The tissue engineering scaffolds imitates the pattern of natural release mechanism of growth factors stored in natural extracellular matrix and employs it to deliver proteins at the target site. The various scaffolds used in delivering a variety of growth factors are described. A study in which insulin like growth factors-1 (IGF1) and Transforming growth factor β(TGF-β) were encapsulated in poly (D, L-lactic-co-glycolic acid) (PLGA) microspheres and then incorporated into polyethylene-based hydrogels for cartilage tissue engineering showed persistent release of these growth factors for 15 days stimulating chondrocyte proliferation [8]. A similar approach for cardiac tissue engineering was studied using the IGF1 and hepatocyte growth factor (HGF) encapsulated into PLGA microspheres. These microspheres were integrated along with human adipose-derived stem cells (ADSCs) into a hydrogel based on tri co-polymer of polypropylene glycol and polyethylene glycol. The results showed an initial burst of release and then a sustained release for 21 days [9]. For the support of the osteoblast proliferation and adhesion, the FGF2 delivery by gelatin microspheres were developed by seeding FGF2 into PCL-PLLA scaffolds. In a study involving rhBMP7 (bone morphogenic factor) incorporated nanospheres which were immobilized into NF scaffolds for use in the treatment of bone damage. The result showed that the bone formation with rhBMP-7 nanospheres were significantly more than that of rhBMP7 absorbed scaffold [10]. Protein delivery through scaffolds is a sophisticated task and involves lots of hardships. Further researches are going on to develop more efficient scaffold matrices for efficacious protein delivery. DNA delivery for protein expression followed by intracellular expression of these biomolecules separately resulted in enhanced formation and differentiation of the cells to a tissue of a specific lineage in tissue regeneration and wound healing therapies.

4. SCAFFOLDS FOR DNA DELIVERY:

Scaffolds for gene delivery are an impressive approach to manipulate intracellular environment for directing cell function. This advancement in using scaffold for enhancing transgene expression resulted in greater control over expression and delivery to promote tissue regeneration. For delivery of nucleic acids (e.g., DNA, RNA, siRNA), the vectors used must evade the immune system and infiltrate the cell’s microenvironment. The vector must evade before it is degraded by the lysosome and nucleic acids must dissociate from this vector to induce expression of the encoded gene. Viral vectors proved to be more efficient than non-viral vectors. Adenovirus and retrovirus are most commonly used viral vectors whereas lentivirus proved to be a promising and efficient vector. But viral vectors elicit more inflammatory response [11] compared to either naked plasmid or non-viral vectors which possess low transfection efficiency.

The factors to be considered while selecting vectors for delivery to promote tissue formation are,

· Immune response elicited by the vector,

· Target cells for drug delivery,

· Duration of expression and

· Vector stability.

Initially, the transgene expression was limited due to the immune response [12] but regeneration can be potentially influenced by local inflammatory responses [13]. The intensity and time period of gene expression must be optimized to prevent side effects due to excessive protein activity. The optimization can be done with the help of inducible promoters which are either tissue specific or molecule activated to control the expression [14]. Novel vectors were designed for drug delivery using a variety of strategies such as rational design [15] and high throughput screening [16]. Since the vectors are used for scaffold-based delivery, the interactions between the vector and material should be assessed. There should be an optimal affinity between the vector and material.

The advantages of using scaffolds for direct delivery of vectors are

· The transgene expression can be localized at the site of implantation,

· Protection from extracellular barriers, immune system attack and degradation by serum nucleases or proteases.

· It increases the half-life of the vector.

· The ability to maintain effective vector level for a period of time to improve gene transfer efficiency.

The vector and material are designed to regulate interactions between vector and matrix for the subsequent release of the DNA from the system. The scaffolds were categorized into two, based on mechanisms by which the DNA gets impregnated: (i) DNA Encapsulation in the scaffolds and (ii) vector Immobilization.

4.1 DNA ENCAPSULATION IN SCAFFOLDS:

The important criteria to be beware of in scaffold encapsulation method is the compatibility between scaffold fabrication method and the vector integrity. The scaffolds are fabricated either by hydrophobic or hydrophilic polymers.

4.1.1. Scaffolds fabricated from hydrophobic polymers:

Hydrophobic polymer scaffolds release vector sequentially through polymer degradation, dissolution of vector and consequent diffusion from the polymer [17]. When implanted, the polymer is wetted by the body fluids resulting in initial release of vectors that are weakly associated with surface. In case of degradable polymers, for instance PLG, is degraded by the fluid and the entrapped vector are released by diffusion. On the other hand, fluid penetrates through pre-existing network of pores in the non-degradable materials (such as poly (ethylene-covinyl acetate), EVAc), dissolving the entrapped vector and is released by diffusion [18]. To encapsulate plasmid into porous tissue engineering scaffolds without using organic solvents, a process named gas foaming/particulate leaching was employed. In this method, plasmid, a porogen and PLG microspheres are mixed and then foamed [19]. This method provided a versatile approach for encapsulating DNA in scaffolds for promoting transgene expression in cranial defect or spinal cord injury or subcutaneous implantation. A more sustained release of the plasmid was observed when the DNA was pre-encapsulated into polymer microspheres before foaming. Subcutaneous implantation of plasmid loaded scaffolds exhibited transgene expression lasting for more than 3 months.

The gas foaming process was also used to deliver polyplexes comprising cationic polymer polyethylenimine (PEI), porogen and a plasmid complex. These polyplexes reduced the amount of DNA required for delivering and also had influence in the release profile. Porogen to polymer ratio directly influenced the release of the complexes. When this PEI/DNA-loaded scaffold was subcutaneously implanted, the transfected cells were noticed up to 3 months at the site of implantation [20]. By dissolving PLG in plasmid with glycofurol (biocompatible water-miscible solvent) an injectable scaffold was developed [21]. Once injected, the solvent from the site of injection diffuses to form a solid polymeric implant. 100% DNA incorporation efficiency was exhibited by this technique.

4.1.2. Hydrogel (hydrophilic polymer fabrication):

The release from hydrogel occurs through either diffusion alone (non-degradable hydrogels), or a combination of hydrogel degradation and vector diffusion. Hydrogels possess high encapsulation efficiencies. Based on the type of hydrogel, different mechanisms can be designed for degradation like hydrolysis, cell secreted enzymes or manipulation by mesh size, ion exchange and interaction strength. Collagen was the first tissue engineering scaffold used for gene delivery. It can be used to prepare hydrogels, sponges [22] etc. for delivery applications. Collagen based scaffolds were used to induce in vivo transgene expression in bone regeneration, muscle repair, optic nerve repair and wound healing application by employing with various viral and non-viral vectors. Atelocollagen could be used as a potent alternative for loading DNA and siRNA since it elicits less immune response than collagen but the release rate was slow with steady prolonged transgene expression for two months [23]. Atelocollagen delivery of siRNA was able to attain gene inhibition up to 20 days [24]. One disadvantage is that since viruses have the tendency to bind with ECM proteins such as collagen, the delivery of viral vectors from hydrogel gets complicated. But still it showed persistent expression. A research involving gelatin cationized with ethylenediamine to bind DNA within the gel was examined for release kinetics and the results depicted the persistence of DNA for 7-10 days [25]. The canarypox viruses were entrapped in gelatin sponges and implanted which showed transgene expression up to 96 days [26]. PEG was combined with lactic acid, caprolactone, fumarate, and hyaluronic acid to form hydrogels with varied functionality and degradation rates for gene delivery. A steady release kinetics was observed.

4.2 VECTOR IMMOBILIZATION:

Vector immobilization is an imitation of the natural process in which virus binds to extracellular matrix [27] resulting in co-localization of vector and adhered cells with the matrix. Similarly, vectors were immobilized and then fabricated on the scaffold surface for gene delivery applications. Hydrophilic and ionic surfaces showed more efficiency compared to hydrophobic and non-ionic surfaces respectively. Recently PEG was immobilized on the surface and used for gene delivery. The study showed that the PEG surfaces stabilized the vector conformation resulting in increased gene expression. This study on hydrophilic surfaces led to protein coating of PLG scaffolds to increase hydrophilicity enabling more efficient gene delivery. PLG was also employed with cationic groups, such as PEI, cetyl trimethyl ammonium bromide (CTAB), and PLL to more effectively bind plasmid [28]. Another immobilization approach is by precipitating vectors onto the scaffold surface. For instance, in bone tissue engineering, calcium phosphates (CaP) were precipitated onto the polymer scaffolds and used as transfection reagents [29]. Plasmid is co-precipitated with CaP and used for expression. The study showed that there was 90% of the plasmid bound to CaP at maximal transgene expression conditions [30].

An alternate approach for all these nonspecific immobilization strategies was achieved by the specific binding of vectors with pre-fabricated scaffolds using biotin–avidin and antibody–antigen binding. For instance, HA-based hydrogels surface immobilized with neutravidin had the ability to bind biotinylated PEI/DNA complexes [31]. Dense gene vectors surfaces were achieved in specific binding resulting in high persistence of vectors on the scaffolds exhibiting increased gene expression. These advancements encompassing scaffolds for gene delivery aided in controlled and sustained expression of the transgene for the production of proteins and peptides to enhance induction and promotion of tissue regeneration, wound healing etc.

5. SCAFFOLDS FOR BIOMOLECULE DELIVERY IN HARD TISSUE ENGINEERING:

Most of the fractures heal without any intervention barring a few serious bone defects like severe nonunion fractures; bone tumor resections etc. do not possess the template for regeneration and needs medicinal intervention. Owing to the disadvantages in autograft and allograft, new treatment strategy involving scaffolds are developed.

The characteristics of scaffolds used for bone tissue engineering are,

1. A bone remodeling permitting biodegradability.

2. Mechanical stability and growth factors/cells carrier.

3. Osteoconductive properties to guide bone in the implant.

4. Macro-porosity to facilitate cell ingrowth into composite.

The methods used to treat bone fractures are autografts and allografts. But they elicit certain problems. For instance,

· High infection rate

· The process of harvesting bone cells from iliac is painful

· The recovery period is long and post-operative

· They are avascular and not made of viable cells

· The failure rate is 25-30% in this treatment

Scaffolds can be used to overwhelm these issues. Different materials can be used for making scaffolds in bone tissue regeneration. Polymers with calcium phosphate minerals like hydroxyapatite or other compounds like carbon nanotubes can be used to synthesize scaffolds. These scaffolds should be biocompatible, bioresorbable with controlled degradation and resorption rate. Biocompatible materials such as bio glass and ceramic are preferred as they have an advantage to integrate themselves to the surrounding tissues. Owing to this property, the host reactions at the implant site is minimized. The various materials used for synthesizing scaffolds are listed below.

Natural polymers:

· Collagen.

· Hyaluronic acid (HA)

· Carboxymethyl cellulose (CMC)

· Chitosan

Synthetic polymers:

· Polylactide (PLA) and its related copolymers polylactide-co-glycolide (PLGA)

· Polycaprolactone (PCL)

· Poly propylene fumarate (PPF)

· Polyanhydrides-eg.Poly(1,6-bis-(p-carboxyphenoxy hexane)-co-(sebacic anhydride) (PANH)

Composite materials:

1. Ceramics-Hydroxyapatite (HAP),-tricalcium phosphate (-TCP) and biphasic calcium phosphate (BCP).

2. Bio-glasses-the original form was based on Na₂O-CaO-SiO₂-P₂O₅. phosphate-based, silicate-based, and borate-based bio-glasses are prevalent in the market. They are important for bone tissue regeneration. One of the important problem in bio-glass is its byproduct glass. The drawbacks in bio-glasses are

· slow degradation rate

· At the site of implantation there is increase of Na+and Ca₂+concentration

· The scarce sintering ability of the glass makes the 3D processing of the material into a scaffold difficult.

5.1. DRUG DELIVERY IN OSTEOMYELITIS:

Osteomyelitis is characterized by an infection in the bone with subsequent inflammation and bone loss, caused by microorganisms such as staphylococcus aureus, Pseudomonas, Enterobacteriaceae, Salmonella, Clostridium and Pasteurella multocida resulting in conditions such as diabetic foot. Conventional treatments for osteomyelitis include surgical debridement, removal of implant and necrotic tissue, blood supply and soft tissue restoration and oral/systemic antibiotic administration. The removal of infected bone tissues involves surgery which can lead to further infections. The antibiotic therapy was proposed as an alternative but glycopeptide drugs like vancomycin, daptinomycin, and teicoplanin had limited bone tissue penetration [32]. Oral therapy with fluroquinone drugs for osteomyelitis was also inefficient [33]. Inert materials based antibiotic loaded carrier such as PMMA beads infused with antibiotics were used to treat arthroplasties and musculoskeletal infection. There was sustained release of drugs at local administration but it triggered immune response. The drugs Vancomycin, gentamicin, rifampicin, and moxifloxamicin were loaded into scaffold materials such as cements, bioglasses and polymer systems and were studied for elution kinetics and antimicrobial effects. The results showed high and sustained elution rate with antimicrobial effect intact [34,35,36]. Thus, biodegradable scaffolds were synthesized for efficient and sustained drug delivery in the treatment of osteomyelitis: for instance,

· Proteins such as collagens from skin or tendons of animals help in mineralization and regeneration of bone. So, they are used in the manufacture of scaffolds for osteomyelitis.

· The compatibility of synthetic polymers such as PLA, PLGA, PCL and their copolymers with several antibiotics is high and they provide sustained release of the drugs with slow degradation rate.

· Bone graft materials and substitutes: The addition of antibiotic is easy.it is done either by mixing its powder with bone graft or submerging the bone graft in antibiotic solution. Calcium sulphate has low immune reaction, structural properties and easy reabsorption. So, it is used in treatment of osteomyelitis.

Osteomyelitis stands as an exemplar where a locally delivered antibiotic integrated with biodegradable polymer scaffold can be used in efficacious treatment of a severe infection for bone regeneration.

5.2. BIOMOLECULE DELIVERY IN PERIODONTAL REGENERATION:

Periodontium encompassing of cementum, periodontal ligament (PDL), and alveolar bone acts as an anchorage and support for teeth to remain fixed in mandible and maxilla. If periodontium degrades, it will lead to periodontal disease resulting in tooth loss. The methods used for periodontium regeneration are bone autografts, allografts, cell occlusive barrier membranes, bio-glass, beta-tricalcium phosphate (βTCP), polytetrafluorethylene, poly lactic-co-glycolic acid (PLGA), collagen membranes, and enamel matrix derivatives. Fibroblast growth factor-2 (FGF2), PDGF, IGF-1, BMP and TGFβ are the growth factors which support in the proliferation of cells to form periodontium. BMPs can effectively induce ectopic bone formation. In a study, the collagen sponges were loaded with BMP-12 and used for the treatment of periodontal ligament. it revealed enhanced periodontal ligament regeneration. When BMP-2 was co-applied with BMP-7, the alveolar regeneration did not improve. When administered alone alveolar regeneration improved. The delivery of BMP-7 alone showed significant growth of the cementum. Cementogenesis improved when BMP-2 was delivered with slow dissolving collagen than fast collagen gel delivery [37,38].

5.3. BIOMOLECULE DELIVERY IN TEMPOROMANDIBULAR JOINTS (TMJ):

The mandible is connected to the temporal bone of the skull by TMJ (a disc that sits between two cartilage layers). Any defect in it results in temporomandibular joints disorder. In a study, a rabbit was treated with BMP-2 encapsulated in collagen carrier and it demonstrated increased cartilage regeneration in the rabbit [39]. Another study in which both cartilaginous and osseous components from a single rat population bone-marrow derived mesenchymal stem cells (rMSCs) were loaded into hydrogel matrix. A solution containing PBS and a biocompatible UV photo initiator was used to dissolve Poly (ethylene glycol) diacrylate (PEGDA) and MSCs were suspended in it. The photopolymerized osteochondral construct was implanted in the dorsum of severe combined immunodeficient (SCID) mice. The constructs expressed cartilage specific glycosaminoglycans (GAG) in chondrogenic layer and bony mineral nodules in osseous layers. Increasing the cell-seeding density in this approach resulted in 4-folds increase in tissue maturation in both the layer after 12 weeks of subcutaneous implantation.

5.4. BIO-MOLECULE DELIVERY IN TOOTH DECAY:

Tooth decay is one of the most common disease persistent mostly among children. It is an infection of acidogenic bacteria leading to partial or complete loss of tooth structure. The dental materials used for treatment are metal alloys, cements, acid-based polymer products and synthetic materials. Amalgam and formocresol used in pulp therapy are also used in dental treatment but they are potent carcinogens. The hard tissues of the tooth are dentin, enamel and cementum. The signaling molecules which play a major role in the regulation of interactions between ectoderm and mesenchyme for the tooth development belongs to transforming growth factor-β (TGF-β), fibroblast growth factor (FGF), Hedgehog, and Wnt families. The expression of TGFβ1, TGFβ2 and TGFβ3 results in the accumulation of odontoblasts in the dentin matrix. TGFC has a role to play in the pulp cell regulation and alginate gels help in cellular wound healing. A study which involved fabrication of alginate gels using calcium chloride and TGFβ1 solutions for dental repair. The result showed that the TGFβ1 loaded gels showed two types of responses, reactionary and reparative dentinogenesis. Another study in which 2 μg of BMP-2 was delivered in collagen sponges to amputated pulp in dogs resulted in mineralized osteodentine-like tissue with embedded osteodentinocytes after 70 days of implantation [40].

5.5. BIOMOLECULE DELIVERY IN CRANIAL TISSUE:

Cranial sutures are the soft tissue present between the calvarial joints (joints in the skull cap) and immobile joints of the skull. TGF-β3 is expressed in high levels during suture patency and when the skull matures, TGF-β3 expression decreases and TGF-β2 expression increases. Any defect in this signaling process leads to a condition called craniosynostosis, characterized by visible craniofacial disfigurations with severe neurological problems. Since there is a need for sustained long-term expression of cytokines for craniofacial development, PLGA and polycaprolactone (PCL) scaffolds were widely used. PLGA can be fabricated in many forms including sheets, blocks, microspheres, and nanofibers with required degradation rates. growth factors and growth hormones such as BMPs, TGFβs, FGFs, and insulin were encapsulated for controlled drug delivery. A study which involves microencapsulation of TGF-β3 in PLGA microspheres and embedded in thermosensitive hydrogels resulted in a sustained release of TGF-β3 for 36 days [41]. In another study, an implant consisting of bone marrow derived stem cells and TGF-β3 encapsulated in PLGA microspheres in a pre-sized collagen sponge carrier was designed for the development of cranial tissues and it resulted in the formation of a bone-tissue interface similar to that of cranial sutures. Also, there was a decrease in bone deposition.

6. SCAFFOLDS FOR BIOMOLECULE DELIVERY IN NEURAL TISSUE ENGINEERING:

The brain tissue damages resulted in critical health conditions such as traumatic brain injury (TBI), Parkinson’s and Alzheimer’s disease. The neural tissue damage can also result in spinal cord injury(SCI) and peripheral nerve injury. The tissue repair mechanisms for curing these diseases are induced with certain tissue growth factors, cytokines etc. as depicted below.

· Neurotrophins: They are family of growth factors (nerve growth factor (NGF), neurotrophin-3(NT-3), neurotrophin-4/5 and brain derived neurotrophic factor (BNDF)).

· Other growth factors are ciliary neurotrophic factor (CNTF), fibroblast growth factors (acidic and basic, aFGF and bFGF), transforming growth factor β (TGF-β), and glial

· derived neurotrophic factor (GDNF).

· Anti-Inflammatory Drugs: Dexamethasone, α-melanocyte.

The release of all these factors into the respective nerve tissues was aided by scaffolds which should have high efficacy in release kinetics and drug loading capacities. The scaffolds which are used for targeted delivery of drugs for nerve tissue repair are detailed.

Some synthetic polymers are used as scaffolds for drug delivery in neural tissue are,

· Poly (ethylene glycol)/Poly (ethylene oxide) (PEG),

· Poly (ethylene-co-vinyl acetate) (EVA),

· Poly (lactic-co-glycolic acid) (PLGA),

· Poly (2-hydroxyethyl methacrylate) and Poly (2-hydroxyethyl methacrylate-co-methyl methacrylate) and

· Poly pyrroles

Natural materials used as scaffold for drug delivery in neural tissue are,

· Agarose/Alginate,

· Methylcellulose/Nitrocellulose,

· Collagen,

· Dextran and

· Fibrin.

The addition of growth factors in the scaffolds aids in controlled release of the drugs. The release of drugs from scaffold will reduce the damage to the nerve cells and preserve proper functioning. A study compared three different materials methylcellulose, collagen, and laminin containing ECM preparation (Biomatrix) for synthesizing scaffolds and employed in peripheral nerve repair. The study showed methylcellulose was more efficient for delivering platelet derived and insulin like growth factor for peripheral nerve tissue repair [42]. A study involving the controlled release of various growth factors such as NGF, NT-3, BDNF, and GDNF from EVA rods demonstrated initial burst and then sustained release for 30 days [43]. Identical approach for treatment of SCL was developed using scaffolds. Collagen scaffolds was used to deliver NT-3 to the site of injury and the result showed improved recovery suggesting sustained release of drug from the scaffold preserved the biological activity. Another study involving photopolymerized PEG and PEG–PLA scaffolds were used to deliver drugs for the treatment of SCL resulting in drug release for 50 days and 14 days respectively in vitro. PEG-PLA scaffolds were used for the release of NGF in vitro as illustrated by neurite extension of PC12 cells, showed longer drug delivery time period for 20 days [44].

An affinity-based delivery system was developed using heparin (HBDS), which can deliver any protein which binds to heparin. This system was initially developed to deliver basic fibroblast growth factor (bFGF) [45] and lately it was conjugated with fibrin for the treatment of peripheral and central nervous system injuries. HBDS conjugated with fibrin and NT-3 were used for the treatment of SCL and results showed that there was a promotion in neuronal fiber sprouting. Immobilized drug delivery systems were developed by tethering proteins to various materials such as including pHEMA, PPy, and polydimethylsiloxane (PDMS). For instance, NGF was tethered to these materials for the delivery into injured bone tissue.

Microspheres can also be coated with drug and incorporated into scaffold made from other materials for treating nerve tissue repair. Some examples of microsphere incorporated scaffold used were,

· Magnetically aligned collagen conduits for treatment of peripheral nerve injuries.

· chitin/chitosan composite scaffolds with PLGA microspheres in neural tissue engineering.

These are some of the ways by which scaffolds can be employed for delivering drugs in the treatment of critical health conditions caused due to nerve tissue damage.

7. SCAFFOLDS FOR BIOMOLECULE DELIVERY IN VASCULAR TISSUE ENGINEERING:

Vascular disease is one of the top killer disease in the world and is continuously expanding. Fibroblast growth factor-1 (FGF-1) and vascular endothelial growth factor (VEGF) are some of the endothelial chemokines which has been used for drug delivery in the treatment of vascular tissue. the release rate of the scaffold can be reduced by adding the cross-linked fibrin and growth factors bound to them. Heparin was also integrated to fibrin gels to control the growth release characteristics. A study, which compared the cell growth when insulin was immobilized on synthetic polymer and non-specifically absorbed into synthetic polymers the immobilized insulin showed increased cell growth than non-specifically absorbed insulin [46]. In a study, electro-spun low molecular weight heparin was grafted with polyethylene glycol which is present within poly(lactide-co- glycolide) (PLGA) showed enhanced heparin retention along with the growth factors [47]. Angiogenic stimulation and inhibition aids in the balance of proliferative state of normal blood vessels. The cellular environment releases specific growth factors for the stimulation of angiogenesis. These factors trigger proliferation, migration, vascular tube formation and maturation. The angiogenic factors involved in the revascularization process are vascular endothelial growth factor (VEGF), Fibroblast growth factors (FGFs), Platelet-derived growth factor (PDGF), Angiopoietins, Transforming growth factor-β (TGF-β), Hepatocyte growth factor(HGF), neurotrophin nerve growth factor (NGF), erythropoietin and insulin-like growth factor (IGF).

STRATEGIES FOR ANGIOGENESIS IN VASCULAR TISSUE:

1. A single angiogenic growth factor or encoded gene is directly injected into the required tissue site. The growth factors involved in this strategy are Topical recombinant human PDGF-BB, VEGF and FGF-2.

Disadvantage: Double-blinded, randomized, controlled trials of this strategy did not have sufficient efficacy for approval

2. The stem cells which had the potential to develop into vascular tissues were isolated from bone marrow then cultured and injected into areas of ischemia tissues. The cells which were used for this process are mesenchymal stem cells (MSCs), hematopoietic stem cells (HSCs) and EPCs, VEGF, SDF-1 and GM-CSF.

Disadvantage: Growth delivery is insufficient, intermediate dosing and unresponsiveness to specific growth factor. Many growth factors are involved in the vessel formation in highly regulated and timed manner. For stem cell therapy, there is an extensive cell death after implantation and lack of well described mechanism for angiogenesis.

As solution for many challenges, the combination of two growth factors or stem cells and growth factor with a flexible, suitable delivery platform was developed. Fibrin glue is combination of fibrinogen and thrombin (which is derived from human plasma) can be used for the treatment of vascular disease [48]. When applied subcutaneously Fibrin glue can stimulate the growth of capillaries without growth factors. This may be due to the formation of fibrinopeptides. The natural biomaterials like collagen, gelatin and alginate are also used as drug carriers. In case of collagens, the addition of glycosaminoglycans changes the crosslinking density. So, it reduces the pore size. The burst release is controlled by reducing the pore size. Alginate is composed of negatively charged α-L-guluronic and β-D-mannuronic acid residues linked by 1-4 glycosidic bonds. Alginate can be used to form microspheres which can be used to entrap proteins for sustained release. Synthetic polymers have found fewer opportunities in the therapeutic angiogenesis as drug delivery vehicles.

8. BIOMOLECULES DELIVERY USING ELECTROSPUN SCAFFOLDS:

Electrospinning is a widespread potential polymer processing technique for applications in drug delivery and tissue engineering. Electrospinning is a process which utilizes electrostatic forces to generate polymeric fibers. Scaffolds with nanoscale topography and high porosity identical to extracellular matrix can be engineered using electrospinning technique to improvise cell adhesion, drug loading and transfer properties. Biodegradable, non-biodegradable as well as natural materials can be electro-spun enabling great flexibility in choosing materials for drug delivery. These electro-spun matrices can be used for controlled release of proteins, drugs as well as DNA either employing biodegradable (diffusion+material degradation) or non-biodegradable (diffusion alone) materials. Additionally, different loading techniques can be utilized in electrospinning, for instance, embedded drug, encapsulated drug etc. for finer drug release control.

The electrospun scaffolds can be used for,

i. Drug delivery,

ii. DNA delivery and

iii. Growth factor delivery.

8.1. DRUG DELIVERY:

8.1.1. TETRACYCLINE HYDROCHLORIDE

The tetracycline hydrochloride from three electro-spun mats composing of poly (ethylene-co-vinyl acetate) (PEVA), PLA and a 50:50 blend of both polymers was studied for tetracycline release.The tetracycline hydrochloride (5 or 25 wt%) and polymer (14% w/v) were dissolved in a chloroform/methanol solution to prepare the mats. The results showed that rate of drug release was quicker with PEVA than either the 50:50 PEVA/PLA blend or PLA alone. Additionally, these electro-spun mats showed smooth release over a period of 5 days eliminating initial high bursts seen with other films [49].

8.1.2. BITERAL:

The potential of PCL electro-spun mats loaded with Biteral drug for preventing abdominal adhesion was studied in a rat model. PCL (13% w/v) in chloroform/DMF (30:70) was electro-spun into a fibrous mat which was covered fully with Biteral solution. The drug was absorbed into mat and was only located on the electro-spun fiber surfaces [50]. The results demonstrated the release of almost 80% of the drug after first 3 hours and a complete release in almost 18 hours. The persistence and extent of abdominal adhesion was reduced significantly with the use of this PCL mat.

8.1.3. MEFOXIN

Electrospun PLGA and PLGA/PEG-b-PLA/PLA (80:15:5 by wt%) mats with embedded Mefoxin antibiotic were examined for the controlled release of the drug. The polymer and the antibiotic were dissolved in DMF to embed drug within the electro-spun fibers [51]. The results demonstrated that the PLGA/PEG-b-PLA/PLA blend gave a more sustained release than PLGA alone.

8.1.4. BCNU:

The anticancer drug BCNU was incorporated into electro-spun PEG-PLLA mats to study the release kinetics of BCNU from electro-spun mats on the growth of rat Glioma C6 cells [52]. It was observed that the free BCNU lost its anticancer activity in 48 hours whereas BCNU loaded PEG-PLLA exhibited over 72 hours of anticancer activity corroborating that the drug embedded in polymer secured it from degradation and perpetuated anticancer activity.

8.1.5. PACLITAXEL:

The anticancer drug Paclitaxel was encapsulated into an electro-spun PLGA mat for examining release kinetics in the treatment of C6 gliomas. The encapsulation efficiency in electro-spun mats was over 90% and both PLGA micro and nanofibers were examined [53]. The study showed faster release rate in nanofibers which was around 80% in 61 days release period whereas it was only 60% in microfibers. Cytotoxicity study showed 70% of C6 glioma cells were killed after 72 hours depending upon the drug loading.

8.1.6. IBUPROFEN:

Ibuprofen- loaded PLA nanofibrous scaffolds helped in the attachment and proliferation of human epidermal keratinocytes and human dermal fibroblasts. Cell-seeded ibuprofen-loaded PLA bandages has been used for rescuing wound contracture and increased blood vessel formation [54].

8.1.7. GINSENOSIDE-Rg3:

Hypertrophic scarring is a disease which results due to a disorder of dermal fibroblast proliferation. PLA fibrous mats with both nano and microfibers were used for the delivery of drug ginsenoside-Rg3 to treat this disease in rabbits. And the result showed that the drug delivery was prolonged over a period of 3 months [55]. The hypertrophic scars were also reduced in rabbits.

8.1.8. ANTIMICROBIAL PEPTIDES

The antimicrobial peptide bacteriocin was incorporated into blends of PDLLA and poly(ethylene oxide) (PEO) for antibiotic resistance related issues in cutaneous wounds. The antimicrobial peptide nisin was electro-spun into a nanofibrous PEO:PDLLA (50:50) and was used for cutaneous wound dressing. This dressing reduced the bacterial burden caused by Staphylococcus aureus [56].

8.2. DNA DELIVERY:

The DNA was incorporated into electro-spun PLGA scaffolds and the release kinetics were studied. Plasmid DNA was encapsulated into michelles comprising of a copolymer (PLA-PEG-PLA) resulting in encapsulated DNA nanoparticles. The micelles were electro-spun by dissolving them in a solution containing DMF and PLGA to produce PLGA fibers with encapsulated DNA nanoparticles. This study demonstrated around 20% of the encapsulated DNA was released after a 7 days period [57].

8.3. GROWTH FACTOR DELIVERY:

8.3.1. β-NERVE GROWTH FACTOR:

Chew et al. stabilized β-nerve growth factor in BSA from a €-caprolactone and ethyl ethylene phosphate (PCLEEP) copolymer to study its release. PCLEEP owing to its hydrophobic backbone showed slow degradation rate at a mass loss of 7% over 3 months demonstrating sustained release of NGF for this time period [58].

8.3.2. BONE MORPHOGENIC FACTOR:

Human bone marrow derived mesenchymal stem cells (hMSCs) were cultivated on the scaffolds (silk/PEO/BMP-2, silk/PEO/nHAP, silk/PEO/nHAP/BMP-2) containing osteogenic media for 31 days. This electrospun silk fibroin containing BMP-2 supports hMSC growth and differentiation for in vitro bone formation. The results showed that the scaffolds containing BMP-2 produced higher calcium deposition and bone formation than ones without BMP-2 [59].

8.3.3. PLATELET DERIVED GROWTH FACTOR:

The chemotaxis of mesenchymal stem cells (MSCs) towards platelet derived growth factors PDGF-AB, PDGF-BB and a mixture of chemokines (SDF-1α, CXCL16, MIP-1α, MIP-1β, and RANTES) were assessed. The results showed that the efficacious stimulation of MSC migration was done by PDGF-BB. When this growth factor was adsorbed with electrospun PCL/col/HA scaffold, it resulted in sustained release of PDGF-BB for over 8 weeks [60].

8.3.4. BASIC FIBROBLAST GROWTH FACTOR:

bFGF was fabricated into PLGA nanofibers using facile blending and electrospinning (group 1) and complicated coaxial electrospinning (group 2). Both group 1 and group 2 possessed similar efficiency for protein encapsulation and sustained release for 1-2 weeks favoring bone marrow stem cell attachment and proliferation. But group 1 exhibited an additional increase in collagen production indicating fibroblastic differentiation [61].

The fibers with core-shell morphology which was developed by new nozzle configurations showed promising contribution to the sustained biomolecule release as well as in alleviating initial bursts seen in polymer/biomolecule blends. Still researches are going on to unveil the true potential of electrospinning in biomolecule delivery applications.

9. CONCLUSION:

As reported above, lots of remarkable and innovative methods were developed for providing sustained release of biomolecules from scaffolds for various therapeutic conditions. These studies demonstrated the efficacious potential of such methods for the treatment of tissue related diseases and gives an insight into more advanced potential strategies. Still, some issues such as the requirement of combining multiple materials should be addressed for more efficient controlled release of biomolecules from scaffolds. Also, implementing appropriate combination of biomolecules in the scaffolds will be of great interest for further development. Each and every aspect of the intracellular environment developed by the scaffold must be considered for the promotion of tissue formation and enhancement of more efficacious biomolecule releasing scaffolds holds great promise for a large number of applications in tissue engineering and regenerative medicine.

10. ACKNOWLEDGEMENTS:

We would like to acknowledge Dr.I.Manjubala for guiding us in writing this review article as a part of Tissue Engineering–J component project work.

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Received on 10.04.2018 Modified on 05.05.2018

Research J. Pharm. and Tech 2018; 11(10): 4719-4730.

DOI: 10.5958/0974-360X.2018.00861.2

S.NO	GROWTH FACTORS	ABBREVIATION	THERAPY	ACTIVITY
1.	Vascular endothelial growth factor	VEGF	Angiogenesis	Migration, survival, and proliferation of endothelial cells (ECs)
2.	Basic fibroblast growth factor Fibroblast growth factor-2	bFGF-2 FGF-2 FGF	Angiogenesis Bone regeneration Wound healing	Migration, survival, and proliferation of endothelial cells (ECs) and other type of cells Osteoblasts proliferation. General wound healing stimulant
3.	Platelet derived growth factor	PDGF	Angiogenesis Bone regeneration Wound healing	Promotes the maturation of blood vessels by the recruiting smooth muscle cells Osteoblasts proliferation. Active in all stages of healing process
4.	Angiopoietin-1	Ang-1	Angiogenesis	Strengthens EC-smooth muscle cell interaction
5.	Angiopoietin-2	Ang-2	Angiogenesis	Weakens EC-smooth muscle cell interaction
6.	Placental growth factor	PIGF	Angiogenesis	Stimulation of angiogenesis
7.	Transforming growth factor	TGF TGF-β TGF-β	Angiogenesis Bone regeneration Wound healing	Stabilization of new blood vessels by promotion of matrix deposition Proliferation and differentiation of bone Promotes keratinocyte migration, ECM synthesis and remodeling, and differentiation of epithelial cells
8.	Bone morphogenetic protein	BMP	Bone regeneration	Differentiation of bone forming cells
9.	Insulin-like growth factor	IGF-1	Bone regeneration	Synthesis of bone matrix and stimulates proliferation of osteoblasts
10.	Epidermal growth factor	EGF	Wound healing	Mitogenic for keratinocytes