold formulations. Besides this, these formulations are also helpful in the incorporation of foreign DNA particles into the host cells and provide efficient transduction, high gene expression, minimum immunogenic reactions or mutagenesis of transfected cells. A schematic diagram of biomaterial-based scaffold formulation applications against various diseases is shown in Figure 3 and Tables 7 -- 10. Biomaterial-based scaffold formulations have been used as barriers to prevent restenosis or thrombosis, and to improve the healing response following tissue injury. For example, thin hydrogel layer provides a barrier to prevent platelets, 776

coagulation factors and plasma proteins from contacting the vascular wall. Poly (ethylene glycol-co-lactic acid) diacrylate hydrogels prevent fibrin deposition and fibroblast attachment at the tissue surface, resist protein adsorption and diffusion as well as minimise cell adhesion. Zervantonakis et al. developed a microfluidic-based hydrogel for tumour cell intravasation into the blood stream and endothelial barrier function [45]. Na et al. prepared injectable hydrogel of hyaluronic acid A embedded in mildly crosslinked alginate (HA/mcALG hydrogel), which acts as an injectable tissue adhesion barrier [46]. Kumar et al. developed flexible and microporous chitosan hydrogel/nano-zinc oxide composite bandages, which showed enhanced wound



Table 6. Important characteristics of analytical techniques. Analytical techniques Ultraviolet-visible spectrophotometry


Fluorescence spectrophotometer

Atomic absorption photometer

Enzyme-linked immunosorbent assay

Characteristics of analytical techniques Uses light in the visible and adjacent (near-UV and near-infrared (NIR)) ranges The absorption or reflectance in the visible range directly affects the perceived colour of the chemicals involved In this region of the electromagnetic spectrum, molecules undergo electronic transitions, while absorption measures transitions from the ground state to the excited state It is a type of electromagnetic spectroscopy that analyses fluorescence from a sample It involves using a beam of light, usually ultraviolet light, that excites the electrons in molecules of certain compounds and causes them to emit light The species is first excited, by absorbing a photon, from its ground electronic state to one of the various vibrational states in the excited electronic state Use of absorption spectrometry to assess the concentration of an analyte in a sample It is a spectroanalytical procedure for the quantitative determination of chemical elements employing the absorption of optical radiation (light) by free atoms in the gaseous state It is a test that uses antibodies and colour change to identify a substance A liquid sample is added onto a stationary solid phase with special binding properties and is followed by multiple liquid reagents that are sequentially added, incubated and washed followed by some optical change The qualitative reading usually based on detection of intensity of transmitted light by spectrophotometry

Data taken from [86,87].

healing and helped for faster re-epithelialisation and collagen deposition [47]. Industrial applications of biomaterial-based scaffold formulations

6.2

Biomaterial-based scaffold formulations have been used as absorbents for industrial effluents. Health hazards substances like dioxins cause carcinogenicity, immunotoxicity or endocrine disruption and these hazardous substances can be successfully removed by DNA of Salmon milt hydrogel beads. Approaches using immobilised biological materials in hydrogels are used in different branches of the food industry, especially in the production of fermented beverages. Deriase et al. optimised the process parameters for the continuous ethanol production by Kluyveromyces lactis immobilised cells in hydrogel copolymer carrier. Hydrogels are also used for glucose monitoring, detection of vanillin and determining the extent of degradation of paper artworks [48]. Hadizadeh et al. (2013) prepared lactose imprinted hydrogels by using methacrylamide and ethylene glycol dimethacrylate and finally used as a sorbent for separation of lactose from milk [49].

Commercial status of biomaterial-based scaffold formulations

7.

Currently, various biomaterial-based scaffold formulations are running successfully in global market due to their unique

characteristic properties. Various scaffold formulations take place in the market to control or treat numerous diseases. If we compare all the scaffold formulations, the hydrogel and microsphere-based products are more in the market as compared with porous matrix and nano-fibre-based products. Nowadays, nano-fibre becomes an attractive choice for delivery of the various bioactive agents to the target place. Table 11 shows some biomaterial-based scaffold products along with their major research applications. 8.

Conclusion and future directions

Multi-functional, biodegradable, biocompatible and biomimetic polymeric scaffolds have been intensively studied for decades to offer implantable devices for tissue regeneration. The tissue engineering tactic was originally imagined to discourse the gap between patients waiting for donor tissue and the number of donors really available. The field of biomaterials has played a critical role in the development as well as improvement of tissue-engineered products. Few biomaterial-based scaffold products are available commercially, particularly for cell, drug and gene delivery. Most of the scaffold formulations studied are static in the investigation stage and are yet to be approved for clinical use. These polymeric devices improve its cell adhesive characteristics or actively induce cell migration, proliferation and differentiation. Surface-engineered and drug-releasing scaffold


777


Cells

Drugs/Bioactive agents

Gene/DNA/Plasmid


Incorporated into porous scaffold

3D porous matrixs

Nano-fiber mesh

Hydrogels

Microspheres

Glau coma Diabetes

ACNE

Skin disease

Hyper tension

Bone defects

Liver disease

Carti lage defects

Spinal cord injury Heart disease

Cancer

Figure 3. Schematic diagram of biomaterial-based scaffolds applications against diseases.

formulations can be realistically considered to simulate the ECM environment, so expediting the tissue-regeneration process. The use of adhesion peptides and growth factors provides adequate signals to the cells for inducing and maintaining them in their desired differentiation stage as well as their survival and growth. An example would be to incorporate growth factors in scaffolds for different types of cells such as 778

endothelial progenitor cells in an attempt to repair a fullthickness osteochondral defect in rabbits. Furthermore, the combination of drugs (i.e., inflammatory inhibitors and/or antibiotics), into biomaterial-based scaffolds may be used to avoid infection after surgery and treat the disease effectively. Our main goal is reducing the number of patients by developing more advanced biomaterials as well as bioreactors, and as



Table 7. Applications of 3D porous matrix for drug/cell/gene delivery at various sites of the body organ.


Polymers

Drug/cell/gene

Applications

k-carrageenan, Poloxamer 407

Model drug

Hydroxy propyl methyl cellulose

Metformin hydrochloride

Silicon

Triclosan

Eudragit, Polycarbophil

Indomethacin

Hydroxy propyl methyl cellulose

Propranolol HCl

Polyoxyethylated oleic acid glycerides

Model drug

Chitosan, xanthan gum

Propranolol HCl

Alginate, PLGA

Model drug

Chitosan

Quinacrine

Poly(lactide-co-glycolide)

Endothelial progenitor cells

Calcium sulphate/alginate

Mesenchymal stem cells

Gelatin Hyaluronic acid

Human adipose-derived stem cells Chondrocytes

Calcium phosphate alginate


PLGA/Pluronic F127 Poly(hydroxyethyl methacrylate)-gelatin

pDNA Chondrocyte

Hyaluronic acid


Collagen

Recombinant DNA

Polyurethane

Endothelial cell

Poly(1,8-octane diol-co-citric acid)

Cardiac cells

research leads to more knowledge of the cell, drug as well as gene-signalling mechanisms required to barrier the chain of various diseases [50]. 9.

Expert opinion

Biomaterial-based scaffold formulations (3D porous matrix, nano-fibre, hydrogels and microspheres) have several properties that make them ideal for drug/cell/gene delivery to the target site into the body. The ability to incorporate precise drug amounts over varied concentrations, the ability to prevent metabolic degradation of the encapsulated active biomolecules and the ability to sustained and controlled release mark

Act as optimum physicomechanical matrix for potential buccal drug delivery [15] Act as an effective carrier for controlled delivery of drug [88] Act as an effective carrier for the sustained delivery of antibacterial agents with an enhanced inhibitory activity [89] Potential application as colon-specific drug delivery systems with pH- and time-dependent drug release profile [90] Prolong the release of drug and shift the release pattern approach to zero order [91] Well-tolerated exhibiting cell viabilities and can be used to modulate the release of drugs [92] Showed prolonged and sustained drug release from these carrier system [93] Showed increased matrix tolerance and reduced burst effect [94] Potential as a depot drug delivery system for water-soluble drugs [95] Successfully repaired a full-thickness osteochondral defect in rabbits [96] Showed better reconstruct the craniofacial bone in rats [97] Provide support the proliferation and delivery of encapsulated cells [98] Improved the clinical efficiency in osteoarthritis treatment [99] Supported proliferation of cells and their subsequent differentiation [100] Effective gene delivery for 3D tissue formation [101] Used as a matrix for cell attachment and proliferation in 3D environment in cartilage--tissue engineering [102] Useful in the case of cartilage repair, wound healing and large vessel replacement [103] Used to deliver genes of choice to localised subgroups of specific cells of interest [104] Supported to vascular cells in three-dimensional bioreactor-based culture conditions [105] Meet the mechanical and biological parameters needed for cardiac culture [106]

them an attractive candidate to control and treat various diseases. Several polymers like chitosan, alginate, starch, dextran, polyvinyl pyrrolidone, polyvinyl alcohol, polybenzimidazole, poly (vinyl alcohol), polyurethane, polystyrene, nylon 6, polycarbonate, cellulose acetate and gelatin are successfully used for fabrication of scaffold formulations. Several drugs, cells and genes have been evaluated for delivery with biomaterialbased scaffold formulations, including indomethacin, tacrolimus, ciprofloxacin, naltrexone, adipose-derived stem cells, bone marrow-mesenchymal stem cell, adult brain-derived neural stem/progenitor cells, etc. 3D porous matrix (an interconnected open porous structure) incorporated with drug, cell, gene, growth factors,


779


Table 8. Applications of nano-fibres for drug/cell/gene delivery at various sites of the body organ.


Polymers

Drug/cell/gene

Applications

Polycaprolactone

Vitamin B12

Poly(D,L-lactic-co-glycolic acid)

siRNA

Collagen, Chitosan

5-fluorouracil

Gelatin Poly(ester amide)

Basic fibroblast growth factor Nitroxyl radical model compound

Cellulose (PQ-4) Poly vinyl alcohol

Model drug

Poly(vinyl pyrrolidone)

Silver

Polyvinyl alcohol

Silver

Poly(L-lactide-co-glycolide)

Tacrolimus

Tecophilic polymer

Silver(I)-imidazole cyclophane

Poly(lactic-co-glycolic acid) Poly(3-capro lactone)

Adipose-derived mesenchymal stem cells Embryonic stem (ES) cells

Chitosan

Rat calvarial osteoblast

Poly(lactic-co-glycolic acid)

Human marrow stromal cell

Silk fibroin

Mesenchymal stem cell

Collagen

Human dental pulp stem cell

Poly(lactic-co-glycolic acid) Poly(lactic-co-glycolic acid) Poly(3-capro lactone)

Islet cell from human embryonic stem cell Anterior cruciate ligament Rabbit chondrocytes

DNA, histones and proteins have been implanted into the body in chronic disease condition. This allows high drugloading and cell seeding density. It also provides suitable platform for growing the cells into the body. Various bioactive molecules such as metformin hydrochloride, triclosan, indomethacin, quinacrine, cardiac cells, endothelial progenitor cells, endothelial cell, chondrocytes, mesenchymal stem cells, pDNA, recombinant DNA successfully delivered through this systems to control and treat various diseases such as osteochondral bone defects, in cartilage-tissue engineering, in diabetes, in cardiac failure, etc. Nano-fibres (fibres with diameter < 1000 nm) applications have increased immensely as a tool for drug delivery system against various diseases. The continuously increasing applications on nano-fibres and their use for delivering various drugs, 780

Increased entrapment efficiency and showed sustained release behaviour [107] Exhibited up to 50% EGFP gene-silencing activity after 48 h post-transfection on H1299 cells [108] Potential application in cancer therapy as a drug-delivery agent in post-surgical treatment of cancer [109] Promote angiogenesis [110] Showed complete release of drug within 5 days [111] Showed antibacterial activity against Gram-negative bacteria Escherichia coli and Gram-positive bacteria Staphylococcus aureus [112] Showed high water stability and significant antibacterial efficacy against all three types of bacteria [113] Significantly inhibited E. coli (gram-positive) and S. aureus (gram-negative) bacteria and displayed high mechanical stability [114] Useful for prevention of recurrent pulmonary venous obstruction [115] Effective against E. coli, Pseudomonas aeruginosa, S. aureus, Candida albicans, Aspergillus niger and Saccharomyces cerevisiae [116] The novel layered scaffold has the potential for improving tendon healing [117] Lead to the development of a better strategy for nerve injury repair [118] Promoted bone healing and regeneration within 10 days in rat [119] Showed in vivo bone formation into athymic mice [120] Exhibited osteogenic and chondrogenic differentiation within 5 weeks [121,122] Newly derived pulp tissue was seen in the 6 weeks in mice [122] Continuous release insulin for 7 weeks in mice [123] Growth of ligament tissue within 1 week [124] Growth of new cells within 3 weeks in rabbit [125]

proteins and DNA prove its importance and accessibility as drug carriers. This delivery system is easily applied on the target site and its effectiveness along with minimum dosing frequency are exhibited. They have advantages of high drugloading efficiency, minimum toxicity, encapsulated wide range of drugs, better targeting and does not need surgery as most current implants do. They can be further turned into different kinds of drug delivery systems for all types of drug delivery routes, such as for transdermal administration, oral administration, pulmonary administration, subcutaneous implant or for dissolution into a liquid media for administration, such as a suspension or solution or by parenteral/ intramuscular or intracavernosum injection and so on. Hydrogels (3D hydrophilic polymeric structures) are capable of absorbing massive amounts of water or biological fluids along



Table 9. Applications of hydrogels for drug/cell/gene delivery at various sites of the body organ.


Polymers

Drug/cell/gene

Sodium alginate, Poloxamer

Moxifloxacin hydrochloride

Chitosan glutamate

Lidocaine hydrochloride

Sterculia gum Chitosan glutamate

Ciprofloxacin Insulin

Chitosan

Diclofenac-sodium

Poloxamers, polycarbophil

Clotrimazole

Carbopol

Rutin

Gelatin

Simvastatin

Guar gum

Ibuprofen

Carboxymethyl chitosan Alginate

Ornidazole Dental-derived mesenchymal stem cells PC12 cells

Poly ethylene glycol

Applications Therapeutically efficacious, stable and increases the residence time of the drug in the ocular cavity [126] Provide relief of aphthosis or other painful mouth diseases [127] Successfully cure diverticulitis [128] Protect insulin from enzymatic degradation in acidic stomach [129] Showed a controlled drug release pattern (zero order) reaching 34.6 -- 39.7% after 6 h [130] Showed increased and prolonged antifungal activity of clotrimazole [131] Increases total protein levels and improves skin wound healing in rats [132] Observed the therapeutic effect for fracture healing [133] Showed controlled delivery of Ibuprofen to colon [134] Achieved colon targeted delivery of ornidazole [135] Showed cartilage regeneration, with a high capacity for chondrogenic differentiation [136] Provide a temporary neurogenic environment that supports cell survival during encapsulation [137] Cell viability was assessed in nude mice [138]

Chitosan Collagen Chitosan Collagen

Skeletal muscle satellite cells

Hyaluronic acid, PEG derivative

Human adipose-derived stem cells

Gelatin

Renal cells

Hyaluronan Methyl cellulose

Adult brain-derived neural stem/progenitor cells

k-carrageenan

Human adipose stem cells

Silanized hydroxypropyl methylcellulose

Bone marrow-mesenchymal stem cell

Chitosan

Adipose-derived stem cells

Adult human mesenchymal stem cells

with satisfactory mechanical strength as well as physical integrity. It involved naturally derived materials such as alginate, fibrin and gelatin, but recently, physical hydrogels based on synthetic polymers like poly (nisopropylacrylamide, NIPAAm), pluronic poly(ethylene oxide)-poly propylene oxide (PEO-PPO)--PEO triblock copolymers, poly(lactic-coglycolic acid (PLGA)--PEO--PLGA triblock copolymers and polyphosphazenes exhibiting temperature-dependent sol--gel phase-transition behaviour have been widely used. Above critical concentrations, these hydrogels show a sol state at room temperature, but reversibly change into a gel state at body temperature. These physical hydrogels exhibited sufficient mechanical strength to hold the proliferating and differentiating

Showed orthopaedic tissue regeneration by allowing minimally invasive delivery of progenitor cells in microenvironments [139] High cell viability for up to 7 days in hydrogels and an ideal living dressing system for wound-healing applications [140] Provided functional stability to damaged kidneys in preclinical models [141] Showed significant reduction in cavitation, improved graft survival, increased oligodendrocytic differentiation in rats [142] Provide the basis for new successful approaches for the treatment of cartilage defects [143] Preserved cardiac function and attenuate left ventricular remodelling during an 8-week follow-up study in a rat model of myocardial infarction [144] Can be maintained in culture for extended periods [145]

cells with providing a structural support for a desired period as the tissue is regenerated. Various bioactive molecules are potentially administered through stimuli-sensitive hydrogels by various routes on the target site. Microspheres (spherical empty particles) loaded with drugs as doxorubicin, retinoic acid and ganciclovir have been recognised to be more operative than consistent intravitreal drug delivery in solution. They have the advantage of lasting longer than a single injection, increased therapeutic efficacy, longer duration of action and delivering a wide range of drugs to the target site. These injectable porous scaffold microspheres could be used for microcarrier suspension culture of cells, as well as injection of the cell/


781


Table 10. Applications of microspheres for drug/cell/gene delivery at various sites of the body organ.


Polymers

Drug/cell/gene

Guar gum, Tragacanth

Diclofenac sodium

Hydroxyapatite

Gentamicin

L-lysine,

Insulin

poly ester amide

Applications

Polylactide, gelatin

Fenretinide

PLGA

Infliximab

Hydroxypropyl methylcellulose

Flurbiprofen

Poly[La-(Glc-Leu)]

Naltrexone

Eudragit S-100

Aceclofenac

Starch

Lovastatin

Poly(butylene succinate)

Levodopa

Alginate

Human bone mesenchymal stem cells

Fibrin, agarose, gellan gum

Progenitor cells

Chitosan, Heparin

Neural stem cells

N-methacrylate glycol chitosan


Eudragit S-100

Bone-marrow-mesenchymal stem cells Adipose-derived mesenchymal stem cells

Gelatin

Agarose


Alginate


Chitosan

Adipose-derived mesenchymal stem cells Mesenchymal stem cells

Chitosan, heparin

microsphere constructs into a tissue defect site and provide a great advantage for cell therapy in many aspects. Proceeding to injection, the porous structure would permit sufficient cell seeding in and out of the matrix. After injection in vivo, the porous matrix would permit infiltration of cells and ingrowth of tissue from the host, facilitating the regeneration process. The study of biomaterial-based scaffold formulations is exhilarating with novel approaches to drug/cell/gene delivery being developed all the time. The field of biomaterials 782

Showed good mucoadhesive property, targeting the absorption site to thrive oral drug delivery [146] Showed controlled release of gentamicin can minimise significantly bacterial adhesion and prevent biofilm formation against Staphylococcus epidermidis [147] Effectively suppress the blood glucose level in diabetic rats for 10 h [148] Showed marked bioadhesion to the tumour cell membranes [149] Infliximab did not change significantly after encapsulation into microspheres [150] Showed excellent sustained drug delivery from the system [151] An excellent technique for the long-term treatment of alcohol dependence [152] Offered an exciting mode of drug delivery to colon in the chronopharmacological treatment of rheumatoid arthritis [153] Showed good physical stability under storage for 12 months [154] Improved the entrapment efficiency and bioavailability of drug [155] Showed promising modality for repair and regeneration of craniofacial, axial and appendicular bone defects [156] Showed significant impact on the ability of stem cells to form cartilage [157] Showed good cytocompatibility and supporting neural stem cell attachment and survival [158] Successfully induced cell chondrogenesis and promising strategy for cartilage repair [159] Successfully participate in cutaneous wound healing and sweat-gland repair in mice [160] Showed the potential utility as a cell-culture substrate and vehicle for skin regeneration and soft-tissue reconstruction [161] Showed excellent attachment to cryo-injured epicardium greatly improved perfusion and microvascular density of scar tissue [162] Successfully development of implantable constructs for cartilage repair [163] Showed excellent delivery within an appropriate matrix to repair damaged tissue [164] Improved coronary blood flow and decreased mortality, without sacrificing delivery efficiency [165]

has played a crucial role in the development of tissueengineered products. At present, there is a huge extent of research being performed worldwide on all aspects of tissue engineering/drug or gene delivery. Their market potential proved its ability against various diseases. Scaffolds provide adequate signals to the cells and responsible for their survival and growth. Most of the scaffolds studied are still in the investigation stage and are yet to be approved for clinical use. In future, the main focus is on the development of more patient-compliant sustained and controlled delivery



Table 11. Commercialized biomaterial based scaffold products and their major research applications.


Commercialized scaffold products (formulation type) Aquamere (hydrogels) Arestin (microsphere) Apligraf (hydrogels) Biomend (porous matrix) Cervidil (hydrogels) CONSTA (microsphere) Collagraft (porous matrix) Curasol (hydrogels) Decapeptyl (microsphere) Enantone Depot (microsphere) Enantone-Gyn (microsphere) Gelfoam (hydrogel) Healos (nanofibre) Hycore-R (hydrogels) Hycore-V (hydrogels) Lupron Depot (microsphere) Nutropin (microsphere) Parlodel LAR (microsphere) RISPERDAL (microsphere) Revitix (porous matrix) Sandostatin (microsphere) Smart C (hydrogels) Somatuline (microsphere) SQZ Gel (hydrogels) Suprecur (microsphere) Surgifoam (porous matrix) Trelstar (microsphere) Trenantone (microsphere) Vivitrol (microsphere) VCTO1 (porous matrix)

Major research applications

Useful in skincare, topical and oral drug delivery [1] Useful in the treatment of periodontitis [26] Useful as artificial skin products [1] Useful in the regeneration of periodontal tissue [2] Useful in initiation and/or continuation of cervical ripening [1] Useful in the treatment of schizophrenia and bipolar I disorder [26] Useful in the treatment of long bone fractures [2] Useful in wound dressing [1] Useful in the treatment of prostate cancer [26] Useful in the treatment of prostate cancer/endometriosis [26] Useful in the treatment of prostate cancer/endometriosis [26] Used as a haemostatic device [1] Useful as a bone graft substitute in spinal fusions 166 Useful in the treatment of rectal infections [1] Useful in the treatment of vaginal infections [1] Useful in the treatment of alcohol dependence [26] Useful in the treatment of growth deficiencies [26] Useful in the treatment of parkinsonism [26] Useful in the treatment of schizophrenia and bipolar I disorder [6] Useful as topical cosmetic product [2] Useful in the treatment of acromegaly [6] Useful in the development of ophthalmic, buccal, nasal, vaginal and transdermal [1] Useful in the treatment of acromegaly [26] Useful in the treatment of hypertension [1] Useful in the treatment of endometriosis [26] Useful as an absorbable implant in neuro-, thoracic, and ocular surgery [2] Useful in the treatment of prostate cancer [26] Useful in the treatment of prostate cancer/endometriosis [26] Useful in the treatment of alcohol dependence [26] Useful as an antimicrobial wound dressing [2]

systems against various diseases by modification in polymers, manufacturing technologies as well as carrier systems.

Department of Biotechnology (DBT), New Delhi, India and Punjab Technical University, Jalandhar.

Acknowledgement

Declaration of interest

Authors AK Goyal (under IYBA scheme; BT/01/IYBA/ 2009 dated 24/05/2010) and T Garg are thankful to

The authors state no conflict of interest and have received no payment in preparation of this manuscript.


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Affiliation

Tarun Garg†1 & Amit K Goyal2 Author for correspondence 1 Senior Research Scientist, ISF College of Pharmacy, Department of Pharmaceutics, Moga, Punjab, India Tel: +91 814 626 5364; E-mail: [email protected] 2 ISF College of Pharmacy, Department of Pharmaceutics, Moga, Punjab, India †

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Cardiovascular pharmacogenomics: current status and future directions.

Metformin pharmacogenomics: current status and future directions.

Palliative radiotherapy: current status and future directions.

Antagonizing leptin: current status and future directions.

HPV vaccine: Current status and future directions.

GenBank: current status and future directions.

Transgenic fish: present status and future directions.

Photodynamic therapy: current status and future directions.

Honey and Cancer: Current Status and Future Directions.

Genistein and cancer: current status, challenges, and future directions.

Intestine and multivisceral transplantation: current status and future directions.

Molecular cancer prevention: Current status and future directions.

Protein misfolding cyclic amplification (PMCA): Current status and future directions.

Minimally invasive central pancreatectomy: current status and future directions.

γ-Secretase modulators: current status and future directions.

Vaccines against invasive Salmonella disease: current status and future directions.

Hepatic applications of endoscopic ultrasound: Current status and future directions.

The Cobb Collection: current status and future research directions.

DNA barcoding to fishes: current status and future directions.

Outpatient diabetes clinical decision support: current status and future directions.

Cancer stem cells: current status and future directions.

Medical models of teleoncology: current status and future directions.

Early pregnancy diagnosis in bovines: current status and future directions.

Image-Guided Hydrodynamic Gene Delivery: Current Status and Future Directions.