tissue engineering , with significant research inputs over the last decades , has emerged as a potential tool to regenerate damaged and diseased tissues .
as one of the key components in tissue engineering approach , growth factors provide chemical cues to stem cells , regulating their biological responses and tissue differentiation . while the basic biological functions of growth factors and their endogenic roles in tissue development and repair process have relatively been well studied , the use of growth factors in tissue engineering regime has recently gained great interest .
growth factors are a potential agent to target specific tissue reactions because of their regulatory roles in cellular functions , including adhesion , proliferation , migration , and differentiation in the epithelium , bone , and soft connective tissues and nerves
.   fibroblast growth factor ( fgf ) is a representative growth factor which has shown the potential effects on the repair and regeneration of tissues [ 26 ] .
it was originally identified as a protein capable of promoting fibroblast proliferation and is now known to comprise 22 members .
fgfs exert multiple functions through the binding into and activation of fibroblast growth factor receptors ( fgfrs ) , and the main signaling through the stimulation of fgfrs is the ras / map kinase pathway . with their potential biological functions ,
fgfs have been utilized for the regeneration of damaged tissues , including skin , blood vessel , muscle , adipose , tendon / ligament , cartilage , bone , tooth , and nerve .
then , the prospective source of fgf for the tissue regeneration is used with recombinant human fgf family .
in fact , many previous studies administered the fgfs directly to the wound sites , like other growth factors .
however , free - fgfs are readily degradable in vivo , leading to loss of biological activity and functions [ 79 ] . to gain satisfactory performance , fgfs are adsorbed onto or encapsulated within materials to secure biological activity in a sustained and controllable manner .
although many types of materials have been developed to carry fgfs and elicit their therapeutic efficacy in vitro and in vivo , more sustained , controlled , and targeted delivering system still remain a challenge .   here , we review the cellular biology of fgfs and their functions in cell proliferation , migration , differentiation , and angiogenesis and address the current development of biomaterials - based delivery systems of fgfs and their applications for tissue regeneration , including skin , blood vessel , muscle , adipose , tendon / ligament , cartilage , bone , tooth , and nerve .
fgf , which was first discovered in pituitary extracts in 1973 , is widely expressed in cells and tissues .
acidic fgf ( fgf1 ) and basic fgf ( fgf2 ) were originally isolated from the brain and pituitary gland as growth factors for fibroblasts . since then , at least 22 distinct fgfs have been identified or isolated .
many fgf genes have been identified in vertebrates , including ten fgfs in zebrafish ( fgf24 , 6 , 8 , 10 , 17a , 17b , 18 , 24 ) , six in xenopus ( fgf24 , 810 ) , 13 in chickens ( fgf14 , 810 , 12 , 13 , 16 , 1820 ) , 22 in mice ( fgf118 , 2023 ) and humans ( fgf114 , 1623 ) , whereas only three drosophila fgf genes and two caenorhabditis elegans fgf genes have been observed in invertebrates .
human fgfs contain 22 members : fgf1 , fgf2 , fgf3 ( int2 ) , fgf4 , fgf5 , fgf6 , fgf7 ( kgf ) , fgf8 ( aigf ) , fgf9 , fgf10 , fgf11 , fgf12 , fgf13 , fgf14 , fgf16 , fgf17 , fgf18 , fgf19 , fgf20 , fgf21 , fgf22 , and fgf23 .  
four family members do not bind with fgfr as fgf homologous factors ( fgf11 , fgf12 , fgf13 , and fgf14 ) and are more correctly referred to as fgf homologous factors .
in addition , there is no human fgf15 gene ; the gene orthologous to mouse fgf15 is fgf19 . by phylogenetic analysis
, the human fgf gene family can be divided into seven subfamilies : fgf1 , fgf4 , fgf7 , fgf8 , fgf9 , fgf11 , and fgf19 ( figure 1 ) .
the fgf1 , fgf4 , fgf7 , fgf8 , fgf9 , fgf11 , and fgf19 subfamilies comprise fgf1 and 2 , fgf4 , 5 , and 6 , fgf3 , 7 , 10 , and 22 , fgf8 , 17 , and 18 , fgf9 , 16 , and 20 , fgf11 , 12 , 13 , and 14 , and fgf19 , 21 , and 23 , respectively .
in contrast to phylogenetic analysis , gene location analysis indicates that the human fgf gene family can be divided into six subfamilies : fgf1/2/5 , fgf3/4/6/19/21/23 , fgf7/10/22 , fgf8/17/18 , fgf9/16/20 , and fgf11/12/13/14 .
members of the fgf8 , fgf9 , and fgf11 subfamilies are similar to those of the fgf7/10/22 , fgf8/17/18 , fgf9/16/20 , and fgf11/12/13/14 subfamilies in the gene location analysis .
the molecular weight of fgfs in vertebrates varies from 17 to 34  kda , while the drosophila fgf is 84  kda .
all members of the family share a conserved sequence of 120 amino acids that show 16%65% sequence identity .
fgfs have various biological functions both in vivo and in vitro , including roles in mitogenesis , cellular migration , differentiation , angiogenesis , and wound healing .  
fgfs exert their physiological roles through binding fgfr and regulate developmental pathways , controlling events such as mesoderm patterning in the early embryo through development of multiple organ systems .
the mammalian fgf family is composed of 18 ligands that elicit their actions through four highly conserved transmembrane tyrosine kinase receptors ( fgfr1 , fgfr2 , fgfr3 , and fgfr4 ) .
four fgfrs have been identified in humans and mice and encode receptor tyrosine kinases ( ca .
800 amino acids ) that contain an extracellular ligand - binding domain with three immunoglobulin domains ( i , ii , and iii ) , a transmembrane domain , and a split intracellular tyrosine kinase domain .
fgfrs are expressed on many different cell types and regulate key cell behaviors , such as proliferation , differentiation , and survival , which make fgf signaling susceptible to subversion by cancer cells . unlike other growth factors , fgfs act in concert with heparin or heparan sulfate proteoglycan ( hspg ) to activate fgfrs and induce the pleiotropic responses that lead to a variety of cellular responses induced by this large family of growth factors .  
several germline fgf mutations have been identified in human disease , including loss - of - function mutations , and gain - of - function mutations .
for instance , loss - of - function in fgf3 is involved with hereditary deafness , leading to total inner ear agenesis in humans .
degradation of fgf8 by loss - of - function leads to kallmann 's syndrome ( kal1 ) , a developmental disorder characterized by anosmia and hypogonadism .
fgf10 loss - of - function causes lacrimo - auditory - dento - digital ( ladd ) syndrome , which is characterized by hearing loss , dental anomalies , and lacrimal and salivary gland hypoplasia .
gain - of - function mutations in fgf23 have been identified in hypophosphataemic rickets .  

table 1 summarizes the location , receptor , and therapeutic application of the fgf family .
activated fgfrs mediate signaling by recruiting specific molecules that bind to phosphorylated tyrosine at the cytosolic part of the receptor , triggering a number of signaling pathways leading to specific cellular responses .
these then serve as docking sites for the recruitment of sh2 ( src homology-2 ) or ptb ( phosphotyrosine binding ) domains of adaptors docking proteins or signaling enzymes .
signaling complexes are formed and recruited to the active receptors resulting in a cascade of phosphorylation events .
the best understood pathways are the ras / map kinase pathway , pi3 kinase / akt pathway , and plc pathway .  
figure 2 schematically describes the three pathways of the fgf signal , the ras / map kinase pathway , pi3 kinase / akt pathway , and plc pathway .
mitogen - activated protein ( map ) kinases are serine / threonine - specific protein kinases that respond to extracellular stimuli ( mitogens ) and regulate various cellular activities such as gene expression , mitosis , differentiation , and cell survival / apoptosis .
c - jun n - terminal kinase ( jnk ) , extracellular signal regulated kinase ( erk ) , and p38 mitogen - activated kinase are examples of effectors map kinase .
interestingly , the activation of erk 1/2 and p38 in response to fgf has been observed in all cell types , while the activities of other signal transduction pathways vary depending on the cell type .   to date , the main pathway of the fgf signal is the ras / map kinase pathway , which contains many signaling proteins . a key event in the fgf signaling pathway is phosphorylation of the tyrosine residues of the docking protein , fibroblast growth factor receptor substrate 2 ( frs2 ) , which provides new binding sites for direct or indirect recruitment of proteins that are responsible for both activation and attenuation of signaling [ 23 , 24 ] .
frs2 recruits a complex consisting of an adaptor protein , the guanine nucleotide exchange factor 2 ( grb2 ) , the son of sevenless ( sos ) , the tyrosine phosphatase ( shp2 ) , and the docking protein , grb2-associated binding protein 1 ( gab1 ) .
formation of the frs2 signaling complex results in activation of ras / map kinase   as well as pi3 kinase / akt pathways .
the ras - map kinase pathway has been implicated in cell growth and differentiation in many studies .  
lax et al .   showed that fgf signals induce a map kinase mediated negative feedback loop that causes threonine phosphorylation of frs2a , leading to a reduction of its tyrosine phosphorylation and decreased recruitment of grb2 .
receptor tyrosine kinases also induce negative signals via activation of the sprouty proteins that inhibit the recruitment of grb2-sos complexes to frs2 and the receptor and attenuate the ras - map kinase pathway .
members of the sef and map kinase phosphatase families are other negative modulators of fgf signaling , while xflrt3 , a member of a leucine - rich - repeat transmembrane protein family , is a novel positive modulator .
expression of xflrt3 is induced by fgf and down - regulated after inhibition of fgf signaling .
thus , fgf signaling is modulated by both positive and negative mechanisms , and subtle modulations in the signal are important determinants of the biological response during development .
similar to the ras / map kinase pathway , the phosphoinositide 3 ( pi3 ) kinase / akt pathway is initiated by forming an frs2 signaling complex .
gab1 consists of a pleckstrin homology domain , a proline - rich region , and multiple tyrosine phosphorylation sites that serve as binding sites for the sh2 domains .
the p110 catalytic subunit of pi3 kinase is in a complex with an adaptor protein ( p85 ) that has two sh2 domains ; thus , p85 binds to phosphorylated tyrosine residues in gab1 adaptor protein .
phosphoinositide - dependent kinase and the anti - apoptotic protein kinase akt are activated downstream of the pi3 kinase .  
the pi3 kinase / akt pathway is implicated in cell survival and cell fate determination , as well as the pi3 kinase / apkc signaling cascade in cell polarity control .
.   showed that gab1 is required for stimulation of the akt pathway by fgf .
one of the target molecules for activated fgfr is phospholipase c gamma ( plc ) , which binds to the phosphorylated tyr-766 of the receptor and then becomes tyrosine phosphorylation of plc , resulting in plc activation .
activated plc hydrolyzes phosphatidylinositol , generating inositol triphosphate ( ip3 ) and diacylglycerol ( dag ) .
ip3 is a cellular second messenger that facilitates the release of calcium from the endoplasmic reticulum .
increased levels of calcium in the cytosol and dag together activate protein kinase c ( pkc ) .
the physiological relevance of this pathway is not obvious since its disruption does not abolish either mitogenesis   or cell differentiation .
however , some data indicate that it may be necessary for adhesion , at least in some cell types .
as stated above , fgfs exert their physiological roles by binding to high affinity tyrosine kinase fgfrs on the surface of the target cell .
therefore , the function of fgfs depends on the fgf signal pathway between the fgf family and fgfrs .
many studies have reported that fgfs have functions such as cell proliferation , migration , differentiation , and angiogenesis in various cells and tissues .  
cell proliferation by fgfs has been reported in many cell types , including endothelial cells , stem cells , and epithelial cells .
fgf1 is a proliferative factor for human preadipocytes that may be important to the overall regulation of human adipogenesis .
in addition , fgf1 leads to an increase in the proliferation of iec-6 , caco-2 , and ht-29 cell lines with fgf2 and fgf7 .
fgf2 induces cell proliferation after flia - specific gene transfer in mice   and stimulates the proliferation and survival of neuroepithelial cells isolated from the telencephalon and mesencephelon of e10 mice .
fgf4 knockout mouse embryos experience postimplantation lethality owing to the necessity of fgf4 for trophoblast proliferation .
fgf10 play a role in the pathogenesis of prostate cancer via facilitation of epithelial cell proliferation .
fgf18 has also been shown to stimulate the proliferation of cultured mouse primary osteoblasts , osteoblastic mc3t3-e1 cells , primary chondrocytes , and prechondrocytic atdc5 cells , although it inhibited the differentiation and matrix synthesis of these cells .
tissue formation during embryonic development , wound healing , and immune responses all require the orchestrated movement of cells in particular directions to specific locations .
cells often migrate in response to and toward specific external signals in a process known as chemotaxis .
apparently , both fgf1 and fgf2 play important roles in the migration of cochlear ganglion neurons in mice .
fgf2 induces cell migration after flia - specific gene transfer in mice   and stimulates cell migration of mouse embryonic limb myogenic cells such as fgf4 .
fgf7 is known to stimulate migration and plasminogen activity ( pa ) of normal human keratinocytes .
similar to fgf2 , fgf8 is a potent chemoattractant in the migration of mesencephalic neural crest cells .   in developmental biology
, cellular differentiation is the process by which a less specialized cell becomes a more specialized cell type .
differentiation occurs numerous times during the development of multicellular organisms as they change from a single zygote to a complex system of tissues and cell types .
specifically , adult stem cells divide and create fully - differentiated daughter cells during tissue repair and normal cell turnover .
differentiation dramatically changes the size , shape , membrane potential , metabolic activity , and responsiveness of a cell to signals .  
fgf1 and fgf2 play important roles in the initial differentiation of cochlear ganglion neurons in mice .
fgf7 is essential for the morphogenesis of suprabasal keratinocytes and establishment of the normal program of keratinocyte differentiation .
exogenous fgf20 stimulates the differentiation of monkey stem cells into dopaminergic neurons after treatment in vitro .
angiogenesis is the process of the formation of new blood vessels from pre - existing vessels .
this process plays a key role in various physiological and pathological conditions such as embryonic development , wound repair , inflammation , and tumor growth .
angiogenesis is a multistep process that begins with the degradation of the basement membrane by activated endothelial cells that migrate and proliferate , leading to the formation of solid endothelial cell sprouts into the stromal space .
next , vascular loops are formed and capillary tubes develop with the formation of tight junctions and deposition of new basement membrane .  
numerous inducers of angiogenesis have been identified , including members of the vascular endothelial growth factor ( vegf ) family , angiopoietins , transforming growth factor - alpha and -beta ( tgf - alpha and beta ) , platelet - derived growth factor ( pdgf ) , tumor necrosis factor - alpha ( tnf - alpha ) , interleukins , chemokines , and members of the fibroblast growth factor ( fgf ) family .
however , only a limited number of the 22 members of the fgf family have been investigated for their angiogenic potential in vitro and in vivo .
specifically , fgf1 and fgf2 induce the promotion of endothelial cell proliferation and the physical organization of endothelial cells into tube - like structures .
fgf1 and fgf2 are more potent angiogenic factors than vascular endothelial growth factor ( vegf ) or platelet - derived growth factor ( pdgf ) .
due to their specific biological functions and roles , fgfs have the potential for application to induce the regeneration of a wide spectrum of tissues , including skin , blood vessel , muscle , adipose , tendon / ligament , cartilage , bone , tooth , and nerve tissues .
indeed , many previous studies have evaluated the administration of fgfs directly to the sites of wounds , similar to that of other growth factors . however , when free fgf solutions are injected in vivo , they rapidly lose their biological functional activity , primarily due to diffusional loss and/or enzymatic inactivation / degradation [ 79 ] . therefore , to gain satisfactory performance , a large amount of fgfs with a continuous dose for the determined period is required .  
conversely , when fgfs are adsorbed onto or encapsulated within materials , their degradation risk can be largely protected while securing the biological activity .
therefore , to make full use of the fgfs , it is essential to develop appropriate materials and substrates to contain and deliver them to defective regions , after which allowing their release at a controllable and sustainable rate .
a wide range of biomaterials including synthetic and natural polymers and even tissue matrices have been studied as candidate materials to carry fgfs and elicit their therapeutic efficacy in vitro and/or in vivo .  
while their functional activity has been demonstrated in different types of cells in vitro and/or target tissues in vivo , there have been relatively few , if any , reports addressing the delivery mechanism . in this part , we review the applications of fgfs in concert with medical materials for tissue regeneration .
specific targets include skin , cartilage , bone , blood vessel , muscle , tendon / ligament , and nerve .
the materials are developed to specifically conjugate with fgfs or encapsulate within the structure and are engineered in the form of hydrogels or porous scaffolds or nano- and microparticulates .   
generally , the most common scaffold materials range from polymers ( synthetic or natural ) and ceramics to their composites , which can be chosen depending on the target tissues of concern .
many natural polymers such as collagen , alginate , fibrin , silk , chitosan , and glycosaminoglycans ( gags ) are biologically well defined , tissue compatible , and degradable ; therefore , they are regarded as feasible materials for the intake of growth factors within the structures .
such polymers are easily engineered into porous scaffolds by dissolution in water - based solutions and subsequent freeze - drying [ 63 , 64 ] . because they dissolve well in water ,
when incorporated within the structure , growth factors are released through the scaffold when they come into contact with the fluid . in most cases , bulk diffusion is the dominant method of releasing growth factors , which is led by water permeation and can be accelerated by collapse of the polymer network .  
due to their comprising a class of proteins or polysaccharides , natural polymers contain a large number of ionic groups .
apart from chitosan , which is highly positively charged , all of the other proteins mentioned above preserve a large number of negatively charged groups . therefore , depending on their charge characteristics ( basic or acidic ) , fgfs can form charge - charge interactions with natural polymers . in such cases ,
the fgf - incorporated scaffolds show sustained and long - term delivery of fgfs if their structure is maintained without collapse [ 65 , 66 ] .
more specific biochemical interactions between the fgfs and natural proteins are favored in maintenance of the stability of fgfs , which can be exploited by utilizing some binding sites of fgfs , such as the heparin binding site [ 8 , 67 , 68 ] .
collagen scaffolds mixed with heparin hold fgf2 within the structure better than scaffolds without heparin , which is beneficial for long - term fgf delivery .
hydrogel - type scaffolds that absorb a high level of water within their pore structure and are mechanically meta - stable gel matrices will be discussed separately in the following section .  
synthetic polymers primarily those that are degradable , such as poly(lactic acid ) , poly(glycolic acid ) , poly(caprolactone ) , and their copolymers have also been well developed into porous scaffolds by many possible processing routes . unlike natural polymers ,
this class of polymers can be dissolved in nonaqueous organic solvents that would otherwise melt at elevated temperatures , and most growth factors , including fgfs , are very susceptible to degradation when incorporated during the scaffold processing stages .
therefore , surface modification of the scaffolds is recommended to carry the growth factors . through chemical treatment of the surface of synthetic polymers such as amination or carboxylation
, growth factors can be covalently coupled via the formation of an amide linkage [ 70 , 71 ] .
however , in such cases , growth factors are only on the scaffold surface , which limits the continual biological action of fgfs and long - term targeting while influencing the initial adhesive reactions of cells .   apart from the polymer groups , bioceramics are promising candidate materials for grafting defects of hard tissues , such as bones and teeth [ 7279 ] . many publications and clinical trials have already demonstrated the high performance of some bioceramics including calcium phosphates and bioactive glasses / glass ceramics [ 73 , 77 , 79 ] .
therefore , for hard tissue regeneration , there is a potential need to use bioceramics in concert with growth factors including fgfs , leading to some pioneering studies on the incorporation of fgfs within bone grafts [ 80 , 81 ] .
however , most currently applicable bioceramics can only be obtained following heat treatment ( generally above a thousand degree ) , which limits the direct loading of growth factors within the scaffolds .
nevertheless , some unique properties such as granular morphology ( grain boundaries ) , surface charges , crystallography , and micropores are known to significantly alter the adsorption of proteins and their release behaviors [ 8285 ] , which suggests the possible manipulation of fgfs on bioceramic scaffolds that is different from the case in polymers .  
one promising form of bioceramics is self - setting or hardening cements , such as calcium phosphate cements that quickly harden when they come in contact with water - based solutions and are easily moldable and applicable in an injectable type [ 78 , 115 ] .
while their applications in hard tissue reconstruction have a long history , the need for using this class of material for drug delivery including growth factors has emerged recently .
fgf - incorporating cements that target bones and teeth are considered to hold great promise ; however , additional studies evaluating their use are necessary .
natural polymers such as collagen , gelatin , fibrin , and glycosaminoglycans are the most commonly used hydrogels for tissue regeneration and drug delivery .
these hydrogels largely mimic the native extracellular matrices ( ecms ) of tissues , and cells recognize the hydrogel molecules in a manner similar to the recognition that occurs under in vivo conditions .
when fgfs are loaded within the hydrogels of natural polymers , they play a role in controlling cell processes , such as cell division , migration , and differentiation . because the growth factors easily bind to ecm components such as heparan sulfate proteoglycans and fibronectin [ 118 , 119 ] , these compounds are often combined with hydrogel matrices .
the bound growth factors are secured within the hydrogel matrices until released by an enzymatic reaction or hydrolytic cleavage .  
collagen hydrogels form a fibrillar network , which is also degradable enzymatically through the action of collagenase .
however , recent studies have reported the recombinant fusion of growth factors into well - known collagen binding sequences such as the sequence from collagenase , von willerbrand factor , or fibronectin .
gelatin is a denatured form of collagen that has been investigated as a good candidate carrier of fgfs .
depending on the charge characteristics ( acidic or basic ) and biodegradability of gelatin , the release profile of fgfs can vary greatly . in the case of fgf2 ,
fibrin hydrogel can be formed through the spontaneous polymerization of fibrinogen in the presence of thrombin protease .
the fgf2 noncovalently bound to the fibrin network was able to provide growth factor - specific bioactivity , such as enhancing endothelial cell proliferation .
gags are strongly anionic polymer hydrogels that can absorb a large amount of water while preserving good mechanical integrity .
similar to collagen , gags bind growth factors noncovalently but very stably and then further release them through the enzymatic cleavage reaction .
the structure of gags is often modified to provide sites of covalent binding for biomecules , such as adhesive proteins and growth factors .  
synthetic hydrogel polymers can be formed with various compositions , including poly(ethylene glycol ) ( peg ) , poly(vinyl alcohol ) ( pva ) , and poly(hydroxyethyl methacrylate ) ( phema ) . during preparation of the synthetic hydrogels , various target binding domains or cleavage domains such as mmp - cleavable sequences and growth factor
binding ligands can be introduced , and these ultimately play a role in covalently linking growth factors such as fgfs and further allowing cleavage of the network to release fgfs . when compared to the natural polymer hydrogels , which have limited properties to control , the development of synthetic polymer hydrogels to retain the functional and some cell / tissue responsive properties are now increasing rapidly .
indeed , cell responsive functional groups can be tuned to deliver specific molecules including fgfs , and the mechanical and degradation properties can be manipulated to be responsive to surrounding conditions , such as ph , temperature , light , and mechanical load [ 7 , 128 ] . as one example , a peg hydrogel system was engineered to be crosslinked through a photoreaction , during which time physical properties such as the degree of swelling could be modulated .
the release of fgf2 from the hydrogel was highly dependent on the crosslinking density , which was mediated by the swelling ability .   when compared to the structured porous scaffolds or hydrogels , particulate forms with sizes at the nano- ( tens to hundreds of nanometers ) and micron - scale ( a few micrometers ) have gained interest for specialized delivery of growth factors , requiring a system to be delivered through the blood stream and oral administration , and within intracellular compartments [ 129132 ] .
many different formulations of natural and synthetic polymeric materials for this purpose have been reported , and these primarily include liposomes , micelles , dendrimers , microspheres , nanospheres , and nanoshells .
the common process in production of those particulates requires the formation of droplets within solutions that were conditioned to disperse individual particles while preserving the morphological ( spherical ) integrity .
the size and composition of the particulates are easily controllable , which ultimately determines the release profile of growth factors .  
as described in the previous section , natural polymers such as gelatin and collagen are possible candidates for in situ encapsulation of fgfs during the formation of particulates , where the charge interaction of natural polymers with fgfs must be considered .
similarly , synthetic polymers can be exploited in the form of nanoshells ( or nanocapsules ) , in which hydrophobic polymers comprise an outer shell that surrounds an inner water - based portion that contains hydrophilic growth factors [ 89 , 133 ] . depending on the polymer composition and outer shell thickness , the release profile of growth factors can be controlled . due to their sizes being far greater or less than one hundred nanometers ,
dna designed to encode fgfs can be encapsulated within the nanocapsules to form a complex and then further transfected to the target cells to elicit the biological functionality of fgfs .   when compared to the polymeric compositions , inorganic particulates for growth factor delivery have been relatively less studied .
recent attention has been given to the mesoporous silica nanoparticles , which have a large amount of mesopores with sizes of about 210  nm [ 136 , 137 ] .
growth factors can be entrapped within the mesopores of the particles and then delivered into the target tissues or cells .
one of the widely available inorganic materials for gene delivery is calcium phosphate ( cap ) nanoparticles because the calcium ion can easily bind with negatively charged nucleic acids , which then forms a cap - dna complex .
intracellular delivery of genes that encode fgfs can be implemented using inorganic nanoparticulate delivery systems [ 138 , 139 ] .  
while nano- and microparticulates are easily manipulated to take up growth factors and can be safely implemented into defect sites , the scaffolds and hydrogels can provide 3d matrices for cells to adhere , migrate , populate , and differentiate .
therefore , particulates carrying growth factors are often embedded within the matrices to act as cell scaffolding and produce therapeutic effects [ 139 , 140 ] , which is an effective method of fully utilizing the roles and functions of fgfs in cells and tissues for regenerative therapy and tissue engineering .
fgfs have the biological activity of stimulating the proliferation of fibroblasts and angiogenesis , which facilitates potential use in skin wound healing . both fgf1 and fgf2
are known to be highly released by damaged endothelial cells and macrophages at wound sites , and if fgf2 activity is blocked , wound angiogenesis is almost completely impaired .
fgf7 and fgf10 play a role in stimulation of the migration and proliferation of keratinocytes .  
however , owing to the short half - like of free fgfs , the use of delivery systems has been proposed . among the fgfs
, application studies of wound healing and skin regeneration have primarily been conducted on fgf2 . because acidic gelatin is highly negatively charged , it can hold fgf2 well by forming an ionic complex with gelatin . the 2  mg
freeze - dried gelatin microspheres were soaked in 20  l aqueous solution of fgf2 ( 10  mg / ml with an isoelectric point 9.6 ) .
while the free - fgf2 administered in vivo ( 10 , 50 , and 100  g ) to guinea pigs with full - thickness skin defect could not induce sufficient dermal wound healing , gelatin microspheres incorporated with fgf2 greatly accelerated dermal tissue regeneration . using a synthetic hydrogel of chitosan , which is photocrosslinkable , fgf2-incorporation and sustained release
the fgf2-incorporated hydrogel was photocrosslinked and injected into the skin wound of healing - impaired diabetic ( db / db ) mice .
the involvement of fgf2 within chitosan hydrogel was shown to greatly improve wound closure in terms of granulation tissue formation , capillary formation , and epithelialization of the wounds .
the angiogenesis and blood vessel formation induced by the treatment of fgfs have been relatively well studied .
the most commonly used carrier systems for fgfs are natural polymers including gelatin , which can be prepared to have different charge statuses ( negative for acidic and positive for basic gelatin ) .
tabata et al . prepared gelatin hydrogels with different water contents and charge statuses and then incorporated them with fgf2 . when the complex was implanted subcutaneously into mice , the most significant neovascularization was induced in the fgf2-incorporating acidic gelatin with a low water content .
this was largely due to the effect of released fgf2 , which was manipulated to be sustained and controlled from the gelatin hydrogel .  
a sustained release of fgf2 and its stimulation of human umbilical vein endothelial cell ( huvec ) growth were reported using the heparinized collagen matrix , where the heparin was first immobilized to collagen , and fgf2 was then bound to the heparinized collagen because the fgf2 has a heparin - binding domain .
the heparin was also immobilized onto synthetic polymer plga scaffold to allow a high affinity to fgf2 .
the fgf2 loading was greatly enhanced , and the release from the heparinized - plga was controllable . when subcutaneously implanted in vivo , the fgf2-loaded scaffold effectively induced blood vessel formation .  
synthetic polymer plga microspheres were also used to incorporate fgf2 and its sustained - release , which was then combined with alginate porous scaffold . when implanted in the rat peritoneum , the fgf2-plga administered group showed accelerated vascularization , with a 4-fold increase in penetrating capillaries when compared to the fgf2-free group occurring .
muscle regeneration has also been greatly controlled by the fgfs which are abundant in regenerating areas of muscles [ 144 , 145 ] .
fgf6 is of particular interest because it is muscle specific and highly upregulated during muscle regeneration .
fgf2 was demonstrated to promote the recruitment of skeletal muscle satellite cells using a single myofiber culture model .
doukas et al . used a gene delivery system that encodes fgf2 and fgf6 for the repair of skeletal muscle .
specifically , plasmid and adenovirus vectors were immobilized in a collagen - gelatin mixture that was then delivered to muscle wounds .
they found early muscle angiogenic response and subsequent arteriogenesis , and muscle repair was also greatly enhanced showing regenerating myotubes with specific markers expressions .
although there have been some controversies regarding the critical role of fgfs in muscle repair , it is largely accepted that their roles in muscle regeneration are closely related to the revascularization process .
kawaguchi et al . reported the induction of de novo adipogenesis in mouse subcutis in response to the injection of fgf2-matrigel mixture , which is associated with vascular formation .
additionally , matrigel matrix containing gelatin microspheres incorporated with fgf2 was used for the controlled release of fgf2 and induction of adipogenesis . at 6 weeks after subcutaneous implantation in mice , the fgf2-incorporating group showed significantly higher formation of adipose tissue accompanied with angiogenesis when compared to that treated with free fgf2 .
the adipogenesis was dose dependent ( 0.01 , 0.1 , and 1  g fgf2 ) and the best result was obtained in the 0.1  g fgf2-incorporated group , suggesting that the use of polymer carrier incorporating an appropriate level of fgf2 provides a tool for adipose tissue engineering .
gelatin microspheres incorporating fgf2 were also used in other studies of adipose tissue regeneration [ 93 , 94 ] .
kimura et al . showed that fgf2-incorporated gelatin microspheres enabled preadipocytes to differentiate adipose tissue formation .
they further showed that collagen scaffold containing gelatin - fgf2 microspheres stimulated adipogenesis in a rabbit fat defect in response to the treatment of fgf2-complex scaffold .   following injury ( ~during one week ) of tendons and ligaments
, the level of fgf2 and its receptors has been shown to increase in vivo , with fgf2 playing a significant role in the recruitment of progenitor cell differentiation and the repair process [ 150 , 151 ] .
for applications of human bone marrow stem cells ( hbmscs ) in the repair of tendons and ligaments , the effects of fgf2 on their proliferation and differentiation were investigated .
specifically , when the fgf2 concentration was low ( 3  ng / ml ) , the action was positive in terms of triggering both cell proliferation and expression of genes related to tendon and ligament tissue .
however , treatment with a high dose ( 30  ng / ml ) did not show any beneficial effects on the hbmscs .
targeting anterior cruciate ligament ( acl ) , fgf2 was incorporated into a gelatin hydrogel and then mixed with pla woven fabric . following implantation of fgf2-combined materials wrapped with a collagen membrane in the tibia and femur of rabbit , acl and bone
one recent study showed the possibility of tendon tissue engineering using bmscs and a delivery system encapsulating fgf2 within electrospun nanofiber that was combined with silk microfiber fabric .
the behavior of bmscs , including their proliferation and tendogenic differentiation , was significantly promoted upon fgf2-loaded fibrous scaffolds .
while the differentiated traits of chondrocytes are generally lost during expansion in monolayer culture , chondrocytes treated with 5  ng / ml fgf2 within the poly(glycolic acid ) fibrous scaffold and further expanded ( up to 2000 fold ) were shown to redifferentiate to recover the chondrocytes phenotypes .
when fgf2 was impregnated within collagen sponge ( soaked in 80  g / ml pbs for 24  h at 4c ) and implanted subcutaneously in nude mice , the cartilage regeneration was remarkably accelerated with cartilage tissues that were immature at 2 weeks and almost mature at 4 weeks . with regard to the mesenchymal stem cells ( mscs ) ,
fgf2 have been shown to modulate cell growth and maintain the undifferentiated state of stem cells , facilitating the expansion of stem cells . moreover , its regulation of chondrogenic differentiation of bone marrow stem cells ( bmscs ) has been demonstrated .
chiou et al . also reported that the treatment of fgf2 in either bmscs or adipose stem cells ( ascs ) significantly enhanced chondrogenesis , where cell proliferation was shown to increase dose dependently .
specifically , they found that fgf2 at lower concentration ( 10  ng / ml ) enhanced chondrogenic differentiation , while the effect was negated at higher concentration ( 50  ng / ml ) , suggesting that the use of fgf2 at an appropriate dose is beneficial for cartilage repair .   to induce slow release of fgf2 from the scaffold , fgf2
while free fgf2 showed a rapid in vivo clearance ( ~3% remained after 24  h ) , the level of gelatin - impregnated fgf2 remained approximately 44% and 18% at days 3 and 14 , respectively , suggesting that the gelatin held the fgf2 effectively .
the fgf2-containing gelatin was preadministrated in an ear - shaped polymer scaffold that also contained chondrocytes for 1 week . when the tissue - engineered construct was implanted subcutaneously in mice ,
significantly improved chondrogenic and neovascularization traits were observed , suggesting the importance of sustained fgf2 release in cartilage tissue engineering . to augment the repair of tracheal stenosis , an animal study in rabbit
the results demonstrated greatly enhanced chondrogenesis with cartilage accumulation in the engineered tracheal wall at three months after implantation .
the combinatory approach of fgf2 with polymer scaffold was also conducted by ma et al .
, who coated the pla scaffold surface with collagen and fgf2 . to accomplish this ,
the pla surface was first chemically treated to covalently graft collagen molecules and then physically coated with collagen solution containing fgf2 .
cultured bmscs derived from rats within a hyaluronate - based polymer scaffold with or without fgf2 .
they found that the presence of fgf2 strongly enhanced the expression of osteogenic markers and mineralization , demonstrating a possible role in bone regeneration .
tabata et al . studied the role of fgf2 in a rabbit skull defect model .
gelatin hydrogel was also used to incorporate fgf2 ( 100  g ) to form a polyionic complex and function as an effective carrier . at 12 weeks
, the implants showed dramatic improvement in defect closure , bone mineral density , and bone regeneration in groups treated with varying doses ( 2 to 200  g ) of fgf2-gelatin when compared to an untreated group .  
the improvement of bone cell proliferation and differentiation by the fgf2 was also observed in an experiment using hydroxyapatite ( ha ) porous granules incorporating fgf2 .
the fgf2 administrated at 0.25  m to 100  mg of ha granules was shown to remain at approximately 80% after release for 3 days .
specifically , the osteoblastic differentiation of cells ( mc3t3-e1 ) such as alkaline phosphatase ( alp ) activity and mrna levels of bone - related genes ( osteocalcin , collagen i and alp ) were shown to be significantly up - regulated by the fgf2 treatment , suggesting that ha also may preserve the biological activity of fgf2 .
a recent study also showed that the in vivo bone formation in rats greatly improved in response to the use of an appropriate carrier of fgf2 .
collagen was made into a hybrid membrane with nanobioactive glass , which was impregnated with fgf2 ( 100  g ) and then implanted within a rat calvarium defect .
the nanobioactive glass - amended hybrid scaffold greatly enhanced the defect closure and bone formation , and the fgf2-treated group displayed further improvements , showing the synergistic effect of fgf2 with a bioactive inorganic component .  
the use of fgf2 in dental implants has also been shown to influence the formation of bone around ti - based metals .
fgf2 suspended in matrigel was administrated to the surface of ti and then implanted in ovariectomized rats .
the matrigel used was shown to prolong the life span of fgf2 upon sustained release for up to 21 days in vitro .
the implant samples after three months were found to induce great enhancement in new - bone formation ( 2-fold ) and mechanical stability ( 3-fold ) on the matrigel - fgf2-treated group when compared to groups treated with fgf2 or matrigel alone .  
the treatment of osteochondral complex tissue was also investigated in vivo using a composite scaffold made of ha / collagen incorporating fgf2 .
either 0.5  g ( 50  l from 10  g / ml ) or 5  g ( 50  l from 100  g / ml ) fgf2 was impregnated within the ha / collagen scaffold and then implanted into the osteochondral defect in a rabbit femoral trochlear groove of the knee . during the periods of implantation through 3 to 24 weeks , the 0.5  g fgf2-treated scaffold group displayed greatly enhanced bone regeneration and satisfactory cartilage regeneration , suggesting that the ha / collagen composite is a good candidate for delivering fgf2 during the regeneration of osteochondral defect .   while there has been some consensus regarding improvement of the proliferative potential of osteoblasts ,
some adverse effects of fgf2 on the osteogenic differentiation and mineralization have also been identified .
investigated the influence of different fgfs ( fgf2 , fgf4 , and fgf6 ) on the behavior of human primary osteoblasts .
they demonstrated that all fgfs treated in culture medium at 0.7  m induced osteoblast proliferation but inhibited alp activity and mineralization .
however , when vitamin d was co - administrated with fgfs , the alp and mineralization were greatly enhanced , suggesting the combinatory use of fgfs with other mineralizing agents for bone induction and bone tissue engineering .
coadministration of fgf2 with melatonin to the ti implants showed promotion of osseointegration during 4 weeks of implantation in rat tibia .  
apart from the use of fgf2 , which has been the most widely studied , fgf1 was also shown to stimulate angiogenesis as well as osteogenesis in vitro .
fgf1 was entrapped within the ha - fibrin composite scaffold , which was further implanted in rats subcutaneously . at 2 and 4
weeks postimplantation , the fgf1-containing scaffolds was found to stimulate angiogenesis with blood vessel formation and the expression of osteogenic markers ( osteopontin and osteocalcin ) , suggesting that ha - fibrin scaffold incorporating fgf1 is effective for bone repair .
additionally , periodontal ligament tissue has been shown to be regenerated by the action of fgf2 contained in a gelatin carrier .
tan et al . reported that a periodontal defect in dogs could be significantly regenerated by treatment with mscs that were transfected with fgf2 . at eight weeks after implantation in nonhuman primates , the fgf2-gelatin group showed significant regeneration of periodontal tissues in a dose - dependent manner .
shirakata et al . demonstrated that treatment with fgf2 induced the promotion of periodontal healing in two - wall intrabony defects in dogs , suggesting that they were a possible candidate for replacement of the established benchmarks enamel matrix derivative or platelet - derived growth factor combined tricalcium phosphate .  
as one of the neurotrophic factors , fgfs have been shown to enhance the in vitro survival and neurite extension of various types of neurons as well as in vivo wound healing and neuronal functions .  
fgf2 is known to exist at the blood - brain barrier in matrix - bound and soluble form , and it is produced by astrocytes and has autocrine effects on astrocytes proliferation and stellation .
when compared to the soluble form , fgf2 bound to matrices has improved half life , and its effects on astrocytes can be potentiated [ 160 , 161 ] .
covalently linked fgf2 onto a nanofiber polymer scaffold and observed significant autocrine expression of fgf2 and the modulation of astrocytes - neuron interactions , suggesting the utility of the system in nerve injury and disease .   for the repair of spinal cord injury ( sci ) , marquet et al . developed a porous pla modified with copolymer and combined with fibrin glue containing fgf2 and found that the scaffold allowed cell migration and angiogenesis in the transected spinal cord of rats , suggesting the potential for the use of the system for sci , although more clear examination and functional studies must be conducted to confirm these findings . due to the difficulty of the proteins in penetrating the blood - spinal cord barrier , a local delivery system consisting of poly(ethylene glycol)-modified fgf2 and using an intrathecal delivery has recently been developed by kang et al . .
also developed a polymer nanocomposite hydrogel fgf1 that has also been shown to have neuroprotective functions in the repair of sci .
tsai et al . demonstrated that rat sci treated with fgf1 showed significant functional recovery .
they also used a synthetic polymer and hydrogel of fibrin or collagen to carry growth factors such as fgf1 and nt-3 , and showed that fgf1 involvement significantly improved the axonal regeneration of vestibular neurons .  
for the peripheral nerve regeneration , fgf2 was loaded within a polymer tube channel and implanted in the 15 mm gap of the sciatic nerve .
the results revealed the in vitro sustained release of fgf2 with biological activity , and in vivo regeneration of nerve cables bridging nerve stumps .
similar to other tissue applications , anionic gelatin was also used to embed fgf2 and induce sustainable delivery to the intratemporal facial nerve .
the results at six weeks after implantation demonstrated that facial nerve functions such as facial movements , electrophysiology and histological morphology , were greatly improved .
fibroblast growth factors ( fgfs ) that signal through fgf receptors ( fgfrs ) regulate a wide range of biological functions , including cell proliferation , survival , migration , and differentiation . among the signal pathways ,
ras / map kinase is known to be predominant in the case of fgfs . while the biological functions of fgfs are largely implicated in many types of cells in vitro through this signaling pathway
biomaterial - based systems , including delivery carriers of fgfs and scaffolds of stem cells regulated by the fgfs functions , have recently been potentially developed and shown to have many good results in vivo .
future clinical applications of fgfs in the regeneration of tissues , including skin , muscle , tendon / ligament , bone , tooth , and nerve tissues will be realized when their biological functions are maximized by the appropriate use of biomaterials and stem cells .
 fibroblast growth factors ( fgfs ) that signal through fgf receptors ( fgfrs ) regulate a broad spectrum of biological functions , including cellular proliferation , survival , migration , and differentiation . 
 the fgf signal pathways are the ras / map kinase pathway , pi3 kinase / akt pathway , and plc pathway , among which the ras / map kinase pathway is known to be predominant . 
 several studies have recently implicated the in vitro biological functions of fgfs for tissue regeneration . however , to obtain optimal outcomes in vivo , it is important to enhance the half - life of fgfs and their biological stability . 
 future applications of fgfs are expected when the biological functions of fgfs are potentiated through the appropriate use of delivery systems and scaffolds . 
 this review will introduce the biology and cellular functions of fgfs and deal with the biomaterials based delivery systems and their current applications for the regeneration of tissues , including skin , blood vessel , muscle , adipose , tendon / ligament , cartilage , bone , tooth , and nerve tissues . 
1. Introduction
2. Biology of FGF
3. FGF Signaling
4. Biological Functions of FGFs
5. Delivery Systems for FGFs
6. Tissue-Specific Applications
7. Concluding Remarks