for the purposes of promoting an understanding of the principles of the present disclosure , reference will now be made to the embodiments illustrated in the drawings , and specific language will be used to describe the same . it will nevertheless be understood that no limitation of the scope of this disclosure is thereby intended . while the concepts of the present disclosure are illustrated and described in detail in the figures and the description herein , results in the figures and their description are to be considered as exemplary and not restrictive in character . it is understood that only the illustrative embodiments are shown and described and that all changes and modifications that come within the spirit of the disclosure are desired to be protected . unless defined otherwise , the scientific and technology nomenclatures have the same meaning as commonly understood by a person in the ordinary skill in the art pertaining to this disclosure . as disclosed herein , in at least one embodiment , a gag - based polymeric system can be prepared by following two strategies : ( i ) chemical covalent bonding and ( ii ) physically entrapped and / or entangled ( non - covalent ) strategies as shown in fig1 . in at least one embodiment , gag polysaccharides can be modified in predictable synthetic routes to control the properties of the resulting materials , including modifications leading to hydrophobicity and biological activities . in part , the present disclosure provides for a composition comprising at least one monomeric unit of ha functionalized by at least one functional group moiety . chemical modifications of ha can be targeted to three functional groups : the glucuronic acid carboxylic acid , the primary and secondary hydroxyl groups , and the n - acetyl group ( following deamidation ). in some embodiments , compositions of ha in the present disclosure are provided that may be represent by formula i , ii , iii and iv ( fig2 ). carboxylates in a ha backbond can be modified by carbodiimide - mediated reactions , esterification , and amidation . hydroxyls in a ha backbond can be modified by etherification , divinylsulfone crosslinking , esterification , and bisepoxide crosslinking . additionally , converting diols to aldehydes can be achieved through periodate oxidation of ha . finally , deacetylation of the n - acetyl group of ha recovers an amino group which can then react with an acid using the same amidation . the functional groups r 1 , r 2 , r 3 , r 4 , and r 5 may include any one of or a combination of haloacetates , dihydrazides , amines , thiols , carboxylic acids , aldehydes , ketones , active hydrogen sites on aromatic ring , dienes , azide isothiocyanates , isocyanates , acyl azides , n - hydroxysuccinimide ( nhs ) esters , sulfo - nhs , sulfonyl chloride , epoxides , carbonates , aryl halides , imidoesters , carbodiimides ( e . g . n , n ′- dicyclohexylcarbodiimide ( dcc ) and 1 - ethyl - 3 -( 3 - dimethylaminopropyl ) carbodiimide ( edc )), alkylphosphate compounds , anhydrides , fluorophenyl esters , hydroxymethyl phosphines , guanidino groups , iodoacetyl derivatives , maleimides , aziridines , acryloyl derivatives , arylating agents , disulfide derivatives , vinylsulfone , phenylthioester , cisplatins , diazoacetates , carbonyl diimidazoles , oxiranes , n , n ′- disuccinimidyl carbonates , n - hydroxylsuccinimidyl chloroformates , alkyl halogens , hydrazines , alkynes , and phosphorus - bound chlorine . the functionalized polysaccharides may include the combination of ha and cs . ha may be used in an amount ranging from 0 . 1 % to 99 % by weight . ha has an average molecular weight in the range of 10 - 3 , 000 kilo daltons , preferably 1000 - 3000 kilo daltons . sulfated gags may be used in an amount ranging from 0 . 1 % to 99 % by weight . cross - linkers can be both synthetic polymers and natural polymers . the natural polymers may include any one of or a combination of fibrin , collagen , matrigel , elastin , elastin - like peptides , albumin , natural poly ( amino acids ) ( e . g . cyanophycin , poly ( lysine ), and poly ( γ - glutamic acid )), polysaccharides ( e . g . chitosan , dextran , chondroitin sulfate , agarose , alginate , methylcellulose , and heparin ), α - cyclodextrin ( cd ), β - cd , γ - cd , and blends thereof . synthetic polymers may include any one of or a combination of poly ( aliphatic ester ) ( e . g . poly ( lactide ) ( pla )), poly ( ε - caprolactone ) ( pcl ), poly ( glycolic acid ) ( pga ), poly ( lactic - co - glycolic acid ) ( plga ), poly ( trimethylene carbonate ) ( ptmc ), polydioxanone ( pds ), poly ( ortho ester ), polyanhydrides , poly ( anhydride - co - imide ), poly ( anhydride - esters ), polyurethanes ( e . g . degrapols ), poly ( amides ), poly ( esteramides ), poly ( orthoesters ), poly ( dioxanones ), poly ( acetals ), poly ( ketals ), poly ( carbonates ), poly ( orthocarbonates ), poly ( hydroxylbutyrates ), poly ( hydroxyl - valerats ), poly ( alkylene oxalates ), poly ( alkylene succunates ), poly ( malic acid ), poly ( amino acids ), poly ( vinylpyrolidone ), poly ( hydroxycellulose ), poly ( glycerol sebacate ), poly ( ethylene imine ), poly ( acrylic acid )( paa ), poly ( n , n ′- diethylaminoethyl methacrylate ) ( pdeaema ), polyethylene glycol ( peg ), poly ( propylene oxide ) ( ppo ), peo - b - ppo block copolymers ( e . g . pluronics or poloxamers , and tetronic ), poly ( vinyl alcohol ) ( pva ), poly ( n - isopropylacrylamide ) ( pnipam ), poly ( n , n - diethylacrylamide ) ( pdeaam ), poly ( oxazolines ) ( e . g . poly ( 2 - methyloxazoline and poly ( 2 - ethyl - 2 - oxazoline )), oligo ( poly ( ethylene glycol ) fumarates ), poly ( propylene fumarate ), poly ( alkyl cyanoacrylates ), poly ( acrylic amide ), synthetic poly ( amino acids ) ( e . g . poly ( l - glutamic acid ) ( l - pga ) and poly ( aspartic acid )), poly ( phosphazenes ), poly ( phosphoesters ), and blends thereof . a cross - linker may include a homo - or hetero - functional modifier of the following formula : a - cross - linker - z in some embodiment , one of a and z is a moiety selected from the group consisting of hydroxyls , thiols , aminos , alkyls , alkenyls , alkoxysulfonate , arylsulfonate , heteroarylsulfonate , azides , maleimido , propargyl , haloacetate , dihydrazide , amines , carboxylic acids , aldehydes , ketones , active hydrogen sites on aromatic ring , dienes , azide isothiocyanates , isocyanates , acyl azides , ether nhs esters , sulfo - nhs , pentafluorophenyl ( pfp ), azlactones , sulfonyl chloride , epoxides , carbonates , aryl halides , imidoesters , biotin , carbodiimides ( e . g . dcc and edc ), alkylphosphate compounds , anhydrides , fluorophenyl esters , hydroxymethyl phosphine , guanidino groups , iodoacetyl derivatives , maleimides , aziridines , acryloyl derivatives , arylating agents , disulfide derivatives , vinylsulfone , phenylthioester , cisplatin , diazoacetate , carbonyl diimidazole , oxiranes , n , n ′- disuccinimidyl carbonate , n - hydroxylsuccinimidyl chloroformate , alkyl halogens , hydrazine , maleimide , alkyne , and phosphorus - bound chlorine . structures of a cross - linker may be any one of or a combination of linear , dendrimers - like , star - shaped , hyper - branched , combed , brushed , cross - linked architectures , fibers , microspheres , and nanoparticles . examples are shown in fig4 . the number of arms in star - shaped polymers may be two or more . fibers may comprise any one of or a combination of polymer fibers , carbon fibers , and ceramic fibers . microspheres and nanoparticles may comprise any one of or a combination of polymer micro / nanospheres , iron oxide , silica , gold , and mesoporous silica nanoparticles . a cross - linker with homo - or hetero - functional groups can be prepared by addition or chain growth polymerizations , coordination polymerizations , and condensation or step growth polymerizations . addition or chain growth polymerizations include free radical polymerization , controlled - living radical polymerization ( e . g . atom transfer radical polymerization ( atrp ), reversible addition fragmentation transfer ( raft ) polymerization , and nitroxide - mediated radical polymerization ( nmp )), cationic polymerizations , anionic polymerizations and the like . the conjugation of functionalized ha and cross linkers can be achieved by any one of or the combination of the following reactions : carbodiimide - mediated reactions , esterification , amidation , aldehyde and ketone reactions , active hydrogen reactions , photo - chemical reaction , azide - alkyne cycloaddition ( e . g . copper - catalyzed azide - alkyne cycloaddition ( cuaac ), copper - free azide - alkyne huisgen cycloaddition or strain - promoted azide - alkyne cycloaddition ( spacc )), thiol - based click reaction ( thiol - yne , thiol - ene , thiol - isocyanate , thiol - michael addition ), diels - alder reactions , tetrazole cycloaddition , nitrile oxide cycloaddition , oxime / hydrazone formation , enzymatic crosslinking , and coordination chemistry , and ligand exchange reactions . examples are shown in fig3 . the conjugated linkages may include any one of or a combination of isothiourea , isourea , amide , sulfonamide , secondary amine , sulfonamide , shift - base , secondary amino - methyl , carbamate , aryl amine , amidine , amide , phosphoramidate , guanidine , substituted imidocarbonate , thioether , aryl thioether , disulfide , sulfonate , thiosulfonyl , ester , ether , carbamate , hydrazone , diazo , triazoles , iodinated compound , carbohydrates , amino acid esters bond , cycloalkene , oxime triazole , and triazoline . physical interactions may include any one of or a combination of hydrophobic interactions , hydrophilic interactions , hydrogen bonding , electrostatic interactions , and van der waal interactions . examples are shown in fig5 . the gag - based system can be used as a cellular matrix for 3d cell culture and tissue engineering as well as a delivery vehicle for therapeutic agents including but not limited to cells , growth factors , and small molecules . cells to be encapsulated within the engineered composite may comprise any one of or the combination of , but not limited to , mesenchymal stem cells , osteoblast , chondrocytes , adipocyte , fibroblast , hepatocytes , enterocytes , urothelial cells , blood cells , skin cells , endothelial cells , nerve cells , sex cells , and cancer cells . example 1 : ha / cs / poly ( ethylene glycol ) diacrylate composite hydrogel as hmscs niches we developed a novel biomimetic hydrogel system as a tunable stem cell niche through the combination of thiolated ha ( ha - sh ) and thiolated cs ( cs — sh ) cross - linked with poly ( ethylene glycol ) diacrylate ( pegda ) as shown in fig6 . the combination of ha and cs offers a cell - friendly microenvironment found in native tissues , whereas the selection of peg is based on its established biocompatibility and chemical versatility . the efficient control of various hydrogel properties is demonstrated by simply varying the molecular weight of peg . a thiol - ene click reaction was selected due to its high efficiency in aqueous media , reliability , lack of by - products , unsubstantial degradation of the polysaccharide backbone , and without the use of any metal catalysts [ 22 ] . to our knowledge , this is the first report that tricopolymers of ha , cs , and peg in the forms of natural / synthetic composite hydrogels were fabricated through thiol - ene click chemistry . we showed the efficient control of various hydrogel properties ( e . g ., swelling , modulus , and gelation time ) by simply varying the peg molecular weight ( mw ). we also demonstrated that the composite hydrogels could support 3d encapsulation of human mscs ( hmscs ) with high viability as well as tunable cell - hydrogel interactions by varying the properties of hydrogels . the ability of fine - tuning hydrogel properties paves the way for further optimization of cellular responses to promote in situ tissue regeneration . hyaluronic acid sodium salt ( ha ) with molecular weight ( mw ) of 2 - 3 million daltons and chondroitin sulfate sodium salt ( cs ) with average mw 10 - 30k daltons were purchased from carbosynth limited ( berkshire , uk ). 1 - ethyl - 3 -( 3 - dimethylaminopropyl ) carbodiimide ( edc ), and polyethylene glycol diacrylate ( pegda ) with mw of 700 , 3400 , and 8000 were purchased from alfa aesar ( ward hill , mass .). pegda700 was passed through a short column containing basic alumina to remove the inhibitor before use . pegda3400 and pegda8000 were precipitated in diethyl ether twice to remove the inhibitor before use . peg divinylsulfone , ( mw 3500 daltons ) and 4 arm peg vinylsulfone ( mw 20000 daltons ), were purchased from jenkem technology usa inc . n - hyroxysuccinimide ( nhs ), cystamine dihydrochloride , 5 , 5 ′- dithiobis ( 2 - nitrobenzoic acid ) ( ellman &# 39 ; s reagent ), dl - dithiotreitol ( dtt ), and 2 -( n - morpholino ) ethanesulfonic acid ( mes ) were purchased from sigma - aldrich ( st . louis , mo .). 10 × phosphate buffer saline ( pbs ) was purchased from fisher bioreagents ( pittsburgh , pa .). penicillin - streptomycin ( pen strep ), ethidium homodimer - 1 ( ethd - 1 ), and calcein am were purchased from life technologies ( carlsbad , calif .). carboxyl groups in ha and cs were functionalized to thiol groups by a simple two - step reaction scheme which is depicted in fig7 . in a typical procedure , ha ( 1 g , 2 . 5 mmol ) was dissolved in 100 ml mes buffer ( 0 . 1 m mes , ph 6 . 0 ) in a 45 ° c . oil bath and allowed to dissolve overnight . edc ( 2 . 4 g , 12 . 5 mmol ) and nhs ( 3 . 9 g , 34 mmol ) were added and allowed to react for 2 h . then cystamine dihydrochloride ( 5 . 65 g , 25 mmol ) was added to the mixture and allowed to react overnight while stirring . the reaction mixture was exhaustively dialyzed ( mwco of 12 - 14 , 000 ) against distilled 0 . 1m nacl for 60 h , 25 % ethanol for 12 h and distilled ( di ) water for 12 h . after dialysis , the product , ha - conjugated cystamine ( ha - s — s — nh 2 ), was lyophilized and kept at in − 20 ° c . ha - conjugated cystamine ( 0 . 25 g ) was dissolved in pbs with a concentration of 5 mg / ml then dtt ( 0 . 75 g , 4 . 8 mmol ) was added to the flask and ph was adjusted to 7 . 4 with 1 m naoh . nacl was added to produce a 5 % w / v solution after 20 h and the modified ha was precipitated in 10 - fold ethanol three times . the precipitation was dissolved in h 2 o at a concentration of approximately 5 mg / ml and the purified product was freeze - dried and kept at − 80 ° c . freezer . in a typical procedure , cs ( 1 g , 2 mmol ) was dissolved in 40 ml mes buffer ( 0 . 1m mes , ph 6 . 0 ). edc ( 3 . 086 g , 16 . 2 mmol ) and nhs ( 2 . 280 g , 19 . 8 mmol ) were added to the flask and allowed to react for 2 h . following the 2 h activation step , the ph was raised to 7 . 2 using 1 m naoh . cystamine dihydrochloride ( 4 . 460 g , 20 mmol ) was subsequently added to the solution and allowed to react for overnight . the cs - conjugated cystamine ( cs — s — s — nh 2 ) was exhaustively dialyzed ( mwco of 12 - 14 , 000 ) against distilled 0 . 1m nacl for 60 h , 25 % ethanol for 12 h and di water for 12 h and then was lyophilized . the cs — s — s — nh 2 was reduced using dtt as the same procedure of reduction of ha . the structure of functionalized - ha or cs were confirmed 1 h nmr spectroscopy ( d 2 o , bruker arx 400 mhz ) and the degree of thiolation was also confirmed with an ellman &# 39 ; s assay using l - cysteine as the standard . the structures of ha and cs - conjugated cystamines were confirmed by proton nuclear magnetic resonance ( 1 h nmr ) spectroscopy , with the degree of modification ( ds ) estimated from integration of methylene protons relative to the n - acetyl methyl protons in fig8 . by varying the molar ratios of ha / edc / nhs , variable ds values ( 4 . 1 - 43 . 1 %) were obtained ( table 1 , which shows the effects of edc concentration on degree of substitution ( ds ) of — nh 2 .). for example , molar ratios of 1 : 5 : 13 . 5 and 1 : 10 : 13 . 5 yielded ha - conjugated cystamines with ds of 27 % and 43 . 1 %, respectively . therefore , cs / edc / nhs molar ratio of 1 : 10 : 13 . 5 was selected for cs modification . subsequently , reduction of ha and cs - conjugated cystamines by dithiotreitol ( dtt ) resulted in free thiol groups . the two - pot reaction allows for efficient control of dtt reduction reactions and minimizes the oxidation of thiols resulting in the disulfide formation and the insolubility of ha or cs . ds of both amine and thiol groups were controlled by simply adapting the ph of reaction mixture or the amount of dtt . the results of ds determined by the ellman &# 39 ; s assay and 1 h nmr were consistent and listed in table 2 , which shows the effects of dtt concentration and ph on ds of — sh . 1 h nmr spectra ( fig9 ) showed the decrease of proton signals from the cystamines disulfide pendants . peak at d is attributed to proton signals of methylenes (— ch 2 ch 2 nh 2 ). peaks at a , b , a ′ and b ′ correspond to resonances of the two side chain methylenes (— ch 2 ch 2 sh ). by altering the ph of reaction mixture , the thiol group ds of cs — sh could be controlled from 17 . 7 % to 36 . 0 % ( table 2 ). the ph at 7 . 2 was selected to offer reactive thiolate and avoid the possible degradation of thiolated ha . by altering the amount of dtt , the thiol group ds of ha - sh with 5 . 2 % and 15 . 3 % respectively were obtained ( table 2 ). for the remaining studies , ha - sh with a thiol group ds of 5 . 3 % was employed because lower substituted ha hydrogels have been shown to display higher cellular bioactivity [ 23 ]. cs — sh with a thiol group ds of 23 . 5 % was employed . moreover , the residual — nh 2 groups allow for additional functionalization of the hydrogels if desired . crosslinking of ha - sh and cs — sh mixtures was achieved using pegda as shown in fig6 . to study the effects of chain length of cross - linker , the mw of pegda was altered to distribute either 700 da , 3400 da , or 8000 da within hydrogel while the molar ratio of thiol groups to acrylate groups was kept at 1 . 07 and all other conditions were fixed ( table 3 , which shows the composition of hcp hydrogels ). hydrogels were formed by simple mixing of ha - sh , cs — sh , and pegda in pbs at 37 ° c . specifically , 1 % ha - sh ( 199 μl ) and 5 % cs — sh ( 217 μl ) were mixed in the microcentrifuge tube . either 5 % pegda 700 ( 26 . 8 μl ), 20 % pegda 3400 ( 33 . 8 μl ), or 33 % pegda 8000 ( 46 μl ), was added to the microcentrifuge tube to create ha / cs / pegda 700 ( hcp700 ), ha / cs / pegda 3400 ( hcp3400 ), and ha / cs / pegda8000 ( hcp8000 ) respectively . hydrogel samples were imaged using an fei nova nanosem field emission scanning electron microscope ( fei company , hillsboro oreg .) using et ( everhart - thornley ) dectector or the high - resolution thorough - the lens ( tld ) detector operating at 5 kv accelerating voltage , spot 3 , ˜ 5 . 0 mm working distance and 30 mm aperture . the composite hydrogels exhibited interconnected porous structures with micron - sized pores as shown in sem micrographs ( fig1 ). generally , hcp700 hydrogels possessed larger pores than hcp3400 and hcp8000 hydrogels presumably due to lower cross - linking density of hydrogels . the time to form a gel ( denoted as gelation time ) is defined as the time when the gel , in an inverted state , shows no fluidity for 1 min [ 24 ] ( fig1 ). the experiment was performed in triplicate . the gelation times decreased with increasing molecular weight of pegda ( p & lt ; 0 . 05 , n = 3 ) ( fig1 a ). hcp700 , hcp3400 and hcp8000 hydrogels exhibited a gelation time of ˜ 30 min , ˜ 18 min , and ˜ 16 min respectively . for swelling tests , hydrogel samples (˜ 0 . 35 ml ) were prepared as described above . the hydrogels were freeze - dried and weighed ( w d ). subsequently , 1 ml of pbs was applied on top of the hydrogels and then the samples were incubated at 37 ° c . for 24 h to reach the swelling equilibrium . the excess pbs was then aspirated away and the remaining saturated hydrogels were weighed ( w s ). the experiments were performed in triplicate and the degree of swelling of the hydrogels was expressed as (( w s − w d )/ w d )× 100 %. all data are presented as mean ± standard deviation . the swelling ratio is another important parameter for hydrogels , which is associated with hydrogel mechanical properties including strength and flexibility . all the composite hydrogels were highly swollen in water resulting from the hydrophilicity of peg , ha and cs molecules , and the maintenance of cross - linked ha / cs / peg networks . as shown in fig1 b , the swelling ratio decreased significantly with the increase of mw of pegda from 700 da to 3400 da ( p & lt ; 0 . 05 , n = 3 ). although not significant , a similar trend was also found while increasing mw of pegda from 3400 da to 8000 da . tricomponent hydrogels ( 350 μl ) with different peg molecular weights ( 700 , 3400 , and 8000 ) were prepared in a syringe with the end cut off . fitc - dextran ( mw = 70k , 20 μg ) was added to each hydrogel precursor solution . the syringe was then placed in a 37 ° c . incubator . the hydrogels were equilibrated for 2 h at 37 ° c . after the two hours the hydrogel was then dispensed into cell culture inserts ( 12 mm diameter with 3 μm pore size ( corning incorporated , usa ) in a 12 well plate . the hydrogels were then submerged with pbs and the well plate was placed into a 37 ° c . water bath . at specified time intervals , 1 ml of the solution from individual wells was withdrawn and replaced with pre - heated water . the amounts of released fitc - dextran were determined by fluorescence measurements ( excitation at 485 nm , emission at 528 nm ). fig1 c shows the percent cumulative release profiles of fitc - dextran from different composite hydrogels as a function of time at 37 ° c . after 58 hours , the fitc - dextran encapsulated in hcp 700 , hcp3400 , and hcp8000 hydrogels resulted in about 84 . 3 %, 77 . 6 %, and 72 . 2 % cumulative release of total fitc - dextran , respectively . the release of fitc - dextran from hcp hydrogels was mainly controlled by the diffusion . the release rate correlated well with the hydrogel pore size as a functional of pegda molecular weight . increase of pegda chain length generates greater crosslinking density resulting in smaller pore size and slower release of fitc - dextran . this observation was consistent with our swelling ratio results . rheological experiments were carried out with a new discovery series hybrid rheometer ( dhr )- 3 ( ta ) using parallel plate ( 20 mm diameter , 0 °) in the oscillatory mode . oscillatory time , frequency , and strain sweeps were performed at 37 ° c ., and the storage ( g ′) and loss ( g ″) moduli were recorded . 450 μl of gel precursor solution was mixed by vortexing at room temperature for 15 s before loading on to the rheometer . hydrogels were cast between the lower peltier plate ( preheated at 37 ° c .) and upper parallel plate . the 20 mm parallel plate geometry was set to a gap of 1000 μm . each hydrogel sample was used for only one test . strain sweeps and frequency sweeps were performed in duplicate and the data represents the average of the two tests . 1 % strain and a frequency of 1 hz were used for the time sweeps , with the same 20 mm parallel plate for 4800 s . time sweep tests were performed in triplicate and the data represents the average of the three tests with corresponding standard deviation . the composite hydrogels showed tunable rheological and mechanical properties by varying the pegda chain length . a strain sweep from 0 . 1 % to 10 % strain was conducted at a frequency of 1 . 0 hz ( chosen arbitrarily ) on a formed gel . fig1 a showed constant storage modulus g ′ values as strain is varied up to 10 % strain , indicating that strains in the range 0 . 1 % to 10 % was in the linear - viscoelastic regime ( lve )[ 25 ]. consequently , a strain of 1 % was selected for the subsequent frequency sweep tests . at low frequencies from 0 . 1 to 10 rad / s , g ′ does not change ( fig1 b ) indicating the solid - like nature of the gel . as a result , a frequency of 1 hz was selected for future tests based on a favorable torque signal at that frequency . a time sweep was performed under the condition that the strain was small enough to be in the lve and the frequency was a value under which the instrument provides a clear and favorable torque signal . therefore , 1 % strain , 1 hz , and 37 ° c . were determined to be the appropriate conditions for the hydrogel time sweep tests . ha / cs / pegda formed a gel and reached stability in ˜ 45 min ( fig1 c ). however , the hydrogels formed rapidly and the crossover of g ′ and g ″ was not observed ( g ″ was too low to measure for the extent of the tests ). as noted in fig1 c , the gels have a solid - like viscoelastic behavior . extensive rheology testing showed that by increasing the mw of the pegda , a significant increase in the hydrogel storage modulus ( p & lt ; 0 . 05 , fig1 d ) was observed , which is in good agreement with the conclusion derived from the sem data . accordingly , hcp700 hydrogels were the weakest with a g ′ value of 410 ± 30 pa , whereas hcp8000 hydrogels were the strongest with a g ′ value of 1810 ± 106 pa . storage modulus of hcp3400 hydrogels was 1300 ± 190 pa , approximately triple that of hcp700 hydrogels . hmscs were obtained from lonza . cells were cultured in growth medium containing dmem ( life technologies , catalog # 11885084 ) supplemented with 10 % fetal bovine serum ( fbs , atlanta biologicals ), 1 % glutamine ( life technologies , catalog # 25030081 ), and 1 % penicillin / streptomycin ( life technologies , catalog # 15140122 ) at 37 ° c . with 5 % co 2 . for the cell encapsulation in the hydrogels , cell pellets were obtained by centrifugation , followed by a brief resuspension with hydrogel solution through pipetting up and down gently to ensure a homogeneous distribution . then cell suspension with the density of 20 , 000 cells / 25 μl was transferred to an inverted pre - cut 1 ml syringe and incubated at 37 ° c . for 45 min . ( fig1 ). subsequently , hydrogels were injected to 24 well tissue culture plate and incubated in growth medium . the medium was changed every other day thereafter . the viability of the cells encapsulated in hydrogels after 1 and 5 days &# 39 ; culture was investigated by the live / dead viability assay ( life technologies , catalog # l3224 ) according to the manufacturer &# 39 ; s instructions . media were removed from all the samples , washed twice with sterile pbs and stained with calcein - am and ethidium homodimer - 1 solution . samples in staining solution were incubated at room temperature for 30 min . imaging was performed with a confocal microscope ( nikon air ). mesenchymal stem cells ( mscs ) offer a promising cell source for musculoskeletal regenerative engineering due to their ease availability , high expansion capacity , and multipotency . thus , hmscs were encapsulated in the composite hydrogels and characterized for cell viability and cellular responses . confocal images using a live / dead assay demonstrated that all composite hydrogels supported progressive cellular growth with high viability (& gt ; 95 %) during cell culture ( fig1 and fig1 ). cells encapsulated within hydrogels displayed an overall spherical morphology , which is in line with previous literature reports for 3d hydrogel cell culture [ 26 ]. actin immunostaining was performed with the actin cytoskeleton and focal adhesion staining kit ( millipore ). briefly , cell / hydrogels were fixed in 4 % paraformaldehyde and blocked with 1 % bsa in pbs . then samples were stained with tritc - conjugated for phalloidin f - actin and with dapi for nuclei . imaging was performed with a confocal microscope ( nikon a1r ). interestingly , hmscs responded to differences in the mechanical properties of hydrogels as evidenced from the changes in actin cytoskeleton . in specific , hmscs encapsulated in hcp700 hydrogels showed less defined actin fibers as compared to those in hcp3400 and hcp8000 hydrogels ( fig1 ). furthermore , cortical actin protrusions into the surrounding hydrogel matrix were only observed with hmscs encapsulated in hcp3400 and hcp8000 hydrogels . hmsc cells were incubated with a cell permeable focal adhesion kinase ( fak ) phosphorylation biosensor and were washed gently with pbs ( 3 × 1 ml ). after fak biosensors were successfully delivered into hmscs , they were encapsulated in hcp hydrogels . the dynamic monitoring of focal adhesion kinase ( fak ) activity was conducted to examine cell - hydrogel interactions using fluorescence lifetime imaging microscopy ( flim )[ 27 ]. the dynamic monitoring of fak activity using flim confirmed a similar finding where stiffened hydrogels exhibited increased fak phosphorylation of hmscs ( fig1 ). it is important to note that cells in different tissues are tuned to the specific mechanical environments in which they reside [ 28 ]. the prepared hcp hydrogels were freeze - dried , sterilized for 20 min under uv light , and pre - incubated in the growth medium containing dmem ( life technologies , catalog # 11885084 ) which was supplemented with 10 % fetal bovine serum ( fbs , atlanta biologicals ), 1 % glutamine ( life technologies , catalog # 25030081 ), and 1 % penicillin / streptomycin ( life technologies , catalog # 15140122 ). hmscs were seeded on the surface of the lyophilized hydrogel scaffolds with a density of 20 , 000 cells / per scaffold and were pre - incubated at 37 ° c . with 5 % co 2 for 1 h . subsequently , the lyophilized hydrogel scaffolds seeded with hmscs were cultured in 500 μl growth medium at 37 ° c . with 5 % co 2 for 1 day . cell seeded hydrogel scaffolds were dehydrated sequentially with 50 , 70 , 80 , 90 , 95 , and 100 % ethanol for 10 minutes each and then were freezer dried . the samples were then cut to expose the cross - sectional area as well as the surface , and then sputter coated with platinum for 60 seconds . the hydrogels were examined on a scanning electron microscope ( nova nanosem ) at 3 . 0 kv . the lyophilized hcp hydrogel scaffolds were shown to have porous structures ( fig1 a , c ) and to be able to support cell growth on the surface of scaffolds ( fig1 a , b ). pore structure was roughly 40 to 50 μm in diameter . example 2 . ha / cs / poly ( ethylene glycol ) divinyl sulfone ( pegvs ) composite hydrogel as primary chondrocytes niches in all experiments , the molar ratio — sh /- ene concentration was held constant . specifically , 1 % ha - sh ( 192 μl ), 5 % cs — sh ( 199 . 2 μl ), and poly ( ethylene glycol ) divinyl sulfone ( pegvs ) 3400 ( 52 . 8 μl ) were mixed at 37 ° c . in the microcentrifuge tube to create ha / cs / pegvs 3400 ( hcpv3400 ). rheological experiments were carried out with a new discovery series hybrid rheometer ( dhr )- 3 ( ta ) using parallel plate ( 20 mm diameter , 0 °) in the oscillatory mode . oscillatory time , frequency , and strain sweeps were performed at 37 ° c ., and the storage ( g ′) and loss ( g ″) moduli were recorded . 450 μl of gel precursor solution was mixed by vortexing at room temperature for 15 s before loading on to the rheometer . hydrogels were cast between the lower peltier plate ( preheated at 37 ° c .) and upper parallel plate . the 20 mm parallel plate geometry was set to a gap of 1000 μm . each hydrogel sample was used for only one test . time sweep tests were performed under 1 % strain , 1 hz , and 37 ° c . conditions ( n = 4 ). the data represents the average of the three tests with corresponding standard deviation . due to more reactive vinyl sulfone moiety , pegvs demonstrates significantly shorter gelation time and higher storage modulus compared to pegda ( fig2 ). chondrocyte cultures were prepared from pig articular cartilage [ 29 ]. shavings of cartilage were removed from the outside of the articular cartilage , such that contamination with bone cells or other connective tissue cells could be avoided . the pig cartilage was finely chopped and the chondrocytes were released from their extracellular matrix by sequential digestion at 37 ° c . with collagenase . cells obtained from the collagenase digests were pooled and passed through a sterile 60 fan aperture nylon screen ( nitex ) to remove any undigested cartilage fragments . primary chondrocytes were cultured in growth medium containing dmem ( life technologies , catalog # 11885084 ) supplemented with 20 % fetal bovine serum ( fbs , atlanta biologicals ), and 1 % penicillin / streptomycin ( life technologies , catalog # 15140122 ) at 37 ° c . with 5 % co 2 . for the cell encapsulation in the hydrogels , cell pellets were obtained by centrifugation , followed by a brief resuspension with hydrogel solution through pipetting up and down gently to ensure a homogeneous distribution . then cell suspension with the density of 20 , 000 cells / 25 μl was transferred to an inverted pre - cut 1 ml syringe and incubated at 37 ° c . for 45 min . ( fig1 ). subsequently , hydrogels were injected to a 24 well tissue culture plate and incubated in growth medium . the medium was changed every other day thereafter . actin immunostaining was performed with the actin cytoskeleton and focal adhesion staining kit ( millipore ). briefly , cell / hydrogels were fixed in 4 % paraformaldehyde and blocked with 1 % bsa in pbs . then samples were stained with tritc - conjugated phalloidin for f - actin , with aggrecan antibody ( h - 300 ) ( santa cruz biotech ) for aggrecan , and with dapi for nuclei . imaging was performed with a confocal microscope ( nikon air ). confocal images using a live / dead assay demonstrated that all composite hydrogels supported cellular growth with high viability after a 21 - day culture ( fig2 a ). primary chondrocytes are easy to de - differentiate after passages . here we show that our hydrogel can maintain chondrocytesm morphology after 21 days indicated by the staining of aggrecan ( fig2 b ). example 3 : ha / cs / 4 - arm poly ( ethylene glycol ) divinyl sulfone ( 4pegvs ) composite hydrogel as hmsc niches in all experiments , the molar ratio — sh /- ene concentration was held constant . specifically , 1 % ha - sh ( 140 μl ), 5 % cs — sh ( 150 μl ), and 25 % 4 - arm peg ) divinyl sulfone ( 4pegvs ) 20k ( 113 μl ) were mixed in the 1 . 5 ml centrifuge tube to create ha / cs / 4pegvs 3400 ( hcpv3400 ). rheological experiments were carried out with a new discovery series hybrid rheometer ( dhr )- 3 ( ta ) using parallel plate ( 20 mm diameter , 0 °) in the oscillatory mode . oscillatory time , frequency , and strain sweeps were performed at 37 ° c ., and the storage ( g ′) and loss ( g ″) moduli were recorded . 450 μl of gel precursor solution was mixed by vortexing at room temperature for 15 s before loading on to the rheometer . hydrogels were cast between the lower peltier plate ( preheated at 37 ° c .) and upper parallel plate . the 20 mm parallel plate geometry was set to a gap of 1000 μm . each hydrogel sample was used for only one test . time sweep tests were performed under 1 % strain , 1 hz , and 37 ° c . conditions ( n = 4 ). the data represents the average of the three tests with corresponding standard deviation . compared with 2 - arm pegvs , 4 - arm pegvs demonstrates significantly shorter gelation time and higher storage modulus compared to pegda ( fig2 ). hmscs were cultured in growth medium at 37 ° c . with 5 % co 2 . for the cell encapsulation in the hydrogels , cell pellets were obtained by centrifugation , followed by a brief resuspension with hydrogel solution through pipetting up and down gently to ensure a homogeneous distribution . then the cell suspension with a density of 20 , 000 cells / 25 μl was transferred to an inverted pre - cut 1 ml syringe and incubated at 37 ° c . for 30 min . ( fig1 ). subsequently , hydrogels were injected to a 24 well tissue culture plate and incubated in growth medium . the medium was changed every other day thereafter . confocal images using a live / dead assay demonstrated that all composite hydrogels supported cellular growth with high viability (& gt ; 95 %) after 21 days ( fig2 a ). cells encapsulated within hydrogels displayed an overall spherical morphology ( fig2 b ), which is in line with previous literature reports for 3d hydrogel cell culture [ 26 ]. treatment of osteochondral defects encompassing injury or degeneration to both the articular cartilage as well as the underlying subchondral bone presents a significant medical challenge . current treatment options including autografts and allografts suffer from limited availability and risk of immunogenicity , respectively . the long term goal of this work is to develop an integrated scaffold system for treatment of osteochondral defects via in situ regeneration of bone , cartilage and the bone - cartilage interface ( fig2 ). hydrogels provide an attractive biomaterial platform for regeneration of cartilage . in the present disclosure , we have developed a novel composite hydrogel that includes thiolated ha and cs cross - linked with peg to mimic cartilage . the bone mimetic is based on plga / nano - hydroxyapatite composite scaffolds [ 30 ]. here , integration between hydrogels and plga 3d scaffolds was achieved using a novel multifunctional ha . ha was chemically functionalized with both — sh and — nh 2 groups to form two functional arms : one arm to covalently bond to a cross - linker to form hydrogels and the second arm bonding to the plga 3d scaffold surface . this multifunctional ha provides a biologically active and mechanically functional bridge with scaffolds . poly ( lactide - co - glycolide ) ( plga ) 85 : 15 was purchased from lakeshore biomaterials . plga microspheres were obtained using the emulsion - solvent evaporation method . plga 3d scaffolds were fabricated by heat sintering at an optimized condition [ 31 ]. the ha - sh , cs — sh and pegda solution ( pbs ) was placed onto plga 3d scaffold surface for 45 min at 37 ° c . plga 3d scaffold - hydrogel integration was lyophilized and then sputter coated with platinum for 60 seconds . the integration were examined on a scanning electron microscope ( nova nanosem ) at 3 . 0 kv . sem images of plga 3d scaffolds - hydrogel integration was shown in fig2 . differences among groups were assessed by one - way anova with bonferroni post hoc correction to identify statistical differences among three treatments . a p - value of 0 . 05 was set as the criteria for statistical significance . graphs are annotated where values are represented as * 0 . 05 . additional disclosure is found in appendix - a , filed herewith , entirety of which is incorporated herein by reference into the present disclosure . those skilled in the art will recognize that numerous modifications can be made to the specific implementations described above . the implementations should not be limited to the particular limitations described . other implementations may be possible . in addition , all references cited herein are indicative of the level of skill in the art and are hereby incorporated by reference in their entirety . deng m , james r , laurencin c t , kumbar s g . nanostructured polymeric scaffolds for orthopaedic regenerative engineering . nanobioscience , ieee transactions on . 2012 ; 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4 : 2 .
disclosed herein is a cross - linked polymeric system comprising thiolated hyaluronic acid , thiolated chondroitin sulfate , and functionalized polyethylene glycol , wherein said functionalized peg cross - links thiolated ha and thiolated cs . methods of fabrication and utilization of the same are also claimed . this polymeric system may be used as an engineered biocompatible cellular matrix for 3d cell culture , tissue engineering and regenerative medicine applications .